Hi there,
I am looking at using a modified ddRAD protocol in a genus of plants with a genome approximately 1000Mbp in size. I do not have a Pippin Prep available, so instead I have been playing around using double-ampure size selection. I use a 0.65-0.75x ratio on the initial digest (with EcoRI and MseI) and after adapter ligation and PCR amplification my library size is between 400-600bp (or close to, only based on gel photos).
Using the available genomes of the most closely related organisms I can find, Allium and Asparagus, I estimate that this would be between 30k-100k ddRAD sites in my genus.
However, I note that size selection in ddRAD is often focused on a +- of about 50bp. I am worried that with the wideness of the AMpure will give more fragments than I expect, and my coverage would be too low.
I am looking to pool ~120 or so samples on a Hiseq 2000 lane and am worried that I might be on the low end of coverage.
Cheers,
Todd
I am looking at using a modified ddRAD protocol in a genus of plants with a genome approximately 1000Mbp in size. I do not have a Pippin Prep available, so instead I have been playing around using double-ampure size selection. I use a 0.65-0.75x ratio on the initial digest (with EcoRI and MseI) and after adapter ligation and PCR amplification my library size is between 400-600bp (or close to, only based on gel photos).
Using the available genomes of the most closely related organisms I can find, Allium and Asparagus, I estimate that this would be between 30k-100k ddRAD sites in my genus.
However, I note that size selection in ddRAD is often focused on a +- of about 50bp. I am worried that with the wideness of the AMpure will give more fragments than I expect, and my coverage would be too low.
I am looking to pool ~120 or so samples on a Hiseq 2000 lane and am worried that I might be on the low end of coverage.
Cheers,
Todd
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