Genotype-Independent Near Whole Genome Next Generation Assays for HCV Resistance Evaluation How Do We Test for HCV Ravs?
How do we test for HCV RAVs? A Technology Based Presentation
Genotype-Independent Near Whole Genome Next Generation Assays for HCV Resistance Evaluation
Anita Howe, Ph.D. Centre for Excellence in HIV/AIDS British Columbia, Canada Objectives
1. Overview of key virology assays
2. Sanger population sequencing and RECall
3. Near Whole Genome HCV Next Generation Sequencing (NGS)
4. Random Primer NGS assay for Mixed Infection
5. Probe Enrichment Key HCV Assays for Resistance Testing
Viral Load Genotyping Sequencing Phenotypic Assays Branched DNA Line-Probe Sanger Population ® VERSANT HCV RNA Hybridization Sequencing F.L. Stable Replicons 3.0 branched DNA VERSANT® HCV High throughput • GT1a_H77 (Bayer/Siemens) GENOTYPE 2.0 [LIPA] LOD ~20% • GT1b_con1 (Innogenetics) • GT 2a_JFH1 RT-PCR Clonal Sequencing • GT 3a_S52 • ABBOTT REAL- RT-PCR Labor intensive • GT 4a_ED43 TIME HCV RT-PCR ABBOTT REAL-TIME HCV • GT 5a_SA1 (Abbott) Linkage of mutations GENOTYPE II (Abbott) • GT 6a_consensus • HCV SUPERQUANT (National Genetics Allele-specific Real-time Institute) Direct Sequencing Chimeric/Transient • COBAS AmpliPrep/ TRUGENE DIRECT DNA PCR Replicons in Limit to known mutations COBAS TaqMan HCV SEQUENCING (Bayer/ • GT1a_H77 Siemens) TEST (Roche • GT1b_con1 Molecular Systems) • GT2a_JFH1 Serotyping Next Generation Transcription- MUREX HCV Sequencing Infectious HCV SEROTYPING (Abbott) Mediated Illumina, Ion Torrent, 454 • GT1a_H77 Amplification (Roche), PyroMark (Qiagen), • GT 2a_JFH1 VERSANT HCV RNA ABI SOLiD, SMRT (Pac Bio) (Siemens) Ø Sensitive Reporter Assay Ø 5’UTR/Core/NS5B Ø GT1 – 6 Ø Medium-high throughput SEAP for NS3 Ø High costs Ø 5’UTR/Core Ø Limited subtype Ø 9.6 – 108 IU/mL information Enzymatic assays NS3, NS5B RECall Web Based Sequence Analysis
http://pssm.cfenet.ubc.ca/account/login Woods et al. (2012) JCM 50.6:1936-1942 Sanger Population Sequencing
Uptake of Q80K Screening (Feb 2014 – March 2015) Genotype Switch in Treatment Naïve PWIDS Observed Between 1999 – 2004 (mean = 4.2 yr) # Tests per month Tests #
Proportion of infections in British Columbia, Canada that displayed reduced susceptibility to simeprevir 1.0
Joy et al. (2016) 5th Can Hep C Symposium. Montreal. Canada Illumina Next Generation Sequencing
Host RNAs and DNAs Target N.A. HCV RNA amplification
Library Preparation
Data Analysis
Cluster Amplification on a Flow Cell Sequencing by Synthesis with FL-NTPs Image Paired-end sequences http://www.illumina.com/technology/next-generation-sequencing/paired-end-sequencing_assay.html MiCall Pipeline
Preliminary “seeding” 1a 1b Illumina Paired- HCV GT/Subtype end reads in References from Genbank 3a FASTQ 4d
Sample-specific Active Seed Consensus References 1a
Illumina Paired- 1b end reads in 3a FASTQ 4d
Re-map sample sequences using the sample-specific Remove poor consensus as references consensus sequences and seed references
Muta on Report FDA references e.g. 1b_R30H Genotype-Independent Near Whole Genome HCV NGS Assays
Oligo dA20 RT-primer
288 9250 WG-1 amplicon
288 8640 WG-2 amplicon
Limitations of Assays used for HCV Resistance: • Genotype-dependent; prior GT knowledge is needed to generate sequences • One target gene at a Time =>$$$ and long turn around time • Inaccurate genotype/subtype identification Accuracy Whole Genome NGS vs. Sanger Sequencing NGS Basecall Sanger Basecall SangerBasecall
• Overall mean nucleotide, amino acid and Q80K concordance are 98.8%, 99.6% and 100%, respectively. Limit of Quantification
2 pure plasmid clones at conserved positions 100 plasma samples. All sequenced positions within HCV
Error Rate = 100% - Coefficient of variation frequency of the most common a.a. at that position
variants with 0.5% prevalence = noise
CV dramatically increases in minority variants with <0.1% mean prevalence
The lower limit of quantification is 0.5% How many reads do we need for detecting variants with a 2% prevalence? Variant Frequency < 0.5% 0.5% < Variant Frequency < 2%
2% < Variant Frequency < 20% Variant Frequency > 20%
A A Frequency Fold-Difference from Mean Meanfrom Frequency Fold-Difference A A
Read Coverage Read Coverage Sequence Coverage of HCV Genes Genotype Spectrum
GT1 - 6 plasma samples with viral load from 3.5 to >7 IU/mL
# Samples NS3 NS5a NS5b Genotype∆ GT Subtypes A empted # Passing (%) # Passing (%) # Passing (%)
78 1a 72 (92%) 72 (92%) 72 (92%) 1 11 1b 11 (100%) 11 (100%) 11 (100%) 3 1e 2 (67%) 2 (67%) 2 (67%) 2 20 2, 2a, 2b, 2c 19 (95%) 19 (95%) 19 (95%) 3 76* 3, 3a 62 (82%) 62 (82%) 60 (79%) 4, 4a, 4d, 4e, 4 20* 16 (80%) 13 (65%) 8 (40%) 4r, 4N, 4t 5 3 5a 3 (100%) 3 (100%) 3 (100%) 6a, 6e, 6h, 6 21 21 (100%) 20 (95%) 18 (86%) 6k, 6l, 6t
*~50% of the samples did not have viral load information Viral Load
GT1 – GT6 Clinical Samples
log10 HCV RNA (IU/mL) NS3 NS5A NS5B >7 25/25 (100%) 25/25 (100%) 25/25 (100%)
6.6 – 7.0 33/34 (97%) 33/34 (97%) 33/34 (97%)
6.1 – 6.5 28/28 (100%) 28/28 (100%) 28/28 (100%)
5.6 – 6.0 26/26 (100%) 26/26 (100%) 26/26 (100%)
5.1 – 5.5 21/25 (84%) 21/25 (84%) 16/25 (64%)
3.5 - 5.0 8/18 (44%) 9/18 (50%) 8/18 (44%)
Success rates for serum samples or samples with multiple freezing and thawing cycles might be lower Receiver Operator Characteristic
• Threshold for variant calls Optimal Conditions for • RAVs selected for analysis RAV Analysis • EC50 fold-shifts etc.
disease (e.g. non-SVR) without disease (e.g. SVR) TP / (TP + FN) + (TP / TP
SVR Non- SVR Non-SVR SVR +RAV TP FP -RAV FN TN
Test (threshold) TN / (FN+TN) How does the choice of RAVs affect ROC?
Any polymorphisms at resistance loci Removing impactful RAVs
Condition 1: NS5A RAVs: M28A/G/T/V, Adding Q30D/E/G/H/R/L, L31I/M/F/V, H58D and polymorphic Y93any Condition 2. as condition 1 but skip M28V, variants with Q30H/L, L31M no resistance Condition 3: any polymorphisms from GT1a_H77 i.e. M28Any, Q30any, L31any, H58any, Y93any Condition 4: as condition 1 but add K24any, A92any, R44any and R78any Clinical Samples
• 97 samples were examined between September 2015 – March 2016 - 63 GT1a, 13 GT1b, 7 GT2, 12 GT3, 2 GT4 and 1 GT6 • Treatment status unknown but majority were likely from virologic failures • >60% of the samples had NS5A RAVs; 80% of which were resistant to ALL approved NS5A drugs Mixed Infection Among PWIDs
Cunningham, E. B. et al. (2015) Mixed HCV infection and reinfection in people who inject drugs—impact on therapy Nat. Rev. Gastroenterol. Hepatol. doi:10.1038/nrgastro.2015.36 Random Priming Next Generation Assay
Random Primer Genotypes and Reproducibility Random Priming NGS
Mixtures of plasma samples at 10:90, 50:50 and 90:10 nominal ratios. Viral load ~5 log10 IU/mL 2 replicates from the mixtures at 10:90 were evaluated for reproducibility
• The Random Priming NGS assay accurately identified mixed- infected samples containing GT1 - 6 genotypes/subtypes • Linearity of detection was observed in mixed-infected samples with 10:90, 50:50 and 90:10 ratios • Good reproducibility: the average difference between replicates was <1% Ratio was expressed in the same order of the GT mixes e.g. 10:90 for 1a vs.1b A Natural Mixed Infection Case Detected by Random Priming NGS
Background: This is a patient who was thought to be infected with GT1b as determined by LiPA and Sanger Population Sequencing GT2b was identified by Sanger Population Sequencing at virologic failure after DAA treatment
Subsequent sequencing using Random Priming NGS showed that this subject in fact had a mixed infection at baseline. GT1b was cleared but GT2b remained
GT1b GT2b GT 2b Temporal viral dynamics in a Subject with mixed infection Random Priming NGS
GT3a GT3a GT3a GT1a GT1a GT1a
GT1a
GT1a GT3a Use of Capture Probes to Enrich Target Sequences
sequencing library of randomly primed cDNA HCV-specific probes = human & others (biotinylated) = HCV 1. hybridize probes to target sequences
2. bind beads to probes
3. capture probe- magnetic beads, bound beads with streptavidin coated magnet 61518A − HCV_S10
Enrichment61525A − HCV_S9 of HCV Sequences61516A − HCV_S2 61515A − HCV_S1 56587A − HCV_S2 56585A − HCV_S1 56587A − HCV_S2 56585A − HCV_S1 pool3 − HCV_S5 pool2 − HCV_S4 pool1 − HCV_S3 3x4 − HCV_S12 2x6 − HCV_S11 5x6 − HCV_S8 4x6 − HCV_S7 4x5 − HCV_S6 15_Jul_24_S1S2_checkMiseq15_Dec_02_checkMiseq proportion of total reads hitting references proportion15_Dec_02_checkMiseq of total reads hitting references proportion of total reads hitting references 61515A−HCV_S1
proportion of total reads hitting references without proportion of total reads hitting references 61515A−HCV_S1 proportion of total reads hitting references proportion of total reads hitting references proportion proportion of total reads hitting references proportion using HCV capture probes 61516A−HCV_S2 of total reads hitting references proportion probes 100 61516A−HCV_S2 0 0 0 15_Jul_24_S1S2_checkMiseq pool1−HCV_S3 15_Jul_24_S1S2_checkMiseq
pool1−HCV_S3 15_Dec_02_checkMiseq reference genomes % of reads 56585Apool2−HCV_S1HCV_S4 phiX174 75 phiX174 mapping to 25 pool2−HCV_S4 25 25 reference pool3−HCV_S5 phiX174EcoliE. coli Propionibacterium acnes genomes pool3−HCV_S5 EcoliPacnes(skin bacterium) 4x5−HCV_S6 Paracoccus denitrificans 50 PdenitrificansPacneshg38(probable reagent contaminant) 50 50 50 4x5−HCV_S6 4x6−HCV_S7 Pacneshg38HCVhuman (hg38) HCV 4x6−HCV_S7 hg38HCVHIV1 5x6−HCV_S8 HCVHIV1GBvirusCHIV 2575 75 75 5x6−HCV_S8 not_referencedGBvirusCGB virus C 61525A−HCV_S9 not_referencednot referenced 61525A56587A−HCV_S2HCV_S9 1000 61518A−HCV_S10 100 100 samples: 1 2 3 4 5 6 61518A7 8− HCV_S109 10 11 12 13 14 2x6−HCV_S11 not_referenced HCV hg38 Pacnes Pdenitrificans Ecoli phiX174 not_referenced HCV hg38 Pacnes Pdenitrificans Ecoli phiX174 not_referenced GBvirusC HIV1 HCV hg38 Pacnes 2x6Ecoli −HCV_S11phiX174 3x4−HCV_S12 3x4−HCV_S12 0 25 50 75 100 0 0 25 50 75 proportion of 25 total reads50 hitting75 references 100 100 proportionproportion of total reads hitting referencesreferences Coverage in HCV NS5A with vs. without Probe Enrichment GT1a and GT3a in 90:10 mix Read Depth
NS5A Amino Acid Position
GT1a + Probe GT1a GT3a + Probe GT3a Total NS5A Reads 4,377,073 797,111 742,769 66,116 Fold Increase 5.5 11.2 Mean Reads 9,814 1,787 1,651 147 Maxium Reads 18129 3190 4046 221 Minium Reads 3286 945 568 59 Summary
• The near Whole Genome NGS assay represents an efficient tool for the evaluation of HCV resistance in all genotypes
• The lower limit of minority variant detection (threshold) is 0.5%
• Viral titer and quality of the samples are important factors that determine sequencing success
• ROC can be used to optimize the thresholds/RAVs used for prediction of treatment outcomes
• Random Primer NGS provides an agnostic approach to interrogate potential mixed-infections
• Sensitivity of the Random Primer NGS assay may be improved with the use of capture probes Acknowledgement
Centre for Excellence in HIV/AIDS • Chanson Brumme Merck Research Laboratory • Winnie Dong • Ping Qiu • Celia Chui • Wei Bo • Weiyan Dong • Rick Stevens • Jeff Joy • Art Poon • Don Kirby Vancouver VIDUS Cohort • Vera Tai Patients and Investigators • Conan Woods • Richard Harrigan