WikiJournal of Science/A broad introduction to RNA-Seq/XML

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    <full_title>WikiJournal of Science/A broad introduction to RNA-Seq</full_title>
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     <title>A broad introduction to RNA-Seq</title>
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RNA-Seq, named as an abbreviation of "RNA sequencing" and sometimes spelled RNA-seq, RNAseq, or RNASeq, uses next-generation sequencing (NGS) to reveal the presence and quantity of ribonucleic acid (RNA) in a biological sample at a given moment.      RNA-Seq is used to analyze the continuously changing cellular transcriptome (Figure 1).  Specifically, RNA-Seq facilitates the ability to look at alternative gene spliced transcripts, post-transcriptional modifications, gene fusion, mutations/single nucleotide polymorphisms (SNPs) and changes in gene expression over time, or differences in gene expression in different groups or treatments.    In addition to messenger RNA (mRNA) transcripts, RNA-Seq can look at different populations of RNA to include total RNA, small RNA, such as microRNA (miRNA), transfer RNA (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, in situ sequencing of fixed tissue, and native RNA molecule sequencing with single-molecule real-time sequencing.    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''.   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.  thumb|500px|left|''Summary of RNA-Seq.'' Within the organism, genes are transcribed and (in a [[w:eukaryote|eukaryotic organism) spliced to produce mature mRNA transcripts (red). The mRNA is extracted from the organism, fragmented and reverse-transcribed into stable double-stranded (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.  ]]
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