How to interpret De Novo Sequence from Trinity

Question

[progistar]

Hello,

I have a quick question about de novo sequence from paired-ends RNA-seq using Trinity. Many researchers use six-frame translation to get open reading frames from de novo sequences. In my opinion, however, the de novo sequences are the sequence of transcripts so it is more natural to use three-frame translation. Is anyone can explain reason why they use six-frame translation?

Thanks.

Answer 1

[lieven.sterck]

If you have (older) RNAseq data that will likely be unstranded data. Meaning it you have no idea whether the 'sense' or 'anti-sense' version of the gene has been sequenced. Hence you will have to look at all 6 frames.

Nowadays most RNAseq is stranded, you thus know you have the sense version of the gene and can indeed refrain yourself to only the 3 forward frames .

On a related note: you are always for better of running some sort of ORF detection tool on your data rather than just translate the transcripts in frames and look for the longest ORF. Those tools will usually analyse both strands and bring back the most likely ORF.

[progistar]

Thanks for your comment! It is really helpful advice! Now I can make a decision for my data.