Align to a single gene -- best procedure

Question

[marongiu.luigi]

Hello,

I have a set of files derived from a WGS experiment. Using Qualimap, I found a mean coverage of about 30x, which was expected.

I now need to concentrate on a single gene, so I aligned these files against the reference for that gene instead that all the human genome. Now the coverage is about 1000x. Something is not right.

So, my question is: what is the best practice to align for a specific reference? Shall I align the fastq files against the reference of the gene of interest (as I have done now)? Or against the whole human genome (since the main experiment was on human WGS) and extract from there?

Thank you

[GenoMax]

Something is not right.

Are you just looking for confirmation since you already know what you should do?

Or against the whole human genome (since the main experiment was on human WGS) and extract from there?

[marongiu.luigi]

Microfuge confirmed my fear: the aligner did not work properly and I should have used the second approach. So essentially, yes I was looking for a confirmation.

Answer 1

[microfuge]

I would strongly be for aligning to whole genome and not to a specific gene (unless it was targeted sequencing and not WGS). In absence of a complete reference the aligner would force reads from other regions on to partial reference.