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getzlab / rnaseqc

Fast, efficient RNA-Seq metrics for quality control and process optimization
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library type #65

Closed leungman426 closed 2 years ago

leungman426 commented 2 years ago

could you clarify in more details what RF, rf, FR and fr mean respectively for the --stranded options?

agraubert commented 2 years ago

Sure! @francois-a can confirm here, but my understanding is that these options refer to the order that reads are placed in the original FASTQ files produced by your sequencer. RF/rf indicates that the first read in a pair is sequenced from the reverse strand, followed by its forward strand mate (rf -- reverse forward). This is the format produced by Illumina and other sequencers. FR/fr indicates the opposite -- that the first read in a pair is the forward strand, followed by its reverse strand mate (fr -- forward reverse). In either case, the uppercased and lowercased options mean the same, it's just a preference.

RNA-SeQC uses these options as an error check to ignore reads which have an invalid mapping. Reads which exhibit an illegal combination of the strand orientation option (rf/fr), the read's strand flag (forward/reverse), and the strand that a read was mapped to are ignored. This is usually a very small percentage of mapped reads, but can indicate an issue with your aligner configuration if there is a significant proportion of these discarded reads.