Early MinION™ nanopore single-molecule sequencing technology enables the characterization of hepatitis B virus genetic complexity in clinical samples

Abstract

Until recently, the method of choice to characterize viral diversity consisted in cloning PCR amplicons of full-length viral genomes and Sanger-sequencing of multiple clones. However, this is extremely laborious, time-consuming, and low-throughput. Next generation short-read sequencing appears also limited by its inability to directly sequence full-length viral genomes. The MinION™ device recently developed by Oxford Nanopore Technologies can be a promising alternative by applying long-read single-molecule sequencing directly to the overall amplified products generated in a PCR reaction. This new technology was evaluated by using hepatitis B virus (HBV) as a model. Several previously characterized HBV-infected clinical samples were investigated including recombinant virus, variants that harbored deletions and mixed population. Original MinION device was able to generate individual complete 3,200-nt HBV genome sequences and to identify recombinant variants. MinION was particularly efficient in detecting HBV genomes with multiple large in-frame deletions and spliced variants concomitantly with non-deleted parental genomes. However, an average-12% sequencing error rate per individual reads associated to a low throughput challenged single-nucleotide resolution, polymorphism calling and phasing mutations directly from the sequencing reads. Despite this high error rate, the pairwise identity of MinION HBV consensus genome was consistent with Sanger sequencing method. MinION being under continuous development, further studies are needed to evaluate its potential use for viral infection characterization.

Fig 1. Schematic representation of the recombinant HBV strain B5584.

The HBV genome is represented in a circular form and the HBV open reading frames are shown. Breakpoint positions for D (red, dotted)/E (blue, striped) genomic recombination are indicated with arrows and positions are given relative to an HBV genotype A reference sequence (GenBank AM282986).

Fig 2. Phasing of the S gene mutations Thr116Asn and Thr118Ser in the HBV genome.

Each bar represents one read and the dots indicate a deletion. Both single nucleotide variants were called by MinION (A) and direct Sanger sequencing (B). The number of MinION reads called for each of the single nucleotide variant patterns (C).

Fig 3. Alignment of nanopore reads of B6260.

Complete and partial Nanopore sequence reads were aligned with the corresponding Sanger consensus sequence used as reference. The viral core, surface, polymerase and X proteins are indicated and positions provided according to the reference sequence AM282986. Four different types of Minion reads are shown: nearly complete HBV genome (blue); reads showing a 123-nt deletion (positions 2,968–3,090) in the PreS1 region (purple); reads with the 123-nt and an additional 24-nt deletion (positions 501–524) in the “a determinant” of the S region (green); and unclassified partial reads (yellow).