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The Genomics Behind the Amerithrax Investigation

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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 HOW WE WERE ABLE TO IDENTIFY SPECIFIC GENETIC SIGNATURE THAT COULD LINK THE LETTERS AND BE USED FOR ATTRIBUTION ARE THEY THE SAME? CHALLENGE IN THE AMERITHRAX INVESTIGATION PRINCIPLES DO NOT APPLY TO ALL ORGANISMS - IN VIEW OF WHAT CLAIRE JUST TALKED ABOUT. COMES OF AGE - NEW ON THE FLY LESSON 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 The Science behind the Amerithrax
New York post letter spore preparation 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 All isolates were identified as B. anthracis Ames (strain) Would the genome sequence of these B. anthracis isolates differ, and could this information be used for attribution? 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 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 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 50898 ORF transporter putative A AAAAAAAAAAAAAAAA 35:36:35:35:36:36:36:36:35:27:36:36:36:36:37:36 A AAAAAAAAAAAAAAAA 34:36:35:35:36:36:36:36:35:35:36:36:36:36:35:36 A AAAAAAAAAAAAAAAA 18:36:35:35:36:36:36:36:34:36:36:35:36:34:35:36 T TTTTTTTTTTTTTTTT 13:36:36:35:36:36:36:34:35:38:35:34:36:34:37:34 G GGGGGGGGGGGGGGGG 17:35:35:35:34:37:36:36:32:26:34:34:33:36:22:35 * T TTTTTTTTTTTTTTTT 11:36:35:33:34:36:31:34:35:19:31:35:31:36:33:34 C CCCCCCCCCCCCCCCC 33:34:35:35:36:36:34:35:32:35:36:35:36:36:35:36 C CCCCCCCCCCCCCCC 34:35:35:36:34:36:36:29:34:36:35:35:36:35:29 A AAAAAAAAAAAAAAA 36:34:34:36:40:34:36:32:26:36:34:36:36:32:26 T TTTTTTTTTTTTTTT 36:35:35:36:35:36:36:36:25:36:37:35:36:29:30 T TTTTTTTTTTTTTTT 36:35:34:36:33:36:36:35:15:36:35:32:36:33:30 REFERENCE: B. anthracis Ames Ancestor A AAAAAAAAAA 29:29:37:31:51:22:45:40:35:15 A AAAAAAAAAA 25:41:37:34:51:27:45:40:34:28 A AAAAAAAAAA 29:41:37:34:45:27:45:40:34:15 T TTTTTTTTTT 25:41:45:33:45:21:45:25:34:27 G GGGGGGGGGG 33:41:38:33:45:16:45:24:34:27 * C CCCCCCCCCC 33:37:40:37:45:16:18:45:35:28 C CCCCCCCCCC 33:37:40:38:45:18:4:45:35:23 C CCCCCCCCC 33:37:37:37:41:15:4:45:35 A AAAAAAAAA 27:45:37:37:51:21:4:51:34 T TTTTTTTTT 24:45:37:37:41:24:4:45:34 T 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 The Amerithrax investigation
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

10 Morphological variants analyzed
Origin Type Sequencing Leahy Letter Wild Type 12 X A Closed B C E/Opaque NY Post Letter 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
Genomics JigSaw Puzzle analogy Could the population composition be the ID that can be used to make a match? Institute for Genome Sciences University of Maryland School of Medicine

17 Morphological variants analyzed
Origin Type Sequencing Leahy Letter Wild Type 12 X A Closed B C E/Opaque NY Post Letter 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
NO POLYMORPHIC SITE WERE DETECTED AT FIRST Institute for Genome Sciences University of Maryland School of Medicine

19 Morphological variants A
Ames Ancestor Leahy Letter A PCR reaction - point away from each others PCR assay Institute for Genome Sciences University of Maryland School of Medicine

20 Morphological variants A
Ames Ancestor Daschle Letter A Leahy Letter A CAN’T EXPLAIN THE PHENOTYPE WITH THE GENOTYPE NY Post Letter A ORF1573: polysaccharide deacetylase ORF1572: conserved hypothetical protein 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 TTTTTTTTT :40:47:38:36:36:47:40:42 T TTTTTTTTT :40:47:38:36:36:47:40:44 C CCCCCCCCC :45:47:38:36:36:45:47:47 T TTTTTTTTT :40:47:34:36:24:44:47:41 T TTTTTTTTT :40:47:38:36:36:49:47:47 * C CCCCCCCCC :44:47:38:36:36:49:49:47 C CCCCCCCCC :40:47:38:36:36:47:40:47 T TTTTTTTTT :40:47:32:36:36:47:40:47 T TTTTTTTTT :40:47:34:36:36:47:40:47 A AAAAAAAAA :45:47:38:36:36:47:40:47 T TTTTTTTTT :40:44:34:36:38:45:40:47 REFERENCE: B. anthracis Ames Ancestor T TTTTTTTTTTTTTTTTT 45:40:45:41:24:51:34:27:33:37:34:4:51:36:36:36:21 T TTTTTTTTTTTTTTTTT 45:40:45:41:28:51:33:31:33:37:33:4:51:36:36:36:18 C CCCCCCCCCCCCCCCCC 51:49:49:41:31:51:34:33:34:37:38:15:51:36:36:36:17 T TTTTTTTTTTTTTTTTT 51:49:49:51:33:51:36:31:34:37:38:15:51:36:36:36:34 T TTTTTTTTTTTTTTTTT 51:49:49:51:34:51:38:33:40:37:45:4:51:36:36:35:33 * T TTTTTTTTTTTTTTTTT 51:45:45:51:34:51:33:36:40:37:45:4:51:36:36:35:24 C CCCCCCCCCCCCCCCCC 45:36:45:51:31:51:33:36:37:37:40:15:45:36:35:35:20 T TTTTTTTTTTTTTTTTT 45:36:38:51:30:51:31:36:36:37:40:4:45:34:36:36:28 T TTTTTTTTTTTTTTTT- 45:36:45:45:31:51:31:38:36:37:34:15:45:39:36:29:0 A AAAAAAAAAAAAAAAAA 45:36:45:45:31:51:28:38:33:37:34:18:41:39:34:34:18 T TTTTTTTTTTTTTTTTTT 45:34:45:45:31:51:28:38:33:37:34:19:41:28:34:36:35:28 COV: 17 QUAL: 38.47 PCR/sequencing based assay aimed at amplifying and resequencing a 466 bp sequence surrounding the region SNP found in each variant B analyzed Institute for Genome Sciences University of Maryland School of Medicine

22 Morphological variants B
spoOF KinB NO POLYMORPHIC SITE WERE DETECTED AT FIRST 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 47197 ORF05728 sensor histidine kinase A AAAAAAAAA :47:47:47:44:47:19:36:35 A AAAAAAAAA :47:47:41:47:44:19:41:35 T TTTTTTTTT :47:47:36:47:47:21:47:34 G GGGGGGGGG :36:47:47:47:47:22:44:30 T TTTTTTTTT :41:47:47:44:47:17:38:30* A AAAAAAAAA :41:49:41:36:47:27:38:26 G GGGGGGGGG :36:41:36:47:47:14:47:44 A AAAAAAAAA :47:47:44:41:36:20:47:33 A AAAAAAAAA :47:47:47:36:47:20:47:33 T TTTTTTTTT :47:47:47:41:47:20:47:33 G GGGGGGGGG :47:41:36:41:47:23:47:35 REFERENCE: B. anthracis Ames Porton A 319 AAAAAAAAAA 4:36:37:36:36:36:31:34:36:33 A 299 AAAAAAAAAA 4:36:37:36:36:36:28:29:36:21 T 290 TTTTTTTTTT 4:34:36:36:36:28:28:24:36:28 G 308 GGGGGGGGGG 18:34:36:36:36:27:28:26:36:31 T 291 TTTTTTTTTT 4:34:36:35:36:29:28:22:36:31 * G 311 GGGGGGGGGG 4:34:36:36:36:34:28:33:36:34 G 309 GGGGGGGGGG 4:35:37:36:36:36:20:34:36:35 A 309 AAAAAAAAAA 4:34:31:36:36:36:32:29:36:35 A 295 AAAAAAAAAA 4:34:31:36:36:36:34:17:34:33 T 317 TTTTTTTTTT 15:35:29:34:36:34:36:23:40:35 G 313 GGGGGGGGGG 4:35:34:34:36:36:33:32:35:34 COV: 10 CBQUAL: 311 QUAL: 31.1 PCR/sequencing based assay aimed at amplifying and resequencing a 533 bp sequence surrounding the region 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
PCR/sequencing based assay aimed at amplifying and resequencing a 533 bp sequence surrounding the region 264 bp in frame deletion. Institute for Genome Sciences University of Maryland School of Medicine

25 Morphological variants C/D - Leahy Letter
Wild Type MEMEGMEVFPIDKDIKEVFCSHLKNNRHQFVENWKNKMIISDKDPFRLEV 50 Morphotype D 1 MEMEGMEVFPIDKDIKEVFCSHLKNNRHQFVENWKNKMIISDKDPFRLEV 50 Wild Type VQNGEDLLEFIIELIMEEKDINYLQPLCEKIAIERAGADANIGDFVYNAN 100 orphotype D VQNGEDLLEFIIELIMEEKDINYLQPLCEKIAIERAGADANIGDFVYNAN 100 Wild Type VGRNELFEAMCELDVSARELKPIMNQIHTCFDKLIYYTVLKYSEIISRNL 150 Morphotype D VGRNELFEAMCELDVSARELKPIMNQIHTCFDKLIYYTVLKYSEIISRNL 150 Wild Type EEKQQYINETHKERLTILGQMSASFVHEFRNPLTSIMGFVKLLKADHPSL 200 Morphotype D EEKQQYINETHKERLTILGQMSASFVHEFRN Wild Type SYLDIISHELDQLNFRISQFLFVSKKEMWNESESFWLNDLFQDIIQFLYP 250 Morphotype D Wild Type SLVNANVSIEKNLPYPIPLTGYRSEVRQVFLNILMNSIDALESMKEERKI 300 Morphotype D PLTGYRSEVRQVFLNILMNSIDALESMKEERKI 214 Wild Type IIDVFEEDQSIRIVIKNNGPMIPAENVETIFEPFVTTKKLGTGIGLFVCK 350 Morphotype D IIDVFEEDQSIRIVIKNNGPMIPAENVETIFEPFVTTKKLGTGIGLFVCK 264 Wild Type QIVEKHNGSIMCRSDDDWTEFQIAFQK* 378 Morphotype D QIVEKHNGSIMCRSDDDWTEFQIAFQK* 292 PCR/sequencing based assay aimed at amplifying and resequencing a 533 bp sequence surrounding the region 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 REFGBAAA0205 response regulator putative R: ATCAATATATGCTTGATAGTTTAAGTATTGGAAAAGATAGTTTTGATAAAGTAGATTCACT GE: ATCAATATATGCTTGATAGTT TTGATAAAGTAGATTCACTG PCR/sequencing based assay aimed at amplifying and resequencing a 503 bp sequence surrounding the region Institute for Genome Sciences University of Maryland School of Medicine

27 Morphological variants E - Leahy Letter
GBAAA MIVSVKGNEQITKMLNDWYIEIRARHVGKAHNLKLEIDQKIHNIEEDQNL 50 LL MIVSVKGNEQITKMLNDWYIEIRARHVGKAHNLKLEIDQKIHNIEEDQNL 50 LL MIVSVKGNEQITKMLNDWYIEIRARHVGKAHNLKLEIDQKIHNIEEDQNL ************************************************** GBAAA LLYYALLDFRHQYMLDSLSIGKDSFDKVDSLGVPADQFLQYYYHFFKAIH 100 LL LLYYALLDFRHQYMLD SFDKVDSLGVPADQFLQYYYHFFKAIH 93 LL LLYYALLDFRHQYMLDS---LKDSFDKVDSLGVPADQFLQYYYHFFKAIH **************** *************************** GBAAA SNITGDFTSAKEHYNQAELLLKHIPDEIEHAEFRFKLSTFHYHIYKPLAA 150 LL SNITGDFTSAKEHYNQAELLLKHIPDEIEHAEFRFKLSTFHYHIYKPLAA 143 LL SNITGDFTSAKEHYNQAELLLKHIPDEIEHAEFRFKLSTFHYHIYKPLAA ************************************************** GBAAA IKEATKAKDIFKKHAGYETNIGLCDNLIGLACTHLKQFEEAEEHFITAIN 200 LL IKEATKAKDIFKKHAGYETNIGLCDNLIGLACTHLKQFEEAEEHFITAIN 193 LL IKEATKAKDIFKKHAGYETNIGLCDNLIGLACTHLKQFEEAEEHFITAIN ************************************************** GBAAA TFKKSGKEKNITFVRHNLGLMYSGQNLSELAIRYLSEVTQELPKDYKAIF 250 LL TFKKSGKEKNITFVRHNLGLMYSGQNLSELAIRYLSEVTQELPKDYKAIF 243 LL TFKKSGKEKNITFVRHNLGLMYSGQNLSELAIRYLSEVTQELPKDYKAIF ************************************************** GBAAA IKAREHMKIGESKETYNLIVKGLEICKELKNEEYEHHFLILEKLNQKVSA 300 LL IKAREHMKIGESKETYNLIVKGLEICKELKNEEYEHHFLILEKLNQKVSA 293 LL IKAREHMKIGESKETYNLIVKGLEICKELKNEEYEHHFLILEKLNQKVSA ************************************************** GBAAA DELEKTIKTGISYFKRENLHEYVQEYAKKLAVLFHQENNRSKASDYFYLS 350 LL DELEKTIKTGISYFKRENLHEYVQEYAKKLAVLFHQENNRSKASDYFYLS 343 LL DELEKTIKTGISYFKRENLHEYVQEYAKKLAVLFHQENNRSKASDYFYLS ************************************************** GBAAA HQAEEQNFEKEALK* 365 LL HQAEEQNFEKEALK* 358 LL HQAEEQNFEKEALK* 362 *************** PCR/sequencing based assay aimed at amplifying and resequencing a 466 bp sequence surrounding the region

28 Morphotypes/genotypes
Variant Mutation Locus Function A Duplication rRNA-D, Polysaccharide deacetylase and GBAA0151 unknown B SNP Upstream spoOF Initation of Sporulation C/D SNP/INDEL Sensor histidine kinase Phosphorylation of SpoOF/SpoOA E INDEL (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 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 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 2001-2002
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 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 Acknowledgments IGS/TIGR FBI USAMRIID NAU NSF NIH/NIAID
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 ) National Institute of Allergy and Infectious Diseases (N01-AI15447) National Science Foundation Institute for Genome Sciences University of Maryland School of Medicine


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