RNA sequencing (RNA-seq)
RNA sequencing can be used to detect and quantify coding and non-coding RNAs, for studies of differential gene expression and studies of alternate splicing.
In RNA sequencing (RNA-seq), next generation sequencing (NGS) is used to detect the presence and quantity of all RNA molecules in a patient sample. This type of analysis is sometimes referred to as transcriptomics.
RNA-seq currently has limited applications through the NHS Genomic Medicine Service. Exploring the clinical applications of RNA-seq is, however, an active area of research globally.
RNA-seq may be particularly useful:
- for the detection of fusion transcripts in cancer patients;
- for patients with a strong suspicion of a rare genetic disorder, in whom all DNA sequencing options (including whole genome sequencing (WGS)) have been exhausted; and
- for studying a range of types of small regulatory RNA that may be sequenced alongside mRNA, for example microRNAs (involved in gene repression), long non-coding RNAs (involved in the regulation of gene expression) or small nuclear RNAs (which regulate splicing).
How does it work?
The first part of the RNA-seq process (the library preparation stage) includes the steps outlined below.
- A sample is taken from a tissue relevant to the patient’s condition (for example, from muscle if the patient has a muscular dystrophy phenotype).
- RNA is extracted from the patient’s sample.
- The RNA may be treated to enrich the amount of messenger RNA (mRNA) present.
- The RNA is converted to complementary DNA (cDNA) through reverse transcription. RNA can be fragmented before or after this step.
- For mRNA analysis, cDNA adapter ligation is used. This means that short DNA sequences (adapters) are added to enable processing during NGS. When samples from different patients are analysed in tandem, cDNA is also tagged with individualised DNA barcodes for sample identification.
After the library preparation steps detailed above, massively parallel sequencing of the cDNA and quality control of the sequencing data take place, as they would for other short-read sequencing applications. Alignment, variant calling and annotation are also performed, but these are reliant on different bioinformatics tools to those used for NGS of DNA.
Advantages and limitations of RNA-seq
- It can detect changes in gene expression, the use of different transcripts and alternative splicing.
- It may uncover non-coding variants not reported through DNA sequencing applications. These variants would be present in DNA WGS data, but may be of unknown significance if the impact on the RNA molecule is unknown, so they may not be reported.
- It can be used to detect fusion genes because analysis is not limited by pre-designed probe sequences.
- It is better than microarray-based assays for accurately measuring large differences in gene expression levels (the read depth can be adjusted, giving the technique a greater dynamic range).
- It can be used to characterise a range of RNA types, including mRNA but also small regulatory RNAs.
- It can detect allele-specific expression (where only one copy of the gene is active), for example due to skewed X-inactivation or genomic imprinting.
- It is not yet available in the NHS Genomic Medicine Service.
- It is likely that virtual panels would be used for the analysis of RNA-seq data. This means that, although sequencing data would be generated for the whole transcriptome, only RNA transcribed from genes known to be associated with the patient’s features would be analysed.
- It ideally requires a sample from an appropriate patient tissue, as peripheral blood lymphocytes may not express the gene(s) of interest.
- RNA is less stable than DNA, so tissue samples should be transported rapidly on dry ice or in a special medium (for more information, contact your local Genomic Laboratory Hub).
- Interpretation of non-coding variants and low-level alternative transcripts may be challenging, potentially producing many variants of uncertain significance.
- RNA-seq protocols for testing mRNA (to study gene expression) may not be optimal for studying small regulatory RNAs.
- The clinical relevance of many small regulatory RNAs is unclear.
- Reference standards and control materials used to help standardise and develop assays are not as readily available as they are for DNA-based testing.
- It is currently a bespoke test, and reporting times are therefore likely to be longer than for most tests.
Currently within the Genomic Medicine Service there is limited RNA-seq testing. Where it is carried out:
- one tube of PAXgene blood in an RNA tube is required; and
- testing should be agreed with the laboratory in advance.
- The Cresko Lab, University of Oregon: The RNA-seqlopedia
- Byron S, Van Keuren-Jensen KR, Engelthaler DM and others. ‘Translating RNA sequencing into clinical diagnostics: opportunities and challenges‘. Nature Reviews Genetics 2016: volume 17, pages 257-271. doi: 10.1038/nrg.2016.10