Genotyping by Sequencing in silico

[rosatoc]

Hi
For testing a given reference genome against different restriction enzymes I've received a recommendation to first mask the repetitive DNA sequence before running the in silico digest. Can anyone suggest a software or script for doing this (I am not a bioinformaticist, but I do have friends We have CLC Bio and we have biopieces currently available to use.
Many thanks for any suggestions.

[ophurtado]

I have always heard that there is nothing like collecting empirical data. Simulation might provide some guidance but the actual results (if resources allow it) might be more powerful.
Just a thought.

[rosatoc]

Hi ophurtado. I agree that often experience is the best teacher, and I look forward to learning. Until I get the go-ahead from my boss to spend money to gain the experience I am investigating ways of looking at options regarding choice of restriction enzyme by doing in silico digests. Eventually I expect to be able to correlate in silico data to actual data. Thanks for the reminder

[swbarnes2]

You can download masked genomes from ensembl, I think.

I'm not sure why you would want to mask then, though. If that DNA is really there, and will really be cut, and will really make pieces of a certain size, don't you need to know that?

[rosatoc]

Hi swbarnes2
The GBS strategy is used as a method of reduced representation library construction. Of course there are good and not-so-good things about this method, but it is being used successfully in a number of different agricultural genotyping projects. Often the choice for one enzyme (whether single or double digest design) is methylation sensitive. As I understand it, this is to 'skip' repetitive DNA regions and focus in on genic regions.

Follow-up experiments might include using differernt REs or going into the untranslated areas for SNP discovery.

As the core facility where I work will be working initially with researchers who are at the beginning of SNP discovery, we expect to focus in on areas of the genome where we would (hopefully) determine a novel SNP (marker) in a QTL of interest to the breeders.

At the beginning it is always a mystery, and I may realize my naivite as the process evolves.


Thanks for the tip about ensembl.