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