RNA-Seq methods for transcriptome analysis

Abstract

Deep sequencing has been revolutionizing biology and medicine in recent years, providing single base-level precision for our understanding of nucleic acid sequences in high throughput fashion. Sequencing of RNA, or RNA-Seq, is now a common method to analyze gene expression and to uncover novel RNA species. Aspects of RNA biogenesis and metabolism can be interrogated with specialized methods for cDNA library preparation. In this study, we review current RNA-Seq methods for general analysis of gene expression and several specific applications, including isoform and gene fusion detection, digital gene expression profiling, targeted sequencing and single-cell analysis. In addition, we discuss approaches to examine aspects of RNA in the cell, technical challenges of existing RNA-Seq methods, and future directions. WIREs RNA 2017, 8:e1364. doi: 10.1002/wrna.1364 For further resources related to this article, please visit the WIREs website.

FIGURE 1

Methods for strand-specific RNA-Seq. (a) Ligation of the 3′ preadenylated and 5′ adapters. “xxx” indicates barcode. (b) Labeling of the second strand with dUTP, followed by enzymatic degradation. (c) The Peregrine method involves template-switch attachment of the 3′ adapter. (d) BrAD-Seq captures the 3′ adapter by taking advantage of terminal breathing of double-stranded DNA.

FIGURE 2

Targeted RNA-Seq by target capture. (a) The Capture-Seq method is based on capture of regions of interest by hybridization of RNA-Seq libraries to DNA oligonucleotide probes. (b) TARDIS is based on hybridization of input RNA to DNA oligonucleotide probes. The enrichment step is followed by the construction of a directional RNA-Seq library by ligation of 3′ and 5′ adapters.

FIGURE 3

Targeted RNA-Seq by amplicon sequencing. (a) PCR method using gene specific primers with overhangs containing sequences for common primers. (b) PCR methods using a pair of gene-specific primers followed by ligation of adapters with sequences necessary for sequencing. (c) Archer methods for detection of gene fusion. Sequence for a common primer is introduced by adapter ligation and is followed by nested PCR with gene-specific primers. MBC, Molecular Barcoded. (d) Detection of the TCR variable region. The sequence of a common primer is introduced during reverse transcription, and RT is followed by nested PCR with gene-specific primers. (e) Digital encoding of targeted mRNAs. Reverse transcription will introduce molecular indexes and sequences for common primers. Nested PCR follows, where gene-specific primers capture targeted sequences.

FIGURE 4

RNA-Seq of single cells. (a) Reverse transcription with oligo-dT primers and a universal primer sequence is followed by poly(A) tailing. After PCR amplifications, standard RNA-Seq libraries are prepared. (b) Reverse transcription incorporates a universal primer sequence. Template switching of reverse transcription is followed by annealing of the oligonucleotide with the sequence for a second PCR primer. (c) cDNA synthesis introduces the T7 promoter sequence at the 5′ end. After second strand cDNA synthesis, cRNA copies are generated by in vitro transcription. Finally, the second adapter is ligated to the 3′ end of the cRNA and libraries are constructed by PCR amplification. (d) Single-cell MALBAC RNA-Seq. Primers with seven random nucleotides at the 3′ end are annealed to cDNA and extended. Amplicons are looped to protect them from being further amplified. Ten cycles of quasilinear amplification are followed by exponential PCR.