RNA sequencing

Usually RNA is first converted into cDNA (complementary DNA) with a reverse transcriptase enzyme, and then a second strand synthesis reaction is performed so DNA sequencing techniques can be applied to double stranded DNA copies of RNA transcripts. Since this method can lose information from the 5' and 3' ends of the transcript, other methods have been developed that omit the second strand synthesis reaction and ligate adapters to the cDNA for sequencing reactions. Sequencing adapters are consistent sequences that, when applied to flank the cDNA fragments, produce a cDNA library.

RNA sequencing is replacing gene expression arrays to analyze the spectrum and abundance of transcripts in a given cell or tissue type at a given time. The technique called RNA-Seq, also known as whole transcriptome shotgun sequencing, generates cDNA and uses it in next-generation sequencing.

UK-based Oxford Nanopore Technologies devised a system to directly sequence RNA with a device called the MinION, in which electrical current is applied across a nanoscale molecular pore and current fluctuations detect the RNA sequence as the RNA molecule snakes through the pore. This RNA sequencing device was used by NASA on the International Space Station because NASA is interested in using it to identify onboard microorganisms and to monitor changes in human health or microbiomes; NASA is also interested in the possibility of detecting life based on DNA or RNA elsewhere in the universe.

RNA-Seq delivers an unbiased and unprecedented high-resolution view of the global transcriptional landscape, which allows an affordable and accurate approach for gene expression quantification and differential gene expression analysis between multiple groups of samples. RNA-Seq can identify novel and previously unexpected transcripts without the need for a reference genome, allowing de novo assembly of new transcriptome that is not previously studied before. It also enables the discovery of novel gene structures, alternatively spliced isoforms, gene fusions, SNPs/InDel, and allele-specific expression (ASE).RNA-Seq is a sensitive tool for gene expression profiling. Compared to microarray, RNA-Seq offers a digital read that is more accurate for all gene expression.

Abstract

Over the past decade, RNA sequencing (RNA-seq) has become an indispensable tool for transcriptome-wide analysis of differential gene expression and differential splicing of mRNAs. However, as next-generation sequencing technologies have developed, so too has RNA-seq. Now, RNA-seq methods are available for studying many different aspects of RNA biology, including single-cell gene expression, translation (the translatome) and RNA structure (the structurome). Exciting new applications are being explored, such as spatial transcriptomics (spatialomics). Together with new long-read and direct RNA-seq technologies and better computational tools for data analysis, innovations in RNA-seq are contributing to a fuller understanding of RNA biology, from questions such as when and where transcription occurs to the folding and intermolecular interactions that govern RNA function.