featureCounts, strand specificity, reversely stranded

Question

[tonja.r]

I am working with featureCounts and got a bit confused by -s parameter. I have strand specific RNAseq data. So, I guess my option for -s would 1 but why would somebody use 2 (reversely stranded)?

-s <int>      Indicate if strand-specific read counting should be performed.
                  It has three possible values:  0 (unstranded), 1 (stranded) and
                  2 (reversely stranded). 0 by default.

Answer 1

[Thibault D.]

Hi,

It depends on the library used in the sequencing protocol. There is a wide range of library types : ff-firststrand, ff-secondstrand, ff-unstranded, fr-firststrand, fr-secondstrand, fr-unstranded ... A read and its mate will not map to the genome in the same way according to the library stranding.

As featureCounts cares about the stranding of a read, it is important to specify the correct -s parameter.

PS: I know it might be a bit off topic, but this manual page from sailfish provides a nice overview of the ways a read and its mate can map on the genome according to libraries.

[tonja.r]

I checked my data with infer_experiment.py script, inside RSeQC package and found out following configuration. Does it mean that I have to use -s2 for featureCounts and I have fr-secondstrand?

2. +-,-+

• read mapped to '+' strand indicates parental gene on '-' strand
• read mapped to '-' strand indicates parental gene on '+' strand