1 / 36

The Genomics Behind the Amerithrax Investigation

Institute for Genome Sciences University of Maryland School of Medicine. 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

vernon
Download Presentation

The Genomics Behind the Amerithrax Investigation

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Institute for Genome Sciences University of Maryland School of Medicine 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 21201

  2. 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 The Science behind the Amerithrax

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

  4. 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 The Amerithrax investigation

  5. 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 The Amerithrax investigation

  6. Institute for Genome Sciences University of Maryland School of Medicine B. anthracis Ames Ancestor: the reference 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

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

  8. Institute for Genome Sciences University of Maryland School of Medicine SNP analysis in B. anthracis genomes >ID642-16 (94989bp) 200731 c-->t 50878 COV: 16 QUAL: 31.5 NON-SYNONYMOUS (2): GGC - G ---> GAC - D (from start: 5 bp/848 bp) AMES : GAAGAAGCGCTAATAAAATGCCCATTACAAGACTCCCTTCG 200751 |||||||||||||||||||| |||||||||||||||||||| QUERY : GAAGAAGCGCTAATAAAATGTCCATTACAAGACTCCCTTCG 50898 ORF01519 transporter putative 50873 A 564 AAAAAAAAAAAAAAAA 35:36:35:35:36:36:36:36:35:27:36:36:36:36:37:36 50874 A 569 AAAAAAAAAAAAAAAA 34:36:35:35:36:36:36:36:35:35:36:36:36:36:35:36 50875 A 550 AAAAAAAAAAAAAAAA 18:36:35:35:36:36:36:36:34:36:36:35:36:34:35:36 50876 T 545 TTTTTTTTTTTTTTTT 13:36:36:35:36:36:36:34:35:38:35:34:36:34:37:34 50877 G 517 GGGGGGGGGGGGGGGG 17:35:35:35:34:37:36:36:32:26:34:34:33:36:22:35 *50878 T 504 TTTTTTTTTTTTTTTT 11:36:35:33:34:36:31:34:35:19:31:35:31:36:33:34 50879 C 559 CCCCCCCCCCCCCCCC 33:34:35:35:36:36:34:35:32:35:36:35:36:36:35:36 50880 C 515 CCCCCCCCCCCCCCC 34:35:35:36:34:36:36:29:34:36:35:35:36:35:29 50881 A 508 AAAAAAAAAAAAAAA 36:34:34:36:40:34:36:32:26:36:34:36:36:32:26 50882 T 513 TTTTTTTTTTTTTTT 36:35:35:36:35:36:36:36:25:36:37:35:36:29:30 50883 T 498 TTTTTTTTTTTTTTT 36:35:34:36:33:36:36:35:15:36:35:32:36:33:30 REFERENCE: B. anthracis Ames Ancestor 200726 A 334 AAAAAAAAAA 29:29:37:31:51:22:45:40:35:15 200727 A 362 AAAAAAAAAA 25:41:37:34:51:27:45:40:34:28 200728 A 347 AAAAAAAAAA 29:41:37:34:45:27:45:40:34:15 200729 T 341 TTTTTTTTTT 25:41:45:33:45:21:45:25:34:27 200730 G 336 GGGGGGGGGG 33:41:38:33:45:16:45:24:34:27 *200731 C 334 CCCCCCCCCC 33:37:40:37:45:16:18:45:35:28 200732 C 318 CCCCCCCCCC 33:37:40:38:45:18:4:45:35:23 200733 C 284 CCCCCCCCC 33:37:37:37:41:15:4:45:35 200734 A 307 AAAAAAAAA 27:45:37:37:51:21:4:51:34 200735 T 291 TTTTTTTTT 24:45:37:37:41:24:4:45:34 200736 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

  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? Institute for Genome Sciences University of Maryland School of Medicine The Amerithrax investigation

  10. Morphological variants analyzed Strategy - Discover genetic differences by comparing the genome sequences to Ames Ancestor. Institute for Genome Sciences University of Maryland School of Medicine

  11. Institute for Genome Sciences University of Maryland School of Medicine 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

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

  13. Morphological variants isolation

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

  15. 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 Morphological variants characteristics

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

  17. Morphological variants analyzed Strategy - Discover genetic differences by comparing the genome sequences to Ames Ancestor. Institute for Genome Sciences University of Maryland School of Medicine

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

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

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

  21. >ID161revcom-1 (295550bp) 5065092 T-->C 8836 COV: 9 QUAL: 43 AMES : CCAAATTCCTTCCTATTCTTTCTTATTTTACCGTTTCCTAT 5065112 |||||||||||||||||||| |||||||||||||||||||| B : CCAAATTCCTTCCTATTCTTCCTTATTTTACCGTTTCCTAT 8856 Intergenic 286720 T 373 TTTTTTTTT 47:40:47:38:36:36:47:40:42 286719 T 375 TTTTTTTTT 47:40:47:38:36:36:47:40:44 286718 C 388 CCCCCCCCC 47:45:47:38:36:36:45:47:47 286717 T 360 TTTTTTTTT 47:40:47:34:36:24:44:47:41 286716 T 387 TTTTTTTTT 47:40:47:38:36:36:49:47:47 *286715 C 387 CCCCCCCCC 41:44:47:38:36:36:49:49:47 286714 C 372 CCCCCCCCC 41:40:47:38:36:36:47:40:47 286713 T 365 TTTTTTTTT 40:40:47:32:36:36:47:40:47 286712 T 367 TTTTTTTTT 40:40:47:34:36:36:47:40:47 286711 A 380 AAAAAAAAA 44:45:47:38:36:36:47:40:47 286710 T 368 TTTTTTTTT 44:40:44:34:36:38:45:40:47 REFERENCE: B. anthracis Ames Ancestor 5065087 T 595 TTTTTTTTTTTTTTTTT 45:40:45:41:24:51:34:27:33:37:34:4:51:36:36:36:21 5065088 T 598 TTTTTTTTTTTTTTTTT 45:40:45:41:28:51:33:31:33:37:33:4:51:36:36:36:18 5065089 C 639 CCCCCCCCCCCCCCCCC 51:49:49:41:31:51:34:33:34:37:38:15:51:36:36:36:17 5065090 T 668 TTTTTTTTTTTTTTTTT 51:49:49:51:33:51:36:31:34:37:38:15:51:36:36:36:34 5065091 T 673 TTTTTTTTTTTTTTTTT 51:49:49:51:34:51:38:33:40:37:45:4:51:36:36:35:33 *5065092 T 654 TTTTTTTTTTTTTTTTT 51:45:45:51:34:51:33:36:40:37:45:4:51:36:36:35:24 5065093 C 628 CCCCCCCCCCCCCCCCC 45:36:45:51:31:51:33:36:37:37:40:15:45:36:35:35:20 5065094 T 614 TTTTTTTTTTTTTTTTT 45:36:38:51:30:51:31:36:36:37:40:4:45:34:36:36:28 5065095 T 593 TTTTTTTTTTTTTTTT- 45:36:45:45:31:51:31:38:36:37:34:15:45:39:36:29:0 5065096 A 607 AAAAAAAAAAAAAAAAA 45:36:45:45:31:51:28:38:33:37:34:18:41:39:34:34:18 5065097 T 642 TTTTTTTTTTTTTTTTTT 45:34:45:45:31:51:28:38:33:37:34:19:41:28:34:36:35:28 COV: 17 QUAL: 38.47 Institute for Genome Sciences University of Maryland School of Medicine Morphological variants B - NY Post Letter SNP found in each variant B analyzed

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

  23. >ID25-2 (934628bp) 2139122 g-->a 47177 COV: 9 CB_QVal: 352 QUAL: 39.11NON-SYNONYMOUS (2): TGG - W ---> TAG - _ (from start: 685 bp/1130 bp) AMES : CGTATCGAAAAAGGAAATGTGGAATGAATCAGAAAGTTTTT 2139142 C : CGTATCGAAAAAGGAAATGTAGAATGAATCAGAAAGTTTTT 47197 ORF05728 sensor histidine kinase 47172 A 369 AAAAAAAAA 47:47:47:47:44:47:19:36:35 47173 A 368 AAAAAAAAA 47:47:47:41:47:44:19:41:35 47174 T 373 TTTTTTTTT 47:47:47:36:47:47:21:47:34 47175 G 361 GGGGGGGGG 41:36:47:47:47:47:22:44:30 47176 T 358 TTTTTTTTT 47:41:47:47:44:47:17:38:30* 47177 A 352 AAAAAAAAA 47:41:49:41:36:47:27:38:26 47178 G 359 GGGGGGGGG 47:36:41:36:47:47:14:47:44 47179 A 351 AAAAAAAAA 36:47:47:44:41:36:20:47:33 47180 A 371 AAAAAAAAA 47:47:47:47:36:47:20:47:33 47181 T 365 TTTTTTTTT 36:47:47:47:41:47:20:47:33 47182 G 364 GGGGGGGGG 47:47:41:36:41:47:23:47:35 REFERENCE: B. anthracis Ames Porton 2139117 A 319 AAAAAAAAAA 4:36:37:36:36:36:31:34:36:33 2139118 A 299 AAAAAAAAAA 4:36:37:36:36:36:28:29:36:21 2139119 T 290 TTTTTTTTTT 4:34:36:36:36:28:28:24:36:28 2139120 G 308 GGGGGGGGGG 18:34:36:36:36:27:28:26:36:31 2139121 T 291 TTTTTTTTTT 4:34:36:35:36:29:28:22:36:31 * 2139122 G 311 GGGGGGGGGG 4:34:36:36:36:34:28:33:36:34 2139123 G 309 GGGGGGGGGG 4:35:37:36:36:36:20:34:36:35 2139124 A 309 AAAAAAAAAA 4:34:31:36:36:36:32:29:36:35 2139125 A 295 AAAAAAAAAA 4:34:31:36:36:36:34:17:34:33 2139126 T 317 TTTTTTTTTT 15:35:29:34:36:34:36:23:40:35 2139127 G 313 GGGGGGGGGG 4:35:34:34:36:36:33:32:35:34 COV: 10 CBQUAL: 311 QUAL: 31.1 Institute for Genome Sciences University of Maryland School of Medicine Morphological variants C - Leahy Letter SNP results in Amber stop codon and a truncated protein

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

  25. 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------------------- 181 • Wild Type 201 SYLDIISHELDQLNFRISQFLFVSKKEMWNESESFWLNDLFQDIIQFLYP 250 • Morphotype D 182 -------------------------------------------------- 182 • Wild Type 251 SLVNANVSIEKNLPYPIPLTGYRSEVRQVFLNILMNSIDALESMKEERKI 300 • Morphotype D 182 -----------------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 Morphological variants C/D - Leahy Letter

  26. INDEL-REF-1:172510 21 bp ID3 (12311 bp) INS on REFGBAAA0205 response regulator putative R: ATCAATATATGCTTGATAGTTTAAGTATTGGAAAAGATAGTTTTGATAAAGTAGATTCACT GE: ATCAATATATGCTTGATAGTT------------------------------------------TTGATAAAGTAGATTCACTG Institute for Genome Sciences University of Maryland School of Medicine Morphological variants E - Leahy Letter

  27. GBAAA0205 1 MIVSVKGNEQITKMLNDWYIEIRARHVGKAHNLKLEIDQKIHNIEEDQNL 50 LL18 1 MIVSVKGNEQITKMLNDWYIEIRARHVGKAHNLKLEIDQKIHNIEEDQNL 50 LL19 1 MIVSVKGNEQITKMLNDWYIEIRARHVGKAHNLKLEIDQKIHNIEEDQNL 50 ************************************************** GBAAA0205 51 LLYYALLDFRHQYMLDSLSIGKDSFDKVDSLGVPADQFLQYYYHFFKAIH 100 LL18 51 LLYYALLDFRHQYMLD-------SFDKVDSLGVPADQFLQYYYHFFKAIH 93 LL19 51 LLYYALLDFRHQYMLDS---LKDSFDKVDSLGVPADQFLQYYYHFFKAIH 97 **************** *************************** GBAAA0205 101 SNITGDFTSAKEHYNQAELLLKHIPDEIEHAEFRFKLSTFHYHIYKPLAA 150 LL18 94 SNITGDFTSAKEHYNQAELLLKHIPDEIEHAEFRFKLSTFHYHIYKPLAA 143 LL19 98 SNITGDFTSAKEHYNQAELLLKHIPDEIEHAEFRFKLSTFHYHIYKPLAA 147 ************************************************** GBAAA0205 151 IKEATKAKDIFKKHAGYETNIGLCDNLIGLACTHLKQFEEAEEHFITAIN 200 LL18 144 IKEATKAKDIFKKHAGYETNIGLCDNLIGLACTHLKQFEEAEEHFITAIN 193 LL19 148 IKEATKAKDIFKKHAGYETNIGLCDNLIGLACTHLKQFEEAEEHFITAIN 197 ************************************************** GBAAA0205 201 TFKKSGKEKNITFVRHNLGLMYSGQNLSELAIRYLSEVTQELPKDYKAIF 250 LL18 194 TFKKSGKEKNITFVRHNLGLMYSGQNLSELAIRYLSEVTQELPKDYKAIF 243 LL19 198 TFKKSGKEKNITFVRHNLGLMYSGQNLSELAIRYLSEVTQELPKDYKAIF 247 ************************************************** GBAAA0205 251 IKAREHMKIGESKETYNLIVKGLEICKELKNEEYEHHFLILEKLNQKVSA 300 LL18 244 IKAREHMKIGESKETYNLIVKGLEICKELKNEEYEHHFLILEKLNQKVSA 293 LL19 248 IKAREHMKIGESKETYNLIVKGLEICKELKNEEYEHHFLILEKLNQKVSA 297 ************************************************** GBAAA0205 301 DELEKTIKTGISYFKRENLHEYVQEYAKKLAVLFHQENNRSKASDYFYLS 350 LL18 294 DELEKTIKTGISYFKRENLHEYVQEYAKKLAVLFHQENNRSKASDYFYLS 343 LL19 298 DELEKTIKTGISYFKRENLHEYVQEYAKKLAVLFHQENNRSKASDYFYLS 347 ************************************************** GBAAA0205 351 HQAEEQNFEKEALK* 365 LL18 344 HQAEEQNFEKEALK* 358 LL19 348 HQAEEQNFEKEALK* 362 *************** Morphological variants E - Leahy Letter

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

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

  30. Institute for Genome Sciences University of Maryland School of Medicine Making the match 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

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

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

  33. 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. Population genetics - the unique signature

  34. Crime committed Fall 2001 Variants identified 2001-2002 Variants genetically characterized 2002-2003 Assay developed and validated 2003-2006 All repository collections and analysis completed mid 2007 Preparation for indictment 2008 Unfortunate and tragic dealth of the suspect 2008 Timeline

  35. Making the match and attribution 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

  36. Institute for Genome Sciences University of Maryland School of Medicine Acknowledgments 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 USAMRIID Patricia Worsham NAU Paul Keim NSF Rita Colwell Maryanna Henckart NIH/NIAID Maria Giovanni Federal Bureau of Investigation (J-FBI-02-016) National Institute of Allergy and Infectious Diseases (N01-AI15447) National Science Foundation

More Related