ddRAD RE selection Peterson et al.

[SNPsaurus]

I take it you don't have a reference to search and get the actual distribution of sites?

Without that, there are quite a few unknowns that could affect your final result. For example, there are many more SbfI sites in some fish compared to others even though they have similar GC % and genome size. But, you can still try to predict it as best you can!

Clearly you want more than 40,000 loci for your less-frequent cutter and then use the more-frequent cutter to get a portion of those. PstI gives you 300k sites, SbfI gives you 10k sites. I wouldn't want to go with a purely GC 6-cutter (~100k sites) since that probably targets non-random sequences more than a mixed site, and same with a pure AT 8-cutter like PacI. So I think your safest bet is PstI, combined with a balanced (AT/GC) 4-cutter that will typically cut every 300 bp, then get out the Pippin and make your size selection. If you cut a bit away from the peak of 4-cutter size distribution (say 150-200 insert size) then you can pick up the 15% of fragments you want and have a large enough region to try to avoid locus drop-outs from the different size selections.

You may need to devote more sequencing per sample depending on what proportion of your loci you want to be above the 25X read depth. Right now you have 40k x 25 x 96 = 96M reads. But you will probably see a 4-fold range in sequencing depth per sample, and a wide range of depths between different loci. You probably have to toss 25% of the reads as being repetitive as well. So for markers with complete data, the samples with lower reads may only have 5k loci above 25X depth.

If you want to start thinking about analysis, here is a good video from Cornell about some of the packages (and a warning about population statistics at the end): http://www.cornell.edu/video/rad-gbs...ing-strategies

[maxbangs]

My lab is about to embark on ddRAD following the Peterson et al. protocol. We are trying to decide on restriction enzymes (RE) to try. We want to get around 40,000 loci from single end sequencing with 25x coverage with 96 individuals per lane of HiSeq2000. The organism we are looking at is ~2.42Gb with 38% GC content.

The problem I have now is choosing a set of RE to give us said number of loci in a reasonable size selection. Does anyone have any suggestions on what combination to try?