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HTSeq count (reverse count with paired-end data)

To which strand should a pair be counted to? I'm confused counting reads in paire-end RNA-seq data.

https://drive.google.com/file/d/0B8-ZAuZe8jldb3FJVXlZQkNxbjA/edit?usp=sharing

htseq rna-seq
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updated by Ram Ram • written by biotech biotech

If you are using HTSeq count, you should also be providing a GTF/GFF file as a reference against which the reads are counted.

According to the manual:

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

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updated by Ram Ram • written by komal.rathi komal.rathi

Of course I'm using it.

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written by biotech biotech

Probably the strand of read1, but it depends on how you made your library.

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written by Devon Ryan Devon Ryan

1 answer

The read counting depends on the library protocol. For example, in case the applied protocol is non-strand specific or strand-specific forward, then the read pair is counted, since the first read in the pair has alignment strand which matches the gene strand. In case the protocol is strand-specific reverse the read is not counted. There is a good explanation about strand-specific RNA-seq here.

There are tools which allow to estimate the strand-specificity based on the alignment data. For example, this one.

Hope this helps.

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updated by Ram Ram • written by madk00k madk00k

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