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RNA-Seq

Summary of RNA-Seq. Within the organism, genes are transcribed and (in an 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.[1]

RNA-Seq (named as an abbreviation of RNA sequencing) is a technique that uses next-generation sequencing to reveal the presence and quantity of RNA molecules in a biological sample, providing a snapshot of gene expression in the sample, also known as transcriptome.[2][3]

Specifically, 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.[4] 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.[5] 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,[6] 3' mRNA-sequencing, in situ sequencing of fixed tissue, and native RNA molecule sequencing with single-molecule real-time sequencing.[7] 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.[8]

Prior to RNA-Seq, gene expression studies were done with hybridization-based microarrays. Issues with microarrays include cross-hybridization artifacts, poor quantification of lowly and highly expressed genes, and needing to know the sequence a priori.[9] Because of these technical issues, transcriptomics transitioned to sequencing-based methods. These progressed from Sanger sequencing of Expressed sequence tag libraries, to chemical tag-based methods (e.g., serial analysis of gene expression), and finally to the current technology, next-gen sequencing of complementary DNA (cDNA), notably RNA-Seq.

First, cellular mRNA is extracted and fragmented into smaller mRNA sequences, which undergo reverse transcription. The resulting cDNAs are sequenced on a Next Generation Sequencing (NGS) platform. The results of such sequencing allow the generation of transcriptomic sequencing genomic maps.
Experimental transcriptome sequencing technique (RNA-seq).
  1. ^ Lowe R, Shirley N, Bleackley M, Dolan S, Shafee T (May 2017). "Transcriptomics technologies". PLOS Computational Biology. 13 (5): e1005457. Bibcode:2017PLSCB..13E5457L. doi:10.1371/journal.pcbi.1005457. PMC 5436640. PMID 28545146.
  2. ^ Chu Y, Corey DR (August 2012). "RNA sequencing: platform selection, experimental design, and data interpretation". Nucleic Acid Therapeutics. 22 (4): 271–4. doi:10.1089/nat.2012.0367. PMC 3426205. PMID 22830413.
  3. ^ Cite error: The named reference wang2009 was invoked but never defined (see the help page).
  4. ^ Cite error: The named reference maher2009 was invoked but never defined (see the help page).
  5. ^ Ingolia NT, Brar GA, Rouskin S, McGeachy AM, Weissman JS (July 2012). "The ribosome profiling strategy for monitoring translation in vivo by deep sequencing of ribosome-protected mRNA fragments". Nature Protocols. 7 (8): 1534–50. doi:10.1038/nprot.2012.086. PMC 3535016. PMID 22836135.
  6. ^ Alpern D, Gardeux V, Russeil J, Mangeat B, Meireles-Filho AC, Breysse R, et al. (April 2019). "BRB-seq: ultra-affordable high-throughput transcriptomics enabled by bulk RNA barcoding and sequencing". Genome Biology. 20 (1): 71. doi:10.1186/s13059-019-1671-x. PMC 6474054. PMID 30999927.
  7. ^ Lee JH, Daugharthy ER, Scheiman J, Kalhor R, Yang JL, Ferrante TC, et al. (March 2014). "Highly multiplexed subcellular RNA sequencing in situ". Science. 343 (6177): 1360–3. Bibcode:2014Sci...343.1360L. doi:10.1126/science.1250212. PMC 4140943. PMID 24578530.
  8. ^ Thind AS, Monga I, Thakur PK, Kumari P, Dindhoria K, Krzak M, et al. (November 2021). "Demystifying emerging bulk RNA-Seq applications: the application and utility of bioinformatic methodology". Briefings in Bioinformatics. 22 (6). doi:10.1093/bib/bbab259. PMID 34329375.
  9. ^ Kukurba KR, Montgomery SB (April 2015). "RNA Sequencing and Analysis". Cold Spring Harbor Protocols. 2015 (11): 951–69. doi:10.1101/pdb.top084970. PMC 4863231. PMID 25870306.
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