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

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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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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

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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

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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.

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Reporting System

Sentosa® SQ HIV Genotyping Assay

Variant frequencyKey mutations

Subtyping information

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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).

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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:

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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

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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.