Strand-Specific RNA-Seq Provides Greater Resolution of Transcriptome Profiling

Abstract

RNA-Seq is a recently developed sequencing technology, that through the analysis of cDNA allows for unique insights into the transcriptome of a cell. The data generated by RNA-Seq provides information on gene expression, alternative splicing events and the presence of non-coding RNAs. It has been realised non-coding RNAs are more then just artefacts of erroneous transcription and play vital regulatory roles at the genomic, transcriptional and translational level. Transcription of the DNA sense strand produces antisense transcripts. This is known as antisense transcription and often results in the production of non-coding RNAs that are complementary to their associated sense transcripts. Antisense tran-scription has been identified in bacteria, fungi, protozoa, plants, invertebrates and mammals. It seems that antisense tran-scriptional 'hot spots' are located around nucleosome-free regions such as those associated with promoters, indicating that it is likely that antisense transcripts carry out important regulatory functions. This underlines the importance of identifying the presence and understanding the function of these antisense non-coding RNAs. The information concerning strand ori-gin is often lost during conventional RNA-Seq; capturing this information would substantially increase the worth of any RNA-Seq experiment. By manipulating the input cDNA during the template preparation stage it is possible to retain this vital information. This forms the basis of strand-specific RNA-Seq. With an ability to unlock immense portions of new in-formation surrounding the transcriptome, this cutting edge technology may hold the key to developing a greater under-standing of the transcriptome.

Keywords: Antisense RNA; Next-generation sequencing; Non-coding; Pervasive transcription.; RNA; Transcriptome.

Fig. (1)

Different methods of template preparation for strand-specific RNA-Seq. A. Strand-specific 3’-end RNA-Seq. Reads that align with a stretch of adenines at the end of the transcript are sense transcripts originating from the DNA antisense strand. Reads that align with a stretch of thymines at the front of the transcript are antisense transcripts originating from the DNA sense strand [54]. B. Single-stranded adapter ligation. Adapters are ligated directly to ss cDNA using T4 DNA ligase. As there is only one strand, stranded information is retained [56]. C. FRT-Seq. Poly(A) RNA is selected and fragmented. Adapters are ligated to the 3’ and 5’ end. The adapters consist of two regions; for the 3’ adapter the light purple region hybridizes to the flowcell surface and the sequencing primers anneal to the dark purple region. Similarly for the 5’ adapter the dark blue region hybridizes to the flowcell and the sequencing primers anneal to the light blue region. The fragments undergo reverse transcription on the flowcell surface then proceed to sequencing [59]. D. Bisulfite Treatment. By applying bisulfite mix to the RNA strand, all cytidine residues are converted to uridine. Through subsequent alignment with converted sense and antisense strands, the strand of origin can be identified. Reads from sense transcripts will align with the converted DNA sense (plus) strand, but not with the converted DNA antisense (minus) strand or either of the unconverted strands. Reads from antisense transcripts will align with the converted DNA antisense (minus) strand, but not converted DNA sense (plus) strand or either of the unconverted strands [19]. E. dUTP second strand. During second strand synthesis dUTP are added to the mix rather than dTTPs. These residues are removed by the addition of UNG (also known as UDG), destroying the strand. Only one strand proceeds to sequencing [63].