Whole Genome Sequencing of Hepatitis A Virus Using a PCR-Free Single-Molecule Nanopore Sequencing Approach

Abstract

Hepatitis A virus (HAV) is one of the most common causes of acute viral hepatitis in humans. Although HAV has a relatively small genome, there are several factors limiting whole genome sequencing such as PCR amplification artefacts and ambiguities in de novo assembly. The recently developed Oxford Nanopore technologies (ONT) allows single-molecule sequencing of long-size fragments of DNA or RNA using PCR-free strategies. We have sequenced the whole genome of HAV using a PCR-free approach by direct reverse-transcribed sequencing. We were able to sequence HAV cDNA and obtain reads over 7 kilobases in length containing almost the whole genome of the virus. The comparison of these raw long nanopore reads with the HAV reference wild type revealed a nucleotide sequence identity between 81.1 and 96.6%. By de novo assembly of all HAV reads we obtained a consensus sequence of 7362 bases, with a nucleotide sequence identity of 99.0% with the genome of the HAV strain pHM175/18f. When the assembly was performed using as reference the HAV strain pHM175/18f a consensus with a sequence similarity of 99.8 % was obtained. We have also used an ONT amplicon-based assay to sequence two fragments of the VP3 and VP1 regions which showed a sequence similarity of 100% with matching regions of the consensus sequence obtained using the direct cDNA sequencing approach. This study showed the applicability of ONT sequencing technologies to obtain the whole genome of HAV by direct cDNA nanopore sequencing, highlighting the utility of this PCR-free approach for HAV characterization and potentially other viruses of the Picornaviridae family.

Retrospective Detection and Complete Genomic Sequencing of Canine morbillivirus in Eurasian Otter (Lutra lutra) Using Nanopore Technology

“…NGS can be optimized for rapid sample preparation and real-time sequence analysis, as well as the rapid identification of certain pathogens [ 42 , 43 , 44 , 45 , 46 , 47 ]. Amplicon-based NGS sequencing is an increasingly preferred method in the rapid detection and genomic characterization of specific pathogens, with high specificity and sensitivity [ 48 , 49 , 50 , 51 , 52 ].…”

Transcriptional and epi-transcriptional dynamics of SARS-CoV-2 during cellular infection

“…Long-read sequencing platforms can generate reads spanning the length of these sgRNA and are thus better suited to transcriptomic characterization of its highly nested transcriptome. One such platform is the MinION (Oxford Nanopore Technologies [ONT]), which can sequence either native RNA or cDNA directly without requirement for PCR amplification, therefore reducing PCR-induced biases in estimation of expression levels (Garalde et al, 2018;Batista et al, 2020). Furthermore, RNA modifications induce changes in ONT signal, which enable exploration of the epi-transcriptome using direct RNA (dRNA) sequencing (Garalde et al, 2018;Kim et al, 2020).…”

Role of biomaterials in the diagnosis, prevention, treatment, and study of corona virus disease 2019 (COVID-19)

“…For example, it was reported that 382 nucleotides of the SARS-CoV-2 genome had been deleted in Singapore, which was caused by false-negative reports when target sequences were defined based on deleted region [126]. Recently, singlemolecule nanopore (SMN) sequencing or third-generation platform was developed by Oxford Nanopore Technologies (ONT), which makes it possible to sequence long-size fragments of DNA/RNA [127]. SMN technique can detect changes in the nucleotides of DNA and RNA strand by passing them through a nanopore protein, which results in the production of a detectable electrical signal.…”

“…SMN technique can detect changes in the nucleotides of DNA and RNA strand by passing them through a nanopore protein, which results in the production of a detectable electrical signal. Recently, SMN sequencing for detecting Hepatitis C [128], Hepatitis A [127], and Influenza virus A [129] were reported. However, because SMN is more time-consuming than other POC platforms, it is not currently considered a primary method for detecting SARS-CoV-2.…”