RNA sequencing: from tag-based profiling to resolving complete transcript structure

Abstract

Technological advances in the sequencing field support in-depth characterization of the transcriptome. Here, we review genome-wide RNA sequencing methods used to investigate specific aspects of gene expression and its regulation, from transcription to RNA processing and translation. We discuss tag-based methods for studying transcription, alternative initiation and polyadenylation events, shotgun methods for detection of alternative splicing, full-length RNA sequencing for the determination of complete transcript structures, and targeted methods for studying the process of transcription and translation. With the ensemble of technologies available, it is now possible to obtain a comprehensive view on transcriptome complexity and the regulation of transcript diversity.

A comprehensive analysis of 3′ end sequencing data sets reveals novel polyadenylation signals and the repressive role of heterogeneous ribonucleoprotein C on cleavage and polyadenylation

“…Many experimental protocols to capture transcript 3 ′ ends and enable studies of the dynamics of polyadenylation have been developed (for review, see de Klerk et al 2014), and consequently, a few databases of 3 ′ end processing sites are available (Lee et al 2007;Derti et al 2012;You et al 2014). However, none of these databases has used the entire set of 3 ′ end sequencing data available to date, and thus, their coverage is limited.…”

Alternative mRNA transcription, processing, and translation: insights from RNA sequencing

“…CAGE still represents the basic technology for the detection of TSSs. Recently, several highthroughput CAGE methods, such as DeepCAGE, have been developed [14]. These transcriptome-wide studies suggest that TSS use is highly tissue specific [4,[15][16][17][18] and that the number of alternative TSSs differs by tissue type, with the hippocampus accounting for a larger number of TSSs than any other tissue [18,19].…”

“…These auxiliary RNA-binding proteins (RBPs) are not part of the spliceosomal machinery but can enhance or suppress alternative splicing by interfering with it [36][37][38][39]. Various crosslinking and RNA immunoprecipitation techniques, followed by next-generation sequencing, have been developed to map RNA-protein interactions in vivo [14]. An early goal of these studies was the identification of RNA-binding sites.…”

“…x TIGS-1175; No. of Pages 12 accurate detection and quantification of PASs [14]. These transcriptome-wide studies have deepened our understanding of APA, providing information on newly discovered PASs, elucidating the impact of APA on gene expression, and discovering new APA regulatory mechanisms.…”

Comparison of gloverin gene expression patterns between domesticated and wild silkworms

“…Whole-genome mRNA sequencing (RNA-Seq) technologies have been a significant advance for high-throughput transcriptome analyses, as they can generate hundreds of millions reads in a single sequencing run (Castillo et al, 2015;Ozsolak and Milos, 2011;Wang et al, 2009). This is more sensitive, quantitative and efficient, and it has higher reproducibility compared to previously use hybridizationbased microarray techniques (Castillo et al, 2015;de Klerk et al, 2014). RNA-Seq has already produced exciting and novel information in the study of various diseases (Costa et al, 2013;Castillo et al, 2015;Xuan et al, 2013).…”