Plus and minus strands inverted

[will-rowe]

Hi there,

Thank you for making a great script available. I have been using it on a recent batch of sequencing data but have noticed that the plus and minus stranded bigwigs are the opposite to what we were expecting.

After checking through the script, it looks as though the F and f flags are the wrong way around at lines 20 and 22 in the bam_divide function, what do you think? I.E for forward strands, samtools command should be 'samtools view -F 20' and for reverse, 'samtools view -f 16'

[kepbod]

In general, we used Illumina Truseq Stranded Kit (dUPT method) to carry out the stranded sequencing, and the first strand cDNAs (reverse complementary to sample RNAs) were actually sequenced. So in the final bam file, if it is labeled with 16, it stands for plus strand in our case. If your sequencing protocol is different with ours, please feel free to modify it. 😄

[will-rowe]

Ahh that makes sense, I hadn't considered dUPT libraries. Thank you!