Cufflinks XS tag

[Mchicken]

Hey guys,

I have an issue with my Sam files I want to use as input for Cufflinks. Unfortunately I trucated the lines of my Sam file right after the 11th column (due to disk space) and by this clipped off the XS tag, which is a requirement by Cufflinks for spliced alignments.

I found this post on SEQanswers: http://seqanswers.com/forums/showthread.php?p=69643

According to the last 5 answers in this thread, if I have a strand-specific library I should be able to set the XS flag own my own by:

1) For the R1 (forward read): Setting a + if the R1 read aligned + (watson) and setting a - if the read aligned - (crick)

2) For the R2 (reverse read): Setting a + if the R2 read aligned - (crick) and setting a - if the read aligned + (watson)

Could anybody confirm this assumption?

Thanks
Mchicken

[Brian Bushnell]

That is correct for what Cufflinks calls "firststrand" libraries. "secondstrand" libraries are the opposite.

[Mchicken]

So what I see in the IGV-Browser is, that all the R1 reads map on the gene-strand (coding strand) and all the R2 reads map on the opposite strand.

So is this now firststrand or secondstrand?

I read this: https://dbrg77.wordpress.com/2015/03...-tuxedo-suite/

But I am still not sure.