Getting RNA sequence from RNA-Seq reads; some questions

Question

[R.Blues]

Hello everyone,

Although this question has been addressed before, I would like to bring it up again, as I would need it for a study and the state-of-the-art may have changed.

I am working with a reference genome and different clones. I have RNA-Seq data from these clones. I would like to do a DE transcript analysis, but also to determinate the sequence of certain already known transcripts in these clones (so I cannot merely extract the sequences using the reference genome).

Which would be the best way for addressing this?

I guess I could assembly the reads using Trinity, and obtain the sequence of these transcripts, while doing the DE analysis using the TopHat+Cufflinks Suite + DeSeq. Is this the most correct way to act, taking into account that I want this study for a paper? What would you do? Do you know of similar examples already done in previous papers? I have taken a look, but I have not been lucky.

Thank you for your time, and sorry for bothering you.

Kind regards.

Answer 1

[Devon Ryan]

If the clones are very similar to the reference sequence then just do the alignment as normal and then generate the consensus sequence on each clone from the results (search this site for "consensus sequence BAM"). While you could use something like Trinity to assemble the transcripts, if you happen to know that the sequences are very similar then the former method would both be faster and yield good enough results.

[R.Blues]

Alright! Thank you very much, Devon!! :)