HTSeq count stranded setting
Entering edit mode
5 months ago


In HTSeq's manual for the stranded parameter, they state:

"For stranded=no, a read is considered overlapping with a feature regardless of whether it is mapped to the same or the opposite strand as the feature. For stranded=yes and single-end reads, the read has to be mapped to the same strand as the feature. For paired-end reads, the first read has to be on the same strand and the second read on the opposite strand. For stranded=reverse, these rules are reversed."

I interpreted the line " For stranded=yes and single-end reads, the read has to be mapped to the same strand as the feature." as meaning single-end data only needs the stranded=yes parameter whether it is reverse or not. Is this interpretation correct, or should I be using stranded=reverse for single-end data also if it is in fact reverse? The wording here is slightly confusing, since they only mention the reverse option after talking about paired-end data.

I usually work with paired-end data and am currently working on a small project involving single-end data, so my apologies if this seems like a simple question.

Thank you in advance to anyone who can help with this.

RNA-seq • 482 views
Entering edit mode
5 months ago

the wording is extraordinarily confusing and has probably lead to numerous non-reproducible analyses and publications.

the wording conflates the strandedness yes/no with the directionality forward/reverse whenever it is yes stranded

the proper naming would be


alas, instead, we get the misleading nomenclature of:

  • stranded=no means unstranded
  • stranded=yes means stranded in the forward direction
  • stranded=reverse also means stranded but in the reverse direction

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