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

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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

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Sentosa SQ HIV Genotyping Assay
Design Concept and Targets Map

Protease
HIV Genome and RT Integrase
P66 (NNRTI)
Codon 1-335
p51 (NRTI)
Codon 1-250

reverse transcriptase Codon 1-289

Codon 1-99
Integrase

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.
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Sentosa SQ HIV Genotyping Assay
Major Characteristics
Parameter
Core Technology
RNA Extraction and Library Prep
Turn Around Time
Specimen type
Specimen pre-treatment
Number of tests per kit
Format
HIV Genotypes coverage
Carryover contamination control
Sequence data analysis
Characteristic
Next-Generation Sequencing (NGS), PGM platform
Automated
~27 hours
EDTA Plasma or Serum
Not required
60 clinical samples
4x16 (4 runs, 15 samples + 1 system control in each
run)
HIV-1 Group M (subtypes A to K)
Uracil-DNA glycosylase system
Fully automatic
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Sentosa SQ HIV Genotyping Assay Workflow
TAT is ~27 hours with hands-on time ~3.5 hours
1 2 3 4 5
Step

RNA Data Analysis and

Library Preparation Template Preparation Sequencing
Extraction Reporting
Time

2 hrs. 45 min 8.5 hrs. 7 hrs. 5.5 hrs. 3.5 hrs.

Plasma / serum samples
Instruments

ST401i ST401e

Sentosa SX101 Sentosa ST401 Sentosa SQ

Sentosa SQ301
System: Reporter Server
Sentosa ST401i and
Sentosa ST401e
PX1
Veriti Dx
Plate
Cycler**
Sealer*
Reagents

Sentosa SX Sentosa SQ 318 Chip

Sentosa SQ HIV Kit
Virus Total Sentosa ST Template
Genotyping Assay
Nucleic Acid Kit Sentosa SQ
(4x16)
Plus II (4x16) Kit Sequencing Kit

Sentosa Link
ware
Soft

Sentosa SX101 Software Sentosa SQ Suite Sentosa SQ Reporter

* PX1 PCR Plate Sealer, Bio-Rad (Mat. No: 181-4000). Not available from Vela Diagnostics The power of standardized versatility | 4
** Veriti Dx 96-well Cycler, Life Technologies (Mat. No: 4452300). Not available from Vela Diagnostics
Sentosa SQ HIV Genotyping Assay
Reporting System

Key mutations Variant frequency

Subtyping information

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 Mutation Detection Rate
98.74% for the Sentosa SQ HIV Genotyping Assay
Number of Mutations Detection 95% Confidence
HIV Gene Test
Mutations Detected rate Interval
Sentosa SQ HIV
199 199 100.00% 98.11 100.00%
Genotyping Assay
Protease
TruGene HIV-1
199 180 90.45% 85.57 93.80%
Genotyping Kit
Sentosa SQ HIV
435 427 98.16% 96.41 99.07%
Reverse Genotyping Assay
Transcriptase TruGene HIV-1
435 324 74.48% 70.18 78.35%
Genotyping Kit
Sentosa SQ HIV
634 626 98.74% 97.53 99.36%
Genotyping Assay
Overall
TruGene HIV-1
634 504 79.50% 79.02 79.62%
Genotyping Kit
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).
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Performance of the Assays
Drug resistance mutations detection
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:

Gene Mutation Percentage Resistance to / Effect
M184V 48,7% (54/111) 3TC, FTC (NRTI), ddl
K103N 29.7% (33/111) NVP and EFV (NNRTI)
Reverse
Y181C 27,9% (31/111) NVP, ETR, RPV, EFV (NNRTI)
Transcriptase
G190A 18.9% (21/111) NVP, EFV (NNRTI)
D67N 18.9% (21/111) AZT, d4T (NRTI), ddI
Increases the replication fitness of
M36I 91.9% (102/111)
viruses with PI-resistance mutations
Increases the replication fitness of
K20R 21.6% (24/111)
Protease viruses with PI-resistance mutations
Either reduce PI susceptibility or
L10I 20.7% (23/111) increase the replication of viruses
containing PI-resistance mutations

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Comparison of Two Assays
Summary

TruGene HIV-1 Sentosa SQ HIV
Genotyping Kit Genotyping Assay
Technology CLIP-sequencing (ABI 9700) NGS (PGM)
RNA Extraction Manual Automated
Protease (codons 1-99),
Protease (codons 4-99) and
Targets RT (codons 1-335) and
RT (codons 38-248)
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
20% 5%
limit
Heavy, individual patient
Labor intensity Moderate
sequence run
Cost per test $150 $200

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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.

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