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

How do we test for HCV RAVs? A Technology Based Presentation

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

Genotyping Line-Probe Hybridization VERSANT® HCV GENOTYPE 2.0 [LIPA] (Innogenetics)

RT-PCR ABBOTT REAL-TIME HCV GENOTYPE II (Abbott) Direct Sequencing TRUGENE DIRECT DNA SEQUENCING (Bayer/Siemens) Serotyping MUREX HCV SEROTYPING (Abbott) Ø  5’UTR/Core/NS5B Ø GT1 – 6 Ø  Limited subtype

information

Sequencing

Sanger Population Sequencing High throughput LOD ~20% Clonal Sequencing Labor intensive Linkage of mutations Allele-specific Real-time PCR Limit to known mutations Next Generation Sequencing Illumina, Ion Torrent, 454 (Roche), PyroMark (Qiagen), ABI SOLiD, SMRT (Pac Bio) Ø Sensitive Ø Medium-high throughput Ø High costs

Phenotypic Assays

F.L. Stable Replicons •  GT1a_H77 •  GT1b_con1 •  GT 2a_JFH1 •  GT 3a_S52 •  GT 4a_ED43 •  GT 5a_SA1 •  GT 6a_consensus

Chimeric/Transient Replicons in •  GT1a_H77 •  GT1b_con1 •  GT2a_JFH1

Infectious HCV •  GT1a_H77 •  GT 2a_JFH1 Reporter Assay SEAP for NS3 Enzymatic assays NS3, NS5B

Viral Load Branched DNA VERSANT® HCV RNA 3.0 branched DNA (Bayer/Siemens) RT-PCR •  ABBOTT REAL-

TIME HCV RT-PCR (Abbott)

•  HCV SUPERQUANT (National Genetics Institute) •  COBAS AmpliPrep/COBAS TaqMan HCV TEST (Roche Molecular Systems) Transcription-Mediated Amplification VERSANT HCV RNA (Siemens) Ø  5’UTR/Core Ø  9.6 – 108 IU/mL

RECall Web Based Sequence Analysis

http://pssm.cfenet.ubc.ca/account/login Woods et al. (2012) JCM 50.6:1936-1942

Sanger Population Sequencing

Genotype Switch in Treatment Naïve PWIDS Observed Between 1999 – 2004 (mean = 4.2 yr)

1.0

Proportion of infections in British Columbia, Canada that displayed reduced susceptibility to simeprevir

# Te

sts

per m

onth

Uptake of Q80K Screening (Feb 2014 – March 2015)

Joy et al. (2016) 5th Can Hep C Symposium. Montreal. Canada

Illumina Next Generation Sequencing

Host RNAs and DNAs

HCV RNA

http://www.illumina.com/technology/next-generation-sequencing/paired-end-sequencing_assay.html

Target N.A. amplification

Cluster Amplification on a Flow Cell

Sequencing by Synthesis with FL-NTPs Image Paired-end sequences

Library Preparation

Data Analysis

HCV GT/Subtype References from Genbank

Sample-specific Consensus

Active Seed References

1a

1b

3a

4d

Preliminary “seeding”

Re-map sample sequences using the sample-specific consensus as references

1a

4d

1b

3a

Illumina Paired-end reads in FASTQ

Remove poor consensus sequences and seed references

MiCall Pipeline

Muta%on  Report  FDA  references  e.g.  1b_R30H  

Illumina Paired-end reads in FASTQ

Genotype-Independent Near Whole Genome HCV NGS Assays

WG-­‐1  amplicon  288 9250

Oligo dA20 RT-primer

WG-­‐2  amplicon  288 8640

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

•  Overall mean nucleotide, amino acid and Q80K concordance are 98.8%, 99.6% and 100%, respectively.

NGS Basecall S

ange

r Bas

ecal

l

Limit of Quantification

100 plasma samples. All sequenced positions within HCV

Coefficient of variation

CV dramatically increases in minority variants with <0.1% mean prevalence

2 pure plasmid clones at conserved positions

The lower limit of quantification is 0.5%

variants with 0.5% prevalence = noise

Error Rate = 100% - frequency of the most common a.a. at that position

How many reads do we need for detecting variants with a 2% prevalence?

Read Coverage Read Coverage

A A

Freq

uenc

y Fo

ld-D

iffer

ence

from

Mea

n

Variant Frequency < 0.5% 0.5% < Variant Frequency < 2%

2% < Variant Frequency < 20% Variant Frequency > 20%

Sequence Coverage of HCV Genes

Genotype Spectrum GT1 - 6 plasma samples with viral load from 3.5 to >7 IU/mL

Genotype∆   #  Samples  ABempted   GT  Subtypes   NS3  

#  Passing  (%)  NS5a  

#  Passing  (%)  NS5b  

#  Passing  (%)  

1  78   1a   72  (92%)   72  (92%)   72  (92%)  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     20*   4,  4a,  4d,  4e,  4r,  4N,  4t   16  (80%)   13  (65%)   8  (40%)  

5   3   5a   3  (100%)   3  (100%)   3  (100%)  

6   21   6a,  6e,  6h,  6k,  6l,  6t   21  (100%)   20  (95%)   18  (86%)  

*~50% of the samples did not have viral load information

Viral Load

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%)  

GT1 – GT6 Clinical Samples

Success rates for serum samples or samples with multiple freezing and thawing cycles might be lower

Receiver Operator Characteristic

•  Threshold for variant calls•  RAVs selected for analysis •  EC50 fold-shifts etc.

Optimal Conditions for RAV Analysis

Test (threshold)

disease (e.g. non-SVR) without disease (e.g. SVR)

Non-­‐SVR    

SVR  

+RAV   TP   FP  

-­‐RAV   FN   TN  

Non-SVR SVR

TP /

(TP

+ FN

)

TN / (FN+TN)

Any polymorphisms at resistance loci

Adding polymorphic variants with no resistance

Condition 1: NS5A RAVs: M28A/G/T/V, Q30D/E/G/H/R/L, L31I/M/F/V, H58D and Y93any Condition 2. as condition 1 but skip M28V, Q30H/L, L31M 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

Removing impactful RAVs

How does the choice of RAVs affect ROC?

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

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

Mixed Infection Among PWIDs

Random Priming Next Generation Assay

Random Primer

•  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

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

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 GT 2b

GT2b

Temporal viral dynamics in a Subject with mixed infection Random Priming NGS

GT1a GT3a

GT1a

GT3a

GT1a

GT3a

GT3a GT1a

GT1a

Use of Capture Probes to Enrich Target Sequences

sequencing library of randomly primed cDNA

= human & others = HCV

HCV-specific probes (biotinylated)

magnetic beads, streptavidin coated

2. bind beads to probes

3. capture probe-bound beads with magnet

1. hybridize probes to target

sequences

Enrichment of HCV Sequences

% of reads mapping to

reference genomes

3x4−HC

V_S

12

2x6−HC

V_S

11

61518A−H

CV

_S10

61525A−H

CV

_S9

5x6−HC

V_S

8

4x6−HC

V_S

7

4x5−HC

V_S

6

pool3−HC

V_S

5

pool2−HC

V_S

4

pool1−HC

V_S

3

61516A−H

CV

_S2

61515A−H

CV

_S1

0

25

50

75

100

proportion of total reads hitting references

phiX174

Ecoli

Pacneshg38H

CV

HIV

1G

BvirusC

not_referenced

15_Dec_02_checkM

iseq proportion of total reads hitting references

3x4−HCV_S12

2x6−HCV_S11

61518A−HCV_S10

61525A−HCV_S9

5x6−HCV_S8

4x6−HCV_S7

4x5−HCV_S6

pool3−HCV_S5

pool2−HCV_S4

pool1−HCV_S3

61516A−HCV_S2

61515A−HCV_S1

0 25 50 75 100

proportion of total reads hitting references

phiX174EcoliPacneshg38HCVHIV1GBvirusCnot_referenced

15_Dec_02_checkMiseq proportion of total reads hitting references

phiX174

E. coli Propionibacterium acnes (skin bacterium)

human (hg38) HCV HIV GB virus C not referenced

56

58

7A−

HC

V_

S2

56

58

5A−

HC

V_

S1

0

25

50

75

100

proportion of total reads hitting references

ph

iX

17

4E

co

li

Pd

en

itrifica

ns

Pa

cn

es

hg

38

HC

Vn

ot_

re

fe

re

nce

d

15_Ju

l_24_S

1S

2_ch

eckM

iseq

p

ro

po

rtio

n o

f to

tal read

s h

ittin

g referen

ces

56587A−

HC

V_S

2

56585A−

HC

V_S

10

25

50

75

100

proportion of total reads hitting references

phiX

174

Ecoli

Pdenitrificans

Pacnes

hg38

HC

Vnot_referenced

15_Ju

l_24_S

1S

2_ch

eckM

iseq

p

ro

po

rtio

n o

f to

tal read

s h

ittin

g referen

ces

reference genomes

1 samples: 2 3 4 5 6 9 7 10 8 11 12 13 14 0

25

50

75

100

using  HCV  capture  probes   without  probes  

3x4−HCV_S12

2x6−HCV_S11

61518A−HCV_S10

61525A−HCV_S9

5x6−HCV_S8

4x6−HCV_S7

4x5−HCV_S6

pool3−HCV_S5

pool2−HCV_S4

pool1−HCV_S3

61516A−HCV_S2

61515A−HCV_S1

0 25 50 75 100

proportion of total reads hitting references

phiX174EcoliPacneshg38HCVHIV1GBvirusCnot_referenced

15_Dec_02_checkMiseq proportion of total reads hitting references

56587A−HCV_S2

56585A−HCV_S1

0 25 50 75 100

proportion of total reads hitting references

phiX174EcoliPdenitrificansPacneshg38HCVnot_referenced

15_Jul_24_S1S2_checkMiseq proportion of total reads hitting references

Paracoccus denitrificans (probable reagent contaminant)

Coverage  in  HCV  NS5A  with  vs.  without  Probe  Enrichment  GT1a  and  GT3a  in  90:10  mix  

Rea

d D

epth

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  

NS5A Amino Acid Position

GT1a + Probe GT3a + Probe GT1a GT3a

•  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

Summary

Acknowledgement

Centre for Excellence in HIV/AIDS •  Chanson Brumme •  Winnie Dong •  Celia Chui •  Weiyan Dong •  Jeff Joy •  Art Poon •  Don Kirby •  Vera Tai •  Conan Woods •  Richard Harrigan

Merck Research Laboratory •  Ping Qiu •  Wei Bo •  Rick Stevens

Vancouver VIDUS Cohort Patients and Investigators