Validate Novel Isoforms Generated By Cuffcompare

Question

[Ge]

When I compare the assembled transcripts with a reference annotation with cuffcompare, one of the output files is cuff_in.tmap. According to class code column in this file, we can see the relationship between Cufflinks transcripts with reference transcripts. So when the class code is j, it is potentially novel isoform, but how can we validate it is actually a novel isoform?

Thanks!!

Answer 1

[JC]

Well, computationally speaking you can check if the isoform is validated in your sample seeing the reads that align that position (check for mapping errors or incorrect splice sites). Also you can compare with the genes in other species. But for publication, many reviewers will ask you for a RT-PCR validating your isoform.