2pm 2016 NAR V4 - lung · Identify new mechanisms of drug resistance " Existing and new drugs ! Identify preexisting resistance " Repurposed and new drugs ! Microevolution in the

Posey: The Imipact of Genomics Era on Mtb Research

2/26/16-‐TB Genomics

1

The Impact of Genomics Era on Mycobacterium tuberculosis Research

Jamie Posey, PhD Applied Research Team Lead

National Center for HIV/AIDS, Viral Hepatitis, STD, and TB Prevention Division of Tuberculosis Elimination

NGS Platforms

MOLECULAR EPIDEMIOLOGY



2

Cluster 1

~ 100 Patients

DRUG RESISTANCE

Conventional Drug Susceptibility Testing

q  Growth based §  Time consuming (week to month) §  Laborious §  Infrastructure

q  Liquid or solid media §  Equivalent concentrations?

q  Critical concentration

q  Minimum inhibitory concentration



3

Drug Resistant Survey

q  Based on phenotypic assays §  Some countries perform routine DST §  Resource-limited areas (once every 3-5 years) §  Lack infrastructure

q  Can we use molecular assays? §  Do we have the knowledge §  What tools are needed

rpoB Surveillance

q  Rifamycin resistance §  Role of mutations

q  Frequency of mutations §  Population level §  Assay development

q  Importance of data §  Calculate sensitivity and specificity of assays §  Silent mutations §  How much phenotypic resistance is missed §  What is the affect on patient outcome

Fusion Primers for Ion Torrent PGM

RRDR



4

Rifampin Resistant Determining Region (RRDR) of rpoB

rpoB Surveillance

q  14 months §  >10,000 isolates

q  Mutations §  411 isolates (35 unique mutations) §  Ser531Leu (184 isolates) §  Silent mutations (94 isolates)

RESEARCH



5

Areas to Address

q  Identify new mechanisms of drug resistance §  Existing and new drugs

q  Identify preexisting resistance §  Repurposed and new drugs

q  Microevolution in the patient

q  Compensatory mutations

29 44

26

19

36 63

46 44

B B

B B

B

B

A

C

C G C31998G58 bp upstream Rv0029

C TC1663856T

acn

C T C51403T Rv0047c G AG1674048A

fabG1

C T C118832T Rv0102 T CT1877958C

pks7

C T C247984T Rv0207c C GC1888075G

pks9

T C T362962CPE_PGRS5

T CT2087076C

171 bp upstream of Rv1838c

C G C477188G Rv0398c C TC2372126T

Rv2112c

C A C480678A mmpL1 G CG2402463C

Rv2142c

A GA649974G

ubiE T CT2614547C

46 bp upstream Rv2339

C T C761147T rpoB G AG2751471A

Rv2449c

G C G765719C rpoC G AG2958534A

Rv2631

C G C799139G Rv0698 G AG3126489A

Rv2819c

G AG905686A

Rv0811c G AG3137406A

echA16

C A C926861APE_PGRS13

C TC3213150T

lepB

C GC1023436G

betP A CA3377940C

PPE46

T GT1093459G

PE_PGRS17

A CA3380380C

PPE47

G AG1114491A

Rv0997 A GA3416480G

Rv3055

C TC1208858T

Rv1084 C TC3455434T

Rv3088

C TC1231660T

Rv1104 G CG3608047C

Rv3230c

A CA1246730C

bpoB C TC3764285T

PPE56

C TC1266797T

Rv1139c G AG3765280A

PPE56

C TC1309314T

fdxC C AC3777772A

spoU

A CA1320356C

papA3 A GA4026439G

5 bp upstream Rv3585

G AG1353888A

tagA C AC4037284A

PE_PGRS59

G AG1421085A

Rv1272c T AT4072484A

Rv3633

G CG4084482C

topA

A GA4314271G

bfrB



6

Identify New Mechanisms of Resistance

fabG1 inhA

L203L A silent mutation in mabA confers isoniazid resistance on Mycobacterium tuberculosis. Ando H, Miyoshi-Akiyama T, Watanabe S, Kirikae T.

inhA Transcript Levels

CLINICAL MANAGEMENT



7

Starting Material

Sputum Dx Culture Subculture

WGS versus Targeted Approach

q  WGS §  Need a culture §  High molecular weight DNA (1—5 ug) §  Analyze about 90—99% of genome §  Low to medium throughput

q  Targeted loci §  Could possibly start with processed sputum §  PCR based §  Lower quality and quantity of DNA §  Only analyze the areas amplified §  High throughput

Sensitivity and Specificity of Loci

Drug Gene(s) Sensitivity (%) Specificity (%)

RIF rpoB 97.1 93.6

INH inhA, katG 90.6 100

EMB embB 78.6 93.1

FQ gyrA 81.6 97.7

KAN rrs, eis 86.5 96.1

AMK rrs 90.0 98.8

CAP rrs, tlyA 60.9 87.3

MDR rpoB, inhA, katG 90.8 94.7



8

Retrospective DR Study and MDDR Service Sensitivity and Specificity of Loci

Drug Gene(s) Sensitivity (%) Specificity (%)

RIF rpoB 97.1 97.4

INH inhA, katG 86.0 99.1

EMB embB 78.8 94.3

FQ gyrA 79.0 99.6

KAN rrs, eis 86.7 99.6

AMK rrs 90.9 98.4

CAP rrs, tlyA 55.2 91.0

Molecular Detection of Drug Resistance

q  Original assay §  8 single PCR reactions §  Sanger DNA sequencing §  16 sequencing reactions

q  High throughput assay §  NGS – Ion Torrent PGM §  Multiplex PCR §  Barcoded – 96 samples per assay

Targeted Loci

DRUG Gene Region Rifampin rpoB 176 and RRDR Isoniazid katG 315

inhA Promoter Ethambutol embB ERDR

Pyrazinamide pncA Promoter and ORF Fluoroquinolones gyrA QRDR

gyrB QRDR Kanamycin eis Promoter

rrs 1401 Amikacin rrs 1401

Capreomycin rrs 1401 tlyA Promoter and ORF



9

Workflow

2

13Ion AmpliSeq™ Library Preparation User Guide

Methods

Workflow

Amplify targets(page 15)

Partially digest primer sequences (page 17)

Ligate adapters to the amplicons and purify (page 17)

Option 1: Equalize the

library(page 19)

Option 2: Quantify the unamplified

library by qPCR

(page 21)

Option 3: Quantify the amplified

library with the Qubit® 2.0

Fluorometer or Agilent® 2100 Bioanalyzer® instrument (page 23)

Combine libraries (optional) and proceed to template preparation.

A P1

AP1Adapters

Amplify targets usingIon AmpliSeq™ Primer Pool

Genomic DNA

Partially digest primer sequences

Ligate adapters P1

XBarcode Adapters

OR

Barcoded libraryP1X

OR

Nonbarcoded library

Pilot Study

q  8 and 48 samples

q  Use crude DNA preps

q  Compare Ion Torrent data to Sanger sequence

Number of Reads Per Sample

Sample Number of Reads MLB2 100,039

MLB18 55,909 MLB36 40,246

MLB138 5,715 MLB149 51,144 MLB176 4,525 MLB207 98,238 MLB224 50,865



10

>

MID Ref'Pos Type Ref'Base Called'BaseSNP'% Feature'Name Amino'Acid'ChangeDepthMLB2 4247730 SNP G C 97.70% embB G406A 1196MLB224 4247578 SNP G A 99.20% embB L355L 1658MLB224 4247646 SNP A C 95.90% embB E378A 560MLB138 7585 SNP G C 90.90% gyrA S95T 220MLB149 7585 SNP G C 90.30% gyrA S95T 944MLB176 7585 SNP G C 72.00% gyrA S95T 100MLB176 7582 SNP A G 84.00% gyrA D94G 100MLB18 7585 SNP G C 90.00% gyrA S95T 964MLB2 7585 SNP G C 89.80% gyrA S95T 2538MLB224 7585 SNP G C 91.80% gyrA S95T 1279MLB36 7585 SNP G C 89.20% gyrA S95T 869MLB36 7570 SNP C T 99.50% gyrA A90V 830MLB18 2155168 SNP C G 99.60% katG S315T 1308MLB2 2155168 SNP C G 99.60% katG S315T 2475MLB36 2155168 SNP C G 99.30% katG S315T 957MLB18 2288827 SNP C T 78.30% pncA V139M 2702MLB2 2288935 SNP A G 99.80% pncA Y103H 8046MLB224 2288733 SNP G A 98.70% pncA A170V 1475MLB36 2288826 SNP A G 78.90% pncA V139A 1882MLB176 761161 SNP T C 60.00% rpoB L452P 25MLB18 761155 SNP C T 99.10% rpoB S450L 900MLB2 761155 SNP C T 99.50% rpoB S450L 1577

SNP Report

SNP Report

MLB36 761155 SNP C T 99.10% rpoB S450L 680MLB2 1473246 SNP A G 99.80% rrs 5051MLB207 1473183 SNP A G 35.00% rrs 20MLB36 1473246 SNP A G 99.80% rrs 1658MLB138 1917972 SNP A G 93.10% tlyA L11L 159MLB149 1917972 SNP A G 96.40% tlyA L11L 863MLB176 1917972 SNP A G 97.40% tlyA L11L 76MLB176 1918523 SNP G A 98.90% tlyA G195D 89MLB18 1917972 SNP A G 97.00% tlyA L11L 943MLB2 1917972 SNP A G 96.60% tlyA L11L 1511MLB207 1917972 SNP A G 97.40% tlyA L11L 1337MLB224 1917972 SNP A G 96.30% tlyA L11L 934MLB36 1917972 SNP A G 96.50% tlyA L11L 818



11

Number of Reads Per Sample (48)

16,000

32,000

48,000

# R

eads

0

500

1000

1500

2000

2500

3000

0 1 2 3 4 5 6 7 8 9 10 11 12

Coverage Depth

0

10

20

30

40

50

60

70

80

90

100

0 1 2 3 4 5 6 7 8 9 10 11 12

Coverage Depth

# R

eads



12

Summary and Next Steps q  Pilot project

§  8 samples – completed and 100% agreement with Sanger data §  48 samples – analyzing data

q  Quality and quantity of DNA

q  Determine the minimal coverage and number of reads

q  Scale up to 96 samples

q  Test processed specimens

METAGENOMICS

Starting Material

Sputum Dx Culture Subculture



13

Synthetic Dilution Series Strategy

•  Pure gDNA dilution to extinction •  Mtb, STEC, C diff

•  gDNA/gRNA dilution to extinction in complex background •  Mtb/Sputum •  STEC/Stool •  Influenza/A549 RNA •  OC43 Coronavirus/A549 RNA

•  Targeted enrichment study •  Background depletion study

Mtb Sputum Synthetic Sets

•  Create seven sets of dilutions (100 x 50 µL ea.) in a constant background of Sputum gDNA (25 ng µL-1)

Ct 40 Mtb IS6110 RT-PCR

•  Dilution 0 – Unspiked Sputum Ct >40

•  Dilution 1 – 10% Mtb Ct 13.0 ±0.16

•  Dilution 2 – 1% Mtb Ct 16.7 ±0.12

•  Dilution 3 – 0.1% Mtb Ct 20.1 ±0.05

•  Dilution 4 – 0.01% Mtb Ct 23.5 ±0.14

•  Dilution 5 – 0.001% Mtb Ct 27.1 ±0.15

•  Dilution 6 – 0.0001% Mtb Ct 30.4 ±0.12

1 2 3 4 5 6

5 aliquots of each dilution sampled at random assayed in triplicate RT-PCR

0

DNA Targeted Sequence Enrichment Strategy

•  Commercial and lab developed kits •  Agilent SureSelect •  Roche NimbleGen SeqCap •  NuGEN Select •  Custom assays

•  Three initial designs for each approach •  Mtb H37Rv •  STEC O157:H7 Sakai •  Influenza H3N2 A/Texas/50/2012



14

Agilent SureSelect

Roche NimbleGen SeqCap

Roche Targeted Sequence Enrichment Project Workflow

MTb gDNA + Sputum gDNA at different ratios

MTb % 0.0001 0.001 0.01 0.1 1 10

KAPA-Based Library Prep

Sequence Capture

MiSeq Sequencing and Analysis

All mixtures @ 25 ng uL-1

0



15

Mtb Sputum Roche TE Analysis – Pre Treatment/ No Enrichment Read Count Normalized

0

1000000

2000000

3000000

4000000

5000000

6000000

7000000

8000000

9000000

0 10 1 0.1 0.01 0.001 0.0001

Num

ber o

f Reads

% Mtb DNA in Original Sample

Other

Mtb

Human

QC

Mtb Sputum Roche TE analysis – Pre Treatment/ No Enrichment

5814354, 96%

554, 0%

268994, 4%

No Spike

5013170, 93%

137646, 3%

243280, 4%

10%

5261796, 95%

12682, 0%

253198, 5%

1%

5376692, 95%

1548, 0%

284728, 5%

0.1%

6501390, 95%

214, 0% 323650, 5%

0.01%

6171228, 95%

104, 0%

315366, 5%

0.001%

6497154, 95%

72, 0% 374982,

5%

0.0001%

Mtb Sputum Roche TE analysis - Pre Treatment/ No Enrichment

0

1

2

3

4

5

6

7

0 10 1 0.1 0.01 0.001 0.0001

Fold Gen

ome Co

verage

Mtb in Original Sample

Average Read Depth

0

20

40

60

80

100

0 10 1 0.1 0.01 0.001 0.0001

% Gen

ome Co

vered

% Mtb in Original Sample

Percent Coverage (Reference Base pairs Covered)



16

Mtb Sputum Roche TE analysis - Pre Treatment/ No Enrichment

0

1000

2000

3000

4000

5000

6000

1000

193000

385000

577000

769000

961000

1153000

1345000

1537000

1729000

1921000

2113000

2305000

2497000

2689000

2881000

3073000

3265000

3457000

3649000

3841000

4033000

4225000

H37Rv Genome PosiXon

10% Mtb

0 500

1000 1500 2000 2500 3000 3500 4000 4500

1000

193000

385000

577000

769000

961000

1153000

1345000

1537000

1729000

1921000

2113000

2305000

2497000

2689000

2881000

3073000

3265000

3457000

3649000

3841000

4033000

4225000


1% Mtb

0 2000 4000 6000 8000 10000 12000 14000 16000

1000

178000

355000

532000

709000

886000

1063000

1240000

1417000

1594000

1771000

1948000

2125000

2302000

2479000

2656000

2833000

3010000

3187000

3364000

3541000

3718000

3895000

4072000

4249000


0.1% Mtb Gene: rrs (16S) Gene: rrl (23S) Gene: rrf (5S) At decreasing levels of Mtb genome

sequence present in samples – mappers appear to erroneously attribute other 16S sequence in background sputum to the reference genome

Mtb Sputum Roche Post Enrichment Analysis – Read Count Normalized

0

1000000

2000000

3000000

4000000

5000000

6000000

7000000

8000000

9000000

0 1 0.1 0.01 0.001 0.0001

Num

ber o

f Reads


Other

Mtb

Human

QC

4318994, 84%

389712, 8%

407228, 8%

0.001%

1878156, 39%

2566948, 54%

344742, 7%

0.01%

533198, 13%

3227798, 81%

245416, 6%

0.1%

417872, 9%

4037224, 85%

272644, 6%

1%

6577598, 93%

37984, 1% 431280, 6%

No Spike

Mtb Sputum Roche Post Treatment Analysis

5194026, 89%

114054, 2%

533266, 9%

0.0001%



17

Mtb Sputum Roche Post Enrichment Analysis

1.37

184.33

148.8

115.61

17.6 7.58

0 20 40 60 80 100 120 140 160 180 200

0 1 0.1 0.01 0.001 0.0001

Fold Gen

ome Co

verage


Average Read Depth

5.36

99.4 99.31 99.18 96.77

57.72

0 10 20 30 40 50 60 70 80 90 100

0 1 0.1 0.01 0.001 0.0001

% Gen

ome Co

verage


Percent Coverage (Reference Base Pairs Covered)

Mtb Sputum Roche Post Enrichment Analysis

Gene: rrs (16S) 1471846 – 1473382 Gene: rrl (23S) 1473658 – 1476795 Gene: rrf (5S) 1476899 – 1477013

0

5000

10000

15000

1000

23

4000

46

7000

70

0000

93

3000

11

6600

0 13

9900

0 16

3200

0 18

6500

0 20

9800

0 23

3100

0 25

6400

0 27

9700

0 30

3000

0 32

6300

0 34

9600

0 37

2900

0 39

6200

0 41

9500

0

No Spike

0 1000 2000 3000 4000 5000 6000

1000

24

7000

49

3000

73

9000

98

5000

12

3100

0 14

7700

0 17

2300

0 19

6900

0 22

1500

0 24

6100

0 27

0700

0 29

5300

0 31

9900

0 34

4500

0 36

9100

0 39

3700

0 41

8300

0

1%

0 1000 2000 3000 4000 5000

1000

26

1000

52

1000

78

1000

10

4100

0 13

0100

0 15

6100

0 18

2100

0 20

8100

0 23

4100

0 26

0100

0 28

6100

0 31

2100

0 33

8100

0 36

4100

0 39

0100

0 41

6100

0

0.1%

0 2000 4000 6000 8000 10000 12000 14000 16000

1000

24

7000

49

3000

73

9000

98

5000

12

3100

0 14

7700

0 17

2300

0 19

6900

0 22

1500

0 24

6100

0 27

0700

0 29

5300

0 31

9900

0 34

4500

0 36

9100

0 39

3700

0 41

8300

0

0.01%

0

5000

10000

15000

20000

1000

24

7000

49

3000

73

9000

98

5000

12

3100

0 14

7700

0 17

2300

0 19

6900

0 22

1500

0 24

6100

0 27

0700

0 29

5300

0 31

9900

0 34

4500

0 36

9100

0 39

3700

0 41

8300

0

0.001%

0 5000 10000 15000 20000 25000 30000

1000

24

7000

49

3000

73

9000

98

5000

12

3100

0 14

7700

0 17

2300

0 19

6900

0 22

1500

0 24

6100

0 27

0700

0 29

5300

0 31

9900

0 34

4500

0 36

9100

0 39

3700

0 41

8300

0

0.0001%

Mtb Sputum Roche TE Coverage

0

20

40

60

80

100

0 10 1 0.1 0.01 0.001 0.0001

% Gen

ome Co

vered


Percent Coverage (Reference Base pairs Covered) No Enrichment

5.36

99.4 99.31 99.18 96.77

57.72

0

20

40

60

80

100

0 1 0.1 0.01 0.001 0.0001

% Gen

ome Co

verage


Percent Coverage (Reference Base Pairs Covered) Post Enrichment



18

Mtb Sputum Roche Average Depth

0 1 2 3 4 5 6 7

0 10 1 0.1 0.01 0.001 0.0001

Fold Gen

ome Co

verage


Average Read Depth No Enrichment

1.37

184.33

148.8

115.61

17.6 7.58

0 20 40 60 80 100 120 140 160 180 200

0 1 0.1 0.01 0.001 0.0001

Fold Gen

ome Co

verage


Average Read Depth Post Enrichment

Drug Resistant Genes

Drug Resistant Genes



19

0 20 40 60 80

100

0 1 2 3 4 5 6

BLR 3

S95A

0 20 40 60 80 100

0 1

BLR 4

D94A S95T

0 20 40 60 80 100

0 1

BLR 2

D94A S95T

0 20 40 60 80

100

0 14 15 21

BLR 1

A90V D94N S95T

% Reads

% Reads

% Reads

% Reads

0 20 40 60 80

100

0 1 3 10 16

AQR 1

D94G S95T

0 20 40 60 80 100

0 1 3 5 6 8 9 10 11 12

AQR 2

D94Y D94G S95T

0 20 40 60 80

100

0 3 8 10 13 15 18 21

AQR 3

G88C D89G A90V D94N D94G S95T

0 20 40 60 80 100

1 2 3 4 6 7 8 9 13

AQR 4

A90V D94N D94A S95T

% Reads

% Reads

% Reads

% Reads

Molecular Epidemiology

Drug Resistance

Metagenomics



20

Acknowledgements q  Laboratory Branch / Applied Research Team

§  Melisa Willby §  Paige Chopra §  Paul Grwzybowski §  Lauren Cowan

q  Metagenomics Group §  Chris Hopkins §  Eishita Tyagi §  Scott Burns

q  Core Facility §  Mike Frace §  Scott Sammons §  Kristen Knipe

Documents

2pm 2016 NAR V4 - lung · Identify new mechanisms of drug resistance " Existing and new drugs ! Identify preexisting resistance " Repurposed and new drugs ! Microevolution in the