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Old 11-07-2015, 06:03 PM   #5
lukeSeq
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Location: Australia

Join Date: Oct 2015
Posts: 3
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Hi Kerplunk,

Thanks for that reply, much appreciated! The genomes I'd be working on are around 300MB. I guess I could afford to sequence quite a few whole genomes with respectable coverage, but I'm interested in getting the costs down low enough to do masses of biological replication (it's amazing how many studies have 1 or a couple of replicates - surely this opens the door to artefacts that happen to look like biology, e.g. incomplete BiS conversion in sample A but not sample B, not to mention the fact that A and B are differentially methylated purely by chance?).

I do think methylation will be mostly in CpGs (like CCGG for MspI), so you're right that it'd be good to use a cutter that enriched for these regions, just as proposed in RRBS. Thus I was thinking of cutting with MspI and some other generic, non-methylation sensitive cutter, possibly after doing some in-silico trials in the genomes of close relatives.

p.s. for interested readers, have a look at this new methods paper which is very similar to what I had in mind, yet slightly distinct. Doesn't use BiS treatment, just cuts with a methyl-sensitive enzyme, and looks for RAD tags that drop out in some individuals http://onlinelibrary.wiley.com/doi/1...10X.12435/full
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