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

Join Date: Mar 2015
Posts: 5

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.

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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