RNA-Seq

Summary of RNA-Seq. Within the organism, genes are transcribed and (in a eukaryotic organism) spliced to produce mature mRNA transcripts (red). The mRNA is extracted from the organism, fragmented and copied into stable ds-cDNA (blue). The ds-cDNA is sequenced using high-throughput, short-read sequencing methods. These sequences can then be aligned to a reference genome sequence to reconstruct which genome regions were being transcribed. This data can be used to annotate where expressed genes are, their relative expression levels, and any alternative splice variants.

RNA-Seq (short for RNA sequencing) is a next-generation sequencing (NGS) technique used to quantify and identify RNA molecules in a biological sample, providing a snapshot of the transcriptome at a specific time. It enables transcriptome-wide analysis by sequencing cDNA derived from RNA. Modern workflows often incorporate pseudoalignment tools (such as Kallisto and Salmon) and cloud-based processing pipelines, improving speed, scalability, and reproducibility.

RNA-Seq facilitates the ability to look at alternative gene spliced transcripts, post-transcriptional modifications, gene fusion, mutations/SNPs and changes in gene expression over time, or differences in gene expression in different groups or treatments. In addition to mRNA transcripts, RNA-Seq can look at different populations of RNA to include total RNA, small RNA, such as miRNA, tRNA, and ribosomal profiling. RNA-Seq can also be used to determine exon/intron boundaries and verify or amend previously annotated 5' and 3' gene boundaries. Recent advances in RNA-Seq include single cell sequencing, bulk RNA sequencing, 3' mRNA-sequencing, in situ sequencing of fixed tissue, and native RNA molecule sequencing with single-molecule real-time sequencing. Other examples of emerging RNA-Seq applications due to the advancement of bioinformatics algorithms are copy number alteration, microbial contamination, transposable elements, cell type (deconvolution) and the presence of neoantigens.