evolution of next generation sequencing technology: ready ...€¦ · anti-microbial resistance...
TRANSCRIPT
3rd December 2015, Union Meeting Cape Town
Evolution of Next Generation Sequencing
Technology: Ready for Patient Management?
Timothy Rodwell MD, PhD, MPH
Senior Scientific Officer at FIND
Clinical Utility of NGS for TB
Diagnostics
• High Throughput Rapid drug susceptibility testing (RDST) for
guiding therapeutics
Anti-Microbial Resistance (AMR) Surveillance
• “Culture-free” surveillance for guiding regional and
national rollout of new drug regimens and molecular
diagnostics
Treatment monitoring for Drug resistant TB
• Detection and monitoring of low frequency drug
resistance alleles
Contact Tracing and Transmission Mapping
2
Clinical Utility of NGS for TB
Diagnostics
• High Throughput Rapid drug susceptibility testing (RDST) for
guiding therapeutics
Anti-Microbial Resistance (AMR) Surveillance
• “Culture-free” surveillance for guiding regional and
national rollout of new drug regimens and molecular
diagnostics
Treatment monitoring for Drug resistant TB
• Detection and monitoring of low frequency drug
resistance alleles
Contact Tracing and Transmission Mapping
3
Whole Genome vs Targeted NGS for RDST
Whole Genome Sequencing
Strengths
• Full genome sequenced
• Comprehensive solution
Weaknesses
• Slower
• Can’t yet get Mtb WGS direct from
sputum consistently
• More expensive
• More complicated bioinformatics
4
Targeted Next Gen Sequencing
Strengths
• Up to 200 gene targets sequenced
• Sequence DNA direct from sputum
• Faster
• Less expensive
Weaknesses
• Need some knowledge of targets
• Not comprehensive
Steps in an NGS Pipeline
5
DNA Extraction Amplification and Library Prep NGS Sequencing
Mutation Detection Clinical Interpretation
NGS for Rapid DST of Drug Resistant TB
Advantages • Fast (relative to phenotypic DST)
• “Open” platform
• Easily and quickly adaptable to new genes and mutations
• Comprehensive (compared to array/probe-based platforms)
• High throughput
Disadvantages
• Slower relative to array/probe-based molecular diagnostics
• Higher cost relative to array/probe-based molecular diagnostics
• High throughput
6
Status of NGS for Rapid DST of Clinical Samples
Today
• US - PSQ is in use for Rapid DST of clinical samples at
state and national level (AFB+ sputa and clinical TB Cx)
• UK – Illumina WGS is in use for comprehensive clinical
and epidemiological characterization of clinical strains -
Proof of concept study in eight clinical labs to
demonstrate that the method is feasible and cost-
effective
• Germany – Research Center Borstel provides Illumina
WGS analysis for RDST of MDR-TB clinical samples
from TB Cx
7
Near Future • Global NGS solution suitable for high throughput RDST
direct from sputum and deployable in reference
laboratories in LMICs
- Simplified
- Standardized
- Validated
- Minimum expertise to operate
8
Status of NGS for Rapid DST of Clinical Samples
Steps in an NGS Pipeline
9
DNA Extraction Amplification and Library Prep NGS Sequencing
Mutation Detection Clinical Interpretation
Proposed Simplified, Standard Pipeline for Targeted NGS
10
Automated DNA Extraction Simplified PCR/library Prep + Targeted NGS
Variant Analysis,
interpretation
and archiving
Proposed Simplified, Standard Pipeline for Targeted NGS
11
Automated DNA Extraction
System Format # Identified Sample Type Technology Capacity Processing
Time
Manual 3 Diverse Extraction
columns
single 4-20mins
Semi-automated 3 Diverse Magnetic bead 1-12 25-70mins
Fully automated 12 Diverse Magnetic bead 1-96 25-120mins
Completed a landscape analysis of commercially available clinical sample processing platforms
Developing TPPs
Proposed Simplified, Standard Pipeline for Targeted NGS
12
Simplified PCR/library Prep + Targeted NGS
Multiple Groups Developing Solutions
• Translational Genomics Research Institute (TGen) & UCSD
• Rutgers University & CDC
• Thermo Fisher Scientific
• bioMérieux
13
Targeted NGS for RDST from Sputum: A Proof of
Principal Study (PI Dave Engelthaler)
Goal
Goal
• Demonstrate that targeted NGS could be used to detect ~90%
of XDR-TB phenotypes from remnant Mtb DNA extracted
directly from TB patient sputa
• Demonstrate that (R/S) mixed populations as low as 1% could
be detected in clinical samples using targeted NGS
Methods
Design
• Retrospective lab study of remnant DNA extracted previously from
patient sputa as part of a large prospective diagnostics trial1
Patient Population
• Drug resistant TB suspects
• Moldova NTP
NGS Targets
• katG – Isoniazid resistance
• inhA promoter – Isoniazid resistance
• rpoB – Rifampicin resistance
• gyrA – Fluoroquinolone resistance
• rrs – Amikacin, Kanamycin and Capreomycin resistance
• eis promoter – Kanamycin resistance
1Catanzaro etc al. PLoS ONE, 2015
Results – Success of Targeted NGS
unpublished data not shown
Heteroresistance/Mixed Populations
The presence of mixtures of susceptible and
resistant phenotypes in the same population
May originate from “superinfections” of multiple
strains with differing phenotypes or from evolution of
original infecting strain into multiple lineages in situ
Selection of resistant strains in the presence of the
drug can result in treatment failure.
The sooner resistance alleles are detected the
more likely we can alter patient treatment to prevent
treatment failure
SMOR: Single Molecule Over-lapping Reads
Read 1
Read 2
Colman et al, PloS ONE, 2015
SMOR Error Calculation
Standard baseline error for Illumina ≈ 1X10-2
SMOR Error = error on both reads: Product rule
Read 1 T A C Read 1
Read 2 T A G
T A G
1x10-2 * 1x10-2 = 1x10-4
1x10-2
Colman et al, PloS ONE, 2015
SMOR analysis
Clinical Heteroresistance Analysis
10%
1% 0.1%
Colman et al, PloS ONE, 2015
Unpublished data not shown
INH - Mixtures
Colman et al, PloS ONE, 2015
Unpublished data not shown
RIF - Mixtures
2Colman et al, PloS ONE, 2015
Unpublished data not shown
Next Steps
Add new targets
• pncA and ahpC added
• Mutations for predicting phenotypic resistance
to PA-184, Bedaquiline and Delamanid being
developed
Optimize running conditions
simplify workflow
Proposed Simplified, Standard Pipeline for Targeted NGS
25
Automated DNA Extraction Simplified PCR/library Prep + Targeted NGS
Variant Analysis,
interpretation
and archiving
Proposed Simplified, Standard Pipeline for Targeted NGS
26
• Developing a solution for automated NGS bioinformatics
• Cloud-based
• TB WGS Specific
• No expert knowledge required for use
DTBE CDC and
DAIDS NIAID
Conclusions
High concordance between phenotypic DST, NGS RDST results
and pyrosequencing
Using the SMOR methodology we were able to detect mixed
populations (S/R) with high confidence direct from patient sputa
which has novel clinical ramifications
Targeted NGS appears to be valid means of doing “culture-free”
RDST but will need some optimization to simplify it sufficiently for
global deployment in LMICs
28
Acknowledgements
FIND
Claudia Denkinger
David Dolinger
NGS Development
David Engelthaler, Rebecca Colman & NGS Team (TGen)
Antonino Catanzaro, Donald Catanzaro, Valeriu Crudu (UCSD & Moldova)
David Alland (Rutgers University)
Jamie Posey (CDC)
ReSeqTB
DTBE CDC and
DAIDS NIAID