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The power of standardized versatility 0|
Next Generation Sequencing for
Detection of Drug Resistance Mutations
in HIV-1
1st Asia Pacific AIDS & Co-Infections Conference
Hong Kong, 17-19 May 2016
The power of standardized versatility 1|
Study Synopsis
Objective:
To evaluate the performance of the Sentosa® SQ HIV Genotyping Assay (4x16)
and compare two sequencing-based HIV-1 drug resistance monitoring systems:
1) CLIP-based system (TruGene HIV-1 Genotyping Kit, SIEMENS)
2) Novel Next Generation Sequencing (NGS)-based test (Sentosa SQ HIV-1
Genotyping Assay (4x16), Vela Diagnostics).
Clinical site:Ramathibodi Hospital Mahidol University, Bangkok, Thailand
Study population & sample size:Human EDTA plasma specimens from 111 HIV positive patients
The power of standardized versatility 2|
Design Concept and Targets Map
Sentosa SQ® HIV Genotyping Assay
Targets: Protease, Reverse Transcriptase and Integrase genes
Number of amplicons: 2
Amplicons lengths: ~1500 bp (Protease and RT) and ~1000 bp (Integrase)
The amplicons cover 86 known Nucleoside RT Inhibitor (NRTI), Non-Nucleoside RT
Inhibitor (NNRTI), Protease Inhibitor (PI) and Integrase Inhibitor (INI) resistance
mutations.
Protease
and RT IntegraseHIV Genome
Codon 1-99
Codon 1-250
Codon 1-335
p51 (NRTI)
P66 (NNRTI)
Codon 1-289
Integrase
reverse transcriptase
The power of standardized versatility 3|
Major Characteristics
Sentosa® SQ HIV Genotyping Assay
Parameter Characteristic
Core Technology Next-Generation Sequencing (NGS), PGM platform
RNA Extraction and Library Prep Automated
Turn Around Time ~27 hours
Specimen type EDTA Plasma or Serum
Specimen pre-treatment Not required
Number of tests per kit 60 clinical samples
Format4x16 (4 runs, 15 samples + 1 system control in each
run)
HIV Genotypes coverage HIV-1 Group M (subtypes A to K)
Carryover contamination control Uracil-DNA glycosylase system
Sequence data analysis Fully automatic
The power of standardized versatility 4|
Sentosa® SQ HIV Genotyping Assay WorkflowTAT is ~27 hours with hands-on time ~3.5 hours
Template Preparation SequencingData Analysis and
ReportingLibrary Preparation
RNA
Extraction
2 31 4 5
Ste
p
* PX1 PCR Plate Sealer, Bio-Rad (Mat. No: 181-4000). Not available from Vela Diagnostics
** Veriti Dx 96-well Cycler, Life Technologies (Mat. No: 4452300). Not available from Vela Diagnostics
Sentosa® SX101 SoftwareSo
ftw
are
Sentosa® SQ Suite Sentosa® SQ Reporter
Sentosa® Link
Sentosa® SX
Virus Total
Nucleic Acid
Plus II (4x16) Kit
Sentosa® SQ HIV
Genotyping Assay
(4x16)
Sentosa® ST Template
Kit
Sentosa® SQ 318 Chip
Kit
Re
ag
en
ts
Sentosa® SQ
Sequencing Kit
Sentosa® ST401
System:
Sentosa® ST401i and
Sentosa® ST401e
Sentosa® SQ301 Sentosa® SQ
Reporter ServerSentosa® SX101
PX1
Plate
Sealer*
Veriti Dx
Cycler**
Ins
tru
me
nts
ST401i ST401e
Pla
sm
a /
seru
m s
am
ple
s
Tim
e
2 hrs. 45 min 8.5 hrs. 7 hrs. 5.5 hrs. 3.5 hrs.
The power of standardized versatility 5|
Reporting System
Sentosa® SQ HIV Genotyping Assay
Variant frequencyKey mutations
Subtyping information
The power of standardized versatility 6|
Mutation Detection Rate
98.74% for the Sentosa SQ HIV Genotyping Assay
HIV Gene Test Number of
Mutations
Mutations
Detected
Detection
rate
95% Confidence
Interval
Protease
Sentosa® SQ HIV Genotyping Assay
199 199 100.00% 98.11 – 100.00%
TruGene HIV-1
Genotyping Kit 199 180 90.45% 85.57 – 93.80%
Reverse
Transcriptase
Sentosa® SQ HIV
Genotyping Assay 435 427 98.16% 96.41 – 99.07%
TruGene HIV-1
Genotyping Kit 435 324 74.48% 70.18 – 78.35%
Overall
Sentosa® SQ HIV
Genotyping Assay 634 626 98.74% 97.53 – 99.36%
TruGene HIV-1 Genotyping Kit
634 504 79.50% 79.02 – 79.62%
• 647 drug resistance mutations were detected (199 mutations in the Protease gene, 435
mutations in the RT gene and 13 mutations in the Integrase gene).
• 130 mutations were detected by the Sentosa SQ HIV Genotyping Assay, that were not
found by TruGene.
• 8 mutations were not detected by the Sentosa SQ HIV Genotyping Assay (but detected by
TruGene).
The power of standardized versatility 7|
Performance of the Assays
Drug resistance mutations detection
Gene Mutation Percentage Resistance to / Effect
Reverse Transcriptase
M184V 48,7% (54/111) 3TC, FTC (NRTI), ddl
K103N 29.7% (33/111) NVP and EFV (NNRTI)
Y181C 27,9% (31/111) NVP, ETR, RPV, EFV (NNRTI)
G190A 18.9% (21/111) NVP, EFV (NNRTI)
D67N 18.9% (21/111) AZT, d4T (NRTI), ddI
Protease
M36I 91.9% (102/111) Increases the replication fitness of viruses with PI-resistance mutations
K20R 21.6% (24/111) Increases the replication fitness of viruses with PI-resistance mutations
L10I 20.7% (23/111) Either reduce PI susceptibility or increase the replication of viruses containing PI-resistance mutations
All HIV strains were carrying 1 or multiple drug resistance mutations in 61 AA
positions of the RT gene, 16 AA positions of the Protease gene and 9 AA
positions of the Integrase gene.
The most prevalent mutations:
The power of standardized versatility 8|
Comparison of Two AssaysSummary
TruGene HIV-1 Genotyping Kit
Sentosa® SQ HIV
Genotyping Assay
Technology CLIP-sequencing (ABI 9700) NGS (PGM)
RNA Extraction Manual Automated
Targets Protease (codons 4-99) and
RT (codons 38-248)
Protease (codons 1-99), RT (codons 1-335) and
Integrase (codons 1-289)
HIV-1 Genotype coverage Subtype B (can detect non-B) Group M (subtypes A to K)
Sequence data analysis Semi-automatic Fully automatic
Viral population detection limit
≥20% ≥5%
Labor intensity Heavy, individual patient
sequence run Moderate
Cost per test $150 $200
The power of standardized versatility 9|
Conclusion
The newly developed Sentosa SQ HIV Genotyping NGS
workflow appears as a promising new tool for detecting
clinically relevant HIV variants.
Given its high sensitivity (up to 5% mutation frequency)
compared to Sanger sequencing based systems and the
comparatively short turnaround time the workflow offers
relevant improvements in HIV drug resistance monitoring
systems.