Use of illumina deep sequencing technology to differentiate hepatitis C virus variants

Abstract

Hepatitis C virus (HCV) is a positive-strand enveloped RNA virus that shows diverse viral populations even in one individual. Though Sanger sequencing has been used to determine viral sequences, deep sequencing technologies are much faster and can perform large-scale sequencing. We demonstrate the successful use of Illumina deep sequencing technology and subsequent analyses to determine the genetic variants and amino acid substitutions in both treatment-naïve (patient 1) and treatment-experienced (patient 7) isolates from HCV-infected patients. As a result, almost the full nucleotide sequence of HCV was detectable for patients 1 and 7. The reads were mapped to the HCV reference sequence. The coverage was 99.8% and the average depth was 69.5× for patient 7, with values of 99.4% (coverage) and 51.1× (average depth) for patient 1. In patient 7, amino acid (aa) 70 in the core region showed arginine, with methionine at aa 91, by Sanger sequencing. Major variants showed the same amino acid sequence, but minor variants were detectable in 18% (6/34 sequences) of sequences, with replacement of methionine by leucine at aa 91. In NS3, 8 amino acid positions showed mixed variants (T72T/I, K213K/R, G237G/S, P264P/S/A, S297S/A, A358A/T, S457S/C, and I615I/M) in patient 7. In patient 1, 3 amino acid positions showed mixed variants (L14L/F/V, S61S/A, and I586T/I). In conclusion, deep sequencing technologies are powerful tools for obtaining more profound insight into the dynamics of variants in the HCV quasispecies in human serum.

Fig 1

Evaluation of the quality of libraries. (A) The library was well refined for patient 7, but the primer and adaptor dimers were mixed in the libraries of patients 1 and 9, obtained using Bioanalyzer (Agilent). The horizontal axis shows the DNA size, and the vertical axis shows the quantity of DNA. The blue arrows indicate the desired products, and the red arrows indicate the primer or adaptor dimers. The peaks of the wave at 35 bp and 10,380 bp express the marker. (B) Amplification plots for patients 1 (treatment naïve) and 7 (treatment experienced), showing the presence of the HCV genome in the libraries by quantitative PCR with StepOnePlus (Applied Biosystems).

Fig 2

Mapping to the HCV reference genome. For patient 1, 6,303 reads were mapped to MD5-1. The coverage was 99.4%, and the average depth was 51.1×. For patient 7, 8,583 reads were aligned to MD2-2. The coverage was 99.8%, and the average depth was 69.5%.

Fig 3

Amino acid substitutions of aa 70 and 91 in the core region (A) and aa 2209 to 2248 of the NS5A ISDR (B). Mutations in these regions were reported to affect the outcome of IFN-based therapies for chronic hepatitis C patients. The lower two lines show the nucleotide sequence and amino acid sequence of HCV-J. Amino acid abbreviations: F, phenylalanine; S, serine; Y, tyrosine; C, cysteine; W, tryptophan; L, leucine; P, proline; H, histidine; Q, glutamine; R, arginine; I, isoleucine; M, methionine; T, threonine; N, asparagine; K, lysine; V, valine; A, alanine; D, aspartic acid; E, glutamic acid; G, glycine.