Mutations of Bacteria From Virus Sensitivity to Virus Resistance S. E. Luria and M. Delbrück.
Next Generation Sequencing for Detection of Drug Resistance Mutations...
Transcript of Next Generation Sequencing for Detection of Drug Resistance Mutations...
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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.