clinical testing of mycobacterium tuberculosisby ngs: two ...60. 70. 80. 90. 100. sensitivty....
TRANSCRIPT
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Clinical Testing of Mycobacterium tuberculosis by NGS: Two Years Strong
Kimberlee Musser, PhDChief, Bacterial DiseasesWadsworth Center
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Why NGS on TB?
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2010 2011 2012 2013 2014 2015 2016
TB Cases* 954 910 866 872 787 765 768
DR-TB 63 74 67 54 62 61 54
MDR-TB 14 20 19 8 11 6 10
XDR-TB 0 2 2 0 2 0 1
* 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
0.0
2.0
4.0
6.0
8.0
10.0
Perc
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DR-TB MDR-TB
2010
2011
2012
2013
2014
2015
2016
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Whole-genome sequencing for TB?
• Goals for TB WGS:– Utilize as soon as possible in testing
algorithm to impact patient treatment– Expand molecular resistance prediction– Provide more comprehensive results
• mixed infections, heteroresistance, typing
– Assess costs and staff time
2013- Wadsworth Center Public Health Genomics Center (PHGC) funding announcement
2014- PHGC funding to test 60 TB isolates by WGS
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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
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TB Bioinformatics Pipeline
Bioinformatics
KrakenK-mer
matching
Detect spacers
SNP calling with indels
SNP calling ignore indels
Map to Reference Genome
Importing into LIMS
MTBC member ID
SpoligotypingPrediction of antibiotic resistance
Strain typing-relatedness
Report
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Imagine shredding a whole book into millions of shreds…then trying to put it back together in the right order
How do we apply quality control to this complicated method?
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Extract DNA
Library Preparation
DNAsequencing
Nextera XT
QC QC QC
QC
Bioinformaticpipeline
QC
Controls, QC, and more QC…
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What’s in a pipeline?• Species ID• Spoligotype derivation
• Resistance Prediction– Single Nucleotide
Polymorphism (SNP)– Insertion/ deletions
• Phylogenetic Analysis
• Results & Reporting
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CLEP QC Guidance
• Minimum base calling of Q20 (99% base call accuracy)
• Minimum average 40x depth of coverage
• All QC metrics must be documented and monitored over time
• All software updates that affect key processes should be revalidated
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So many moving parts…
Bio-informaticsNGS
Core
TB Lab
Molecular TB
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High-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 =Current validated pipeline
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Putting it all together…• Detailed SOP or SOPs
– All QC, limitations, step by step details
• Reporting– Interpretation, disclaimers,
examples• Quality Control
– Metrics, criteria, controls• Validation
– Specificity– Reproducibility (Inter- and Intra-)– Accuracy verification
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Import of pipeline
LIMS Final Laboratory
Reportmutations
Interpretation
Separating SOPs makes future assay development more streamlined
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SOP QC Examples…
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Retrospective study: Fluoroquinolone comparison
Validating against other molecular tests
Validating against DST
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Retrospective Study: Isoniazid comparison
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50
60
70
80
90
100
Sens
itivt
yMolecular INH Resistance Prediction
katG315
katG 315 inhA -15
2016 2017 Future
pyrosequencing WGS
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What have we learned in 2+ years…
• Communication with NGS Core and bioinformaticians is critical!
• Discordance almost always is determined to be due to AST
• Not that many surprises, but continual improvement
• TB Control Epidemiologists and Regional NY colleagues love this data!
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2015
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What have we learned from NYS CLEP and CLIA Surveys?
• This type of testing is new to everyone• Special internal audit with our QA Officer helpful• Memos stating assay developers when training
documentation doesn’t make sense• Able to utilize CDC Model Performance Evaluation
Program (MPEP) for Internal Quality Assurance• Tracking and QC reagents and Log • Documenting pipeline updates
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Looking back…what would we do differently?
Consider instrumentation redundancy, utilize pivot tables to manage data, talk as much as possible about pipeline needs, talk more to TB controllers, develop training and competency documents, reagent logs and assessments initially
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Future• Direct Sputum testing research
• WGS pipeline (2017)–Added drugs AST for MDR
strains–WGS targets linezolid,
clofazimine, PAS, bedaquiline–thyA stop mutation- PAS
resistance
• Third pipeline update (2018)– New mutations– New mutation category
(unclassified)– Externally facing pipeline
• Discontinuing DST very conservative approach on strains with no markers for resistance
• Funding– NIH R03- Evaluate TB WGS directly
on sputum specimens– NIH grant award Nanopore
MinION TB– APHL/CDC RFA to perform TB WGS
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How about Antimicrobial Resistance?
• To determine a novel carbapenemasemechanism
• To detect an IMP variant (other than IMP1)• To assess other resistance genes• (To determine relatedness)
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Why NGS on TB?
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What if there were also random pages included in the pile that needed to stay with the book
PlasmidPlasmid
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Along came an ARLN fellow…
Identification of Antimicrobial Resistance Genes Using:1. ResFinder2. ARG-ANNOT3. NCBI AR Database
Species ID CheckMulti-locus Sequence
Typing
Illumina Sequencing ReadsCollect all dataCharacterization of isolatesRepeat some initial testingBuild pipeline and validateAssess WGSSummarizeCollaborate
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AcknowledgementsCore TB WGS TeamVincent Escuyer Kim MusserTanya Halse Joseph SheaPascal Lapierre
ARLN TeamElizabeth Nazarian, Kara Mitchell,Emily Snavely
MYCOBACTERIOLOGY LABMOLECULAR BACTERIOLOGY LAB
APPLIED GENOMIC TECHNOLOGIES CORE
BIOINFORMATICS COREMike Palumbo Erica Lasek-Nesselquist, Wolfgang Haas
CLIMSColleen Walsh, Alok Mehta
Wadsworth CLEP Reviewers, CLEP staff
NYC PHLJennifer Rakeman, John Kornblum, Jamie LemonWadsworth Center Jill Taylor, Victoria Derbyshire, Ron Limberger
R03 NIH- Use of whole genome sequencing for tuberculosis diagnostics *New Funding NIH MinION TB project
CDCAngela Starks, Jamie Posey
APHLAnne Gaynor
NYS TB Control, NYC TB Control
National Center for HIV/AIDS, Viral Hepatitis, STD, and TB Prevention
Wadsworth Center, NYSDOHPublic Health Genomics Initiative
Establishment of Mycobacterium tuberculosis complex WGS Reference Centers APHL/CDC