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The Genomics Behind the Amerithrax Investigation Claire M. Fraser-Liggett, Jacques Ravel, David A. Rasko Institute for Genome Sciences Department of Microbiology.

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Presentation on theme: "The Genomics Behind the Amerithrax Investigation Claire M. Fraser-Liggett, Jacques Ravel, David A. Rasko Institute for Genome Sciences Department of Microbiology."— Presentation transcript:

1 The Genomics Behind the Amerithrax Investigation Claire M. Fraser-Liggett, Jacques Ravel, David A. Rasko Institute for Genome Sciences Department of Microbiology and Immunology University of Maryland School of Medicine Baltimore, MD Institute for Genome Sciences University of Maryland School of Medicine

2 The Science behind the Amerithrax Obtained B. anthracis spore preparations (powder) from the letters and other sources (buildings, victims...) Goal: establish origin or a linkages between events, people, places and processes Finding unique features using traditional DNA-based analysis Institute for Genome Sciences University of Maryland School of Medicine

3 New York post letter spore preparation The Science behind the Amerithrax Leahy letter spore preparation Institute for Genome Sciences University of Maryland School of Medicine

4 The Amerithrax investigation Traditional genotyping methods did not achieve high discriminative power Would the genome sequence of these B. anthracis isolates differ, and could this information be used for attribution? All isolates were identified as B. anthracis Ames (strain) Institute for Genome Sciences University of Maryland School of Medicine

5 The Amerithrax investigation Obtained DNA from B. anthracis recovered from the spinal fluid of Mr. Stevens Sequenced the genome of B. anthracis Florida Need to compare the genome sequence to that of an appropriate reference Porton Down strain was not an appropriate reference Sequenced the genome of B. anthracis Ames Ancestor Institute for Genome Sciences University of Maryland School of Medicine

6 fully virulent (pXO1, pXO2) isolated from a dead Beefmaster heifer in Texas in 1981 used worldwide for vaccine challenge studies sequenced and closed (AE017334) chromosome : 5,227,419 bp pXO1 : 181,677 bp pXO2 : 94,830 bp B. anthracis Ames Ancestor: the reference Institute for Genome Sciences University of Maryland School of Medicine

7 SNP analysis in B. anthracis genomes Institute for Genome Sciences University of Maryland School of Medicine

8 SNP analysis in B. anthracis genomes >ID (94989bp) c-->t COV: 16 QUAL: 31.5 NON-SYNONYMOUS (2): GGC - G ---> GAC - D (from start: 5 bp/848 bp) AMES : GAAGAAGCGCTAATAAAATGCCCATTACAAGACTCCCTTCG |||||||||||||||||||| |||||||||||||||||||| QUERY : GAAGAAGCGCTAATAAAATGTCCATTACAAGACTCCCTTCG ORF01519 transporter putative A 564 AAAAAAAAAAAAAAAA 35:36:35:35:36:36:36:36:35:27:36:36:36:36:37: A 569 AAAAAAAAAAAAAAAA 34:36:35:35:36:36:36:36:35:35:36:36:36:36:35: A 550 AAAAAAAAAAAAAAAA 18:36:35:35:36:36:36:36:34:36:36:35:36:34:35: T 545 TTTTTTTTTTTTTTTT 13:36:36:35:36:36:36:34:35:38:35:34:36:34:37: G 517 GGGGGGGGGGGGGGGG 17:35:35:35:34:37:36:36:32:26:34:34:33:36:22:35 *50878 T 504 TTTTTTTTTTTTTTTT 11:36:35:33:34:36:31:34:35:19:31:35:31:36:33: C 559 CCCCCCCCCCCCCCCC 33:34:35:35:36:36:34:35:32:35:36:35:36:36:35: C 515 CCCCCCCCCCCCCCC 34:35:35:36:34:36:36:29:34:36:35:35:36:35: A 508 AAAAAAAAAAAAAAA 36:34:34:36:40:34:36:32:26:36:34:36:36:32: T 513 TTTTTTTTTTTTTTT 36:35:35:36:35:36:36:36:25:36:37:35:36:29: T 498 TTTTTTTTTTTTTTT 36:35:34:36:33:36:36:35:15:36:35:32:36:33:30 REFERENCE: B. anthracis Ames Ancestor A 334 AAAAAAAAAA 29:29:37:31:51:22:45:40:35: A 362 AAAAAAAAAA 25:41:37:34:51:27:45:40:34: A 347 AAAAAAAAAA 29:41:37:34:45:27:45:40:34: T 341 TTTTTTTTTT 25:41:45:33:45:21:45:25:34: G 336 GGGGGGGGGG 33:41:38:33:45:16:45:24:34:27 * C 334 CCCCCCCCCC 33:37:40:37:45:16:18:45:35: C 318 CCCCCCCCCC 33:37:40:38:45:18:4:45:35: C 284 CCCCCCCCC 33:37:37:37:41:15:4:45: A 307 AAAAAAAAA 27:45:37:37:51:21:4:51: T 291 TTTTTTTTT 24:45:37:37:41:24:4:45: T 293 TTTTTTATT 24:45:45:37:41:24:4:45:35 COV: 10 QUAL: 33.4 Access to genome coverage, sequence quality scores, and sequence in a single file Institute for Genome Sciences University of Maryland School of Medicine

9 Surprisingly, no differences were found between Florida isolate and Ames ancestor sequences No differences in over 5 million base pairs - these two genome sequences were identical What did it mean? Could genomics still help in making a match? The Amerithrax investigation Institute for Genome Sciences University of Maryland School of Medicine

10 Morphological variants analyzed OriginTypeSequencing Leahy LetterWild Type12 X Leahy LetterAClosed Leahy LetterB12 X Leahy LetterC12 X Leahy LetterE/Opaque12 X NY Post LetterWild TypeClosed NY Post LetterA12 X NY Post LetterBClosed Strategy - Discover genetic differences by comparing the genome sequences to Ames Ancestor. Institute for Genome Sciences University of Maryland School of Medicine

11 Wild Type isolates from letters When the closed genome of B. anthracis Ames Ancestor was compared to the closed genome of a wild type isolate of the NY Post letter and the draft genome of a wild type isolate of the Leahy Letter, no polymorphisms were found. The genome sequences were 100% identical at each of the 5,227,419 bp compared. Accessing differences through a combination of microbiological analyses and whole genome sequencing Institute for Genome Sciences University of Maryland School of Medicine

12 Morphological variants isolation Visual differential tests Colony morphology, color, sporulation efficiency for each spore samples Institute for Genome Sciences University of Maryland School of Medicine

13 Morphological variants isolation

14 Morphological variants characteristics Institute for Genome Sciences University of Maryland School of Medicine

15 Morphological variants characteristics All four morphotypes were found in the spores preparations recovered from each of the four letters This was a key step in the investigation, as it linked the two events in New York and Washington DC Physical examination of the spore preparation originally showed different quality in the New York and Washington DC letters Institute for Genome Sciences University of Maryland School of Medicine

16 Morphological variants isolation Could the population composition be the ID that can be used to make a match? Genomics Institute for Genome Sciences University of Maryland School of Medicine

17 Morphological variants analyzed OriginTypeSequencing Leahy LetterWild Type12 X Leahy LetterAClosed Leahy LetterB12 X Leahy LetterC12 X Leahy LetterE/Opaque12 X NY Post LetterWild TypeClosed NY Post LetterA12 X NY Post LetterBClosed Strategy - Discover genetic differences by comparing the genome sequences to Ames Ancestor. Institute for Genome Sciences University of Maryland School of Medicine

18 Morphological variants A Institute for Genome Sciences University of Maryland School of Medicine

19 Morphological variants A Leahy Letter A Ames Ancestor PCR assay Institute for Genome Sciences University of Maryland School of Medicine

20 Morphological variants A NY Post Letter A Leahy Letter A Daschle Letter A ORF1573: polysaccharide deacetylase ORF1572: conserved hypothetical protein Ames Ancestor These three morphotypes A are morphologically identical, but genetically different. All three are present in the letters Institute for Genome Sciences University of Maryland School of Medicine

21 Morphological variants B - NY Post Letter >ID161revcom-1 (295550bp) T-->C 8836 COV: 9 QUAL: 43 AMES : CCAAATTCCTTCCTATTCTTTCTTATTTTACCGTTTCCTAT |||||||||||||||||||| |||||||||||||||||||| B : CCAAATTCCTTCCTATTCTTCCTTATTTTACCGTTTCCTAT 8856 Intergenic T 373 TTTTTTTTT 47:40:47:38:36:36:47:40: T 375 TTTTTTTTT 47:40:47:38:36:36:47:40: C 388 CCCCCCCCC 47:45:47:38:36:36:45:47: T 360 TTTTTTTTT 47:40:47:34:36:24:44:47: T 387 TTTTTTTTT 47:40:47:38:36:36:49:47:47 * C 387 CCCCCCCCC 41:44:47:38:36:36:49:49: C 372 CCCCCCCCC 41:40:47:38:36:36:47:40: T 365 TTTTTTTTT 40:40:47:32:36:36:47:40: T 367 TTTTTTTTT 40:40:47:34:36:36:47:40: A 380 AAAAAAAAA 44:45:47:38:36:36:47:40: T 368 TTTTTTTTT 44:40:44:34:36:38:45:40:47 REFERENCE: B. anthracis Ames Ancestor T 595 TTTTTTTTTTTTTTTTT 45:40:45:41:24:51:34:27:33:37:34:4:51:36:36:36: T 598 TTTTTTTTTTTTTTTTT 45:40:45:41:28:51:33:31:33:37:33:4:51:36:36:36: C 639 CCCCCCCCCCCCCCCCC 51:49:49:41:31:51:34:33:34:37:38:15:51:36:36:36: T 668 TTTTTTTTTTTTTTTTT 51:49:49:51:33:51:36:31:34:37:38:15:51:36:36:36: T 673 TTTTTTTTTTTTTTTTT 51:49:49:51:34:51:38:33:40:37:45:4:51:36:36:35:33 * T 654 TTTTTTTTTTTTTTTTT 51:45:45:51:34:51:33:36:40:37:45:4:51:36:36:35: C 628 CCCCCCCCCCCCCCCCC 45:36:45:51:31:51:33:36:37:37:40:15:45:36:35:35: T 614 TTTTTTTTTTTTTTTTT 45:36:38:51:30:51:31:36:36:37:40:4:45:34:36:36: T 593 TTTTTTTTTTTTTTTT- 45:36:45:45:31:51:31:38:36:37:34:15:45:39:36:29: A 607 AAAAAAAAAAAAAAAAA 45:36:45:45:31:51:28:38:33:37:34:18:41:39:34:34: T 642 TTTTTTTTTTTTTTTTTT 45:34:45:45:31:51:28:38:33:37:34:19:41:28:34:36:35:28 COV: 17 QUAL: SNP found in each variant B analyzed Institute for Genome Sciences University of Maryland School of Medicine

22 Morphological variants B KinB spoOF Institute for Genome Sciences University of Maryland School of Medicine

23 Morphological variants C - Leahy Letter >ID25-2 (934628bp) g-->a COV: 9 CB_QVal: 352 QUAL: 39.11NON-SYNONYMOUS (2): TGG - W ---> TAG - _ (from start: 685 bp/1130 bp) AMES : CGTATCGAAAAAGGAAATGTGGAATGAATCAGAAAGTTTTT C : CGTATCGAAAAAGGAAATGTAGAATGAATCAGAAAGTTTTT ORF05728sensor histidine kinase A 369AAAAAAAAA 47:47:47:47:44:47:19:36: A 368 AAAAAAAAA 47:47:47:41:47:44:19:41: T 373 TTTTTTTTT 47:47:47:36:47:47:21:47: G 361 GGGGGGGGG 41:36:47:47:47:47:22:44: T 358 TTTTTTTTT 47:41:47:47:44:47:17:38:30* A 352 AAAAAAAAA 47:41:49:41:36:47:27:38: G 359 GGGGGGGGG 47:36:41:36:47:47:14:47: A 351 AAAAAAAAA 36:47:47:44:41:36:20:47: A 371 AAAAAAAAA 47:47:47:47:36:47:20:47: T 365 TTTTTTTTT 36:47:47:47:41:47:20:47: G 364 GGGGGGGGG 47:47:41:36:41:47:23:47:35 REFERENCE: B. anthracis Ames Porton A319AAAAAAAAAA4:36:37:36:36:36:31:34:36: A299AAAAAAAAAA4:36:37:36:36:36:28:29:36: T290TTTTTTTTTT4:34:36:36:36:28:28:24:36: G308GGGGGGGGGG18:34:36:36:36:27:28:26:36: T291TTTTTTTTTT4:34:36:35:36:29:28:22:36:31 * G311GGGGGGGGGG4:34:36:36:36:34:28:33:36: G309GGGGGGGGGG4:35:37:36:36:36:20:34:36: A309AAAAAAAAAA4:34:31:36:36:36:32:29:36: A295AAAAAAAAAA4:34:31:36:36:36:34:17:34: T317TTTTTTTTTT15:35:29:34:36:34:36:23:40: G313GGGGGGGGGG4:35:34:34:36:36:33:32:35:34 COV: 10 CBQUAL: 311 QUAL: 31.1 SNP results in Amber stop codon and a truncated protein Institute for Genome Sciences University of Maryland School of Medicine

24 Morphological variants C/D - Leahy Letter Institute for Genome Sciences University of Maryland School of Medicine

25 Morphological variants C/D - Leahy Letter Wild Type 1 MEMEGMEVFPIDKDIKEVFCSHLKNNRHQFVENWKNKMIISDKDPFRLEV 50 Morphotype D 1 MEMEGMEVFPIDKDIKEVFCSHLKNNRHQFVENWKNKMIISDKDPFRLEV 50 Wild Type 51 VQNGEDLLEFIIELIMEEKDINYLQPLCEKIAIERAGADANIGDFVYNAN 100 orphotype D 51 VQNGEDLLEFIIELIMEEKDINYLQPLCEKIAIERAGADANIGDFVYNAN 100 Wild Type 101 VGRNELFEAMCELDVSARELKPIMNQIHTCFDKLIYYTVLKYSEIISRNL 150 Morphotype D 101 VGRNELFEAMCELDVSARELKPIMNQIHTCFDKLIYYTVLKYSEIISRNL 150 Wild Type 151 EEKQQYINETHKERLTILGQMSASFVHEFRNPLTSIMGFVKLLKADHPSL 200 Morphotype D 151 EEKQQYINETHKERLTILGQMSASFVHEFRN Wild Type 201 SYLDIISHELDQLNFRISQFLFVSKKEMWNESESFWLNDLFQDIIQFLYP 250 Morphotype D Wild Type 251 SLVNANVSIEKNLPYPIPLTGYRSEVRQVFLNILMNSIDALESMKEERKI 300 Morphotype D PLTGYRSEVRQVFLNILMNSIDALESMKEERKI 214 Wild Type 301 IIDVFEEDQSIRIVIKNNGPMIPAENVETIFEPFVTTKKLGTGIGLFVCK 350 Morphotype D 215 IIDVFEEDQSIRIVIKNNGPMIPAENVETIFEPFVTTKKLGTGIGLFVCK 264 Wild Type 351 QIVEKHNGSIMCRSDDDWTEFQIAFQK* 378 Morphotype D 265 QIVEKHNGSIMCRSDDDWTEFQIAFQK* 292 Institute for Genome Sciences University of Maryland School of Medicine

26 Morphological variants E - Leahy Letter INDEL-REF-1: bp ID3 (12311 bp) INS on REFGBAAA0205response regulator putative R: ATCAATATATGCTTGATAGTTTAAGTATTGGAAAAGATAGTTTTGATAAAGTAGATTCACT GE: ATCAATATATGCTTGATAGTT TTGATAAAGTAGATTCACTG Institute for Genome Sciences University of Maryland School of Medicine

27 Morphological variants E - Leahy Letter GBAAA MIVSVKGNEQITKMLNDWYIEIRARHVGKAHNLKLEIDQKIHNIEEDQNL 50 LL18 1 MIVSVKGNEQITKMLNDWYIEIRARHVGKAHNLKLEIDQKIHNIEEDQNL 50 LL19 1 MIVSVKGNEQITKMLNDWYIEIRARHVGKAHNLKLEIDQKIHNIEEDQNL 50 ************************************************** GBAAA LLYYALLDFRHQYMLDSLSIGKDSFDKVDSLGVPADQFLQYYYHFFKAIH 100 LL18 51 LLYYALLDFRHQYMLD SFDKVDSLGVPADQFLQYYYHFFKAIH 93 LL19 51 LLYYALLDFRHQYMLDS---LKDSFDKVDSLGVPADQFLQYYYHFFKAIH 97 **************** *************************** GBAAA SNITGDFTSAKEHYNQAELLLKHIPDEIEHAEFRFKLSTFHYHIYKPLAA 150 LL18 94 SNITGDFTSAKEHYNQAELLLKHIPDEIEHAEFRFKLSTFHYHIYKPLAA 143 LL19 98 SNITGDFTSAKEHYNQAELLLKHIPDEIEHAEFRFKLSTFHYHIYKPLAA 147 ************************************************** GBAAA IKEATKAKDIFKKHAGYETNIGLCDNLIGLACTHLKQFEEAEEHFITAIN 200 LL IKEATKAKDIFKKHAGYETNIGLCDNLIGLACTHLKQFEEAEEHFITAIN 193 LL IKEATKAKDIFKKHAGYETNIGLCDNLIGLACTHLKQFEEAEEHFITAIN 197 ************************************************** GBAAA TFKKSGKEKNITFVRHNLGLMYSGQNLSELAIRYLSEVTQELPKDYKAIF 250 LL TFKKSGKEKNITFVRHNLGLMYSGQNLSELAIRYLSEVTQELPKDYKAIF 243 LL TFKKSGKEKNITFVRHNLGLMYSGQNLSELAIRYLSEVTQELPKDYKAIF 247 ************************************************** GBAAA IKAREHMKIGESKETYNLIVKGLEICKELKNEEYEHHFLILEKLNQKVSA 300 LL IKAREHMKIGESKETYNLIVKGLEICKELKNEEYEHHFLILEKLNQKVSA 293 LL IKAREHMKIGESKETYNLIVKGLEICKELKNEEYEHHFLILEKLNQKVSA 297 ************************************************** GBAAA DELEKTIKTGISYFKRENLHEYVQEYAKKLAVLFHQENNRSKASDYFYLS 350 LL DELEKTIKTGISYFKRENLHEYVQEYAKKLAVLFHQENNRSKASDYFYLS 343 LL DELEKTIKTGISYFKRENLHEYVQEYAKKLAVLFHQENNRSKASDYFYLS 347 ************************************************** GBAAA HQAEEQNFEKEALK* 365 LL HQAEEQNFEKEALK* 358 LL HQAEEQNFEKEALK* 362 ***************

28 Morphotypes/genotypes VariantMutationLocusFunction ADuplication rRNA-D, Polysaccharide deacetylase and GBAA0151 unknown BSNP Upstream spoOF Initation of Sporulation C/DSNP/INDEL Sensor histidine kinase Phosphorylation of SpoOF/SpoOA EINDEL (9/21 bp) pXO1 response regulator Dephosphorylation of SpoOF Institute for Genome Sciences University of Maryland School of Medicine

29 Morphotypes/genotypes Institute for Genome Sciences University of Maryland School of Medicine

30 Develop and validate quantitative PCR assays for each of the genotypes (genetic variation) Screen a repository of nearly 1,100 samples All four mutations were only found in one source RMR-1029 USMARIID No other samples with three hits, a few with two or one hits Making the match Institute for Genome Sciences University of Maryland School of Medicine

31 RMR-1029 Institute for Genome Sciences University of Maryland School of Medicine

32 Population genetics - the unique signature Science Magazine - August 2008

33 Population genetics - the unique signature Population genetics - a minor subpopulation is unique to the spore preparation recovered from the letters. These polymorphisms could be used to screen spore preparations, not single B. anthracis colonies on a plate. Link the NY and Washington DC letters.

34 Timeline Crime committed Fall 2001 Variants identified Variants genetically characterized Assay developed and validated All repository collections and analysis completed mid 2007 Preparation for indictment 2008 Unfortunate and tragic dealth of the suspect 2008

35 The scientific investigation was key in identification of the source material More traditional police investigation was required to narrow down the list of potential perpetrators Microbial Forensics - A new field (attribution/exclusion) - Different scientific standards Making the match and attribution

36 Acknowledgments National Institute of Allergy and Infectious Diseases (N01-AI15447) National Institute of Allergy and Infectious Diseases (N01-AI15447) IGS/TIGR David Rasko Lingxia Jiang Regina Cer Steven Salzberg Claire Fraser-Liggett IGS/TIGR David Rasko Lingxia Jiang Regina Cer Steven Salzberg Claire Fraser-Liggett FBI Jason Bannan Mark Wilson Richard Langham Scott Stanley Scott Decker Matt Feinberg FBI Jason Bannan Mark Wilson Richard Langham Scott Stanley Scott Decker Matt Feinberg USAMRIID Patricia Worsham USAMRIID Patricia Worsham Federal Bureau of Investigation (J-FBI ) NIH/NIAID Maria Giovanni NIH/NIAID Maria Giovanni NSF Rita Colwell Maryanna Henckart NSF Rita Colwell Maryanna Henckart National Science Foundation NAU Paul Keim NAU Paul Keim Institute for Genome Sciences University of Maryland School of Medicine


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