Reconstructing an incomplete transcript of interest

Question

[Emily Wasson]

Hi there,

I am in need of some advice.

Background: I have performed a genome-guided transcriptome assembly by aligning multiple RNAseq reads to a reference genome using HiSAT2 and assembling using StringTie. I then use TransDecoder to obtain the CDS of these transcripts. From these, I have identified a few transcripts of interest (candidate genes), however, quite a few of these are fragmented/incomplete.

My question is as follows: Is there a way of obtaining complete CDS of these transcripts of interest by using the above-mentioned resources? I'm wondering if it would be possible to map reads to these transcripts of interest and then try to reassemble using the mapped reads..has anyone tried such an approach?

Any reading material on the topic would also be very much appreciated.

Answer 1

[shelkmike]

You can try programs like TASR or Mapsembler2. They map reads to a contig and extend this contig.
However, if I were you, I would first try a different approach: to assemble that transcriptome with Trinity and rnaSPAdes and see whether one of these programs assembled the transcripts of interest completely.
The approach with TASR and Mapsembler is a bit dangerous, because if there is a close paralog of your gene that has higher expression, you may obtain a chimera of these two genes.