Using Direct RNA Nanopore Sequencing to Deconvolute Viral Transcriptomes

Abstract

The genomes of DNA viruses encode deceptively complex transcriptomes evolved to maximise coding potential within the confines of a relatively small genome. Defining the full range of viral RNAs produced during an infection is key to understanding the viral replication cycle and its interactions with the host cell. Traditional short-read (Illumina) sequencing approaches are problematic in this setting, due to the difficulty of assigning short reads to individual RNAs in regions of transcript overlap and to the biases introduced by the required recoding and amplification steps. Additionally, different methodologies may be required to analyse the 5' and 3' ends of RNAs, which increases both cost and effort. The advent of long-read nanopore sequencing simplifies this approach by providing a single assay that captures and sequences full length RNAs, either in cDNA or native RNA form. The latter is particularly appealing as it reduces known recoding biases whilst allowing more advanced analyses such as estimation of poly(A) tail length and the detection of RNA modifications including N6-methyladenosine. Using herpes simplex virus (HSV)-infected primary fibroblasts as template we provide a step-by-step guide to the production of direct RNA sequencing libraries suitable for sequencing using Oxford Nanopore Technologies platforms and provide a simple computational approach to deriving a high-quality annotation of the HSV transcriptome from the resulting sequencing data.

Direct RNA nanopore sequencing of SARS-CoV-2 extracted from critical material from swabs

“…Next-generation sequencing technologies can be used to detect the nucleotide sequences in analyzed samples within a short duration and at an affordable cost (18). However, these approaches depend on both the harvest and extraction protocols of the sample, which significantly influence the coverage and depth of the sequencing reads (21, 22).…”

“…Instead, direct RNA-seq can help identify the native sequences without contamination by artefacts that are usually introduced in the in vitro amplification step, even though it requires a higher input concentration compared to cDNA sequencing methods (21) In this context, swab RNA, which has a lower concentration and is also more fragmented than in vitro RNA, may not be suitable for performing direct sequencing.…”

“…However, these approaches depend on both the harvest and extraction protocols of the sample, which significantly influence the coverage and depth of the sequencing reads (21,22).…”

De novo Sequencing of Novel Mycoviruses From Fusarium sambucinum: An Attempt on Direct RNA Sequencing of Viral dsRNAs

“…Since the sequence accuracy of the DRS reads is not high enough compared with that of the DNA ion tolerance sequencing (MinION DNA, error rate <5%), virologists have only used DRS to detect viruses in clinical samples, to analyse the RNA modification of viral genomes, and to investigate complex viral genomic structures (Quick et al, 2016;Keller et al, 2018;Kim et al, 2019;Lewandowski et al, 2019;Wongsurawat et al, 2019;Depledge and Wilson, 2020). This method has so far not been used successfully for sequence determination in virus research.…”

“…The average length of DRS reads obtained in this study (672.5 nt) was significantly shorter than those reported in other studies. The plausible reason is the use of different RNA templates: we used dsRNA molecules in this study, whereas previous studies relied on ssRNAs (Quick et al, 2016;Keller et al, 2018;Kim et al, 2019;Lewandowski et al, 2019;Wongsurawat et al, 2019;Depledge and Wilson, 2020). Because the Oxford Nanopore DRS method is designed for mRNA sequencing, here the viral dsRNAs were required to be physically modified into a suitable form [ssRNA with poly(A)] by heat-denaturation in the presence of DMSO.…”