whole genome sequencing - weebly · 2018. 9. 4. · the road map deciphering the biology of...

Biomolecular diagnostics, gene sequencing and whole genome

sequencing

Rob Warren

Classic molecular epidemiology

IS6110 – DNA fingerprinting (between 0 and 26 loci)

Spoligotyping (1 locus)

MIRU-VNTR (24 loci)

The Road Map

Deciphering the biology of Mycobacterium tuberculosisfrom the complete genome sequenceNature 393, 537-544 (11 June 1998)

Two new families of glycine-rich proteins with a repetitive structure that may represent a source of antigenic variation.

4,411,529 base pairs 4,000 genes

Whole genome sequencing (Illumina)Sample preparation Cluster generation on flow cell

Sequencing and imagingData analysis

Shear DNALigate adapters

DNA extraction

Mapping/Aligning Reads to a Reference

Reference genome

Trimmed ReadsX100

X

X

X

X

X

X

X

USAP

BWA

Smalt

NOVOAlign

GATK

SAMTools

Filtered Variants used in downstream

applications

Alignment

Variant calling

Phylogeny

Out-of-Africa migration and Neolithic coexpansion of Mycobacterium tuberculosis with modern humans.Nat Genet. 2013 Oct;45(10):1176-82

Global Phylogeny of Mycobacterium tuberculosis

Transmission

House hold contacts

IS6110 RFLP

WGS

100% transmission

(ngd < 2)

J Clin Microbiol. 2002 Apr;40(4):1277-82.Evolution of the IS6110-based restriction fragment length polymorphism pattern during the transmission of Mycobacterium tuberculosis.

6/12 < 10 SNVs

50% transmission

6/12 > 45 SNVs≤ 2 band differences

0 band differences

https://www.ncbi.nlm.nih.gov/pubmed/11923345

Relapse / Reinfection

1

2

IS6110

RFLP

WGS

25/25 relapse

(ngd < 2)

Clin Infect Dis. 2014 Jun;58(12):1676-83.The temporal dynamics of relapse and reinfection tuberculosis after successful treatment: a retrospective cohort study.

22/25 relapse

(< 5 SNVs)

https://www.ncbi.nlm.nih.gov/pubmed/?term=Warren+R+and+Marx+F


WGS

2/25 reinfection

(mixed

infection)

First episode

isolate

Second episode

isolate

Patient Isolate number Isolate number

IS6110evolution

Total

variation

23 3459 4203 no 757

24 5835 6283 no 833

Heterogeneous /

underlying

population

(rpoB S531L)

(katG S315T)


WGS 1/25 20 SNVs

M. tuberculosis K93 L1 M. tuberculosis T17 L1 M. tuberculosis T92 L1 M. tuberculosis M4100A L2 M. tuberculosis T85 L2

318 (2) 1696 (0) 5557 (1) 4664 (0) 5408 (0) 3954 (0)

M. tuberculosis T67 L2 5602 (4) 3752 (0) 1994 (1) 1522 (0) 1389 (0) 1492 (0) 5577 (1) 3835 (0) 6420 (0) 5159 (0) 1411 (2) 1376 (3) 6826 (0) 7812 (0) 3327 (0) 3726 (0)

M. tuberculosis 910079 L3 M. tuberculosis K49 L3

1775 (35) 1725 (31) 4954 (1) 3857 (0) 2091 5790 (1) 3778 (1) 3205 (2) 3901 (0) 7403 (1) 6434 (0) 340 (0) 123 (0) 2415 (0) 2778 (1) 682 (1) 4885 (1) 3667 (0) 2626 (0) 757 3439 2971 1663 (41) 3773 (46) 298 (1) 1291 1653 (2) 770 (0) 2167 (5) 2621 (3) 644 (0) 1485 (1) 1457 (4) 1454 (3)

M. tuberculosis 4783-04 L4 M. tuberculosis H37Rv M. tuberculosis K37 L4

1159 (1) 903 (2) 5084 (0) 5946 (0) 2069 (0) 5181 (0) 4913 (0) 4224 (0) 7484 (1) 6314 (1) 4564 8156 (2) 6798 (0) 7464 3840 (0) 4759 (0) 7182 (7) 5130 (4) 7793 (1) 6558 (0) 6072 (0) 4402 (0)

M. canettii

M. tuberculosis K93 L1 M. tuberculosis T17 L1 M. tuberculosis T92 L1 M. tuberculosis M4100A L2 M. tuberculosis T85 L2

318 (2) 1696 (0) 5557 (1) 4664 (0) 5408 (0) 3954 (0)

M. tuberculosis T67 L2 5602 (4) 3752 (0) 1994 (1) 1522 (0) 1389 (0) 1492 (0) 5577 (1) 3835 (0) 6420 (0) 5159 (0) 1411 (2) 1376 (3) 6826 (0) 7812 (0) 3327 (0) 3726 (0)

M. tuberculosis 910079 L3 M. tuberculosis K49 L3

1775 (35) 1725 (31) 4954 (1) 3857 (0) 2091 5790 (1) 3778 (1) 3205 (2) 3901 (0) 7403 (1) 6434 (0) 340 (0) 123 (0) 2415 (0) 2778 (1) 682 (1) 4885 (1) 3667 (0) 2626 (0) 757 3439 2971 1663 (41) 3773 (46) 298 (1) 1291 1653 (2) 770 (0) 2167 (5) 2621 (3) 644 (0) 1485 (1) 1457 (4) 1454 (3)

M. tuberculosis 4783-04 L4 M. tuberculosis H37Rv M. tuberculosis K37 L4

1159 (1) 903 (2) 5084 (0) 5946 (0) 2069 (0) 5181 (0) 4913 (0) 4224 (0) 7484 (1) 6314 (1) 4564 8156 (2) 6798 (0) 7464 3840 (0) 4759 (0) 7182 (7) 5130 (4) 7793 (1) 6558 (0) 6072 (0) 4402 (0)

M. canettii

(1)

N = 16 (1993 to 1997)

katG 315 (AGC to ACC)rpoB 531 (TCG to TTG)embB 306 (ATG to ATA)

rrs 513 (CAG to CCG)

Transmission chain: Beijing outbreak

Transmission chain: Beijing outbreak

J Infect Dis. 1999 Nov;180(5):1608-15.Transmission of a multidrug-resistant Mycobacterium tuberculosis strain resembling "strain W" among noninstitutionalized, human immunodeficiency virus-seronegative patients.


N Engl J Med. 2017 Jan 19;376(3):243-253. doi: 10.1056/NEJMoa1604544.Transmission of Extensively Drug-Resistant Tuberculosis in South Africa.Shah NS1, Auld SC1, Brust JC1, Mathema B1, Ismail N1, Moodley P1, Mlisana K1, Allana S1, Campbell A1, Mthiyane T1, Morris N1, Mpangase P1, van der Meulen H1, Omar SV1, Brown TS1, Narechania A1, Shaskina E1, Kapwata T1, Kreiswirth B1, Gandhi NR1.

Transmission = 84%

https://www.ncbi.nlm.nih.gov/pubmed/28099825https://www.ncbi.nlm.nih.gov/pubmed/?term=Shah NS[Author]&cauthor=true&cauthor_uid=28099825https://www.ncbi.nlm.nih.gov/pubmed/?term=Auld SC[Author]&cauthor=true&cauthor_uid=28099825https://www.ncbi.nlm.nih.gov/pubmed/?term=Brust JC[Author]&cauthor=true&cauthor_uid=28099825https://www.ncbi.nlm.nih.gov/pubmed/?term=Mathema B[Author]&cauthor=true&cauthor_uid=28099825https://www.ncbi.nlm.nih.gov/pubmed/?term=Ismail N[Author]&cauthor=true&cauthor_uid=28099825https://www.ncbi.nlm.nih.gov/pubmed/?term=Moodley P[Author]&cauthor=true&cauthor_uid=28099825https://www.ncbi.nlm.nih.gov/pubmed/?term=Mlisana K[Author]&cauthor=true&cauthor_uid=28099825https://www.ncbi.nlm.nih.gov/pubmed/?term=Allana S[Author]&cauthor=true&cauthor_uid=28099825https://www.ncbi.nlm.nih.gov/pubmed/?term=Campbell A[Author]&cauthor=true&cauthor_uid=28099825https://www.ncbi.nlm.nih.gov/pubmed/?term=Mthiyane T[Author]&cauthor=true&cauthor_uid=28099825https://www.ncbi.nlm.nih.gov/pubmed/?term=Morris N[Author]&cauthor=true&cauthor_uid=28099825https://www.ncbi.nlm.nih.gov/pubmed/?term=Mpangase P[Author]&cauthor=true&cauthor_uid=28099825https://www.ncbi.nlm.nih.gov/pubmed/?term=van der Meulen H[Author]&cauthor=true&cauthor_uid=28099825https://www.ncbi.nlm.nih.gov/pubmed/?term=Omar SV[Author]&cauthor=true&cauthor_uid=28099825https://www.ncbi.nlm.nih.gov/pubmed/?term=Brown TS[Author]&cauthor=true&cauthor_uid=28099825https://www.ncbi.nlm.nih.gov/pubmed/?term=Narechania A[Author]&cauthor=true&cauthor_uid=28099825https://www.ncbi.nlm.nih.gov/pubmed/?term=Shaskina E[Author]&cauthor=true&cauthor_uid=28099825https://www.ncbi.nlm.nih.gov/pubmed/?term=Kapwata T[Author]&cauthor=true&cauthor_uid=28099825https://www.ncbi.nlm.nih.gov/pubmed/?term=Kreiswirth B[Author]&cauthor=true&cauthor_uid=28099825https://www.ncbi.nlm.nih.gov/pubmed/?term=Gandhi NR[Author]&cauthor=true&cauthor_uid=28099825

Social networks 30%

Transmission through Migration: Atypical Beijing

Atypical

Beijing

Other

Atypical

Beijing

J Clin Microbiol. 2015 Nov;53(11):3650-3. Molecular Epidemiological Interpretation of the Epidemic of Extensively Drug-Resistant Tuberculosis in South Africa.


Precision Medicine

https://platform.reseqtb.org/

H37Rv gene associated antibiotic resistance

aftA,embC ethambutol

ahpC compensates for KatG deficit

ahpC upstream compensates for KatG deficit

ald cycloserine

ald upstream cycloserine

alr cycloserine

alr upstream cycloserine

atpE bedaquiline

atpE upstream bedaquiline

blaC beta-lactams

blaC upstream beta-lactams

cycA cycloserine

cycA upstream cycloserine

dacB2 beta-lactams

dacB2 upstream beta-lactams

ddlA cycloserine

ddlA upstream cycloserine

ddn delamanid, PA-824

ddn upstream delamanid, PA-824

dfrA PAS, co-trimoxazole

dfrA upstream PAS, co-trimoxazole

eccB5 upstream moxifloxacin, gatifloxacin, ofloxacin, levofloxacin

eccB5,eccC5 moxifloxacin, gatifloxacin, ofloxacin, levofloxacin

eis kanamycin

eis upstream kanamycin

embA upstream ethambutol

embA,embB ethambutol

embR ethambutol

embR upstream ethambutol

erm(37) clarithromycin

erm(37) upstream clarithromycin

ethA ethionamide, prothionamide, thioacetazone

ethA upstream ethionamide, prothionamide, thioacetazone

ethR ethionamide, prothionamide, thioacetazone

fabG1 isoniazid, ethionamide, prothionamide

fabG1 upstream isoniazid, ethionamide, prothionamide

fbiA upstream delamanid, PA-824

fbiA,B delamanid, PA-824

fbiC delamanid, PA-824

fbiC upstream delamanid, PA-824

fgd1 delamanid, PA-824

fgd1 upstream delamanid, PA-824

folC PAS

folP1 PAS, co-trimoxazole

folP1 upstream PAS, co-trimoxazole

folP2 co-trimoxazole

folP2 upstream co-trimoxazole

furA isoniazid

furA upstream isoniazid

gidB streptomycin

gidB upstream streptomycin

gpsI pyrazinamide

gpsI upstream pyrazinamide

gyrA moxifloxacin, gatifloxacin, ofloxacin, levofloxacin

gyrA upstream moxifloxacin, gatifloxacin, ofloxacin, levofloxacin

gyrB moxifloxacin, gatifloxacin, ofloxacin, levofloxacin

gyrB upstream moxifloxacin, gatifloxacin, ofloxacin, levofloxacin

hadA upstream thioacetazone

hadA,hadB,hadC thioacetazone

inbR isoniazid

inbR upstream isoniazid

inhA isoniazid, ethionamide, prothionamide

inhA upstream isoniazid, ethionamide, prothionamide

kasA isoniazid

katG isoniazid

katG upstream isoniazid

ldtA beta-lactams

ldtA upstream beta-lactams

ldtB beta-lactams

ldtB upstream beta-lactams

ldtD beta-lactams

ldtD upstream beta-lactams

ldtE beta-lactams

ldtE upstream beta-lactams

lprQ beta-lactams

lprQ upstream beta-lactams

mmaA2 thioacetazone

mmaA2 upstream thioacetazone

mmaA3 isoniazid, ethionamide, prothionamide

mmaA3 upstream isoniazid, ethionamide, prothionamide

mmaA4 thioacetazone


mmpL5,mmpS5 bedaquiline, clofazimine

mmpR bedaquiline, clofazimine

mmpR upstream bedaquiline, clofazimine

mshA isoniazid, ethionamide, prothionamide

mshA upstream isoniazid, ethionamide, prothionamide

mshB isoniazid

mshB upstream isoniazid

mshC isoniazid, ethionamide, prothionamide

mshC upstream isoniazid, ethionamide, prothionamide

mymA isoniazid, ethionamide, prothionamide

mymA upstream isoniazid, ethionamide, prothionamide

nat isoniazid

nat upstream isoniazid

ndh isoniazid, ethionamide, prothionamide, clofazimine, thioridazine

ndh upstream isoniazid, ethionamide, prothionamide, clofazimine, thioridazine

nudC isoniazid, ethionamide, prothionamide

nudC upstream isoniazid, ethionamide, prothionamide

panD pyrazinamide

pbpA beta-lactams

pepQ bedaquiline, clofazimine

pepQ upstream bedaquiline, clofazimine

pncA pyrazinamide

pncA upstream pyrazinamide

ponA1 rifampicin, rifabutin, beta-lactams

ponA1 upstream rifampicin, rifabutin, beta-lactams

pykA cycloserine

pykA upstream cycloserine

rpfB upstream clarithromycin

rpfB, ksgA clarithromycin

rplC linezolid

rplC upstream linezolid

rpoA compensates for rpoB mutations

rpoA upstream compensates for rpoB mutations

rpoB rifampicin, rifabutin

rpoB upstream rifampicin, rifabutin

rpoC compensates for rpoB mutations

rpoC upstream compensates for rpoB mutations

rpsA pyrazinamide

rpsA upstream pyrazinamide

rpsL streptomycin

rpsL upstream streptomycin

rrl linezolid, capreomycin, clarithromycin

rrl upstream linezolid, capreomycin, clarithromycin

rrs streptomycin, amikacin, kanamycin, capreomycin

rrs upstream streptomycin, amikacin, kanamycin, capreomycin

Rv0565c ethionamide, prothionamide

Rv0565c upstream ethionamide, prothionamide

Rv1979c bedaquiline, clofazimine

Rv1979c upstream bedaquiline, clofazimine

Rv2671 PAS

Rv2671 upstream PAS

Rv2731 pyrazinamide

Rv3169 pyrazinamide

serB2 clofazimine

serB2 upstream clofazimine

sigI isoniazid

sigI upstream isoniazid

tap streptomycin, PAS

tap upstream streptomycin, PAS

thyA PAS

thyA upstream PAS

thyX compensates for ThyA deficit

thyX upstream compensates for ThyA deficit

tlyA capreomycin

ubiA ethambutol

ubiA upstream ethambutol

whiB7 streptomycin, kanamycin, clarithromycin, PAS

whiB7 upstream streptomycin, kanamycin, clarithromycin, PAS





ald cycloserine


alr cycloserine


atpE bedaquiline


blaC beta-lactams


cycA cycloserine


dacB2 beta-lactams


ddlA cycloserine








eis kanamycin




embR ethambutol















folC PAS





furA isoniazid


gidB streptomycin


gpsI pyrazinamide








inbR isoniazid




kasA isoniazid

katG isoniazid


ldtA beta-lactams


ldtB beta-lactams


ldtD beta-lactams


ldtE beta-lactams


lprQ beta-lactams


mmaA2 thioacetazone




mmaA4 thioacetazone







mshB isoniazid






nat isoniazid






panD pyrazinamide

pbpA beta-lactams



pncA pyrazinamide




pykA cycloserine




rplC linezolid








rpsA pyrazinamide


rpsL streptomycin










Rv2671 PAS

Rv2671 upstream PAS

Rv2731 pyrazinamide

Rv3169 pyrazinamide

serB2 clofazimine


sigI isoniazid




thyA PAS

thyA upstream PAS



tlyA capreomycin

ubiA ethambutol








ald cycloserine


alr cycloserine


atpE bedaquiline


blaC beta-lactams


cycA cycloserine


dacB2 beta-lactams


ddlA cycloserine








eis kanamycin




embR ethambutol















folC PAS





furA isoniazid


gidB streptomycin


gpsI pyrazinamide








inbR isoniazid




kasA isoniazid

katG isoniazid


ldtA beta-lactams


ldtB beta-lactams


ldtD beta-lactams


ldtE beta-lactams


lprQ beta-lactams


mmaA2 thioacetazone




mmaA4 thioacetazone







mshB isoniazid






nat isoniazid






panD pyrazinamide

pbpA beta-lactams



pncA pyrazinamide




pykA cycloserine




rplC linezolid








rpsA pyrazinamide


rpsL streptomycin










Rv2671 PAS

Rv2671 upstream PAS

Rv2731 pyrazinamide

Rv3169 pyrazinamide

serB2 clofazimine


sigI isoniazid




thyA PAS

thyA upstream PAS



tlyA capreomycin

ubiA ethambutol




rpoB codon rpoB base change No of isolates RIF MIC range RBT MIC range Xpert

1 WT WT 27 2 Probe B

6 513 A>C 2 >50 - >100 0.5 – 1.0 Probe B

7 513 A>T 2 >50 - >100 >2 Probe B

8 515 6bp deletion 2 >100 >2 Probe B

9 516 3bp deletion 4 >1 - T 15 100 >2 Probe D

21 526 CA>TG 1 >1 20 - >100 >2 Probe D

23 526 A>T 4 100

27 531 CG>TT 2 >100 >2 Probe E

28 533 T>C 10 5 1A + G>T 1 >100

31 513 + 516 C>G + A>T 1 >100 >2 Probe B only

32 512 + 516 C>G + A>G 1 >1 T 1 >1

608 Mutations (SNPs and in/dels) at 397 positions

A Global Perspective on Pyrazinamide Resistance: Systematic Review and Meta-Analysis. PLoS One. 2015 Jul 28;10(7):e0133869

Pyrazinamide genotypic DST

Terizidoneresistance

Heteroresistance

Drug-susceptible population Mixed population

Drug pressure

Spontaneous mutation

Mixed population Resistant population

Drug pressurex x

x x

xx

x Mutant outcompetes

progenitor

Mutant grows better than progenitor

Gold standard Accurate Lengthy

Limited number of markers detected in 1 reaction Detects some

INH and RIF

Standard of care Detects RIF only

Sequencing of the entire genome of the organism

Expensive Requires culture Detects any known

resistance marker

Detecting Heteroresistance

PCR/Line Probe Assay (MTBDRplus)

Isoniazid (INH) Sensitive

Clinical Example (Day 1)

First Line Drugs – Agar Culture Based:

Isoniazid (INH) Resistant

Follow up culture based DST (Day 24)

Additional TestsNHLS Results:LPA: SusceptibleCulture: Resistant

Susceptible

Requested DNA sequencing of KatG gene and inhA promoter (Stellenbosch University)

Sanger sequencing

Whole Genome Sequencing

Automated pipeline analysis

Susceptible

katG

Resistant population

Susceptiblepopulation

When does heteroresistance become clinically relevant?

Mixed population1%

Mixed population50%

Mutant population90%

Mixed population < 1%

Phenotypically Resistant

Phenotypically Resistant

Phenotypically Resistant????

Phenotypically Suceptible

Targeted deep sequencing• Based on WGS technology

• Amplification of a target region of the genome

>50,000 x

X

X

Reference

Error rate

7 5 6 0 7 5 6 5 7 5 7 0 7 5 7 5 7 5 8 0 7 5 8 5 7 5 9 0 7 5 9 5 7 6 0 0 7 6 0 5 7 6 1 0 7 6 1 5 7 6 2 0 7 6 2 5 7 6 3 0

0 .0

0 .1

0 .2

0 .3

0 .4

0 .5

0 .6

0 .7

0 .8

0 .9

1 .0

R 6 7 4 7 _ Q R D R o f g y r A

A lle le p o s itio n in Q R D R o f g y rA

All

ele

pe

rc

en

tag

e (

%)

A

C

G

T

Allele positions in the QRDR of gyrA

All

ele

pe

rce

nta

ge (

%)

"T" mutant (0,17%) codon 90

(position 7570)

7 5 6 0 7 5 6 5 7 5 7 0 7 5 7 5 7 5 8 0 7 5 8 5 7 5 9 0 7 5 9 5 7 6 0 0 7 6 0 5 7 6 1 0 7 6 1 5 7 6 2 0 7 6 2 5 7 6 3 0

0

1 0

2 0

3 0

4 0

5 0

6 0

7 0

8 0

9 0

1 0 0

R 6 9 7 1 _ Q R D R o f g y r A


All

ele

pe

rc

en

tag

e (

%)

A

C

G

T"T" mutant (68,18%)

codon 90 (position 7570)


All

ele

pe

rce

nta

ge (

%)

7 5 6 0 7 5 6 5 7 5 7 0 7 5 7 5 7 5 8 0 7 5 8 5 7 5 9 0 7 5 9 5 7 6 0 0 7 6 0 5 7 6 1 0 7 6 1 5 7 6 2 0 7 6 2 5 7 6 3 0

0

1 0

2 0

3 0

4 0

5 0

6 0

7 0

8 0

9 0

1 0 0

R 7 5 1 5 _ Q R D R o f g y r A


All

ele

pe

rc

en

tag

e (

%)

A

C

G

T

"T" mutant (86,14%)

codon 90


All

ele

pe

rce

nta

ge (

%)

28/08/2009 02/10/2009

27/10/2009

DST = S DST = R

DST = R

gyrA 90 T allele @ 99.91%


gyrA 94 C allele @ 0.07%




Limitations

• Time to result

• Data analysis

• Cost

• Lack of knowledge on drug-resistance mutations

• Ability to detect heteroresistance

• 10 to 15% of the genome is not analyses

Acknowledgements

whole genome sequencing - weebly · 2018. 9. 4. · the road map deciphering the biology of...

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