Long-Read Sequencing – A Powerful Tool in Viral Transcriptome Research

Abstract

Long-read sequencing (LRS) has become increasingly popular due to its strengths in de novo assembly and in resolving complex DNA regions as well as in determining full-length RNA molecules. Two important LRS technologies have been developed during the past few years, including single-molecule, real-time sequencing by Pacific Biosciences, and nanopore sequencing by Oxford Nanopore Technologies. Although current LRS methods produce lower coverage, and are more error prone than short-read sequencing, these methods continue to be superior in identifying transcript isoforms including multispliced RNAs and transcript-length variants as well as overlapping transcripts and alternative polycistronic RNA molecules. Viruses have small, compact genomes and therefore these organisms are ideal subjects for transcriptome analysis with the relatively low-throughput LRS techniques. Recent LRS studies have multiplied the number of previously known transcripts and have revealed complex networks of transcriptional overlaps in the examined viruses.

Exploring prokaryotic transcription, operon structures, rRNA maturation and modifications using Nanopore-based native RNA sequencing

“…As ONT-based native RNA-seq holds the capacity to sequence full-length transcripts, to identify RNA base modifications and to detect molecular heterogeneity in a transcriptome, the technology found widespread attention 16 . Recently, the technology was exploited to sequence viral RNA genomes 17–20 to gain insights into viral and eukaryotic transcriptomes 19,21–23 and to detect RNA isoforms in eukaryotes 24,25 . However, prokaryotic transcriptomes have not been characterized on the genome-wide level by native RNA-seq approaches so far as prokaryotic RNAs lack a poly(A) tail which is required to capture the RNA and feed it into the nanopore.…”

A beginner’s guide for FMDV quasispecies analysis: sub-consensus variant detection and haplotype reconstruction using next-generation sequencing

“…Nanopore direct RNA sequencing has no limit in reading length and is independent from cDNA synthesis and amplification prior to sequencing. Thus, it seems to be an excellent tool for full-length sequencing of viral quasispecies and transcriptomes [50,51]. However, despite its enormous potential, this technology has a very high error rate (∼10%).…”

Systematic characterization of non‐coding RNAs in triple‐negative breast cancer

“…Due to the complexity and diversity of lncRNAs, the biogenesis and regulation mechanism of different lncRNAs have not been completely summarized. In the future years, by virtue of the advanced techniques, including chromatin isolation by ChIRP‐Seq, CRISPR and CLIP, 71‐73 it is worthy of believing that the mechanism of biogenesis and synthesis of lncRNAs will be further deeply cognized.…”