RNA sequencing, or RNA-seq, is a method for mapping and quantifying the total amount of RNA transcripts in a cell at any given time, otherwise known as the transcriptome, for any organism that has a genomic DNA sequence assembly. Compared to microarrays that detect and quantify transcripts by hybridization against known sequences, RNA-seq directly sequences transcripts and is especially well-suited for de novo discovery of RNA splicing patterns and for determining unequivocally the presence or absence of lower abundance class RNAs. RNA-seq is performed by reverse-transcribing an RNA sample into cDNA followed by high throughput DNA sequencing. Most data is produced in the format of either single reads or paired-end reads. In the format of single reads each sequence read comes from one end of a randomly primed cDNA molecule (and represent one end of one cDNA segment), while paired-end reads are obtained as pairs from both ends of a randomly primed cDNA (and represent two opposite ends of one cDNA segment). The resulting sequence reads are then informatically mapped onto the genome sequence (Alignments). The current mappers (TopHat and STAR) have the ability to map reads to annotated and unannotated genomic regions. Reads mapped to annotated or novel RNA splice junctions are (Splice Sites). Earlier versions of this software did not map reads to unannotated genomic regions.

Some RNA-seq protocols do not specify the coding strand. As a result, there can be ambiguity at loci where both strands are transcribed.

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