RNA sequencing (RNA-seq) is a new tool in the genetic diagnostic laboratory made possible by the advent of low-cost, high-throughput, next-generation sequencing technology. Historically, RNA studies were limited to gene expression using microarray technology for the detection of gene rearrangements and quantification of gene expression for predefined genes. RNA-seq allows detection of qualitative and quantitative changes in RNA expression across the genome in clinical samples and is increasingly being used as an adjunct to diagnostic exome sequencing and whole-genome sequencing.
How It Works
RNA-seq works by sequencing every RNA molecule in a sample and measuring the expression of genes by counting the number of times its transcripts have been sequenced. The resulting information can be used to identify aberrant transcript events (genes expressed at aberrant levels), aberrantly spliced genes (where RNA transcripts are inaccurately cleaved to remove introns to become mature mRNA), and expressed rare variants where only 1 of the 2 copies of a gene is active while the other is silent.
A typical RNA-seq workflow begins with tissue sample selection followed by complementary DNA synthesis of total RNA by reverse transcription (Figure). Complementary DNA is then fragmented and sequencing adaptors are added to each end of these fragments, which are subjected to massively parallel sequencing to obtain sequence reads from one or both ends (single or paired-end sequencing, respectively). After sequencing, the resulting reads are assembled using either a reference-guided approach, ie, based on a template DNA blueprint, or de novo, ie, reference free using the information contained in the reads alone.
