Adaptation of Oxford Nanopore technology for hepatitis C whole genome sequencing and identification of within-host viral variants

Abstract

Background Hepatitis C (HCV) and many other RNA viruses exist as rapidly mutating quasi-species populations in a single infected host. High throughput characterization of full genome, within-host variants is still not possible despite advances in next generation sequencing. This limitation constrains viral genomic studies that depend on accurate identification of hemi-genome or whole genome, within-host variants, especially those occurring at low frequencies. With the advent of third generation long read sequencing technologies, including Oxford Nanopore Technology (ONT) and PacBio platforms, this problem is potentially surmountable. ONT is particularly attractive in this regard due to the portable nature of the MinION sequencer, which makes real-time sequencing in remote and resource-limited locations possible. However, this technology (termed here ‘nanopore sequencing’) has a comparatively high technical error rate. The present study aimed to assess the utility, accuracy and cost-effectiveness of nanopore sequencing for HCV genomes. We also introduce a new bioinformatics tool (Nano-Q) to differentiate within-host variants from nanopore sequencing. Results The Nanopore platform, when the coverage exceeded 300 reads, generated comparable consensus sequences to Illumina sequencing. Using HCV Envelope plasmids (~ 1800 nt) mixed in known proportions, the capacity of nanopore sequencing to reliably identify variants with an abundance as low as 0.1% was demonstrated, provided the autologous reference sequence was available to identify the matching reads. Successful pooling and nanopore sequencing of 52 samples from patients with HCV infection demonstrated its cost effectiveness (AUD$ 43 per sample with nanopore sequencing versus $100 with paired-end short read technology). The Nano-Q tool successfully separated between-host sequences, including those from the same subtype, by bulk sorting and phylogenetic clustering without an autologous reference sequence (using only a subtype-specific generic reference). The pipeline also identified within-host viral variants and their abundance when the parameters were appropriately adjusted. Conclusion Cost effective HCV whole genome sequencing and within-host variant identification without haplotype reconstruction are potential advantages of nanopore sequencing.

Historical Perspective on the Discovery of the Quasispecies Concept

“…Detectability of a mutation may also depend on the accompanying mutations in the same genome and other genomes. Fortunately, advances in deep sequencing, with increasingly lower cutoff values for mutant detection and new methods for sequencing entire viral genomes (44), should contribute to clarifying if a residue responsible for one phenotype is present at low frequency in the mutant spectrum of the virus exhibiting a different (or the opposite) phenotype.…”

Exploring the complexity of RNA virus infections using viral genomics

“…In an extensive body of work spanning over three years, we demonstrated the suitability of utilising ONT for RNA virus sequencing when certain conditions were met (using sequencing depth to balance the sequencing error). 34 In other words, we showed that for the same sample, the consensus sequences generated by ONT and Illumina platforms were similar if the ONT consensus was built from at least 300 individual reads or more. 34 We also developed a novel bioinformatics tool named Nano-Q (https:// github.com/PrestonLeung/Nano-Q) which uses a hierarchal clustering algorithm, to differentiate within host variants from ONT sequencing while adjusting for sequencing errors.…”

“…34 In other words, we showed that for the same sample, the consensus sequences generated by ONT and Illumina platforms were similar if the ONT consensus was built from at least 300 individual reads or more. 34 We also developed a novel bioinformatics tool named Nano-Q (https:// github.com/PrestonLeung/Nano-Q) which uses a hierarchal clustering algorithm, to differentiate within host variants from ONT sequencing while adjusting for sequencing errors. 34 To test our workflow, we mixed plasmid clones with known HCV sequence inserts in known proportions to recreate a "variant mix" and then applied the new ONT based workflow to see if it can identify constituent plasmid HCV sequences and their proportions correctly.…”

“…34 We also developed a novel bioinformatics tool named Nano-Q (https:// github.com/PrestonLeung/Nano-Q) which uses a hierarchal clustering algorithm, to differentiate within host variants from ONT sequencing while adjusting for sequencing errors. 34 To test our workflow, we mixed plasmid clones with known HCV sequence inserts in known proportions to recreate a "variant mix" and then applied the new ONT based workflow to see if it can identify constituent plasmid HCV sequences and their proportions correctly. We demonstrated that our workflow reproduced all variants in the mix even when one constituent had an abundance as low as 0.1%.…”

Key SARS-CoV-2 Mutations of Alpha, Gamma, and Eta Variants Detected in Urban Wastewaters in Italy by Long-Read Amplicon Sequencing Based on Nanopore Technology

“…This method is characterised by high stringency: of the 972,348 reads obtained in total for the three pools after the filters (reads preserving both the 5 and 3 adaptor, consistent with the PCR amplicon product size, with an average quality equal or above 8, and aligning to the SARS-CoV-2 reference sequence) only 319,300 (32.8%) could be assigned. The Oxford Nanopore MinION, third generation sequencing technology, has several advantages such as portability, long read length, user friendliness, speed of sequencing, capability for multiplexing samples, and it has been demonstrated that Nanopore sequencing can identify low frequency variants in a mix of sequences [29]. However, it is important to underline that reverse transcription and PCR amplification in complex matrices display variable degrees of efficiency on different molecular targets and may significantly affect the final proportions of the amplified sequences.…”