The Genomics Behind the Amerithrax Investigation Claire M. Fraser-Liggett, Jacques Ravel, David A....

The Genomics Behind the Amerithrax Investigation

Claire M. Fraser-Liggett, Jacques Ravel, David A. RaskoInstitute for Genome Sciences

Department of Microbiology and ImmunologyUniversity of Maryland School of Medicine

Baltimore, MD 21201

Institute for Genome SciencesUniversity of Maryland School of Medicine

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


New York post letter spore preparation

The Science behind the Amerithrax

Leahy letter spore preparation


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)



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


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


SNP analysis in B. anthracis genomes


SNP analysis in B. anthracis genomes>ID642-16 (94989bp) 200731 c-->t 50878 COV: 16 QUAL: 31.5NON-SYNONYMOUS (2): GGC - G ---> GAC - D (from start: 5 bp/848 bp)AMES : GAAGAAGCGCTAATAAAATGCCCATTACAAGACTCCCTTCG 200751 |||||||||||||||||||| ||||||||||||||||||||QUERY : GAAGAAGCGCTAATAAAATGTCCATTACAAGACTCCCTTCG 50898ORF01519 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 36:35:34:36:33:36:36:35:15:36:35:32:36:33:30REFERENCE: 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 24:45:45:37:41:24:4:45:35COV: 10 QUAL: 33.4

Access to genome coverage, sequence quality scores, and sequence in a single file


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?



Morphological variants analyzed

Origin Type Sequencing

Leahy Letter Wild Type 12 X

Leahy Letter A Closed

Leahy Letter B 12 X

Leahy Letter C 12 X

Leahy Letter E/Opaque 12 X

NY Post Letter Wild Type Closed

NY Post Letter A 12 X

NY Post Letter B Closed

Strategy - Discover genetic differences by comparing the genome sequences to Ames Ancestor.


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


Morphological variants isolation

Visual differential tests

Colony morphology, color, sporulation efficiency for each spore samples


Morphological variants characteristics


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



Could the population composition be the ID that can be used to make a match?

Genomics


Morphological variants analyzed

Origin Type Sequencing

Leahy Letter Wild Type 12 X

Leahy Letter A Closed

Leahy Letter B 12 X

Leahy Letter C 12 X

Leahy Letter E/Opaque 12 X

NY Post Letter Wild Type Closed

NY Post Letter A 12 X

NY Post Letter B Closed

Strategy - Discover genetic differences by comparing the genome sequences to Ames Ancestor.


Morphological variants A



Leahy Letter ALeahy Letter A

Ames AncestorAmes Ancestor

PCR assayPCR assay



NY Post Letter ANY Post Letter A

Leahy Letter ALeahy Letter A

Daschle Letter ADaschle Letter A

ORF1573: polysaccharide deacetylaseORF1573: polysaccharide deacetylase

ORF1572: conserved hypothetical proteinORF1572: conserved hypothetical protein

Ames AncestorAmes Ancestor

These three morphotypes A are morphologically identical, but genetically different. All three are present in the letters


Morphological variants B - NY Post Letter

>ID161revcom-1 (295550bp) 5065092 T-->C 8836 COV: 9 QUAL: 43AMES : CCAAATTCCTTCCTATTCTTTCTTATTTTACCGTTTCCTAT 5065112 |||||||||||||||||||| |||||||||||||||||||| B : CCAAATTCCTTCCTATTCTTCCTTATTTTACCGTTTCCTAT 8856Intergenic 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 44:40:44:34:36:38:45:40:47REFERENCE: 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

SNP found in each variant B analyzedSNP found in each variant B analyzed


Morphological variants B

KinB

spoOF


Morphological variants C - Leahy Letter

>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 47:47:47:41:47:44:19:41:3547174 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

SNP results in Amber stop codon and a truncated proteinSNP results in Amber stop codon and a truncated protein


Morphological variants C/D - Leahy Letter


Morphological variants C/D - Leahy Letter

Wild Type 1 MEMEGMEVFPIDKDIKEVFCSHLKNNRHQFVENWKNKMIISDKDPFRLEV 50Morphotype D 1 MEMEGMEVFPIDKDIKEVFCSHLKNNRHQFVENWKNKMIISDKDPFRLEV 50 Wild Type 51 VQNGEDLLEFIIELIMEEKDINYLQPLCEKIAIERAGADANIGDFVYNAN 100orphotype D 51 VQNGEDLLEFIIELIMEEKDINYLQPLCEKIAIERAGADANIGDFVYNAN 100

Wild Type 101 VGRNELFEAMCELDVSARELKPIMNQIHTCFDKLIYYTVLKYSEIISRNL 150Morphotype D 101 VGRNELFEAMCELDVSARELKPIMNQIHTCFDKLIYYTVLKYSEIISRNL 150

Wild Type 151 EEKQQYINETHKERLTILGQMSASFVHEFRNPLTSIMGFVKLLKADHPSL 200Morphotype 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


Morphological variants E - Leahy Letter

INDEL-REF-1:172510 21 bp ID3 (12311 bp) INS on REFGBAAA0205 response regulator putative

R: ATCAATATATGCTTGATAGTTTAAGTATTGGAAAAGATAGTTTTGATAAAGTAGATTCACTGE: ATCAATATATGCTTGATAGTT------------------------------------------TTGATAAAGTAGATTCACTG


Morphological variants E - Leahy Letter

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 ***************

Morphotypes/genotypes

Variant Mutation Locus Function

A Duplication

rRNA-D,Polysaccharide

deacetylase and GBAA0151

unknown

B SNP Upstream spoOFInitation of Sporulation

C/D SNP/INDEL Sensor histidine kinase

Phosphorylation of

SpoOF/SpoOA

E INDEL (9/21 bp)pXO1

response regulator

Dephosphorylation of SpoOF


Morphotypes/genotypes


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


RMR-1029


Population genetics - the unique signature

Science Magazine - August 2008

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.

Timeline

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 2007Preparation for indictment 2008

Unfortunate and tragic dealth of the suspect 2008

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

AcknowledgmentsAcknowledgments

National Institute of Allergy and Infectious Diseases(N01-AI15447)National Institute of Allergy and Infectious Diseases(N01-AI15447)

IGS/TIGRDavid RaskoLingxia JiangRegina CerSteven SalzbergClaire Fraser-Liggett

IGS/TIGRDavid RaskoLingxia JiangRegina CerSteven SalzbergClaire Fraser-Liggett

FBIJason BannanMark WilsonRichard LanghamScott StanleyScott DeckerMatt Feinberg

FBIJason BannanMark WilsonRichard LanghamScott StanleyScott DeckerMatt Feinberg

USAMRIIDPatricia Worsham

USAMRIIDPatricia Worsham

Federal Bureau of Investigation (J-FBI-02-016)Federal Bureau of Investigation (J-FBI-02-016)

NIH/NIAIDMaria Giovanni

NIH/NIAIDMaria Giovanni

NSFRita ColwellMaryanna Henckart

NSFRita ColwellMaryanna Henckart

National Science FoundationNational Science Foundation

NAUPaul Keim

NAUPaul Keim


The Genomics Behind the Amerithrax Investigation Claire M. Fraser-Liggett, Jacques Ravel, David A....

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