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NEXT GENERATION SEQUENCING

FOR

HIV DRUG RESISTANCE TESTING

Ekawat PasomsubUnit of Virology and Molecular Microbiology,

Department of Pathology, Ramathibodi Hospital

ekawat.pas@mahidol.ac.th

1

HIV infection (HIV and Host interaction)

3

DNADEOXYRIBONUCLEIC ACID

5

Nitrogen Bases: A C G T

PhosphateDeoxyriboseRNA = Ribonucleic acid (use Uracil instead of Thymidine)

Reverse transcriptase = NO proof reading enzyme activity

ARV Treatment

Adverse Drug Reaction

Drug Resistance

First-lin

e A

RV

Seco

nd

-line A

RV

Seco

nd

-line A

RV

Our Mission:To make first-line ARV last longer

Missing just two drug

doses

virus and drug resistance

causing treatment failure

and waste of financial resources.

Factors for patients to take ARVs on : ADR & Life style

Start First-line ARV : CD4<350 cells/ul)

Over 13-15 years

With in 3 months

Poor adherence

Strict adherence

CLINICAL APPLICATIONS FOR

THERAPY ON HIV INFECTION

RT

Protease

Pressure: anti-retroviral drug

Wild Type(Susceptible)

Mutant(Resistant)

HIV life cycle and Patient

drug adherence

Wild Type Mutant

ในภาวะทีผูป้่วยไม่ไดร้บัยาตา้นไวรสั

HIV แบบใดมีความสามารถในการแบ่งตวัไดด้กีว่ากนั?

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LTRgag

pol

env

LTR

nef

RT

RNaseH Integrase

vif

vpuvpr

tat

rev

gp120

gp41

IDR

U3

Pr

3 major genes in the HIV genome:

• gag – codes for the structural proteins

• pol – codes for the viral enzymes protease (Pr),

reverse transcriptase (RT), and integrase

• env – codes for the envelope glycoproteins

GenotypePhenotype Virtual Phenotype

GENOTYPIC ASSAY Investigate the nucleotide sequence of an HIV on the

region that control the Protease and Reverse Transcriptase enzyme

The region that usually mutation that are known to be associated with resistance

Comparing with nucleotide

sequence of HIV wild-type

Ref – AGTCTGCAGTAGATC

Pt - AGTGTGCAGAAGATC

GENEOBJECTS 4.1

© 2007 Siemens Medical Solutions Diagnostics. All rights reserved

Bi-directional sequencing

Reference

Patient

TRUGENE HIV-1 RESISTANCE REPORT

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Resistance associated

RT mutations

Resistance associated

PR mutations

No

evidence of

resistance

Possible

resistance Resistance

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Resistance rule

NNNNEXTEXTEXTEXT GENERATIONGENERATIONGENERATIONGENERATION SEQUENCINGSEQUENCINGSEQUENCINGSEQUENCING

????????????

Picotiter plate > 100,000 wellsApproximately 70,000 beads filled in the wellsEach bead will produce 500 bpPer run would be 35 Million bp

POOLED SAMPLE

WORKFLOW

ตดั DNA เป็น ชิ�นเล็กๆ ตดิ adapter เชื�อม adapter กบั bead

เพิ�มปรมิาณ DNA หาลาํดบัเบส (sequencing)

TARGET RESEQUENCING

Target gene Amp.

Reference genome

SEQUENCING Standard sequencing

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Next generation sequencing

AGCTACGTCGTACGTACGTACGT AGCTACGTCGTACGTACGTA

ACGTCGTACGT

ACGTCGTACGTACGT

GTCGTACGTACGTACGT

TACGTCGTACGTACGT

AGCTACGTCGTAC

WHAT WE HAVE TO DO IS

1. Start to make sequence preparation

with the “BAM file” (Binary

alignment file)

2. Prepare the file by UNIX OS

3. Have to do the “Sequence/Genotype

Quality (QUAL/GQ)”

4. Have to make multiple sequence

using “depth”

5. Use multiple sequences to make the

resistance report

Bam file

Multiple HIV-1 sequences

Drug resistance interpretation

BAM file

Variant call format (vcf)

Quality control

Filter by depth (20%, 10%, 5%)

Sequences

Drug resistance report

samtools

samtools

samtools

Ramascore

WHY “NGS”?23

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HIV-1 deep sequencing

Library preparation

Template

preparation

Sequencing

Data analysi

s

Sanger sequencing

Ref

SEQUENCING

Standard sequencing

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Next generation sequencing

AGCTACGTCGTACGTACGTACGT AGCTACGTCGTACGTACGTA

ACGTCGTACGT

ACGTCGTACGTACGT

GTCGTACGTACGTACGT

TACGTCGTACGTACGT

AGCTACGTCGTAC

Minor peak cannot be

distinguished from noise

Selection of resistant HIVIncomplete suppression

Inadequate potency

Inadequate drug levels

Inadequate adherence

Pre-existing resistance

Vir

al

loa

d

Time

Drug-susceptible variants

Drug-resistant variantsTreatment begins

SURVIVAL STRATEGYC

ON

FID

EN

TIA

L -

AB

L S

A -

14

/11

/20

12

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Metzner K, Paredes R, CHAIN Training slides

Sanger Sequencing

NGS

≥ 20%

≥ 1%

Drug X Drug X Resistance

No drug X

• Results:

– Screenshot from AVA software showing a D30N mutation at 3.46% prevalence

– D30N: signature mutation for Nelfinavir, Protease Inhibitor

ULTRA DEEP SEQUENCING OF TARGET REGIONS

DETECTION OF HIV DRUG RESISTANCE MUTATIONS

Simen et al., HIV poster 2009

K103

Viroseq Vela

Assay 2856 2857 2858 Mutation

Viroseq A A A -

Vela C A A K103Q

Ref A A A -

Vela

Pos2856

Total count : 4653

A : 2881 (62%,1422+,1459-)

C : 1672 (36%,805+,867-)

G : 99 (2%,29+,70-)

T : 1 (0%,0+,1-)

ENVA-1510

1st Gen Sequencing 2nd Gen Sequencing

First Generation

(Sanger Sequencing) Second Generation (454)

70,000 bead

400 base pair/read

a million molecules of SS DNA

after clonal amplification/bead

Base calling error!

No Base calling

BASE CALLING AND QUASISPECIES

SANGER POPULATION BASED VS. NGS VARIANTS

POPULATION BASED

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A T A C A G

A T A C A G

A G G C A GA T

G

A

G

C A G

Variant calling

Codon 1 Codon 2 Codon 1 Codon 2

Pos 1 Pos 2

ATA (I) CAG (Q)

ATA (I) CAG (Q)

AGG (R) CAG (Q)

Pos 1 Pos 2

ATA (I) CAG (Q)

ATG (M)

AGG (R)

AGA (R)

Mutation list: 1I/M/R, 2Q Mutation list: 1I/R, 2Q

Virtual

codons

Virtual mutation (false positive)

NGS Variants Population sequencingSanger population sequencing

HIV-1 Genotypic drug resistance testing

Confounding factors

- Amplification of PR,RT gene

Comparing detected DRMs in PR and RT gene associated with resistance of

PIs NRTIs and NNRTIs

-DRMs are based on update 2015 of IAU-USA

COMPARISON OF SANGER AND NGSCONCEPTUAL FRAMEWORK

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↓

HIV deep sequencing

↓

ARV regimen

↓

Patient management

TruGeneDeep sequencing

Output : Comparison result

TruGene HIV-1 Genotyping kit HIV-1 deep sequencing

Samples : 120 HIV-Infected patients

Mutations listDrug resistance mutations

1. Performance of HIV-1 Deep sequencing prototype assay

120 plasma samples from HIV-1 infected subjects were tested by Sanger sequencing.

N= 120Sanger sequencing

Successful Unsuccessful

Deep

sequencing prototype

Successful 109 2

Unsuccessful 8 1

109 Clinical samples

Comparison DRMs detection

Sanger sequencing vs Deep sequencing

Based on the 2015 edition of IAS-USA drug resistance mutation list

Overall : The number of Drug Resistance Mutations (DRMs)

7818 130

Deep SequencingSanger

Total number of DRMs = 919

2. Comparison DRMs detection between Sanger sequencing and

Deep sequencing

Your personal performance score was 339.The maximum score achievable was 340.

The full details of the scoring system and a full analysis of test results from all centers is

provided in the final report.

On behalf of the AACC Academy, I wish to personally

congratulate you on winning one of the NACB’s

Distinguished Abstract Awards for your abstract entitled

“Next Generation Sequencing-based HIV-1 Drug Resistance

Monitoring System.”

Your abstract was one of 29 (out of 1024 accepted for the

2016 AACC Annual Meeting) selected for scientific

excellence by a panel of Academy Fellows.

SANGER VS ULTRA DEEP

TECHNOLOGY IN HIV DR

AmpliconsRNA ExtractionRT-PCR

A: >20%

B: < 20%

Sanger (TruGene) Ultra Deep Tech (454)

NGS: 454, Roche

A: >20%

Sequencing (2,000 copies/mL)

B: < 20%A: >20% B: < 20%

emPCR & Sequencing (20,000 copies/mL)

DetectableUndetectable DetectableDetectable

1

1 2 3

3

2

DEEP SEQUENCING REPORT

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Mutation list

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DEEP SEQUENCING REPORT

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43

44

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ขอ้ใดไดป้ระโยชนจ์าก NEXT GENERATION SEQUENCING

กบั การตรวจการดื�อตอ่ยาของเชื�อ HIV นอ้ยที�สุด?

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กาํลงัจะเริ�มกินยาใหม่

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NEXT GENERATION SEQUENCING

FOR

HIV DRUG RESISTANCE TESTING

Ekawat PasomsubUnit of Virology and Molecular Microbiology,

Department of Pathology, Ramathibodi Hospital

ekawat.pas@mahidol.ac.th

48