Identification and Characterization of HCV Genotype 1L by Whole Genome

Sequencing

A.P. Davis, X. Zhang, A.M. Tigges, K. Kelliher, E.Z. Zhang, J.L. Dorrian, D.J. Bartels, T.L. Kieffer, J.C. Sullivan

Vertex Pharmaceuticals Incorporated, Cambridge, MA, USA

Presented at the 8th International Workshop on Hepatitis C: Resistance & New Compounds, June 27-28, 2013, Boston, MA, USA

Disclosures

● APD, XZ, AMT, KK, EZ, JLD, DJB, TLK, and JCS are current employees of Vertex Pharmaceuticals Incorporated.

● These individuals may own stock or stock options in their respective companies.

● This research was sponsored by Vertex Pharmaceuticals Incorporated and Janssen Pharmaceuticals.

Introduction

● Two Phase 3 clinical studies conducted in Europe assessed telaprevir in combination with pegylated interferon and ribavirin in 1,402 patients with genotype 1 HCV infection, who primarily had HCV subtypes 1a and 1b.1,2

● Subtyping was initially done by Inno-LiPA assay for the OPTMIZE study and TRUGENE for the REALIZE study, followed by HCV NS3-4a sequencing for both studies

● From these studies, two genotype 1 clinical samples could not be subtyped by HCV NS3-4a sequencing

● Massively parallel sequence analyses of the nearly full length genome was performed on these two samples in order to determine the subtype

1. Zeuzem et. al. N Engl J Med 2011;364:2417-28. 2. Buti et al. Presented at the 63rd Annual Meeting of the American Association for the Study of Liver Diseases (AASLD), Boston, MA, November 9-13, 2012; Abstract LB8.

Inno-LiPA and TRUGENE

● Inno-LiPA - amplified 5’ non-coding (NC) region, reverse-hybridized to type specific probes and visualize using alkaline phosphatase and chromogen

● TRUGENE- 5’ NC is amplified, then sequenced

NS3-4a

Population Sequencing of HCV NS3-4a Region

Amino acid 1 695

Comparison Subtyping Methods: INNO-LiPA and NS3-4a Sequencing Treatment Naïve HCV Patients (OPTIMIZE Study)1

• 99.6% (737/740) Subtyped by LiPA 5’NC genotyping • 99.5% (736/740) Subtyped by NS3-4a Sequence • 98.5% (733/740) Subtype Consensus Between Assays • 100% Subtyped by at least 1 Assay Subtyping Discrepancies

LiPA 5’NC

NS3-4a Sequence

1 1a 1 1a 1 1b 1a 1b 1a 1b 1a 1b 1b 1a

LiPA 5’ NC NS3-4a Sequence 1a Amplified, unable to subtype 1a Unable to amplify 1b Unable to amplify 1b Unable to amplify

NS3 Sequence Failures

1. Buti et al. Presented at the 63rd Annual Meeting of the American Association for the Study of Liver Diseases (AASLD), Boston, MA, November 9-13, 2012; Abstract LB8.

Phylogenetic Analysis of Two Untyped GT 1 Patients by NS3-4a Sequencing

Patient Screening Genotype

Sequencing Primers n=11

1 1a (LiPA) 4 successful

2 1 (TRUGENE) 5 successful

NS3-4a Population Sequence

Patient 2

Patient 1

Considered Sequencing Methods

• Primer walking • Ideal for shorter DNA fragments 1.3-7 kb

• Shot-gun (Sanger)

• Cost per base more expensive than next generation

• Next Generation Sequencing

• High coverage, lower cost per base

Methods: Quality Score Trimming and Filtering

Read position

40 30 20

Qua

lity

Sco

re

Read position

Qua

lity

Sco

re 40

30

20

Quality Box Plot of Reads, Patient 1

Post Filtering: Quality Box Plot of Reads, Patient 1

1,845,770 reads (Removed 50%)

Patient 1 : 3,736,418 reads

Reference guided assembly (i.e. human)

Methods: Viral Whole Genome Sequencing (WGS) Requires de novo Assembly

©2013 Vertex Pharmaceuticals Incorporated 10

de novo assembly (i.e. viral)

Reads

Reference guided assembly

Consensus Sequence

• De novo assembly using VICUNA1

Methods: Assembly Summary

• Mapped contigs to reference using NCBI command line BLAST2

• Made mock reference using both set of sequences

• Aligned original quality trimmed traces to consensus reference using BWA and Samtools3,4.

Con1(1b) Con1(1b)

E2 NS5A

1. Yang et. al. BMC Genomics 2012;13:475. 2. Camacho et. al. BMC Bioinformatics 2008; 10:421 3. Li et.al. Bioinformatics, 2010; 26: 589-95. 4. Li et. al. Bioinformatics 2009 25, 2078-9.

Results: Average 10,000-Fold Coverage Obtained

Patient 1

Theoretical Minority Variant Detection 0.007-0.015%

20,000

10,000 Cov

erag

e (#

of r

eads

)

30,000

40,000

50,000

94

Conclusions: Current State of Genotype 1L

• 28 G1L NS5B sequences in Genbank

Identified during screening study in Cameroon

• The two patients in this study are from UK and France

• This study met criteria for changing subtype 1L from provisional to ‘confirmed’

1. Li et al. J Gen Virol 2013; May 15, 2013, ePub ahead of Print.

• Full-length G1L genome recently became available from 3 individuals in Cameroon1