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Old 04-19-2018, 09:26 AM   #3
Reid
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Location: Flagstaff, Arizona, USA

Join Date: Mar 2015
Posts: 5
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Thanks for your response, nucacidhunter.

What I gathered from your response is that the pilot test I propose using a MiSeq will be useful mainly for providing an estimate of the number of restriction fragments I am actually attempting to sequence, given that I currently only have theoretical estimates via in-silico digests. In that case, it makes sense that there should be no problem in using a MiSeq to assess the number of restriction fragments I achieve through my double digest. Knowing fragment number will then allow me to calculate the number of samples to assess in parallel on a higher throughput Illumina machine. Thanks for that clarification.

Regarding the first portion of your reply:

While plants do maintain a number of methylated sequence contexts, CpG appears to remain an important sequence context for studies of differential methylation in plants.

See:
Gugger, P.F., Fitz‐Gibbon, S., PellEgrini, M. and Sork, V.L., 2016. Species‐wide patterns of DNA methylation variation in Quercus lobata and their association with climate gradients. Molecular ecology, 25(8), pp.1665-1680

That said, I am using HindIII and Taq(alpha)I for my double digests, as MspI is sensitive to certain methylation contexts present in the conifer genome I study.
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