Read orientation originated from a library was made by dUTP method

Question

[seta]

Hi all,

I've got Illumina sequencing reads that generated by the TruSeq Stranded mRNA Sample Prep Kit. Based on Trinity's guide, the first read (/1) of fragment pair is sequenced as anti-sense (reverse) and the second read (/2) is in the sense strand by dUTP sequencing method. However, the sequencing center told us the reads made as forward-reverse (dUTP). So, it sounds some discrepancy with the location of first read on the sense or anti-sense strand. Could anybody please clear me about it.

Thanks

Answer 1

[Devon Ryan]

For TruSeq kits (well all dUTP kits), read #2 dictates the strand. I assume that there was just a communication mix up with the core (we core facility folks are fallible!).

For what it's worth, I haven't seen a dataset where read #1 dictated the strand in quite a while.

[seta]

Thanks Devon. Such a mistake from core may have bad output for me as a learner in this field. Sorry, I'm a bit confused with the concept of orientation as FR or RF on reads generated by dUTP method. Based on this link, the concept of read orientation --RF for running Trinity tool on dUTP reads is different from --rf for running bowtie or RSEM on them. Could you please clear me on this issue? why there is such difference?

Thanks

[Devon Ryan]

"FR" and "RF" don't really have any meaning outside of specific tools. In trinity, the decided to make "RF" mean that read #1 is on the antisense strand and read 2 is on the sense strand. Every tool will have its own method of dictating this.

[seta]

Thanks friend. It's strange that the concept of orientation is different for every tool. Sorry for confusing, but, we cannot say read orientation by the UTP method is reverse-forward?

[Devon Ryan]

That and whether coordinates are 0 or 1 based are the two biggest stumbling blocks in bioinformatics :P