How do you extract regions of interest when you have massive exome data?

[Katherine_B]

Hi everyone,

I am new to bioinformatics and I have been having trouble with exome data. I've practised using standalone BLAST+ and I've understood how to create databases with my own sequences, BLAST another set of data, return values using outfmt, etc. However, I still cannot for the life of me understand how to use exome data (sequences contain gaps) and blast them when I have a set of CDS genes that I want to find. i.e. I have exome data for 10 fish species, and I want to find their genes associated with aquaporins (sequences I have using zebrafish reference).

I've tried everything but I just get errors, not can I figure out how to show my query sequences in the output. (using Windows 10 if that matters).

[GenoMax]

BLAST is probably not the right tool to use in this case. You really should be aligning using a NGS aligner. Since you are limited to using windows 10 (would be much better to switch to unix or bash on windows 10) you could give BBMap suite a try (it will run on windows as long as you have Java). Create indexes with the genes you have and then align using BBMap.

[Katherine_B]

Do you mean that I align my exome data for species A with the whole genome for zebrafish using NGS aligner? Some of the exons in the data are very small, like 10bp long only. Won't that cause problems as it could align to other 10bp regions (very likely for the code to repeat) or even align to introns by chance?

[GenoMax]

So you don't have a reference genome for species you are working with? Did you use a capture array or some other method?

[Katherine_B]

I have a reference genome which is broken into exons (this data was given to me and I am to handle it). I guess I should call it a reference exome to be specific.
I have exome data for other species - I have gene A that I want to find in all species using their exome data. So I tried blasting the CDS of gene A, I even tried blasting the individual exons of gene A against the exome data for species 1, 2, 3... etc. I was comparing the lables for the reference exome which is fine, I can find the information easily on Ensembl, but the labels for individual species just does not make sense at all to me. Ideally, if I have CDS of gene A, and I know it has 5 exons, I was truly hoping that after BLASTing species 1, I would get 5 sequences that match to regions for gene A.
Was not happening...