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DMNO 10-24-2017 06:48 PM

"Stranded" data may actually be unstranded?

I have some paired-end RNA-seq data that I was told were "stranded", but nothing about whether it's RF or FR.

I aligned some randomly sampled pairs of reads to the reference genome of the organism. I see that 50% of the read 1s map to the "+" strand, the other 50% to the "-" strand. Same as the read 2s.

Does this mean that the data is actually unstranded?

P.S. I can't get QoRTs to work on my data, so I can't use it to tell me whether the data is stranded or not.

Thank you!

Michael.Ante 10-24-2017 11:41 PM


did you take the transcripts' strand-orientation into account? Try using RSeQC's infer-experiment tool. It gives you a good way to assess if your library was stranded and in which orientation.



DMNO 10-25-2017 06:01 AM

Thank you very much, Michael!

The output of is:

Fraction of reads failed to determine: 0.0098
Fraction of reads explained by "1++,1--,2+-,2-+": 0.0089
Fraction of reads explained by "1+-,1-+,2++,2--": 0.9813

From what I understand, this means that the library is RF?

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