These alignments serve as a basis for many types of downstream analysis, such as calculating transcript /gene expression, differential gene expression, detection of novel splice junctions and isoforms, signal visualization in the genomic browsers, and so forth. The Basic Protocol performs the most common analysis task - alignment of the reads to the references genome. RNA-seq data from the Next Generation Sequencing platforms such as Illumina or Ion Torrent consists of millions of relatively short sequences (“reads”) representing fragments of the original RNA molecules. Most of the options described in these Protocols can be combined to generate all the necessary output files in a single STAR job.īasic Protocol: Mapping RNA-seq reads to the reference genome In this unit we describe computational protocols that produce various output files, use different RNA-seq datatypes, and utilize different mapping strategies. STAR generates output files that can be used for many downstream analyses such as transcript/gene expression quantification, differential gene expression, novel isoform reconstruction, signal visualization, and so forth. STAR can align spliced sequences of any length with moderate error rates providing scalability for emerging sequencing technologies. In addition to detecting of annotated and novel splice junctions, STAR is capable of discovering more complex RNA sequence arrangements, such as chimeric and circular RNA. The STAR ( Dobin et al, 2013) software package enables highly accurate and ultra-fast alignment of RNA-seq reads to a reference genome. derived from the non-contiguous regions of the genome. This task presents more challenges than alignment of genomic DNA reads because RNA sequences are often spliced, i.e. One of the foundational steps in the RNA-seq data analysis is mapping (alignment) of the large sets of sequenced reads to a reference genome. Recent advances in the high-throughput sequencing have made sequencing of RNA transcripts (RNA-seq) an attractive tool for the studies of the transcriptome at single nucleotide resolution.
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