Whole Genome Next Genera/on Sequencing of TB in a Public Health Laboratory: A New Diagnos/c Era
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1 Whole Genome Next Genera/on Sequencing of TB in a Public Health Laboratory: A New Diagnos/c Era March 31, 2016 Kimberlee Musser, PhD Chief, Bacterial Diseases Wadsworth Center 900,000 sq. O. state- of- the- art- facili/es- 5 loca/ons ~700 staff, >150 doctoral level scien/sts $25 million in external grant funding Laboratories in four scien/fic divisions: Environmental Health, Infec/ous Disease, Gene/cs, Transla/onal Medicine Division of Infec/ous Diseases Viral Diseases Virology Bacterial Diseases Bacteriology Bloodborne Diseases Bloodborne Virus Myco3c & Parasi3c Diseases Parasitology Viral Replica3on and Vector Biology Arbovirology Viral Encephali/s Mycobacteriology Diagnos/c Immunology Mycology Rabies Enteric Virus Biodefense Roles of the Wadsworth Center Bacteriology and Mycobacteriology Laboratories Reference services Outbreak and hospital investigations, surveillance Specialized testing Applied research (NIH, CDC, contracts) Development of diagnostics, algorithms Evaluation of FDA approved tests Retrospective analysis Shared Services 1
2 March 31, TB Cases : 786 cases; 201 (NYS); 581 (NYC) Focus on con/nual improvement of TB Diagnos/cs NAAT (isolates and specimens): MTBC real- 3me PCR since 2007 MTBC differen3a3on real- 3me PCR since 2010 TB posi/ve- resistance assessment: rpob gene pyrosequencing since 2009 katg gene pyrosequencing since 2010 inha promoter pyrosequencing since 2012 gyra, gyrb genes pyrosequencing since 2013 Non- tuberculous mycobacteria (NTM): MAC real- 3me PCR since 2011 M. abscessus real- 3me PCR since 2014 NTM ID rpob, hsp65, 16S gene sequence analysis since 2012 MALDI- TOF MS since 2013 Whole- genome sequencing for TB? Wadsworth Center Public Health Genomics Center (PHGC) funding announcement PHGC funding to test 60 TB isolates by WGS Goals for TB WGS: U/lize as soon as possible in tes/ng algorithm to impact pa/ent treatment Expand molecular resistance predic/on Provide more comprehensive results mixed infec/ons, heteroresistance, typing Assess costs and staff /me 2
3 March 31, Isolates Solid MGITs Primary specimens sputum other Where to start? Worst case scenario- Day 0 Flag Posi/ve MGIT Culture Whole Genome Sequencing TB- Assay Development 40X Depth and 100% Coverage Extract TB DNA Library Prepara/on DNA sequencing Bioinforma/cs Nextera XT Sequencing on MiSeq, 2 x 250bp PE reads March 31, Sequencing Depth: Essen/ally the number of /mes the base was read; measure of confidence in correct call Can be given as a genome average We are aiming for 40X 8X Depth ATTGC ATTGC TTGCATAAAATTC ATTGCAT TGCATAAATTC ATTGCAT ATTG TGCATAAAT Coverage: A percentage that describes how much of the genome was sequenced Best 100% 67% Coverage 3
4 March 31, TB DNA Prepara/on for WGS InstaGene matrix (Chelex resin) The Chelex matrix binds to PCR inhibitors rather than DNA, preventing DNA loss due to irreversible DNA binding. Fastprep tissue homogenizer Good enough yield to provide reliable WGS data even with 0 day MGIT Success! March 31, Library Prepara3on is Another Key Factor Votintseva et al. suggested using 15 cycle library preparation 2015 paper about WGS of early positive MGIT Sample Method stock ng/ul Avg depth M. bovis BCG (0day) 12 cycle library prep 15 cycle library prep coverage % Avg coverage % depth InstaGene FAIL FAIL InstaGene FAIL FAIL InstaGene FAIL FAIL Success! TB Bioinforma3cs Pipeline Bioinforma/cs Pascal Lapierre, PhD Map to Reference Genome Kraken K- mer matching Detect spacers SNP calling with indels SNP calling ignore indels MTBC member ID Spoligotyping Predic3on of an3bio3c resistance Strain typing- relatedness Report Impor3ng into LIMS 4
5 March 31, High Confidence Muta/ons Bioinforma/cs Evaluating 8 Drug Classes 12 loci Hundreds of potential SNPs Map to Reference Genome SNP calling with indels Additional loci throughout the genome frameshifts insertions deletions Predic3on of an3bio3c resistance Decision making- retrospec3ve tes3ng, literature, experience March 31, Valida3on of TB WGS for isolates Specificity, intra-assay and inter-assay reproducibility Retrospective testing (96 isolates from 8 year period selected based on DST ) Prospective testing (50 isolates that were received in the lab between 6/11/15 and 9/15/15) Evaluation of each drug ~50 page valida3on document Lots of data to assess and track over /me! 5
6 Retrospec/ve study: Fluoroquinolone comparison Valida/ng against other molecular tests Valida/ng against DST March 31, Retrospec/ve Study: Isoniazid comparison March 31, Drug Resistance Predic/on WGS genotype predic3on vs. DST phenotype * Current EMB FLQ INH PZA RIF SM KAN ETA Sensi3vity Specificity PPV (Res) NPV (Susc) Concordance /8 Drugs have >90% concordance Predictions will be improved as we gather more data Increased support for new genes or new mutations to be included as high confidence *phenotype based on the cri3cal concentra3ons used at WC 6
7 March 31, Why we don t think we can live without TB WGS... March 31, Prospec/ve specimens (received 7/3-7/13/15)! Results as of 8/3/15:! Current!method!results! WGS!results! High! Frameshift! Pyro! Genome! Ave! confidence! and/or!large! Sample! DST! spoligo! ID! spoligo! results! coverage! Depth! mutations! deletions! detected! detected! IDR15D invalid;! No! ND! 98.36! 91.05! Mtb! None! none! S00034! 51087! PZA!S! mutations! gyra! Ala90Val;! IDR15D pending;! Unknown! ND! ND! 98.46! 75.32! Mtb! gyra! none! 52024! PZA!S! (new)! Asp94Gly! (FLQ)! embb! Met305Val! (EMB);! rpob!(+ttc)! gyra! IDR15D indframe! pending! ND! ND! 98.82! 79.08! Mtb! Ser91Pro! S00034! 52248! insertion! (FLQ);! (RIF)! katg! Ser315Thr! (INH)! No Results WGS complete March 31, Results! as of 8/18/15:! Current!method!results! WGS!results! High! Frameshift! Pyro! Genome! Ave! confidence! and/or!large! Sample! DST! spoligo! ID! spoligo! results! coverage! Depth! mutations! deletions! detected! detected! IDR15D PanD No! ND! 98.36! 91.05! Mtb! None! none! S00034! 51087! Susceptible! mutations! gyra! Ala90Val;! IDR15D pending;! gyra:! Unknown! pending! 98.46! 75.32! Mtb! gyra! none! 52024! PZA!S! Asp94Gly! (new)! Asp94Gly! (FLQ)! rpob:! embb! insertion! Met305Val! of!3!bases! (EMB);! rpob!(+ttc)! IDR15D at!codon! gyra! indframe! pending! ND! 98.82! 79.08! Mtb! S00034! 52248! 514;!katG:! Ser91Pro! insertion! Ser315Thr (FLQ);!katG! (RIF)! ;!gyra:! Ser315Thr! Ser91Pro! (INH)! Some Results WGS complete 7
8 March 31, Results! as of 10/8/15:! Current!method!results! WGS!results! High! Frameshift! Sample! DST! Pyro! results! Genome! Ave! spoligo! coverage! Depth! ID! confidence! and/or!large! mutations! deletions! spoligo! detected! detected! IDR15D PanD No! 51087! Susceptible! mutations! ND! 98.36! 91.05! Mtb! None! none! S00034! IDR15D 52024! IDR15D 52248! gyra:! FLQ! S02467! 98.46! 75.32! Mtb! Asp94Gly! rpob:! insertion! 1 st!line! of!3!bases! invalid;!2 nd! at!codon! S00034! 98.82! 79.08! Mtb! line! 514;!katG:! pending! Ser315Thr;! gyra:! Ser91Pro! gyra! Ala90Val;! Unknown! gyra! none! (new)! Asp94Gly! (FLQ)! embb! Met305Val! (EMB);! rpob!(+ttc)! gyra! indframe! S00034! Ser91Pro! insertion!(rif)! (FLQ);!katG! Ser315Thr! (INH)! More Results WGS complete March 31, Heteroresistance March 31, Turn- around Time Imagine this is 4-5 days! 8
9 March 31, We have found that TB WGS provides: 1- MDR identification in ~ 2 weeks or less on TB cases (some that are not even known as cases) weeks-months before DST available 2- Resolution/ early identification of mixed samples (NTM/TB) 3- Resolves inconclusive identifications MTB complex due to missing RD regions 4- Resolves issues where pyrosequencing or Sanger sequencing will FAIL due to deletion in target genes 5- Finds mutations outside of pyrosequencing region of target genes 6- Finds mutations in 2 nd line drugs which would never have been found when 1 st line drugs are susceptible 8- Resolving spoligotyping issues 9- Can identify heteroresistance 10- Predicts resistance when DST is invalid. WGS is even more valuable because the normal time to susceptibility results is pushed back. In some cases these specimens turn out to have contamination so that DST can never be completed and is canceled. March 31, Ongoing TB WGS Ac/vi/es Received conditional approval to offer this test on 2/8/16! Finalizing LIMs import, reporting Performing TB WGS 1x/ week- TAT 7 days Created guidance document CLEP NIH R03- Evaluate TB WGS directly on sputum specimens APHL/CDC RFA to perform TB WGS on every case of TB in NYS and NYC- completed first 34 March 31, Whole Genome Sequencing of TB: A One Stop Shop WGS Single assay Species identification Genotyping (more accurate) Drug resistance mutations (more comprehensive) COST Es/mated around $100- $200 per sample TURN- AROUND TIME DNA prepara/on (1 days) WGS result (4-5 days) Reality for TB Cultures! 9
10 March 31, 2016 Acknowledgements 28 MYCOBACTERIOLOGY LAB Vincent Escuyer Donna Kohlerschmidt Michelle Isabelle Susan Wolfe Dennis Biggins BACTERIOLOGY LAB Joe Shea Tanya Halse Tammy Quinlan Jus/ne Edwards Linda Gebhardt APPLIED GENOMIC TECHNOLOGIES CORE Mas Schudt Patrick VanRoey Pascal Lapierre Mike Palumbo Establishment of Mycobacterium tuberculosis complex WGS Reference Centers James Posey Na/onal Center for HIV/AIDS, Viral Hepa//s, STD, and TB Preven/on R03 NIH- Use of whole genome sequencing for tuberculosis diagnos/cs Wadsworth Center, NYSDOH Public Health Genomics Ini/a/ve Ques3ons? hkp://biocomicals.blogspot.com/2011_05_01_archive.html March 31,
11 March 31, Rapid Diagnosis of Mycobacterium tuberculosis with WGS Faster turn- around /me More comprehensive results Detect mixed infec/ons Many predictors of drug resistance Emerging resistance Cost effec/ve Replace exis/ng assays (real- /me PCR, pyrosequencing, spoligotyping) Staff /me savings March 31,
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