We prepared un-stranded RNA-Seq library (SMART-Seq2) and sequenced in Illumina platform. We mapped with STAR (by adding XS tag) and assembled using cufflinks RABT assembly. Now after comparing with the reference annotation with gffcompare we found 5% of transcripts are coming from the opposite strand of the sense strand. Now I am little confused, according to literature/previous discussions, we know in un-stranded library we loose the information about strand. Thus detecting anti-sense RNA is not a straight forward process with this protocol. But after performing the assembly using RABT method are we dissolving the limitation of un-stranded protocol. If not why I am seeing only 5% from the anti-sense strand.
Thanks in advance.
Hi Sourav,
Please read our paper here: https://academic.oup.com/gigascience/article/7/8/giy093/5057872
we have highlighted the transcripts sensitivity issues in cufflinks.
Best wishes
Shab
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