May 1, 2017

Implementing whole‐genome sequencing on clinical isolates of Mycobacterium 

tuberculosis in New York State

Kimberlee Musser, PhDChief, Bacterial DiseasesWadsworth Center

May 1, 2017 2

Why perform WGS on Mycobacterium tuberculosis?

• Faster turnaround 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

May 1, 2017 3

2009 2010 2011 2012 2013 2014 2015

TB Cases* 1007 954 910 864 872 784 766

DR‐TB 80 63  74  67 54 109  115 

MDR‐TB 12 14 20 19 8  12 7 

XDR‐TB 0 0 2 2 0 2 0

* National rank #3 or #4 each year by number of cases

TB in New YorkUniversal FAST TRACK program:

• Implemented in 1993• Rapid detection of MTBC from a priority group 

of highly infectious NY patients with newly diagnosed AFB smear positive sputum

May 1, 2017 4

TB positive‐ resistance assessment:

rpoB gene pyrosequencing 2009katG gene pyrosequencing 2010inhA promoter pyrosequencing 2012gyrA, gyrB genes pyrosequencing 2013

NAAT (isolates and specimens):

MTBC  real‐time PCR 2007MTBC species ID real‐time PCR 2010

Focus on continual improvement of TB Diagnostics

Non‐tuberculous mycobacteria (NTM):

MAC real‐time PCR 2011M. abscessus real‐time PCR 2014

NTM IDrpoB, hsp65, 16S gene sequence analysis 2012MALDI‐TOF MS 2013

May 1, 2017 5

Developing a TB WGS Assay

• Starting material Day 0 MGIT• Compare DNA preparation methods• Nextera XT/ MiSeq• Build Pipeline• LIMS/ Epidemiology Reporting (ECLRS)• Validation Plan

May 1, 2017 6

• 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

TB DNA Preparation for WGS

Success!

• Working with TB is a challenge!• Comparison of CTAB, Zymo Research Kit, 

other methods• Review literature 

May 1, 2017 7

Whole Genome SequencingNext Generation Technologies

Extract TB DNA

Nextera XT/ Miseq WGS Bioinformatics

12 to 15 cycles

May 1, 2017 8TB BIOINFORMATICS PIPELINE

Bioinformatics

KrakenK-mer

matching

Detect spacers (allow 1 SNP)

SNP calling with indels, major

deletions

SNP calling ignore indels

Map to Reference Genome

1 2 3 4

Importing into Importing into CLIMS

MTBC member ID Spoligotyping

Prediction of antibiotic resistance

Build consensus for

phylogeny

Report

May 1, 2017 9

May 1, 2017 10High-confidence mutations Drug Locus Codon/NT positionRifampin (RIF) rpoB 251, 511, 513, 516, 522, 

526, 531, 533, 572 

Isoniazid (INH) katGoxyR‐ahpC promoter regionmabA promoter regionmabAinhA

279, 315, 394, 525 ‐81‐17, ‐15, ‐820394

Pyrazinamide (PZA) pncA/pncA promoter region Any nonsynonymous change

Ethambutol (EMB) embB 306, 406, 497

Streptomycin (SM) rrsrpsL

512, 513, 516, 90643, 88

Kanamycin/Amikacin (KAN/AMI)

rrs 1401

Kanamycin (KAN) eis promoter region ‐10, ‐37

Fluroquinolones(FLQ)

gyrAgyrB

74, 90, 91, 94510

Ethionamide (ETH) mabA promoter regionmabAethA

‐17, ‐15, ‐8203Frameshift/STOP

Red =New pipeline

May 1, 2017 11

Galaxy output to email:1‐ CLIMS report2‐ Access report3‐ Individual text files for each specimen

45 min/sample one at a time  ‐‐‐‐‐‐‐<30 min/samples 6 at a time!

May 1, 2017 12Comprehensive Pipeline Results

identification

spoligotype

Mapping statistics

May 1, 2017 13

Import of pipeline 

CLIMS Final Laboratory Report

mutations

Interpretation

May 1, 2017 14

Validation of TB WGS

• SOP, reporting language, interpretation, QC, assay controls, metrics

• Specificity, intra-assay and inter-assay reproducibility

• Retrospective testing• Prospective testing• Evaluate each drug

May 1, 2017 15TIMELINE

• 2015‐ RFA: Establishment of MTBC WGS Reference Centers/Pilot Project; NIH R03 grant TB WGS sputum

• Jan 2016‐ Established NYS guidelines for validation of NGS‐based methods for isolates of infectious agents

• Feb 2016‐ Validation package submitted to NYS

Routine WGS of all NYS TB Patients > a year!

2013‐Wadsworth Center Public Health Genomics Center (PHGC) funding announcement

2014‐ PHGC funding to test 60 TB isolates by WGS‐pilot study 

2014:   ‐ Bioinformatics work began‐Development and Validation

May 1, 2017 16

SPECIES AND STRAIN IDENTIFICATION

• 535 M. tuberculosis• 9 M. bovis• 6 M. bovis-BCG• 8 M. africanum• 2 M. orygis

527 pan-susceptible (83%)

112 resistant (17%) 14 MDR 1 XDR 58 other resistance 26 SM mono-resistant 13 2nd line drug mono-

resistant

RESISTANCE PROFILING

Based on WGS & DST, if available.

Prospective Testing Results

May 1, 2017 17Prospective Testing Results

May 1, 2017 18

608 samples had WGS-generated spoligotype

High levels of concordance with blot/ luminex spoligotype when available (n=204)

88.2% perfect matches (n=180) 10.8% 1 spacer off (n=22)* 1% 2 spacers off (n=2)*

Spoligotyping

*Several known discrepancies due to differences in methods of spacer detection

May 1, 2017 19

Euro-American (53%)

Beijing (23%)

CAS (6.3%)

EAI (13.3%)

Other species (4.4%)

SNP Analysis

May 1, 2017 20

Isoniazid Resistance Predictions: Improved by WGS*

Pipeline identifies high confidence mutations in 4 loci, plus katGframeshift mutations & gene

deletions

Sensitivity of INH resistance detection increased from 72% to 88%

PREDICTION OF DRUG RESISTANCE PROFILES

5th loci added in new pipeline

May 1, 2017 21

Current Success of Pipeline for Resistance Prediction – 8 Drugs

Current EMB FLQ INH PZA RIF SM KAN ETASensitivity    0.80 0.97 0.87 0.88 1.00 0.71 1.00 0.57Specificity 0.99 1.00 0.99 0.98 0.97 1.00 1.00 0.97PPV (Res) 0.89 1.00 0.98 0.83 0.80 1.00 1.00 0.92

NPV (Susc) 0.98 0.99 0.96 0.98 1.00 0.92 1.00 0.76

Concordance 0.97 0.99 0.96 0.96 0.97 0.93 1.00 0.80

PREDICTION OF DRUG RESISTANCE PROFILES

New pipelineConcordance 0.97  0.99    0.97    0.97    0.97   0.93    0.99    0.92  

May 1, 2017 22

Turnaround Time is Improving

WGS results have completed faster than first-line DST in 168/212 cases.

This continues to improve!

Turnaround time (days)

Extraction – WGS Result Receipt – WGS Result

# Isolates Received

# Primary Received

NYS NYC NYS NYC

January (15‐31)

19 0 13 11 28 28

February (1‐29)

49 3 9 8 23 28

March      (1‐22)

42 1 7 7 16 17

May 1, 2017 23

What does it really cost?

Core charge‐$180/ TB WGS MiSeqCore charge‐$120/ TB WGS NextSeq

May 1, 2017 24What have we learned?

We can’t live without TB WGS in NYS

Interesting findings:

• Invalid DST caused by mixed sample

• Increased resolution for TB investigations (VISIT POSTERS 42 and 49)

• Heteroresistance, emerging resistance

• Linking specimens in LIMs with variation in demographics

• katG mutation identified not detected by HAIN test

• 2 sisters, one developed mutation to RIF

May 1, 2017 25

Accepted in ~4 weeks!

May 1, 2017 26Whole Genome Sequencing of TB:A “One Stop Shop”

WGS

Single assay

Species identification

Genotyping (more accurate)Drug resistance mutations (more comprehensive)

COST

~$250 per sample

TURN‐AROUND TIME

WGS result (7 days)

Future‐ Direct specimen WGSProviding PipelineImproving the assay

AcknowledgementsCore TB WGS TeamVincent Escuyer Kim MusserTanya Halse Joseph SheaPascal Lapierre

MYCOBACTERIOLOGY LABDonna KohlerschmidtMichelle IsabelleSusan WolfeDennis Biggins

MOLECULAR BACTERIOLOGY LAB

Tammy Quinlan Justine EdwardsLinda Gebhardt Samantha WirthAPPLIED GENOMIC TECHNOLOGIES CORE

BIOINFORMATICS COREMike Palumbo Erica Lasek-NesselquistCLIMSColleen Walsh, Alok Mehta

NYC PHLJennifer Rakeman, John Kornblum

National Center for HIV/AIDS, Viral Hepatitis, STD, and TB Prevention

Wadsworth Center, NYSDOHPublic Health Genomics Initiative

R03 NIH- Use of whole genome sequencing for tuberculosis diagnostics

Establishment of Mycobacterium tuberculosis complex WGS Reference Centers

Patrick VanRoey Zhen ZhangMatt Shudt Helen LingMelissa Leisner Nathalie Boucher

NYS TB ControlNYC TB Control

May 1, 2017 28

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 2nd line drugs which would never have been found when 1st 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.

May 1, 2017 29

TB MGIT

56°C 15 min

15 sec 2X

Centrifuge

200 ul

Centrifuge

Proteins, inhibitors, etc...

WGS