Whole Genome Sequencing for TB Diagnostics. Kimberlee Musser, PhD Chief, Bacterial Diseases Wadsworth Center
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1 Whole Genome Sequencing for TB Diagnostics Kimberlee Musser, PhD Chief, Bacterial Diseases Wadsworth Center
2 900,000 sq. ft. state-of-the-art-facilities- 5 locations ~700 staff, >150 doctoral level scientists $25 million in external grant funding Laboratories in four scientific divisions: Environmental Health, Infectious Disease, Genetics, Translational Medicine Division of Infectious Diseases Viral Diseases Virology Bacterial Diseases Bacteriology Bloodborne Diseases Bloodborne Virus Mycotic & Parasitic Diseases Parasitology Viral Replication and Vector Biology Arbovirology Viral Encephalitis Mycobacteriology Diagnostic Immunology Mycology Rabies Enteric Virus Biodefense Research Laboratories PI- grant funded programs
3 November 15, Roles of the Wadsworth Center Bacteriology and Mycobacteriology Laboratories Reference services Outbreak and hospital investigations Specialized testing Support of disease surveillance and epidemiology investigations Preparedness and response Applied research (NIH, CDC, contracts) Samples SNP alignment Posi ons with SNPs
4 November 15, TB Background Caused by Mycobacterium tuberculosis and other MTBC species Roughly one third of the world s population is infected with TB 2013: 9 million new infections, 1.5 million deaths Second only to HIV/AIDS as a worldwide killer
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7 November 15,
8 November 15, Rapid Diagnosis of Mycobacterium tuberculosis with WGS Faster turn-around time More comprehensive results Detect mixed infections Many predictors of drug resistance Emerging resistance Cost effective Replace existing assays (real-time PCR, pyrosequencing, spoligotyping) Staff time savings
9 24-48 hrs Testing Algorithm November 15, Primary specimens/ isolates Heat-killed Processed/decontaminated WGS real-time PCR MTBC/MAC AFB Smear Culture Growth No Growth rpob, katg, inha Pyrosequencing Pos MTBC MTBC member real-time PCR real-time PCR MTBC/MAC NTM MTBC MALDI-TOF rpob, hsp65 DST 1 st line drugs Identification of mutation (pnca, rpob, katg, inha, gyra/b) Resistant Susceptible DST 2 nd line drugs
10 Whole Genome Sequencing Next Generation Sequencing Extract TB DNA DNA sequencing Bioinformatics Sequencing on MiSeq, 2 x 250bp PE reads
11 November 15, Where to start? Isolates Solid MGITs Primary specimens sputum other
12 November 15, Optimizing TB isolate preparation for WGS Assess methods used in lab Research TB WGS methods Assess worse case scenario 1-2 ml MGIT early MGIT positive (Day 0-3 flagged positive) Ease of use, cost DNA concentration Ultimately- WGS 40X depth and close to 100% coverage
13 November 15, Breaking TB Open is Critical for DNA Extraction Important TB Characteristics ~24 hour doubling time TB clumps together Unique cell wall Rich in lipids (>60%) Mycolic acids Initial Methods Tested Typical bacterial extraction Zymo Research Kit Meant for tough to lyse fungi/ bacteria CTAB method Ideal for plant cell nucleic acid extraction/ MTB DNA yield too low, labor intensive, WGS variable results CDC
14 November 15, InstaGene Matrix and Tissue Homogenizer 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!
15 Whole Genome Sequencing Next Generation Sequencing 40X Depth and 100% Coverage Extract TB DNA Library Preparation DNA sequencing Bioinformatics Nextera XT Sequencing on MiSeq, 2 x 250bp PE reads
16 November 15, Successful WGS Depth: Essentially the number of times 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
17 November 15, Library Preparation is Another Key Factor Votintseva et al. suggested using 15 cycle library preparation 2015 paper about WGS of early positive MGIT 12 cycle library prep 15 cycle library prep Sample Method stock ng/ul Avg depth coverage % M. bovis BCG (0day) Avg depth coverage % InstaGene FAIL FAIL InstaGene FAIL FAIL InstaGene FAIL FAIL
18 November 15, Bioinformatics Pipeline Pascal Lapierre, PhD Michael Palumbo, PhD
19 TB Bioinformatics Pipeline Bioinformatics Map to Reference Genome Kraken K-mer matching Detect spacers SNP calling with indels SNP calling ignore indels MTBC member ID Spoligotyping Prediction of antibiotic resistance Strain typingrelatedness Report Importing into LIMS
20 November 15, Evaluating 8 Drug Classes 12 loci Hundreds of potential SNPs Additional loci throughout the genome frameshifts insertions deletions
21 November 15, Validation of TB WGS for isolates SOP, reports, interpretation, QC, assay controls, metrics Specificity, intra-assay and inter-assay reproducibility Retrospective testing Prospective testing Evaluate each drug
22 November 15,
23 November 15, Fluoroquinolone comparison
24 November 15, Isoniazid comparison
25 November 15, Prospective Testing
26 November 15, First Batch of Prospective specimens Results as of 8/3/15: Current method results WGS results Sample IDR IDR IDR DST invalid; PZA S pending; PZA S Pyro results No mutations spoligo Genome coverage Ave Depth ID High confidence mutations detected Frameshift and/or large deletions detected spoligo ND Mtb None none S00034 ND ND Mtb pending ND ND Mtb gyra Ala90Val; gyra Asp94Gly (FLQ) embb Met305Val (EMB); gyra Ser91Pro (FLQ); katg Ser315Thr (INH) none rpob (+TTC) in-frame insertion (RIF) Unknown (new) S00034 No Results WGS complete
27 November 15, First Batch of Prospective specimens Results as of 8/18/15: Current method results WGS results Sample IDR IDR IDR DST Pan- Susceptible pending; PZA S pending Pyro results No mutations gyra: Asp94Gly rpob: insertion of 3 bases at codon 514; katg: Ser315Thr ; gyra: Ser91Pro spoligo Genome coverage Ave Depth ID High confidence mutations detected Frameshift and/or large deletions detected spoligo ND Mtb None none S00034 pending Mtb ND Mtb gyra Ala90Val; gyra Asp94Gly (FLQ) embb Met305Val (EMB); gyra Ser91Pro (FLQ); katg Ser315Thr (INH) none rpob (+TTC) in-frame insertion (RIF) Unknown (new) S00034 Some Results WGS complete
28 November 15, First Batch of Prospective specimens Results as of 10/8/15: Current method results WGS results Sample IDR IDR IDR DST Pan- Susceptible FLQ 1 st line invalid; 2 nd line pending Pyro results No mutations gyra: Asp94Gly rpob: insertion of 3 bases at codon 514; katg: Ser315Thr; gyra: Ser91Pro spoligo Genome coverage Ave Depth ID High confidence mutations detected Frameshift and/or large deletions detected spoligo ND Mtb None none S00034 S Mtb S Mtb gyra Ala90Val; gyra Asp94Gly (FLQ) embb Met305Val (EMB); gyra Ser91Pro (FLQ); katg Ser315Thr (INH) none rpob (+TTC) in-frame insertion (RIF) Unknown (new) S00034 More Results WGS complete
29 November 15, Heteroresistance
30 November 15, Turn-Around Time Imagine this is 4-5 days!
31 November 15, WGS prediction spoligotypes and genotyping with increased resolution NYS
32 Can we develop one assay capable of generating the same results...and more? Can we do it in <1 week? XDR Case (November 2014) Days from specimen receipt
33 XDR Case (November 2014) Spoligotype: S00062 ( ) Lineage Euro-American M. tuberculosis X1 family Drug Resistant phenotype: FLQ (OFL,LVX, MX) RIF INH SM EMB PZA RBT KAN AMI CAP (11%)
34 XDR Case (November 2014) Days from specimen receipt TB WGS Predicted Drug Resistant phenotype: FLQ (OFL,LVX, MX) RIF, INH, SM, EMB PZA, RBT, KAN, AMI CAP (11%)
35 November 15, Exciting Anecdotal Findings 1- MDR identification in ~ 2 weeks on not even known as TB case 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- Clears up 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.
36 November 15, What does it really cost?
37 TB WGS Reports Whole genome sequencing
38 Molecular Drug Susceptibility Prediction- Whole Genome Sequencing Rifampin rpob: Isoniazid katg: Mutation present: Ser531Trp suggests resistance Note: XX% of isolates in our in-house evaluation of XX clinical isolates with this mutation are resistant. Mutation present: Ser315Thr suggests resistance Note: XX% of isolates in our in-house evaluation of XX clinical isolates with this mutation are resistant. inha: Mutation present: C-15T suggests resistance Note: XX% of isolates in our in-house evaluation of XX clinical isolates with this mutation are resistant. Pyrazinamide pnca: Mutation present: Trp68Arg suggests resistance Ethambutol embb: Mutation present: Met306Ile suggests resistance Note: XX% of isolates in our in-house evaluation of XX clinical isolates with this mutation are resistant. Streptomycin rrs: Mutation present A>C at 513 suggests resistance Note: XX% of isolates in our in-house evaluation of XX clinical isolates with this mutation are resistant. Ofloxacin gyra gyrb Kanamycin eis: rrs: No Mutation No mutation No mutation No mutation Ethionamid inha: Mutation: C-15T suggests resistance Note: XX% of isolates in our in-house evaluation of XX clinical isolates with this mutation are resistant. Disclaimer: A negative result (e.g. no mutation) does not rule out contributory mutations present elsewhere in the genome. CULTURE MUST BE PERFORMED FOR FINAL SUSCEPTIBILITY RESULT.
39 November 15, Whole Genome Sequencing of TB: A One Stop Shop WGS Single assay Species identification Genotyping (more accurate) Drug resistance mutations (more comprehensive) COST Estimated around $100-$200 per sample TURNAROUND TIME DNA preparation (1 days) WGS result (4-5 days) Reality for TB Cultures! Next challenge TB specimens
40 November 15, Future Directions WGS TB Finalize validation and implement WGS for TB culture testing Evaluate TAT, sample numbers Finalizing pipeline Data interpretation/ notes LIMS importing NCBI (server access?) Data Storage/ assessing data over time TB Primary specimens
41 November 15, 2015 Acknowledgements 41 MYCOBACTERIOLOGY LAB Vincent Escuyer Donna Kohlerschmidt Michelle Isabelle Susan Wolfe Dennis Biggins BACTERIOLOGY LAB Joe Shea Tanya Halse Tammy Quinlan Justine Edwards Linda Gebhardt APPLIED GENOMIC TECHNOLOGIES CORE Matt Schudt Patrick VanRoey Pascal Lapierre Mike Palumbo FUNDING Wadsworth Center, NYSDOH Public Health Genomics Initiative James Posey National Center for HIV/AIDS, Viral Hepatitis, STD, and TB Prevention R03 NIH- Use of whole genome sequencing for tuberculosis diagnostics
42 Wadsworth Center Master of Science in Laboratory Sciences November 15, NEW YORK STATE DEPARTMENT OF HEALTH Training in public health investigation, environmental assessment, Genetic and infectious disease testing, and novel technologies Imagine a career: Protecting and improving the health of our communities Diagnosing diseases of public health importance Developing cutting edge testing methods
43 Wadsworth Center NEW YORK STATE DEPARTMENT OF HEALTH Master of Science in Laboratory Sciences November 15, Month Full-time intensive program: 27 didactic credits 8 eight-week rotations through different scientific focus areas 1 Capstone research project Tuition and Scholarships: No tuition fees Maximum of 4 scholarships of $10,000 will be awarded each year based on academic merit. * Applications due: March 2016
44 Questions?
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