RNA sequencing: advances, challenges and opportunities

Abstract

Powered by improving next-generation sequencing capabilities, RNA-seq studies continue to provide knowledge about the quantitative and qualitative aspects of transcriptomes in both prokaryotes and eukaryotes. Advances have been made in areas including the cataloguing of sense and antisense transcripts, alternative splicing events, fused transcripts and transcription initiation sites in physiologically normal and disease settings. RNA-seq studies traditionally rely on sequencing of cDNA libraries that are generated from RNAs through various reverse transcription and sample preparation strategies. Biases and artefacts introduced by these experimental steps may hinder some applications. Recent technological developments, including direct RNA sequencing, may alleviate some of the limitations of current RNA-seq approaches and enable new paths of research into transcriptomics. Although several technological barriers still remain, major advances towards reliable analyses of RNAs from limited cell quantities have recently been achieved, paving the way towards transcriptome profiling at the single-cell level.