Question about hisat2 parameter "--no-templatelen-adjustment"

Question

[ChrisT]

Hello,

I am quite new at analysing NGS data, but I am trying to set up a pipeline for my lab. We have performed single end sequencing using Ion Torrent Proton to analyse differential gene expression (through QuantSeq 3' mRNA-seq). I am testing alignment using the Hisat2 aligner, however I am not sure whether I need to use the parameter --no-templatelen-adjustment. I read the manual and paper describing the aligner, but it is not quite clear for me what this parameter does exactly. Can anyone help?

Thank you in advance.

[Bastien Hervé]

I would say it is not totally clear for me neither

Samtools definition of TLEN :

If all segments are mapped to the same reference, the unsigned observed template length equals the number of bases from the leftmost mapped base to the rightmost mapped base.

--no-templatelen-adjustment

Disables template length adjustment for RNA-seq reads.

In your case as you're working with single end, I think that the size of the template is equal to the size of your read

I do not know the effects of disabling the template length adjustment

But in any case if you don't understand an option, do not use it

Some info : https://github.com/infphilo/hisat2/issues/116

---

Edit :

Template length adjustment for RNA-seq reads is used to fit TLEN to the transcript length template due to splicing events

Using the --no-templatelen-adjustment you will got a template length based on the reference sequence (which in not what you want in typical RNA-Seq analysis).

But some tools as Manta (issue above) needs this template length information to the reference.

In conclusion, do not use it

[ChrisT]

Thank you very much for your help!