The present invention provides compositions, kits and methods for rapid identification and quantification of bacteria by molecular mass and base composition analysis.

Description

51 parts

›RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No. 11/409,535, filed April 21, 2006, which is a continuation-in-part of U.S. application Ser. No. 11/060,135, filed Feb. 17, 2005 which claims the benefit of priority to U.S. Provisional Application Ser. No. 60/545,425 filed Feb. 18, 2004; U.S. Provisional Application Ser. No. 60/559,754, filed Apr. 5, 2004; U.S. Provisional Application Ser. No. 60/632,862, filed Dec. 3, 2004; U.S. Provisional Application Ser. No. 60/639,068, filed Dec. 22, 2004; and U.S. Provisional Application Ser. No. 60/648,188, filed Jan. 28, 2005. U.S. application Ser. No. 11/409,535 is a also continuation-in-part of U.S. application Ser. No. 10/728,486, filed Dec. 5, 2003 now U.S. Pat. No. 7,718,354 which claims the benefit of priority to U.S. Provisional Application Ser. No. 60/501,926, filed Sep. 11, 2003. U.S. application Ser. No. 11/409,535 also claims the benefit of priority to: U.S. Provisional Application Ser. No. 60/674,118, filed Apr. 21, 2005; U.S. Provisional Application Ser. No. 60/705,631, filed Aug. 3, 2005; U.S. Provisional Application Ser. No. 60/732,539, filed Nov. 1, 2005; and U.S. Provisional Application Ser. No. 60/773,124, filed Feb. 13, 2006. Each of the above-referenced U.S. Applications is incorporated herein by reference in its entirety. Methods disclosed in U.S. application Ser. Nos. 09/891,793, 10/156,608, 10/405,756, 10/418,514, 10/660,122, 10/660,996, 10/660,997, 10/660,998, 10/728,486, 11/060,135, and 11/073,362, are commonly owned and incorporated herein by reference in their entirety for any purpose.

›STATEMENT OF GOVERNMENT SUPPORT

This invention was made with United States Government support under CDC contracts RO1 CI000099-01. The United States Government has certain rights in the invention.

›SEQUENCE LISTING

The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled DIBIS0083USC3SEQ.txt, created on Mar. 6, 2007 which is 252 Kb in size. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety.

›FIELD OF THE INVENTION

The present invention provides compositions, kits and methods for rapid identification and quantification of bacteria by molecular mass and base composition analysis.

›BACKGROUND OF THE INVENTION

A problem in determining the cause of a natural infectious outbreak or a bioterrorist attack is the sheer variety of organisms that can cause human disease. There are over 1400 organisms infectious to humans; many of these have the potential to emerge suddenly in a natural epidemic or to be used in a malicious attack by bioterrorists (Taylor et al. Philos. Trans. R. Soc. London B. Biol. Sci., 2001, 356, 983-989). This number does not include numerous strain variants, bioengineered versions, or pathogens that infect plants or animals.

Much of the new technology being developed for detection of biological weapons incorporates a polymerase chain reaction (PCR) step based upon the use of highly specific primers and probes designed to selectively detect certain pathogenic organisms. Although this approach is appropriate for the most obvious bioterrorist organisms, like smallpox and anthrax, experience has shown that it is very difficult to predict which of hundreds of possible pathogenic organisms might be employed in a terrorist attack. Likewise, naturally emerging human disease that has caused devastating consequence in public health has come from unexpected families of bacteria, viruses, fungi, or protozoa. Plants and animals also have their natural burden of infectious disease agents and there are equally important biosafety and security concerns for agriculture.

A major conundrum in public health protection, biodefense, and agricultural safety and security is that these disciplines need to be able to rapidly identify and characterize infectious agents, while there is no existing technology with the breadth of function to meet this need. Currently used methods for identification of bacteria rely upon culturing the bacterium to effect isolation from other organisms and to obtain sufficient quantities of nucleic acid followed by sequencing of the nucleic acid, both processes which are time and labor intensive.

Mass spectrometry provides detailed information about the molecules being analyzed, including high mass accuracy. It is also a process that can be easily automated. DNA chips with specific probes can only determine the presence or absence of specifically anticipated organisms. Because there are hundreds of thousands of species of benign bacteria, some very similar in sequence to threat organisms, even arrays with 10,000 probes lack the breadth needed to identify a particular organism.

The present invention provides oligonucleotide primers and compositions and kits containing the oligonucleotide primers, which define bacterial bioagent identifying amplicons and, upon amplification, produce corresponding amplification products whose molecular masses provide the means to identify bacteria, for example, at and below the species taxonomic level.

›SUMMARY OF THE INVENTION · 1 of 8

The present invention provides compositions, kits and methods for rapid identification and quantification of bacteria by molecular mass and base composition analysis.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 456.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1261.

Another embodiment is an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 456 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1261.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 288.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1269.

Another embodiment is an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 288 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1269.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 698.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1420.

Another embodiment is an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 698 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1420.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 217.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1167

Another embodiment is an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 217 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1167.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 399.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1041.

Another embodiment is an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 399 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1041.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 430.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1321.

Another embodiment is an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 430 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1321.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 174.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 853.

Another embodiment is an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 174 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 853.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 172.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1360.

Another embodiment is an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 172 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1360.

Another embodiment is a kit comprising an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 456 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1261 and further comprising one or more primer pairs wherein each member of said one or more primer pairs is of a length of 14 to 35 nucleobases and has 70% to 100% sequence identity with the corresponding member from the group of primer pairs represented by SEQ ID NOs: 288:1269, 698:1420, 217:1167, 399:1041, 430:1321, 174:853, and 172:1360.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 681.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1022.

Another embodiment is an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 681 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1022.

›SUMMARY OF THE INVENTION · 2 of 8

One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 315.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1379.

Another embodiment is an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 315 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1379.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 346.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 955.

Another embodiment is an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 346 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 955.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 504.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1409.

Another embodiment is an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 504 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1409.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 323.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1068.

Another embodiment is an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 323 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1068.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 479.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 938.

Another embodiment is an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 479 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 938.

Another embodiment is a kit comprising an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 681 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1022 and further comprising one or more primer pairs wherein each member of said one or more primer pairs is of a length of 14 to 35 nucleobases and has 70% to 100% sequence identity with the corresponding member from the group of primer pairs represented by SEQ ID NOs: 315:1379, 346:955, 504:1409, 323:1068, 479:938.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 583.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 923.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 454.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1418.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 250.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 902.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 384.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 878.

›SUMMARY OF THE INVENTION · 3 of 8

One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 694.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1215.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 194.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1173.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 375.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 890.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 656.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1224.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 618.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1157.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 302.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 852.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 199.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 889.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 596.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1169.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 150.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1242.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 166.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1069.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 166.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1168.

›SUMMARY OF THE INVENTION · 4 of 8

Another embodiment is a kit comprising an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 583 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 923 and further comprising one or more primer pairs wherein each member of said one or more primer pairs is of a length of 14 to 35 nucleobases and has 70% to 100% sequence identity with the corresponding member from the group of primer pairs represented by SEQ ID NOs: 454:1418, 250:902, 384:878, 694:1215, 194:1173, 375:890, 656:1224, 618:1157, 302:852, 199:889, 596:1169, 150:1242, 166:1069 and 166:1168.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 437.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1137.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 530.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 891.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 474.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 869.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 268.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1284.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 418.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1301.

An embodiment is an oligonucleotide primer pair comprising a forward and a reverse primer, each comprising between 13 and 35 linked nucleotides in length, designed to generate an amplicon that is between about 45 and about 200 linked nucleotides in length, wherein said forward primer comprises at least 80% complementarity to a first region within nucleotides 1-286 of a reference sequence, said reference sequence being a sequence extraction of coordinates 830671-831072 of Genbank gi number 21281729 (ttccacgaaacagatgaa gaaattaacaaaaaagcgcacgctattttcaaacatggaatgactccaattatttgtgttggtgaaacagacgaagagc gtgaaagtggtaaagctaacgatgttgtaggtgagcaagttaagaaagctgttgcaggtttatctgaagatcaacttaa atcagttgtaattgcttatgagccaatctgggcaatcggaactggtaaatcatcaacatctgaagatgcaaatgaaatgt gtgcatttgtacgtcaaactattgctgacttatcaagcaaagaagtatcagaagcaactcgtattcaatatggtggtagtg ttaaacctaacaacattaaagaatacatggcacaaactgatattgatggggcattagtaggtggc (SEQ ID NO.: 1465)), and wherein said reverse primer comprises at least 80% complementarity to a second region within nucleotides 1-286 of said reference sequence.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 318.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1300.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 440.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1076.

›SUMMARY OF THE INVENTION · 5 of 8

One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 219.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1013.

Another embodiment is a kit comprising an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 437 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1137 and further comprising one or more primer pairs wherein each member of said one or more primer pairs is of a length of 14 to 35 nucleobases and has 70% to 100% sequence identity with the corresponding member from the group of primer pairs represented by SEQ ID NOs: 530:891, 474:869, 268:1284, 418:1301, 318:1300, 440:1076 and 219:1013.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 325.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1163.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 278.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1039.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 465.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1037.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 148.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1172.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 190.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1254.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 266.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1094.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 508.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1297.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 259.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1060.

Another embodiment is a kit comprising an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 325 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1163 and further comprising one or more primer pairs wherein each member of said one or more primer pairs is of a length of 14 to 35 nucleobases and has 70% to 100% sequence identity with the corresponding member from the group of primer pairs represented by SEQ ID NOs: 278:1039: 465:1037, 148:1172, 190:1254, 266:1094, 508:1297 and 259:1060.

›SUMMARY OF THE INVENTION · 6 of 8

One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 376.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1265.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 267.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1341.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 705.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1056.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 710.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1259.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 374.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1111.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 545.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 978.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 249.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1095.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 195.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1376.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 311.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1014.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 365.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1052.

One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 527.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1071.

›SUMMARY OF THE INVENTION · 7 of 8

One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 490.

Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1182.

Another embodiment is a kit comprising an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 376 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1265 and further comprising one or more primer pairs wherein each member of said one or more primer pairs is of a length of 14 to 35 nucleobases and has 70% to 100% sequence identity with the corresponding member from the group of primer pairs represented by SEQ ID NOs: 267:1341, 705:1056, 710:1259, 374:1111, 545:978, 249:1095, 195:1376, 311:1014, 365:1052, 527:1071 and 490:1182.

In some embodiments, either or both of the primers of a primer pair composition contain at least one modified nucleobase such as 5-propynyluracil or 5-propynylcytosine for example.

In some embodiments, either or both of the primers of the primer pair comprises at least one universal nucleobase such as inosine for example.

In some embodiments, either or both of the primers of the primer pair comprises at least one non-templated T residue on the 5′-end.

In some embodiments, either or both of the primers of the primer pair comprises at least one non-template tag.

In some embodiments, either or both of the primers of the primer pair comprises at least one molecular mass modifying tag.

In some embodiments, the present invention provides primers and compositions comprising pairs of primers, and kits containing the same, and methods for use in identification of bacteria. The primers are designed to produce amplification products of DNA encoding genes that have conserved and variable regions across different subgroups and genotypes of bacteria.

Some embodiments are kits that contain one or more of the primer pair compositions. In some embodiments, each member of the one or more primer pairs of the kit is of a length of 14 to 35 nucleobases and has 70% to 100% sequence identity with the corresponding member from any of the primer pairs listed in Table 2.

Some embodiments of the kits contain at least one calibration polynucleotide for use in quantitiation of bacteria in a given sample, and also for use as a positive control for amplification.

Some embodiments of the kits contain at least one anion exchange functional group linked to a magnetic bead.

In some embodiments, the present invention also provides methods for identification of bacteria. Nucleic acid from the bacterium is amplified using the primers described above to obtain an amplification product. The molecular mass of the amplification product is measured. Optionally, the base composition of the amplification product is determined from the molecular mass. The molecular mass or base composition is compared with a plurality of molecular masses or base compositions of known analogous bacterial identifying amplicons, wherein a match between the molecular mass or base composition and a member of the plurality of molecular masses or base compositions identifies the bacterium. In some embodiments, the molecular mass is measured by mass spectrometry in a modality such as electrospray ionization (ESI) time of flight (TOF) mass spectrometry or ESI Fourier transform ion cyclotron resonance (FTICR) mass spectrometry, for example. Other mass spectrometry techniques can also be used to measure the molecular mass of bacterial bioagent identifying amplicons.

In some embodiments, the present invention is also directed to a method for determining the presence or absence of a bacterium in a sample. Nucleic acid from the sample is amplified using the composition described above to obtain an amplification product. The molecular mass of the amplification product is determined. Optionally, the base composition of the amplification product is determined from the molecular mass. The molecular mass or base composition of the amplification product is compared with the known molecular masses or base compositions of one or more known analogous bacterial bioagent identifying amplicons, wherein a match between the molecular mass or base composition of the amplification product and the molecular mass or base composition of one or more known bacterial bioagent identifying amplicons indicates the presence of the bacterium in the sample. In some embodiments, the molecular mass is measured by mass spectrometry.

In some embodiments, the present invention also provides methods for determination of the quantity of an unknown bacterium in a sample. The sample is contacted with the composition described above and a known quantity of a calibration polynucleotide comprising a calibration sequence. Nucleic acid from the unknown bacterium in the sample is concurrently amplified with the composition described above and nucleic acid from the calibration polynucleotide in the sample is concurrently amplified with the composition described above to obtain a first amplification product comprising a bacterial bioagent identifying amplicon and a second amplification product comprising a calibration amplicon. The molecular masses and abundances for the bacterial bioagent identifying amplicon and the calibration amplicon are determined. The bacterial bioagent identifying amplicon is distinguished from the calibration amplicon based on molecular mass and comparison of bacterial bioagent identifying amplicon abundance and calibration amplicon abundance indicates the quantity of bacterium in the sample. In some embodiments, the base composition of the bacterial bioagent identifying amplicon is determined.

›SUMMARY OF THE INVENTION · 8 of 8

In some embodiments, the present invention provides methods for detecting or quantifying bacteria by combining a nucleic acid amplification process with a mass determination process. In some embodiments, such methods identify or otherwise analyze the bacterium by comparing mass information from an amplification product with a calibration or control product. Such methods can be carried out in a highly multiplexed and/or parallel manner allowing for the analysis of as many as 300 samples per 24 hours on a single mass measurement platform. The accuracy of the mass determination methods in some embodiments of the present invention permits allows for the ability to discriminate between different bacteria such as, for example, various genotypes and drug resistant strains of Staphylococcus aureus.

›BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing summary of the invention, as well as the following detailed description of the invention, is better understood when read in conjunction with the accompanying drawings which are included by way of example and not by way of limitation.

FIG. 1 : process diagram illustrating a representative primer pair selection process.

FIG. 2 : process diagram illustrating an embodiment of the calibration method.

FIG. 3 : common pathogenic bacteria and primer pair coverage. The primer pair number in the upper right hand corner of each polygon indicates that the primer pair can produce a bioagent identifying amplicon for all species within that polygon.

FIG. 4 : a representative 3D diagram of base composition (axes A, G and C) of bioagent identifying amplicons obtained with primer pair number 14 (a precursor of primer pair number 348 which targets 16S rRNA). The diagram indicates that the experimentally determined base compositions of the clinical samples (labeled NHRC samples) closely match the base compositions expected for Streptococcus pyogenes and are distinct from the expected base compositions of other organisms.

FIG. 5 : a representative mass spectrum of amplification products indicating the presence of bioagent identifying amplicons of Streptococcus pyogenes, Neisseria meningitidis , and Haemophilus influenzae obtained from amplification of nucleic acid from a clinical sample with primer pair number 349 which targets 23S rRNA. Experimentally determined molecular masses and base compositions for the sense strand of each amplification product are shown.

FIG. 6 : a representative mass spectrum of amplification products representing a bioagent identifying amplicon of Streptococcus pyogenes , and a calibration amplicon obtained from amplification of nucleic acid from a clinical sample with primer pair number 356 which targets rplB. The experimentally determined molecular mass and base composition for the sense strand of the Streptococcus pyogenes amplification product is shown.

FIG. 7 : a representative mass spectrum of an amplified nucleic acid mixture which contained the Ames strain of Bacillus anthracis , a known quantity of combination calibration polynucleotide (SEQ ID NO: 1464), and primer pair number 350 which targets the capC gene on the virulence plasmid pX02 of Bacillus anthracis . Calibration amplicons produced in the amplification reaction are visible in the mass spectrum as indicated and abundance data (peak height) are used to calculate the quantity of the Ames strain of Bacillus anthracis.

›DEFINITIONS · 1 of 8

As used herein, the term “abundance” refers to an amount. The amount may be described in terms of concentration which are common in molecular biology such as “copy number,” “pfu or plate-forming unit” which are well known to those with ordinary skill. Concentration may be relative to a known standard or may be absolute.

As used herein, the term “amplifiable nucleic acid” is used in reference to nucleic acids that may be amplified by any amplification method. It is contemplated that “amplifiable nucleic acid” also comprises “sample template.”

As used herein the term “amplification” refers to a special case of nucleic acid replication involving template specificity. It is to be contrasted with non-specific template replication (i.e., replication that is template-dependent but not dependent on a specific template). Template specificity is here distinguished from fidelity of replication (i.e., synthesis of the proper polynucleotide sequence) and nucleotide (ribo- or deoxyribo-) specificity. Template specificity is frequently described in terms of “target” specificity. Target sequences are “targets” in the sense that they are sought to be sorted out from other nucleic acid. Amplification techniques have been designed primarily for this sorting out. Template specificity is achieved in most amplification techniques by the choice of enzyme. Amplification enzymes are enzymes that, under conditions they are used, will process only specific sequences of nucleic acid in a heterogeneous mixture of nucleic acid. For example, in the case of Qβ replicase, MDV-1 RNA is the specific template for the replicase (D. L. Kacian et al., Proc. Natl. Acad. Sci. USA 69:3038 [1972]). Other nucleic acid will not be replicated by this amplification enzyme. Similarly, in the case of T7 RNA polymerase, this amplification enzyme has a stringent specificity for its own promoters (Chamberlin et al., Nature 228:227 [1970]). In the case of T4 DNA ligase, the enzyme will not ligate the two oligonucleotides or polynucleotides, where there is a mismatch between the oligonucleotide or polynucleotide substrate and the template at the ligation junction (D. Y. Wu and R. B. Wallace, Genomics 4:560 [1989]). Finally, Taq and Pfu polymerases, by virtue of their ability to function at high temperature, are found to display high specificity for the sequences bounded and thus defined by the primers; the high temperature results in thermodynamic conditions that favor primer hybridization with the target sequences and not hybridization with non-target sequences (H. A. Erlich (ed.), PCR Technology, Stockton Press [1989]).

As used herein, the term “amplification reagents” refers to those reagents (deoxyribonucleotide triphosphates, buffer, etc.), needed for amplification, excluding primers, nucleic acid template, and the amplification enzyme. Typically, amplification reagents along with other reaction components are placed and contained in a reaction vessel (test tube, microwell, etc.).

As used herein, the term “analogous” when used in context of comparison of bioagent identifying amplicons indicates that the bioagent identifying amplicons being compared are produced with the same pair of primers. For example, bioagent identifying amplicon “A” and bioagent identifying amplicon “B”, produced with the same pair of primers are analogous with respect to each other. Bioagent identifying amplicon “C”, produced with a different pair of primers is not analogous to either bioagent identifying amplicon “A” or bioagent identifying amplicon “B”.

As used herein, the term “anion exchange functional group” refers to a positively charged functional group capable of binding an anion through an electrostatic interaction. The most well known anion exchange functional groups are the amines, including primary, secondary, tertiary and quaternary amines.

The term “bacteria” or “bacterium” refers to any member of the groups of eubacteria and archaebacteria.

As used herein, a “base composition” is the exact number of each nucleobase (for example, A, T, C and G) in a segment of nucleic acid. For example, amplification of nucleic acid of Staphylococcus aureus strain carrying the lukS-PV gene with primer pair number 2095 (SEQ ID NOs: 456:1261) produces an amplification product 117 nucleobases in length from nucleic acid of the lukS-PV gene that has a base composition of A35 G17 C19 T46 (by convention—with reference to the sense strand of the amplification product). Because the molecular masses of each of the four natural nucleotides and chemical modifications thereof are known (if applicable), a measured molecular mass can be deconvoluted to a list of possible base compositions. Identification of a base composition of a sense strand which is complementary to the corresponding antisense strand in terms of base composition provides a confirmation of the true base composition of an unknown amplification product. For example, the base composition of the antisense strand of the 139 nucleobase amplification product described above is A46 G19 C17 T35.

As used herein, a “base composition probability cloud” is a representation of the diversity in base composition resulting from a variation in sequence that occurs among different isolates of a given species. The “base composition probability cloud” represents the base composition constraints for each species and is typically visualized using a pseudo four-dimensional plot.

In the context of this invention, a “bioagent” is any organism, cell, or virus, living or dead, or a nucleic acid derived from such an organism, cell or virus. Examples of bioagents include, but are not limited, to cells, (including but not limited to human clinical samples, bacterial cells and other pathogens), viruses, fungi, protists, parasites, and pathogenicity markers (including but not limited to: pathogenicity islands, antibiotic resistance genes, virulence factors, toxin genes and other bioregulating compounds). Samples may be alive or dead or in a vegetative state (for example, vegetative bacteria or spores) and may be encapsulated or bioengineered. In the context of this invention, a “pathogen” is a bioagent which causes a disease or disorder.

›DEFINITIONS · 2 of 8

As used herein, a “bioagent division” is defined as group of bioagents above the species level and includes but is not limited to, orders, families, classes, clades, genera or other such groupings of bioagents above the species level.

As used herein, the term “bioagent identifying amplicon” refers to a polynucleotide that is amplified from a bioagent in an amplification reaction and which 1) provides sufficient variability to distinguish among bioagents from whose nucleic acid the bioagent identifying amplicon is produced and 2) whose molecular mass is amenable to a rapid and convenient molecular mass determination modality such as mass spectrometry, for example.

As used herein, the term “biological product” refers to any product originating from an organism. Biological products are often products of processes of biotechnology. Examples of biological products include, but are not limited to: cultured cell lines, cellular components, antibodies, proteins and other cell-derived biomolecules, growth media, growth harvest fluids, natural products and bio-pharmaceutical products.

The terms “biowarfare agent” and “bioweapon” are synonymous and refer to a bacterium, virus, fungus or protozoan that could be deployed as a weapon to cause bodily harm to individuals. Military or terrorist groups may be implicated in deployment of biowarfare agents.

In context of this invention, the term “broad range survey primer pair” refers to a primer pair designed to produce bioagent identifying amplicons across different broad groupings of bioagents. For example, the ribosomal RNA-targeted primer pairs are broad range survey primer pairs which have the capability of producing bacterial bioagent identifying amplicons for essentially all known bacteria. With respect to broad range primer pairs employed for identification of bacteria, a broad range survey primer pair for bacteria such as 16S rRNA primer pair number 346 (SEQ ID NOs: 202:1110) for example, will produce an bacterial bioagent identifying amplicon for essentially all known bacteria.

The term “calibration amplicon” refers to a nucleic acid segment representing an amplification product obtained by amplification of a calibration sequence with a pair of primers designed to produce a bioagent identifying amplicon.

The term “calibration sequence” refers to a polynucleotide sequence to which a given pair of primers hybridizes for the purpose of producing an internal (i.e.: included in the reaction) calibration standard amplification product for use in determining the quantity of a bioagent in a sample. The calibration sequence may be expressly added to an amplification reaction, or may already be present in the sample prior to analysis.

The term “clade primer pair” refers to a primer pair designed to produce bioagent identifying amplicons for species belonging to a clade group. A clade primer pair may also be considered as a “speciating” primer pair which is useful for distinguishing among closely related species.

The term “codon” refers to a set of three adjoined nucleotides (triplet) that codes for an amino acid or a termination signal.

In context of this invention, the term “codon base composition analysis,” refers to determination of the base composition of an individual codon by obtaining a bioagent identifying amplicon that includes the codon. The bioagent identifying amplicon will at least include regions of the target nucleic acid sequence to which the primers hybridize for generation of the bioagent identifying amplicon as well as the codon being analyzed, located between the two primer hybridization regions.

As used herein, the terms “complementary” or “complementarity” are used in reference to polynucleotides (i.e., a sequence of nucleotides such as an oligonucleotide or a target nucleic acid) related by the base-pairing rules. For example, for the sequence “5′-A-G-T-3′,” is complementary to the sequence “3′-T-C-A-5′.” Complementarity may be “partial,” in which only some of the nucleic acids' bases are matched according to the base pairing rules. Or, there may be “complete” or “total” complementarity between the nucleic acids. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands. This is of particular importance in amplification reactions, as well as detection methods that depend upon binding between nucleic acids. Either term may also be used in reference to individual nucleotides, especially within the context of polynucleotides. For example, a particular nucleotide within an oligonucleotide may be noted for its complementarity, or lack thereof, to a nucleotide within another nucleic acid strand, in contrast or comparison to the complementarity between the rest of the oligonucleotide and the nucleic acid strand.

The term “complement of a nucleic acid sequence” as used herein refers to an oligonucleotide which, when aligned with the nucleic acid sequence such that the 5′ end of one sequence is paired with the 3′ end of the other, is in “antiparallel association.” Certain bases not commonly found in natural nucleic acids may be included in the nucleic acids of the present invention and include, for example, inosine and 7-deazaguanine. Complementarity need not be perfect; stable duplexes may contain mismatched base pairs or unmatched bases. Those skilled in the art of nucleic acid technology can determine duplex stability empirically considering a number of variables including, for example, the length of the oligonucleotide, base composition and sequence of the oligonucleotide, ionic strength and incidence of mismatched base pairs. Where a first oligonucleotide is complementary to a region of a target nucleic acid and a second oligonucleotide has complementary to the same region (or a portion of this region) a “region of overlap” exists along the target nucleic acid. The degree of overlap will vary depending upon the extent of the complementarity.

In context of this invention, the term “division-wide primer pair” refers to a primer pair designed to produce bioagent identifying amplicons within sections of a broader spectrum of bioagents For example, primer pair number 352 (SEQ ID NOs: 687:1411), a division-wide primer pair, is designed to produce bacterial bioagent identifying amplicons for members of the Bacillus group of bacteria which comprises, for example, members of the genera Streptococci, Enterococci , and Staphylococci . Other division-wide primer pairs may be used to produce bacterial bioagent identifying amplicons for other groups of bacterial bioagents.

›DEFINITIONS · 3 of 8

As used herein, the term “concurrently amplifying” used with respect to more than one amplification reaction refers to the act of simultaneously amplifying more than one nucleic acid in a single reaction mixture.

As used herein, the term “drill-down primer pair” refers to a primer pair designed to produce bioagent identifying amplicons for identification of sub-species characteristics or confirmation of a species assignment. For example, primer pair number 2146 (SEQ ID NOs: 437:1137), a drill-down Staphylococcus aureus genotyping primer pair, is designed to produce Staphylococcus aureus genotyping amplicons. Other drill-down primer pairs may be used to produce bioagent identifying amplicons for Staphylococcus aureus and other bacterial species.

The term “duplex” refers to the state of nucleic acids in which the base portions of the nucleotides on one strand are bound through hydrogen bonding the their complementary bases arrayed on a second strand. The condition of being in a duplex form reflects on the state of the bases of a nucleic acid. By virtue of base pairing, the strands of nucleic acid also generally assume the tertiary structure of a double helix, having a major and a minor groove. The assumption of the helical form is implicit in the act of becoming duplexed.

As used herein, the term “etiology” refers to the causes or origins, of diseases or abnormal physiological conditions.

The term “gene” refers to a DNA sequence that comprises control and coding sequences necessary for the production of an RNA having a non-coding function (e.g., a ribosomal or transfer RNA), a polypeptide or a precursor. The RNA or polypeptide can be encoded by a full length coding sequence or by any portion of the coding sequence so long as the desired activity or function is retained.

The terms “homology,” “homologous” and “sequence identity” refer to a degree of identity. There may be partial homology or complete homology. A partially homologous sequence is one that is less than 100% identical to another sequence. Determination of sequence identity is described in the following example: a primer 20 nucleobases in length which is otherwise identical to another 20 nucleobase primer but having two non-identical residues has 18 of 20 identical residues (18/20=0.9 or 90% sequence identity). In another example, a primer 15 nucleobases in length having all residues identical to a 15 nucleobase segment of a primer 20 nucleobases in length would have 15/20=0.75 or 75% sequence identity with the 20 nucleobase primer. In context of the present invention, sequence identity is meant to be properly determined when the query sequence and the subject sequence are both described and aligned in the 5′ to 3′ direction. Sequence alignment algorithms such as BLAST, will return results in two different alignment orientations. In the Plus/Plus orientation, both the query sequence and the subject sequence are aligned in the 5′ to 3′ direction. On the other hand, in the Plus/Minus orientation, the query sequence is in the 5′ to 3′ direction while the subject sequence is in the 3′ to 5′ direction. It should be understood that with respect to the primers of the present invention, sequence identity is properly determined when the alignment is designated as Plus/Plus. Sequence identity may also encompass alternate or modified nucleobases that perform in a functionally similar manner to the regular nucleobases adenine, thymine, guanine and cytosine with respect to hybridization and primer extension in amplification reactions. In a non-limiting example, if the 5-propynyl pyrimidines propyne C and/or propyne T replace one or more C or T residues in one primer which is otherwise identical to another primer in sequence and length, the two primers will have 100% sequence identity with each other. In another non-limiting example, Inosine (I) may be used as a replacement for G or T and effectively hybridize to C, A or U (uracil). Thus, if inosine replaces one or more C, A or U residues in one primer which is otherwise identical to another primer in sequence and length, the two primers will have 100% sequence identity with each other. Other such modified or universal bases may exist which would perform in a functionally similar manner for hybridization and amplification reactions and will be understood to fall within this definition of sequence identity.

As used herein, “housekeeping gene” refers to a gene encoding a protein or RNA involved in basic functions required for survival and reproduction of a bioagent. Housekeeping genes include, but are not limited to genes encoding RNA or proteins involved in translation, replication, recombination and repair, transcription, nucleotide metabolism, amino acid metabolism, lipid metabolism, energy generation, uptake, secretion and the like.

As used herein, the term “hybridization” is used in reference to the pairing of complementary nucleic acids. Hybridization and the strength of hybridization (i.e., the strength of the association between the nucleic acids) is influenced by such factors as the degree of complementary between the nucleic acids, stringency of the conditions involved, and the T m of the formed hybrid. “Hybridization” methods involve the annealing of one nucleic acid to another, complementary nucleic acid, i.e., a nucleic acid having a complementary nucleotide sequence. The ability of two polymers of nucleic acid containing complementary sequences to find each other and anneal through base pairing interaction is a well-recognized phenomenon. The initial observations of the “hybridization” process by Marmur and Lane, Proc. Natl. Acad. Sci. USA 46:453 (1960) and Doty et al., Proc. Natl. Acad. Sci. USA 46:461 (1960) have been followed by the refinement of this process into an essential tool of modern biology.

The term “in silico” refers to processes taking place via computer calculations. For example, electronic PCR (ePCR) is a process analogous to ordinary PCR except that it is carried out using nucleic acid sequences and primer pair sequences stored on a computer formatted medium.

›DEFINITIONS · 4 of 8

As used herein, “intelligent primers” are primers that are designed to bind to highly conserved sequence regions of a bioagent identifying amplicon that flank an intervening variable region and, upon amplification, yield amplification products which ideally provide enough variability to distinguish individual bioagents, and which are amenable to molecular mass analysis. By the term “highly conserved,” it is meant that the sequence regions exhibit between about 80-100%, or between about 90-100%, or between about 95-100% identity among all, or at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% of species or strains.

The “ligase chain reaction” (LCR; sometimes referred to as “Ligase Amplification Reaction” (LAR) described by Barany, Proc. Natl. Acad. Sci., 88:189 (1991); Barany, PCR Methods and Applic., 1:5 (1991); and Wu and Wallace, Genomics 4:560 (1989) has developed into a well-recognized alternative method for amplifying nucleic acids. In LCR, four oligonucleotides, two adjacent oligonucleotides which uniquely hybridize to one strand of target DNA, and a complementary set of adjacent oligonucleotides, that hybridize to the opposite strand are mixed and DNA ligase is added to the mixture. Provided that there is complete complementarity at the junction, ligase will covalently link each set of hybridized molecules. Importantly, in LCR, two probes are ligated together only when they base-pair with sequences in the target sample, without gaps or mismatches. Repeated cycles of denaturation, hybridization and ligation amplify a short segment of DNA. LCR has also been used in combination with PCR to achieve enhanced detection of single-base changes. However, because the four oligonucleotides used in this assay can pair to form two short ligatable fragments, there is the potential for the generation of target-independent background signal. The use of LCR for mutant screening is limited to the examination of specific nucleic acid positions.

The term “locked nucleic acid” or “LNA” refers to a nucleic acid analogue containing one or more 2′-O, 4′-C-methylene-β-D-ribofuranosyl nucleotide monomers in an RNA mimicking sugar conformation. LNA oligonucleotides display unprecedented hybridization affinity toward complementary single-stranded RNA and complementary single- or double-stranded DNA. LNA oligonucleotides induce A-type (RNA-like) duplex conformations. The primers of the present invention may contain LNA modifications.

As used herein, the term “mass-modifying tag” refers to any modification to a given nucleotide which results in an increase in mass relative to the analogous non-mass modified nucleotide. Mass-modifying tags can include heavy isotopes of one or more elements included in the nucleotide such as carbon-13 for example. Other possible modifications include addition of substituents such as iodine or bromine at the 5 position of the nucleobase for example.

The term “mass spectrometry” refers to measurement of the mass of atoms or molecules. The molecules are first converted to ions, which are separated using electric or magnetic fields according to the ratio of their mass to electric charge. The measured masses are used to identity the molecules.

The term “microorganism” as used herein means an organism too small to be observed with the unaided eye and includes, but is not limited to bacteria, virus, protozoans, fungi; and ciliates.

The term “multi-drug resistant” or multiple-drug resistant”refers to a microorganism which is resistant to more than one of the antibiotics or antimicrobial agents used in the treatment of said microorganism.

The term “multiplex PCR” refers to a PCR reaction where more than one primer set is included in the reaction pool allowing 2 or more different DNA targets to be amplified by PCR in a single reaction tube.

The term “non-template tag” refers to a stretch of at least three guanine or cytosine nucleobases of a primer used to produce a bioagent identifying amplicon which are not complementary to the template. A non-template tag is incorporated into a primer for the purpose of increasing the primer-duplex stability of later cycles of amplification by incorporation of extra G-C pairs which each have one additional hydrogen bond relative to an A-T pair.

The term “nucleic acid sequence” as used herein refers to the linear composition of the nucleic acid residues A, T, C or G or any modifications thereof, within an oligonucleotide, nucleotide or polynucleotide, and fragments or portions thereof, and to DNA or RNA of genomic or synthetic origin which may be single or double stranded, and represent the sense or antisense strand

As used herein, the term “nucleobase” is synonymous with other terms in use in the art including “nucleotide,” “deoxynucleotide,” “nucleotide residue,” “deoxynucleotide residue,” “nucleotide triphosphate (NTP),” or deoxynucleotide triphosphate (dNTP).

The term “nucleotide analog” as used herein refers to modified or non-naturally occurring nucleotides such as 5-propynyl pyrimidines (i.e., 5-propynyl-dTTP and 5-propynyl-dTCP), 7-deaza purines (i.e., 7-deaza-dATP and 7-deaza-dGTP). Nucleotide analogs include base analogs and comprise modified forms of deoxyribonucleotides as well as ribonucleotides.

The term “oligonucleotide” as used herein is defined as a molecule comprising two or more deoxyribonucleotides or ribonucleotides, preferably at least 5 nucleotides, more preferably at least about 13 to 35 nucleotides. The exact size will depend on many factors, which in turn depend on the ultimate function or use of the oligonucleotide. The oligonucleotide may be generated in any manner, including chemical synthesis, DNA replication, reverse transcription, PCR, or a combination thereof. Because mononucleotides are reacted to make oligonucleotides in a manner such that the 5′ phosphate of one mononucleotide pentose ring is attached to the 3′ oxygen of its neighbor in one direction via a phosphodiester linkage, an end of an oligonucleotide is referred to as the “5′-end” if its 5′ phosphate is not linked to the 3′ oxygen of a mononucleotide pentose ring and as the “3′-end” if its 3′ oxygen is not linked to a 5′ phosphate of a subsequent mononucleotide pentose ring. As used herein, a nucleic acid sequence, even if internal to a larger oligonucleotide, also may be said to have 5′ and 3′ ends. A first region along a nucleic acid strand is said to be upstream of another region if the 3′ end of the first region is before the 5′ end of the second region when moving along a strand of nucleic acid in a 5′ to 3′ direction. All oligonucleotide primers disclosed herein are understood to be presented in the 5′ to 3′ direction when reading left to right. When two different, non-overlapping oligonucleotides anneal to different regions of the same linear complementary nucleic acid sequence, and the 3′ end of one oligonucleotide points towards the 5′ end of the other, the former may be called the “upstream” oligonucleotide and the latter the “downstream” oligonucleotide. Similarly, when two overlapping oligonucleotides are hybridized to the same linear complementary nucleic acid sequence, with the first oligonucleotide positioned such that its 5′ end is upstream of the 5′ end of the second oligonucleotide, and the 3′ end of the first oligonucleotide is upstream of the 3′ end of the second oligonucleotide, the first oligonucleotide may be called the “upstream” oligonucleotide and the second oligonucleotide may be called the “downstream” oligonucleotide.

›DEFINITIONS · 5 of 8

In the context of this invention, a “pathogen” is a bioagent which causes a disease or disorder.

As used herein, the terms “PCR product,” “PCR fragment,” and “amplification product” refer to the resultant mixture of compounds after two or more cycles of the PCR steps of denaturation, annealing and extension are complete. These terms encompass the case where there has been amplification of one or more segments of one or more target sequences.

The term “peptide nucleic acid” (“PNA”) as used herein refers to a molecule comprising bases or base analogs such as would be found in natural nucleic acid, but attached to a peptide backbone rather than the sugar-phosphate backbone typical of nucleic acids. The attachment of the bases to the peptide is such as to allow the bases to base pair with complementary bases of nucleic acid in a manner similar to that of an oligonucleotide. These small molecules, also designated anti gene agents, stop transcript elongation by binding to their complementary strand of nucleic acid (Nielsen, et al. Anticancer Drug Des. 8:53 63). The primers of the present invention may comprise PNAs.

The term “polymerase” refers to an enzyme having the ability to synthesize a complementary strand of nucleic acid from a starting template nucleic acid strand and free dNTPs.

As used herein, the term “polymerase chain reaction” (“PCR”) refers to the method of K. B. Mullis U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,965,188, hereby incorporated by reference, that describe a method for increasing the concentration of a segment of a target sequence in a mixture of genomic DNA without cloning or purification. This process for amplifying the target sequence consists of introducing a large excess of two oligonucleotide primers to the DNA mixture containing the desired target sequence, followed by a precise sequence of thermal cycling in the presence of a DNA polymerase. The two primers are complementary to their respective strands of the double stranded target sequence. To effect amplification, the mixture is denatured and the primers then annealed to their complementary sequences within the target molecule. Following annealing, the primers are extended with a polymerase so as to form a new pair of complementary strands. The steps of denaturation, primer annealing, and polymerase extension can be repeated many times (i.e., denaturation, annealing and extension constitute one “cycle”; there can be numerous “cycles”) to obtain a high concentration of an amplified segment of the desired target sequence. The length of the amplified segment of the desired target sequence is determined by the relative positions of the primers with respect to each other, and therefore, this length is a controllable parameter. By virtue of the repeating aspect of the process, the method is referred to as the “polymerase chain reaction” (hereinafter “PCR”). Because the desired amplified segments of the target sequence become the predominant sequences (in terms of concentration) in the mixture, they are said to be “PCR amplified.” With PCR, it is possible to amplify a single copy of a specific target sequence in genomic DNA to a level detectable by several different methodologies (e.g., hybridization with a labeled probe; incorporation of biotinylated primers followed by avidin-enzyme conjugate detection; incorporation of 32P-labeled deoxynucleotide triphosphates, such as dCTP or dATP, into the amplified segment). In addition to genomic DNA, any oligonucleotide or polynucleotide sequence can be amplified with the appropriate set of primer molecules. In particular, the amplified segments created by the PCR process itself are, themselves, efficient templates for subsequent PCR amplifications.

The term “polymerization means” or “polymerization agent” refers to any agent capable of facilitating the addition of nucleoside triphosphates to an oligonucleotide. Preferred polymerization means comprise DNA and RNA polymerases.

As used herein, the terms “pair of primers,” or “primer pair” are synonymous. A primer pair is used for amplification of a nucleic acid sequence. A pair of primers comprises a forward primer and a reverse primer. The forward primer hybridizes to a sense strand of a target gene sequence to be amplified and primes synthesis of an antisense strand (complementary to the sense strand) using the target sequence as a template. A reverse primer hybridizes to the antisense strand of a target gene sequence to be amplified and primes synthesis of a sense strand (complementary to the antisense strand) using the target sequence as a template.

The primers are designed to bind to highly conserved sequence regions of a bioagent identifying amplicon that flank an intervening variable region and yield amplification products which ideally provide enough variability to distinguish each individual bioagent, and which are amenable to molecular mass analysis. In some embodiments, the highly conserved sequence regions exhibit between about 80-100%, or between about 90-100%, or between about 95-100% identity, or between about 99-100% identity. The molecular mass of a given amplification product provides a means of identifying the bioagent from which it was obtained, due to the variability of the variable region. Thus design of the primers requires selection of a variable region with appropriate variability to resolve the identity of a given bioagent. Bioagent identifying amplicons are ideally specific to the identity of the bioagent.

Properties of the primers may include any number of properties related to structure including, but not limited to: nucleobase length which may be contiguous (linked together) or non-contiguous (for example, two or more contiguous segments which are joined by a linker or loop moiety), modified or universal nucleobases (used for specific purposes such as for example, increasing hybridization affinity, preventing non-templated adenylation and modifying molecular mass) percent complementarity to a given target sequences.

›DEFINITIONS · 6 of 8

Properties of the primers also include functional features including, but not limited to, orientation of hybridization (forward or reverse) relative to a nucleic acid template. The coding or sense strand is the strand to which the forward priming primer hybridizes (forward priming orientation) while the reverse priming primer hybridizes to the non-coding or antisense strand (reverse priming orientation). The functional properties of a given primer pair also include the generic template nucleic acid to which the primer pair hybridizes. For example, identification of bioagents can be accomplished at different levels using primers suited to resolution of each individual level of identification. Broad range survey primers are designed with the objective of identifying a bioagent as a member of a particular division (e.g., an order, family, genus or other such grouping of bioagents above the species level of bioagents). In some embodiments, broad range survey intelligent primers are capable of identification of bioagents at the species or sub-species level. Other primers may have the functionality of producing bioagent identifying amplicons for members of a given taxonomic genus, clade, species, sub-species or genotype (including genetic variants which may include presence of virulence genes or antibiotic resistance genes or mutations). Additional functional properties of primer pairs include the functionality of performing amplification either singly (single primer pair per amplification reaction vessel) or in a multiplex fashion (multiple primer pairs and multiple amplification reactions within a single reaction vessel).

As used herein, the terms “purified” or “substantially purified” refer to molecules, either nucleic or amino acid sequences, that are removed from their natural environment, isolated or separated, and are at least 60% free, preferably 75% free, and most preferably 90% free from other components with which they are naturally associated. An “isolated polynucleotide” or “isolated oligonucleotide” is therefore a substantially purified polynucleotide.

The term “reverse transcriptase” refers to an enzyme having the ability to transcribe DNA from an RNA template. This enzymatic activity is known as reverse transcriptase activity. Reverse transcriptase activity is desirable in order to obtain DNA from RNA viruses which can then be amplified and analyzed by the methods of the present invention.

The term “ribosomal RNA” or “rRNA” refers to the primary ribonucleic acid constituent of ribosomes. Ribosomes are the protein-manufacturing organelles of cells and exist in the cytoplasm. Ribosomal RNAs are transcribed from the DNA genes encoding them.

The term “sample” in the present specification and claims is used in its broadest sense. On the one hand it is meant to include a specimen or culture (e.g., microbiological cultures). On the other hand, it is meant to include both biological and environmental samples. A sample may include a specimen of synthetic origin. Biological samples may be animal, including human, fluid, solid (e.g., stool) or tissue, as well as liquid and solid food and feed products and ingredients such as dairy items, vegetables, meat and meat by-products, and waste. Biological samples may be obtained from all of the various families of domestic animals, as well as feral or wild animals, including, but not limited to, such animals as ungulates, bear, fish, lagamorphs, rodents, etc. Environmental samples include environmental material such as surface matter, soil, water, air and industrial samples, as well as samples obtained from food and dairy processing instruments, apparatus, equipment, utensils, disposable and non-disposable items. These examples are not to be construed as limiting the sample types applicable to the present invention. The term “source of target nucleic acid” refers to any sample that contains nucleic acids (RNA or DNA). Particularly preferred sources of target nucleic acids are biological samples including, but not limited to blood, saliva, cerebral spinal fluid, pleural fluid, milk, lymph, sputum and semen.

As used herein, the term “sample template” refers to nucleic acid originating from a sample that is analyzed for the presence of “target” (defined below). In contrast, “background template” is used in reference to nucleic acid other than sample template that may or may not be present in a sample. Background template is often a contaminant. It may be the result of carryover, or it may be due to the presence of nucleic acid contaminants sought to be purified away from the sample. For example, nucleic acids from organisms other than those to be detected may be present as background in a test sample.

A “segment” is defined herein as a region of nucleic acid within a target sequence.

The “self-sustained sequence replication reaction” (3SR) (Guatelli et al., Proc. Natl. Acad. Sci., 87:1874-1878 [1990], with an erratum at Proc. Natl. Acad. Sci., 87:7797 [1990]) is a transcription-based in vitro amplification system (Kwok et al., Proc. Natl. Acad. Sci., 86:1173-1177 [1989]) that can exponentially amplify RNA sequences at a uniform temperature. The amplified RNA can then be utilized for mutation detection (Fahy et al., PCR Meth. Appl., 1:25-33 [1991]). In this method, an oligonucleotide primer is used to add a phage RNA polymerase promoter to the 5′ end of the sequence of interest. In a cocktail of enzymes and substrates that includes a second primer, reverse transcriptase, RNase H, RNA polymerase and ribo- and deoxyribonucleoside triphosphates, the target sequence undergoes repeated rounds of transcription, cDNA synthesis and second-strand synthesis to amplify the area of interest. The use of 3SR to detect mutations is kinetically limited to screening small segments of DNA (e.g., 200-300 base pairs).

As used herein, the term “sequence alignment” refers to a listing of multiple DNA or amino acid sequences and aligns them to highlight their similarities. The listings can be made using bioinformatics computer programs.

›DEFINITIONS · 7 of 8

In context of this invention, the term “speciating primer pair” refers to a primer pair designed to produce a bioagent identifying amplicon with the diagnostic capability of identifying species members of a group of genera or a particular genus of bioagents. Primer pair number 2249 (SEQ ID NOs: 430:1321), for example, is a speciating primer pair used to distinguish Staphylococcus aureus from other species of the genus Staphylococcus.

As used herein, a “sub-species characteristic” is a genetic characteristic that provides the means to distinguish two members of the same bioagent species. For example, one viral strain could be distinguished from another viral strain of the same species by possessing a genetic change (e.g., for example, a nucleotide deletion, addition or substitution) in one of the viral genes, such as the RNA-dependent RNA polymerase. Sub-species characteristics such as virulence genes and drug-are responsible for the phenotypic differences among the different strains of bacteria.

As used herein, the term “target” is used in a broad sense to indicate the gene or genomic region being amplified by the primers. Because the present invention provides a plurality of amplification products from any given primer pair (depending on the bioagent being analyzed), multiple amplification products from different specific nucleic acid sequences may be obtained. Thus, the term “target” is not used to refer to a single specific nucleic acid sequence. The “target” is sought to be sorted out from other nucleic acid sequences and contains a sequence that has at least partial complementarity with an oligonucleotide primer. The target nucleic acid may comprise single- or double-stranded DNA or RNA. A “segment” is defined as a region of nucleic acid within the target sequence.

The term “template” refers to a strand of nucleic acid on which a complementary copy is built from nucleoside triphosphates through the activity of a template-dependent nucleic acid polymerase. Within a duplex the template strand is, by convention, depicted and described as the “bottom” strand. Similarly, the non-template strand is often depicted and described as the “top” strand.

As used herein, the term “T m ” is used in reference to the “melting temperature.” The melting temperature is the temperature at which a population of double-stranded nucleic acid molecules becomes half dissociated into single strands. Several equations for calculating the T m of nucleic acids are well known in the art. As indicated by standard references, a simple estimate of the T m value may be calculated by the equation: T m =81.5+0.41(% G+C), when a nucleic acid is in aqueous solution at 1 M NaCl (see e.g., Anderson and Young, Quantitative Filter Hybridization, in Nucleic Acid Hybridization (1985). Other references (e.g., Allawi, H. T. & SantaLucia, J., Jr. Thermodynamics and NMR of internal G. T mismatches in DNA. Biochemistry 36, 10581-94 (1997) include more sophisticated computations which take structural and environmental, as well as sequence characteristics into account for the calculation of T m .

The term “triangulation genotyping analysis” refers to a method of genotyping a bioagent by measurement of molecular masses or base compositions of amplification products, corresponding to bioagent identifying amplicons, obtained by amplification of regions of more than one gene. In this sense, the term “triangulation” refers to a method of establishing the accuracy of information by comparing three or more types of independent points of view bearing on the same findings. Triangulation genotyping analysis carried out with a plurality of triangulation genotyping analysis primers yields a plurality of base compositions that then provide a pattern or “barcode” from which a species type can be assigned. The species type may represent a previously known sub-species or strain, or may be a previously unknown strain having a specific and previously unobserved base composition barcode indicating the existence of a previously unknown genotype.

As used herein, the term “triangulation genotyping analysis primer pair” is a primer pair designed to produce bioagent identifying amplicons for determining species types in a triangulation genotyping analysis.

The employment of more than one bioagent identifying amplicon for identification of a bioagent is herein referred to as “triangulation identification.” Triangulation identification is pursued by analyzing a plurality of bioagent identifying amplicons produced with different primer pairs. This process is used to reduce false negative and false positive signals, and enable reconstruction of the origin of hybrid or otherwise engineered bioagents. For example, identification of the three part toxin genes typical of B. anthracis (Bowen et al., J. Appl. Microbiol., 1999, 87, 270-278) in the absence of the expected signatures from the B. anthracis genome would suggest a genetic engineering event.

In the context of this invention, the term “unknown bioagent” may mean either: (i) a bioagent whose existence is known (such as the well known bacterial species Staphylococcus aureus for example) but which is not known to be in a sample to be analyzed, or (ii) a bioagent whose existence is not known (for example, the SARS coronavirus was unknown prior to April 2003). For example, if the method for identification of coronaviruses disclosed in commonly owned U.S. patent Ser. No. 10/829,826 (incorporated herein by reference in its entirety) was to be employed prior to April 2003 to identify the SARS coronavirus in a clinical sample, both meanings of “unknown” bioagent are applicable since the SARS coronavirus was unknown to science prior to April, 2003 and since it was not known what bioagent (in this case a coronavirus) was present in the sample. On the other hand, if the method of U.S. patent Ser. No. 10/829,826 was to be employed subsequent to April 2003 to identify the SARS coronavirus in a clinical sample, only the first meaning (i) of “unknown” bioagent would apply since the SARS coronavirus became known to science subsequent to April 2003 and since it was not known what bioagent was present in the sample.

›DEFINITIONS · 8 of 8

The term “variable sequence” as used herein refers to differences in nucleic acid sequence between two nucleic acids. For example, the genes of two different bacterial species may vary in sequence by the presence of single base substitutions and/or deletions or insertions of one or more nucleotides. These two forms of the structural gene are said to vary in sequence from one another. In the context of the present invention, “viral nucleic acid” includes, but is not limited to, DNA, RNA, or DNA that has been obtained from viral RNA, such as, for example, by performing a reverse transcription reaction. Viral RNA can either be single-stranded (of positive or negative polarity) or double-stranded.

The term “virus” refers to obligate, ultramicroscopic, parasites that are incapable of autonomous replication (i.e., replication requires the use of the host cell's machinery). Viruses can survive outside of a host cell but cannot replicate.

The term “wild-type” refers to a gene or a gene product that has the characteristics of that gene or gene product when isolated from a naturally occurring source. A wild-type gene is that which is most frequently observed in a population and is thus arbitrarily designated the “normal” or “wild-type” form of the gene. In contrast, the term “modified”, “mutant” or “polymorphic” refers to a gene or gene product that displays modifications in sequence and or functional properties (i.e., altered characteristics) when compared to the wild-type gene or gene product. It is noted that naturally-occurring mutants can be isolated; these are identified by the fact that they have altered characteristics when compared to the wild-type gene or gene product.

As used herein, a “wobble base” is a variation in a codon found at the third nucleotide position of a DNA triplet. Variations in conserved regions of sequence are often found at the third nucleotide position due to redundancy in the amino acid code.

›DETAILED DESCRIPTION OF EMBODIMENTS · 1 of 9

A. Bioagent Identifying Amplicons

The present invention provides methods for detection and identification of unknown bioagents using bioagent identifying amplicons. Primers are selected to hybridize to conserved sequence regions of nucleic acids derived from a bioagent, and which bracket variable sequence regions to yield a bioagent identifying amplicon, which can be amplified and which is amenable to molecular mass determination. The molecular mass then provides a means to uniquely identify the bioagent without a requirement for prior knowledge of the possible identity of the bioagent. The molecular mass or corresponding base composition signature of the amplification product is then matched against a database of molecular masses or base composition signatures. A match is obtained when an experimentally-determined molecular mass or base composition of an analyzed amplification product is compared with known molecular masses or base compositions of known bioagent identifying amplicons and the experimentally determined molecular mass or base composition is the same as the molecular mass or base composition of one of the known bioagent identifying amplicons. Alternatively, the experimentally-determined molecular mass or base composition may be within experimental error of the molecular mass or base composition of a known bioagent identifying amplicon and still be classified as a match. In some cases, the match may also be classified using a probability of match model such as the models described in U.S. Ser. No. 11/073,362, which is commonly owned and incorporated herein by reference in entirety. Furthermore, the method can be applied to rapid parallel multiplex analyses, the results of which can be employed in a triangulation identification strategy. The present method provides rapid throughput and does not require nucleic acid sequencing of the amplified target sequence for bioagent detection and identification.

Despite enormous biological diversity, all forms of life on earth share sets of essential, common features in their genomes. Since genetic data provide the underlying basis for identification of bioagents by the methods of the present invention, it is necessary to select segments of nucleic acids which ideally provide enough variability to distinguish each individual bioagent and whose molecular mass is amenable to molecular mass determination.

Unlike bacterial genomes, which exhibit conservation of numerous genes (i.e. housekeeping genes) across all organisms, viruses do not share a gene that is essential and conserved among all virus families. Therefore, viral identification is achieved within smaller groups of related viruses, such as members of a particular virus family or genus. For example, RNA-dependent RNA polymerase is present in all single-stranded RNA viruses and can be used for broad priming as well as resolution within the virus family.

In some embodiments of the present invention, at least one bacterial nucleic acid segment is amplified in the process of identifying the bacterial bioagent. Thus, the nucleic acid segments that can be amplified by the primers disclosed herein and that provide enough variability to distinguish each individual bioagent and whose molecular masses are amenable to molecular mass determination are herein described as bioagent identifying amplicons.

In some embodiments of the present invention, bioagent identifying amplicons comprise from about 45 to about 150 nucleobases (i.e. from about 45 to about 200 linked nucleosides), although both longer and short regions may be used. One of ordinary skill in the art will appreciate that the invention embodies compounds of 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75,76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, and 150 nucleobases in length, or any range therewithin.

It is the combination of the portions of the bioagent nucleic acid segment to which the primers hybridize (hybridization sites) and the variable region between the primer hybridization sites that comprises the bioagent identifying amplicon. Thus, it can be said that a given bioagent identifying amplicon is “defined by” a given pair of primers.

In some embodiments, bioagent identifying amplicons amenable to molecular mass determination which are produced by the primers described herein are either of a length, size or mass compatible with the particular mode of molecular mass determination or compatible with a means of providing a predictable fragmentation pattern in order to obtain predictable fragments of a length compatible with the particular mode of molecular mass determination. Such means of providing a predictable fragmentation pattern of an amplification product include, but are not limited to, cleavage with chemical reagents, restriction enzymes or cleavage primers, for example. Thus, in some embodiments, bioagent identifying amplicons are larger than 150 nucleobases and are amenable to molecular mass determination following restriction digestion. Methods of using restriction enzymes and cleavage primers are well known to those with ordinary skill in the art.

In some embodiments, amplification products corresponding to bioagent identifying amplicons are obtained using the polymerase chain reaction (PCR) that is a routine method to those with ordinary skill in the molecular biology arts. Other amplification methods may be used such as ligase chain reaction (LCR), low-stringency single primer PCR, and multiple strand displacement amplification (MDA). These methods are also known to those with ordinary skill.

B. Primers and Primer Pairs

In some embodiments, the primers are designed to bind to conserved sequence regions of a bioagent identifying amplicon that flank an intervening variable region and yield amplification products which provide variability sufficient to distinguish each individual bioagent, and which are amenable to molecular mass analysis. In some embodiments, the highly conserved sequence regions exhibit between about 80-100%, or between about 90-100%, or between about 95-100% identity, or between about 99-100% identity. The molecular mass of a given amplification product provides a means of identifying the bioagent from which it was obtained, due to the variability of the variable region. Thus, design of the primers involves selection of a variable region with sufficient variability to resolve the identity of a given bioagent. In some embodiments, bioagent identifying amplicons are specific to the identity of the bioagent.

›DETAILED DESCRIPTION OF EMBODIMENTS · 2 of 9

In some embodiments, identification of bioagents is accomplished at different levels using primers suited to resolution of each individual level of identification. Broad range survey primers are designed with the objective of identifying a bioagent as a member of a particular division (e.g., an order, family, genus or other such grouping of bioagents above the species level of bioagents). In some embodiments, broad range survey intelligent primers are capable of identification of bioagents at the species or sub-species level. Examples of broad range survey primers include, but are not limited to: primer pair numbers: 346 (SEQ ID NOs: 202:1110), 347 (SEQ ID NOs: 560:1278), 348 SEQ ID NOs: 706:895), and 361 (SEQ ID NOs: 697:1398) which target DNA encoding 16S rRNA, and primer pair numbers 349 (SEQ ID NOs: 401:1156) and 360 (SEQ ID NOs: 409:1434) which target DNA encoding 23S rRNA.

In some embodiments, drill-down primers are designed with the objective of identifying a bioagent at the sub-species level (including strains, subtypes, variants and isolates) based on sub-species characteristics which may, for example, include single nucleotide polymorphisms (SNPs), variable number tandem repeats (VNTRs), deletions, drug resistance mutations or any other modification of a nucleic acid sequence of a bioagent relative to other members of a species having different sub-species characteristics. Drill-down intelligent primers are not always required for identification at the sub-species level because broad range survey intelligent primers may, in some cases provide sufficient identification resolution to accomplishing this identification objective. Examples of drill-down primers include, but are not limited to: confirmation primer pairs such as primer pair numbers 351 (SEQ ID NOs: 355:1423) and 353 (SEQ ID NOs: 220:1394), which target the pX01 virulence plasmid of Bacillus anthracis . Other examples of drill-down primer pairs are found in sets of triangulation genotyping primer pairs such as, for example, the primer pair number 2146 (SEQ ID NOs: 437:1137) which targets the arcC gene (encoding carmabate kinase) and is included in an 8 primer pair panel or kit for use in genotyping Staphylococcus aureus , or in other panels or kits of primer pairs used for determining drug-resistant bacterial strains, such as, for example, primer pair number 2095 (SEQ ID NOs: 456:1261) which targets the pv-luk gene (encoding Panton-Valentine leukocidin) and is included in an 8 primer pair panel or kit for use in identification of drug resistant strains of Staphylococcus aureus.

A representative process flow diagram used for primer selection and validation process is outlined in FIG. 1 . For each group of organisms, candidate target sequences are identified ( 200 ) from which nucleotide alignments are created ( 210 ) and analyzed ( 220 ). Primers are then designed by selecting appropriate priming regions ( 230 ) to facilitate the selection of candidate primer pairs ( 240 ). The primer pairs are then subjected to in silico analysis by electronic PCR (ePCR) ( 300 ) wherein bioagent identifying amplicons are obtained from sequence databases such as GenBank or other sequence collections ( 310 ) and checked for specificity in silico ( 320 ). Bioagent identifying amplicons obtained from GenBank sequences ( 310 ) can also be analyzed by a probability model which predicts the capability of a given amplicon to identify unknown bioagents such that the base compositions of amplicons with favorable probability scores are then stored in a base composition database ( 325 ). Alternatively, base compositions of the bioagent identifying amplicons obtained from the primers and GenBank sequences can be directly entered into the base composition database ( 330 ). Candidate primer pairs ( 240 ) are validated by testing their ability to hybridize to target nucleic acid by an in vitro amplification by a method such as PCR analysis ( 400 ) of nucleic acid from a collection of organisms ( 410 ). Amplification products thus obtained are analyzed by gel electrophoresis or by mass spectrometry to confirm the sensitivity, specificity and reproducibility of the primers used to obtain the amplification products ( 420 ).

Many of the important pathogens, including the organisms of greatest concern as biowarfare agents, have been completely sequenced. This effort has greatly facilitated the design of primers for the detection of unknown bioagents. The combination of broad-range priming with division-wide and drill-down priming has been used very successfully in several applications of the technology, including environmental surveillance for biowarfare threat agents and clinical sample analysis for medically important pathogens.

Synthesis of primers is well known and routine in the art. The primers may be conveniently and routinely made through the well-known technique of solid phase synthesis. Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems (Foster City, Calif.). Any other means for such synthesis known in the art may additionally or alternatively be employed.

In some embodiments primers are employed as compositions for use in methods for identification of bacterial bioagents as follows: a primer pair composition is contacted with nucleic acid (such as, for example, bacterial DNA or DNA reverse transcribed from the rRNA) of an unknown bacterial bioagent. The nucleic acid is then amplified by a nucleic acid amplification technique, such as PCR for example, to obtain an amplification product that represents a bioagent identifying amplicon. The molecular mass of each strand of the double-stranded amplification product is determined by a molecular mass measurement technique such as mass spectrometry for example, wherein the two strands of the double-stranded amplification product are separated during the ionization process. In some embodiments, the mass spectrometry is electrospray Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR-MS) or electrospray time of flight mass spectrometry (ESI-TOF-MS). A list of possible base compositions can be generated for the molecular mass value obtained for each strand and the choice of the correct base composition from the list is facilitated by matching the base composition of one strand with a complementary base composition of the other strand. The molecular mass or base composition thus determined is then compared with a database of molecular masses or base compositions of analogous bioagent identifying amplicons for known viral bioagents. A match between the molecular mass or base composition of the amplification product and the molecular mass or base composition of an analogous bioagent identifying amplicon for a known viral bioagent indicates the identity of the unknown bioagent. In some embodiments, the primer pair used is one of the primer pairs of Table 2. In some embodiments, the method is repeated using one or more different primer pairs to resolve possible ambiguities in the identification process or to improve the confidence level for the identification assignment.

›DETAILED DESCRIPTION OF EMBODIMENTS · 3 of 9

In some embodiments, a bioagent identifying amplicon may be produced using only a single primer (either the forward or reverse primer of any given primer pair), provided an appropriate amplification method is chosen, such as, for example, low stringency single primer PCR (LSSP-PCR). Adaptation of this amplification method in order to produce bioagent identifying amplicons can be accomplished by one with ordinary skill in the art without undue experimentation.

In some embodiments, the oligonucleotide primers are broad range survey primers which hybridize to conserved regions of nucleic acid encoding the hexon gene of all (or between 80% and 100%, between 85% and 100%, between 90% and 100% or between 95% and 100%) known bacteria and produce bacterial bioagent identifying amplicons.

In some cases, the molecular mass or base composition of a bacterial bioagent identifying amplicon defined by a broad range survey primer pair does not provide enough resolution to unambiguously identify a bacterial bioagent at or below the species level. These cases benefit from further analysis of one or more bacterial bioagent identifying amplicons generated from at least one additional broad range survey primer pair or from at least one additional division-wide primer pair. The employment of more than one bioagent identifying amplicon for identification of a bioagent is herein referred to as triangulation identification.

In other embodiments, the oligonucleotide primers are division-wide primers which hybridize to nucleic acid encoding genes of species within a genus of bacteria. In other embodiments, the oligonucleotide primers are drill-down primers which enable the identification of sub-species characteristics. Drill down primers provide the functionality of producing bioagent identifying amplicons for drill-down analyses such as strain typing when contacted with nucleic acid under amplification conditions. Identification of such sub-species characteristics is often critical for determining proper clinical treatment of viral infections. In some embodiments, sub-species characteristics are identified using only broad range survey primers and division-wide and drill-down primers are not used.

In some embodiments, the primers used for amplification hybridize to and amplify genomic DNA, and DNA of bacterial plasmids.

In some embodiments, various computer software programs may be used to aid in design of primers for amplification reactions such as Primer Premier 5 (Premier Biosoft, Palo Alto, Calif.) or OLIGO Primer Analysis Software (Molecular Biology Insights, Cascade, Colo.). These programs allow the user to input desired hybridization conditions such as melting temperature of a primer-template duplex for example. In some embodiments, an in silico PCR search algorithm, such as (ePCR) is used to analyze primer specificity across a plurality of template sequences which can be readily obtained from public sequence databases such as GenBank for example. An existing RNA structure search algorithm (Macke et al., Nucl. Acids Res., 2001, 29, 4724-4735, which is incorporated herein by reference in its entirety) has been modified to include PCR parameters such as hybridization conditions, mismatches, and thermodynamic calculations (SantaLucia, Proc. Natl. Acad. Sci. U.S.A., 1998, 95, 1460-1465, which is incorporated herein by reference in its entirety). This also provides information on primer specificity of the selected primer pairs. In some embodiments, the hybridization conditions applied to the algorithm can limit the results of primer specificity obtained from the algorithm. In some embodiments, the melting temperature threshold for the primer template duplex is specified to be 35° C. or a higher temperature. In some embodiments the number of acceptable mismatches is specified to be seven mismatches or less. In some embodiments, the buffer components and concentrations and primer concentrations may be specified and incorporated into the algorithm, for example, an appropriate primer concentration is about 250 nM and appropriate buffer components are 50 mM sodium or potassium and 1.5 mM Mg 2+ .

One with ordinary skill in the art of design of amplification primers will recognize that a given primer need not hybridize with 100% complementarity in order to effectively prime the synthesis of a complementary nucleic acid strand in an amplification reaction. Moreover, a primer may hybridize over one or more segments such that intervening or adjacent segments are not involved in the hybridization event. (e.g., for example, a loop structure or a hairpin structure). The primers of the present invention may comprise at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% sequence identity with any of the primers listed in Table 2. Thus, in some embodiments of the present invention, an extent of variation of 70% to 100%, or any range therewithin, of the sequence identity is possible relative to the specific primer sequences disclosed herein. Determination of sequence identity is described in the following example: a primer 20 nucleobases in length which is identical to another 20 nucleobase primer having two non-identical residues has 18 of 20 identical residues (18/20=0.9 or 90% sequence identity). In another example, a primer 15 nucleobases in length having all residues identical to a 15 nucleobase segment of primer 20 nucleobases in length would have 15/20=0.75 or 75% sequence identity with the 20 nucleobase primer.

Percent homology, sequence identity or complementarity, can be determined by, for example, the Gap program (Wisconsin Sequence Analysis Package, Version 8 for UNIX, Genetics Computer Group, University Research Park, Madison Wis.), using default settings, which uses the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482-489). In some embodiments, complementarity of primers with respect to the conserved priming regions of viral nucleic acid is between about 70% and about 75% 80%. In other embodiments, homology, sequence identity or complementarity, is between about 75% and about 80%. In yet other embodiments, homology, sequence identity or complementarity, is at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or is 100%.

›DETAILED DESCRIPTION OF EMBODIMENTS · 4 of 9

In some embodiments, the primers described herein comprise at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 98%, or at least 99%, or 100% (or any range therewithin) sequence identity with the primer sequences specifically disclosed herein.

One with ordinary skill is able to calculate percent sequence identity or percent sequence homology and able to determine, without undue experimentation, the effects of variation of primer sequence identity on the function of the primer in its role in priming synthesis of a complementary strand of nucleic acid for production of an amplification product of a corresponding bioagent identifying amplicon.

In one embodiment, the primers are at least 13 nucleobases in length. In another embodiment, the primers are less than 36 nucleobases in length.

In some embodiments of the present invention, the oligonucleotide primers are 13 to 35 nucleobases in length (13 to 35 linked nucleotide residues). These embodiments comprise oligonucleotide primers 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 nucleobases in length, or any range therewithin. The present invention contemplates using both longer and shorter primers. Furthermore, the primers may also be linked to one or more other desired moieties, including, but not limited to, affinity groups, ligands, regions of nucleic acid that are not complementary to the nucleic acid to be amplified, labels, etc. Primers may also form hairpin structures. For example, hairpin primers may be used to amplify short target nucleic acid molecules. The presence of the hairpin may stabilize the amplification complex (see e.g., TAQMAN MicroRNA Assays, Applied Biosystems, Foster City, Calif.).

In some embodiments, any oligonucleotide primer pair may have one or both primers with less then 70% sequence homology with a corresponding member of any of the primer pairs of Table 2 if the primer pair has the capability of producing an amplification product corresponding to a bioagent identifying amplicon. In other embodiments, any oligonucleotide primer pair may have one or both primers with a length greater than 35 nucleobases if the primer pair has the capability of producing an amplification product corresponding to a bioagent identifying amplicon.

In some embodiments, the function of a given primer may be substituted by a combination of two or more primers segments that hybridize adjacent to each other or that are linked by a nucleic acid loop structure or linker which allows a polymerase to extend the two or more primers in an amplification reaction.

In some embodiments, the primer pairs used for obtaining bioagent identifying amplicons are the primer pairs of Table 2. In other embodiments, other combinations of primer pairs are possible by combining certain members of the forward primers with certain members of the reverse primers. An example can be seen in Table 2 for two primer pair combinations of forward primer 16S_EC — 789 — 810_F (SEQ ID NO: 206), with the reverse primers 16S_EC — 880 — 894_R (SEQ ID NO: 796), or 16 S_EC — 882 — 899_R or (SEQ ID NO: 818). Arriving at a favorable alternate combination of primers in a primer depends upon the properties of the primer pair, most notably the size of the bioagent identifying amplicon that would be produced by the primer pair, which preferably is between about 45 to about 150 nucleobases in length. Alternatively, a bioagent identifying amplicon longer than 150 nucleobases in length could be cleaved into smaller segments by cleavage reagents such as chemical reagents, or restriction enzymes, for example.

In some embodiments, the primers are configured to amplify nucleic acid of a bioagent to produce amplification products that can be measured by mass spectrometry and from whose molecular masses candidate base compositions can be readily calculated.

In some embodiments, any given primer comprises a modification comprising the addition of a non-templated T residue to the 5′ end of the primer (i.e., the added T residue does not necessarily hybridize to the nucleic acid being amplified). The addition of a non-templated T residue has an effect of minimizing the addition of non-templated adenosine residues as a result of the non-specific enzyme activity of Taq polymerase (Magnuson et al., Biotechniques, 1996, 21, 700-709), an occurrence which may lead to ambiguous results arising from molecular mass analysis.

In some embodiments of the present invention, primers may contain one or more universal bases. Because any variation (due to codon wobble in the 3 rd position) in the conserved regions among species is likely to occur in the third position of a DNA (or RNA) triplet, oligonucleotide primers can be designed such that the nucleotide corresponding to this position is a base which can bind to more than one nucleotide, referred to herein as a “universal nucleobase.” For example, under this “wobble” pairing, inosine (I) binds to U, C or A; guanine (G) binds to U or C, and uridine (U) binds to U or C. Other examples of universal nucleobases include nitroindoles such as 5-nitroindole or 3-nitropyrrole (Loakes et al., Nucleosides and Nucleotides, 1995, 14, 1001-1003), the degenerate nucleotides dP or dK (Hill et al.), an acyclic nucleoside analog containing 5-nitroindazole (Van Aerschot et al., Nucleosides and Nucleotides, 1995, 14, 1053-1056) or the purine analog 1-(2-deoxy-β-D-ribofuranosyl)-imidazole-4-carboxamide (Sala et al., Nucl. Acids Res., 1996, 24, 3302-3306).

In some embodiments, to compensate for the somewhat weaker binding by the wobble base, the oligonucleotide primers are designed such that the first and second positions of each triplet are occupied by nucleotide analogs that bind with greater affinity than the unmodified nucleotide. Examples of these analogs include, but are not limited to, 2,6-diaminopurine which binds to thymine, 5-propynyluracil (also known as propynylated thymine) which binds to adenine and 5-propynylcytosine and phenoxazines, including G-clamp, which binds to G. Propynylated pyrimidines are described in U.S. Pat. Nos. 5,645,985, 5,830,653 and 5,484,908, each of which is commonly owned and incorporated herein by reference in its entirety. Propynylated primers are described in U.S. Pre-Grant Publication No. 2003-0170682, which is also commonly owned and incorporated herein by reference in its entirety. Phenoxazines are described in U.S. Pat. Nos. 5,502,177, 5,763,588, and 6,005,096, each of which is incorporated herein by reference in its entirety. G-clamps are described in U.S. Pat. Nos. 6,007,992 and 6,028,183, each of which is incorporated herein by reference in its entirety.

›DETAILED DESCRIPTION OF EMBODIMENTS · 5 of 9

In some embodiments, primer hybridization is enhanced using primers containing 5-propynyl deoxy-cytidine and deoxy-thymidine nucleotides. These modified primers offer increased affinity and base pairing selectivity.

In some embodiments, non-template primer tags are used to increase the melting temperature (T m ) of a primer-template duplex in order to improve amplification efficiency. A non-template tag is at least three consecutive A or T nucleotide residues on a primer which are not complementary to the template. In any given non-template tag, A can be replaced by C or G and T can also be replaced by C or G. Although Watson-Crick hybridization is not expected to occur for a non-template tag relative to the template, the extra hydrogen bond in a G-C pair relative to an A-T pair confers increased stability of the primer-template duplex and improves amplification efficiency for subsequent cycles of amplification when the primers hybridize to strands synthesized in previous cycles.

In other embodiments, propynylated tags may be used in a manner similar to that of the non-template tag, wherein two or more 5-propynylcytidine or 5-propynyluridine residues replace template matching residues on a primer. In other embodiments, a primer contains a modified internucleoside linkage such as a phosphorothioate linkage, for example.

In some embodiments, the primers contain mass-modifying tags. Reducing the total number of possible base compositions of a nucleic acid of specific molecular weight provides a means of avoiding a persistent source of ambiguity in determination of base composition of amplification products. Addition of mass-modifying tags to certain nucleobases of a given primer will result in simplification of de novo determination of base composition of a given bioagent identifying amplicon from its molecular mass.

In some embodiments of the present invention, the mass modified nucleobase comprises one or more of the following: for example, 7-deaza-2′-deoxyadenosine-5-triphosphate, 5-iodo-2′-deoxyuridine-5′-triphosphate, 5-bromo-2′-deoxyuridine-5′-triphosphate, 5-bromo-2′-deoxycytidine-5′-triphosphate, 5-iodo-2′-deoxycytidine-5′-triphosphate, 5-hydroxy-2′-deoxyuridine-5′-triphosphate, 4-thiothymidine-5′-triphosphate, 5-aza-2′-deoxyuridine-5′-triphosphate, 5-fluoro-2′-deoxyuridine-5′-triphosphate, O6-methyl-2′-deoxyguanosine-5′-triphosphate, N2-methyl-2′-deoxyguanosine-5′-triphosphate, 8-oxo-2′-deoxyguanosine-5′-triphosphate or thiothymidine-5′-triphosphate. In some embodiments, the mass-modified nucleobase comprises 15 N or 13 C or both 15 N and 13 C.

In some embodiments, multiplex amplification is performed where multiple bioagent identifying amplicons are amplified with a plurality of primer pairs. The advantages of multiplexing are that fewer reaction containers (for example, wells of a 96- or 384-well plate) are needed for each molecular mass measurement, providing time, resource and cost savings because additional bioagent identification data can be obtained within a single analysis. Multiplex amplification methods are well known to those with ordinary skill and can be developed without undue experimentation. However, in some embodiments, one useful and non-obvious step in selecting a plurality candidate bioagent identifying amplicons for multiplex amplification is to ensure that each strand of each amplification product will be sufficiently different in molecular mass that mass spectral signals will not overlap and lead to ambiguous analysis results. In some embodiments, a 10 Da difference in mass of two strands of one or more amplification products is sufficient to avoid overlap of mass spectral peaks.

In some embodiments, as an alternative to multiplex amplification, single amplification reactions can be pooled before analysis by mass spectrometry. In these embodiments, as for multiplex amplification embodiments, it is useful to select a plurality of candidate bioagent identifying amplicons to ensure that each strand of each amplification product will be sufficiently different in molecular mass that mass spectral signals will not overlap and lead to ambiguous analysis results.

C Determination of Molecular Mass of Bioagent Identifying Amplicons

In some embodiments, the molecular mass of a given bioagent identifying amplicon is determined by mass spectrometry. Mass spectrometry has several advantages, not the least of which is high bandwidth characterized by the ability to separate (and isolate) many molecular peaks across a broad range of mass to charge ratio (m/z). Thus mass spectrometry is intrinsically a parallel detection scheme without the need for radioactive or fluorescent labels, since every amplification product is identified by its molecular mass. The current state of the art in mass spectrometry is such that less than femtomole quantities of material can be readily analyzed to afford information about the molecular contents of the sample. An accurate assessment of the molecular mass of the material can be quickly obtained, irrespective of whether the molecular weight of the sample is several hundred, or in excess of one hundred thousand atomic mass units (amu) or Daltons.

In some embodiments, intact molecular ions are generated from amplification products using one of a variety of ionization techniques to convert the sample to gas phase. These ionization methods include, but are not limited to, electrospray ionization (ES), matrix-assisted laser desorption ionization (MALDI) and fast atom bombardment (FAB). Upon ionization, several peaks are observed from one sample due to the formation of ions with different charges. Averaging the multiple readings of molecular mass obtained from a single mass spectrum affords an estimate of molecular mass of the bioagent identifying amplicon. Electrospray ionization mass spectrometry (ESI-MS) is particularly useful for very high molecular weight polymers such as proteins and nucleic acids having molecular weights greater than 10 kDa, since it yields a distribution of multiply-charged molecules of the sample without causing a significant amount of fragmentation.

›DETAILED DESCRIPTION OF EMBODIMENTS · 6 of 9

The mass detectors used in the methods of the present invention include, but are not limited to, Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS), time of flight (TOF), ion trap, quadrupole, magnetic sector, Q-TOF, and triple quadrupole.

D. Base Compositions of Bioagent Identifying Amplicons

Although the molecular mass of amplification products obtained using intelligent primers provides a means for identification of bioagents, conversion of molecular mass data to a base composition signature is useful for certain analyses. As used herein, “base composition” is the exact number of each nucleobase (A, T, C and G) determined from the molecular mass of a bioagent identifying amplicon. In some embodiments, a base composition provides an index of a specific organism. Base compositions can be calculated from known sequences of known bioagent identifying amplicons and can be experimentally determined by measuring the molecular mass of a given bioagent identifying amplicon, followed by determination of all possible base compositions which are consistent with the measured molecular mass within acceptable experimental error. The following example illustrates determination of base composition from an experimentally obtained molecular mass of a 46-mer amplification product originating at position 1337 of the 16S rRNA of Bacillus anthracis . The forward and reverse strands of the amplification product have measured molecular masses of 14208 and 14079 Da, respectively. The possible base compositions derived from the molecular masses of the forward and reverse strands for the B. anthracis products are listed in Table 1.

Among the 16 possible base compositions for the forward strand and the 18 possible base compositions for the reverse strand that were calculated, only one pair (shown in bold) are complementary base compositions, which indicates the true base composition of the amplification product. It should be recognized that this logic is applicable for determination of base compositions of any bioagent identifying amplicon, regardless of the class of bioagent from which the corresponding amplification product was obtained.

In some embodiments, assignment of previously unobserved base compositions (also known as “true unknown base compositions”) to a given phylogeny can be accomplished via the use of pattern classifier model algorithms. Base compositions, like sequences, vary slightly from strain to strain within species, for example. In some embodiments, the pattern classifier model is the mutational probability model. On other embodiments, the pattern classifier is the polytope model. The mutational probability model and polytope model are both commonly owned and described in U.S. patent application Ser. No. 11/073,362 which is incorporated herein by reference in entirety.

In one embodiment, it is possible to manage this diversity by building “base composition probability clouds” around the composition constraints for each species. This permits identification of organisms in a fashion similar to sequence analysis. A “pseudo four-dimensional plot” can be used to visualize the concept of base composition probability clouds. Optimal primer design requires optimal choice of bioagent identifying amplicons and maximizes the separation between the base composition signatures of individual bioagents. Areas where clouds overlap indicate regions that may result in a misclassification, a problem which is overcome by a triangulation identification process using bioagent identifying amplicons not affected by overlap of base composition probability clouds.

In some embodiments, base composition probability clouds provide the means for screening potential primer pairs in order to avoid potential misclassifications of base compositions. In other embodiments, base composition probability clouds provide the means for predicting the identity of a bioagent whose assigned base composition was not previously observed and/or indexed in a bioagent identifying amplicon base composition database due to evolutionary transitions in its nucleic acid sequence. Thus, in contrast to probe-based techniques, mass spectrometry determination of base composition does not require prior knowledge of the composition or sequence in order to make the measurement.

The present invention provides bioagent classifying information similar to DNA sequencing and phylogenetic analysis at a level sufficient to identify a given bioagent. Furthermore, the process of determination of a previously unknown base composition for a given bioagent (for example, in a case where sequence information is unavailable) has downstream utility by providing additional bioagent indexing information with which to populate base composition databases. The process of future bioagent identification is thus greatly improved as more BCS indexes become available in base composition databases.

E. Triangulation Identification

In some cases, a molecular mass of a single bioagent identifying amplicon alone does not provide enough resolution to unambiguously identify a given bioagent. The employment of more than one bioagent identifying amplicon for identification of a bioagent is herein referred to as “triangulation identification.” Triangulation identification is pursued by determining the molecular masses of a plurality of bioagent identifying amplicons selected within a plurality of housekeeping genes. This process is used to reduce false negative and false positive signals, and enable reconstruction of the origin of hybrid or otherwise engineered bioagents. For example, identification of the three part toxin genes typical of B. anthracis (Bowen et al., J. Appl. Microbiol., 1999, 87, 270-278) in the absence of the expected signatures from the B. anthracis genome would suggest a genetic engineering event.

In some embodiments, the triangulation identification process can be pursued by characterization of bioagent identifying amplicons in a massively parallel fashion using the polymerase chain reaction (PCR), such as multiplex PCR where multiple primers are employed in the same amplification reaction mixture, or PCR in multi-well plate format wherein a different and unique pair of primers is used in multiple wells containing otherwise identical reaction mixtures. Such multiplex and multi-well PCR methods are well known to those with ordinary skill in the arts of rapid throughput amplification of nucleic acids. In other related embodiments, one PCR reaction per well or container may be carried out, followed by an amplicon pooling step wherein the amplification products of different wells are combined in a single well or container which is then subjected to molecular mass analysis. The combination of pooled amplicons can be chosen such that the expected ranges of molecular masses of individual amplicons are not overlapping and thus will not complicate identification of signals.

›DETAILED DESCRIPTION OF EMBODIMENTS · 7 of 9

F. Codon Base Composition Analysis

In some embodiments of the present invention, one or more nucleotide substitutions within a codon of a gene of an infectious organism confer drug resistance upon an organism which can be determined by codon base composition analysis. The organism can be a bacterium, virus, fungus or protozoan.

In some embodiments, the amplification product containing the codon being analyzed is of a length of about 35 to about 200 nucleobases. The primers employed in obtaining the amplification product can hybridize to upstream and downstream sequences directly adjacent to the codon, or can hybridize to upstream and downstream sequences one or more sequence positions away from the codon. The primers may have between about 70% to 100% sequence complementarity with the sequence of the gene containing the codon being analyzed.

In some embodiments, the codon base composition analysis is undertaken

In some embodiments, the codon analysis is undertaken for the purpose of investigating genetic disease in an individual. In other embodiments, the codon analysis is undertaken for the purpose of investigating a drug resistance mutation or any other deleterious mutation in an infectious organism such as a bacterium, virus, fungus or protozoan. In some embodiments, the bioagent is a bacterium identified in a biological product.

In some embodiments, the molecular mass of an amplification product containing the codon being analyzed is measured by mass spectrometry. The mass spectrometry can be either electrospray (ESI) mass spectrometry or matrix-assisted laser desorption ionization (MALDI) mass spectrometry. Time-of-flight (TOF) is an example of one mode of mass spectrometry compatible with the analyses of the present invention.

The methods of the present invention can also be employed to determine the relative abundance of drug resistant strains of the organism being analyzed. Relative abundances can be calculated from amplitudes of mass spectral signals with relation to internal calibrants. In some embodiments, known quantities of internal amplification calibrants can be included in the amplification reactions and abundances of analyte amplification product estimated in relation to the known quantities of the calibrants.

In some embodiments, upon identification of one or more drug-resistant strains of an infectious organism infecting an individual, one or more alternative treatments can be devised to treat the individual.

G. Determination of the Quantity of a Bioagent

In some embodiments, the identity and quantity of an unknown bioagent can be determined using the process illustrated in FIG. 2 . Primers ( 500 ) and a known quantity of a calibration polynucleotide ( 505 ) are added to a sample containing nucleic acid of an unknown bioagent. The total nucleic acid in the sample is then subjected to an amplification reaction ( 510 ) to obtain amplification products. The molecular masses of amplification products are determined ( 515 ) from which are obtained molecular mass and abundance data. The molecular mass of the bioagent identifying amplicon ( 520 ) provides the means for its identification ( 525 ) and the molecular mass of the calibration amplicon obtained from the calibration polynucleotide ( 530 ) provides the means for its identification ( 535 ). The abundance data of the bioagent identifying amplicon is recorded ( 540 ) and the abundance data for the calibration data is recorded ( 545 ), both of which are used in a calculation ( 550 ) which determines the quantity of unknown bioagent in the sample.

A sample comprising an unknown bioagent is contacted with a pair of primers that provide the means for amplification of nucleic acid from the bioagent, and a known quantity of a polynucleotide that comprises a calibration sequence. The nucleic acids of the bioagent and of the calibration sequence are amplified and the rate of amplification is reasonably assumed to be similar for the nucleic acid of the bioagent and of the calibration sequence. The amplification reaction then produces two amplification products: a bioagent identifying amplicon and a calibration amplicon. The bioagent identifying amplicon and the calibration amplicon should be distinguishable by molecular mass while being amplified at essentially the same rate. Effecting differential molecular masses can be accomplished by choosing as a calibration sequence, a representative bioagent identifying amplicon (from a specific species of bioagent) and performing, for example, a 2-8 nucleobase deletion or insertion within the variable region between the two priming sites. The amplified sample containing the bioagent identifying amplicon and the calibration amplicon is then subjected to molecular mass analysis by mass spectrometry, for example. The resulting molecular mass analysis of the nucleic acid of the bioagent and of the calibration sequence provides molecular mass data and abundance data for the nucleic acid of the bioagent and of the calibration sequence. The molecular mass data obtained for the nucleic acid of the bioagent enables identification of the unknown bioagent and the abundance data enables calculation of the quantity of the bioagent, based on the knowledge of the quantity of calibration polynucleotide contacted with the sample.

In some embodiments, construction of a standard curve where the amount of calibration polynucleotide spiked into the sample is varied provides additional resolution and improved confidence for the determination of the quantity of bioagent in the sample. The use of standard curves for analytical determination of molecular quantities is well known to one with ordinary skill and can be performed without undue experimentation.

In some embodiments, multiplex amplification is performed where multiple bioagent identifying amplicons are amplified with multiple primer pairs which also amplify the corresponding standard calibration sequences. In this or other embodiments, the standard calibration sequences are optionally included within a single vector which functions as the calibration polynucleotide. Multiplex amplification methods are well known to those with ordinary skill and can be performed without undue experimentation.

›DETAILED DESCRIPTION OF EMBODIMENTS · 8 of 9

In some embodiments, the calibrant polynucleotide is used as an internal positive control to confirm that amplification conditions and subsequent analysis steps are successful in producing a measurable amplicon. Even in the absence of copies of the genome of a bioagent, the calibration polynucleotide should give rise to a calibration amplicon. Failure to produce a measurable calibration amplicon indicates a failure of amplification or subsequent analysis step such as amplicon purification or molecular mass determination. Reaching a conclusion that such failures have occurred is in itself, a useful event.

In some embodiments, the calibration sequence is comprised of DNA. In some embodiments, the calibration sequence is comprised of RNA.

In some embodiments, the calibration sequence is inserted into a vector that itself functions as the calibration polynucleotide. In some embodiments, more than one calibration sequence is inserted into the vector that functions as the calibration polynucleotide. Such a calibration polynucleotide is herein termed a “combination calibration polynucleotide.” The process of inserting polynucleotides into vectors is routine to those skilled in the art and can be accomplished without undue experimentation. Thus, it should be recognized that the calibration method should not be limited to the embodiments described herein. The calibration method can be applied for determination of the quantity of any bioagent identifying amplicon when an appropriate standard calibrant polynucleotide sequence is designed and used. The process of choosing an appropriate vector for insertion of a calibrant is also a routine operation that can be accomplished by one with ordinary skill without undue experimentation.

H. Identification of Bacteria

In other embodiments of the present invention, the primer pairs produce bioagent identifying amplicons within stable and highly conserved regions of bacteria. The advantage to characterization of an amplicon defined by priming regions that fall within a highly conserved region is that there is a low probability that the region will evolve past the point of primer recognition, in which case, the primer hybridization of the amplification step would fail. Such a primer set is thus useful as a broad range survey-type primer. In another embodiment of the present invention, the intelligent primers produce bioagent identifying amplicons including a region which evolves more quickly than the stable region described above. The advantage of characterization bioagent identifying amplicon corresponding to an evolving genomic region is that it is useful for distinguishing emerging strain variants or the presence of virulence genes, drug resistance genes, or codon mutations that induce drug resistance.

The present invention also has significant advantages as a platform for identification of diseases caused by emerging bacterial strains such as, for example, drug-resistant strains of Staphylococcus aureus . The present invention eliminates the need for prior knowledge of bioagent sequence to generate hybridization probes. This is possible because the methods are not confounded by naturally occurring evolutionary variations occurring in the sequence acting as the template for production of the bioagent identifying amplicon. Measurement of molecular mass and determination of base composition is accomplished in an unbiased manner without sequence prejudice.

Another embodiment of the present invention also provides a means of tracking the spread of a bacterium, such as a particular drug-resistant strain when a plurality of samples obtained from different locations are analyzed by the methods described above in an epidemiological setting. In one embodiment, a plurality of samples from a plurality of different locations is analyzed with primer pairs which produce bioagent identifying amplicons, a subset of which contains a specific drug-resistant bacterial strain. The corresponding locations of the members of the drug-resistant strain subset indicate the spread of the specific drug-resistant strain to the corresponding locations.

I. Kits

The present invention also provides kits for carrying out the methods described herein. In some embodiments, the kit may comprise a sufficient quantity of one or more primer pairs to perform an amplification reaction on a target polynucleotide from a bioagent to form a bioagent identifying amplicon. In some embodiments, the kit may comprise from one to fifty primer pairs, from one to twenty primer pairs, from one to ten primer pairs, or from two to five primer pairs. In some embodiments, the kit may comprise one or more primer pairs recited in Table 2.

In some embodiments, the kit comprises one or more broad range survey primer(s), division wide primer(s), or drill-down primer(s), or any combination thereof. If a given problem involves identification of a specific bioagent, the solution to the problem may require the selection of a particular combination of primers to provide the solution to the problem. A kit may be designed so as to comprise particular primer pairs for identification of a particular bioagent. A drill-down kit may be used, for example, to distinguish different genotypes or strains, drug-resistant, or otherwise. In some embodiments, the primer pair components of any of these kits may be additionally combined to comprise additional combinations of broad range survey primers and division-wide primers so as to be able to identify a bacterium.

In some embodiments, the kit contains standardized calibration polynucleotides for use as internal amplification calibrants. Internal calibrants are described in commonly owned U.S. patent application Ser. No. 60/545,425 which is incorporated herein by reference in its entirety.

In some embodiments, the kit comprises a sufficient quantity of reverse transcriptase (if RNA is to be analyzed for example), a DNA polymerase, suitable nucleoside triphosphates (including alternative dNTPs such as inosine or modified dNTPs such as the 5-propynyl pyrimidines or any dNTP containing molecular mass-modifying tags such as those described above), a DNA ligase, and/or reaction buffer, or any combination thereof, for the amplification processes described above. A kit may further include instructions pertinent for the particular embodiment of the kit, such instructions describing the primer pairs and amplification conditions for operation of the method. A kit may also comprise amplification reaction containers such as microcentrifuge tubes and the like. A kit may also comprise reagents or other materials for isolating bioagent nucleic acid or bioagent identifying amplicons from amplification, including, for example, detergents, solvents, or ion exchange resins which may be linked to magnetic beads. A kit may also comprise a table of measured or calculated molecular masses and/or base compositions of bioagents using the primer pairs of the kit.

›DETAILED DESCRIPTION OF EMBODIMENTS · 9 of 9

Some embodiments are kits that contain one or more survey bacterial primer pairs represented by primer pair compositions wherein each member of each pair of primers has 70% to 100% sequence identity with the corresponding member from the group of primer pairs represented by any of the primer pairs of Table 5. The survey primer pairs may include broad range primer pairs which hybridize to ribosomal RNA, and may also include division-wide primer pairs which hybridize to housekeeping genes such as rplB, tufb, rpoB, rpoC, valS, and infb, for example.

In some embodiments, a kit may contain one or more survey bacterial primer pairs and one or more triangulation genotyping analysis primer pairs such as the primer pairs of Tables 8, 12, 14, 19, 21, 23, or 24. In some embodiments, the kit may represent a less expansive genotyping analysis but include triangulation genotyping analysis primer pairs for more than one genus or species of bacteria. For example, a kit for surveying nosocomial infections at a health care facility may include, for example, one or more broad range survey primer pairs, one or more division wide primer pairs, one or more Acinetobacter baumannii triangulation genotyping analysis primer pairs and one or more Staphylococcus aureus triangulation genotyping analysis primer pairs. One with ordinary skill will be capable of analyzing in silico amplification data to determine which primer pairs will be able to provide optimal identification resolution for the bacterial bioagents of interest.

In some embodiments, a kit may be assembled for identification of strains of bacteria involved in contamination of food. An example of such a kit embodiment is a kit comprising one or more bacterial survey primer pairs of Table 5 with one or more triangulation genotyping analysis primer pairs of Table 12 which provide strain resolving capabilities for identification of specific strains of Campylobacter jejuni.

Some embodiments of the kits are 96-well or 384-well plates with a plurality of wells containing any or all of the following components: dNTPs, buffer salts, Mg 2+ , betaine, and primer pairs. In some embodiments, a polymerase is also included in the plurality of wells of the 96-well or 384-well plates.

Some embodiments of the kit contain instructions for PCR and mass spectrometry analysis of amplification products obtained using the primer pairs of the kits.

Some embodiments of the kit include a barcode which uniquely identifies the kit and the components contained therein according to production lots and may also include any other information relative to the components such as concentrations, storage temperatures, etc. The barcode may also include analysis information to be read by optical barcode readers and sent to a computer controlling amplification, purification and mass spectrometric measurements. In some embodiments, the barcode provides access to a subset of base compositions in a base composition database which is in digital communication with base composition analysis software such that a base composition measured with primer pairs from a given kit can be compared with known base compositions of bioagent identifying amplicons defined by the primer pairs of that kit.

In some embodiments, the kit contains a database of base compositions of bioagent identifying amplicons defined by the primer pairs of the kit. The database is stored on a convenient computer readable medium such as a compact disk or USB drive, for example.

In some embodiments, the kit includes a computer program stored on a computer formatted medium (such as a compact disk or portable USB disk drive, for example) comprising instructions which direct a processor to analyze data obtained from the use of the primer pairs of the present invention. The instructions of the software transform data related to amplification products into a molecular mass or base composition which is a useful concrete and tangible result used in identification and/or classification of bioagents. In some embodiments, the kits of the present invention contain all of the reagents sufficient to carry out one or more of the methods described herein.

While the present invention has been described with specificity in accordance with certain of its embodiments, the following examples serve only to illustrate the invention and are not intended to limit the same. In order that the invention disclosed herein may be more efficiently understood, examples are provided below. It should be understood that these examples are for illustrative purposes only and are not to be construed as limiting the invention in any manner.

EXAMPLES

›Examples19

›Example 1

Design and Validation of Primers that Define Bioagent Identifying Amplicons for Identification of Bacteria

For design of primers that define bacterial bioagent identifying amplicons, a series of bacterial genome segment sequences were obtained, aligned and scanned for regions where pairs of PCR primers would amplify products of about 45 to about 150 nucleotides in length and distinguish subgroups and/or individual strains from each other by their molecular masses or base compositions. A typical process shown in FIG. 1 is employed for this type of analysis.

A database of expected base compositions for each primer region was generated using an in silico PCR search algorithm, such as (ePCR). An existing RNA structure search algorithm (Macke et al., Nucl. Acids Res., 2001, 29, 4724-4735, which is incorporated herein by reference in its entirety) has been modified to include PCR parameters such as hybridization conditions, mismatches, and thermodynamic calculations (SantaLucia, Proc. Natl. Acad. Sci. U.S.A., 1998, 95, 1460-1465, which is incorporated herein by reference in its entirety). This also provides information on primer specificity of the selected primer pairs.

Table 2 represents a collection of primers (sorted by primer pair number) designed to identify bacteria using the methods described herein. The primer pair number is an in-house database index number. Primer sites were identified on segments of genes, such as, for example, the 16S rRNA gene. The forward or reverse primer name shown in Table 2 indicates the gene region of the bacterial genome to which the primer hybridizes relative to a reference sequence. In Table 2, for example, the forward primer name 16 S_EC — 1077 — 1106_F indicates that the forward primer (_F) hybridizes to residues 1077-1106 of the reference sequence represented by a sequence extraction of coordinates 4033120.4034661 from GenBank gi number 16127994 (as indicated in Table 3). As an additional example: the forward primer name BONTA_X52066 — 450 — 473 indicates that the primer hybridizes to residues 450-437 of the gene encoding Clostridium botulinum neurotoxin type A (BoNT/A) represented by GenBank Accession No. X52066 (primer pair name codes appearing in Table 2 are defined in Table 3. One with ordinary skill knows how to obtain individual gene sequences or portions thereof from genomic sequences present in GenBank. In Table 2, Tp=5-propynyluracil; Cp=5-propynylcytosine; *=phosphorothioate linkage; I=inosine. T. GenBank Accession Numbers for reference sequences of bacteria are shown in Table 3 (below). In some cases, the reference sequences are extractions from bacterial genomic sequences or complements thereof.

Primer pair name codes and reference sequences are shown in Table 3. The primer name code typically represents the gene to which the given primer pair is targeted. The primer pair name may include specific coordinates with respect to a reference sequence defined by an extraction of a section of sequence or defined by a GenBank gi number, or the corresponding complementary sequence of the extraction, or the entire GenBank gi number as indicated by the label “no extraction.” Where “no extraction” is indicated for a reference sequence, the coordinates of a primer pair named to the reference sequence are with respect to the GenBank gi listing. Gene abbreviations are shown in bold type in the “Gene Name” column.

To determine the exact primer hybridization coordinates of a given pair of primers on a given bioagent nucleic acid sequence and to determine the sequences, molecular masses and base compositions of an amplification product to be obtained upon amplification of nucleic acid of a known bioagent with known sequence information in the region of interest with a given pair of primers, one with ordinary skill in bioinformatics is capable of obtaining alignments of the primers of the present invention with the GenBank gi number of the relevant nucleic acid sequence of the known bioagent. For example, the reference sequence GenBank gi numbers (Table 3) provide the identities of the sequences which can be obtained from GenBank. Alignments can be done using a bioinformatics tool such as BLASTn provided to the public by NCBI (Bethesda, Md.). Alternatively, a relevant GenBank sequence may be downloaded and imported into custom programmed or commercially available bioinformatics programs wherein the alignment can be carried out to determine the primer hybridization coordinates and the sequences, molecular masses and base compositions of the amplification product. For example, to obtain the hybridization coordinates of primer pair number 2095 (SEQ ID NOs: 456:1261), First the forward primer (SEQ ID NO: 456) is subjected to a BLASTn search on the publicly available NCBI BLAST website. “RefSeq_Genomic” is chosen as the BLAST database since the gi numbers refer to genomic sequences. The BLAST query is then performed. Among the top results returned is a match to GenBank gi number 21281729 (Accession Number NC — 003923). The result shown below, indicates that the forward primer hybridizes to positions 1530282.1530307 of the genomic sequence of Staphylococcus aureus subsp. aureus MW2 (represented by gi number 21281729).

Staphylococcus aureus subsp. aureus MW2, complete genome Length=2820462 Features in this part of subject sequence:

Panton-Valentine leukocidin chain F precursor

Score=52.0 bits (26), Expect=2e-05 Identities=26/26 (100%), Gaps=0/26 (0%) Strand=Plus/Plus

The hybridization coordinates of the reverse primer (SEQ ID NO: 1261) can be determined in a similar manner and thus, the bioagent identifying amplicon can be defined in terms of genomic coordinates. The query/subject arrangement of the result would be presented in Strand=Plus/Minus format because the reverse strand hybridizes to the reverse complement of the genomic sequence. The preceding sequence analyses are well known to one with ordinary skill in bioinformatics and thus, Table 3 contains sufficient information to determine the primer hybridization coordinates of any of the primers of Table 2 to the applicable reference sequences described therein.

›Example 2

Sample Preparation and PCR

Genomic DNA was prepared from samples using the DNeasy Tissue Kit (Qiagen, Valencia, Calif.) according to the manufacturer's protocols.

All PCR reactions were assembled in 50 μL reaction volumes in a 96-well microtiter plate format using a Packard MPII liquid handling robotic platform and M. J. Dyad thermocyclers (MJ research, Waltham, Mass.) or Eppendorf Mastercycler thermocyclers (Eppendorf, Westbury, NY). The PCR reaction mixture consisted of 4 units of Amplitaq Gold, 1× buffer II (Applied Biosystems, Foster City, Calif.), 1.5 mM MgCl 2 , 0.4 M betaine, 800 μM dNTP mixture and 250 nM of each primer. The following typical PCR conditions were used: 95° C. for 10 min followed by 8 cycles of 95° C. for 30 seconds, 48° C. for 30 seconds, and 72° C. 30 seconds with the 48° C. annealing temperature increasing 0.9° C. with each of the eight cycles. The PCR was then continued for 37 additional cycles of 95° C for 15 seconds, 56° C. for 20 seconds, and 72° C. 20 seconds.

›Example 3

Purification of PCR Products for Mass Spectrometry with Ion Exchange Resin-Magnetic Beads

For solution capture of nucleic acids with ion exchange resin linked to magnetic beads, 25 μl of a 2.5 mg/mL suspension of BioClone amine terminated superparamagnetic beads were added to 25 to 50 μl of a PCR (or RT-PCR) reaction containing approximately 10 μM of a typical PCR amplification product. The above suspension was mixed for approximately 5 minutes by vortexing or pipetting, after which the liquid was removed after using a magnetic separator. The beads containing bound PCR amplification product were then washed three times with 50 mM ammonium bicarbonate/50% MeOH or 100 mM ammonium bicarbonate/50% MeOH, followed by three more washes with 50% MEOH. The bound PCR amplicon was eluted with a solution of 25 mM piperidine, 25 mM imidazole, 35% MeOH which included peptide calibration standards.

›Example 4

Mass Spectrometry and Base Composition Analysis

The ESI-FTICR mass spectrometer is based on a Bruker Daltonics (Billerica, MA) Apex II 70e electrospray ionization Fourier transform ion cyclotron resonance mass spectrometer that employs an actively shielded 7 Tesla superconducting magnet. The active shielding constrains the majority of the fringing magnetic field from the superconducting magnet to a relatively small volume. Thus, components that might be adversely affected by stray magnetic fields, such as CRT monitors, robotic components, and other electronics, can operate in close proximity to the FTICR spectrometer. All aspects of pulse sequence control and data acquisition were performed on a 600 MHz Pentium II data station running Bruker's Xmass software under Windows NT 4.0 operating system. Sample aliquots, typically 15 μl, were extracted directly from 96-well microtiter plates using a CTC HTS PAL autosampler (LEAP Technologies, Carrboro, N.C.) triggered by the FTICR data station. Samples were injected directly into a 10 μl sample loop integrated with a fluidics handling system that supplies the 100 μl/hr flow rate to the ESI source. Ions were formed via electrospray ionization in a modified Analytica (Branford, Conn.) source employing an off axis, grounded electrospray probe positioned approximately 1.5 cm from the metalized terminus of a glass desolvation capillary. The atmospheric pressure end of the glass capillary was biased at 6000 V relative to the ESI needle during data acquisition. A counter-current flow of dry N 2 was employed to assist in the desolvation process. Ions were accumulated in an external ion reservoir comprised of an rf-only hexapole, a skimmer cone, and an auxiliary gate electrode, prior to injection into the trapped ion cell where they were mass analyzed. Ionization duty cycles greater than 99% were achieved by simultaneously accumulating ions in the external ion reservoir during ion detection. Each detection event consisted of 1 M data points digitized over 2.3 s. To improve the signal-to-noise ratio (S/N), 32 scans were co-added for a total data acquisition time of 74 s.

The ESI-TOF mass spectrometer is based on a Bruker Daltonics MicroTOF™. Ions from the ESI source undergo orthogonal ion extraction and are focused in a reflectron prior to detection. The TOF and FTICR are equipped with the same automated sample handling and fluidics described above. Ions are formed in the standard MicroTOF™ ESI source that is equipped with the same off-axis sprayer and glass capillary as the FTICR ESI source. Consequently, source conditions were the same as those described above. External ion accumulation was also employed to improve ionization duty cycle during data acquisition. Each detection event on the TOF was comprised of 75,000 data points digitized over 75 μs.

The sample delivery scheme allows sample aliquots to be rapidly injected into the electrospray source at high flow rate and subsequently be electrosprayed at a much lower flow rate for improved ESI sensitivity. Prior to injecting a sample, a bolus of buffer was injected at a high flow rate to rinse the transfer line and spray needle to avoid sample contamination/carryover. Following the rinse step, the autosampler injected the next sample and the flow rate was switched to low flow. Following a brief equilibration delay, data acquisition commenced. As spectra were co-added, the autosampler continued rinsing the syringe and picking up buffer to rinse the injector and sample transfer line. In general, two syringe rinses and one injector rinse were required to minimize sample carryover. During a routine screening protocol a new sample mixture was injected every 106 seconds. More recently a fast wash station for the syringe needle has been implemented which, when combined with shorter acquisition times, facilitates the acquisition of mass spectra at a rate of just under one spectrum/minute.

Raw mass spectra were post-calibrated with an internal mass standard and deconvoluted to monoisotopic molecular masses. Unambiguous base compositions were derived from the exact mass measurements of the complementary single-stranded oligonucleotides. Quantitative results are obtained by comparing the peak heights with an internal PCR calibration standard present in every PCR well at 500 molecules per well. Calibration methods are commonly owned and disclosed in U.S. Provisional Patent Application Ser. No. 60/545,425 which is incorporated herein by reference in entirety.

›Example 5 · 1 of 2

De Novo Determination of Base Composition of Amplification Products using Molecular Mass Modified Deoxynucleotide Triphosphates

Because the molecular masses of the four natural nucleobases have a relatively narrow molecular mass range (A=313.058, G=329.052, C=289.046, T=304.046—See Table 4), a persistent source of ambiguity in assignment of base composition can occur as follows: two nucleic acid strands having different base composition may have a difference of about 1 Da when the base composition difference between the two strands is G⇄A (−15.994) combined with C⇄T (+15.000). For example, one 99-mer nucleic acid strand having a base composition of A 27 G 30 C 21 T 21 has a theoretical molecular mass of 30779.058 while another 99-mer nucleic acid strand having a base composition of A 26 G 31 C 22 T 20 has a theoretical molecular mass of 30780.052. A 1 Da difference in molecular mass may be within the experimental error of a molecular mass measurement and thus, the relatively narrow molecular mass range of the four natural nucleobases imposes an uncertainty factor.

The present invention provides for a means for removing this theoretical 1 Da uncertainty factor through amplification of a nucleic acid with one mass-tagged nucleobase and three natural nucleobases. The term “nucleobase” as used herein is synonymous with other terms in use in the art including “nucleotide,” “deoxynucleotide,” “nucleotide residue,” “deoxynucleotide residue,” “nucleotide triphosphate (NTP),” or deoxynucleotide triphosphate (dNTP).

Addition of significant mass to one of the 4 nucleobases (dNTPs) in an amplification reaction, or in the primers themselves, will result in a significant difference in mass of the resulting amplification product (significantly greater than 1 Da) arising from ambiguities arising from the G⇄A combined with C ⇄T event (Table 4). Thus, the same the G⇄A (−15.994) event combined with 5-Iodo-C⇄T (−110.900) event would result in a molecular mass difference of 126.894. If the molecular mass of the base composition A 27 G 30 5-Iodo-C 21 T 21 (33422.958) is compared with A 26 G 31 5-Iodo-C 22 T 20 , (33549.852) the theoretical molecular mass difference is +126.894. The experimental error of a molecular mass measurement is not significant with regard to this molecular mass difference. Furthermore, the only base composition consistent with a measured molecular mass of the 99-mer nucleic acid is A 27 G 30 5-Iodo-C 21 T 21 . In contrast, the analogous amplification without the mass tag has 18 possible base compositions.

Mass spectra of bioagent-identifying amplicons were analyzed independently using a maximum-likelihood processor, such as is widely used in radar signal processing. This processor, referred to as GenX, first makes maximum likelihood estimates of the input to the mass spectrometer for each primer by running matched filters for each base composition aggregate on the input data. This includes the GenX response to a calibrant for each primer.

The algorithm emphasizes performance predictions culminating in probability-of-detection versus probability-of-false-alarm plots for conditions involving complex backgrounds of naturally occurring organisms and environmental contaminants. Matched filters consist of a priori expectations of signal values given the set of primers used for each of the bioagents. A genomic sequence database is used to define the mass base count matched filters. The database contains the sequences of known bacterial bioagents and includes threat organisms as well as benign background organisms. The latter is used to estimate and subtract the spectral signature produced by the background organisms. A maximum likelihood detection of known background organisms is implemented using matched filters and a running-sum estimate of the noise covariance. Background signal strengths are estimated and used along with the matched filters to form signatures which are then subtracted. The maximum likelihood process is applied to this “cleaned up” data in a similar manner employing matched filters for the organisms and a running-sum estimate of the noise-covariance for the cleaned up data.

The amplitudes of all base compositions of bioagent-identifying amplicons for each primer are calibrated and a final maximum likelihood amplitude estimate per organism is made based upon the multiple single primer estimates. Models of all system noise are factored into this two-stage maximum likelihood calculation. The processor reports the number of molecules of each base composition contained in the spectra. The quantity of amplification product corresponding to the appropriate primer set is reported as well as the quantities of primers remaining upon completion of the amplification reaction.

Base count blurring can be carried out as follows. “Electronic PCR” can be conducted on nucleotide sequences of the desired bioagents to obtain the different expected base counts that could be obtained for each primer pair. See for example, ncbi.nlm.nih.gov/sutils/e-pcr/; Schuler, Genome Res. 7:541-50, 1997. In one illustrative embodiment, one or more spreadsheets, such as Microsoft Excel workbooks contain a plurality of worksheets. First in this example, there is a worksheet with a name similar to the workbook name; this worksheet contains the raw electronic PCR data. Second, there is a worksheet named “filtered bioagents base count” that contains bioagent name and base count; there is a separate record for each strain after removing sequences that are not identified with a genus and species and removing all sequences for bioagents with less than 10 strains. Third, there is a worksheet, “Sheet1” that contains the frequency of substitutions, insertions, or deletions for this primer pair. This data is generated by first creating a pivot table from the data in the “filtered bioagents base count” worksheet and then executing an Excel VBA macro. The macro creates a table of differences in base counts for bioagents of the same species, but different strains. One of ordinary skill in the art may understand additional pathways for obtaining similar table differences without undo experimentation.

›Example 5 · 2 of 2

Application of an exemplary script, involves the user defining a threshold that specifies the fraction of the strains that are represented by the reference set of base counts for each bioagent. The reference set of base counts for each bioagent may contain as many different base counts as are needed to meet or exceed the threshold. The set of reference base counts is defined by taking the most abundant strain's base type composition and adding it to the reference set and then the next most abundant strain's base type composition is added until the threshold is met or exceeded. The current set of data was obtained using a threshold of 55%, which was obtained empirically.

For each base count not included in the reference base count set for that bioagent, the script then proceeds to determine the manner in which the current base count differs from each of the base counts in the reference set. This difference may be represented as a combination of substitutions, Si=Xi, and insertions, Ii=Yi, or deletions, Di=Zi. If there is more than one reference base count, then the reported difference is chosen using rules that aim to minimize the number of changes and, in instances with the same number of changes, minimize the number of insertions or deletions. Therefore, the primary rule is to identify the difference with the minimum sum (Xi+Yi) or (Xi+Zi), e.g., one insertion rather than two substitutions. If there are two or more differences with the minimum sum, then the one that will be reported is the one that contains the most substitutions.

Differences between a base count and a reference composition are categorized as one, two, or more substitutions, one, two, or more insertions, one, two, or more deletions, and combinations of substitutions and insertions or deletions. The different classes of nucleobase changes and their probabilities of occurrence have been delineated in U.S. Patent Application Publication No. 2004209260 (U.S. application Ser. No. 10/418,514) which is incorporated herein by reference in entirety.

›Example 6 · 1 of 2

Use of Broad Range Survey and Division Wide Primer Pairs for Identification of Bacteria in an Epidemic Surveillance Investigation

This investigation employed a set of 16 primer pairs which is herein designated the “surveillance primer set” and comprises broad range survey primer pairs, division wide primer pairs and a single Bacillus lade primer pair. The surveillance primer set is shown in Table 5 and consists of primer pairs originally listed in Table 2. This surveillance set comprises primers with T modifications (note TMOD designation in primer names) which constitutes a functional improvement with regard to prevention of non-templated adenylation (vide supra) relative to originally selected primers which are displayed below in the same row. Primer pair 449 (non-T modified) has been modified twice. Its predecessors are primer pairs 70 and 357, displayed below in the same row. Primer pair 360 has also been modified twice and its predecessors are primer pairs 17 and 118.

The 16 primer pairs of the surveillance set are used to produce bioagent identifying amplicons whose base compositions are sufficiently different amongst all known bacteria at the species level to identify, at a reasonable confidence level, any given bacterium at the species level. As shown in Tables 6A-E, common respiratory bacterial pathogens can be distinguished by the base compositions of bioagent identifying amplicons obtained using the 16 primer pairs of the surveillance set. In some cases, triangulation identification improves the confidence level for species assignment. For example, nucleic acid from Streptococcus pyogenes can be amplified by nine of the sixteen surveillance primer pairs and Streptococcus pneumoniae can be amplified by ten of the sixteen surveillance primer pairs. The base compositions of the bioagent identifying amplicons are identical for only one of the analogous bioagent identifying amplicons and differ in all of the remaining analogous bioagent identifying amplicons by up to four bases per bioagent identifying amplicon. The resolving power of the surveillance set was confirmed by determination of base compositions for 120 isolates of respiratory pathogens representing 70 different bacterial species and the results indicated that natural variations (usually only one or two base substitutions per bioagent identifying amplicon) amongst multiple isolates of the same species did not prevent correct identification of major pathogenic organisms at the species level.

Bacillus anthracis is a well known biological warfare agent which has emerged in domestic terrorism in recent years. Since it was envisioned to produce bioagent identifying amplicons for identification of Bacillus anthracis , additional drill-down analysis primers were designed to target genes present on virulence plasmids of Bacillus anthracis so that additional confidence could be reached in positive identification of this pathogenic organism. Three drill-down analysis primers were designed and are listed in Tables 2 and 6. In Table 6, the drill-down set comprises primers with T modifications (note TMOD designation in primer names) which constitutes a functional improvement with regard to prevention of non-templated adenylation (vide supra) relative to originally selected primers which are displayed below in the same row.

Phylogenetic coverage of bacterial space of the sixteen surveillance primers of Table 5 and the three Bacillus anthracis drill-down primers of Table 6 is shown in FIG. 3 which lists common pathogenic bacteria. FIG. 3 is not meant to be comprehensive in illustrating all species identified by the primers. Only pathogenic bacteria are listed as representative examples of the bacterial species that can be identified by the primers and methods of the present invention. Nucleic acid of groups of bacteria enclosed within the polygons of FIG. 3 can be amplified to obtain bioagent identifying amplicons using the primer pair numbers listed in the upper right hand corner of each polygon. Primer coverage for polygons within polygons is additive. As an illustrative example, bioagent identifying amplicons can be obtained for Chlamydia trachomatis by amplification with, for example, primer pairs 346-349, 360 and 361, but not with any of the remaining primers of the surveillance primer set. On the other hand, bioagent identifying amplicons can be obtained from nucleic acid originating from Bacillus anthracis (located within 5 successive polygons) using, for example, any of the following primer pairs: 346-349, 360, 361 (base polygon), 356, 449 (second polygon), 352 (third polygon), 355 (fourth polygon), 350, 351 and 353 (fifth polygon). Multiple coverage of a given organism with multiple primers provides for increased confidence level in identification of the organism as a result of enabling broad triangulation identification.

In Tables 7A-E, base compositions of respiratory pathogens for primer target regions are shown. Two entries in a cell, represent variation in ribosomal DNA operons. The most predominant base composition is shown first and the minor (frequently a single operon) is indicated by an asterisk (*). Entries with NO DATA mean that the primer would not be expected to prime this species due to mismatches between the primer and target region, as determined by theoretical PCR.

Four sets of throat samples from military recruits at different military facilities taken at different time points were analyzed using the primers of the present invention. The first set was collected at a military training center from Nov. 1 to Dec. 20, 2002 during one of the most severe outbreaks of pneumonia associated with group A Streptococcus in the United States since 1968. During this outbreak, fifty-one throat swabs were taken from both healthy and hospitalized recruits and plated on blood agar for selection of putative group A Streptococcus colonies. A second set of 15 original patient specimens was taken during the height of this group A Streptococcus —associated respiratory disease outbreak. The third set were historical samples, including twenty-seven isolates of group A Streptococcus , from disease outbreaks at this and other military training facilities during previous years. The fourth set of samples was collected from five geographically separated military facilities in the continental U.S. in the winter immediately following the severe November/December 2002 outbreak.

›Example 6 · 2 of 2

Pure colonies isolated from group A Streptococcus -selective media from all four collection periods were analyzed with the surveillance primer set. All samples showed base compositions that precisely matched the four completely sequenced strains of Streptococcus pyogenes . Shown in FIG. 4 is a 3D diagram of base composition (axes A, G and C) of bioagent identifying amplicons obtained with primer pair number 14 (a precursor of primer pair number 348 which targets 16S rRNA). The diagram indicates that the experimentally determined base compositions of the clinical samples closely match the base compositions expected for Streptococcus pyogenes and are distinct from the expected base compositions of other organisms.

In addition to the identification of Streptococcus pyogenes , other potentially pathogenic organisms were identified concurrently. Mass spectral analysis of a sample whose nucleic acid was amplified by primer pair number 349 (SEQ ID NOs: 401:1156) exhibited signals of bioagent identifying amplicons with molecular masses that were found to correspond to analogous base compositions of bioagent identifying amplicons of Streptococcus pyogenes (A27 G32 C24 Ti 8), Neisseria meningitidis (A25 G27 C22 T18 Haemophilus influenzae (A28 G28 C25 T20) (see FIG. 5 and Table 7B). These organisms were present in a ratio of 4:5:20 as determined by comparison of peak heights with peak height of an internal PCR calibration standard as described in commonly owned U.S. Patent Application Ser. No: 60/545,425 which is incorporated herein by reference in its entirety.

Since certain division-wide primers that target housekeeping genes are designed to provide coverage of specific divisions of bacteria to increase the confidence level for identification of bacterial species, they are not expected to yield bioagent identifying amplicons for organisms outside of the specific divisions. For example, primer pair number 356 (SEQ ID NOs: 449:1380) primarily amplifies the nucleic acid of members of the classes Bacilli and Clostridia and is not expected to amplify proteobacteria such as Neisseria meningitidis and Haemophilus influenzae . As expected, analysis of the mass spectrum of amplification products obtained with primer pair number 356 does not indicate the presence of Neisseria meningitidis and Haemophilus influenzae but does indicate the presence of Streptococcus pyogenes ( FIGS. 3 and 6 , Table 7B). Thus, these primers or types of primers can confirm the absence of particular bioagents from a sample.

The 15 throat swabs from military recruits were found to contain a relatively small set of microbes in high abundance. The most common were Haemophilus influenza, Neisseria meningitides , and Streptococcus pyogenes. Staphylococcus epidermidis, Moraxella cattarhalis, Corynebacterium pseudodiphtheriticum , and Staphylococcus aureus were present in fewer samples. An equal number of samples from healthy volunteers from three different geographic locations, were identically analyzed. Results indicated that the healthy volunteers have bacterial flora dominated by multiple, commensal non-beta-hemolytic Streptococcal species, including the viridans group streptococci ( S. parasangunis, S. vestibularis, S. mitis, S. oralis and S. pneumoniae ; data not shown), and none of the organisms found in the military recruits were found in the healthy controls at concentrations detectable by mass spectrometry. Thus, the military recruits in the midst of a respiratory disease outbreak had a dramatically different microbial population than that experienced by the general population in the absence of epidemic disease.

›Example 7

Triangulation Genotyping Analysis for Determination of emm-Type of Streptococcus pyogenes in Epidemic Surveillance

As a continuation of the epidemic surveillance investigation of Example 6, determination of sub-species characteristics (genotyping) of Streptococcus pyogenes , was carried out based on a strategy that generates strain-specific signatures according to the rationale of Multi-Locus Sequence Typing (MLST). In classic MLST analysis, internal fragments of several housekeeping genes are amplified and sequenced (Enright et al. Infection and Immunity, 2001, 69, 2416-2427). In classic MLST analysis, internal fragments of several housekeeping genes are amplified and sequenced. In the present investigation, bioagent identifying amplicons from housekeeping genes were produced using drill-down primers and analyzed by mass spectrometry. Since mass spectral analysis results in molecular mass, from which base composition can be determined, the challenge was to determine whether resolution of emm classification of strains of Streptococcus pyogenes could be determined.

For the purpose of development of a triangulation genotyping assay, an alignment was constructed of concatenated alleles of seven MLST housekeeping genes (glucose kinase (gki), glutamine transporter protein (gtr), glutamate racemase (murI), DNA mismatch repair protein (mutS), xanthine phosphoribosyl transferase (xpt), and acetyl-CoA acetyl transferase (yqiL)) from each of the 212 previously emm-typed strains of Streptococcus pyogenes . From this alignment, the number and location of primer pairs that would maximize strain identification via base composition was determined. As a result, 6 primer pairs were chosen as standard drill-down primers for determination of emm-type of Streptococcus pyogenes . These six primer pairs are displayed in Table 8. This drill-down set comprises primers with T modifications (note TMOD designation in primer names) which constitutes a functional improvement with regard to prevention of non-templated adenylation (vide supra) relative to originally selected primers which are displayed below in the same row.

The primers of Table 8 were used to produce bioagent identifying amplicons from nucleic acid present in the clinical samples. The bioagent identifying amplicons which were subsequently analyzed by mass spectrometry and base compositions corresponding to the molecular masses were calculated.

Of the 51 samples taken during the peak of the November/December 2002 epidemic (Table 9A-C rows 1-3), all except three samples were found to represent emm3, a Group A Streptococcus genotype previously associated with high respiratory virulence. The three outliers were from samples obtained from healthy individuals and probably represent non-epidemic strains. Archived samples (Tables 9A-C rows 5-13) from historical collections showed a greater heterogeneity of base compositions and emm types as would be expected from different epidemics occurring at different places and dates. The results of the mass spectrometry analysis and emm gene sequencing were found to be concordant for the epidemic and historical samples.

›Example 8

Design of Calibrant Polynucleotides Based on Bioagent Identifying Amplicons for Identification of Species of Bacteria (Bacterial Bioagent Identifying Amplicons)

This example describes the design of 19 calibrant polynucleotides based on bacterial bioagent identifying amplicons corresponding to the primers of the broad surveillance set (Table 5) and the Bacillus anthracis drill-down set (Table 6).

Calibration sequences were designed to simulate bacterial bioagent identifying amplicons produced by the T modified primer pairs shown in Tables 5 and 6 (primer names have the designation “TMOD”). The calibration sequences were chosen as a representative member of the section of bacterial genome from specific bacterial species which would be amplified by a given primer pair. The model bacterial species upon which the calibration sequences are based are also shown in Table 10. For example, the calibration sequence chosen to correspond to an amplicon produced by primer pair no. 361 is SEQ ID NO: 1445. In Table 10, the forward (_F) or reverse (_R) primer name indicates the coordinates of an extraction representing a gene of a standard reference bacterial genome to which the primer hybridizes e.g.: the forward primer name 16S_EC — 713 — 732_TMOD_F indicates that the forward primer hybridizes to residues 713-732 of the gene encoding 16S ribosomal RNA in an E. coli reference sequence (in this case, the reference sequence is an extraction consisting of residues 4033120-4034661 of the genomic sequence of E. coli K12 (GenBank gi number 16127994). Additional gene coordinate reference information is shown in Table 11. The designation “TMOD” in the primer names indicates that the 5′ end of the primer has been modified with a non-matched template T residue which prevents the PCR polymerase from adding non-templated adenosine residues to the 5′ end of the amplification product, an occurrence which may result in miscalculation of base composition from molecular mass data (vide supra).

The 19 calibration sequences described in Tables 10 and 11 were combined into a single calibration polynucleotide sequence (SEQ ID NO: 1464—which is herein designated a “combination calibration polynucleotide”) which was then cloned into a pCR®—Blunt vector (Invitrogen, Carlsbad, Calif.). This combination calibration polynucleotide can be used in conjunction with the primers of Tables 5 or 6 as an internal standard to produce calibration amplicons for use in determination of the quantity of any bacterial bioagent. Thus, for example, when the combination calibration polynucleotide vector is present in an amplification reaction mixture, a calibration amplicon based on primer pair 346 (16S rRNA) will be produced in an amplification reaction with primer pair 346 and a calibration amplicon based on primer pair 363 (rpoC) will be produced with primer pair 363. Coordinates of each of the 19 calibration sequences within the calibration polynucleotide (SEQ ID NO: 1464) are indicated in Table 11.

›Example 9

Use of a Calibration Polynucleotide for Determining the Quantity of Bacillus Anthracis in a Sample Containing a Mixture of Microbes

The process described in this example is shown in FIG. 2 . The capC gene is a gene involved in capsule synthesis which resides on the pX02 plasmid of Bacillus anthracis . Primer pair number 350 (see Tables 10 and 11) was designed to identify Bacillus anthracis via production of a bacterial bioagent identifying amplicon. Known quantities of the combination calibration polynucleotide vector described in Example 8 were added to amplification mixtures containing bacterial bioagent nucleic acid from a mixture of microbes which included the Ames strain of Bacillus anthracis . Upon amplification of the bacterial bioagent nucleic acid and the combination calibration polynucleotide vector with primer pair no. 350, bacterial bioagent identifying amplicons and calibration amplicons were obtained and characterized by mass spectrometry. A mass spectrum measured for the amplification reaction is shown in FIG. 7 . The molecular masses of the bioagent identifying amplicons provided the means for identification of the bioagent from which they were obtained (Ames strain of Bacillus anthracis ) and the molecular masses of the calibration amplicons provided the means for their identification as well. The relationship between the abundance (peak height) of the calibration amplicon signals and the bacterial bioagent identifying amplicon signals provides the means of calculation of the copies of the pX02 plasmid of the Ames strain of Bacillus anthracis . Methods of calculating quantities of molecules based on internal calibration procedures are well known to those of ordinary skill in the art.

Averaging the results of 10 repetitions of the experiment described above, enabled a calculation that indicated that the quantity of Ames strain of Bacillus anthracis present in the sample corresponds to approximately 10 copies of pX02 plasmid.

›Example 10

Triangulation Genotyping Analysis of Campylobacter Species

A series of triangulation genotyping analysis primers were designed as described in Example 1 with the objective of identification of different strains of Campylobacter jejuni . The primers are listed in Table 12 with the designation “CJST_CJ.” Housekeeping genes to which the primers hybridize and produce bioagent identifying amplicons include: tkt (transketolase), glyA (serine hydroxymethyltransferase), gltA (citrate synthase), aspA (aspartate ammonia lyase), glnA (glutamine synthase), pgm (phosphoglycerate mutase), and uncA (ATP synthetase alpha chain).

The primers were used to amplify nucleic acid from 50 food product samples provided by the USDA, 25 of which contained Campylobacter jejuni and 25 of which contained Campylobacter coli . Primers used in this study were developed primarily for the discrimination of Campylobacter jejuni clonal complexes and for distinguishing Campylobacter jejuni from Campylobacter coli . Finer discrimination between Campylobacter coli types is also possible by using specific primers targeted to loci where closely-related Campylobacter coli isolates demonstrate polymorphisms between strains. The conclusions of the comparison of base composition analysis with sequence analysis are shown in Tables 1 3A-C.

The base composition analysis method was successful in identification of 12 different strain groups. Campylobacter jejuni and Campylobacter coli are generally differentiated by all loci. Ten clearly differentiated Campylobacter jejuni isolates and 2 major Campylobacter coli groups were identified even though the primers were designed for strain typing of Campylobacter jejuni . One isolate (RM4183) which was designated as Campylobacter jejuni was found to group with Campylobacter coli and also appears to actually be Campylobacter coli by full MLST sequencing.

›Example 11

Identification of Acinetobacter baumannii Using Broad Range Survey and Division-Wide Primers in Epidemiological Surveillance

To test the capability of the broad range survey and division-wide primer sets of Table 5 in identification of Acinetobacter species, 183 clinical samples were obtained from individuals participating in, or in contact with individuals participating in Operation Iraqi Freedom (including US service personnel, US civilian patients at the Walter Reed Army Institute of Research (WRAIR), medical staff, Iraqi civilians and enemy prisoners. In addition, 34 environmental samples were obtained from hospitals in Iraq, Kuwait, Germany, the United States and the USNS Comfort, a hospital ship.

Upon amplification of nucleic acid obtained from the clinical samples, primer pairs 346-349, 360, 361, 354, 362 and 363 (Table 5) all produced bacterial bioagent amplicons which identified Acinetobacter baumannii in 215 of 217 samples. The organism Klebsiella pneumoniae was identified in the remaining two samples. In addition, 14 different strain types (containing single nucleotide polymorphisms relative to a reference strain of Acinetobacter baumannii ) were identified and assigned arbitrary numbers from 1 to 14. Strain type 1 was found in 134 of the sample isolates and strains 3 and 7 were found in 46 and 9 of the isolates respectively.

The epidemiology of strain type 7 of Acinetobacter baumannii was investigated. Strain 7 was found in 4 patients and 5 environmental samples (from field hospitals in Iraq and Kuwait). The index patient infected with strain 7 was a pre-war patient who had a traumatic amputation in March of 2003 and was treated at a Kuwaiti hospital. The patient was subsequently transferred to a hospital in Germany and then to WRAIR. Two other patients from Kuwait infected with strain 7 were found to be non-infectious and were not further monitored. The fourth patient was diagnosed with a strain 7 infection in September of 2003 at WRAIR. Since the fourth patient was not related involved in Operation Iraqi Freedom, it was inferred that the fourth patient was the subject of a nosocomial infection acquired at WRAIk as a result of the spread of strain 7 from the index patient.

The epidemiology of strain type 3 of Acinetobacter baumannii was also investigated. Strain type 3 was found in 46 samples, all of which were from patients (US service members, Iraqi civilians and enemy prisoners) who were treated on the USNS Comfort hospital ship and subsequently returned to Iraq or Kuwait. The occurrence of strain type 3 in a single locale may provide evidence that at least some of the infections at that locale were a result of nosocomial infections.

This example thus illustrates an embodiment of the present invention wherein the methods of analysis of bacterial bioagent identifying amplicons provide the means for epidemiological surveillance.

›Example 12

Selection and Use of Triangulation Genotyping Analysis Primer Pairs for Acinetobacter baumanii

To combine the power of high-throughput mass spectrometric analysis of bioagent identifying amplicons with the sub-species characteristic resolving power provided by triangulation genotyping analysis, an additional 21 primer pairs were selected based on analysis of housekeeping genes of the genus Acinetobacter . Genes to which the drill-down triangulation genotyping analysis primers hybridize for production of bacterial bioagent identifying amplicons include anthranilate synthase component I (trpE), adenylate kinase (adk), adenine glycosylase (mutY), fumarate hydratase (fumC), and pyrophosphate phospho-hydratase (ppa). These 21 primer pairs are indicated with reference to sequence listings in Table 14. Primer pair numbers 1151-1154 hybridize to and amplify segments of trpE. Primer pair numbers 1155-1157 hybridize to and amplify segments of adk. Primer pair numbers 1158-1164 hybridize to and amplify segments of mutY. Primer pair numbers 1165-1170 hybridize to and amplify segments of fumC. Primer pair number 1171 hybridizes to and amplifies a segment of ppa. Primer pair numbers: 2846-2848 hybridize to and amplify segments of the parC gene of DNA topoisomerase which include a codon known to confer quinolone drug resistance upon sub-types of Acinetobacter baumannii . Primer pair numbers 2852-2854 hybridize to and amplify segments of the gyrA gene of DNA gyrase which include a codon known to confer quinolone drug resistance upon sub-types of Acinetobacter baumannii . Primer pair numbers 2922 and 2972 are speciating primers which are useful for identifying different species members of the genus Acinetobacter . The primer names given in Table 14A (with the exception of primer pair numbers 2846-2848, 2852-2854) indicate the coordinates to which the primers hybridize to a reference sequence which comprises a concatenation of the genes TrpE, efp (elongation factor p), adk, mutT, fumC, and ppa. For example, the forward primer of primer pair 1151 is named AB_MLST-11-OIF007 — 62 — 91_F because it hybridizes to the Acinetobacter primer reference sequence of strain type 11 in sample 007 of Operation Iraqi Freedom (OIF) at positions 62 to 91. DNA was sequenced from strain type 11 and from this sequence data and an artificial concatenated sequence of partial gene extractions was assembled for use in design of the triangulation genotyping analysis primers. The stretches of arbitrary residues “N”s in the concatenated sequence were added for the convenience of separation of the partial gene extractions (40N for AB_MLST (SEQ ID NO: 1444)).

The hybridization coordinates of primer pair numbers 2846-2848 are with respect to GenBank Accession number X95819. The hybridization coordinates of primer pair numbers 2852-2854 are with respect to GenBank Accession number AY642140. Sequence residue “I” appearing in the forward and reverse primers of primer pair number 2972 represents inosine.

Analysis of bioagent identifying amplicons obtained using the primers of Table 14B for over 200 samples from Operation Iraqi Freedom resulted in the identification of 50 distinct strain type clusters. The largest cluster, designated strain type 11 (ST11) includes 42 sample isolates, all of which were obtained from US service personnel and Iraqi civilians treated at the 28 th Combat Support Hospital in Baghdad. Several of these individuals were also treated on the hospital ship USNS Comfort. These observations are indicative of significant epidemiological correlation/linkage.

All of the sample isolates were tested against a broad panel of antibiotics to characterize their antibodies resistance profiles. As an example of a representative result from antibiotic susceptibility testing, ST11 was found to consist of four different clusters of isolates, each with a varying degree of sensitivity/resistance to the various antibiotics tested which included penicillins, extended spectrum penicillins, cephalosporins, carbepenem, protein synthesis inhibitors, nucleic acid synthesis inhibitors, anti-metabolites, and anti-cell membrane antibiotics. Thus, the genotyping power of bacterial bioagent identifying amplicons, particularly drill-down bacterial bioagent identifying amplicons, has the potential to increase the understanding of the transmission of infections in combat casualties, to identify the source of infection in the environment, to track hospital transmission of nosocomial infections, and to rapidly characterize drug-resistance profiles which enable development of effective infection control measures on a time-scale previously not achievable.

›Example 13

Triangulation Genotyping Analysis and Codon Analysis of Acinetobacter baumannii Samples from Two Health Care Facilities

In this investigation, 88 clinical samples were obtained from Walter Reed Hospital and 95 clinical samples were obtained from Northwestern Medical Center. All samples from both healthcare facilities were suspected of containing sub-types of Acinetobacter baumannii , at least some of which were expected to be resistant to quinolone drugs. Each of the 183 samples was analyzed by the method of the present invention. DNA was extracted from each of the samples and amplified with eight triangulation genotyping analysis primer pairs represented by primer pair numbers: 1151, 1156, 1158, 1160, 1165, 1167, 1170, and 1171. The DNA was also amplified with speciating primer pair number 2922 and codon analysis primer pair numbers 2846-2848 which interrogate a codon present in the parC gene, and primer pair numbers 2852-2854 which bracket a codon present in the gyrA gene. The parC and gyrA codon mutations are both responsible for causing drug resistance in Acinetobacter baumannii . During evolution of drug resistant strains, the gyrA mutation usually occurs before the parC mutation. Amplification products were measured by ESI-TOF mass spectrometry as indicated in Example 4. The base compositions of the amplification products were calculated from the average molecular masses of the amplification products and are shown in Tables 15-18. The entries in each of the tables are grouped according to strain type number, which is an arbitrary number assigned to Acinetobacter baumannii strains in the order of observance beginning from the triangulation genotyping analysis OIF genotyping study described in Example 12. For example, strain type 11 which appears in samples from the Walter Reed Hospital is the same strain as the strain type 11 mentioned in Example 12. Ibis# refers to the order in which each sample was analyzed. Isolate refers to the original sample isolate numbering system used at the location from which the samples were obtained (either Walter Reed Hospital or Northwestern Medical Center). ST=strain type. ND=not detected. Base compositions highlighted with bold type indicate that the base composition is a unique base composition for the amplification product obtained with the pair of primers indicated.

Base composition analysis of the samples obtained from Walter Reed hospital indicated that a majority of the strain types identified were the same strain types already characterized by the OIF study of Example 12. This is not surprising since at least some patients from which clinical samples were obtained in OIF were transferred to the Walter Reed Hospital (WRAIR). Examples of these common strain types include: ST10, ST11, ST12, ST14, ST15, ST16 and ST46. A strong correlation was noted between these strain types and the presence of mutations in the gyrA and parC which confer quinolone drug resistance.

In contrast, the results of base composition analysis of samples obtained from Northwestern Medical Center indicate the presence of 4 major strain types: ST10, ST51, ST53 and ST54. All of these strain types have the gyrA quinolone resistance mutation and most also have the parC quinolone resistance mutation, with the exception of ST35. This observation is consistent with the current understanding that the gyrA mutation generally appears before the parC mutation and suggests that the acquisition of these drug resistance mutations is rather recent and that resistant isolates are taking over the wild-type isolates. Another interesting observation was that a single isolate of ST3 (isolate 841) displays a triangulation genotyping analysis pattern similar to other isolates of ST3, but the codon analysis amplification product base compositions indicate that this isolate has not yet undergone the quinolone resistance mutations in gyrA and parC.

The six isolates that represent species other than Acinetobacter baumannii in the samples obtained from the Walter Reed Hospital were each found to not carry the drug resistance mutations.

The results described above involved analysis of 183 samples using the methods and compositions of the present invention. Results were provided to collaborators at the Walter Reed hospital and Northwestern Medical center within a week of obtaining samples. This example highlights the rapid throughput characteristics of the analysis platform and the resolving power of triangulation genotyping analysis and codon analysis for identification of and determination of drug resistance in bacteria.

›Example 14

Identification of Drug Resistance Genes and Virulence Factors in Staphylococcus aureus

An eight primer pair panel was designed for identification of drug resistance genes and virulence factors of Staphylococcus aureus and is shown in Table 19. The primer sequences are found in Table 2 and are cross-referenced by the primer pair numbers, primer pair names or SEQ ID NOs listed in Table 19.

Primer pair numbers 2256 and 2249 are confirmation primers designed with the aim of high level identification of Staphylococcus aureus . The nuc gene is a Staphylococcus aureus -specific marker gene. The tufB gene is a universal housekeeping gene but the bioagent identifying amplicon defined by primer pair number 2249 provides a unique base composition (A43 G28 C19 T35) which distinguishes Staphylococcus aureus from other members of the genus Staphylococcus.

High level methicillin resistance in a given strain of Staphylococcus aureus is indicated by bioagent identifying amplicons defined by primer pair numbers 879 and 2056. Analyses have indicated that primer pair number 879 is not expected to prime S. sciuri homolog or Enterococcus faecalis/faciem ampicillin-resistant PBP5 homologs.

Macrolide and erythromycin resistance in a given strain of Staphylococcus aureus is indicated by bioagent identifying amplicons defined by primer pair numbers 2081 and 2086.

Resistance to mupriocin in a given strain of Staphylococcus aureus is indicated by bioagent identifying amplicons defined by primer pair number 2313.

Virulence in a given strain of Staphylococcus aureus is indicated by bioagent identifying amplicons defined by primer pair number 2095. This primer pair can simultaneously and identify the pvl (lukS-PV) gene and the lukD gene which encodes a homologous enterotoxin. A bioagent identifying amplicon of the lukD gene has a six nucleobase length difference relative to the lukS-PV gene.

A total of 32 blinded samples of different strains of Staphylococcus aureus were provided by the Center for Disease Control (CDC). Each sample was analyzed by PCR amplification with the eight primer pair panel, followed by purification and measurement of molecular masses of the amplification products by mass spectrometry. Base compositions for the amplification products were calculated. The base compositions provide the information summarized above for each primer pair. The results are shown in Tables 20A and B. One result noted upon un-blinding of the samples is that each of the PVL+identifications agreed with PVL+identified in the same samples by standard PCR assays. These results indicate that the panel of eight primer pairs is useful for identification of drug resistance and virulence sub-species characteristics for Staphylococcus aureus . It is expected that a kit comprising one or more of the members of this panel will be a useful embodiment of the present invention.

›Example 15

Selection and Use of Triangulation Genotyping Analysis Primer Pairs for Staphylococcus aureus

To combine the power of high-throughput mass spectrometric analysis of bioagent identifying amplicons with the sub-species characteristic resolving power provided by triangulation genotyping analysis, a panel of eight triangulation genotyping analysis primer pairs was selected. The primer pairs are designed to produce bioagent identifying amplicons within six different housekeeping genes which are listed in Table 21. The primer sequences are found in Table 2 and are cross-referenced by the primer pair numbers, primer pair names or SEQ ID NOs listed in Table 21.

The same samples analyzed for drug resistance and virulence in Example 14 were subjected to triangulation genotyping analysis. The primer pairs of Table 21 were used to produce amplification products by PCR, which were subsequently purified and measured by mass spectrometry. Base compositions were calculated from the molecular masses and are shown in Tables 22A and 22B.

A total of thirteen different genotypes of Staphylococcus aureus were identified according to the unique combinations of base compositions across the eight different bioagent identifying amplicons obtained with the eight primer pairs. These results indicate that this eight primer pair panel is useful for analysis of unknown or newly emerging strains of Staphylococcus aureus . It is expected that a kit comprising one or more of the members of this panel will be a useful embodiment of the present invention.

›Example 16

Selection and Use of Triangulation Genotyping Analysis Primer Pairs for Members of the Bacterial Genus Vibrio

To combine the power of high-throughput mass spectrometric analysis of bioagent identifying amplicons with the sub-species characteristic resolving power provided by triangulation genotyping analysis, a panel of eight triangulation genotyping analysis primer pairs was selected. The primer pairs are designed to produce bioagent identifying amplicons within seven different housekeeping genes which are listed in Table 23. The primer sequences are found in Table 2 and are cross-referenced by the primer pair numbers, primer pair names or SEQ ID NOs listed in Table 23.

A group of 50 bacterial isolates containing multiple strains of both environmental and clinical isolates of Vibrio cholerae, 9 other Vibrio species, and 3 species of Photobacteria were tested using this panel of primer pairs. Base compositions of amplification products obtained with these 8 primer pairs were used to distinguish amongst various species tested, including sub-species differentiation within Vibrio cholerae isolates. For instance, the non-O1/non-O139 isolates were clearly resolved from the O1 and the O139 isolates, as were several of the environmental isolates of Vibrio cholerae from the clinical isolates.

It is expected that a kit comprising one or more of the members of this panel will be a useful embodiment of the present invention.

›Example 17

Selection and Use of Triangulation Genotyping Analysis Primer Pairs for Members of the Bacterial Genus Pseudomonas

To combine the power of high-throughput mass spectrometric analysis of bioagent identifying amplicons with the sub-species characteristic resolving power provided by triangulation genotyping analysis, a panel of twelve triangulation genotyping analysis primer pairs was selected. The primer pairs are designed to produce bioagent identifying amplicons within seven different housekeeping genes which are listed in Table 24. The primer sequences are found in Table 2 and are cross-referenced by the primer pair numbers, primer pair names or SEQ ID NOs listed in Table 24.

It is expected that a kit comprising one or more of the members of this panel will be a useful embodiment of the present invention.

The present invention includes any combination of the various species and subgeneric groupings falling within the generic disclosure. This invention therefore includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

While in accordance with the patent statutes, description of the various embodiments and examples have been provided, the scope of the invention is not to be limited thereto or thereby. Modifications and alterations of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention.

Therefore, it will be appreciated that the scope of this invention is to be defined by the appended claims, rather than by the specific examples which have been presented by way of example.

Each reference (including, but not limited to, journal articles, U.S. and non-U.S. patents, patent application publications, international patent application publications, gene bank gi or accession numbers, internet web sites, and the like) cited in the present application is incorporated herein by reference in its entirety.

›Tables in the description — 20

TABLE 1 — Possible Base Compositions for B. anthracis 46mer Amplification Product

Calc. Mass	Mass Error	Base	Calc. Mass	Mass Error	Base
Forward	Forward	Composition of	Reverse	Reverse	Composition of
Strand	Strand	Forward Strand	Strand	Strand	Reverse Strand
14208.2935	0.079520	A1 G17 C10 T18	14079.2624	0.080600	A0 G14 C13 T19
14208.3160	0.056980	A1 G20 C15 T10	14079.2849	0.058060	A0 G17 C18 T11
14208.3386	0.034440	A1 G23 C20 T2	14079.3075	0.035520	A0 G20 C23 T3
14208.3074	0.065560	A6 G11 C3 T26	14079.2538	0.089180	A5 G5 C1 T35
14208.3300	0.043020	A6 G14 C8 T18	14079.2764	0.066640	A5 G8 C6 T27
14208.3525	0.020480	A6 G17 C13 T10	14079.2989	0.044100	A5 G11 C11 T19
14208.3751	0.002060	A6 G20 C18 T2	14079.3214	0.021560	A5 G14 C16 T11
14208.3439	0.029060	A11 G8 C1 T26	14079.3440	0.000980	A5 G17 C21 T3
14208.3665	0.006520	A11 G11 C6 T18	14079.3129	0.030140	A10 G5 C4 T27
14208.3890
0.016020
A11 G14 C11 T10
14079.3354	0.007600	A10 G8 C9 T19
14208.4116	0.038560	A11 G17 C16 T2
14079.3579
0.014940
A10 G11 C14 T11
14208.4030	0.029980	A16 G8 C4 T18	14079.3805	0.037480	A10 G14 C19 T3
14208.4255	0.052520	A16 G11 C9 T10	14079.3494	0.006360	A15 G2 C2 T27
14208.4481	0.075060	A16 G14 C14 T2	14079.3719	0.028900	A15 G5 C7 T19
14208.4395	0.066480	A21 G5 C2 T18	14079.3944	0.051440	A15 G8 C12 T11
14208.4620	0.089020	A21 G8 C7 T10	14079.4170	0.073980	A15 G11 C17 T3
—	—	—	14079.4084	0.065400	A20 G2 C5 T19
—	—	—	14079.4309	0.087940	A20 G5 C10 T13

TABLE 2 — Primer Pairs for Identification of Bacteria

Primer			Forward			Reverse
Pair		Forward	SEQ		Reverse	SEQ
Number	Forward Primer Name	Sequence	ID NO:	Reverse Primer Name	Sequence	ID NO:
1	16S_EC_1077_1106_F	GTGAGATGTTG	134	16S_EC_1175_1195_R	GACGTCATCCCCA	809
		GGTTAAGTCCC			CCTTCCTC
		GTAACGAG
2	16S_EC_1082_1106_F	ATGTTGGGTTA	38	16S_EC_1175_1197_R	TTGACGTCATCCC	1398
		AGTCCCGCAAC			CACCTTCCTC
		GAG
3	16S_EC_1090_1111_F	TTAAGTCCCGC	651	16S_EC_1175_1196_R	TGACGTCATCCCC	1159
		AACGATCGCAA			ACCTTCCTC
4	16S_EC_1222_1241_F	GCTACACACGT	114	16_EC_1303_1323_R	CGAGTTGCAGACT	787
		GCTACAATG			GCGATCCG
5	16S_EC_1332_1353_F	AAGTCGGAATC	10	16S_EC_1389_1407_R	GACGGGCGGTGTG	806
		GCTAGTAATCG			TACAAG
6	16S_EC_30_54_F	TGAACGCTGGT	429	16S_EC_105_126_R	TACGCATTACTCA	897
		GGCATGCTTAA			CCCGTCCGC
		CAC
7	16S_EC_38_64_F	GTGGCATGCCT	136	16S_EC_101_120_R	TTACTCACCCGTC	1365
		AATACATGCAA			CGCCGCT
		GTCG
8	16S_EC_49_68_F	TAACACATGCA	152	16S_EC_104_120_R	TTACTCACCCGTC	1364
		AGTCGAACG			CGCC
9	16S_EC_683_700_F	GTGTAGCGGTG	137	16S_EC_774_795_R	GTATCTAATCCTG	839
		AAATGCG			TTTGCTCCC
10	16S_EC_713_732_F	AGAACACCGAT	21	16S_EC_789_809_R	CGTGGACTACCAG	798
		GGCGAAGGC			GGTATCTA
11	16S_EC_785_806_F	GGATTAGAGAC	118	16S_EC_880_897_R	GGCCGTACTCCCC	830
		CCTGGTAGTCC			AGGCG
12	16S_EC_785_810_F	GGATTAGATAC	119	16S_EC_880_897_2_R	GGCGTACTCCCC	830
		CCTGGTAGTCA			AGGCG
		CACGC
13	16S_EC_789_810_F	TAGATACCCTG	206	16S_EC_880_894_R	CGTACTCCCCAGG	796
		GTAGTCCACGC			CG
14	16S_EC_960_981_F	TTCGATGCAAC	672	16S_EC_1054_1073_R	ACGAGCTGACGAC	735
		GCGAAGAACCT			AGCCATG
15	16S_EC_969_985_F	ACGCGAAGAAC	19	16S_EC_1061_1078_R	ACGACCACGAGCTG	734
		CTTACC			ACGAC
16	23S_EC_1826_1843_F	CTGACACCTGC	80	23S_EC_1906_1924_R	GACCGTTATAGTT	805
		CCGGTGC			ACGGCC
17	23S_EC_2645_2669_F	TCTGTCCCTAG	408	23S_EC_2744_2761_R	TGCTTAGATGCTT	1252
		TACGAGAGGAC			TCAGC
		CGG
18	23S_EC_2645_2669_2_F	CTGTCCCTAGT	83	23S_EC_2751_2767_R	GTTTCATGCTTAG	846
		ACGAGAGGACC			ATGCTTTCAGC
		GG
19	23S_EC_493_518_F	GGGGAGTGAAA	125	23S_EC_551_571_R	ACAAAAGGTACGC	717
		GAGATCCTGAA			CGTCACCC
		ACCG
20	23S_EC_493_518_2_F	GGGGAGTGAAA	125	23S_EC_551_571_2_R	ACAAAAGGCACGC	716
		GAGATCCTGAA			CATCACCC
		ACCG
21	23S_EC_971_992_F	CGAGAGGGAAA	66	23S_EC_1059_1077_R	TGGCTGCTTCTAA	1282
		CAACCCAGACC			GCCAAC
22	CAPC_BA_104_131_F	GTTATTTAGCA	139	CAPC_BA_180_205_R	TGAATCTTGAAAC	1150
		CTCGTTTTTAA			ACCATACGTAAC
		TCAGCC			G
23	CAPC_BA_114_133_F	ACTCGTTTTTA	20	CAPC_BA_185_205_R	TGAATCTTGAAAC	1149
		ATCAGCCCG			ACCATACG
24	CAPC_BA_274_303_F	GATTATTGTTA	109	CAPC_BA_349_376_R	GTAACCCTTGTCT	837
		TCCTGTTATGC			TTGAATTGTATTT
		CATTTGAG			GC
25	CAPC_BA_276_296_F	TTATTGTTATC	663	CAPC_BA_358_377_R	GGTAACCCTTGTC	834
		CTGTTATGCC			TTTGAAT
26	CAPC_BA_281_301_F	GTTATCCTGTT	138	CAPC_BA_361_378_R	TGGTAACCCTTGT	1298
		ATGCCATTTG			CTTTG
27	CAPC_BA_315_334_F	CCGTGGTATTG	59	CAPC_BA_361_378_R	TGGTAACCCTTGT	1298
		GAGTTATTG			TCTTTG
28	CYA_BA_1055_1072_F	GAAAGAGTTCG	92	CYA_BA_1112_1130_R	TGTTGACCATGCT	1352
		GATTGGG			TCTTAG
29	CYA_BA_1349_1370_F	ACAACGAAGTA	12	CYA_BA_1447_1426_R	CTTCTACATTTTT	800
		CAATACAAGAC			AGCCATCAC
30	CYA_BA_1353_1379_F	CGAAGTACAAT	64	CYA_BA_1448_1467_R	TGTTAACGGCTTC	1342
		ACAAGACAAAA			AAGACCC
		GAAGG
31	CYA_BA_1359_1379_F	ACAATACAAGA	13	CYA_BA_1447_1461_R	CGGCTTCAAGACC	794
		CAAAAGAAGG			CC
32	CYA_BA_914_937_F	CAGGTTTAGTA	53	CYA_BA_999_1026_R	ACCACTTTTAATA	728
		CCAGAACATG			AGGTTTGTAGCTA
					CAGAC
33	CYA_BA_916_935_F	GGTTTAGTACC	131	CYA_BA_1003_1025_R	CCACTTTTAATAA	768
		AGAACATGC			GGTTTGTAGC
34	INFB_EC_1365_1393_F	TGCTCGTGGTG	524	INFB_EC_1439_1467_R	TGCTGCTTTCGCA	1248
		CACAAGTAACG			TGGTTAATTGCTT
		GAT
		ATTA			CAA
35	LEF_BA_1033_1052_F	TCAAGAAGAAA	254	LEF_BA_1119_1135_R	GAATATCAATTTG	803
		AAGAGC			TAGC
36	LEF_BA_1036_1066_F	CAAGAAGAAAA	44	LEF_BA_1119_1149_R	AGATAAAGAATCA	745
		AGAGCTTCTAA			CGAATATCAATTT
		AAAGAATAC			GTAGC
37	LEF_BA_756_781_F	AGCTTTTGCAT	26	LEF_BA_843_872_R	TCTTCCAAGGATA	1135
		ATTATATCGAG			ATTTATTTCTTG
		CCAC			TTCG
38	LEF_BA_758_778_F	CTTTTGCATATT	90	LEF_BA_843_865_R	AGGATAGATTTAT	748
		ATATCGAGC			TTCTTGTTCG
39	LEF_BA_795_813_F	TTTACAGCTTT	700	LEF_BA_883_900_R	TCTTGACAGCATC	1140
		ATGCACCG			CGTTG
40	LEF_BA_883_899_F	CAACGGATGCT	43	LEF_BA_939_958_R	CAGATAAAGAATC	762
		GGCAAG			GCTCCAG
41	PAG_BA_122_142_F	CAGAATCAAGT	49	PAG_BA_190_209_R	CCTGTAGTAGAAG	781
		TCCCAGGGG			AGGTAAC
42	PAG_BA_123_145_F	AGAATCAAGTT	22	PAG_BA_187_210_R	CCCTGTAGTAGAA	774
		CCCAGGGGTT			GAGGTAACCAC
		AC
43	PAG_BA_269_287_F	AATCTGCTATT	11	PAG_BA_326_344_R	TGATTATCAGCGG	1186
		TGGTCAGG			AAGTAG
44	PAG_BA_655_675_F	GAAGGATATAC	93	PAG_BA_755_772_R	CCGTGCTCCATTT	778
		GGTTGATGTC			TTCAG
45	PAG_BA_753_772_F	TCCTGAAAAAT	341	PAG_BA_849_868_R	TCGGATAAGCTGC	1089
		GGAGCACGG			CACAAGG
46	PAG_BA_763_781_F	TGGAGCACGG	552	PAG_BA_849_868_R	TCGGATAAGCTGC	1089
		CTTCTGATC			CACAAGG
47	RPOC_EC_1018_1045_F	CAAACTTATTA	39	RPOC_EC_1095_1124_R	TCAAGCGCCATTT	959
		AGGTAAGCGTG			CTTTTGGTAAACC
		TTGACT			ACAT
48	RPOC_EC_1018_1045_2_F	CAAAACTTATT	39	RPOC_EC_1095_1124_2_R	TCAAGCGCCATCT	958
		AGGTAAGCGTG			CTTTCGGTAATCC
		TTGACT			ACAT
49	RPOC_EC_114_140_F	TAAGAAGCCGG	158	RPOC_EC_213_232_R	GGCGCTTGTACTT	831
		AAACCATCAAC			ACCGCAC
		TACCG
50	RPOC_EC_2178_2196_F	TGATTCTGGTG	478	RPOC_EC_2225_2246_R	TTGGCCATCAGGC	1414
		CCCGTGGT			CACGCATAC
51	RPOC_EC_2178_2196_2_F	TGATTCCGGTG	477	RPOC_EC_2225_2246_2_R	TTGGCCATCAGAC	1413
		CCCGTGGT			CACGCATAC
52	RPOC_EC_2218_2241_F	CTGGCAGGTAT	81	RPOC_EC_2313_2337_R	CGACCGTGGGTT	790
		GCGTGGTCTGA			GAGATGAAGTAC
		TG
53	RPOC_EC_2218_2241_2_F	CTTGCTGGTAT	86	RPOC_EC_2313_2337_2_R	CGCACCATGCGTA	789
		GCGTGGTCTGA			GAGATGAAGTAC
		TG
54	RPOC_EC_808_833_F	CGTCGGGTGAT	75	RPOC_EC_865_889_R	GTTTTTCGTTGCG	847
		TAACCGTAACA			TACGATGATGTC
		ACCG
55	RPOC_EC_808_833_2_F	CGTCGTGTAAT	76	RPOC_EC_865_891_R	ACGTTTTTCGTTT	741
		AACCGTAACA			TGAACGATAATGC
		ACCG			T
56	RPOC_EC_993_1019_F	CAAAGGTAAGC	41	RPOC_EC_1036_1059_R	CGAACGGCCTGAG	785
		AAGGTCGTTTC			TAGTCAACACG
		CGTCA
57	RPOC_EC_993_1019_2_F	CAAAGGTAAGC	40	RPOC_EC_1036_1059_2_R	CGAACGGCCAGAG	784
		AAGGACGTTTC			TAGTCAACACG
		CGTCA
58	SSPE_BA_115_137_F	CAAGCAAACGC	45	SSPE_BA_197_222_R	TGCACGTCTGTTT	1201
		ACAATCAGAA			CAGTTGCAAATTC
		GC
59	TUFB_EC_239_259_F	TAGACTGCCCA	204	TUFB_EC_283_303_R	GCCGTCCATCTGA	815
		GGACACGCTG			GCAGCACC
60	TUFB_EC_239_259_2_F	TTGACTGCCCA	678	TUFB_EC__283 _303_2_R	GCCGTCCATTTGA	816
		GGTCACGCTG			GCAGCACC
61	TUFB_EC_976_1000_F	AACTACCGTC	4	TUFB_EC_1045_1068_R	GTTGTCGCCAGGC	845
		CGCAGTTCTAC			ATAACCATTTC
		TTCC
62	TUFB_EC_976_1000_2_F	AACTACCGTCC	5	TUFB_EC_1045_1068_2_R	GTTGTCACCAGGC	844
		TCAGTTCTACT			ATTACCATTTC
		TCC
63	TUFB_ECG_985_1012_F	CCACAGTTCTA	56	TUFB_EC_1033_1062_R	TCCAGGCATTACC	1006
		CTTCCGTACTA			ATTTCTACTCCTT
		CTGACG
					CTGG
66	RPLB_EC_650_679_F	GACCTACAGTA	98	RPLB_E739_762_R	TCCAAGTGCTGGT	999
		AGAGGTTCTGT			TTACCCCATGG
		AATTGAACC
67	RPLB_EC_688_710_F	CATCCACACGG	54	RFLB_EC_736_757_R	GTGCTGGTTTACC	842
		TGGTGGTGAAG			CCATGGAGT
		G
68	RPOC_EC_1036_1060_F	CGTGTTGACTA	78	RPOC_EC_1097_1126_R	ATTCAAGAGCCAT	754
		TTCGGGGCGTTC			TTCTTTTGGTAAA
		AG			CCAC
69	RPOB_EC_3762_3790_F	TCAACAACCTC	248	RPOB_EC_3836_3865_R	TTTCTTGAAGAGT	1435
		TTGGAGGTAAA			ATGAGCTGCTCCG
		GCTCAGT			TAAG
70	RPLB_EC_688_710_F	CATCCACACGG	54	RPLB_EC_743_771_R	TGTTTTGTATCCA	1356
		TGGTGGTGAAG			AGTGCTGGTTTAC
		G			CCC
71	VALS_EC_1105_1124_F	CGTGGCGGCGT	77	VALS_EC_1195_1218_R	CGGTACGAACTGG	795
		GGTTATCGA			ATGTCGCCGTT
72	RPOB_EC_1845_1866_F	TATCGCTCAGG	233	RFOB_EC_1909_1929_R	GCTGGATTCGCCT	825
		CGAACTCCAAC			TTGCTACG
73	RPLB_EC_669_698_F	TGTAATGAACC	623	RPLB_BC_735_761_R	CCAAGTGCTGGTT	767
		CTAATGACCAT			TACCCCATGGAGT
		CCACACGG			A
74	RPLB_EC_671_700_F	TAATGAACCCT	169	RPLB_EC_737_762_R	TCCAAGTGCTGGT	1000
		AATGACCATCC			TTACCCCATGGAG
75	SP101_SPET11_1_29_F	AACCTTAATTG	2	SP101_SPET11_92_116_R	CCTACCCAACGTT	779
		GAAAGAAACCC			CACCAAGGGCAG
		AAGAAGT
76	SP101_SPET11_118_147_F	GCTGGTGAAAA	115	SP101_SPET11_213_238_R	TGTGGCCGATTTC	1340
		TAACCCAGATG			ACCACCTGCTCCT
		TCGTCTTC
77	SP101_SPET11_216_243_F	AGCAGGTGGTG	24	SP101_SPET11_308_333_R	TGCCACTTTGACA	1209
		AAATCGGCCAC			ACTCCTGTTGCTG
		ATGATT
78	SP101_SPET11_266_295_F	CTTGTACTTGT	89	SP101_SFET11_355_380_R	GCTGCTTTGATGG	824
		GGCTCACACGG			CTGAATCCCCTTC
		CTGTTTGG
79	SP101_SPET11_322_344_F	GTCAAAGTGGC	132	SF101SFET11_423_441R	ATCCCTGCTTCT	753
		CACGTTTACTG			GCTGCC
		GC
80	SP101_SPET11_358_387_F	GGGGATTCAGC	126	SP101_SPET11_448_473_R	CCAACCTTTTCCA	766
		CATCAAAGCAG			CAACAGAATCAGC
		CTATTGAC
81	SP101_SPET11_600_629_F	CCTTACTTCGA	62	SP101_SPET11_686_714_R	CCCATTTTTTCAC	772
		AACTATGAATC			GCATGCTGAAAAT
		TTTTGGAAG			ATC
82	SP101_SPET11_658_684_F	GGGGATTGATA	127	SP101_SPET11_756_784_R	GATTGGCGATAAA	813
		TCACCATAAG			GTGATATTTTCTA
		AAGAA			AAA
83	SP101_SPET11_776_801_F	TCGCCAATCAA	364	SP101_SPET11_871_896_R	GCCCACCAGAAAG	814
		AACTAAGGGAA			ACTAGCAGGATAA
		TGGC
84	SP101_SPET11_893_921_F	GGGCAACAGCA	123	SP101_SPET11_988_1012_R	CATGACAGCCAAG	763
		GCGGATTGCGA			ACCTCACCCACC
		TTGCGCG
85	SP101_SPET11_1154_1179_F	CAATACCGCAA	47	SP101_SPET11_1251_1277_R	GACCCCAACCTGG	804
		CAGCGGTGGCT			CCTTTTGTCGTTG
		TGGG			A
86	SP101_SPET11_1314_1336_F	CGCAAAAAAAT	68	SP101_SPET11_1403_1431_R	AAACTATTTTTTT	711
		CCAGCTATTAG			AGCTATACTCGAA
		C			CAC
87	SP101_SPET11_1408_1437_F	CGAGTATAGCT	67	SP101_SPET11_1486_1515_R	GGATAATTGGTCG	828
		AAAAAAATAGT			TAACAAGGGATAG
		TTATGACA			TGAG
88	SP101_SPET11_1688_1716_F	CCTATATTAAT	60	SP101_SPET11_1783_1808_R	ATATGATTATCAT	752
		CGTTTACAGAA			TGAACTGCGGCCG
		ACTGGCT
89	SP101_SPET11_1711_1733_F	CTGGCTAAAA	82	SP101_SPET11_1808_1835_R	GCGTGACAGACCT	821
		CTTTGGCAAC			TCTTGAATTGTAA
		GG			TCA
90	SP101_SPET11_1807_1835_F	ATGATTACAAT	33	SP101_SPET11_1901_1927_R	TTGGACCTGTAAT	1412
		TCAAGAAGGTC			CAGCTGAATACTG
		GTCACGC			G
91	SP101_SPET11_1967_1991_F	TAACGGTTATC	155	SP101_SPET11_2062_2083_R	ATTGCCCAGAAAT	755
		ATGGCCCAGAT			CAAATCATC
		GGG
92	SP101_SPET11_2260_2283_F	CAGAGACCGTT	50	SP101_SPET11_2375_2397_R	TCTGGGTGACCTG	1131
		TTATCCTATCA			GTGTTTTAGA
		GC
93	S9101_SPET11_2375_2399_F	TCTAAAACACC	390	SP101_SPET11_2470_2497_R	AGCTGCTAGATGA	747
		AGGTCACCCAG			GCTTCTGCCATGG
		AAG			CC
94	SF101_SPET11_2468_2487_F	ATGGCCATGGC	35	SP101_SPET11_2543_2570_R	CCATAAGGTCACC	770
		AGAAGCTCA			GTCACCATTCAAA
					GC
95	SP101_SPET11_2961_2984_F	ACCATGACAGA	15	SP101_SPET11_3023_3045_R	GGAATTTACCAGC	827
		AGGCATTTTGA			GATAGACACC
		CA
96	SP101_SPET11_3075_3103_F	GATGACTTTTT	108	SP101_SPET11_3168_3196_R	AATCGACGACCAT	715
		AGCTAATGGTC			CTTGGAAAGATTT
		AGGCAGC			CTC
97	SP101_SPET11_3386_3403_F	AGCGTAAAGGT	25	SP101_SPET11_3480_3506_R	CCAGCAGTTACTG	769
		GAACCTT			TCCCCTCATCTTT
					G
98	SP101_SPET11_3511_3535_F	GCTTCAGGAAT	116	SP101_SPET11_3605_3629_R	GGGTCTACACCTG	832
		CAATGATGGAG			CACTTGCATAAC
		CAG
111	RPOB_EC_3775_3803_F	CTTGGAGGTAA	87	RFOB_EC_3829_3858_R	CGTATAAGCTGCA	797
		GTCTCATTTTG			CCATAAGCTTGTA
		GTGGGCA			ATGC
112	VALS_EC_1833_1850_F	CGACGCGCTGC	65	VALS_EC_1920_1943_R	GCGTTCCACAGCT	822
		GCTTCAC			TGTTGCAGAAG
113	RPOB_EC_1336_1353_F	GACCACCTCGG	97	RPOB_EC_1438_1455_R	TTCGCTCTCGGCC	1386
		CAACCGTA			TGGCC
114	TUFB_EC_225_251_F	GCACTATGCAC	111	TUFB_EC_284_309_R	TATAGCACCATCC	930
		ACGTAGATTGT			ATCTGAGCGGCAC
		CCTGG
115	DNAK_EC_428_449_F	CGGCGTACTTC	72	DNAK_EC_503_522_R	CGCGGTCGGCTCG	792
		AACGACAGCCA			TTGATGA
116	VALS_EC_1920_1943_F	CTTCTGCAACA	85	VALS_EC_1948_1970_R	TCGCAGTTCATCA	1075
		AGCTGTGGAAC			GCACGAAGCG
		GC
117	TUFB_EC_757_474_F	AAGACGACCTG	6	TUFB_EC_849_867_R	GCGCTCCACGTCT	819
		CACGGGC			TCACGC
118	23S_EC_2646_2667_F	CTGTTCTTAGT	84	23S_EC_2745_2765_R	TTCGTGCTTAGAT	1389
		ACGAGAGGACC			GCTTTCAG
119	16S_EC_969_985_1P_F	ACGCGAAGAC	19	16S_EC_1061_1078_2P_R	ACGACACGAGCpT	733
		CTTACpC			pGACGAC
120	16S_EC_972_985_2P_F	CGAAGAACpCp	63	16S_EC_1064_1075_2P_R	ACACGAGCpTpGA	727
		TTACC			C
121	16S_EC_972_985_F	CGAAGAACCTT	63	16S_EC_1064_1075_R	ACACGAGCTGAC	727
		ACC
122	TRAN_ILE-	CCTGATAAGGG	61	23S_SC_40_59_R	ACGTCCTTCATCG	740
	RRNH_EC_32_50.2_F				CCTCTGA
123	23S_EC_−7_15_F	GTTGTGAGGTT	140	23S_EC_430_450_R	CTATCGGTCAGTC	799
		AAGCGACTAAG			AGGAGTAT
124	23S_EC_−7_15_F	GTTGTGAGGTT	141	23S_EC_891_910_R	TTGCATCGGGTTG	1403
		AAGCGACTAAG			GTAAGTC
125	23S_EC_430_450_F	ATACTCCTGAC	30	23S_EC_1424_1442_R	AACATAGCCTTCT	712
		TGACCGATAG			CCGTCC
126	23S_EC_891_910_F	GACTTACCAAC	100	23S_SC_1908_1931_R	TACCTTAGGACCG	893
		CCGATGCAA			TTATAGTTACG
127	23S_EC_1424_1442_F	GGACGGAGAAG	117	23S_EC_2475_2494_R	CCAAACACCGCCG	765
		GCTATGTT			TCGATAT
128	23S_EC_1908_1931_F	CGTAACTATAA	73	23S_EC_2833_9852_R	GCTTACACACCCG	826
		CGGTCCTAAGG			GCCTATC
		TA
129	23S_EC_2475_9494_F	ATATCGACGGC	31	TRNA_ASP-	GCGTGACAGGCAG	820
		GGTGTTTGG		RRNH_EC_23_41.2_R	GTATTC
131	16S_EC_−60_−39_F	AGTCTCAAGAG	28	16S_EC_508_525_R	GCTGCTGGCACGG	823
		TGAACACGTAA			AGTTA
132	16S_EC_326_345_F	GACACGGTCCA	95	16S_EC_1041_1058_R	CCATGCAGCACCT	771
		GACTCCTAC			GTCTC
133	16S_EC905_724_F	GATCTGGAGGA	107	16S_EC_1493_3512_R	ACGGTTACCTTGT	739
		ATACCGGTG			TACGACT
134	16S_EC_1268_1287_F	GAGAGCAAGCG	101	TRNA_ALA-	CCTCCTGCGTGCA	780
		GACCTCATA		RRNH_EC_30_40.2_R	AAGC
135	16S_EC_969_985_F	ACGCGAAGAAC	19	16S_EC_1061_1078.2_R	ACAACACGAGCTG	719
		CTTACC			ACGAC
137	165_EC_969_985_F	ACGCGAAGAAC	19	16S_EC_1061_1078.2_I14_R	ACAACACGAGCTG	721
		CCTTACC			ICGAC
138	165_EC_969_985_F	ACGCGAAGAAC	19	16S_EC_1061_107.2_I12_R	ACAACACGAGCIG	718
		CTTACC			CGAC
139	16S_EC_969_985_F	ACGCGAAGAAC	19	16S_EC_1061_1078.2_I11_R	ACAACACGAGITG	722
		CTTACC			ACGAC
140	16S_EC_969_985_F	ACGCGAAGAAC	19	16S_EC_1061_1078.2_I16_R	ACAACACGAGCTG	720
		CTTACC			ACIAC
141	16S_EC_969_985_F	ACGCGAAGAAC	19	16S_EC_1061_1078.2_2I_R	ACAACACGAICTI	723
		CTTACC			ACGAC
142	165_EC_969_985_F	ACGCGAAGAAC	19	16S_EC_1061_1078.2_3I_R	ACAACACIAICTI	724
		CTTACC			ACGAC
143	16S_EC_99_985_F	ACGCGAAGAAC	19	16S_EC_1061_1078.2_4I_R	ACACCACIAICTI	725
		CTTACC			ACIAC
147	23S_EC_2652_2669_F	CTAGTACGAGA	79	23S_EC_2741_2760_R	ACTTAGATGCTTT	743
		GGACCGG			CAGCGGT
158	16S_EC_683_700_F	GTGTAGCGGTG	137	16S_EC_880_894_R	CGTACTCCCCAGG	796
		AAATGCG			CG
159	16S_EC_1100_1116_F	CAACGAGCGCA	42	16S_EC_1174_1188_R	TCCCCACCTTCCT	1019
		ACCCTT			CC
215	SSPE_BA_121_137_F	AACGCACAATC	3	SSPE_BA_197_216_R	TCTGTTTCAGTTG	1132
		AGAAGC			CAAATTC
220	GROL_EC_941_959_F	TGGAAGATCTG	544	GROL_EC_1039_1060_R	CAATCTGCTGACG	759
		GGTCAGGC			GATCTGAGC
221	INFB_EC_1103_1124_F	GTCGTGAAAAC	133	INFB_EC_1174_1191_R	CATGATGGTCACA	764
		GAGCTGGAAGA			ACCGG
222	HFLB_EC_1082_1102_F	TGGCGAACCTG	569	HFLB_EC_1144_1168_R	CTTTCGCTTTCTC	802
		GTGAACGAAGC			GAACTCAACCAT
223	INFB_EC_1969_1994_F	CGTCAGGGTAA	74	INFB_EC_2038_2058_R	AACTTCGCCTTCG	713
		ATTCCGTGAAG			GTCATGTT
		TTAA
224	GROL_EC_219_242_F	GGTGAAAGAAG	128	GROL_EC_328_350_R	TTCAGGTCCATCG	1377
		TTGCCCTCTAA			GGTTCATGCC
		AGC
225	VALS_EC_1105_1124_F	CGTGGCGGCGT	77	VALS_EC_1195_1214_R	ACGAACTGGATGT	732
		GGTTATCGA			CGCCGTT
226	16S_EC_556_575_F	CGGAATTACTG	70	16S_EC_683_700_R	CGCATTTCACCGC	791
		GGCGTAAAG			TACAC
227	RPOC_EC_1256_1277_F	ACCCAGTGCTG	16	RPOC_EC_1295_1315_R	GTTCAAATGCCTG	843
		CTGAACCGTGC			GATACCCA
228	16S_EC_774_795_F	GGGAGCAAACA	122	16S_EC_880_894_R	CGTACTCCCCAGG	796
		GGATTAGATAC			CG
229	RPOC_EC_1584_1604_F	TGGCCCGAAAG	567	RPOC_EC_1623_1643_R	ACGCGGGCATGCA	737
		AAGCTGAGCG			GAGATGCC
230	16S_EC_1082_1100_F	ATGTTGGGTTA	37	16S_EC_1177_1196_R	TGACGTCATCCCC	1158
		AGTCCCGC			ACCTTCC
231	16S_EC_1389_1407_F	CTTGTACACAC	88	16S_EC_1525_1541_R	AAGGAGGTGATCC	714
		CGCCCGTC			AGCC
232	16S_EC_1303_1323_F	CGGATTGGAGT	71	16S_EC_1389_1407_R	GACGGCGGTGTG	808
		CTGCAACTCG			TACAAG
233	23S_EC_23_37_F	GGTGGATGCCT	129	23S_EC_115_130_R	GGGTTTCCCCATT	833
		TGGC			CGG
234	23S_EC_187_207_F	GGGAACTGAAA	121	23S_EC_242_256_R	TTCGCTCGCCGCT	1385
		CATCTAAGTA			AC
235	23S_EC_1602_1620_F	TACCCCAAACC	184	23S_EC_1686_1703_R	CCTTCTCCCGAAG	782
		GACACAGG			TTACG
236	23S_EC_1685_1703_F	CCGTAACTTC	58	23S_EC_1828_1842_R	CACCGGGCAGGCG	760
		GGAGAAGG			TC
237	23S_EC_1827_1843_F	GACGCCTGCCC	99	23S_EC_1929_1949_R	CCGACAAGGAATT	775
		GGTGC			TCGCTACC
238	23S_EC_2434_2456_F	AAGGTACTCCG	9	23S_EC_9490_2511_R	AGCCGACATCGAG	746
		GGGATAACAGG			GGTGCCAAAC
		C
239	23S_EC_2599_2616_F	GACAGTTCGGT	96	23S_SC_2653_2669_R	CCGGTCCTCTCGT	777
		CCCTATC			ACTA
240	23S_EC_2653_2669_F	TAGTACGAGAG	227	23S_EC_2737_2758_R	TTAGATGCTTTCA	1369
		GACCGG			GCACTTATC
241	23S_ES_−68_−44_F	AAACTAGATAA	1	23S_B_5_21_R	GTGCGCCCTTTCT	841
		CAGTAGACATC			AACTT
		AC
242	16S_EC_8_27_F	AGAGTTTGATC	23	16S_SC_342_358_R	ACTGCTGCCTCCC	742
		ATGGCTCAG			GTAG
243	16S_EC_314_332_F	CACTGGAACTG	48	16S_EC_556_575_R	CTTTACGCCCAGT	801
		AGACACGG			AATTCCG
244	16S_EC_518_536_F	CCAGCAGCCGC	57	16S_EC_774_795_R	GTATCTAATCCTG	839
		GGTAATAC			TTTGCTCCC
245	16S_EC_683_700_F	GTGTAGCGGTG	137	16S_EC_967_985_R	GGTAAGGTTCTTC	835
		AAATGCG			GCGTTG
246	16S_EC_937_954_F	AAGCGGTGGAG	7	16S_EC_1220_1240_R	ATTGTAGCACGTG	757
		CATGTGG			TGTAGCCC
247	16S_EC_1195_1213_F	CAAGTCATCAT	46	16S_EC_1525_1541_R	AAGGAGGTGATCC	714
		GGCCCTTA			AGCC
248	16S_EC_8_27_F	AGAGTTTGATC	23	16S_EC_1525_1541_R	AAGGAGGTGATCC	714
		ATGGCTCAG			AGCC
249	23S_EC_1831_1849_F	ACCTGCCCAGT	18	23S_EC_1919_1936_R	TCGCTACCTTAGG	1080
		GCTGGAAG			ACCGT
250	16S_EC_1387_1407_F	GCCTTGTACAC	112	16S_EC_1494_1513_R	CACGGCTACCTTG	761
		ACCTCCCGTC			TTACGAC
251	16S_EC_1390_1411_F	TTGTACACACC	693	16S_EC_1486_1505_R	CCTTGTTACGACT	783
		GCCCGTCATAC			TCACCCC
252	16S_EC_1367_1387_F	TACGGTGAATA	191	16S_EC_1485_1506_R	ACCTTGTTACGAC	731
		CGTTCCCGGG			TTCACCCCA
253	16S_EC_804_822_F	ACCACGCCGTA	14	16S_EC_909_929_R	CCCCCGTCAATTC	773
		AACGATGA			CTTTGAGT
254	16S_EC_791_812_F	GATACCCTGGT	106	16S_EC_886_04_R	GCCTTGCGACCGT	817
		AGTCCACACCG			ACTCCC
255	16S_EC_789_810_F	TAGATACCCTG	206	16S_EC_882_899_R	GCGACCGTACTCC	818
		GTAGTCCACGC			CCAGG
256	16S_EC_1092_1109_F	TAGTCCCGCAA	228	16S_EC_1174_1195_R	GACGTCATCCCCA	810
		CGAGCGC			CCTTCCTCC
257	23S_EC_2586_2607_F	TAGAACGTCGC	203	23S_EC_2658_2677_R	AGTCCATCCCGGT	749
		GAGACAGTTCG			CCTCTCG
258	RNASEP_SA_31_49_F	GAGGAAAGTCC	103	RNASEP_SA_358_379_R	ATAAGCCATGTTC	750
		ATGCTCAC			TGTTCCATC
258	RNASEP_SA_31_49_F	GAGGAAAGTCC	103	RNASEP_EC_345_362_R	ATAAGCCGGGTTC	751
		ATGCTCAC			TGTCG
258	RNASEP_SA_31_49_F	GAGGAAAGTCC	103	RNASEP_BS_363_384_R	GTAAGCCATGTTT	838
		ATGCTCAC			TGTTCCATC
258	RNASEP_BS_43_61_F	GAGGAAAGTCC	104	RNASEP_SA_358_379_R	ATAAGCCATGTTC	750
		ATGCTCGC			TGTTCCATC
258	RNASEP_BS_43_61_F	GAGGAAAGTCC	104	RNASEP_EC_345_362_R	ATAAGCCGGGTTC	751
		ATGCTCGC			TGTCG
258	RNASEP_BS_43_31_F	GAGGAAAGTCC	104	RNASEP_ES_363_384_R	GTAAGCCATGTTT	838
		ATGCTCGC			TGTTCCATC
258	RNASEP_EC_61_77_F	GAGGAAAGTCC	105	RNASEP_SA_358_379_R	ATAAGCCATGTTC	750
		GGGCTC			TGTTCCATC
258	RNASEP_EC_61_77_F	GAGGAAAGTCC	105	RNASEP_EC_345_362_R	ATAAGCCGGGTTC	751
		GGGCTC			TGTCG
258	RNASEP_EC_61_77_F	GAGGAAAGTCC	105	RNASEP_BS_363_384_R	GTAAGCCATGTTT	838
		GGGCTC			TGTTCCATC
259	RNASEP_ES_4331_F	GAGGAAAGTCC	104	RNASEP_ES_363_384_R	GTAAGCCATGTTT	838
		ATGCTCGC			TGTTCCATC
260	RNASEP_EC_61_77_F	GAGGAAAGTCC	105	RNASEP_EC_345_362_R	ATAAGCCGGGTTC	751
		GGGCTC			TGTCG
262	RNASEP_SA_31_49_F	GAGGAAAGTCC	103	RNASEP_SA_358_379_R	ATAAGCCATGTTC	750
		ATGCTCAC			TGTTCCATC
263	16S_EC_1082_1100_F	ATGTTGGGTTA	37	16S_EC_1525_1541_R	AAGGAGGTGATCC	714
		AGTCCCGC			AGCC
264	16S_EC_556_575_F	CGGAATTACTG	70	16S_EC_774_795_R	GTATCTAATCCTG	839
		GGCGTAAAG			TTGCTCCC
265	16S_EC_1082_1100_F	ATGTTGGGTTA	37	16S_EC_1177_1196_10G_R	TGACGCATGCCC	1160
		AGTCCCGC			ACCTTCC
266	16S_EC_1082_1100_F	ATGTTGGGTTA	37	16S_EC_1177_1196_10G_11G_R	TGACGTCATGGCC	1161
		AGTCCCGC			ACCTTCC
268	YAED_EC_513_532_F_MOD	GGTGTTAAATA	130	TRNA_ALA-	AGACCTCCTGCGT	744
		GCCTGGCAG		RRNA_EC_30_49_F_MOD	GCAAAGC
269	16S_EC_1082_1100_F_MOD	ATGTTGGGTTA	37	16S_EC_1177_1196_R_MOD	TGACGCATCCCC	1158
		AGTCCCGC			ACCTTCC
270	23S_EC_2586_2607_F_MOD	TAGAACGTCGC	203	23S_EC_2658_2677_R_MOD	AGTCCATCCCGGT	749
		GAGACAGTTCG			CCTCTCG
272	16S_EC_969_985_F	ACGCGAAGAAC	19	16S_EC_1389_1407_R	GACGGGCGGTGTG	807
		CTTACC			TACAAG
273	16S_EC_683_700_F	GTGTAGCGGTG	137	16S_EC_1303_1323_R	CGAGTTGCAGACT	788
		AAATGCG			GCGATCCG
274	169_EC_49_68_F	TAACACATGCA	152	16S_EC_880_894_R	CGTACTCCCCAGG	796
		AGTCGAACG			CG
275	16S_EC_49_68_F	TAACACATGCA	152	16S_EC_1061_1078_R	ACGACACGAGCTG	734
		AGTCGAACG			ACGAC
277	CYA_BA_1349_1370_F	ACAACGAAGTA	12	CYA_BA_1426_1447_R	CTTCTACATTTTT	800
		CAATACAAGAC			AGCCATCAC
278	16S_EC_1090_1111_2_F	TTAAGTCCCGC	650	16S_EC_1175_1196_R	TGACGTCATCCCC	1159
		AACGAGCGCAA			ACCTTCCTC
279	16S_EC_405_432_F	TGAGTGATGAA	464	16S_EC_507_527_R	CGGCTGCTGGCAC	793
		GGCCTTAGGGT			GAAGTTAG
		TGTAAA
280	GROL_EC_496_518_F	ATGGACAAGGT	34	GROL_EC_577_596_R	TAGCCGCGGTCGA	914
		TGGCAAGGAAG			ATTGCAT
		G
281	GROL_EC_511_536_F	AAGGAAGGCGT	8	GROL_EC_571_593_R	CCGCGGTCGAATT	776
		GATCACCGTTG			GCATGCCTTC
		AAGA
288	RPOB_EC_3802_3821_F	CAGCGTTTCGG	51	RPOB_EC_3862_3885_R	CGACTTGACGGTT	786
		CGAAATGGA			AACATTTCCTG
289	RPOB_EC_3799_3821_F	GGGCAGCGTTT	124	RPOB_EC_3862_3888_R	GTCCGACTTGACG	840
		CGGCGAAATG			GTCAACATTTCCT
		GA			G
290	RPOC_EC_2146_2174_F	CAGGAGTCGTT	52	RPOC_EC_2227_2245_R	ACGCCATCAGGCC	736
		CAACTCGATCT			ACGCAT
		ACATGAT
291	ASPS_EC_405_422_F	GCACAACCTGC	110	ASPS_EC_521_538_R	ACGGCACGAGGTA	738
		GGCTGCG			GTCGC
292	RPOC_EC_1374_1393_F	CGCCGACTTCG	69	RPOC_EC_1437_1455_R	GAGCATCAGCGTG	811
		ACGGTGACC			CGTGCT
293	TUFB_EC_957_979_F	CCACACGCCGT	55	TUFB_EC_1034_1058_R	GGCATCACCATTT	829
		TCTTCAACAAC			CCTTGTCCTTCG
		T
294	16S_EC_7_33_F	GAGAGTTTGAT	102	16S_EC_101_122_R	TGTTACTCACCCG	1345
		CCTGGCTCAGA			TCTGCCACT
		ACGAA
295	VALS_EC_610_649_F	ACCGAGCAAGG	17	VALS_EC_705_727_R	TATAACGCACATC	929
		AGACCAGC			GTCAGGGTGA
344	16S_EC_971_990_F	GCGAAGAACCT	113	16S_EC_1043_1062_R	ACAACCATGCACC	726
		TACCAGGTC			ACCTGTC
346	16S_EC_713_732_TMOD_F	TAGAACACCGA	202	16S_EC_789_809_TMOD_R	TCGTGGACTACCA	1110
		TGGCGAAGGC			GGGTATCTA
347	16S_EC_785_806_TMOD_F	TGGATTAGAGA	560	16S_EC_880_897_TMOD_R	TGGCCGTACTCCC	1278
		CCCTGGTAGTC			CAGGCG
		C
348	16S_EC_960_981_TMOD_F	TTTCGATGCAA	706	16S_EC_1054_1073_TMOD_R	TACGAGCTGACGA	895
		CGCGAAGAACC			CAGCCATG
		T
349	23S_EC_1826_1843_TMOD_F	TCTGACACCTG	401	23S_EC_1906_1924_TMOD_R	TGACCGTTATAGT	1156
		CCCGGTGC			TACGGCC
350	CAPC_BA_274_303_TMOD_F	TGATTATTGTT	476	CAPC_BA_349_376_TMOD_R	TGTAACCCTTGTC	1314
		ATCCTGTTATG			TTTGAATTGTAT
		CCATTTGAG			TGC
351	CYA_BA_1353_1379_TMOD_F	TCGAAGTACAA	355	CYA_BA_1448_1467_TMOD_R	TTGTTAACGGCTT	1423
		TACAAGACAAA			CAAGACCC
		AGAAGG
352	INFB_EC_1365_1393_TMOD_F	TTGCTCGTGGT	687	INFB_EC_1439_1467_TMOD_R	TTGCTGCTTTCGC	1411
		GCACAAGTAAC			ATGGTTAATTGCT
		GGATATTA			TCAA
353	LEF_BA_756_781_TMOD_F	TAGCTTTTGCA	220	LEF_BA_843_872_TMOD_R	TTCTTCCAAGGAT	1394
		TATTATATCGA			AGATTTATTTCTT
		GCCAC			GTTCG
354	RPOC_EC_2218_2241_TMOD_F	TCTGGCAGGTA	405	RPOC_EC_2313_2337_TMOD_R	TCGCACCGTGGGT	1072
		TGCGTGGTCTG			TGAGATGAAGTAC
		ATG
355	SSPE_BA_115_137_TMOD_F	TCAAGCAAACG	255	SSPE_BA_197_222_TMOD_R	TTGCACGTCTGTT	1402
		CACAATCAGAA			TCAGTTGCAAATT
		GC			C
356	RPLB_EC_650_679_TMOD_F	TGACCTACAGT	449	RPLB_EC_739_762_TMOD_R	TTCCAAGTGCTGG	1380
		AAGAGGTTCTG			TTTACCCCATGG
		TAATGAACC
357	RPLB_EC_688_710_TMOD_F	TCATCCACACG	296	RPLB_EC_736_757_TMOD_R	TGTGCTGGTTTAC	1337
		GTGGTGGTGAA			CCCATGGAGT
		GG
358	VALS_EC_1105_1124_TMOD_F	TCGTGGCGGCG	385	VALS_EC_1195_1218_TMOD_R	TCGGTACGAACTG	1093
		TGGTTATCGA			GATGTCGCCGTT
359	RPOB_EC_1845_1866_TMOD_F	TTATCGCTCAG	659	RPOB_EC_1909_1929_TMOD_R	TGCTGGATTCGCC	1250
		GCGAACTCCAA			TTTGCTACG
		C
360	23SEC_2646_2667_TMOD_F	TCTGTTCTTAG	409	23S_EC_2745_2765_TMOD_R	TTTCGTGCTTAGA	1434
		TACGAGAGGAC			TGCTTTCAG
		C
361	16S_EC_1090_1111_2_TMOD_F	TTTAAGTCCCG	697	16S_EC_1175_1196_TMOD_R	TTGACGTCATCCC	1398
		CAACGAGCGCA			CACCTTCCTC
		A
362	RPOB_EC_3799_3821_TMOD_F	TGGGCAGCGTT	581	RPOB_EC_3862_3888_TMOD_R	TGTCCGACTTGAC	1325
		TCGGCGAAATG			GGTCAACATTTCC
		GA			TG
363	RPOC_EC_2146_2174_TMOD_F	TCAGGAGTCGT	284	EPOC_EC_2227_2245_TMOD_R	TACGCCATCAGGC	898
		TCAACTCGATC			CACGCAT
		TACATGAT
364	RPOC_EC_1374_1393_TMOD_F	TCGCCGACTTC	367	RPOC_EC_1437_1455_TMOD_R	TGAGCATCAGCGT	1166
		GACGGTGACC			GCGTGCT
367	TUFB_ECG_957_979_TMOD_F	TCCACACGCCG	308	TUFB_EC_1034_1058_TMOD_R	TGGCATCACCATT	1276
		TTCTTCAACAA			TCCTTGTCCTTCG
		CT
423	SP101_SPET11_893_921_TMOD_F	TGGGCAACAGC	580	SP101_SPET11_988_1012_TMOD_R	TCATGACAGCCAA	990
		AGCGGATTGCG			GACCTCACCCACC
		ATTGCGCG
424	SP101_SPET11_1154_1179 —	TCAATACCGCA	258	SP101_SPET11_1251_1277_TMOD_R	TGACCCCAACCTG	1155
	TMOD_F	ACAGCGGTGGC			GCCTTTTGTCGTT
		TTGGG			GA
425	SP101_SPET11_118_147_TMOD_F	TGCTGGTGAAA	528	SP101_SPET11_213_238_TMOD_R	TTGTGGCCGATTT	1422
		ATAACCCAGAT			CACCACCTGCTCC
		GTCGTCTTC			T
426	SP101_SPET11_1314_1336_TMOD_F	TCGCAAAAAAA	363	SP101_SPET11_1403_1431_TMOD_R	TAAACTATTTTTT	849
		TCCAGCTATTA			TAGCTATACTCGA
		GC			ACAC
427	SP101_SPET11_1408_1437 —	TCGAGTATAGC	359	SP101_SPET11_1486_1515_TMOD_R	TGGATAATTGGTC	1268
	TMOD_F	TTAAAAAAATA			GTAACAAGGGATA
		GTTTATGACA			GTGAG
428	SP101_SPET11_1688_1716_TMOD_F	TCCTATATTAA	334	SP101_SPET11_1783_1808_TMOD_R	TATATGATTATCA	932
		TCGTTTACAGA			TTGAACTGCGGCC
		AACTGGCT			G
429	SP101_SPET11_1711_1733 —	TCTGGCTAAAA	406	SP101_SPET11_1808_1835_TMOD_R	TGCGTGACGACCT	1239
	TMOD_F	CTTTGGCAACG			TCTTGAATTGTAA
		GT			TCA
430	SP101_SPET11_1807_1835_TMOD_F	TATGATTACAA	235	SP101_SPET11_1901_1927_TMOD_R	TTTGGACCTGTAA	1439
		TTCAAGAAGGT			TCAGCTGAATACT
		CGTCACGC			GG
431	SP101_SPET11_1967_1991 —	TTAACGGTTAT	649	SP101_SPET11_2082_2083_TMOD_R	TATTGCCCAGAAA	940
	TMOD_F	CATGGCCCAGA			TCAAATCATC
		TGGG
432	SP101_SPET11_216_243_TMOD_F	TAGCAGGTGGT	210	SP101_SPET11_308_333_TMOD_R	TTGCCACTTTGAC	1404
		GAAATCGGCCA			AACTCCTGTTGCT
		CATGATT			G
433	SP101_SPET11_2260_2283 —	TCAGAGACCGT	272	SP101_SPET11_2375_2397_TMOD_R	TTCTGGGTGACCT	1393
	TMOD_F	TTTATCCTATC			GGTGTTTTAGA
		AGC
434	SP101_SPET11_2375_2399 —	TTCTAAAACAC	675	SP101_SPET11_2470_2497_TMOD_R	TAGCTGCTAGATG	918
	TMOD_F	CAGGTCACCCA			AGCTTCTGCCATG
		GAAG			GCC
435	SP101_SPET11_2468_2487 —	TATGGCCATGG	238	SP101_SPET11_2543_2579_TMOD_R	TCCATAAGGTCAC	1007
	TMOD_F	CAGAAGCTCA			CGTCACCATTCAA
					AGC
436	SP101_SFET11_266_295_TMOD_F	TCTTGTACTTG	417	SP101_SPET11_355_380_TMOD_R	TGCTGCTTTGATG	1249
		TGGCTCACACG			GCTGAATCCCCTT
		GCTGTTTGG			C
437	SP101_SPET11_2961_2984 —	TACCATGACAG	183	SP101_SPET11_3023_3045_TMOD_R	TGGAATTTACCAG	1264
	TMOD_F	AAGGCATTTTG			CGATAGACACC
		ACA
438	SP101_SPET11_3075_3103 —	TGATGACTTTT	473	SP101_SPET11_3168_3196_TMOD_R	TAATCGACGACCA	875
	TMOD_F	TAGCTAATGGT			TCTTGGAAAGATT
		CAGGCAGC			TCTC
439	SP101_SPET11_322_344_TMOD_F	TGTCAAAGTGG	631	SP101_SPET11_423_441_TMOD_R	TATCCCCTGCTTC	934
		CACGTTTACTG			TGCTGCC
		GC
440	SP101_SPET11_3386_3403 —	TAGCGTAAAGG	215	SP101_SPET11_3480_3506_TMOD_R	TCCAGCAGTTACT	1005
	TMOD_F	TGAACCTT			GTCCCCTCATCTT
					TG
441	SP101_SPET11_3511_3535 —	TGCTTCAGGAA	531	SP101_SPET11_3605_3629_TMOD_R	TGGGTCTACACCT	1294
	TMOD_F	TCAATGATGGA			GCACTTGCATAAC
		GCAG
442	SP101_SPET11_358_387_TMOD_F	TGGGGATTCAG	588	SP101_SPET11_448_473_TMOD_R	TCCAACCTTTTCC	998
		CCATCAAAGCA			ACAACAGAATCAG
		GCTATTGAC			C
443	SP101_SPET11_600_628_TMOD_F	TCCTTACTTCG	348	SP101_SPET11_886_714_TMOD_R	TCCCATTTTTTCA	1018
		AACTATGAATC			CGCATGCTGAAAA
		TTTTGGAAG			TATC
444	SP101_SPET11_658_684_TMOD_F	TGGGGATTGAT	589	SP101_SPET11_756_784_TMOD_R	TGATTGGCGATAA	1189
		ATCACCGATAA			AGTGATATTTTCT
		GAAGAA			AAAA
445	SP101_SPET11_776_801_TMOD_F	TTCGCCAATCA	673	SP101_SPET11_871_896_TMOD_R	TGCCCACCAGAAA	1217
		AAACTAAGGGA			GACTAGCAGGATA
		ATGGC			A
446	SP101_SPET11_1_29_TMOD_F	TAACCTTAATT	154	SP101_SPET11_92_116_TMOD_R	TCCTACCCAACGT	1044
		GGAAAGAAACC			TCACCAAGGGCAG
		CAAGAAGT
447	SP101_SPET11_364_385_F	TCAGCCATCAA	276	SP101_SPET11_448_471_R	TACCTTTTCCACA	894
		AGCAGCTATTG			ACAGAATCAGC
448	SP101_SPET11_3085_3104_F	TAGCTAATGGT	216	SP101_SPET11_3170_3194_R	TCGACGACCATCT	1066
		CAGGCAGCC			TGGAAAGATTTC
449	RPLB_EC_990_710_F	TCCACACGGTG	309	RPLB_EC_737_758_R	TGTGCTGGTTTAC	1336
		GTGGTGAAGG			CCCATGGAG
481	BONTA_X52066_538_552_F	TATGGCTCTAC	239	BONTA_X52066_647_660_R	TGTTACTGCTGGA	1346
		TCAA			T
482	BONTA_X5206_538_552P_F	TATpGGCTp	143	BONTA_X52066_647_660P_R	TGTpTpACp	1146
		CpTpACp			TpGCpTpGGAT
		Tp*CpAA
483	BONTA_X5206_701_720_F	GAATAGCAATT	94	BONTA_X5206_759_775_R	TTACTTCTAACCC	1367
		AATCCAAAT			ACTC
484	BONTA_X52066_701_720P_F	GAATpAGCp	91	BONTA_X52066_759_775P_R	TTACpTpTp	1359
		AATpTpAA*			CpTpAACp*Cp
		TpCpCpAAAT			CpACp*TpC
485	BONTA_X5206_450_473_F	TCTAGTAATAA	393	BONTA_X5206_517_539_R	TAACCATTTCGCG	859
		TAGGACCCTCA			TAAGATTCAA
		GC
486	BONTA_X52066_450_473P_F	TCpTpAGTA	142	BONTA_X5206_517_539P_R	TAACCATpTp*	857
		ATAATAGGA*C			Tp*CpGCGTAAGA
		pCpCpTp*			TpTp*CpAA
		C
487	BONTA_X52066_591_620_F	TGAGTCACTTG	463	BONTA_X52066_644_671_R	TCATGTGCTAATG	992
		AAGTTGATACA			TTACTGCTGGATC
		AATCCTCT			TG
608	SSPE_BA_156_168P_F	TGGTpGCpTpA	616	SSPE_BA_243_255P_R	TGCpAGCpTGATp	1241
		GCpATT			TpGT
609	SSPE_BA_75_89P_F	TACpAGAGTpT	192	SSPE_BA_163_177P_R	TGTGCTpTpTpGA	1338
		pTpGCpGAC			ATpGCpT
610	SSPE_BA_150_168P_F	TGCTTCTGGTp	533	SSPE_BA_243_264P_R	TGATTGTTTTGCp	1191
		GCpTpAGCpAT			AGCpTGATpTpGT
		T
611	SSPE_BA_72_89P_F	TGGTACpAGAG	602	SSPE_BA_163_182P_R	TCATTTGTGCTpT	995
		TpTpTpGCpGA			pTpGAATpGCpT
		C
612	SSPE_BA_114_137P_F	TCAAGCAAACG	255	SSPE_BA_196_222P_R	TTGCACGTCpCpG	1401
		CACAATpCpAG			TTTCAGTTGCAAA
		AAGC			TTC
699	SSPE_BA_123_153_F	TGCACAATCAG	488	SSPE_BA_202_231_R	TTTCACAGCATGC	1431
		AAGCTAAGAAA			ACGTCTGTTTCAG
		GCGCAAGCT			TTGC
700	SSPE_BA_156_168_F	TGGTGCTAGCA	612	SSPE_BA_243_255_R	TGCAGCTGATTGT	1202
		TT
701	SSPE_BA_75_89_F	TACAGAGTTTG	179	SSPE_BA_163_177_R	TGTGCTTTGAATG	1338
		CGAC			CT
702	SSPE_BA_150_168_F	TGCTTTCTGGT	533	SSPE_BA_243_264_R	TGATTGTTTTGCA	1190
		GCTAGCATT			GCTGATTGT
703	SSPE_BA_72_89_F	TGGTACAGAGT	600	SSPE_BA_163_182_R	TCATTTGTGCTTT	995
		TTGCGAC			GAATGCT
704	SSPE_BA_146_168_F	TGCAAGCTTCT	484	SSPE_BA_242_267_R	TTGTGATTGTTTT	1421
		GGTGCTAGCAT			GCAGCTGATTGTG
		T
705	SSPE_BA_63_89_F	TGCTAGTTATG	518	SSPE_BA_163_191_R	TCATAACTAGCAT	986
		GTACAGAGTTT			TTGTGCTTTGAAT
		GCGAC			GCT
706	SSPE_BA_114_137_F	TCAAGCAAACG	255	SSPE_BA_196_222_R	TTGCACGTCTGTT	1402
		CACAATCAGAA			TCAGTTGCAAAT
		GC			C
770	PLA_AF053945_7377_7402_F	TGACATCCGGC	442	PLA_AF053945_7434_7462_R	TCTAAATTCCGCA	1313
		TCACGTTATTA			AAGACTTTGGCAT
		TGGT			TA
771	PLA_AF053945_7382_7404_F	TCCGGCTCACG	327	PLA_AF053945_7482_7502_R	TGGTCTGAGTACC	1304
		TTATTATGGTA			TCCTTTGC
		C
772	PLA_AF053945_7481_7503_F	TGCAAAGGAGG	481	PLA_AF053945_7539_7562_R	TATTGGAAATACC	943
		TACTCAGACCA			GGCAGCATCTC
		T
773	PLA_AF053945_7186_7211_F	TTATACCGGAA	657	PLA_AF053945_7257_7280_R	TAATGCGATACTG	879
		ACTTCCCGAAA			GCCTGCAAGTC
		GGAG
774	CAF1_AF053947_33407_33430_F	TCAGTTCCGTT	292	CAF1_AF053947_33494_33514_R	TGCGGGCTGGTTC	1235
		ATCGCCATTGC			AACAAGAG
		AT
775	CAF1_AF053947_33515_33541_F	TCACTCTTACA	270	CAF1_AF053947_33595_33621_R	TCCTGTTTTATAG	1053
		TATAAGGAAGG			CCGCCAAGAGTAA
		CGCTC			G
776	CAF1_AF053947_33435_33457_F	TGGAACTATTG	542	CAF1_AF053947_33499_33517_R	TGATGCGGGCTGG	1183
		CAACTGCTAAT			TTCAAC
		G
777	CAF1_AF053947_33687_33716_F	TCAGGATGGAA	286	CAF1_AF053947_33755_33782_R	TCAAGGTTCTCAC	962
		ATAACCACCAA			CGTTTACCTTAGG
		TTCACTAC			AG
778	INV_U22457_515_539_F	TGGCTCCTTGG	573	INV_U22457_571_598_R	TGTTAAGTGTGTT	1343
		TATGACTCTGC			GCGGCTGTCTTTA
		TTC			TT
779	INV_U22457_699_724_F	TGCTGAGGCCT	525	INV_U22457_753_776_R	TCACGCGACGAGT	976
		GGACCGATTAT			GCCATCCATTG
		TTAC
780	INV_U22457_834_858_F	TTATTTACCTG	664	INV_U22457_942_966_R	TGACCCAAAGCTG	1154
		CACTCCCACAA			AAAGCTTTACTG
		CTG
781	INV_U22457_1558_1581_F	TGGTAACAGAG	597	INV_U22457_1619_1643_R	TTGCGTTGCAGAT	1408
		CCTTATAGGCG			TATCTTTACCAA
		CA
782	LL_NC003143_2366996 —	TGTACCCGCTAA	627	LL_NC003143_2367073_2367097_R	TCTCATCCCGATA	1123
	2367019_F	AGCACTACCAT			TTACCGCCATGA
		CC
783	LL_NC003143_2367172 —	TGGACGGCATC	550	LL_NC003143_2367249_23672	TGGCAACAGCTCA	1272
	2367194_F	ACGATTCTCTA			ACACCTTTGG
		C
874	RPLB_EC_649_679_F	TGICCIACIGT	620	RPLB_EC_739_762_TMOD_R	TTCCAAGTGCTGG	1380
		IIGIGGTTCTG			TTTACCCCATGG
		TAATGAACC
875	RPLB_EC_642_679P_F	TpCpCpTpTpG	646	RPLB_EC_739_762_TMOD_R	TTCCAAGTGCTGG	1380
		ITpGICCIACI			TTTACCCCATGG
		GTIIGIGGTTC
		TGTAATGAACC
876	MECIA_Y14051_3315_3341_F	TTACACATATC	653	MECIA_Y14051_3367_3393_R	TGTGATATGGAGG	1333
		GTGAGCAATGA			TGTAGAAGGTGTT
		ACTGA			A
877	MECA_Y14051_3774_3802_F	TAAAACAAACT	144	MECA_Y14051_3828_3854_R	TCCCAATCTAACT	1015
		ACGGTAACATT			TCCACATACCATC
		GATCGCA			T
878	MECA_Y14051_3645_3670_F	TGAAGTAGAAA	434	MECA_Y14051_3690_3719_R	TGATCCTGAATGT	1181
		TGACTGAACGT			TTATATCTTTAAC
		CCGA			GCCT
879	MECA_Y14051_4507_4530_F	TCAGGTACTGC	288	MECA_Y14051_4555_4581_R	TGGATAGACGTCA	1269
		TATCCACCCTC			TATGAAGGTGTGC
		AA			T
880	MECA_Y14051_4510_4530_F	TGTACTGCTAT	626	MECA_Y14051_4586_4610_R	TATTCTTCGTTAC	939
		CCACCCTCAA			TCATGCCATACA
881	MECA_Y14051_4669_4698_F	TCACCAGGTTC	262	MECA_Y14051_4765_4793_R	TAACCACCCCAAG	858
		AACTCAAAAAA			ATTTATCTTTTG
		ATATTAACA			CCA
882	MECA_Y14051_4520_4530P_F	TCpCpACpCpC	389	MECA_Y14051_4590_4600P_R	TpACpTpCpATpG	1357
		pTpCpAA			CpCpA
883	MECA_Y14051_4520_4530P_F	TCpCpACpCpC	389	MECA_Y14051_4600_4610P_R	TpATpTpCpTpTp	1358
		pTpCpAA			CpGTpT
902	TRPE_AY094355_1467_1491_F	ATGTCGATTGC	36	TRPE_AY094355_1569_1592_R	TGCGCGAGCTTT	1231
		AATCCGTACTT			TATTTGGGTTTC
		GTG
903	TRPE_AY094355_1445_1471_F	TGGATGGCATG	557	TRPE_AY094355_1551_1580_R	TATTTGGGTTTCA	944
		GTGAAATGGAT			TTCCACTCAGATT
		ATGTC			CT
904	TRPE_AY094355_1278_1303_F	TCAAATGTACA	247	TRPE_AY094355_1392_1418_R	TCCTCTTTTCACA	1048
		AGGTGAAGTGC			GGCTCTACTTCAT
		GTGA			C
905	TRPE_AY094355_1064_1086_F	TCGACCTTTGG	357	TRPE_AY094355_1171_1196_R	TACATCGTTTCGC	885
		CAGGAACTAGA			CCAAGATCAATCA
		C
906	TRPE_AY094355_666_688_F	GTGCATGCGGA	135	TRPE_AY094355_769_791_R	TTCAAAATGCGGA	1372
		TACAGAGCAGA			GGCGTATGTG
907	TRPE_AY094355_757_776_F	TGCAAGCGCGA	483	TRPE_AY094355_864_883_R	TGCCCAGGTACAA	1218
		CCACATACG			CCTGCAT
908	RECA_AF251469_43_68_F	TGGTACATGTG	601	RECA_AF251469_140_163_R	TTCAAGTGCTTGC	1375
		CCTTCATTGAT			TCACCATTGTC
		GCTG
909	RECA_AF251469_169_190_F	TGACATGCTTG	446	RECA_AF251469_277_300_R	TGGCTCATAAGAC	1280
		TCCGTTCAGGC			GCGCTTGTAGA
910	PARC_X95819_87_110_F	TGGTGACTCGG	609	PARC_X95819_201_222_R	TTCGGTATAACGC	1387
		CATGTTATGAA			ATCGCAGCA
		GC
911	PARC_X95819_87_110_F	TGGTGACTCGG	609	PARC_X95819_192_219_R	GGTATAACGCATG	836
		CATGTTATGAA			GCAGCAAAAGATT
		GC			TA
912	PARC_X95819_123_147_F	GGCTCAGCCAT	120	PARC_X95819_232_260_R	TCGCTCAGCAATA	1081
		TTAGTTACCGC			ATTCACTATAAGC
		TAT			CGA
913	PARC_X95819_43_63_F	TCAGCGCGTAC	277	PARC_X95819_143_170_R	TTCCCCTGACCTT	1383
		AGTGGGTGAT			CGATTAAAGGATA
					GC
914	OMPA_AY485227_272_301_F	TTACTCCATTA	655	OMPA_AY485227_364_388_R	GAGCTGCGCCAAC	812
		TTGCTTGGTTA			GAATAAATCGTC
		CACTTTCC
915	OMPA_AY485227_379_401_F	TGCGCAGCTCT	509	OMPA_AY485227_492_519_R	TGCCGTAACATAG	1223
		TGGTATCGAGT			AAGTTACCGTTGA
		T			T
916	OMPA_AY485227_311_335_F	TACACAACAAT	178	OMPA_AY485227_424_453_R	TACGTCGCCTTA	901
		GGCGGTAAAGA			ACTTGGTTATATT
		TGG			CAGC
917	OMPA_AY485227_415_441_F	TGCCTCGAAGC	506	OMPA_AY485227_514_546_R	TCGGGCGTAGTTT	1092
		TGAATATAACC			TTAGTAATTAAAT
		AAGTT			CAGAAGT
918	OMPA_AY485227_494_520_F	TCAACGGTAAC	252	OMPA_AY485227_569_596_R	TCGTCGTATTTAT	1108
		TTCTATGTTAC			AGTGACCAGCACC
		TTCTG			TA
919	OMPA_AY485227_551_577_F	TCAAGCCGTAC	257	OMPA_AY485227_658_680_R	TTTAAGCGCCAGA	1425
		GTATTATTAGG			AAGCACCAAC
		TGCTG
920	OMPA_AY485227_555_581_F	TCCGTACGTAT	328	OMPA_AY485227_635_662_R	TCAACACCAGCGT	954
		TATTAGGTGCT			TACCTAAAGTACC
		GGTCA			TT
921	OMPA_AY485227_556_583_F	TCGTACGTATT	379	OMPA_Y485227_659_683_R	TCGTTTAAGCGCC	1114
		ATTAGGTGCTG			AGAAAGCACCAA
		GTCACT
922	OMPA_AY485227_657_679_F	TGTTGGTGCTT	645	OMPA_AY485227_139_765_R	TAAGCCAGCAAGA	871
		TCTGGCGCTTA			GCTGTATAGTTCC
		A			A
923	OMPA_AY485227_660_683_F	TGGTGCTTTCT	613	OMPA_AY485227_786_807_R	TACAGGAGCAGCA	884
		GGCGCTTAAAC			GGCTTCAAG
		GA
924	GYRA_AF100557_4_23_F	TCTGCCCGTGT	402	GYRA_AF100557_119_142_R	TCGAACCGAAGTT	1063
		CGTTGGTGA			ACCCTGACCAT
925	GYRA_AF100557_70_94_F	TCCATTGTTCG	316	GYRA_AF100557_178_201_R	TGCCAGCTTAGTC	1211
		TATGGCTCAAG			ATACGGACTTC
926	GYRB_AB008700_19_40_F	TCAGGTGGCT	289	GYRB_AB008700_111_140_R	TATTGCGGATCAC	941
		TACACGGCGT			CATGATGATATTC
		AG			TTGC
927	GYRB_AB008700_265_292_F	TCTTTCTTGAA	420	GYRB_AB008700_369_395_R	TCGTTGAGATGGT	1113
		TGCTGGTGTAC			TTTTACCTTCGT
		GTATCG			TG
928	GYRB_AB008700_368_394_F	TCAACGAAGGT	251	GYRB_A3008700_466_494_R	TTTGTGAAACAGC	1440
		AAAAACCATCT			GAACATTTTCTTG
		CAACG			GTA
929	GYRB_AB008700_477_504_F	TGTTCGCTGTT	641	GYRB_AB008700_611_632_R	TCACGCGCATCAT	977
		TCACAAACAAC			CACCAGTCA
		ATTCCA
930	GYRB_AB008700_760_787_F	TACTTACTTGA	198	GYRB_AB008700_862_888_R	ACCTGCAATATCT	729
		GAATCCACAAG			AATGCACTCTTAC
		CTGCAA			G
931	WAAA_Z96925_2_29_F	TCTTGCTCTTT	416	WAAA_Z96925_115_138_R	CAAGCGGTTTGCC	758
		CGTGAGTTCAG			TCAAATAGTCA
		TAAATG
932	WAAA_Z96925_286_311_F	TCGATCTGGTT	360	WAAA_Z96925_394_412_R	TGGCACGAGCCTG	1274
		TCATGCGTTT			ACCTGT
939	RPOB_EC_3798_3821_F	TGGGCAGCGTT	581	RPOB_EC_9862_3889_R	TGTCCGACTTGAC	1326
		TCGGCGAAATG			GGTCAGCATTTCC
		GA			TG
940	RPOB_EC_3798_3821_F	TGGGCAGCGTT	581	RPOB_EC_3862_3899_2_R	TGTCCGACTTGAC	1327
		TCGGCGAAATG			GGTTAGCATTTCC
		GA			TG
941	TUFB_EC_275_299_F	TGATCACTGGT	468	TUFB_EC_337_362_R	TGGATGTGCTCAC	1271
		GCTGCTCAGAT			GAGTCTGTGGCAT
		GGA
942	TUFB_EC_251_278_F	TGCACGCCGAC	493	TUFB_EC_337360_R	TATGTGCTCACGA	937
		TATGTTAAGAA			GTTTGCGGCAT
		CATGAT
949	GYRE_AB008700_760_787_F	TACTTACTTGA	198	GYRB_AB008700_862_888_2_R	TCCTGCAATATCT	1050
		GAATCCACAAG			AATGCACTCTTAC
		CTGCAA			G
958	RPOC_EC_2223_2243_F	TGGTATGCGTG	605	RPOC_EC_2329_2352_R	TGCTAGACCTTTA	1243
		GTCTGATGGC			CGTGCACCGTG
959	RPOC_EC_918_938_F	TCTGGATAACG	404	RPOC_EC_1009_1031_R	TCCAGCAGGTTCT	1004
		GTCGTCGCGG			GACGGAAACG
960	RPOC_EC_2334_2357_F	TGCTCGTAAGG	523	RPOC_EC_2380_2403_R	TACTAGACGACGG	905
		GTCTGGCGGAT			GTCAGGTAACC
		AC
961	RPOC_EC_917_938_F	TATTGGACAAC	242	RPOC_EC_1009_1034_R	TTACCGAGCAGGT	1362
		GGTCGTCGCGG			TCTGACGGAAACG
962	RPOB_E2005_2027_F	TCGTTCCTGGA	387	RPOB_EC_2041_2064_R	TTGACGTTGCATG	1399
		ACACGATGACG			TTCGAGCCCAT
		C
963	RPOB_EC_1527_1549_F	TCAGCTGTCGC	282	RPOB_EC_1630_1649_R	TCGTCGCGGACTT	1104
		AGTTCATGGAC			CGAAGCC
		C
964	INFB_EC_1347_1367_F	TGCGTTTACCG	515	INFB_EC_1414_1432_R	TCGGCATCACGCC	1090
		CAATGCGTGC			GTCGTC
965	VALS_EC_1128_1151_F	TATGCTGACCG	237	VALS_EC_1231_1257_R	TTCGCGCATCCAG	1384
		ACCAGTGGTAC			GAGAAGTACATGT
		GT			T
978	RPOC_EC_2145_2175_F	TCAGGAGTCGT	285	RPOC_EC_2228_2247_R	TTACGCCATCAGG	1363
		TCAACTCGATC			CCACGCA
		TACATGATG
1045	CJST_CJ_1668_1700_F	TGCTCGAGTGA	522	CJST_CJ_1774_1799_R	TGAGCGTGTGGAA	1170
		TTGACTTTGCT			AAGGACTTGGATG
		AAATTTAGAGA
1046	CJST_CJ_2171_2197_F	TCGTTTGGTGG	388	CJST_CJ_2283_2313_R	TCTCTTTCAAAGC	1126
		TGGTAGATGAA			ACCATTGCTCATT
		AAAGG			ATAGT
1047	CJST_CJ_584_616_F	TCCAGGACAAA	315	CJST_CJ_663_692_R	TTCATTTTCTGGT	1379
		TGTATGAAAAA			CCAAAGTAAGCAG
		TGTCCAAGAAG			TATC
1048	CJST_CJ_360_394_F	TCCTGTTATCC	346	CJST_CJ_442_476_R	TCAACTGGTTCAA	955
		CTGAAGTAGTT			AAACATTAAGTTG
		AATCAAGTTTG			TAATTGTCC
		TT
1049	CJST_CJ_2636_2668_F	TGCCTAGAAGA	504	CJST_CJ_2753_2777_R	TTGCTGCCATAGC	1409
		TCTTAAAAATT			AAAGCCTACAGC
		TCCGCCAACTT
1050	CJST_CJ_1290_1320_F	TGGCTTATCCA	575	CJST_CJ_1406_1433_R	TTTGCTCATGATC	1437
		AATTTAGATCG			TGCATGAAGCATA
		TGGTTTTAC			AA
1051	CJST_CJ_3267_3293_F	TTTGATTTTAC	707	CJST_CJ_3356_3385_R	TCAAAGAACCCGC	951
		GCCGTCCTCCA			ACCTAATTCATCA
		GGTCG			TTTA
1052	CJST_CJ_5_39_F	TAGGCGAAGAT	222	CJST_CJ_104_137_R	TCCCTTATTTTTC	1029
		ATACAAAGAGT			TTTCTACTACCTT
		ATTAGAAGCT			CGGATAAT
		AGA
1053	CJST_CJ_1080_1110_F	TTGAGGGTATG	681	CJST_CJ_1166_1198_R	TCCCTCATGTTT	1022
		CACCGTCTTT			AAATGATCAGGAT
		TTGATTCTTT			AAAAAGC
1054	CJST_CJ_2060_2090_F	TCCCGGACTTA	323	CJST_CJ_2148_2174_R	TCGATCCGCATCA	1068
		ATATCAATGAA			CCATCAAAAGCAA
		AATTGTGGA			A
1055	CJST_CJ_2869_2895_F	TGAAGCTTGTT	432	CJST_CJ_2979_3007_R	TCCTCCTTGTGCC	1045
		CTTTAGCAGGA			TCAAAACGCATTT
		CTTCA			TTA
1056	CJST_CJ_1880_1910_F	TCCCAATTAAT	317	CJST_CJ_1981_2011_R	TGGTTCTTACTTG	1309
		TCTGCCATTTT			CTTTGCATAAACT
		TCCAGGTAT			TTCCA
1057	CJST_CJ_2185_2212_F	TAGATGAAAAG	208	CJST_CJ_2283_2316_R	TGAATTCTTTCAA	1152
		GGCGAAGTGGC			AGCACCATTGCTC
		TAATGG			ATTATAGT
1058	CJST_CJ_1643_1670_F	TTATCGTTTGT	660	CJST_CJ_1724_1752_R	TGCAATGTGTGCT	1198
		GGAGCTAGTGC			ATGTCAGCAAA
		TTATGC			AAGAT
1059	CJST_CJ_2165_2194_F	TGCGGATCGTT	511	CJST_CJ_2247_2278_R	TCCACACTGGATT	1002
		TGGTGGTTGTA			GTAATTTACCTTG
		GATGAAAA			TTCTTT
1060	CJST_CJ_599_632_F	TGAAAAATGTC	424	CJST_CJ_711_743_R	TCCCGAACAATGA	1024
		CAAGAAGCATA			GTTGTATCAACTA
		GCAAAAAAAGC			TTTTTAC
		A
1061	CJST_CJ_360_393_F	TCCTGTTATCC	345	CJST_CJ_443_477_R	TACAACTGGTTCA	882
		CTGAAGTAGTT			AAAACATTAAGCT
		AATCAAGTTTG			GTAATTGTC
		T
1062	CJST_CJ_2678_2703_F	TCCCCAGGACA	321	CJST_CJ_2760_2787_R	TGTGCTTTTTTTG	1339
		CCCTGAAATTT			CTGCCATAGCAAA
		CAAC			GC
1063	CJST_CJ_1268_1299_F	AGTTATAAACA	29	CJST_CJ_1349_1379_R	TCGGTTTAAGCTC	1096
		CGGCTTTCCTA			TACATGATCGTAA
		TGGCTTATCC			GGATA
1064	CJST_CJ_1680_1713_F	TGATTTTGCTA	479	CJST_CJ_1795_1822_R	TATGTGTAGTTGA	938
		AATTTAGAGAA			GCTTACTACATGA
		ATTGCGGATGA			GC
		A
1065	CJST_CJ_2857_2887_F	TGGCATTTCTT	565	CJST_CJ_2965_2998_R	TGCTTCAAAACGC	1253
		ATGAAGCTTGT			ATTTTTACATTTT
		TCTTTAGCA			CGTTAAAG
1070	RNASEP_BKM_580_599_F	TGCGGGTAGGG	512	RNASEP_BKM_665_686_R	TCCGATAAGCCGG	1034
		AGCTTGAGC			ATTCTGTGC
1071	RNASEP_BKM_616_637_F	TCCTAGAGGAA	333	RNASEP_BKM_665_687_R	TGCCGATAAGCCC	1222
		TGGCTGCCACG			GGATTCTGTGC
1072	RNASEP_BDP_574_592_F	TGGCACGGCCA	561	RNASEP_BDP_616_635_R	TCGTTTCACCCTG	1115
		TCTCCGTG			TCATGCCG
1073	23S_BRM_1110_1129_F	TGCGCGGAAGA	510	23S_BRM_1176_1201_R	TCGCAGGCTTACA	1074
		TGTAACGGG			GAACGCTCTCCTA
1074	23S_BRM_515_536_F	TGCATACAAAC	496	23S_BRM_616_635_R	TCGGACTCGCTTT	1088
		AGTCGGAGCCT			CGCTACG
1075	RNASEP_CLB_459_487_F	TAAGGATAGTG	162	RNASEP_CLB_498_526_R	TGCTCTTACCTCA	1247
		CAACAGAGATA			CCGTTCCACCCTT
		TACCGCC			ACC
1076	RNASEP_CLB_459_487_F	TAAGGATAGTG	162	RNASEP_CLB_498_522_R	TTTACCTCGCCTT	1426
		CAACAGAGATA			TCCACCCTTACC
		TACCGCC
1077	ICD_CXB_93_120_F	TCCTGACCGAC	343	ICD_CXB_172_194_R	TAGGATTTTTCCA	921
		CCATTATTCCC			CGGCGGCATC
		TTTATC
1078	ICD_CXB_92_120_F	TTCCTGACCGA	671	ICD_CXB_172_194_R	TAGGATTTTTCCA	921
		CCCATTATTCC			CGGCGGCATC
		CTTTATC
1079	ICD_CXB_176_198_F	TCGCCGTGGAA	369	ICD_CXB_224_247_R	TAGCCTTTTCTCC	916
		AAATCCTACGC			GGCGTAGATCT
		T
1080	IS1111A_NC002971_6866 —	TCAGTATGTAT	290	IS1111A_NC002971_6928_6954_R	TAAACGTCCGATA	848
	6891_F	CCACCGTAGCC			CCAATGGTTCGCT
		GTC			C
1081	IS1111A_NC002971_7456 —	TGGGTGACATT	594	IS1111A_NC002971_7529_7554_R	TCAACAACACCTC	952
	7483_F	CATCAATTTCA			CTTATTCCCACTC
		TCGTTC
1082	RNASEP_RKP_419_448_F	TGGTAAGAGCG	599	RNASEP_RKP_542_565_R	TCAAGCGATCTAC	957
		TGGTAACA			CCGCATTACAA
1083	RNASEP_RKP_422_443_F	TAAGAGCGCAC	159	RNASEP_RKP_542_565_R	TCAAGCGATCTAC	957
		CGGTAAGTTGG			CCGCATTACAA
1084	RNASEP_RKP_466_491_F	TCCACCAAGAG	310	RNASEP_RKP_542_565_R	TCAAGCGATCTAC	957
		CAAGATCAAAT			CCGCATTACAA
		AGGC
1085	RNASEP_RKP_264_287_F	TCTAAATGGTC	391	RNASEP_RKP_295_321_R	TCTATAGAGTCCG	1119
		GTGCAGTTGCG			GACTTTCCTCGTG
		TG			A
1086	RNASEP_RKP_426_448_F	TGCATACCGGT	497	RNASEP_RKP_542_565_R	TCAAGCGATCTAC	957
		AAGTTGGCAAC			CCGCATTACAA
		A
1087	OMPB_RKP_860_890_F	TTACAGGAAGT	654	OMPB_RKP_972_996_R	TCCTGCAGCTCTA	1051
		TTAGGTGGTAA			CCTGCTCCATTA
		TCTAAAAGG
1088	OMPB_RKP_1192_1221_F	TCTACTGATTT	392	OMPB_RKP_1288_1315_R	TAGCAgCAAAGT	910
		TGGTAATCTTG			TATCACACCTGCA
		CAGCACAG			GT
1089	OMPB_RKP_3417_3440_F	TGCAAGTGGTA	485	OMPB_RKP_3520_3550_R	TGGTTGTAGTTCC	1310
		CTTCAACATGG			TGTAGTTGTTGCA
		GG			TTAAC
1090	GLTA_RKP_1043_1072_F	TGGGACTTGAA	576	GLTA_RKP_1138_1162_R	TGAACATTTGCGA	1147
		GCTATCGCTCT			CGGTATACCCAT
		TAAAGATG
1091	GLTA_RKP_400_428_F	TCTTCTCATCC	413	GLTA_RKP_499_529_R	TGGTGGGTATCTT	1305
		TATGGCTATTA			AGCAATCATTCTA
		TGCTTGC			ATAGC
1092	GLTA_RKP_1023_1055_F	TCCGTTCTTA	330	GLTA_RKP_1129_1156_R	TTGGCGACGGTAT	1415
		AAATAGCAATA			ACCCATAGCTTTA
		GAACTTGAAGC			TA
1093	GLTA_RKP_1043_1072_2_F	TGGAGCTTGAA	553	GLTA_RKP_1138_1162_R	TGAACATTGCGA	1147
		GCTATCGCTCT			CGGTATACCCAT
		AAAGATG
1094	GLTA_RKP_1043_1072_3_F	TGGAACTTGAA	543	GLTA_RKP_1138_1164_R	TGTGAACATTTGC	1330
		GCTCTCGCTCT			GACGGTATACCCA
		TAAAGATG			T
1095	GLTA_RKP_400_428_F	TCTTCTCATCC	413	GLTA_RKP_505_534_R	TGCGATGGTAGGT	1230
		TATGGCTATTA			ATCTTAGCAATCA
		TGCTTGC			TTCT
1096	CTXA_VBC_117_142_F	TCTTATGCCAA	410	CTXA_VBC_194_218_R	TGCCTAACAAATC	1226
		GAGGACAGAGT			CCGTCTGAGTTC
		GAGT
1097	CTXA_VBC_351_377_F	TGTATTAGGGG	630	CTXA_VBC_441_466_R	TGTCATCAAGCAC	1324
		CATACAGTCCT			CCAAAATGAACT
		CATCC
1098	RNASEP_VBC_331_349_F	TCCGCGGAGTT	325	RNASEP_VBC_388_414_R	TGACTTTCCTCCC	1163
		GACTGGGT			CCTTATCAGTCTC
					C
1099	TOXR_VBC_135_158_F	TCGATTAGGCA	362	TOXR_VBC_221_246_R	TTCAAAACCTTGC	1370
		GCAACGAAAGC			TCTCGCCAAACAA
		CG
1100	ASD_FRT_1_29_F	TTGCTTAAAGT	690	ASD_FRT_86_116_R	TGAGATGTCGAAA	1164
		TGGTTTTATTG			AAAACGTTGGCAA
		GTTGGCG			AATAC
1101	ASD_FRT_43_76_F	TCAGTTTTAAT	295	ASD_FRT_129_156_R	TCCATATTGTTGC	1009
		GTCTCGTATGA			ATAAAACCTGTTG
		TCGAATCAAAA			GC
		G
1102	GALE_FRT_168_199_F	TTATCAGCTAG	658	GALE_FRT_241_269_R	TCACCTACAGCTT	973
		ACCTTTTAGGT			TAAAGCCAGCAAA
		AAAGCTAAGC			ATG
1103	GALE_FRT_834_865_F	TCAAAAAGCCC	245	GALE_FRT_901_925_R	TAGCCTTGGCAAC	915
		TAGGTAAAGAG			ATCAGCAAAACT
		ATTCCATATC
1104	GALE_FRT_308_339_F	TCCAAGGTACA	306	GALE_FRT_390_422_R	TCTTCTGTAAAGG	1136
		CTAAACTTACT			GTGGTTTATTATT
		TGAGCTAATG			CATCCCA
1105	IPAH_SGF_258_277_F	TGAGGACCGTG	458	IPAH_SGF_301_327_R	TCCTTCTGATGCC	1055
		TCGCGCTCA			TGATGGACCAGGA
					G
1106	IPAH_SGF_113_134_F	TCCTTGACCGC	350	IPAH_SGF_172_191_R	TTTTCCAGCCATG	1441
		CTTTCCGATAC			CAGCGAC
1107	IPAH_SGF_462_486_F	TCAGACCATGC	271	IPAH_SGF_522_540_R	TGTCACTCCCGAC	1322
		TCGCAGAGAAA			ACGCCA
		CTT
1111	RNASEP_BRM_461_488_F	TAAACCCCATC	147	RNASEP_BRM_542_561_R	TGCCTCGCGCAAC	1227
		GGGAGCAAGAC			CTACCCG
		CGAATA
1112	RNASEP_BRM_325_347_F	TACCCCAGGGA	185	RNASEP_BRM_402_428_R	TCTCTTACCCCAC	1125
		AAGTGCCACAG			CCTTTCACCCTTA
		A			C
1128	HUPB_CJ_113_134_F	TAGTTGCTCAA	230	HUPB_CJ_157_188_R	TCCCTAATAGTAG	1028
		ACAGCTGGGCT			AAATAACTGCATC
					AGTAGC
1129	HUPB_CJ_76_102_F	TCCCGGAGCTT	324	HUPB_CJ_157_188_R	TCCCTAATAGTAG	1028
		TTATGACTAAA			AAATAACTGCATC
		GCAGAT			AGTAGC
1130	HUPB_CJ_102_F	TCCCGGAGCTT	324	HUPB_CJ_114_135_R	TAGCCCAGCTGTT	913
		TTATGACTAAA			TGAGCAACT
		GCAGAT
1151	AB_MLST-11-	TGAGATTGCTG	454	AB_MLST-11-	TTGTACATTTGAA	1418
	OIF007_62_91_F	AACATTTAATG		OIF007_169_203_R	ACAATATGCATGA
		CTGATTGA			CATGTGAAT
1152	AB_MLST-11-	TATTGTTTCAA	243	AB_MLST-11-	TCACAGGTTCTAC	969
	OIF007_185_214_F	ATGTACAAGGT		OIF007_291_324_R	TTCATCAATAATT
		GAAGTGCG			TCCATTGC
1153	AB_MLST-11-	TGGAACGTTAT	541	AB_MLST-11-	TTGCAATCGACAT	1400
	OIF007_260_289_F	CAGGTGCCCCA		OIF007_364_393_R	ATCCATTTCACCA
		AAATTCG			TGCC
1154	AB_MLST-11-	TGAAGTGCGTG	436	AB_MLST-11-	TCCGCCAAAAACT	1036
	OIF007_206_239_F	ATGATATCGAT		OIF007_318_344_R	CCCCTTTTCACAG
		GCACTTGATGT			G
		A
1155	AB_MLST-11-	TCGGTTTAGTA	378	AB_MLST-11-	TTCTGCTTGAGGA	1392
	OIF007_522_552_F	AAAGAACGTAT		OIF007_587_610_R	ATAGTGCGTGG
		TGCTCAACC
1156	AB_MLST-11-	TCAACCTGACT	250	AB_MLST-11-	TACGTTCTACGAT	902
	OIF007_547_571_F	GCGTGAATGGT		OIF007_656_686_R	TTCTTCATCAGGT
		TGT			ACATC
1157	AB_MLST-11-	TCAAGCAGAAG	256	AB_MLST-11-	TACAACGTGATAA	881
	OIF007_601_627_F	CTTTGGAAGAA		OIF007_710_736_R	ACACGACCAGAAG
		GAAGG			C
1158	AB_MLST-11-	TCGTGCCCGCA	384	AB_MLST-11-	TAATGCCGGGTAG	878
	OIF007_1202_1225_F	ATTGCATAAA		OIF007_1266_1296_R	TGCAATCCATTCT
		GC			TCTAG
1159	AB_MLST-11-	TCGTCCCGCA	384	AB_MLST-11-	TGCACCTGCGGTC	1199
	OIG007_1202_1225_F	ATTTGCATAAA		OIF007_1299_1316_R	GAGCG
		GC
1160	AB_MLST-11-	TTGTAGCACAG	694	AB_MLST-11-	TGCCATCCATAAT	1215
	OIF007_1234_1264_F	TCCTGAAAC		OIF007_1335_1362_R	CACGCCATACTGA
					CG
1161	AB_MLST-11-	TAGGTTTACGT	225	AB_MLST-11-	TGCCAGTTTCCAC	1212
	OIF007_1327_1356_F	GATTATGG		OIF007_1422_1448_R	ATTTCACGTTCGT
					G
1162	AB_MLST-11-	TCGTGATTATG	383	AB_MLST-11-	TCGCTTGAGTGTA	1083
	OIF007_1345_1369_F	AA		OIF007_1470_1494_R	GTCATGATTGCG
1163	AB_MLST-11-	TTATGGATGGC	662	AB_MLST-11-	TCGCTTGAGTGTA	1083
	OIF007_1351_1375_F	GT		OIF007_1470_1494_R	GTCATGATTGCG
1164	AB_MLST-11-	TCTTTGCCATT	422	AB_MLST-11-	TCGCTTGAGTGTA	1083
	OIF007_1387_1412_F	GAAGATGACTT		OIF007_1470_1494_R	GTCATAGATTGCG
		AAGC
1165	AB_MLST-11-	TACTAGCGGTA	194	AB_MLST-11-	TGAGTCGGGTTCA	1173
	OIF007_1542_1569_F	AGCTTAAACAA		OIF007_1656_1680_R	CTTACCTGGCA
		GATTGC
1166	AB_MLST-11-	TTGCCAATGAT	684	AB_MLST-11-	TGAGTCGGGTTCA	1173
	OIF007_1566_1593_F	ATTCGTTGGTT		OIF007_1656_1680_R	CTTTACCTGGCA
		AGCAAG
1167	AB_MLST-11-	TCGGCGAAATC	375	AB_MLST-11-	TACCGGAAGCACC	890
	OIF007_1611_1638_F	CGTATTCCTGA		OIF007_1731_1757_R	AGCGACATTAATA
		AAATGA			G
1168	AB_MLST-11-	TACCACTATTA	182	AB_MLST-11-	TGCAACTGAATAG	1195
	OIF007_1726_1752_F	ATGTCGCTGGT		OIF007_1790_1821_R	ATTGCAGTAAGTT
		GCTTC			ATAAGC
1169	AB_MLST-11-	TTATAACTTAC	656	AB_MLST-11-	TGAATTATGCAAG	1151
	OIF007_1792_1826_F	TGCAATCTATT		OIF007_1876_1909_R	AAGTGATCAATTT
		CAGTTGCTG			TCTCACGA
		GTG
1170	AB_MLST-11-	TTATAACTTAC	656	AB_MLST-11-	TGCCGTAACTAAC	1224
	OIF007_1792_1826_F	TGCAATCTATT		OIF007_1895_1927_R	ATAAGAGAATTAT
		CAGTTGCTTGG		GCAAGAA
		TG
1171	AB_MLST-11-	TGGTTATGTAC	618	AB_MLST-11-	TGACGGCATCGA	1157
	OIF007_1970_2002_F	CAAATACTTTG		OIF007_2097_2118_R	ACCACCGTC
		TCTGAAGAGG
1172	RNASEP_BRM_461_488_F	TAAACCCCATC	147	RNASEP_BRM_542_561_2_R	TGCCTCGTGCAAC	1228
		GGGAGCAAGAC			CCACCCG
		CGAATA
2000	CTXB_NC002505_46_70_F	TCAGCGTATGC	278	CTSB_NC002505_132_162_R	TCCGGCTAGAGAT	1039
		ACATGGAACTC			TCTGTATACGAC
		CTC			AATATC
2001	FUR_NC002505_87_113_F	TGAGTGCCAAC	465	FUR_NC002505_205_228_R	TCCGCCTTCAAAA	1037
		ATATCAGTGCT			TGGTGGCGAGT
		GAAGA
2002	FUR_NC002505_87_113_F	TGAGTGCCAAC	465	FUR_NC002505_178_205_R	TCACGATACCTGC	974
		ATATCAGTGCT			ATCATCAAATTG
		GAAGA			GTT
2003	GAPA_NC002505_533_560_F	TCGACAACACC	356	GAPA_NC002505_646_671_R	TCAGAATCGATGC	980
		ATTATCTATGG			CAAATGCGTCATC
2004	GAPA_NC002505_505_19_721_F	TCAATGAACGA	259	GAPA_NC002505_769_798_R	TCCTCTATGCAAC	1046
		CCAACAAGTGA			TTAGTATCAACA
		TTGATG			GAAT
2005	GAPA_NC002505_753_782_F	TGCTAGTCAAT	517	GAPA_NC002505_856_881_R	TCCATCGCAGTCA	1011
		CTATCATTCCG			CGTTTACTGTTGG
		GTTGATAC
2006	GYRB_NC002505_2_32_F	TGCCGGACAAT	501	GYRB_NC002505_109_134_R	TCCACCACCTCAA	1003
		TACGATTCATC			AGACCATGTGGTG
		GAGTATTAA
2007	GYRB_NC002505_123_152_F	TGAGGTGGTGG	460	GYRB_NC002505_199_225_R	TCCGTCATCGCTG	1042
		ATAACTCAATT			ACAGAAACTGAGT
		GATGAAGC			T
2008	GYRB_NC002505_768_794_F	TATGCAGTGGA	236	GYRB_NC002505_832_860_R	TGGAAACCGGCTA	1262
		ACGATGGTTTC			AGTGAGTACCACC
		CAAGA			ATC
2009	GYRB_NC002505_837_860_F	TGGTACTCACT	603	GYRB_NC002505_937_957_R	TCCTTCACGCGCA	1054
		TAGCGGGTTT			TCATCACC
		CG
2010	GYRB_NC002505_934_956_F	TCGGGTGATGA	377	GYRB_NC002505_982_1007_R	TGGCTTGAGAATT	1283
		TGCGCGTGAAG			TAGGATCCGGCAC
		G
2011	GYRB_NC002505_1161_1190_F	TAAAGCCCGTG	148	GYRB_NC002505_1255_1284_R	TGAGTCACCCTCC	1172
		AAATGACTCGT			ACAATGTATAGTT
		CGTAAAGG			CAGA
2012	OMPU_NC002505_275_110_F	TACGCTGACGG	190	OMPU_NC002505_154_180_R	TGCTTCAGCACGG	1254
		AATCAACCAAA			CCACCAACTTCTA
		GCGG			G
2013	OMPU_NC002505_258_283_F	TGACGGCCTAT	451	OMPU_NC002505_346_369_R	TCCGAGACCAGCG	1033
		ACGGTGTTGGT			TAGGTGTAACG
		TTCT
2014	OMPU_NC002505_431_455_F	TCACCGATATC	266	OMPU_NC002505_544_567_R	TCGGTCAGCAAAA	1094
		ATGGCTTACCA			CGGTAGCTTGC
		CGG
2015	OMPU_NC002505_533_557_F	TAGGCGTGAAA	223	OMPU_NC002505_625_651_R	TAGAGAGTAGCCA	908
		GCAAGCTACCG			TCTTCACCGTTGT
		TTT			C
2016	OMPU_NC002505_689_713_F	TAGGTGCTGGT	224	OMPU_NC002505_725_751_R	TGGGGTAAGACGC	1291
		TACGCAGATCA			GGCTAGCATGTAT
		AGA			T
2017	OMPU_NC002505_727_747_F	TACATGCTAGC	181	OMPU_NC002505_811_835_R	TAGCAGCTAGCTC	911
		CGCGTCTTAC			GTAACCAGTGTA
2018	OMPU_NC002505_931_953_F	TACTACTTCAA	193	OMPU_NC002505_1033_1053_R	TTAGAAGTCGTAA	1368
		GCCGAACTTCC			CGTGGACC
		G
2019	OMPU_NC002505_927_953_F	TACTTACTACT	197	OMPU_NC002505_1033_1054_R	TGGTTAGAAGTCG	1307
		TCAAGCCGAAC			TAACGTGGACC
		TTCCG
2020	TCPA_NC002505_48_73_F	TCACGATAAGA	269	TCPA_NC002505_148_170_R	TTCTGCGAATCAA	1391
		AAACCGGTCAA			TCGCACGCTG
		GAGG
2021	TDH_NC004605_265_289_F	TGGCTGACATC	574	TDH_NC004605_357_386_R	TGTTGAAGCTGTA	1351
		CTACATGACTG			CTTGACCTGATTT
		TGA			TACG
2022	VVHA_NC004460_772_802_F	TCTTATTCCAA	412	VVHA_NC004460_862_886_R	TACCAAAGCGTGC	887
		CTTCAAACCGA			ACGATAGTTGAG
		ACTATGACG
2023	23S_EC_2643_2667_F	TGCCTGTTCTT	508	23S_EC_2746_2770_R	TGGGTTTCGCGCT	1297
		AGTACGAGAGG			AGATGCTTTCA
		ACC
2024	16S_EC_713_732_TMOD_F	TAGAACACCCG	202	16S_EC_789_811_R	TGCGTGGACTAC	1240
		ATGGCGAAGGC			AGGGTATCTA
2025	16S_EC_784_806_F	TGGATTAGAGA	560	16S_EC_880_897_TMOD_R	TGGCCGTACTCCC	1278
		CCCTGGTAGTC			CAGGCG
		C
2026	16S_EC_959_981_F	TGTCGATGCAA	634	16S_EC_1052_1074_R	TACGAGCTGACGA	896
		CGCGAAGAACC			CAGCCATGCA
		T
2027	TUFB_EC_956_979_F	TGCACACGCCG	489	TUFB_EC_1034_1058_2_R	TGCATCACCATTT	1204
		TTCTTCAACAA			CCTTGTCCTTCG
		CT
2028	RPOC_EC_2146_2174_TMOD_F	TCAGGAGTCGT	284	RPOC_EC_2227_2249_R	TGCTAGGCCATCA	1244
		TCAACTCGATC			GGCCACGCAT
		TACATGAT
2029	RPOB_EC_1841_1866_F	TGGTTATCGCT	617	RPOB_EC_1909_1929_TMOD_R	TGCTGGATTCGCC	1250
		CAGGCGAACTC			TTTGCTACG
		CAAC
2030	RPLB_EC_650_679_TMOD_F	TGACCTACAGT	449	RPLB_EC_739_763_R	TGCCAAGTGCTGG	1208
		AAGAGGTTCTG			TTTACCCCATGG
		AATGAACC
2031	RPLB_EC_690_710_F	TCCACACGGTG	309	RPLB_EC_737_760_R	TGGGTGCTGGTTT	1295
		GTGGTGAAGG			ACCCCATGGAG
2032	INFB_EC_1366_1393_F	TCTCGTGGTGC	397	INFB_EC_1439_1469_R	TGTGCTGCTTTCG	1335
		ACAAGTAACGG			CATGGTTAATTGC
		ATATTA			TTCAA
2033	VALS_EC_1105_1124_TMOD_F	TCGTGGCGGCG	385	VALS_EC_1195_1219_R	TGGGTACGAACTG	1292
		TGGTTATCGA			GATGTCGCCGTT
2034	SSPE_BA_113_137_F	TGCAAGCAAAC	482	SSPE_BA_197_222_TMOD_R	TTGCACGTCTGTT	1402
		GCACAATCAGA			TCAGTTGCAAATT
		AGC			C
2035	RPOC_EC_2218_2241_TMOD_F	TCTGGCAGGTA	405	RPOC_EC_2313_2338_R	TGGCACCGTGGGT	1273
		TGCGTTGTCTG			TGAGATGAAGTAC
		ATG
2056	MECI-NC003923-	TTTACACATAT	698	MECI-NC003923-41798-	TTGTGATATGGAG	1420
	41798-41609_33_60_F	CGTGAGCAATG		41609_86_113_R	GTGTAGAAGGTGT
		AACTGA			TA
2057	AGR-III_NC003923-	TCACCAGTTTG	263	AGR-III_NC003923-	ACCTGCATCCCTA	730
	2108074-	CCACGTATCTT		2108074-	AACGTACTTGC
	2109508_1_23_F	CAA		2109507_56_79_R
2058	AGR-III_NC003923-	TGAGCTTTTAG	457	AGR-III_NC003923-	TACTTCAGCTTCG	906
	2108074-	TTGACTTTTTC		2108074-	TCCAATAAAAAAT
	2109507_569_596_F	AACAGC		2109507_622_653_R	CACAAT
2059	AGR-III_NC003923-	TTTCACACAGC	701	AGR-III_NC003923-	TGTAGGCAAGTGC	1319
	2108074-	GTGTTTATAGT		2108074-	ATAAGAAATTGAT
	2109507_1024_1052_F	TCTACCA		2109507_1070_1098_R	ACA
2060	AGR-	TGGTGACTTCA	610	AGR-	TCCCCATTAATAA	1021
	I_AJ617706_622_651_F	TAATGGATGAA		I_AJ617706_694_726_R	TTCCACCTACTAT
		GTTGAAGT			CACACT
2061	AGR-	TGGGATTTTAA	579	AGR-	TGGTACTTCAACT	1302
	I_AJ617706_580_611_F	AAAACATTGGT		I_AJ617706_626_655_R	TCATCCATTATGA
		AACATCGCAG			AGTC
2062	AGR-II_NC002745-	TCTTGCAGCAG	415	AGR-II_NC002745-	TTGTTTATTGTTT	1424
	2079448-	TTTATTTGATG		2079448-	CCATATGCTACAC
	2080879_620_651_F	AACCTAAAGT		2080879_700_731_R	ACTTTC
2063	AGR-II_NC002745-	TGTACCCGCTG	624	AGR-II_NC002745-	TCGCCATAGCTAA	1077
	2079448-	AATTAACGAAT		2079448-	GTTGTTTATTGTT
	2080879_649_679_F	TTATACGAC		2080879_715_745_R	TCCAT
2064	AGR-	TGGTATTCTAT	606	AGR-	TGCGCTATCAACG	1233
	IV_AJ617711_931_961_F	TTTGCTGATAA		IV_AJ617711_1004_1035_R	ATTTTGACAATAT
		TGACCTCGC			ATGTGA
2065	AGR-	TGGCACTCTTG	562	AGR-	TCCCATACCTATG	1017
	IV_AJ617711_250_283_F	CCTTTAATATT		IV_AJ617711_309_335_R	GCGATAACTGTCA
		AGTAAACTATC			T
		A
2066	BLAZ_NC002952	TCCACTTATCG	312	BLAZ_NC002952	TGGCCACTTTTAT	1277
	(1913827 . . .	CAAATGGAAAA		(1913827 . . .	CAGCAACCTTACA
	1914672)_68_68_F	TTAAGCAA		1914672)_68_68_R	GTC
2067	BLAZ_NC002952	TGCACTTATCG	494	BLAZ_NC002952	TAGTCTTTTGGAA	926
	(1913827 . . .	CAAATGGAAAA		(1913827 . . .	CACCGTCTTTAAT
	1914672)_68_68_2_F		1914672)_68_68_2_R	TAAAGT
2068	BLAZ_NC002952	TGATACTTCAA	467	BLAZ_NC002952	TGGAACACCGTCT	1263
	(1913827 . . .	CGCCTGCTGCT		(1913827 . . .	TTAATTAAAGTAT
	1914672)_68_68_3_F	TTC		1914672_68_68_3_F	CTCC
2069	BLAZ_NC002952	TATACTTCAAC	232	BLAZ_NC002952	TCTTTTCTTTGCT	1145
	(1913827 . . .	GCCTGCTGCTT		(1913827 . . .	TAATTTTCCATTT
	1914672)_68_68_4_F	TC		1914672)_68_68_4_R	GCGAT
2070	BLAZ_NC002952	TGCAATTGCTT	487	BLAZ_NC002952	TTACTTCCTTACC	1366
	(1913827 . . .	TAGTTTTAAGT		(1913827 . . .	ACTTTTAGTATCT
	1914672)_1_33_F	GCATGTAATTC		1914672)_34_67_R	AAAGCATA
2071	BLAZ_NC002952	TCCTTGCTTTA	351	BLAZ_NC002952	TGGGGACTTCCTT	1289
	(1913827 . . .	GTTTTAAGTGC		(1913827 . . .	ACCACTTTTAGTA
	1914672)_3_34_F	ATGTAATTCAA		1914672)_40_68_R	TCTAA
2072	BSA-A_NC003923-	TAGCGAATGTG	214	BSA-A_NC003923-	TGCAAGGGAAACC	1197
	1304065-	GCTTTACTTCA		1304065-	TAGAATTACAAAC
	1303589_99_125_F	CAATT		1303589_165_193_R
2073	BSA-A_NC003923-	ATCAATTTGGT	32	BSA-A_NC003923-	TGCATAGGAAGG	1203
	1304065-	GGCCAAGAAC		1304065-	TAACACCATAGTT
	1303589_194_218_F	CTGG		1303589_253_278_R
2074	BSA-A_NC003923-	TTGACTGCGGC	679	BSA-A_NC003923-	TAACAACGTTACC	856
	1304065-	ACAACACGGAT		1304065-	TTCGCGATCCACT
	1303589_328_349_F			1303589_388_415_R
2075	BSA-A_NC003923-	TGCTATGGTGT	519	BSA-A_NC003923-	TGTTGTGCCGCAG	1353
	1304065-	TACCTTCCCTA		1304065-	TCAAATATCTAAAT
	1303589_253_278_F	TGCA		1303589_317_344_R
2076	BSA-B_NC003923-	TAGCAACAAAT	209	BSA-B_NC003923-	TGTGAAGAACTTT	1331
	1917149-	ATATCTGAAGC		1917149-	CAAATCTGTGAAT
	1914156_953_982_F	AGCGTACT		1914156_1011_1039_R	CCA
2077	BSA-B_NC003923-	TGAAAAGTATG	426	BSA-B_NC003923-	TCTTCTTGAAAAA	1138
	1917149-	GATTTGAACAA		1917149-	TTGTTGTCCCGAA
	1914156_1050_1081_F	CTCGTGAATA		1914156_1109_1136_R	AC
2078	BSA-B_NC003923-	TCATTATCATG	300	BSA-B_NC003923-	TGGACTAATAACA	1267
	1917149-	CGCCAATGAGT		1917149-	ATGAGCTCATTGT
	1914156_1260_1286_F	GCAGA		1914156_1323_1353_R	ACTGA
2079	BSA-B_NC003923-	TTTCATCTTAT	703	BSA-B_NC003923-	TGAATATGTAATG	1148
	1917149-	CGAGGACCCGA		1917149-	CAAACCAGTCTTT
	1914156_2126_2153_F	ATCGA		1914156_2186_2216_R	GTCAT
2080	ERMA_NC002952-	TCGCTATCTTA	372	ERMA_NC002952-	TGAGTCTACACTT	1174
	55890-	TCGTTGAGAAG		55890-	AGGCTTAGGATGA
	56621_366_392_F	GGATT		56621_487_513_R	A
2081	ERMA_NC002952-	TAGCTATCTTA	217	ERMA_NC002952-	TGAGCATTTTTAT	1167
	55890-	TCGTTGAGAAG		55890-	ATCCATCTCCACC
	56621_366_395_F	GGATTTGC		56621_438_465_R	AT
2082	ERMA_NC002952-	TGATCGTTGAG	470	ERMA_NC002952-	TCTTGGCTTAGGA	1143
	55890-	AAGGGATTTGC		55890-	TGAAAATATAGTG
	56621_374_402_F	GAAAAGA		56621_473_504_R	GTGGTA
2083	ERMA_NC002952-	TGCAAAATCTG	480	ERMA_NC002952-	TCAATACAGAGTC	964
	55890-	CAACGAGCTTT		55890-	TACACTTGGCTTA
	56621_404_427_F	GG		56621_491_520_R	GGAT
2084	ERMA_NC002952-	TCATCCTAAGC	297	ERMA_NC002952-	TGGACGATATTCA	1266
	55890-	CAAGTGTAGAC	55890-	CGGTTTACCCACT
	56621_489_516_F	TCTGTA		56621_586_615_R	TATA
2085	ERMA_NC002952-	TATAAGTGGGT	231	ERMA_NC002952-	TTGACATTTGCA	1397
	55890-	AAACCGTGAAT		55890-	TGCTTCAAAGCCT
	56621_586_614_F	ATCGTGT		56621_640_665_R	G
2086	ERMC_NC005908-	TCTGAACATGA	399	ERMC_NC005908-	TCCGTAGTTTTG	1041
	2004-	TAATATCTTTG		2004-	CATAATTATGGT
	2738_85_116_F	AAATCGGCTC		2738_173_206_R	CTATTTCAA
2087	ERMC_NC005908-	TCATGATAATA	298	ERMC_NC005908-	TTTATGGTCTAT	1429
	2004-	TCTTTGAAATC		2004-	TTCAATGGCAGTT
	2738_90_120_F	GGCTCAGGA		2738_460_189_R	ACGAA
2088	ERMC_NC005908-	TCAGGAAAAGG	283	ERMC_NC005908-	TATGGTCTATTT	936
	2004-	GCATTTTACCC		2004-	CAATGGCAGTTAC
	2738_115_139_F	TTG		2738_161_187_R	GA
2089	ERMC_NC005908-	TAATCGTGGAA	168	ERMC_NC005908-	TCAACTTCTGCC	956
	2004-	TACGGGTTTGC		2004-	ATTAAAAGTAATG
	2738_374_397_F	TA		2738_325_452_R	CCA
2090	ERMC_NC005908-	TCTTTGAAATC	421	ERMC_NC005908-	TGATGGTCTATT	1185
	2004-	GGCTCAGGAAA		2004-	TCAATGGCAGTTA
	2738_101_125_F	AGG		2738_159_188_R	CGAAA
2091	ERMB_Y13600-625-	TGTTGGGAGTA	644	ERMB_Y13600-625-	TCAACAATCAGA	953
	1362_291_321_F	TTCCTTACCAT		1362_352_380_R	TAGATGTCAGACG
		TTAAGCACA			CATG
2092	ERMB_Y13600-625-	TGGAAAGCCAT	536	ERMB_Y13600-625-	TGCAAGAGCAAC	1196
	1362_344_367_F	GCGTCTGACAT		1362_415_437_R	CCTAGTGTTCG
2093	ERMB_Y13600-625-	TGGATATTCAC	556	ERMB_Y13600-625-	TAGGATGAAAGC	919
	1363_404_429_F	CGAACACTAGG		1362_471_493_R	ATTCCGCTGGC
2094	ERMB_Y13600-625-	TAAGCTGCCAG	161	ERMB_Y13600-625-	TCATTCTGTGGTA	989
	1362_465_487_F	CGGAATGCTTT		1362_521_545_R	TGGCGGGTAAGTT
		C
2095	PVLUK_NC003923-	TGAGCTGCATC	456	PVLUK_NC003923-	TGGAAAACTCAT	1261
	1529595-	ACTGTATTGGA		1529595-	GAAATTAAAGTGA
	1531285_688_713_F	TAG		1531285_775_804_R	AAGGA
2096	PVLUK_NC003923-	TGGAACAAAAT	539	PVLUK_NC003923-	TCATTAGGTAAAA	993
	1529595-	AGTCTCTCGGA		1529595-	TGTCTGGACATG
	1531285_1039_1068_F	TTTTGACT		1531285_1095_1125_R	ATCCAA
2097	PVLUK_NC003923-	TGAGTAACATC	461	PVLUK_NC003923-	TCTCATGAAAAAG	1124
	1529595-	CATATTTCTGC		1529595-	GCTCAGGAGATAC
	1531285_908_936_F	CATACGT		1531285_950_978_R	AAG
2098	PVLUK_NC003923-	TCGGAATCTGA	373	PVLUK_NC003923-	TCACACCTGTAAG	968
	1529595-	TGTTCAGTTGT		1529595-	TGAGAAAAAGGTT
	1531285_610_633_F	TT		1531285_654_682_R	TGAT
2099	SA442_NC003923-	TGTCGGTACAC	635	SA442_NC003923-	TTTCCGATCAAC	1433
	2538576-	GATATTCTTCA		2538576-	GTAATGAGATTTC
	25388311_35_F	CGA		2538831_98_124_R	A
2100	SA442_NC003923-	TGAAATCTCAT	427	SA442_NC003923-	TCGTATGACCAGC	1098
	2538576-	TACGTTGCATC		2538576-	TTCGGTACTACTA
	2538831_98_124_F	G		2538831_163_188_R
2101	SA442_NC003923-	TCTCATTACGT	395	SA442_NC003923-	TTTATGACCAGCT	1428
	2538576-	TGCATCGGAAA		2538576-	TCGGTACTACTAA
	2538831_103_126_F	CA		2538831_161_187_R	A
2102	SA442_NC003923-	TAGTACCGAAG	226	SA442_NC003923-	TGATAATGAAGGG	1179
	2538576-	CTGGTCATACG		2538576-	AAACCTTTTTCAC
	2538831_166_188_F	A			G
					2538831_231_257_R
2103	SEA_NC003923-	TGCAGGGAACA	495	SEA_NC003923-	TCGATCGTGACTC	1070
	2052219-	GCTTTAGGCA		2052219-	TCTTTATTTTCAG
	2051456_115_135_F			2051456_173_200_R	TT
2104	SEA_NC003923-	TAACTCTGATG	156	SEA_NC003923-	TGTAATTAACCGA	1315
	2052219-	TTTTTGATGGG		2052219-	AGGTTCTGTAGA
	2051456_572_598_F	AAGGT		2051456_621_651_R	GTATG
2105	SEA_NC003923-	TGTATGGTGGT	629	SEA_NC003923-	TAACCGTTTCCA	861
	2052219-	GTAACGTTACA		2052219-	AGGTACTGTATTT
	2051456_382_414_F	TGATAATAAT		2051456_464_492_R	TGT
		C
2106	SEA_NC003923-	TTGTATGTATG	695	SEA_NC003923-	TAACCGTTTCCAA	862
	2052219-	GTGGTGTAACG		2052219-	AGGTACTGTATTT
	2051456_377_406_F	TTACATGA		2051456_459_492_R	TGTTTACC
2107	SEB_NC002758-	TTTCACATGTA	702	SEB_NC002758-	TCATCTGGTTTAG	988
	2135540-	ATTTTGATATT		2135540-	GATCTGGTTGACT
	2135140_208_137_F	CGCACTGA		2135140_273_298_R
2108	SEB_NC002758-	TATTTCACATG	244	SEB_NC002758-	TGCAACTCATCTG	1194
	2135540-	TAATTTTGATA		2135540-	GTTAGGATCT
	2135140_106_235_F	TTCGCACT		2135140_281_304_R
2109	SEB_NC002758-	TAACAACTCGC	151	SEB_NC002758-	TGTGCAGGCATCA	1334
	2135540-	CTTATGAAACG		2135540-	TGTCATACCAA
	2135140_402_402_F	GGATATA		2135140_402_402_R
2110	SEB_NC002758-	TTGTATGTATG	696	SEB_NC002758-	TTACCATCTTCAA	1361
	2135540-	GTGGTGTAACT		2135540-	ATACCCGAACAG
	2135140_402_402_2_F	GAGCA		2135140_402_402_2_R
2111	SEC_NC003923-	TTAACATGAAG	648	SEC_NC003923-	TGAGTTTGCACTT	1177
	851678-	GAAACCACTTT		851678-	CAAAAGAAATTGT
	852768_546_575_F	GATAATGG		852768_620_647_R	GT
2112	SEC_NC003923-	TGGAATAACAA	546	SEC_NC003923-	TCAGTTTGCACTT	985
	851678-	AACATGAAGGA		851678-	CAAAAGAAATTGT
	852768_537_566_F	AACCACTT		852768_619_647_R	GTT
2113	SEC_NC003923-	TGAGTTTAACA	466	SEC_NC003923-	TCGCCTGGTGCAG	1078
	851678-	GTTCACCATAT		851678-	GCATCATAT
	852768_720_749_F	GAAACAGG		852768_794_815_R
2114	SEC_NC003923-	TGGTATGATAT	604	SEC_NC003923-	TCTTCACACTTTT	1133
	851678-	GATGCCTGCAC		851678-	AGAATCAACCGTT
	852768_787_810_F	CA		852768_853_886_R	TTATTGTC
2115	SED_M28521_657_	TGGTGGTGAAA	615	SED_M28521_	TGTACACCATTTA	1318
	682_F	TAGATAGGACT		741_770_R	TCCACAAATTGAT
		GCTT			TGGT
2116	SED_M28521_	TGGAGGTGTCA	554	SED_M28521_	TGGGCACCATTTA	1288
	690_711_F	ACTCCACACGA		739_770_R	TCCACAAATTGAT
		A			TGGTAT
2117	SED_M28521_	TTGCACAAGCA	683	SED_M28521_	TCGCGCTGTATTT	1079
	833_854_F	AGGCGCTATTT		888_911_R	TTCCTCCGAGA
2118	SED_M28521_	TGGATGTTAAG	559	SED_M28521_	TGTCAATATGAAG	1320
	962_987_F	GGTGATTTTCC		1022_1048_R	GTGCTCTGTGGAT
		CGAA			A
2119	SEA-SEE_NC002952-	TTTACACTACT	699	SEA-SEE_NC002952-	TCATTTATTTCTT	994
	2131289-	TTTATTCATTG		2131289-	CGCTTTTCTGCT
	2130703_16_45_F	CCCTAACG		2130703_71_98_R	AC
2120	SEA-SEE_NC002952-	TGATCATCCGT	469	SEA-SEE_NC002952-	TAAGCACCATATA	870
	2131289-	GGTATAACGAT		2131289-	AGTCTACTTTTTC
	2130703_249_278_F	TTATTAGT		2130703_314_344_R	CCCTT
2121	SEE_NC002952-	TGACATGATAA	445	SEE_NC002952-	TCTATAGGTACT	1120
	2131289-	TAACCGATTGA		2131289-	GTAGTTTGTTTTC
	2130703_409_437_F	CCGAAGA		2130703_485_494_R	CGTCT
2122	SEE_NC002952-	TGTTCAAGAGC	640	SEE_NC002952-	TTTGCACCTTAC	1436
	2131289-	TAGATCTTCAG		2131289-	CGCCAAAGCT
	2130703_525_550_F	GCAA		2130703588_588_R
2123	SEE_NC002952-	TGTTCAAGAGC	639	SEE_NC002952-	TACCTTACCGCC	892
	2131289-	TAGATCTTCAG		2131289-	AAAGCTGTCT
	2130703_525_549_F	GCA		2130703_588_586_2_R
2124	SEE_NC002952-	TCTGGAGGCAC	403	SEE_NC002952-	TCCGTCTATCCA	1043
	2131289-	ACCAAATAAAA		2131289-	CAAGTTAATTGGT
	2130703_381_384_F	CA		2130703_444_471_R	ACT
2125	SEG_NC002758-	TGCTCAACCCG	520	SEG_NC002758-	TAACTCCTCTTCC	863
	1955100-	ATCCTAAATTA		1955100-	TTCAACAGGTGGA
	1954171_225_251_F	GACGA		1954171_321_348_R
2126	SEG_NC002758-	TGGACAATAGA	548	SEG_NC002758-	TGCTTTGTAATC	1260
	1955100-	CAATCACCTTG		1955100-	AGTTCCTGAATAG
	1954171_623_851_F	GATTTACA		1954171_871_702_R	TAACCA
2127	SEG_NC002758-	TGGAGGTTGTT	555	SEG_NC002758-	TGTCTATTGTCGA	1329
	1955100-	GTATGTATGG		1955100-	ATTGTTACCTGTA
	1954171_540_584_F	TGTT		1954171_607_635_R	CAGT
2128	SEG_NC002758-	TACAAAGCAAG	173	SEG_NC002758-	TGATTCAAATGCA	1187
	1955100-	ACACTGGCTC		1955100-	GAACCATCAAACT
	1954171_694_718_F	ACTA		1954171_735_782_R	CG
2129	SEH_NC002953-	TTGCAACTGCT	682	SEH_NC002953-	TAGTGTTGTACCT	927
	60024-	GATTTAGCTCA		60024-	CCATATAGACATT
	60977_449_472_F	GA		60977_547_578_R	CAGA
2130	SEH_NC002953-	TAGAAATCAAG	201	SEH_NC002953-	TTCTGAGCTAAAT	1390
	60024-	GTGATAGTGGC		60024-	ACAGCAGTTGC
	60977_408_434_F	AAATGA		60977_450_473_R
2131	SEH_NC002953-	TCTGAARTGTC	400	SEH_NC002953-	TACCATCTACCC	888
	60024-	TATATGGAGGT		60024-	AACATTAGCACCA
	60977_547_576_F	ACAACACTA		60977_608_634_R	A
2132	SEH_NC002953-	TTCTGAATGTC	677	SEH_NC002953-	TAGCACCAATCAC	909
	60024-	TATATGGAGGT		60024-	CCTTTCCTGT
	60977_546_575_F	ACAACACT		60977_594_616_R
2133	SEI_NC002758-	TCAACTCGAAT	253	SEI_NC002758-	TCACAAGGACCAT	966
	1957830-	TTTCAACAGGT		1957830-	TATAATCAATGCC
	1956949_324_349_F	ACCA		1956949_419_448_R	AA
2134	SEI_NC002758-	TTCAACAGGTA	666	SEI_NC002758-	TGTACAAGGACCA	1316
	1957830-	CCAATGATTTG		1957830-	TTATAATCAATGC
	1956949_336_363_F	ATCTCA		1958949_420_447_R	CA
2135	SEI_NC002758-	TGATCTCAGAA	471	SEI_NC002758-	TCTGGCCCCTCCA	1129
	1957830-	TCTAATAATTG		1957830-	TACATGTATTTAG
	1956949_356_384_F	GGACGAA		1958949_449_474_R
2136	SEI_NC002758-	TCTCAAGGTGA	394	SEI_NC002758-	TGGGTAGGTTTTT	1293
	1957830-	TATTGGTGTAG		1957830-	ATCTGTGACGCCT
	1956949_223_253_F	GTAACTTAA		1958949_290_316_R	T
2137	SEJ_AF053140-	TGTGGAGTAAC	637	SEJ_AF053140-	TCTAGCGGAACAA	1118
	1307_1332_F	ACTGCATGAAA		1381_1404_R	CAGTTCTGATG
		ACAA
2138	SEJ_AF053140-	TAGCATCAGAA	211	SEJ_AF053140-	TCCTGAAGATCTA	1049
	1378_1403_F	CTGTTGTTCCG		1429_1458_R	GTTCTTGAATGGT
		CTAG			TACT
2139	SEJ_AF053140-	TAACCATTCAA	153	SEJ_AF053140-	TAGTCCTTTCTGA	925
	1431_1459_F	GAACTAGATCT		1500_1531_R	ATTTACCATCAA
		TCAGGCA			AGGTAC
2140	SEJ_AF053140-	TCATTCAAGAA	301	SEJ_AF053140-	TCAGGTATGAAAC	984
	1434_1461_F	CTAGATCTTCA		1521_1549_R	ACGATTAGTCCTT
		GGCAAG			TCT
2141	TSST_NC002758-	TGGTTTAGATA	619	TSST_NC002758-	TGTAAAAGCAGGG	1312
	2137564-	ATTCTTTAGGA		2137564-	CTATAATAAGGAC
	2138293_206_236_F	TCTATGCGT		2138293_278_305_R	TC
2142	TSST_NC002758-	TGCGTATAAAA	514	TSST_NC002758-	TGCCCTTTTGTAA	1221
	2137564-	AACACAGATGG		2137564-	AAGCAGGGCTAT
	2138293_232_258_F	CAGCA		2138293_289_313_R
2143	TSST_NC002758-	TCCAAATAAGT	304	TSST_NC002758-	TACTTTAAGGGGC	907
	2137564-	GGCGTTACAAA		2137564-	TATCTTTACCATG
	2138293_382_410_F	TACTGAAA		2138293_448_478_R	AACCT
2144	TSST_NC002758-	TCTTTTACAAA	423	TSST_NC002758-	TAAGTTCCTTCGC	874
	2137564-	AGGGGAAAAG		2137564-	TAGTATGTTGGCT
	2138293_297_325_F	TTGACTT		2138293_347_373_R	T
2145	ARCC_NC003923-	TCGCCGGCAAT	368	ARCC_NC003923-	TGAGTTAAAATGC	1175
	2725050-	GCCATTGGATA		2725050-	GATTGATTTCAGT
	2724595_37_58_F			2724595_97_128_R
2146	ARCC_NC003923-	TGAATAGTGAT	437	ARCC_NC003923-	TCTTCTTCTTCG	1137
	2725050-	AGAACTGTAGG		2725050-	TATAAAAAGGACC
	2724595_131_161_F	CACAATCGT		2724595_214_245_R	AATTGG
2147	ARCC_NC003923-	TTGGTCCTTTT	691	ARCC_NC003923-	TGGTGTTCTAGTA	1306
	2725050-	TATACGAAAGA		2725050-	TAGATTGAGGTAG
	2724595_218_249_F	AGAAGTTGAA		2724595_322_353_R	TGGTGA
2148	AROE_NC003923-	TTGCGAATAGA	686	AROE_NC003923-	TCGAATTCAGCTA	1064
	1674726-	ACGATGGCTCG		1674726-	AATACTTTTCAGC
	1674277_371_393_F	T		1674277_435_464_R	ATCT
2149	AROE_NC003923-	TGGGGCTTTAA	590	AROE_NC003923-	TACCTGCATTAAT	891
	1674726-	ATATTCCAATT		1674726-	CGCTTGTTCATCA
	1674277_30_62_F	GAAGATTTTCA		1674277_155_181_R	A
2150	AROE_NC003923-	TGATGGCAAGT	474	AROE_NC003923-	TAAGCAATACCTT	869
	1674726-	GGATAGGGTAT		1674726-	TACTTGCACCACC
	1674277_204_232_F	AATACAG		1674277_308_335_R	TTG
2151	GLPF_NC003923-	TGCACCGGCTA	491	GLPF_NC003923-	TGCAACAATTAAT	1193
	1296927-	TTAAGAATTAC		1296927-	GCTCCGACAATTA
	297391_270_301_F	TTTGCCAACT		1297391_382_414_R	AAGGATT
2152	GLPF_NC003923-	TGGATGGGGAT	558	GLPF_NC003923-	TAAAGACACCGCT	850
	1296927-	TAGCGGTTACA		1296927-	GGGTTTAAATGTG
	1297391_27_51_F	ATG		1297391_81_108_R	CA
2153	GLPF_NC003923-	TAGCTGGCGCG	218	GLPF_NC003923-	TCACCGATAAATA	972
	1296927-	AAATTAGGTGT		1296927-	AAATACCTAAAGT
	1297391_239_260_F			1297391_323_359_R	TAATGCCATTG
2154	GMK_NC003923-	TACTTTTTTAA	200	GMK_NC003923-	TGATATTGAACTG	1180
	1190906-	AACTAGGGATG		1190906-	GTGTACCATAATA
	119334_91_122_F	CGTTTGAAGC		1191334_166_197_R	GTTGCC
2155	GMK_NC003923-	TGAAGTAGAAG	435	GMK_NC003923-	TCGCTCTCTCAAG	1082
	1190906-	GTGCAAAGCAA		1190906-	TGATCTAAACTTG
	1191334_240_267_F	GTTAGA		1191334_305_333_R	GAG
2156	GMK_NC003923-	TCACCTCCAAG	268	GMK_NC003923-	TGGGACGTAATCG	1284
	1190906-	TTTAGATCACT		1190906-	TATAAATTCATCA
	1191334_301_329_F	TGAGAGA		1191334_403_432_R	TTTC
2157	PTA_NC003923-	TCTTGTTTATG	418	PTA_NC003923-	TGGTACACCTGGT	1301
	628885-	CTGGTAAAGCA		628885-	TTCGTTTTGATGA
	629355_237_263_F	GATGG		629355_314_345_R	TTTGTA
2158	PTA_NC003923-	TGAATTAGTTC	439	PTA_NC003923-	TGCATTGTACCGA	1207
	628885-	AATCATTTGTT		628885-	AGTAGTTCACATT
	629355_141_171_F	GAACGACGT		629355_211_239_R	GTT
2159	PTA_NC003923-	TCCAAACCAGG	303	PTA_NC003923-	TGTTCTGGATTGA	1349
	628885-	TGTATCAAGAA		628885-	TTGCACAATCACC
	629355_328_356_F	CATCAGG		629355_393_422_R	AAAG
2160	TPI_NC003923-	TGCAAGTTAAG	486	TPI_NC003923-	TGAGATGTTGATG	1165
	830671-	AAAGCTGTTGC		830671-	ATTACCAGTTCC
	831072_131_160_F	AGGTTTAT		831072_209_239_R	GATTG
2161	TPI_NC003923-	TCCCACGAAAC	318	TPI_NC003923-	TGGTACAACATCG	1300
	830671-	AGATGAAGAAA		830671-	TTAGCTTTACCAC
	831072_1_34_F	TTAACAAAAAA		831072_97_129_R	TTTCACG
		G
2162	TPI_NC003923-	TCAAACTGGGC	246	TPI_NC003923-	TGGCAGCAATAGT	1275
	830671-	AATCGGAACTG		830671-	TTGACGTACAAAT
	831072_199_227_F	GTAAATC		831072_253_286_R	GCACACAT
2163	YQI_NC003923-	TGAATTGCTGC	440	YQI_NC003923-	TCGCCAGCTAGCA	1076
	378916-	TATGAAAGGTG		378916-	CGATGTCATTTTC
	379431_142_167_F	GCTT		379431_259_284_R
2164	YQI_NC003923-	TACAACATATT	175	YQI_NC003923-	TTCGTGCTGGATT	1388
	378916-	ATTAAAGAGAC		378916-	TTGTCCTTGTCCT
	379431_44_77_F	GGGTTTGAATC		379431_120_145_R
		C
2165	YQI_NC003923-	TCCAGCACGAA	314	YQI_NC003923-	TCCAACCCAGAAC	997
	378916-	TTGCTGCTATG		378916-	CACATACTTTATT
	379431_135_160_F	AAAG		379431_193_221_R	CAC
2166	YQI_NC003923-	TAGCTGGCGGT	219	YQI_NC003923-	TCCATCTGTTAAA	1013
	378916-	ATGGAGAATAT		378916-	CCATCATATACCA
	379431_275_300_F	GTCT		379431_364_396_R	TGCTATC
2167	BLAZ_	TCCACTTATCG	312	BLAZ_	TGGCCACTTTTAT	1277
	(1913827 . . .	CAAATGGAAAA		(1913827 . . .	CAGCAACCTTACA
	1914672)_546_	TTAAGCAA		1914672)_655_	GTC
	575_F			683_R
2168	BLAZ_	TGCACTTATCG	494	BLAZ_	TAGTCTTTTGGAA	926
	(1913827 . . .	CAAATGGAAAA		(1913827 . . .	CACCGTCTTTAAT
	1914672)_546_	TTAAGCAA		1914672)_628_	TAAAGT
	575_2_F			659_R
2169	BLAZ_	TGATACTTCAA	467	BLAZ_	TGGAACACCGTCT	1263
	(1913827 . . .	CGCCTGCTGCT		(1913827 . . .	TTAATTAAAGTAT
	1914672)_507_	TTC		1914672)_622_	CTCC
	531_F			651_R
2170	BLAZ_	TATACTTCAAC	232	BLAZ_	TCTTTTCTTGCT	1145
	(1913827 . . .	GCCTGCTGCTT		1913827 . . .	TAATTTTCCATTT
	1914672)_508_	TC		1914672)_553_	GCGAT
	531_F			583_R
2171	BLAZ_	TGCAATTGCTT	487	BLAZ_	TTACTTCCTTACC	1366
	(1913827 . . .	TAGTTTTAAGT		(1913827 . . .	CTTTTAGTATCT
	1914672)_24_	GCATGTAATTC		1914672)_121_	AAAGCATA
	56_F			154_R
2172	BLAZ_	TCCTTGCTTTA	351	BLAZ_	TGGGGACTTCCTT	1289
	(1913827 . . . 1914672)_	GTTTTAAGTGC		(1913827 . . . 1914672)_	ACCACTTTTAGTA
	26_58_F	ATGTAATTCAA		127_157_R	TCTAA
2173	BLAZ_NC002952-	TCCACTTATCG	312	BLAZ_NC002952-	TGGCCACTTTTAT	1277
	1913827-	CAAATGGAAAA		1913827-	CAGCAACCTTACA
	1914672_546_575_F	TTAAGCAA		1914672_655_683_R	GTC
2174	BLAZ_NC002952-	TGCACTTATCG	494	BLAZ_NC002952-	TAGTCTTTTGGAA	926
	1913827-	CAAATGGAAAA		1913827-	CACCGTCTTTAAT
	1914672_546_575_2_F	TTAAGCAA		1914672_628_659_R	TAAAGT
2175	BLAZ_NC002952-	TGATACTTCAA	467	BLAZ_NC002952-	TGGAACACCGTCT	1263
	1913827-	CGCCTGCTGCT		1913827-	TTAATTAAAGTAT
	1914672_507_531_F	TTC		1914672_622_651_R	CTCC
2176	BLAZ_NC002952-	TATACTTCAAC	232	BLAZ_NC002952-	TCTTTTCTTTGCT	1145
	1913827-	GCCTGCTGCTT		1913827-	TAATTTTCCATTT
	1914672_508_531_F	TC		1914672_553_583_R	GCGAT
2177	BLAZ_NC002952-	TGCAATTGCTT	487	BLAZ_NC002952-	TTACTTCCTTACC	136
	1913827-	TAGTTTTAAGT		1913827-	ACTTTTAGTATCT
	1914672_24_56_F	GCATGTAATTC		1914672_121_154_R	AAAGCATA
2178	BLAZ_NC002952-	TCCTTGCTTTA	351	BLAZ_NC002952-	TGGGGACTTCCTT	1289
	1913827-	GTTTTAAGTGC		1913827-	ACCACTTTTAGTA
	1914672_26_58_F	ATGTAATTCAA		1914672_127_157_R	TCTAA
2247	TUFB_NC002758-	TGTTGAACGTG	643	TUFB_NC002758-	TGTCACCAGCTTC	1321
	615038-	GTCAAATCAAA		615038-	AGCGTAGTCTAA
	616222_693_721_F	GTTGGTG		616222_793_820_R	TAA
2248	TUFB_NC002758-	TCGTGTTGAAC	386	TUFB_NC002758-	TGTCACCAGCTTC	1321
	615038-	GTGGTCAAATC		615038-	AGCGTAGTCTAAT
	616222_690_716_F	AAAGT		616222_793_820_R	AA
2249	TUFB_NC002758-	TGAACGTGGTC	430	TUFB_NC002758-	TGTCACCAGCTTC	1321
	615038-	AAATCAAAGTT		615038-	AGCGTAGTCTAAT
	616222_696_725_F	GGTGAAGA		616222_793_820_R	AA
2250	TUFB_NC002758-	TCCCAGGTGAC	320	TUFB_NC002758-	TGGTTTGTCAGAA	1311
	615038-	GATGTACCTGT		615038-	TCACGTTCTGGAG
	616222_488_513_F	AATC		616222_601_630_R	TTGG
2251	TUFB_NC002758-	TGAAGGTGGAC	433	TUFB_NC002758-	TAGGCATAACCAT	922
	615038-	GTCACACTCCA		615038-	TTCAGTACCTTCT
	616222_945_972_F	TTCTTC		616222_1030_1060_R	GGTAA
2252	TUFB_NC002758-	TCCAATGCCAC	307	TUFB_NC002758-	TTCCATTTCAACT	1382
	615038-	AAACTCGTAA		615038-	AATTCTAATAATT
	616222_333_356_F	CA		616222_424_459_R	CTTCATCGTC
2253	NUC_NC002758-	TCCTGAAGCAA	342	NUC_NC002758-	TACGCTAAGCCAC	899
	894288-	GTGCATTTTAC		894288-	GTCCATATTTATC
	894974_402_424_F	GA		894974_483_509_R	A
2254	NUC_NC002758-	TCCTTATAGGG	349	NUC_NC002758-	TGTTTGTGATGCA	1354
	894288-	ATGGCTATCAG		894288-	TTTGCTGAGCTA
	894974_53_81_F	TAATGTT		894974_165_189_R
2255	NUC_NC002758-	TCAGCAAATGC	273	NUC_NC002758-	TAGTTGAAGTTGC	928
	894288-	ATCACAAACAG		894288-	ACTATATACTGTT
	894974_169_194_F	ATAA		894974_222_250_R	GGA
2256	NUC_NC002758-	TACAAAGGTCA	174	NUC_NC002758-	TAAATGCACTTGC	853
	894288-	ACCAATGACAT		894288-	TTCAGGGCCATAT
	894974_316_345_F	TCAGACTA		89474_396_421_R
2270	RPOB_EC_3798_3821_1_F	TGGCCAGCGCT	566	RPOB_EC_3868_3895_R	TCACGTCGTCCGA	979
		TCGGTGAAATG			CTTCACGGGTCAG
		GA			CT
2271	RPOB_EC_3789_3812_F	TCAGTTCGGCG	294	RPOB_EC_3860_3890_R	TCGTCGGACTTAA	1107
		GTCAGCGCTTC			CGGTCAGCATTTC
		GG			CTGCA
2272	RPOB_EC_3789_3812_F	TCAGTTCGGCG	294	RPOB_EC_3860_3890_2_R	TCGTCCGACTTAA	1102
		GTCAGCGCTTC			CGGTCAGCATTTC
		GG			CTGCA
2273	RPOB_EC_3789_3812_F	TCAGTTCGGCG	294	RPOB_EC_3862_3890_R	TCGTCGGACTTAA	1106
		GTCAGCGCTTC			CGGTCAGCATTTC
		GG			CTG
2274	RPOB_EC_3789_3812_F	TCAGTTCGGCG	294	RPOB_EC_3862_3890_R	TCGTCCGACTTAA	1101
		GTCAGCGCTTC			CGGTCAGCATTTC
		GG			CTG
2275	RPOB_EC_3793_3812_F	TTCGGCGGTCA	674	RPOB_EC_3865_3890_R	TCGTCGGACTTAA	1105
		GCGCTTCGG			CGGTCAGCATTTC
2276	RPOB_EC_3793_3812_F	TTCGGCGGTCA	674	RPOB_EC_3865_3890_R	TCGTCCGACTTAA	1100
		GCGCTTCGG			CGGTCAGCATTTC
2309	MUPR_X75439_1658_1689_F	TCCTTTGATAT	352	MUPR_X75439_1744_1773_R	TCCCTTCCTTAT	1030
		ATTATGCGATG			ATGAGAAGGAAAC
		GAAGGTTGGT			CACT
2310	MUPR_X75439_1330_1353_F	TTCCTCCTTTT	669	MUPR_X75439_1413_1441_R	TGAGCTGGTGCTA	1171
		GAAAGCGACGG			TATGAACAATACC
		TT			AGT
2312	MUPR_X75439_1314_1338_F	TTTCCTCCTTT	704	MUPR_X75439_1381_1409_R	TATATGAACAATA	931
		TGAAAGCGAC			CCAGTTCCTTCTG
		GGTT			AGT
2313	MUPR_X75439_2486_2516_F	TAATTGGGCTC	172	MUPR_X75439_2548_2574_R	TTAATCTGGCGT	1360
		TTTCTCGCTTA			GGAAGTGAAATCG
		AACACCTTA			T
2314	MUPR_X75439_2547_2572_F	TACGATTTCAC	188	MUPR_X75439_2605_2630_R	TCGTCCTCTCGAA	1103
		TTCCGCAGCCA			TCTCCGATATACC
		GATT
2315	MUPR_X75439_2666_2696_F	TGCGTACAATA	513	MUPR_X75439_2711_2740_R	TCAGATATAAATG	981
		CGCTTTATGAA			GAACAAATGGAGC
		ATTTTAACA			CACT
2316	MUPR_X75439_2813_2843_F	TAATCAAGCAT	165	MUPR_X75439_2867_2890_R	TCTGCATTTTTGC	1127
		TGGAAGATGAA			GAGCCTGTCTA
		ATGCATACC
2317	MUPR_X75439_884_914_F	TGACATGGACT	447	MUPR_X75439_977_1007_R	TGTACAATAAGGA	1317
		CCCCCTATATA			GTCACCTTATGTC
		ACTCTTGAG			CCTTA
2318	CTXA_NC002505-	TGGTCTTATGC	608	CTXA_NC002505-	TCGTGCCTAACAA	1109
	1568114-	CAAGAGGACAG		1568114-	ATCCCGTCTGAGT
	1567341_114_142_F	AGTGAGT		1567341_194_221_R	TC
2319	CTXA_NC002505-	TCTTATGCCAA	411	CTXA_NC002505-	TCGTGCCTAACAA	1109
	1568114-	GAGGACAGAGT		1568114-	ATCCCGTCTGAGT
	1567341_117_145_F	GAGTACT		1567341_194_221_R	TC
2320	CTXA_NC002505-	TGGTCTTATGC	608	CTXA_NC002505-	TAACAAATCCCGT	855
	1568114-	CAAGAGGACAG		1568114-	CTGAGTTCCTCTT
	1567341_114_142_F	AGTGAGT		1567341_186_214_R	GCA
2321	CTXA_NC002505-	TCTTATGCCAA	411	CTXA_NC002505-	TAACAAATCCGT	855
	1568114-	GAGGACAGAGT		1568114-	CTGAGTTCCTCTT
	1567341_117_145_F	GAGTACT		1567341_186_214_R	GCA
2322	CTXA_NC002505-	AGGACAGAGTG	27	CTXA_NC002505-	TCCCGTCTGAGTT	1027
	1568114-	AGTACTTTGAC		1568114-	CCTCTTGCATGAT
	1567341_129_156_F	CGAGGT		1567341_180_207_R	CA
2323	CTXA_NC002505-	TGCCAAGAGGA	500	CTXA_NC002505-	TAACAAATCCCGT	855
	1568114-	CAGAGTGAGTA		1568114-	CTGAGTTCCTCTT
	1567341_122_149_F	CTTTGA		1567341_186_214_R	GCA
2324	INV_U22457-74-	TGCTTATTTAC	530	INV_U22457-74-	TGACCCAAAGCT	1154
	3772_831_858_F	CTGCACTCCCA		3772_942_966_R	AAAGCTTTACTG
		CAACTG
2325	INV_U22457-74-	TGAATGCTTAT	438	INV_U22457-74-	TAACTGACCCAAA	864
	3772_827_857_F	TTACCTGCACT		3772_942970_R	GCTGAAAGCTTTA
		CCCACAACT			CTG
2326	INV_U22457-74-	TGCTGGTAACA	526	INV_U22457-74-	TGGGTTGCGTTG	1296
	3772_1555_1581_F	GAGCCTTATAG		3772_1619_1647_R	AGATTATCTTTAC
		GCGCA			CAA
2327	INV_U22457-74-	TGGTAACAGAG	598	INV_U22457-74-	TCATAAGGGTTG	987
	3772_1558_1585_F	CCTTATAGGCG		3772_1622_1652_R	GTTGCAGATTATC
		CATATG			TTTAC
2328	ASD_NC006570-	TGAGGGTTTTA	459	ASD_NC006570-	TGATTCGATCATA	1188
	439714-	TGCTTAAAGTT		439714-	CGAGACATTAAA
	438608_3_37_F	GGTTTTATTGG		438608_54_84_R	CTGAGT
		TT
2329	ASD_NC006570-	TAAAGTTGGTT	149	ASD_NC006570-	TCAAAATCTTTTG	948
	439714-	TTATTGGTTGG		439714-	ATTCGATCATACG
	438608_18_45_F	CGCGGA		438608_66_95_R	AGAC
2330	ASD_NC006570-	TTAAAGTTGGT	647	ASD_NC006570-	TCCCAATCTTTTG	1016
	439714-	TTTATTGGTTG		439714-	ATTCGATCATACG
	438608_17_45_F	GCGCGGA		438608_67_95_R	AGA
2331	ASD_NC006570-	TTTTATGCTTA	709	ASD_NC006570-	TCTGCCTGAGATG	1128
	439714-	AAGTTGGTTTT		439714-	TCGAAAAAACGT
	438608_9_40_F	ATTGGTTGGC		438608_107_134_R	TG
2332	GALE_AF513299_171_200_F	TCAGCTAGACC	280	GALE_AF513299_241_271_R	TCTCACCTACAGC	1122
		TTTTAGGTAAA			TTTAAAGCCAGCA
		GCTAAGCT			AAATG
2333	GALE_AF513299_168_199_F	TTATCAGCTAG	658	GALE_AF513299_245_271_R	TCTCACCTACAG	1121
		ACCTTTTAGGT			TTTAAAGCCAGCA
		AAAGCTAAGC			A
2334	GALE_AF513299_168_199_F	TTATCAGCTAG	658	GALE_AF513299_233_264_R	TACAGCTTTAAAG	883
		ACCTTTTAGGT			CCAGCAAAATGAA
		AAAGCTAAGC			TTACAG
2335	GALE_AF513299_169_198_F	TCCCAGCTAGA	319	GALE_AF513299_252_279_R	TTCAACACTCTCA	1374
		CCTTTTAGGTA			CCTACAGCTTTAA
		AAGCTAAG			AG
2236	PLA_AF053945_7371_7403_F	TTGAGAAGACA	680	PLA_AF053945_7434_7468_R	TACGTATGTAAAT	900
		TCCGGCTCACG			TCCGCAAAGACTT
		TTATTATGGTA			TGGCATTAG
2337	PLA_AF053945_7377_7403_F	TGACATCCGGC	443	PLA_AF053945_7428_7455_R	TCCGCAAAGACTT	1035
		TCACGTTATTA			TGGCATTAGGTGT
		TGGTA			GA
2338	PLA_AF053945_7377_7404_F	TGACATCCGGC	444	PLA_AF053945_7430_7460_R	TAAATTCCGCAAA	854
		TCACGTTATTA			GACTTTGGCATTA
		TGGTAC			GGTGT
2339	CAF_AF053947_33412_33441_F	TCCGTTATCGC	329	CAF_AF053947_33498_33523_R	TAAGAGTGATGC	866
		CATTGCATTAT			GGCTGGTTCAACA
		TTGGAACT
2340	CAF_AF053947_33426_33458_F	TGCATTATTTG	499	CAF_AF053947_33483_33507_R	TGGTTCAACAAG	1308
		GAACTATTGCA			GTTGCCGTTGCA
		ACTGCTAATGC
2341	CAF_AF053947_33407_33429_F	TCAGTTCCGTT	291	CAF_AF053947_33483_33504_R	TTCAACAAGAGTT	1373
		ATCGCCATTGC			GCCGTTGCA
		A
2342	CAF_AF053947_33407_33431_F	TCAGTTCCGTT	293	CAF_AF053947_33494_33517_R	TGATGCGGGCTGG	1184
		ATCGCCATTG			TTCAACAAGAG
		CATT
2344	GAPA_NC_002505_1_28_F_1	TCAATGAACGA	260	GAPA_NC_002505_29_58_R_1	TCCTTTATGCAAC	1060
		TCAACAAGTGA			TTGGTATCAACAG
		TTGATG			GAAT
2472	OMPA_NC000117_68_89_F	TGCCTGTAGGG	507	OMPA_NC000117_145_167_R	TCACACCAAGTAG	967
		AATCCTGCTGA			TGCAAGGATC
		A
2473	OMPA_NC000117_798_821_F	TGATTACCATG	475	OMPA_NC000117_865_893_R	TCAAAACTTGCTC	947
		AGTGGCAAGCA			TAGACCATTTAAC
		AG			TCC
2474	OMPA_NC000117_645_671_F	TGCTCAATCTA	521	OMPA_NC000117_757_777_R	TGTCGCAGCATCT	1328
		AACCTAAAGTC			GTTCCTGC
		GAAGA
2475	OMPA_NC000117_947_973_F	TAACTGCATGG	157	OMPA_NC000117_1011_1040_R	TGACAGGACACAA	1153
		AACCCTTCTTT			TCTGCATGAAGTC
		ACTAG			TGAG
2476	OMPA_NC000117_774_795_F	TACTGGAACAA	196	OMPA_NC000117_871_894_R	TTCAAAAGTTGCT	1371
		AGTCTGCGACC			CGAGACCATTG
2477	OMPA_NC000117_457_483_F	TTCTATCTCGT	676	OMPA_NC000117_511_534_R	TAAAGAGACGTTT	851
		TGGTTTATTCG			GGTAGTTCATTTG
		GAGTT			C
2478	OMPA_NC000117_687_710_F	TAGCCCAGCAC	212	OMPA_NC000117_787_816_R	TTGCCATTCATGG	1406
		AATTTGTGATT			TATTTAAGTGTAG
		CA			AGA
2479	OMPA_NC000117_540_566_F	TGGCGTAGTAG	571	OMPA_NC000117_649_672_R	TTCTTGAACGCGA	1395
		AGCTATTTACA			GGTTTCGATTG
		GACAC
2480	OMPA_NC000117_338_360_F	TGCACGATGCG	492	OMPA_NC000117_417_444_R	TCCTTTAAAATAA	1058
		GAATGGTTCAC			CCGCTAGTAGCTC
		A			CT
2481	OMP2_NC000117_18_40_F	TATGACCAAAC	234	OMP2_NC000117_71_91_R	TCCCGCTGGCAAA	1025
		TCATCAGACGA			TAAACTCG
		G
2482	OMP2_NC000117_354_382_F	TGCTACGGTAG	516	OMP2_NC000117_445_471_R	TGGATCACTGCTT	1270
		GATCTCCTTAT			ACGAACTCAGCTT
		CCTATTG			C
2483	OMP2_NC000117_1297_1319_F	TGGAAAGGTGT	537	OMP2_NC000117_1396_1419_R	TACGTTTGTATCT	903
		TGCAGCTACTC			TCTGCAGAACC
		A
2484	OMP2_NC000117_1465_1493_F	TCTGGTCCAAC	407	OMP2_NC000117_1541_1569_R	TCCTTTCAATGTT	1062
		AAAAGGAACGA			ACAGAAAACTCTA
		TTACAGG			CAG
2485	OMP2_NC000117_44_66_F	TGACGATCTTC	450	OMP2_NC000117_120_148_R	TGTCAGCTAAGC	1323
		GCGGTGACTAG			TAATAACGTTTGT
		T			AGAG
2486	OMP2_NC000117_166_190_F	TGACAGCGAAG	441	OMP2_NC000117_240_261_R	TTGACATCGTCCC	1396
		AAGGTTAGACT			TCTTCACAG
		TGTCC
2487	GYRA_NC000117_514_536_F	TCAGGCATTGC	287	GYRA_NC000117_640_660_R	TGCTGTAGGGAAA	1251
		GGTTGGGATGG			TCAGGGCC
2488	GYRA_NC000117_801_827_F	TGTGAATAAAT	636	GYRA_NC000117_871_893_R	TTGTCAGACTCAT	1419
		CACGATTGATT			CGCGAACATC
		GAGCA
2489	GYRA_NC002952_219_242_F	TGTCATGGGTA	632	GYRA_NC002952_319_345_R	TCCATCCATAGAA	1010
		AATATCACCCT			CCAAAGTTACCTT
		CA			G
2490	GYRA_NC002952_964_983_F	TACAAGCACTC	176	GYRA_NC002952_1024_1041_R	TCGCAGCGTGCGT	1073
		CCAGCTGCA			GGCAC
2491	GYRA_NC002952_1505_1520_F	TCGCCCGCGAG	366	GYRA_NC002952_1546_1562_R	TTGGTGCGCTTGG	1416
		GACGT			CGTA
2492	GYRA_NC002952_59_81_F	TCAGCTACATC	279	GYRA_NC002952_124_143_R	TGGCGATGCACTG	1279
		GACTATGCGAT			GCTTGAG
		G
2493	GYRA_NC002952_216_239_F	TGACGTCATCG	452	GYRA_NC002952_313_333_R	TCCGAAGTTGCCC	1032
		GTAAGTACCAC			TGGCCGTC
		CC
2494	GYRA_NC002952_219_242_2_F	TGTACTCGGTA	625	GYRA_NC002952_308_330_R	TAAGTTACCTTGC	873
		AGTATCACCCG			CCGTCAACCA
		CA
2495	GYRA_NC002952_115_141_F	TGAGATGGATT	453	GYRA_NC002952_220_242_R	TGCGGGTGATACT	1236
		TAAACCTGTTC			TACCGAGTAC
		ACCGC
2496	GYRA_NC002952_517_5_39_F	TCAGGCATTGC	287	GYRA_NC002952_643_663_R	TGCTGTAGGGAAA	1251
		GGTTGGGATGG			TCAGGGCC
		C
2497	GYRA_NC002952_273_2_93_F	TCGTATGGCTC	380	GYRA_NC002952_338_360_R	TGCGGCAGCACTA	1234
		AATGGTGGAG			TCACCATCCA
2498	GYRA_NC000912_257_278_F	TGAGTAAGTT	462	GYRA_NC000912_346_370_R	TCGAGCCGAAGTT	1067
		CCACCCGCACG			CCCTGTCCGTC
		G
2504	ARCC_NC003923-	TAGTpGATpAG	229	ARCC_NC003923-2725050-	TCpTpTpTpCpGT	1116
	2725050-	AACpTpGTAGG		2724595_214_239P_R	ATAAAAAGGACpC
	2724595_135_161P_F	CpACpAATpCp			pAATpTpGG
		GT
2505	PTA_NC003923-	TCTTGTpTpTp	417	PTA_NC003923-628885-	TACpACpCpTGGT	904
	629355_629355_237_263P_F	ATGCpTpGGTA			pTpTpCpGTpTpT
		AAGCAGATGG			pTpGATGATpTpT
					pGTA
2517	CJMLST_ST1_1852_1883_F	TTTGCGGATGA	708	CJMLST_ST1_1945_1977_R	TGTTTTATGTG	1355
		AGTAGGTGCCT			TAGTTGAGCTTAC
		ATCTTTTTGC			TTACTACATGAGC
2518	CJMLST_ST1_2963_2992_F	TGAAATTGCTA	428	CJMLST_ST1_3073_3097_R	TCCCCATCTCCGCA	1020
		CAGGCCCTTTA			AAGACAATAAA
		GGACAAGG
2519	CJMLST_ST1_2350_2378_F	TGCTTTTGATG	535	CJMLST_ST1_2447_2481_R	TCTACAACACT	1117
		GTGATGCAGA			TGATTGTAATTTG
		TCGTTTGG			CCTTGTTCTTT
2520	CJMLST_ST1_654_684_F	TATGTCCAAGA	240	CJMSLT_ST1_725_756_R	TCGGAAACAAAGA	1084
		AGCATAGCAAA			ATTCATTTTCTGG
		AAAAGCAAT			TCCAAA
2521	CJMSLT_ST1_360_395_F	TCCTGTTATTC	347	CJMLST_ST1_454_457_R	TGCTATATGCTAC	1245
		CTGAAGTAGTT			AACTGGTTCAAAA
		AATCAAGTTTG			ACATTAAG
		TTA
2522	CJMSLT_ST1_1231_1258_F	TGGCAGTTTTA	564	CJMSLT_ST1_1312_1340_R	TTTAGCTACTATT	1427
		CAAGGTGCTGT			CTAGCTGCCATTT
		TTCATC			CCA
2523	CJMSLT_ST1_3543_3574_F	TGCTGTAGCTT	529	CJMLST_ST1_3656_3685_R	TCAAAGAACCAGC	1427
		ATCGCGAAATG			ACCTAATTCATCA
		TCTTTGATTT			TTTA
2524	CJMLST_ST1_1_17_F	TAAAACTTTTG	145	CJMSLT_ST1_55_84_R	TGTTCCAATAGCA	1348
		CCGTAATGATG			GTTCCGCCCAAAT
		GGTGAAGATAT			TGAT
2525	CJMSLT_ST1_1312_1342_F	TGGAAATGGCA	538	CJMSLT_ST1_1383_1417_R	TTTCCCCGATC	1432
		GCTAGAATAGT			TAAATTTGGATAA
		AGCTAAAAT			GCCATAGGAAA
2526	CJMSLT_ST1_2254_2286_F	TGGGCCTAATG	582	CJMSLT_ST1_2352_2379_R	TCCAAACGATC	996
		GGCTTAATATC			TGCATCACCATCA
		AATGAAAATTG			AAAG
2527	CJMSLT_ST1_1380_1411_F	TGCTTTCCTAT	534	CJMSLT_ST1_1486_1520_R	TGCATGAAGCATA	1205
		GGCTTATCCAA			AAAACTGTATCAA
		ATTTAGATCG			GTGCTTTTA
2528	CJMLST_ST1_3413_3437_F	TTGTAAATGCC	692	CJMLST_ST1_3511_3542_R	TGCTTGCTCAAAT	1257
		GGTGCTTCAGA			CATCATAAACAAT
		TCC			TAAAGC
2529	CJMSLT_ST1_1130_1156_F	TACGCGTCTTG	189	CJMSLT_ST1_1203_R	TAGGATGAGCATT	920
		AAGCGTTTCGTTA			ATCAGGGAAAGAA
		TGA			AGAATC
2530	CJMSLT_ST1_2840_2872_F	TGGGGCTTTGC	591	CJMSLT_ST1_2940_2973_R	TAGCGATTTCT	917
		TTTATAGTTTT			ACTCCTAGAGTTG
		TTACATTTAAG			AAATTTCAGG
2531	CJMSLT_ST1_2058_2084_F	TATTCAAGGTG	241	CJMSLT_ST1_2131_2162_R	TTGGTTCTTACTT	1417
		GTCCTTTGATG			GTTTTGCATAAAC
		CATGT			TTTCCA
2532	CJMSLT_ST1_553_585_F	TCCTGATGCTC	344	CJMLST_ST1_655_685_R	TATTGCTTTTTTT	942
		AAAGTGCTTTT			GCTATGCTTCTTG
		TTAGATCCTTT			GACAT
2564	GTLA_NC002163-	TCATGTTGAGC	299	GTLA_NC002163-1604930-	TTTTGCTCATGAT	1443
	1604930-	TTAAACCTATA		1604529_352_380_R	CTGCATGAAGCAT
	1604529_306_338_F	GAAGTAAAAGC			AAA
2565	UNCA_NC002163-	TCCCCCACGCT	322	UNCA_NC002163-112166-	TCGACCTGGAGGA	1065
	112166-	TTAATTGTTTT		112647_146_171_R	CGACGTAAAATCA
	112647_80_113_F	ATGATGATTTG			CGACGTAAAATCA
		AG
2566	UNCA_NC002163-	TAATGATGAAT	170	UNCA_NC002316-112166-	TGGGATAACAT	1285
	112166-	TAGGTGCGGGT		112647_294_329_R	TGGTTGGAATATA
	112647_233_305_F	TCTTT			AGCAGAAACATC
2567	PGM_NC002163-	TCTTGATACTT	414	PGM_NC002163-327773-	TCCATCGCCAGTT	1012
	327773-	GTAATGTGGGC		328270_365_396_R	TTTGCATAATCGC
	328270_273_305_F	GATAAATATGT		TAAAAA
2568	TKT_NC002163-	TTATGAAGCGT	661	TKT_NC002163-1569415-	TCAAAACGCATTT	946
	1569415-	GTTCTTTAGCA		1569873_350_383_R	TTACATCTTCGTT
	1569873_255_284_F	GGACTTCA		AAAGGCTA
2570	GTLA_NC002163-	TCGTCTTTTTG	381	GLTA_NC002163-1604930-	TGTTCATGTTTAA	1347
	1604930-	ATTCTTTCCCT		1604529_109_142_R	ATGATCAGGATAA
	1604529_39_68_F	GATAATGC			AAAGCACT
2571	TKT_NC002163-	TGATCTTAAAA	472	TKT_NC002163-1569415-	TGCCATAGCAAAG	1214
	1569415-	ATTTCCGCCAA		1569903_139_162_R	CCTACAGCATT
	1569903_33_62_F	CTTCATTC			CCTACAGCATT
2572	TKT_NC002163-	TAAGGTTTATT	164	TKT_NC002163-1569415-	TACATCTCCTTCG	886
	1569415-	GTCTTTGTGGA		1569903_313_345_R	ATAGAAATTTCAT
	1569903_207_239_F	GATGGGGATTT			TGCTATC
2573	TKT_NC002163-	TAGCCTTTAAC	213	TKT_NC002163-1569415-	TAAGACAAGGTTT	865
	1569415-	GAAAATGTAAA		1569903_449_481_R	TGTGGATTTTTTA
	1569903_350_383_F	AATGCGTTTTG			GCTTGTT
		A
2574	TKT_NC002163-	TTCAAAAACTC	665	TKT_NC002163-1569415-	TTGCCATAGCAAA	1405
	1569415-	CAGGCCATCCT		1569903_139_163_R	GCCTACAGCATT
	1569903_60_92_F	GAAATTTCAAC
2575	GTLA_NC002163-	TCGTCTTTTTG	382	GLTA_NC002163-1604930-	TGCCATTTCCATG	1216
	1604930-	ATTCTTTCCCT		1604529_139_168_R	TACTCTTCTCTAA
	1604529_39_70_F	GATAATGCTC			CATT
2576	GLYA_NC002163-	TCAGCTATTTT	281	GLYA_NC002163-367572-	ATTGCTTCTTACT	756
	367572-	TCCAGGTATCC		368079_476_508_R	TGCTTAGCATAAA
	368079_386_414_F	AAGGTGG			TTTTCCA
2577	GLYA_NC002163-	TGGTGCGAGTG	611	GLYA_NC002163-367572-	TGCTCACCTGCTA	1246
	367572-	CTTATGCTCGT		368079_242_270_R	CAACAAGTCCAGC
	368079_148_174_F	ATTAT			AAT
2578	GLYA_NC002163-	TGTAAGCTCTA	622	GLYA_NC002163-367572-	TTCCACCTTGGAT	1381
	367572-	CAACCCACAAA		368079_384_416_R	ACCTGGAAAAATA
	368079_298_327_F	ACCTTACG			GCTGAAT
2579	GLYA_NC002163-	TGGTGGACATT	614	GLYA_NC002163-367572-	TCAAGCTCTACAC	961
	367572-	TAACACATGGT		368079_52_81_R	CATAAAAAAAGCT
	368079_1_27_F	GCAAA			CTCA
2580	PGM_NC002163-	TGAGCAATGGG	455	PGM_NC002163-327746-	TTTGCTCTCCGCC	1438
	327746-	GCTTTGAAAGA		328270_356_379_R	AAAGTTTCCAC
	328270_254_285_F	AAGAATTTTTA
		AAT
2581	PGM_NC002163-	TGAAAAGGGTG	425	PGM_NC002163-327746-	TGCCCCATTGCTC	1219
	327746-	AAGTAGCAAAT		328270_241_267_R	TCATGATAGTAGT
	328270_153_182_F	GGAGATAG			AGCTAC
2582	PGM_NC002163-	TGGCCTAATGG	568	PGM_NC002163-327746-	TGCACGCAAACGC	1200
	327746-	GCTTAATATCA		328270_79_102_R	TTTACTTCAGC
	328270_19_50_F	ATGAAAATTG
2583	UNCA_NC002163-	TAAGCATGCTG	160	UNCA_NC002163-112166-	TGCCCTTTCTAAA	1220
	112166-	TGGCTTATCGT		112647_196_225_R	AGTCTTGAGTGAA
	112647_114_141_F	GAAATG			GATA
2584	UNCA_NC002163-	TGCTTCGGATC	532	UBCA_NC002163-112166-	TGCATGCTTACTC	1206
	112166-	CAGCAGCACTT	532	112647_88_123_R	AAATCATCATAAA
	112647_3_29_F	CAATA			CAATTAAAGC
2585	ASPA_NC002163-	TTAATTTGCCA	652	ASPA_NC002163-96692-	TGCAAAAGTAACG	1192
	96692-	AAAATGCAACC		97166_403_432_R	GTTACATCTGCTC
	97166_308_335_F	AGGTAG			CAAT
2586	ASPA_NC002163-	TCGCGTTGCAA	370	ASPA_NC002163-966692-	TCATGATAGAACT	991
	96692-	CAAAACTTTCT		97166_316_346_R	ACCTGGTTGCATT
	97166_228_258_F	AAAGTATGT			TTTGG
2587	GLNA_NC002163-	TGGAATGATGA	547	GLNA_NC002163-658085-	TGAGTTTGAACCA	1176
	658085-	TAAAGATTTCG		657609_340_371_R	TTTCAGAGAGCGA
	657609_244_275_F	CAGATAGCTA			ATATCTAC
2588	TKT_NC002163-	TCGCTACAGGC	371	TKT_NC002163-1569415-	TCCCCATCTCCGC	1020
	1569415-	CCTTTAGGACA		1569903_212_236_R	AAAGACAATAAA
	1569903_107_130_F	CAGATAGCTA			ATATCTAC
2589	TKT_NC002163-	TGTTCTTTAGC	642	TKT_NC002163-1569415-	TCCTTGTGCTTCA	1057
	1569415-	AGGACTTCACA		1569903_361_393_R	AAACGCATTTTTA
	1569903_265_296_F	AACTTGATAA			CATTTTC
2590	GLYA_NC002163-	TGCCTATCTTT	505	GLYA_NC002163-367572-	TCCTCTTGGGCCA	1047
	367572-	TTGCTGATATA		368095_317_340_R	CGCAAAGTTTT
	368095_214_246_F	GCACATATTGC			CGCAAAGTTTT
2591	GLYA_NC002163-	TCCTTTGATGC	353	GLYA_NC002163-367572-	TCTTGAGCATTGG	1141
	367572-	ATGTAATTGCT		368095_485_516_R	TTCTTACTTGTTT
	368095_415_444_F	GCAAAAGC			TGCATA
2592	PGM_NC002163_21_54_F	TCCTAATGGAC	332	PGM_NC002163_116_142_R	TCAAACGATCCGC	949
		TTAATATCAAT			ATCACCATCAAAA
		GAAAATTGTGG			G
		G
2593	PGM_NC002163_149_176_F	TAGATGAAAAA	207	PGM_NC002163_247_277_R	TCCCCTTTAAAGC	1023
		GGCGAAGTGGC			ACCATTACTCATT
		TAATGG			ATAGT
2594	GLNA_NC002163-	TGTCCAAGAAG	633	GLNA_NC002163-658085-	TCAAAAACAAAGA
	658085-	CATAGCAAAAA		657609_148_179_R	ATTCATTTTCTGG
	657609_79_106_F	AAGCAA			TCCAAA
2595	ASPA_NC2163-	TCCTGTTATTC	347	ASPA_NC002163-96685-	TCAAGCTATATGC	960
	96685-	CTGAAGTAGTT		97196_467_497_R	TACAACTGGTTCA
	97196_367_402_F	AATCAAGTTTG			AAAAC
		TTA
2596	ASPA_NC002163-	TGCCGTAATGA	502	ASPA_NC002163-96685-	TACAACCTTCGGA	880
	96685-	TAGGTGAAGAT		97196_95_127_R	TAATCAGGATGAG
	97196_1_33_F	ATACAAAGAGT		AATTAAT
2597	ASPA_NC002163-	TGGAACAGGAA	540	ASPA_NC002163-96685-	TAAGCTCCCGTAT	872
	96685-	TTAATTCTCAT		97196_185_210_R	CTTGAGTCGCCTC
	97196_85_117_F	CCTGATTATCC
2598	PGM_NC002163-	TGGCAGCTAGA	563	PGM_NC002163-327746-	TCACGATCTAAAT	975
	327746-	ATAGTAGCTAA		328270_230_261_R	TTGGATAAGCCAT
	328270_165_195_F	AATCCCTAC			AGGAAA
2599	PGM_NC002163-	TGGGTCGTGGT	593	PGM_NC002163-327746-	TTTTGCTCATGAT	1443
	327746-	TTTACAGAAAA		328270_353_381_R	CTGCATGAAGCAT
	328270_252_286_F	TTTCTTATATA			AAA
2600	PGM_NC002163-	TGGGATGAAAA	577	PGM_NC002163-327746-	TGATAAAAAGCAC	1178
	327746-	AGCGTTCTTTT		328270_95_123_R	TAAGCGATGAAAC
	328270_1_30_F	ATCCATGA			AGC
2601	PGM_NC002163-	TAAACACGGCT	146	PGM_NC002163-327746-	TCAAGTGCTTTTA	963
	327746-	TTCCTATGGCT		328270_314_345_R	CTTCTATAGGTTT
	328270_220_250_F	TATCCAAAT			AAGCTC
2602	UNCA_NC002163-	TGTAGCTTATC	628	UNCA_NC002163-112166-	TGCTTGCTCTTTC	1258
	112166-	GCGAAATGTCT		112647_199_229_R	AAGCAGTCTTGAA
	112647_123_152_F	TTGATTTT			TGAAG
2603	UBCA_NC002163-	TCCAGATGGAC	313	UNCA_NC002163-112166-	TCCGAAACTTGTT	1031
	112166-	AAATTTTCTTA		112647_430_461_R	TGTAGCTTTAATT
	112647_333_365_F	GAAACTGATTT			TGAGC
2734	GYRA_AY291534_237_264_F	TCACCCTCATG	265	GYRA_AY291534_268_288_R	TTGCGCCATACGT	1407
		GTGATTCAGCT			ACCATCGT
		GTTTAT
2735	GYRA_AY291534_224_252_F	TAATCGGTAAG	167	GYRA_AY291534_256_285_R	TGCCATACGTACC	1213
		TATCACCCTCA			ATCGTTTCATAAA
		TGGTGAT			CAGC
2736	GYRA_AY291534_170_198_F	TAGGAATTACG	221	GYRA_AY291534_268_288_R	TTGCGCCATACGT	1407
		GCTGATAAAGC			ACCATCGT
		GTATAAA
2737	GYRA_AY291534_224_252_F	TAATCGGTAAG	167	GYRA_AY291534_319_346_R	TATCGACAGATCC	935
		ATCACCCTCAT			AAAGTTACCATGC
		GGTGAT			CC
2738	GYRA_NC002953-7005-	TAAGGTATGAC	163	GYRA_NC002953-7005-	TCTTGAGCCATAC	1142
	9668_166_195_F	ACCGGATAAAT		9668_265_287_R	GTACCATTGC
		CATATAAA			GTACCATTGC
2739	GYRA_NC002953-7005-	TAATGGGTAAA	171	GYRA_NC002953-7005-	TATCCATTGAACC	933
	9668_221_249_F	TATCACCCTCA		9668_316_343_R	AAAGTTACCTTGG
		TGGTGAC			CC
2740	GYRA_NC002953-7005-	TAATGGGTAAA	171	GYRA_NC002953-7005-	TAGCCATACGTAC	912
	9668_221_249_F	TATCACCCTCA		9668_253_283_R	CATTGCTTCATAA
		TGGTGAC			AATAGA
2741	GYRA_NC002953-7005-	TCACCCTCATG	264	GYRA_NC002953-7005-	TCTTGAGCCATAC	1142
	9668_234_261_F	GTGACTCATCT		9668_265_287_R	GTACCATTGC
		ATTTAT
2842	CAPC_AF188935-	TGGGATTATTG	578	CAPC_AF188935-56074-	TGGTAACCCTTGT	1299
	56074-	TTATCCTGTTA		55628_348_378_R	CTTTGAATTGTAT
	55628_271_304_F	TGCCATTTGAG			TTGCA
		A
2843	CAPC_AF188935-	TGATTATTGTT	476	CAPC_AF188935-56074-	TGTAACCCTTGTC	1344
	56074-	ATCCTGTTATG		55628_349_377P_R	TTTGAATpTpGTA
	55628_273_303P_F	CpCpATpTpTp			TpTpTpGC
		GAG
2844	CAPC_AF188935-	TCCGTTGATTA	331	CAPC_AF188935-56074-	TGTTAATGGTAAC	1344
	56074-	TTGTTATCCTG		55628_349_384_R	CCTTGTCTTTGAA
	55628_268_303_F	TTATGCCATTT			TTGTATTTGC
		GAG
2845	CAPC_AF188935-	TCCGTTGATTA	331	CAPC_AF188935-56074-	TAACCCTTGTCTT	860
	56074-	TTGTTATCCTG		55628_337_375_R	TGAATTGTATTTG
	55628_268_303_F	TTATGCCATTT			CAATTAATCCTGG
		GAG
2846	PARC_X95819_33_58_F	TCAAAAAAAT	302	PARC_X95819_121_153_R	TAAAGGATAGCGG	852
		CAGCGCGTACA			TAACTAAATGGCT
		GTGG			GAGCCAT
2847	PARC_X95819_65_92_F	TACTTGGTAAA	199	PARC_X95819_157_178_R	TACCCCAGTTCCC	889
		TACCACCCACA			CTGACCTTC
		TGGTGA
2848	PARC_X95819_69_93_F	TGGTAAATACC	596	PARC_X95819_97_128_R	TGAGCCATGAGTA	1169
		ACCCACACATG			CCATGGCTTCATA
		GTGAC			ACATGC
2849	PARC_NC003997-	TTCCGTAAGTC	668	PARC_NC003997-3362578-	TCCAAGTTTGACT	1001
	3362578-	GGCTAAAACAG		3365001_256_283_R	TAAACGTACCATC
	3365001_181_205_F	TCG			GC
2850	PARC_NC003997-	TGTAACTATCA	621	PARC_NC003997-3362578-	TCGTCAACACTAC	1099
	3362578-	CCCGCACGGTG		3365001_304_335_R	CATTATTACCATG
	3365001_217_240_F	AT			CATCTC
2851	PARC_NC003997-	TGTAACTATCA	621	PARC_NC003997-3362578-	TGACTTAAACGTA	1162
	3362578-	CCCGCACGGTG		3365001_244_275_R	CCATCGCTTCATA
	3365001_217_240_F	AT		TCATCTC
2852	GYRA_AY642140_1_24_F	TAAATCTGCCC	150	GYRA_AY642140_71_100_R	TGCTAAAGTCTTG	1242
		CGTGTCGTTGG			AGCCATACGAACA
		TGAC			ATGG
2853	GYRA_AY642140_26_54_F	TAATCGGTAAA	166	GYRA_AY642140_121_146_R	TCGATCGAACCGA	1069
		TATCACCCGCA			AGTTACCCTGACC
		TGGTGAC
2854	GYRA_AY642140_26_54_F	TAATCGGTAAA	166	GYRA_AY642140_58_89_R	TGAGCCATACGAA	1168
		TATCACCCGCA			CAATGGTTTCATA
		TGGTGAC			AACAGC
2860	CYA_AF065404_1348_1379_F	TCCAACGAAGT	305	CYA_AF065404_1448_1472_R	TCAGCTGTTAACG	983
		ACAATACAAGA			GCTTCAAGACCC
		CAAAAGAAGG
2861	FEF_BA_AF065404_751_781_F	TCGAAAGCTTT	354	LEF_BA_AF065404_843_881_R	TCTTTAAGTTCTT	1144
		TGCATATTATA			CCAAGGATAGATT
		TCGAGCCAC			TATTTCTTGTTCG
2862	LEF_BA_AF065404_762_788_F	TGCATATTATA	498	LEF_BA_AF065404_843_881_R	TCTTTAAGTTCTT	1144
		TCGAGCCACAG			CCAAGGATAGATT
		CATCG			TATTTCTTGTTCG
2917	MUTS_AY698802_106_125_F	TCCGCTGAATC	326	MUTS_AY698802_172_193_R	TGCGGTCTGGCGC	1237
		TGTCGCCGC			ATATAGGTA
2918	MUTS_AY698802_172_192_F	TACCTATATGC	187	MUTS_AY698802_228_252_R	TCAATCTCGACTT	965
		GCCAGACCGC			TTTGTGCCGGTA
2919	MUTS_AY698802_228_252_F	TACCGGCGCAA	186	MUTS_AY698802_314_342_R	TCGGTTTCAGTCA	1097
		AAAGTCGAGAT			TCTCCACCATAAA
		TGG			GGT
2920	MUTS_AY698802_315_342_F	TCTTTATGGTG	419	MUTS_AY698802_413_433_R	TGCCAGCGACAGA	1210
		GAGATGACTGAAA			CCATCGTA
		CCGA
2921	MUTS_AY698802_394_411_F	TGGGCGTGGAA	585	MUTS_AY698802_497_519_R	TCCGGTAACTGGG	1040
		CGTCCAC			TCAGCTCGAA
2922	AB_MLST-11-	TGGGcGATGCT	583	AB_MLST-11-	TAGTATCACCACG	923
	OIF007_991_1018_F	GCgAAATGGTT		OIF007_1110_1137_R	TACACCCGGATCA
		AAAAGA			GT
2927	GAPA_NC002505_694_721_F	TCAATGAACGA	259	GAPA_NC_002505_29_58_R_1	TCCTTTATGCAAC	1060
		CCAACAAGTGA			TTGGTATCAACAG
		TTGATG			GAAT
2928	GAPA_NC002505_694_721_2_F	TCGATGAACGA	361	GAPA_NC002505_769_798_2_R	TCCTTTATGCAAC	1061
		CCAACAAGTGA			TTGGTATCAACCG
		TTGATG			GAAT
2929	GAPA_NC002505_694_721_2_F	TCGATGAACGA	361	GAPA_NC002505_769_798_3_R	TCCTTTATGCAAC	1059
		CCAACAAGTGA			TTAGTATCAACCG
		TTGATG			GAAT
2932	INFB_EC_1364_1394_F	TTGCTCGTGGT	688	INFB_EC_1439_1468_R	TTGCTGCTTTCGC	1410
		GCACAAGTAA			ATGGTTAATCGCT
		CGGATATTAC			TCAA
2933	INFB_EC_1364_1394_2_F	TTGCTCGTGGT	689	INFB_EC_1439_1468_R	TTGCTGCTTTCGC	1410
		GCAIAAGTAA			ATGGTTAATCGCT
		CGGATATIAC			TCAA
2934	INFB_EC_80_110_F	TTGCCCGCGGT	685	INFB_EC_1439_1468_R	TTGCTGCTTTCGC	1410
		GCGGAAGTAAC			ATGGTTAATCGCT
		CGATATTAC			TCAA
2949	ACS_NC002516-	TCGGCGCCTGC	376	ACS_NC002516-970624-	TGGACCACGCCGA	1265
	970624-	CTGATGA		971013_364_383_R	AGAACGG
2950	ARO_NC002516_26883-	TCACCGTGCCG	267	ARO_NC002516-26883-	TGTGTTGTCGCCG	1341
	27380_4_26_F	TTCAAGGAAGA		27380_111_128_R	CGCAG
		G
2951	ARO_NC002516-26883-	TTTCGAAGGGC	705	ARO_NC002516-26883-	TCCTTGGCATACA	1056
	27380_356_377_F	CTTTCGACCTG		27380_459_484_R	TCATGTCGTAGCA
2952	GUA_NC002516-	TGGACTCCTCG	551	GUA_NC002516-4226546-	TCGGCGAACATGG	1091
	4226546-	GTGGTCGC		4226174_127_146_R	CCATCAC
2953	GUA_NC002516-	TGACCAGGTGA	448	GUA_NC002516-4226546-	TGCTTCTCTTCCG	1256
	4226546-	TGGCCATGTTC		4226174_214_233_R	GGTCGGC
	4226174_120_142_F	G
2954	GUA_NC002516-	TTTTGAAGGTG	710	GUA_NC002516-4226546-	TGCTTGGTGGCTT	1259
	4226546-	ATCCGTGCCAA		4226174_265_287_R	CTTCGTCGAA
	4226174_155_178_F	CG
2955	GUA_NC002516-	TTCCTCGGCCG	670	GUA_NC002516-4226546-	TGCGAGGAACTTC	1229
	4226546-	CCTGGC		4226174_288_309_R	ACGTCCTGC
	4226174_190_206_F
2956	GUA_NC002516-	TCGGCCGCACC	374	GUA_NC002516-4226546-	TCGTGGGCCTTGC	1111
	4226546-	TTCATCGAAGT		4226174_355_371_R	CGGT
2957	MUT_NC002516-	TGGAAGTCATC	545	MUT_NC002516-5551158-	TCACGGGCCAGCT	978
	5551158-	AAGCGCCTGGC		5550717_99_116_R	CGTCT
	5550717_5_26_F	AAGCGCCTGGC
2958	MUT_NC002516-	TCGAGCAGGC	358	MUT_NC002516-5551158-	TCACCATGCGCCC	971
	5551158-	GCTGCCG		5550717_256_277_R	GTTCACATA
	5550717_5_26_F
2959	NUO_NC002516-	TCAACCTCGGC	249	NUO_NC002516-2984589-	TCGGTGGTGGTAG	1095
	2984589-	CCGAACCA		2984954_97_117_R	CCGATCTC
2960	NUO_NC002516-	TACTCTCGGTG	195	NUO_NC002516-2984589-	TTCAGGTACAGCA	1376
	2984589-	GAGAAGCTCGC		2984954_301_326_R	GGTGGTTCAGGAT
2961	PPS_NC002516-	TCCACGGTCAT	311	PPS_NC002516-1915014-	TCCATTTCCGACA	1014
	1915014-	GGAGCGCTA		1915383_140_165_R	CGTCGTTGATCAC
	1915383_44_63_P
3962	PPS_NC002516-	TCGCCATCGTC	365	PPS_NC002516-1915014-	TCCTGGCCATCCT	1052
	1915014-	ACCAACCG		1915383_341_360_R	GCAGGAT
	1915383_240_258_F
2963	TRP_NC002516-	TGCTGGTACGG	527	TRP_NC002516-671831-	TCGATCTCCTTG	1071
	671831-	GTCGAGGA		672273_131_150_R	GCGTCCGA
	672273_24_42_F
2964	TRP_NC002516-	TGCACATCGTG	490	TRP_NC002516-671831-	TGATCTCCATGGC	1182
	671831-	TCCAACGTCAC		672273_362_383_R	GCGGATCTT
	672273_261_282_F
2972	AB_MLST-11-	TGGGIGATGCT	592	AB_MLST-11-	TAGTATCACCACG	924
	OIF007_1007_1034_F	GCIAAATGGTT		OIF007_1126_1153_R	TACICCIGGATCA
		AAAAGA			GT
2993	OMPU_NC002505-	TTCCCACCGAT	667	OMPU_NC002505_544_567_R	TCGGTCAGCAAAA	1094
	674828-	ATCATGGCTTA			CGGTAGCTTGC
	675880_428_455_F
2994	GAPA_NC002505-	TCCTCAATGAA	335	GAPA_NC002505-506780-	TTTTCCCTTTATG	1442
	506780	CGAICAACAAG		507937_769_802_R	CAACTTAGTATCA
	507937_691_721_F	TGATTGATG			ACIGGAAT
2995	GAPA_NC002505-	TCCTCIATGAA	339	GAPA_NC002505-506780-	TCCATACCTTTAT	1008
	506780-	ACGAICAACAA		507937_769_803_R	GCAACTTIGTAT
	507937_691_721_2_F	GTGATTGATG			CAACIGGAAT
2996	GAPA_NC002505-	TCCTCGATGAA	396	GAPA_NC002505-506780-	TCGGAAATATTCT	1085
	506780-	CCGACCAACAA		507937_785_817_R	TTCAATACCTT
	507937_692_721_F	GTGATTGATTG			TATGCAACT
2997	GAPA_NC002505-	TCCTCGATGAA	337	GAPA_NC002505-506780-	TCGGAAATATTCT	1085
	506780-	CGAICAACAAG		507937_785_817_R	TTCAATACCTTTA
	507937_691_721_3_F	TIATTGATG			TGCAACT
2998	GAPA_NC002505-	TCCTCAATGAA	336	GAPA_NC002505-506780-	TCGGAAATATTCT	1087
	506780-	TGATCAACAAG		507937_784_817_R	TTCAATICCTTTI
	507937_691_721_4_F	TGATTGATG			TGCAACTT
2999	GAPA_NC002505-	TCCTCIATGAA	340	GAPA_NC002505-506780-	TCGGAAATATTCT	1086
	506780-	IGAICAACAAG		507937_784_817_2_R	TTCAATACCTTTA
	507937_691_721_5_F	TIATTGATG			TGCAACTT
3000	GAPA_NC002505-	TCCTCGATGAA	338	GAPA_NC002505-506780-	TTTCAATACCTTT	1430
	506780-	TGAICAACAAC		507937_769_805_R	GCAACTTIGTATC
	507937_691_721_6_F	AAGTIATTGAT			AACIGGAAT
		G
3001	CTXB_NC002505-	TCAGCATATGC	275	CTXB_NC002505-1566967-	TCCCGGCTAGAGA	1026
	1566967-	ACATGGAACAC		1567341_139_163_R	TTCTGTATACGA
	1567341_46_71_F	CTCA			TTCTGTATACGA
3002	CTXB_NC002505-	TCAGCATATGC	274	CTXB_NC002505-1566967-	TCCGGCTAGAGAT	1038
	1566967-	ACATGGAACAC		1567341_132_162_R	TCTGTATACGAAA
	1567341_46_70_F	CTC			ATATC
3003	CTXB_NC002505-	TCAGCATATGC	274	CTXB_NC002505-1566967-	TGCCGTATACGAA	1225
	1566967-	ACATGGAACAC		1567341_118_150_R	AATATCTTATCAT
	1567341_46_70_F	CTC			TTAGCGT
3004	TUFB_NC002758-	TACAGGCCGTG	180	TUFB_NC002758-615038-	TCAGCGTAGTCTA	982
	615038-	TTGAACGTGG		61622_778_809_R	ATAATTTACGGAA
	616222_684_704_F				CATTTC
3005	TUFB_NC002758-	TGCCGTGTTGA	503	TUFB_NC0027858-615038-	TGCTTCAGCGTAG	1255
	615038-	ACGTGGTCAAA		616222_783_813_R	TCTAATAATTTAC
	616222_688_710_F	T			GGAAC
3006	TUFB_NC0027858-	TGTGGTCAAAT	638	TUFB_NC002758-615038-	TGCGTAGTCTAAT	1238
	615038-	CAAAGTTGGTG		616222_778_807_R	AATTTACGGAACA
	616222_700_726_F	AAGA			TTTC
3007	TUFB_NC002758-	TGGTCAAATCA	607	TUFB_NC002758-615038-	TGCGTAGTCTAAT	1238
	615038-	AAGTTGGTGAAG		616222_778_807_R	AATTTACGGAACA
	616222_702_726_F	AA			TTTC
3008	TUFB_NC002758-	TGAACGTGGTC	431	TUFB_NC002758-615038-	TCACCAGCTTCAG	970
	615038-	AAATCAAAGTT		616222_785_818_R	CGTAGTCTAATAA
	616222_696_726_F	GGTGAAGAA			TTTACGGA
3009	TUFB_NC002758-	TCGTGTTGAAC	386	TUFB_NC002758-615038-	TCTTCAGCGCGTA	1134
	615038-	GTGGTCAAATC		616222_778_812_R	GTCTAATAATTTA
	616222_690_716_F	AAAGT			CGGAACATTTC
3010	MECI-R-NC003923-	TCACATATCGT	261	MECI-R_NC003923-41798-	TGTGATATGGAGG	1332
	41798-41609_36_59_F	GAGCAATGAAC		41609_89_112_R	TGTAGAAGGTG
		TG
3011	MECI-R_NC003923-	TGGGCGTGAGC	584	MECI-R_NC003923-41798-	TGGGATGGAGGTG	1287
	41798-41609_40_66_F	AATGAACTGAT		41609_81_110_R	TAGAAGGTGTTAT
		TATAC			CATC
3012	MECI-R_NC003923-	TGGACACATAT	549	MECI-R_NC003923-41798-	TGGGATGGAGGTG	1286
	41798-	CGTGAGCAATG		41609_81_110_R	TAGAAAGGTGTTA
	41609_33_60_2_F	AACTGA			TCATC
3013	MECI-R_NC003923-	TGGGTTTACAC	595	MECI-R_NC003923-41798-	TGGGGATATGGAG	1290
	41798-41609_29_60_F	ATATCGTGAGC		41609_81_113_R	TGTAGAAGGTGTT
		AATGAACTGA			ATCATC
3014	MUPR_X75439_2409_2513_F	TGGGCTCTTTC	587	MUPR_X75439_2548_2570_R	TCTGGCTGCGGAA	1130
		TCGCTTAAACA			GTGAAATCGT
		CCT
3015	MUPR_X75439_2482_2510_F	TGGGCTCTTTC	586	MUPR_X75439_2547_2568_R	TGGCTGCGGAAGT	1281
		TCGCTTAAACA			GAAATCGTA
		CC
3016	MUPR_X75439_2482_2510_F	TAGATAATTGG	205	MUPR_X75439_2551_2573_R	TAATCTGGCTGCG	876
		GCTCTTTCTCG			GAAGTGAAAT
		CTTAAAC
3017	MUPR_X75439_2490_2514_F	TGGGCTCTTTC	587	MUPR_X75439_2549_2573_R	TAATCTGGCTGCG	877
		TCGCTTAAACA			GAAGTGAAATCG
		CCT
3018	MUPR_X75439_2482_2510_F	TAGATAATTGG	205	MUPR_X75439_2559_2589_R	TGGTATATTCGTT	1303
		GCTCTTTCTCG			AATTAATCTGGCT
		CTTAAAC			GCGGA
3019	MUPR_X75439_2490_2514_F	TGGGCTCTTTC	587	MUPR_X75439_2554_2581_R	TCGTTAATTAATC	1112
		GCTTAAACACC			TGGCTGCGGAAGT
		T			GA
3020	AROE_NC003923-	TGATGGCAAGT	474	AROE_NC003923-1674726-	TAAGCAATACCTT	1378
	1674726-	GGATAGGGTAT		1674277_309_335_R	TACTTGCACCACC
	1674277_204_232_F	AATACAG			ACCT
3021	AROE_NC003923-	TGGCGAGTGGA	570	AROE_NC003923-1674726-	TTCATAAGCAATA	1378
	1674726-	TAGGGTATAAT		1674277_311_339_R	CCCTTTACTTGCA
	1674277_207_232_F	ACAG			CCAC
3022	AROE_NC003923-	TGGCpAAGTpG	572	AROE_NC003923-1674726-	TAAGCAATACCpT	867
	1674726-	GATpAGGGTpA		1674277_311_335P_R	pTpTpACTpTpGC
	1674277_207_232P_F	TpAATpACpAG			pACpCpAC
3023	ARCC_NC003923-	TCTGAAATGAA	398	ARCC_NC003923-2725050-	TCTTCTTCTTTCG	1137
	2725050-	TAGTGATAGAA		2724595_214_245_R	TATAAAAAGGACC
	2724595_124_155_F	CTGTAGGCAC			AATTGG
3024	ARCC_NC003923-	TGAATAGTGAT	437	ARCC_NC003923-2725050-	TCTTCTTTTCGTAT	1139
	2725050-	AGAACTGTAGG		2724595_212_242_R	AAAAAGGACCAAT
	2724595_131_161_F	CACAATCGT			TGGTT
3025	ARCC_NC003923-	TGAATAGTGAT	437	ARCC_NC003923-2725050-	TGCGCTAATTCTT	1232
	2725050-	AGAACTGTAGG		2724595_232_260_R	CAACTTCTTCTTT
	2724595_131_161_F	CACAATCGT			CGT
3026	PTA_NC003923-	TACAATGCTTG	177	PTA_NC003923-628885-	TGTTCTTGATACA	1350
	628885-	TTTATGCTGGTA		629355_322_351_R	CCTGGTTTCGTTT
	629355_231_259_F	AAGCAG			TGAT
3027	PTA_NC003923-	TACAATGCTTG	177	PTA_NC003923-628885-	TGGTACACCTGGT	1301
	62885-	TTTATGCTGGT		629355_314_345_R	TTCGTTTTGATGA
	629355_231_259_F	AAAGCAG			TTTGTA
3028	PTA_NC003923-	TCTTGTTTATG	418	PTA_NC003923-628885-	TGTTCTTGATACA	1350
	628885-	CTGGTAAAAGC		629355_322_351_R	CCTGGTTTCGTTT
	629355_237_263_F	AGATGG			TGAT

TABLE 4 — Molecular Masses of Natural Nucleobases and the Mass-Modified Nucleobase 5-Iodo-C and Molecular Mass Differences Resulting from Transitions

Nucleobase	Molecular Mass	Transition	Molecular Mass
A	313.058	A-->T	−9.012
A	313.058	A-->C	−24.012
A	313.058	A-->5-Iodo-C	101.888
A	313.058	A-->G	15.994
T	304.046	T-->A	9.012
T	304.046	T-->C	−15.000
T	304.046	T-->5-Iodo-C	110.900
T	304.046	T-->G	25.006
C	289.046	C-->A	24.012
C	289.046	C-->T	15.000
C	289.046	C-->G	40.006
5-Iodo-C	414.946	5-Iodo-C-->A	−101.888
5-Iodo-C	414.946	5-Iodo-C-->T	−110.900
5-Iodo-C	414.946	5-Iodo-C-->G	−85.894
G	329.052	G-->A	−15.994
G	329.052	G-->T	−25.006
G	329.052	G-->C	−40.006
G	329.052	G-->5-Iodo-C	85.894

TABLE 5 — Bacterial Primer Pairs of the Surveillance Primer Set

		Forward		Reverse
Primer		Primer		Primer
Pair		(SEQ ID		(SEQ ID
No.	Forward Primer Name	NO:)	Reverse Primer Name	NO:)	Target Gene
346	16S_EC_713_732_TMOD_F	202	16S_EC_789_809_TMOD_R	1110	16S rRNA
10	16S_EC_713_732_F	21	16S_EC_789_809	798	16S rRNA
347	16S_EC_785_806_TMOD_F	560	16S_EC_880_897_TMOD_R	1278	16S rRNA
11	16S_EC_785_806_F	118	16S_EC_880_897_R	830	16S rRNA
348	16S_EC_960_981_TMOD_F	706	16S_EC_1054_1073_TMOD_R	895	16S rRNA
14	16S_EC_960_981_F	672	16S_EC_1054_1073_R	735	16S rRNA
349	23S_EC_1826_1843_TMOD_F	401	23S_EC_1906_1924_TMOD_R	1156	23S rRNA
16	23S_EC_1826_1843_F	80	23S_EC_1906_1924_R	805	23S rRNA
352	INFB_EC_1365_1393_TMOD_F	687	INFB_EC_1439_1467_TMOD_R	1411	infB
34	INFB_EC_1365_1393_F	524	INFB_EC_1439_1467_R	1248	infB
354	RPOC_EC_2218_2241_TMOD_F	405	RPOC_EC_2313_2337_TMOD_R	1072	rpoC
52	RPOC_EC_2218_2241_F	81	RPOC_EC_2313_2337_R	790	rpoC
355	SSPE_BA_115_137_TMOD_F	255	SSPE_BA_197_222_TMOD_R	1402	sspE
58	SSPE_BA_115_137_F	45	SSPE_BA_197_222_R	1201	sspE
356	RPLB_EC_650_679_TMOD_F	232	RPLB_EC_739_762_TMOD_R	592	rplB
66	RPLB_EC_650_679_F	98	RPLB_EC_739_762_R	999	rplB
358	VALS_EC_1105_1124_TMOD_F	385	VALS_EC_1195_1218_TMOD_R	1093	valS
71	VALS_EC_1105_1124_F	77	VALS_EC_1195_1218_R	795	valS
359	RPOB_EC_1845_1866_TMOD_F	659	RPOB_EC_1909_1929_TMOD_R	1250	rpoB
72	RPOB_EC_1845_1866_F	233	RPOB_EC_1909_1929_R	825	rpoB
360	23S_EC_2646_2667_TMOD_F	409	23S_EC_2745_2765_TMOD_R	1434	23S rRNA
118	23S_EC_2646_2667_F	84	23S_EC_2745_2765_R	1389	23S rRNA
17	23S_EC_2645_2669_F	408	23S_EC_2744_2761_R	1252	23S rRNA
361	16S_EC_1090_1111_2_TMOD_F	697	16S_EC_1175_1196_TMOD_R	1398	16S rRNA
3	16S_EC_1090_1111_2_F	651	16S_EC_1175_1196_R	1159	16S rRNA
362	RPOB_EC_3799_3821_TMOD_F	581	RPOB_EC_3862_3888_TMOD_R	1325	rpoB
289	RPOB_EC_3799_3821_F	124	RPOB_EC_3862_3888_R	840	rpoB
363	RPOC_EC_2146_2174_TMOD_F	284	RPOC_EC_2227_2245_TMOD_R	898	rpoC
290	RPOC_EC_2146_2174_F	52	RPOC_EC_2227_2245_R	736	rpoC
367	TUFB_EC_957_979_TMOD_F	308	TUFB_EC_1034_1058_TMOD_R	1276	tufB
293	TUFB_EC_957_979_F	55	TUFB_EC_1034_1058_R	829	tufB
449	RPLB_EC_690_710_F	309	RPLB_EC_737_758_R	1336	rplB
357	RPLB_EC_688_710_TMOD_F	296	RPLB_EC_736_757_TMOD_R	1337	rplB
67	RPLB_EC_688_710_F	54	RPLB_EC_736_757_R	842	rplB

TABLE 6 — Drill-Down Primer Pairs for Confirmation of Identification of Bacillus anthracis

		Forward		Reverse
Primer		Primer		Primer
Pair		(SEQ ID		(SEQ ID
No.	Forward Primer Name	NO:)	Reverse Primer Name	NO:)	Target Gene
350	CAPC_BA_274_303_TMOD_F	476	CAPC_BA_349_376_TMOD_R	1314	capC
24	CAPC_BA_274_303_F	109	CAPC_BA_349_376_R	837	capC
351	CYA_BA_1353_1379_TMOD_F	355	CYA_BA_1448_1467_TMOD_R	1423	cyA
30	CYA_BA_1353_1379_F	64	CYA_BA_1448_1467_R	1342	cyA
353	LEF_BA_756_781_TMOD_F	220	LEF_BA_843_872_TMOD_R	1394	lef
37	LEF_BA_756_781_F	26	LEF_BA_843_872_R	1135	lef

TABLE 8 — Triangulation Genotyping Analysis Primer Pairs for Group A Streptococcus Drill-Down

Primer		Forward Primer		Reverse Primer
Pair No.	Forward Primer Name	(SEQ ID NO:)	Reverse Primer Name	(SEQ ID NO:)	Target Gene
442	SP101_SPET11_358_387_TMOD_F	588	SP101_SPET11_448_473_TMOD_R	998	gki
80	SP101_SPET11_358_387_F	126	SP101_SPET11_448_473_TMOD_R	766	gki
443	SP101_SPET11_600_629_TMOD_F	348	SP101_SPET11_686_714_TMOD_R	1018	gtr
81	SP101_SPET11_600_629_F	62	SP101_SPET11_686_714_R	772	gtr
426	SP101_SPET11_1314_1336_TMOD_F	363	SP101_SPET11_1403_1431_TMOD_R	849	murI
86	SP101_SPET11_1314_1336_F	68	SP101_SPET11_1403_1431_R	711	murI
430	SP101_SPET11_1807_1835_TMOD_F	235	SP101_SPET11_1901_1927_TMOD_R	1439	mutS
90	SP101_SPET11_1807_1835_F	33	SP101_SPET11_1901_1927_R	1412	mutS
438	SP101_SPET11_3075_3103_TMOD_F	473	SP101_SPET11_3168_3196_TMOD_R	875	xpt
96	SP101_SPET11_3075_3103_F	108	SP101_SPET11_3168_3196_R	715	xpt
441	SP101_SPET11_3511_3535_TMOD_F	531	SP101_SPET11_3605_3629_TMOD_R	1294	yqiL
98	SP101_SPET11_3511_3535_F	116	SP101_SPET11_3605_3629_R	832	yqiL

TABLE 9A — Base Composition Analysis of Bioagent Identifying Amplicons of Group A Streptococcus samples from Six Military Installations Obtained with Primer Pair Nos. 426 and 430

	emm-type by				murI	mutS
# of	Mass	emm-Gene	Location		(Primer Pair	(Primer Pair
Instances	Spectrometry	Sequencing	(sample)	Year	No. 426)	No. 430)
48	3	3	MCRD San	2002	A39 G25 C20 T34	A38 G27 C23 T33
2	6	6	Diego		A40 G24 C20 T34	A38 G27 C23 T33
1	28	28	(Cultured)		A39 G25 C20 T34	A38 G27 C23 T33
15	3	ND			A39 G25 C20 T34	A38 G27 C23 T33
6	3	3	NHRC San	2003	A39 G25 C20 T34	A38 G27 C23 T33
3	5, 58	5	Diego-		A40 G24 C20 T34	A38 G27 C23 T33
6	6	6	Archive		A40 G24 C20 T34	A38 G27 C23 T33
1	11	11	(Cultured)		A39 G25 C20 T34	A38 G27 C23 T33
3	12	12			A40 G24 C20 T34	A38 G26 C24 T33
1	22	22			A39 G25 C20 T34	A38 G27 C23 T33
3	25, 75	75			A39 G25 C20 T34	A38 G27 C23 T33
4	44/61, 82, 9	44/61			A40 G24 C20 T34	A38 G26 C24 T33
2	53, 91	91			A39 G25 C20 T34	A38 G27 C23 T33
1	2	2	Ft.	2003	A39 G25 C20 T34	A38 G27 C24 T32
2	3	3	Leonard		A39 G25 C20 T34	A38 G27 C23 T33
1	4	4	Wood		A39 G25 C20 T34	A38 G27 C23 T33
1	6	6	(Cultured)		A40 G24 C20 T34	A38 G27 C23 T33
11	25 or 75	75			A39 G25 C20 T34	A38 G27 C23 T33
1	25, 75, 33,	75			A39 G25 C20 T34	A38 G27 C23 T33
	34, 4, 52, 84
1	44/61 or 82	44/61			A40 G24 C20 T34	A38 G26 C24 T33
	or 9
2	5 or 58	5			A40 G24 C20 T34	A38 G27 C23 T33
3	1	1	Ft. Sill	2003	A40 G24 C20 T34	A38 G27 C23 T33
2	3	3	(Cultured)		A39 G25 C20 T34	A38 G27 C23 T33
1	4	4			A39 G25 C20 T34	A38 G27 C23 T33
1	28	28			A39 G25 C20 T34	A38 G27 C23 T33
1	3	3	Ft.	2003	A39 G25 C20 T34	A38 G27 C23 T33
1	4	4	Benning		A39 G25 C20 T34	A38 G27 C23 T33
3	6	6	(Cultured)		A40 G24 C20 T34	A38 G27 C23 T33
1	11	11			A39 G25 C20 T34	A38 G27 C23 T33
1	13	94**			A40 G24 C20 T34	A38 G27 C23 T33
1	44/61 or 82	82			A40 G24 C20 T34	A38 G26 C24 T33
	or 9
1	5 or 58	58			A40 G24 C20 T34	A38 G27 C23 T33
1	78 or 89	89			A39 G25 C20 T34	A38 G27 C23 T33
2	5 or 58	ND	Lackland	2003	A40 G24 C20 T34	A38 G27 C23 T33
1	2		AFB		A39 G25 C20 T34	A38 G27 C24 T32
1	81 or 90		(Throat		A40 G24 C20 T34	A38 G27 C23 T33
1	78		Swabs)		A38 G26 C20 T34	A38 G27 C23 T33
3***	No detection				No detection	No detection
7	3	ND	MCRD San	2002	A39 G25 C20 T34	A38 G27 C23 T33
1	3	ND	Diego		No detection	A38 G27 C23 T33
1	3	ND	(Throat		No detection	No detection
1	3	ND	Swabs)		No detection	No detection
2	3	ND			No detection	A38 G27 C23 T33
3	No detection	ND			No detection	No detection

TABLE 9B — Base Composition Analysis of Bioagent Identifying Amplicons of Group A Streptococcus samples from Six Military Installations Obtained with Primer Pair Nos. 438 and 441

	emm-type by				xpt	yqiL
# of	Mass	emm-Gene	Location		(Primer Pair	(Primer Pair
Instances	Spectrometry	Sequencing	(sample)	Year	No. 438)	No. 441)
48	3	3	MCRD San	2002	A30 G36 C20 T36	A40 G29 C19 T31
2	6	6	Diego		A30 G36 C20 T36	A40 G29 C19 T31
1	28	28	(Cultured)		A30 G36 C20 T36	A41 G28 C18 T32
15	3	ND			A30 G36 C20 T36	A40 G29 C19 T31
6	3	3	NHRC San	2003	A30 G36 C20 T36	A40 G29 C19 T31
3	5, 58	5	Diego-		A30 G36 C20 T36	A40 G29 C19 T31
6	6	6	Archive		A30 G36 C20 T36	A40 G29 C19 T31
1	11	11	(Cultured)		A30 G36 C20 T36	A40 G29 C19 T31
3	12	12			A30 G36 C19 T37	A40 G29 C19 T31
1	22	22			A30 G36 C20 T36	A40 G29 C19 T31
3	25, 75	75			A30 G36 C20 T36	A40 G29 C19 T31
4	44/61, 82, 9	44/61			A30 G36 C20 T36	A41 G28 C19 T31
2	53, 91	91			A30 G36 C19 T37	A40 G29 C19 T31
1	2	2	Ft.	2003	A30 G36 C20 T36	A40 G29 C19 T31
2	3	3	Leonard		A30 G36 C20 T36	A40 G29 C19 T31
1	4	4	Wood		A30 G36 C19 T37	A41 G28 C19 T31
1	6	6	(Cultured)		A30 G36 C20 T36	A40 G29 C19 T31
11	25 or 75	75			A30 G36 C20 T36	A40 G29 C19 T31
1	25, 75, 33,	75			A30 G36 C19 T37	A40 G29 C19 T31
	34, 4, 52, 84
1	44/61 or 82	44/61			A30 G36 C20 T36	A41 G28 C19 T31
	or 9
2	5 or 58	5			A30 G36 C20 T36	A40 G29 C19 T31
3	1	1	Ft. Sill	2003	A30 G36 C19 T37	A40 G29 C19 T31
2	3	3	(Cultured)		A30 G36 C20 T36	A40 G29 C19 T31
1	4	4			A30 G36 C19 T37	A41 G28 C19 T31
1	28	28			A30 G36 C20 T36	A41 G28 C18 T32
1	3	3	Ft.	2003	A30 G36 C20 T36	A40 G29 C19 T31
1	4	4	Benning		A30 G36 C19 T37	A41 G28 C19 T31
3	6	6	(Cultured)		A30 G36 C20 T36	A40 G29 C19 T31
1	11	11			A30 G36 C20 T36	A40 G29 C19 T31
1	13	94**			A30 G36 C20 T36	A41 G28 C19 T31
1	44/61 or 82	82			A30 G36 C20 T36	A41 G28 C19 T31
	or 9
1	5 or 58	58			A30 G36 C20 T36	A40 G29 C19 T31
1	78 or 89	89			A30 G36 C20 T36	A41 G28 C19 T31
2	5 or 58	ND	Lackland	2003	A30 G36 C20 T36	A40 G29 C19 T31
1	2		AFB		A30 G36 C20 T36	A40 G29 C19 T31
1	81 or 90		(Throat		A30 G36 C20 T36	A40 G29 C19 T31
1	78		Swabs)		A30 G36 C20 T36	A41 G28 C19 T31
3***	No detection				No detection	No detection
7	3	ND	MCRD San	2002	A30 G36 C20 T36	A40 G29 C19 T31
1	3	ND	Diego		A30 G36 C20 T36	A40 G29 C19 T31
1	3	ND	(Throat		A30 G36 C20 T36	No detection
1	3	ND	Swabs)		No detection	A40 G29 C19 T31
2	3	ND			A30 G36 C20 T36	A40 G29 C19 T31
3	No detection	ND			No detection	No detection

TABLE 9C — Base Composition Analysis of Bioagent Identifying Amplicons of Group A Streptococcus samples from Six Military Installations Obtained with Primer Pair Nos. 438 and 441

	emm-type by				gki	gtr
# of	Mass	emm-Gene	Location		(Primer Pair	((Primer Pair
Instances	Spectrometry	Sequencing	(sample)	Year	No. 442)	No. 443)
48	3	3	MCRD San	2002	A32 G35 C17 T32	A39 G28 C16 T32
2	6	6	Diego		A31 G35 C17 T33	A39 G28 C15 T33
1	28	28	(Cultured)		A30 G36 C17 T33	A39 G28 C16 T32
15	3	ND			A32 G35 C17 T32	A39 G28 C16 T32
6	3	3	NHRC San	2003	A32 G35 C17 T32	A39 G28 C16 T32
3	5, 58	5	Diego-		A30 G36 C20 T30	A39 G28 C15 T33
6	6	6	Archive		A31 G35 C17 T33	A39 G28 C15 T33
1	11	11	(Cultured)		A30 G36 C20 T30	A39 G28 C16 T32
3	12	12			A31 G35 C17 T33	A39 G28 C15 T33
1	22	22			A31 G35 C17 T33	A38 G29 C15 T33
3	25, 75	75			A30 G36 C17 T33	A39 G28 C15 T33
4	44/61, 82, 9	44/61			A30 G36 C18 T32	A39 G28 C15 T33
2	53, 91	91			A32 G35 C17 T32	A39 G28 C16 T32
1	2	2	Ft.	2003	A30 G36 C17 T33	A39 G28 C15 T33
2	3	3	Leonard		A32 G35 C17 T32	A39 G28 C16 T32
1	4	4	Wood		A31 G35 C17 T33	A39 G28 C15 T33
1	6	6	(Cultured)		A31 G35 C17 T33	A39 G28 C15 T33
11	25 or 75	75			A30 G36 C17 T33	A39 G28 C15 T33
1	25, 75, 33,	75			A30 G36 C17 T33	A39 G28 C15 T33
	34, 4, 52, 84
1	44/61 or 82	44/61			A30 G36 C18 T32	A39 G28 C15 T33
	or 9
2	5 or 58	5			A30 G36 C20 T30	A39 G28 C15 T33
3	1	1	Ft. Sill	2003	A30 G36 C18 T32	A39 G28 C15 T33
2	3	3	(Cultured)		A32 G35 C17 T32	A39 G28 C16 T32
1	4	4			A31 G35 C17 T33	A39 G28 C15 T33
1	28	28			A30 G36 C17 T33	A39 G28 C16 T32
1	3	3	Ft.	2003	A32 G35 C17 T32	A39 G28 C16 T32
1	4	4	Benning		A31 G35 C17 T33	A39 G28 C15 T33
3	6	6	(Cultured)		A31 G35 C17 T33	A39 G28 C15 T33
1	11	11			A30 G36 C20 T30	A39 G28 C16 T32
1	13	94**			A30 G36 C19 T31	A39 G28 C15 T33
1	44/61 or 82	82			A30 G36 C18 T32	A39 G28 C15 T33
	or 9
1	5 or 58	58			A30 G36 C20 T30	A39 G28 C15 T33
1	78 or 89	89			A30 G36 C18 T32	A39 G28 C15 T33
2	5 or 58	ND	Lackland	2003	A30 G36 C20 T30	A39 G28 C15 T33
1	2		AFB		A30 G36 C17 T33	A39 G28 C15 T33
1	81 or 90		(Throat		A30 G36 C17 T33	A39 G28 C15 T33
1	78		Swabs)		A30 G36 C18 T32	A39 G28 C15 T33
3***	No detection				No detection	No detection
7	3	ND	MCRD San	2002	A32 G35 C17 T32	A39 G28 C16 T32
1	3	ND	Diego		No detection	No detection
1	3	ND	(Throat		A32 G35 C17 T32	A39 G28 C16 T32
1	3	ND	Swabs)		A32 G35 C17 T32	No detection
2	3	ND			A32 G35 C17 T32	No detection
3	No detection	ND			No detection	No detection

TABLE 11 — Primer Pair Gene Coordinate References and Calibration Polynucleotide Sequence Coordinates within the Combination Calibration Polynucleotide Coordinates of

	Gene Extraction			Calibration Sequence in
Bacterial	Coordinates	Reference GenBank GI		Combination Calibration
Gene and	of Genomic or Plasmid	No. of Genomic (G) or	Primer	Polynucleotide (SEQ ID
Species	Sequence	Plasmid (P) Sequence	Pair No.	NO: 1464)
16S E. coli	4033120 . . . 4034661	16127994 (G)	346	16 . . . 109
16S E. coli	4033120 . . . 4034661	16127994 (G)	347	83 . . . 190
16S E. coli	4033120 . . . 4034661	16127994 (G)	348	246 . . . 353
16S E. coli	4033120 . . . 4034661	16127994 (G)	361	368 . . . 469
23S E. coli	4166220 . . . 4169123	16127994 (G)	349	743 . . . 837
23S E. coli	4166220 . . . 4169123	16127994 (G)	360	865 . . . 981
rpoB E. coli.	4178823 . . . 4182851	16127994 (G)	359	1591 . . . 1672
	(complement strand)
rpoB E. coli	4178823 . . . 4182851	16127994 (G)	362	2081 . . . 2167
	(complement strand)
rpoC E. coli	4182928 . . . 4187151	16127994 (G)	354	1810 . . . 1926
rpoC E. coli	4182928 . . . 4187151	16127994 (G)	363	2183 . . . 2279
infB E. coli	3313655 . . . 3310983	16127994 (G)	352	1692 . . . 1791
	(complement strand)
tufB E. coli	4173523 . . . 4174707	16127994 (G)	367	2400 . . . 2498
rplB E. coli	3449001 . . . 3448180	16127994 (G)	356	1945 . . . 2060
rplB E. coli	3449001 . . . 3448180	16127994 (G)	449	1986 . . . 2055
valS E. coli	4481405 . . . 4478550	16127994 (G)	358	1462 . . . 1572
	(complement strand)
capC	56074 . . . 55628	6470151 (P)	350	2517 . . . 2616
B. anthracis
(complement strand)
cya	156626 . . . 154288	4894216 (P)	351	1338 . . . 1449
B. anthracis
(complement strand)
lef	127442 . . . 129921	4894216 (P)	353	1121 . . . 1234
B. anthracis
sspE	226496 . . . 226783	30253828 (G)	355	1007-1104
B. anthracis

TABLE 12 — Campylobacter Genotyping Primer Pairs Primer

Pair		Forward Primer		Reverse Primer
No.	Forward Primer Name	(SEQ ID NO:)	Reverse Primer Name	(SEQ ID NO:)	Target Gene
1053	CJST_CJ_1080_1110_F	681	CJST_CJ_1166_1198_R	1022	gltA
1047	CJST_CJ_584_616_F	315	CJST_CJ_663_692_R	1379	glnA
1048	CJST_CJ_360_394_F	346	CJST_CJ_442_476_R	955	aspA
1049	CJST_CJ_2636_2668_F	504	CJST_CJ_2753_2777_R	1409	tkt
1054	CJST_CJ_2060_2090_F	323	CJST_CJ_2148_2174_R	1068	pgm
1064	CJST_CJ_1680_1713_F	479	CJST_CJ_1795_1822_R	938	glyA

TABLE 14A — Triangulation Genotyping Analysis Primer Pairs for Identification of Sub-species characteristics (Strain Type) of Members of the Bacterial Genus Acinetobacter

Primer		Forward Primer		Reverse Primer
Pair No.	Forward Primer Name	(SEQ ID NO:)	Reverse Primer Name	(SEQ ID NO:)
1151	AB_MLST-11-OIF007_62_91_F	454	AB_MLST-11-OIF007_169_203_R	1418
1152	AB_MLST-11-OIF007_185_214_F	243	AB_MLST-11-OIF007_291_324_R	969
1153	AB_MLST-11-OIF007_260_289_F	541	AB_MLST-11-OIF007_364_393_R	1400
1154	AB_MLST-11-OIF007_206_239_F	436	AB_MLST-11-OIF007_318_344_R	1036
1155	AB_MLST-11-OIF007_522_552_F	378	AB_MLST-11-OIF007_587_610_R	1392
1156	AB_MLST-11-OIF007_547_571_F	250	AB_MLST-11-OIF007_656_686_R	902
1157	AB_MLST-11-OIF007_601_627_F	256	AB_MLST-11-OIF007_710_736_R	881
1158	AB_MLST-11-OIF007_1202_1225_F	384	AB_MLST-11-OIF007_1266_1296_R	878
1159	AB_MLST-11-OIF007_1202_1225_F	384	AB_MLST-11-OIF007_1299_1316_R	1199
1160	AB_MLST-11-OIF007_1234_1264_F	694	AB_MLST-11-OIF007_1335_1362_R	1215
1161	AB_MLST-11-OIF007_1327_1356_F	225	AB_MLST-11-OIF007_1422_1448_R	1212
1162	AB_MLST-11-OIF007_1345_1369_F	383	AB_MLST-11-OIF007_1470_1494_R	1083
1163	AB_MLST-11-OIF007_1351_1375_F	662	AB_MLST-11-OIF007_1470_1494_R	1083
1164	AB_MLST-11-OIF007_1387_1412_F	422	AB_MLST-11-OIF007_1470_1494_R	1083
1165	AB_MLST-11-OIF007_1542_1569_F	194	AB_MLST-11-OIF007_1656_1680_R	1173
1166	AB_MLST-11-OIF007_1566_1593_F	684	AB_MLST-11-OIF007_1656_1680_R	1173
1167	AB_MLST-11-OIF007_1611_1638_F	375	AB_MLST-11-OIF007_1731_1757_R	890
1168	AB_MLST-11-OIF007_1726_1752_F	182	AB_MLST-11-OIF007_1790_1821_R	1195
1169	AB_MLST-11-OIF007_1792_1826_F	656	AB_MLST-11-OIF007_1876_1909_R	1151
1170	AB_MLST-11-OIF007_1792_1826_F	656	AB_MLST-11-OIF007_1895_1927_R	1224
1171	AB_MLST-11-OIF007_1970_2002_F	618	AB_MLST-11-OIF007_2097_2118_R	1157
2846	PARC_X95819_33_58_F	302	PARC_X95819_121_153_R	852
2847	PARC_X95819_33_58_F	199	PARC_X95819_157_178_R	889
2848	PARC_X95819_33_58_F	596	PARC_X95819_97_128_R	1169
2852	GYRA_AY642140_−1_24_F	150	GYRA_AY642140_71_100_R	1242
2853	GYRA_AY642140_26_54_F	166	GYRA_AY642140_121_146_R	1069
2854	GYRA_AY642140_26_54_F	166	GYRA_AY642140_58_89_R	1168
2922	AB_MLST-11-OIF007_991_1018_F	583	AB_MLST-11-OIF007_1110_1137_R	923
2972	AB_MLST-11-OIF007_1007_1034_F	592	AB_MLST-11-OIF007_1126_1153_R	924

TABLE 14B — Triangulation Genotyping Analysis Primer Pairs for Identification of Sub-species characteristics (Strain Type) of Members of the Bacterial Genus Acinetobacter

	Forward		Reverse
Primer	Primer		Primer
Pair No.	(SEQ ID NO:)	SEQUENCE	(SEQ ID NO:	SEQUENCE
1151	454	TGAGATTGCTGAACATTTAATGCTGATTGA	1418	TTGTACATTTGAAACAATATGCATGACATGTGAAT
1152	243	TATTGTTTCAAATGTACAAGGTGAAGTGCG	969	TCACAGGTTCACTTCATCAATAATTTCCATTGC
1153	541	TGGAACGTTATCAGGTGCCCCAAAAATTCG	1400	TTGCAATCGACATATCCATTTCACCATGCC
1154	436	TGAAGTGCGTGATGATATCGATGCACTTGATGTA	1036	TCCGCCAAAAACTCCCCTTTTCACAGG
1155	378	TCGGTTTAGTAAAAGAACGTATTGCTCAACC	1392	TTCTGCTTGAGGAATAGTGCGTGG
1156	250	TCAACCTGACTGCGTGAATGGTTGT	902	TACGTTCTACGATTTCTTCATCAGGTACATC
1157	256	TCAAGCAGAAGCTTTGGAAGAAGAAGG	881	TACAACGTGATAAACACGACCAGAAGC
1158	384	TCGTGCCCGCAATTTGCATAAAGC	878	TAATGCCGGGTAGTGCAATCCATTCTTCTAG
1159	384	TCGTGCCCGCAATTTGCATAAAGC	1199	TGCACCTGCGGTCGAGCG
1160	694	TTGTAGCACAGCAAGGCAAATTTCCTCAAAC	1215	TGCCATCCATAATCACGCCATACTGACG
1161	225	TAGGTTTACGTCAGTATGGCGTGATTATGG	1212	TGCCAGTTTCCACATTTCACGTTCGTG
1162	383	TCGTGATTATGGATGGCAACGTGAA	1083	TCGCTTGAGTGTACTGATGATTGCG
1163	662	TTATGGATGGCAACGTGAAACGCGT	1083	TCGCTTGAGTGTAGTCATGATTGCG
1164	422	TCTTTGCCATTGAAGATGACTTAAGC	1083	TCGCTTGAGTGTAGTCATGATTGCG
1165	194	TACTAGCGGTAAGCTTAAACAAGATTGC	1173	TGAGTCGGGTTCACTTTACCTGGCA
1166	684	TTGCCAATGATATTCGTTGGTTAGCAAG	1173	TGAGTCGGGTTCACTTTACCTGGCA
1167	375	TCGGCGAAATCCGTATTCCTGAAAATGA	890	TACCGGAAGCACCAGCGACATTAATAG
1168	182	TACCACTATTAATGTCGCTGGTGCTTC	1195	TGCAACTGAATAGATTGCAGTAAGTTATAAGC
1169	656	TTATAACTTACTGCAATCTATTCAGTTGCTTGGT	1151	TGAATTATGCAAGAAGTGATCAATTTTCTCACGA
		G
1170	656	TTATAACTTACTGCAATCTATTCAGTTGCTTGGT	1224	TGCCGTAACTAACATAAGAGAATTATGCAAGAA
		G
1171	618	TGGTTATGTACCAAATACTTTGTCTGAAGATGG	1157	TGACGGCATCGATACCACCGTC
2846	302	TCCAAAAAAATCAGCGCGTACAGTGG	852	TAAAGGATAGCGGTAACTAAATGGCTGAGCCAT
2847	199	TACTTGGTAAATACCACCCACATGGTGA	889	TACCCCAGTTCCCCTGACCTTC
2848	596	TGGTAAATACCACCCACATGGTGAC	1169	TGAGCCATGAGTACCATGGCTTCATAACATGC
2852	150	TAAATCTGCCCGTGTCGTCGGTGAC	1242	TGCTAAAGTCTTGAGCCATACGAACAATGG
2853	166	TAATCGGTAAATATCACCCGCATGGTGAC	1069	TCGATCGAACCGAAGTTACCCTGACC
2854	166	TAATCGGTAAATATCACCCGCATGGTGAC	1168	TGAGCCATACGAACAATGGTTTCATAAACAGC
2922	583	TGGGCGATGCTGCGAAATGGTTAAAAGA	923	TAGTATCACCACGTACACCCGCATCAGT
2972	592	TGGGIGATGCTGCIAAATGGTTAAAAGA	924	TAGTATCACCACGTACICCIGGATCAGT

TABLE 19 — Primer Pairs for Identification of Drug Resistance Genes and Virulence Factors in Staphylococcus aureus

		Forward		Reverse
Primer		Primer		Primer
Pair		(SEQ ID		(SEQ ID	Target
No.	Forward Primer Name	NO:)	Reverse Primer Name	NO:)	Gene
879	MECA_Y14051_4507_4530_F	288	MECA_Y14051_4555_4581_R	1269	mecA
2056	MECI-R_NC003923-41798-	698	MECI-R_NC003923-41798-	1420	MecI-R
	41609_33_60_F		41609_86_113_R
2081	ERMA_NC002952-55890-	217	ERMA_NC002952-55890-	1167	ermA
	56621_366_395_F		56621_438_465_R
2086	ERMC_NC005908-2004-	399	ERMC_NC005908-2004-	1041	ermC
	2738_85_116_F		2738_173_206_R
2095	PVLUK_NC003923-1529595-	456	PVLUK_NC003923-1529595-	1261	Pv-luk
	1531285_688_713_F		1531285_775_804_R
2249	TUFB_NC002758-615038-	430	TUFB_NC002758-615038-	1321	tufB
	616222_696_725_F		616222_793_820_R
2256	NUC_NC002758-894288-	174	NUC_NC002758-894288-	853	Nuc
	894974_316_345_F		894974_396_421_R
2313	MUPR_X75439_2486_2516_F	172	MUPR_X75439_2548_2574_R	1360	mupR

TABLE 20A — Drug Resistance and Virulence Identified in Blinded Samples of Various Strains of Staphylococcus aureus with Primer Pair Nos. 2081, 2086, 2095 and 2256

	Primer	Primer		Primer
Sample	Pair No.	Pair No.	Primer Pair No.	Pair No.
Index No.	2081 (ermA)	2086 (ermC)	2095 (pv-luk)	2256 (nuc)
CDC0010	−	−	PVL−/lukD+	+
CDC0015	−	−
PVL+/lukD+
+
CDC0019	−	+	PVL−/lukD+	+
CDC0026	+	−	PVL−/lukD+	+
CDC0030	+	−	PVL−/lukD+	+
CDC004	−	−
PVL+/lukD+
+
CDC0014	−	+
PVL+/lukD+
+
CDC008	−	−	PVL−/lukD+	+
CDC001	+	−	PVL−/lukD+	+
CDC0022	+	−	PVL−/lukD+	+
CDC006	+	−	PVL−/lukD+	+
CDC007	−	−	PVL−/lukD+	+
CDCVRSA1	+	−	PVL−/lukD+	+
CDCVRSA2	+	+	PVL−/lukD+	+
CDC0011	+	−	PVL−/lukD+	+
CDC0012	−	−
PVL+/lukD−
+
CDC0021	+	−	PVL−/lukD+	+
CDC0023	+	−	PVL−/lukD+	+
CDC0025	+	−	PVL−/lukD+	+
CDC005	−	−	PVL−/lukD+	+
CDC0018	+	−
PVL+/lukD−
+
CDC002	−	−	PVL−/lukD+	+
CDC0028	+	−	PVL−/lukD+	+
CDC003	−	−	PVL−/lukD+	+
CDC0013	−	−
PVL+/lukD+
+
CDC0016	−	−	PVL−/lukD+	+
CDC0027	+	−	PVL−/lukD+	+
CDC0029	−	−
PVL+/lukD+
+
CDC0020	−	+	PVL−/lukD+	+
CDC0024	−	−	PVL−/lukD+	+
CDC0031	−	−	PVL−/lukD+	+

TABLE 21 — Primer Pairs for Triangulation Genotyping Analysis of Staphylococcus aureus

		Forward		Reverse
Primer		Primer		Primer
Pair		(SEQ ID		(SEQ ID	Target
No.	Forward Primer Name	NO:)	Reverse Primer Name	NO:)	Gene
2146	ARCC_NC003923-2725050-	437	ARCC_NC003923-2725050-	1137	arcC
	2724595_131_161_F		2724595_214_245_R
2149	AROE_NC003923-1674726-	530	AROE_NC003923-1674726-	891	aroE
	1674277_30_62_F		1674277_155_181_R
2150	AROE_NC003923-1674726-	474	AROE_NC003923-1674726-	869	aroE
	1674277_204_232_F		1674277_308_335_R
2156	GMK_NC003923-1190906-	268	GMK_NC003923-1190906-	1284	gmk
	1191334_301_329_F		1191334_403_432_R
2157	PTA_NC003923-628885-	418	PTA_NC003923-628885-	1301	pta
	629355_237_263_F		629355_314_345_R
2161	TPI_NC003923-830671-	318	TPI_NC003923-830671-	1300	tpi
	831072_1_34_F		831072_97_129_R
2163	YQI_NC003923-378916-	440	YQI_NC003923-378916-	1076	yqi
	379431_142_167_F		379431_259_284_R
2166	YQI_NC003923-378916-	219	YQI_NC003923-378916-	1013	yqi
	379431_275_300_F		379431_364_396_R

TABLE 22A — Triangulation Genotyping Analysis of Blinded Samples of Various Strains of Staphylococcus aureus with Primer Pair Nos. 2146, 2149, 2150 and 2156

Sample		Primer Pair No.	Primer Pair No.	Primer Pair No.	Primer Pair No.
Index No.	Strain	2146 (arcC)	2149(aroE)	2150 (aroE)	2156 (gmk)
CDC0010	COL	A44 G24 C18 T29	A59 G24 C18 T51	A40 G36 C13 T43	A50 G30 C20 T32
CDC0015	COL	A44 G24 C18 T29	A59 G24 C18 T51	A40 G36 C13 T43	A50 G30 C20 T32
CDC0019	COL	A44 G24 C18 T29	A59 G24 C18 T51	A40 G36 C13 T43	A50 G30 C20 T32
CDC0026	COL	A44 G24 C18 T29	A59 G24 C18 T51	A40 G36 C13 T43	A50 G30 C20 T32
CDC0030	COL	A44 G24 C18 T29	A59 G24 C18 T51	A40 G36 C13 T43	A50 G30 C20 T32
CDC004	COL	A44 G24 C18 T29	A59 G24 C18 T51	A40 G36 C13 T43	A50 G30 C20 T32
CDC0014	COL	A44 G24 C18 T29	A59 G24 C18 T51	A40 G36 C13 T43	A50 G30 C20 T32
CDC008	????	A44 G24 C18 T29	A59 G24 C18 T51	A40 G36 C13 T43	A50 G30 C20 T32
CDC001	Mu50	A45 G23 C20 T27	A58 G24 C18 T52	A40 G36 C13 T43	A51 G29 C21 T31
CDC0022	Mu50	A45 G23 C20 T27	A58 G24 C18 T52	A40 G36 C13 T43	A51 G29 C21 T31
CDC006	Mu50	A45 G23 C20 T27	A58 G24 C18 T52	A40 G36 C13 T43	A51 G29 C21 T31
CDC0011	MRSA252	A45 G24 C18 T28	A58 G24 C19 T51	A41 G36 C12 T43	A51 G29 C21 T31
CDC0012	MRSA252	A45 G24 C18 T28	A58 G24 C19 T51	A41 G36 C12 T43	A51 G29 C21 T31
CDC0021	MRSA252	A45 G24 C18 T28	A58 G24 C19 T51	A41 G36 C12 T43	A51 G29 C21 T31
CDC0023	ST:110	A45 G24 C18 T28	A59 G24 C18 T51	A40 G36 C13 T43	A50 G30 C20 T32
CDC0025	ST:110	A45 G24 C18 T28	A59 G24 C18 T51	A40 G36 C13 T43	A50 G30 C20 T32
CDC005	ST:338	A44 G24 C18 T29	A59 G23 C19 T51	A40 G36 C14 T42	A51 G29 C21 T31
CDC0018	ST:338	A44 G24 C18 T29	A59 G23 C19 T51	A40 G36 C14 T42	A51 G29 C21 T31
CDC002	ST:108	A46 G23 C20 T26	A58 G24 C19 T51	A42 G36 C12 T42	A51 G29 C20 T32
CDC0028	ST:108	A46 G23 C20 T26	A58 G24 C19 T51	A42 G36 C12 T42	A51 G29 C20 T32
CDC003	ST:107	A45 G23 C20 T27	A58 G24 C18 T52	A40 G36 C13 T43	A51 G29 C21 T31
CDC0013	ST:12	ND	A59 G24 C18 T51	A40 G36 C13 T43	A51 G29 C21 T31
CDC0016	ST:120	A45 G23 C18 T29	A58 G24 C19 T51	A40 G37 C13 T42	A51 G29 C21 T31
CDC0027	ST:105	A45 G23 C20 T27	A58 G24 C18 T52	A40 G36 C13 T43	A51 G29 C21 T31
CDC0029	MSSA476	A45 G23 C20 T27	A58 G24 C19 T51	A40 G36 C13 T43	A50 G30 C20 T32
CDC0020	ST:15	A44 G23 C21 T27	A59 G23 C18 T52	A40 G36 C13 T43	A50 G30 C20 T32
CDC0024	ST:137	A45 G23 C20 T27	A57 G25 C19 T51	A40 G36 C13 T43	A51 G29 C22 T30
CDC0031	***	No product	No product	No product	No product

TABLE 22B — Triangulation Genotyping Analysis of Blinded Samples of Various Strains of Staphylococcus aureus with Primer Pair Nos. 2146, 2149, 2150 and 2156 Note: *** The sample CDC0031 was identified as Staphylococcus scleiferi as indicated in Example 14. Thus, the triangulation genotyping primers designed for Staphylococcus aureus would generally not be expected to prime and produce amplification products of this organism. Tables 22A and 22B indicate that amplification products are obtained for this organism only with primer pair numbers 2157 and 2161.

Sample		Primer Pair No.	Primer Pair No.	Primer Pair No.	Primer Pair No.
Index No.	Strain	2157 (pta)	2161 (tpi)	2163 (yqi)	2166 (yqi)
CDC0010	COL	A32 G25 C23 T29	A51 G28 C22 T28	A41 G37 C22 T43	A37 G30 C18 T37
CDC0015	COL	A32 G25 C23 T29	A51 G28 C22 T28	A41 G37 C22 T43	A37 G30 C18 T37
CDC0019	COL	A32 G25 C23 T29	A51 G28 C22 T28	A41 G37 C22 T43	A37 G30 C18 T37
CDC0026	COL	A32 G25 C23 T29	A51 G28 C22 T28	A41 G37 C22 T43	A37 G30 C18 T37
CDC0030	COL	A32 G25 C23 T29	A51 G28 C22 T28	A41 G37 C22 T43	A37 G30 C18 T37
CDC004	COL	A32 G25 C23 T29	A51 G28 C22 T28	A41 G37 C22 T43	A37 G30 C18 T37
CDC0014	COL	A32 G25 C23 T29	A51 G28 C22 T28	A41 G37 C22 T43	A37 G30 C18 T37
CDC008	unknown	A32 G25 C23 T29	A51 G28 C22 T28	A41 G37 C22 T43	A37 G30 C18 T37
CDC001	Mu50	A33 G25 C22 T29	A50 G28 C22 T29	A42 G36 C22 T43	A36 G31 C19 T36
CDC0022	Mu50	A33 G25 C22 T29	A50 G28 C22 T29	A42 G36 C22 T43	A36 G31 C19 T36
CDC006	Mu50	A33 G25 C22 T29	A50 G28 C22 T29	A42 G36 C22 T43	A36 G31 C19 T36
CDC0011	MRSA252	A32 G25 C23 T29	A50 G28 C22 T29	A42 G36 C22 T43	A37 G30 C18 T37
CDC0012	MRSA252	A32 G25 C23 T29	A50 G28 C22 T29	A42 G36 C22 T43	A37 G30 C18 T37
CDC0021	MRSA252	A32 G25 C23 T29	A50 G28 C22 T29	A42 G36 C22 T43	A37 G30 C18 T37
CDC0023	ST:110	A32 G25 C23 T29	A51 G28 C22 T28	A41 G37 C22 T43	A37 G30 C18 T37
CDC0025	ST:110	A32 G25 C23 T29	A51 G28 C22 T28	A41 G37 C22 T43	A37 G30 C18 T37
CDC005	ST:338	A32 G25 C24 T28	A51 G27 C21 T30	A42 G36 C22 T43	A37 G30 C18 T37
CDC0018	ST:338	A32 G25 C24 T28	A51 G27 C21 T30	A42 G36 C22 T43	A37 G30 C18 T37
CDC002	ST:108	A33 G25 C23 T28	A50 G28 C22 T29	A42 G36 C22 T43	A37 G30 C18 T37
CDC0028	ST:108	A33 G25 C23 T28	A50 G28 C22 T29	A42 G36 C22 T43	A37 G30 C18 T37
CDC003	ST:107	A32 G25 C23 T29	A51 G28 C22 T28	A41 G37 C22 T43	A37 G30 C18 T37
CDC0013	ST:12	A32 G25 C23 T29	A51 G28 C22 T28	A42 G36 C22 T43	A37 G30 C18 T37
CDC0016	ST:120	A32 G25 C24 T28	A50 G28 C21 T30	A42 G36 C22 T43	A37 G30 C18 T37
CDC0027	ST:105	A33 G25 C22 T29	A50 G28 C22 T29	A43 G36 C21 T43	A36 G31 C19 T36
CDC0029	MSSA476	A33 G25 C22 T29	A50 G28 C22 T29	A42 G36 C22 T43	A36 G31 C19 T36
CDC0020	ST:15	A33 G25 C22 T29	A50 G28 C21 T30	A42 G36 C22 T43	A36 G31 C18 T37
CDC0024	ST:137	A33 G25 C22 T29	A51 G28 C22 T28	A42 G36 C22 T43	A37 G30 C18 T37
CDC0031	***	A34 G25 C25 T25	A51 G27 C24 T27	No product	No product

TABLE 23 — Primer Pairs for Triangulation Genotyping Analysis of Members of the Bacterial Genus Vibrio

		Forward		Reverse
Primer		Primer		Primer
Pair		(SEQ ID		(SEQ ID	Target
No.	Forward Primer Name	NO:)	Reverse Primer Name	NO:)	Gene
1098	RNASEP_VBC_331_349_F	325	RNASEP_VBC_388_414_R	1163	RNAse P
2000	CTXB_NC002505_46_70_F	278	CTXB_NC002505_132_162_R	1039	ctxB
2001	FUR_NC002505_87_113_F	465	FUR_NC002505_205_228_R	1037	fur
2011	GYRB_NC002505_1161_1190_F	148	GYRB_NC002505_1255_1284_R	1172	gyrB
2012	OMPU_NC002505_85_110_F	190	OMPU_NC002505_154_180_R	1254	ompU
2014	OMPU_NC002505_431_455_F	266	OMPU_NC002505_544_567_R	1094	ompU
2323	CTXA_NC002505-1568114-	508	CTXA_NC002505-1568114-	1297	ctxA
	1567341_122_149_F		1567341_186_214_R
2927	GAPA_NC002505_694_721_F	259	GAPA_NC_002505_29_58_R	1060	gapA

TABLE 24 — Primer Pairs for Triangulation Genotyping Analysis of Members of the Bacterial Genus Pseudomonas

		Forward		Reverse
Primer		Primer		Primer
Pair		(SEQ ID		(SEQ ID	Target
No.	Forward Primer Name	NO:)	Reverse Primer Name	NO:)	Gene
2949	ACS_NC002516-970624-	376	ACS_NC002516-970624-	1265	acsA
	971013_299_316_F		971013_364_383_R
2950	ARO_NC002516-26883-	267	ARO_NC002516-26883-	1341	aroE
	27380_4_26_F		27380_111_128_R
2951	ARO_NC002516-26883-	705	ARO_NC002516-26883-	1056	aroE
	27380_356_377_F		27380_459_484_R
2954	GUA_NC002516-4226546-	710	GUA_NC002516-4226546-	1259	guaA
	4226174_155_178_F		4226174_265_287_R
2956	GUA_NC002516-4226546-	374	GUA_NC002516-4226546-	1111	guaA
	4226174_242_263_F		4226174_355_371_R
2957	MUT_NC002516-5551158-	545	MUT_NC002516-5551158-	978	mutL
	5550717_5_26_F		5550717_99_116_R
2959	NUO_NC002516-2984589-	249	NUO_NC002516-2984589-	1095	nuoD
	2984954_8_26_F		2984954_97_117_R
2960	NUO_NC002516-2984589-	195	NUO_NC002516-2984589-	1376	nuoD
	2984954_218_239_F		2984954_301_326_R
2961	PPS_NC002516-1915014-	311	PPS_NC002516-1915014-	1014	pps
	1915383_44_63_F		1915383_140_165_R
2962	PPS_NC002516-1915014-	365	PPS_NC002516-1915014-	1052	pps
	1915383_240_258_F		1915383_341_360_R
2963	TRP_NC002516-671831-	527	TRP_NC002516-671831-	1071	trpE
	672273_24_42_F		672273_131_150_R
2964	TRP_NC002516-671831-	490	TRP_NC002516-671831-	1182	trpE
	672273_261_282_F		672273_362_383_R

Claims as granted

15 claims

Classifications

5 codes

IPC · International Patent Classification

Section C — Chemistry; metallurgy

C12P19/34
C07H21/04

USPC · US Patent Classification

536/24.33435/6435/91.2

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