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Old 01-10-2019, 12:42 PM   #1
rflrob
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Location: Berkeley, CA

Join Date: May 2010
Posts: 50
Default Low-concentration, AT rich genome sequencing

Hi all,
Wondering what the current consensus is on recommended protocols for gDNA library prep from small concentrations (1-5ng) of an AT rich genome (Dictyostelium discoideum, ~23% GC). Empirically, we've had better luck with the protocol from Baym et al 2015[1] than the official Nextera XT protocol, but that only gets us to about 35% GC. Others have tweeted about reducing the extension temperature [2], or using a different (not commercially available?) transposase [3], but I thought I'd ask the hive mind prior to setting off on my own experiments.

I'd love to be able to do a PCR-free protocol, but the experiment I'm planning doesn't give more than about 5ng of gDNA, so that's off the table for all the kits I've seen.

Thanks!


[1] https://journals.plos.org/plosone/ar...l.pone.0128036

[2] https://twitter.com/masonry_stoves/s...29693978722304

[3] https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5240201/
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