Read counts from SAM file mapped to de novo assembled transcripts using HTSeq-count

[alan_sm]

Hello,

Very sorry for cross posting it on other blog site but I'm under pressure to sort this out.

I tried using HTSeq-count to extract read counts per transcript from the SAM file (generated using Bowtie2 and only uniquely aligned reads were considered) mapped to de novo assembled transcripts (for DE analysis). I made GTF file for the assembled transcripts FASTA file with a Perl script. Here are few lines of my GTF file.

Locus_47_Transcript_16/31_Confidence_0.158_Length_1485 AssembledTranscriptome exon 1 1485 . + . gene_id "AssemTrans1"; transcript_id "Locus_47_Transcript_16/31_Confidence_0.158_Length_1485";

Locus_58_Transcript_85/85_Confidence_0.017_Length_650 AssembledTranscriptome exon 1 650 . + . gene_id "AssemTrans1"; transcript_id "Locus_58_Transcript_85/85_Confidence_0.017_Length_650";

Transcript start is by default 1 and end is the length of the transcript and Strand is + for all.

It looks like it works great but I'm not sure if this is the right way to do it. Don't know if I have to worry about what Simon Anders as mentioned "If you must align against the transcriptome, make sure that you count for genes, not transcripts, and remove reads mapping to transcripts from more than one gene."

Any thoughts/comments/suggestions are much appreciated.

Thanks,
Alan

[dpryan]

Simon's comment is very much something to keep in mind, though the importance of that and how feasible the normal pipeline are will depend on the particulars of your experiment. As a biologist, it's easiest for me to understand changes at the gene level. Changes at the transcript level are undoubtedly important, but I honestly don't think that we have a good handle on how to really interpret the meaningfulness of most of these. Perhaps in whatever organism you work things are different, but without further details I don't think anyone could really say.

[kurban910]

hello @alan_sm
you said you made GTF file for the assembled transcripts FASTA file with a Perl script. could you please share the script?
once i really needed to make GTF file myself but could not, because i am not familiar with the perl scripting. if you will here is my email : [email protected]
thanks