Abstract
Advancing interventions to tackle the huge global burden of hepatitis B virus (HBV) infection depends on improved insights into virus epidemiology, transmission, within-host diversity, drug resistance and pathogenesis, all of which can be advanced through the large-scale generation of full-length virus genome data. Here we describe advances to a protocol that exploits the circular HBV genome structure, using isothermal rolling-circle amplification to enrich HBV DNA, generating concatemeric amplicons containing multiple successive copies of the same genome. We show that this product is suitable for Nanopore sequencing as single reads, as well as for generating short-read Illumina sequences. Nanopore reads can be used to implement a straightforward method for error correction that reduces the per-read error rate, by comparing multiple genome copies combined into a single concatemer and by analysing reads generated from plus and minus strands. With this approach, we can achieve an improved consensus sequencing accuracy of 99.7% and resolve intra-sample sequence variants to form whole-genome haplotypes. Thus while Illumina sequencing may still be the most accurate way to capture within-sample diversity, Nanopore data can contribute to an understanding of linkage between polymorphisms within individual virions. The combination of isothermal amplification and Nanopore sequencing also offers appealing potential to develop point-of-care tests for HBV, and for other viruses.
Highlights
In the context of clinical and public health settings, hepatitis B virus (HBV) sequencing can provide information that is useful in characterizing virus genotype, potential transmission networks, drug and vaccine resistance, and aspects of the dynamics of infection[5,7,8]
Following CL, genomes were amplified by the use of primers (Fig. 1A(iii) and rolling circle amplification (RCA; Fig. 1A(iv))[15,16]
We confirmed an increase in HBV DNA after rolling circle amplification’ (RCA) by comparing extracted DNA to RCA products using qPCR (Suppl Methods 1)
Summary
In the context of clinical and public health settings, HBV sequencing can provide information that is useful in characterizing virus genotype, potential transmission networks, drug and vaccine resistance, and aspects of the dynamics of infection[5,7,8]. While error-corrected Nanopore consensus sequences may be sufficiently accurate for many uses, raw-read accuracy remains a concern if it is to be used for the assessment of within-sample (between-molecule) diversity. One strategy to reduce error rates from single source molecules is to create concatemeric (chain-like) successive copies of each template, so that a single concatemer contains several reads of each base from the original molecule This approach has been demonstrated in the circularization of 16 S bacterial DNA sequences followed by ‘rolling circle amplification’ (RCA) using a high-fidelity DNA polymerase[12]. HBV isolates have previously been sequenced with Nanopore technology using full-length and sub-genomic PCR approaches to enrich for HBV sequences[13,14] Whilst these approaches worked well in the studies when applied to high viral load samples, in both publications correction was only possible at the consensus
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