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