ve-SEQ: Robust, unbiased enrichment for streamlined detection and whole-genome sequencing of HCV and other highly diverse pathogens

Abstract

The routine availability of high-depth virus sequence data would allow the sensitive detection of resistance-associated variants that can jeopardize HIV or hepatitis C virus (HCV) treatment. We introduce ve-SEQ, a high-throughput method for sequence-specific enrichment and characterization of whole-virus genomes at up to 20% divergence from a reference sequence and 1,000-fold greater sensitivity than direct sequencing. The extreme genetic diversity of HCV led us to implement an algorithm for the efficient design of panels of oligonucleotide probes to capture any sequence among a defined set of targets without detectable bias. ve-SEQ enables efficient detection and sequencing of any HCV genome, including mixtures and intra-host variants, in a single experiment, with greater tolerance of sequence diversity than standard amplification methods and greater sensitivity than metagenomic sequencing, features that are directly applicable to other pathogens or arbitrary groups of target organisms, allowing the combination of sensitive detection with sequencing in many settings.

Characterization of the Specificity, Functionality, and Durability of Host T‐Cell Responses Against the Full‐Length Hepatitis E Virus

“…For the samples from volunteers with a history of acute HEV infection, HEV genotyping was performed at Public Health England Reference Laboratories or performed in‐house by nested reverse‐transcriptase PCR (RT‐PCR) on 10 μL of purified RNA using the Access kit (Promega, Southampton, UK) with primers as described . In 1 patient, HEV viremia was detected by RNA sequencing analysis as described . For the samples from chronically infected patients, HEV RNA was detected by a nested RT‐PCR as described .…”

“…(43) In 1 patient, HEV viremia was detected by RNA sequencing analysis as described. (44) For the samples from chronically infected patients, HEV RNA was detected by a nested RT-PCR as described. (45) Nucleotide sequences were deduced from bulk PCR products and genotyped by comparison with HEV genotype 3 subtype reference sequences.…”

Clinical and biological insights from viral genome sequencing

“…Highly variable pathogens, particularly those that have widely divergent genetic lineages or genotypes, such as HCV 97 and norovirus, cause problems for PCR amplification, such as primer amplification 27,92 and primer mismatches 86 . Careful primer design may help to mitigate these problems, but novel variants remain problematic.…”

Comparison of next Generation Sequencing Technologies for the Comprehensive Assessment of Full-Length Hepatitis C Viral Genomes

“…This may originate through variability in the degree of match between probe or primer sets to the target viral sequence and therefore differences in the efficiency of target capture or amplification. We have recently shown that introducing probes to better represent known sequence variation can reduce bias in coverage due to probe-target divergence to zero (10). However, incomplete coverage was not a consistent problem for any of the capture methods, which in fact provided substantially greater depth of HCV coverage than metagenomic methods that were probe independent.…”

“…(i) Oxford. As described previously (10,12), low-quality bases were trimmed from demultiplexed sequences using QUASR v7.01 (www.bioconductor.org/packages/release/bioc/html /QuasR.html), and adapter sequences were removed using CutAdapt v1.7.1 (http://cutadapt.readthedocs.io/en/stable/index.html). Human sequences were excluded by mapping to the HG19 human reference genomes with Bowtie v2.2.4 (http://bowtie-bio.sourceforge.net/index.shtml), and HCV-derived reads were aligned to a local BLAST database of 165 HCV genomes collated by the ICTV (International Committee on the Taxonomy of Viruses).…”