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 Mutaon 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 Sanger Basecall • 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% - of variation Coefficient 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 from Mean 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 ABempted # 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 Enrichment61525A of HCV Sequences61516A 61515A 56587A 56585A 56587A 56585A pool3 pool2 pool1 3x4 2x6 5x6 4x6 4x5 15_Jul_24_S1S2_checkMiseq15_Dec_02_checkMiseq − − − − − − − − − − − − HCV_S10 HCV_S12 HCV_S11 proportion of total reads hitting references − − proportion of total reads− hitting− references HCV_S5 HCV_S4 HCV_S3 HCV_S9 HCV_S8 HCV_S7 HCV_S6 15_Dec_02_checkMiseqHCV_S2 HCV_S1 HCV_S2 HCV_S1 proportion of total readsHCV_S2 hittingHCV_S1 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 of total reads hitting references using HCV capture probes 61516A−HCV_S2 proportion of total reads hitting references probes 100 61516A−HCV_S2 0 0 0 15_Jul_24_S1S2_checkMiseq 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) 4x5−HCV_S6 50 50 4x6−HCV_S7 Pacneshg38HCVhuman (hg38) HCV 4x6−HCV_S7 hg38HCVHIV1 25 5x6−HCV_S8 HCVHIV1GBvirusCHIV 75 75 75 5x6−HCV_S8 not_referencedGBvirusCGB virus C 61525A−HCV_S9 not_referencednot referenced 61525A56587A−HCV_S2HCV_S9 0 61518A−HCV_S10 100 100 100 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