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NEXT GENERATION SEQUENCING
FOR
HIV DRUG RESISTANCE TESTING
Ekawat PasomsubUnit of Virology and Molecular Microbiology,
Department of Pathology, Ramathibodi Hospital
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HIV infection (HIV and Host interaction)
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DNADEOXYRIBONUCLEIC ACID
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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 -
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/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?
1. ใชก้บัผูท้ี�ตดิเชื�อมานาน 10 ปีแลว้แตไ่มเ่คยไดร้บัยา
2. ใชก้บัผูป้่วยที�หยุดยาไปชว่งเวลาหนึ�ง (1 เดอืน) และ
กาํลงัจะเริ�มกินยาใหม่
3. ใชก้บัผูป้่วยที�เปลี�ยนยามาแลว้ 3 สูตร
4. ใชก้บัผูป้่วยที�เปลี�ยนโรงพยาบาลในการรกัษา46
NEXT GENERATION SEQUENCING
FOR
HIV DRUG RESISTANCE TESTING
Ekawat PasomsubUnit of Virology and Molecular Microbiology,
Department of Pathology, Ramathibodi Hospital
48
