Nex ext gen ener erat ion seque uenc ncing ng t o t o det - - PowerPoint PPT Presentation

▶
nex ext gen ener erat ion seque uenc ncing ng t o t o det
SMART_READER_LITE
LIVE PREVIEW

Nex ext gen ener erat ion seque uenc ncing ng t o t o det - - PowerPoint PPT Presentation

Jan 23, 2024 165 likes •586 views

Nex ext gen ener erat ion seque uenc ncing ng t o t o det et er erm ine HI HI V-1 1 corec ecep ept or t ro ropism Martin Dumer Apr 11, 2013 Genotypic tropism testing Plasma virus RNA (first choice) Standard Sanger Sequencing

slide-1
SLIDE 1

Nex ext gen ener erat ion seque uenc ncing ng t o t o det et er erm ine HI HI V-1 1 corec ecep ept or t ro ropism

Martin Däumer

Apr 11, 2013

slide-2
SLIDE 2

Genotypic tropism testing

Standard Sanger Sequencing

Plasma virus RNA

(first choice)

Proviral DNA

(Low or suppressed pVL)

Next-Generation Sequencing

Plasma virus RNA (first choice) Proviral DNA

(Low or suppressed pVL)

slide-3
SLIDE 3

Genotypic tropism testing

Standard Sanger Sequencing

  • Availability in most HIV laboratories
  • Low costs
  • Technically simple
  • Low sensitivity in terms of detecting minority non-R5 variants

(might be somewhat improved by triplicate parallel testing)

Next-Generation Sequencing

  • Only few HIV laboratories
  • Higher costs
  • Complex workflow
  • Sensitivity comparable to that of phenotypic assays

... but: things change rapidly

slide-4
SLIDE 4

Limitations of standard sequencing

Blood plasma RNA

RNA extraction

cDNA

Reverse Transcription/ Polymerase Chain Reaction

PCR products

  • 2. Nested PCR

Sequencing Reaction

Purification (optional)

Sequencer

Purification (optional)

M Sample

Sensitivity for m inor variants: > 2 0 %

slide-5
SLIDE 5

Swenson LC et al., 2011. J Infect Dis 203:237–245

Virologic responses of patients stratified by the percentage of non-R5 variants detected by deep sequencing at screening

Of these, only 37% were detected by population sequencing

MOTIVATE-1,-2, A4001029

slide-6
SLIDE 6

Hunting for the needle in the haystack

slide-7
SLIDE 7

Technical hurdles in population sequencing

Insertion Deletion

slide-8
SLIDE 8

„Ultra-deep-sequencing“

...PQIYMDDHTRE...

Standard Sanger-sequencing

...PQIYVDDHTRE... ...PQIYMDDHTRE... ...PQIYMDDHTRE... ...PQIYMDDHTRE... ...PQIYMDDHTRE... ...PQIYMDDHTRE...

Ultra-deep sequencing

slide-9
SLIDE 9

The players

slide-10
SLIDE 10

Requirements for a routine diagnostic-capable NGS system

฀Gain in time and/or information ฀easy sample preparation / simple workflow ฀acceptable „turnaround times“ ฀data volume adjusted to diagnostic need ฀cost effectiveness ฀„ready-to-go“ bioinformatics (does that really exist?)

slide-11
SLIDE 11

V3 V3 deep eep sequencing

Strategies

“hybrid”-approach (amplicon/ shotgun), fragmentation of large amplicons

Library preparation: Nebulization (Roche), Tagmentation (Nextera), Fragmentation (NEB) Sonification (Covaris)

amplicon sequencing approach

Library preparation: Standard PCR fusion primers plus pooling, Fluidigm access array (4 primer approach) TruSeq amplicon (4 primer approach)

slide-12
SLIDE 12

 ~ 1.000.000 „reads“ (sequences) in 10,5h  ~ 500 Mio bases / run  ~ 450 nts read length (amplicon)

“454” GS FLX

slide-13
SLIDE 13

Amplicon sequencing approach

Prim er ers s label eled ed w it h MI Ds s / Roche

V3

slide-14
SLIDE 14

Amplicon generation

  • Sample preparation
  • cDNA
  • PCR
  • Purification
  • Quantitation
  • Equimolar Pooling

GS FLX Chemistry

  • emPCR
  • Breaking
  • Enrichment

GS FLX Sequencing

“454” GS FLX / junior Workflow

cDNA Plasma

Complex workflow

slide-15
SLIDE 15

฀ Illumina’s benchtop sequencer ฀ easy sample preparation ฀ current specification: ~ 7-8 Gb output 2x12-15mio reads of 250 nts length each 37h run time Moderate sequencing costs

MiSeq

slide-16
SLIDE 16

NexteraTM “tagmentation”

slide-17
SLIDE 17
  • Easy library preparation
  • Fast - less than 20 minutes hands-on time
  • Only 1ng DNA per sample needed
  • Indices for up to 96 samples
  • Normalization step included

Library preparation using NexteraTM XT “tagmentation”

slide-18
SLIDE 18

ENV

Experimental setup

IN PRRT whole genome

slide-19
SLIDE 19

37

2 x 250 nts

Illumina MiSeq workflow

3

slide-20
SLIDE 20

… putting things together

Reference sequences PRRT IN ENV

„mapping“

slide-21
SLIDE 21

Coverage

PR/RT ENV

~7500 full V3 loops

slide-22
SLIDE 22

Coverage distribution

S1-1 S1-2 S1-3 S2-1 S2-2 S2-3 S3-1 S3-2 S3-3

Coverages per replicate and gene, blue: PR/RT, red: ENV

slide-23
SLIDE 23

geno2pheno[454]

slide-24
SLIDE 24

geno2pheno[454] output

slide-25
SLIDE 25

geno2pheno[454] plot prediction

Proportion Prediction value

X4 R5

slide-26
SLIDE 26

geno2pheno[454] read/variant distribution

slide-27
SLIDE 27

MiSeq 454

X4 minority variants

MiSeq vs. 454 FLX

slide-28
SLIDE 28
slide-29
SLIDE 29

Number of sequencing errors, substitutions, deletions, and insertions

Archer J et al., 2012, PLoS ONE 7: e49602. doi:10.1371/journal.pone.0049602

slide-30
SLIDE 30
  • riginal test

HIV-1 coreceptor tropism determination and virologic outcome

patient

slide-31
SLIDE 31

Viral RNA vs proviral DNA in V3 Next-Generation sequencing

slide-32
SLIDE 32

Proviral DNA seen through the UDS loupe

Patient F, seroconverter (IB neg), 3.8 mio. cop/ml

Viral RNA Proviral DNA

slide-33
SLIDE 33

Viral RNA Proviral DNA „archived diversity“

Patient F, IB complete (week 16), 76.000 cop/ml

Proviral DNA seen through the UDS loupe

slide-34
SLIDE 34

Swenson LC et al., Clin Infect Dis. 2013 FPR: 3,5 12/156: 7,7%

slide-35
SLIDE 35

Cost per sample calculation Illumina MiSeq

(reagents only)

20 samples per week PRRT plus env V3 plus Integrase: 3.400 nts Coverage: 20.000 X sum: 1,4 Gb, spare capacity: 6-7 Gb Sample preparation: €34,-/sample* Sequencing: €50,-/sample* total €84.-/sample (incl. of VAT)

*based on Illumina‘s list prices in Germany

slide-36
SLIDE 36

Patient THB* 1954

1 10 100 1000 10000 100000 50 100 150 200 250 300 350 400

CD4+ abs Viruslast [cop/mL]

AZT,3TC,ABC DRV/ r

RAL, MVC

TPV/ r AZT,3TC,ABC,TDF

DRV/ r RAL, ETR

diverse ART

slide-37
SLIDE 37

3D l landscap ape p plot t ing of X4/ R5 p populat at ions

proportion cut off 3,5% FPR

slide-38
SLIDE 38

Com par arat at ive lan andscape p plot t ing

  • f X4/ R5 populat

at ions

slide-39
SLIDE 39
slide-40
SLIDE 40

Alexander Thielen

Institute for Immunology and Genetics,

Bettina Spielberger

Laboratory Dr. Thiele, Kaiserslautern,

Kirsten Becker

Germany

Anna Memmer Thomas Alef Bernhard Thiele

Acknowledgements

Thank you!

Thomas Berg, Patrick Braun, Martin Däumer, Josef Eberle, Robert Ehret, Rolf Kaiser, Klaus Korn, Claudia Kücherer, Harm Müller, Christian Noah, Martin Obermeier, Martin Stürmer, Alexander Thielen, Hauke Walter, Eva Wolf

Thomas Lengauer

MPI for Informatics, Saarbrücken, Germany