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Old 12-26-2014, 01:43 PM   #1
zaratieg
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Default AT rich region depletion

We are having some trouble with our ChIP-seqs: we seem to be completely blind to AT rich regions. When I check the GC distribution in the ChIP and Input reads I see that the input has maybe a 2% difference with the theoretical genomic reads, and in the IP reads the difference has increased to between 5 and 8%.

We sonicate with a bioruptor, so I get that we won't ever get close to Covaris style temperature control. But what befuddles me is how the IP could have depleted AT rich sequences even more.

Library prep was exactly the same for input and IP samples starting with cross-link reversal (65C o/n), then NEBnext library prep with only bead size selection, no gel purifications, and final amplification with 2x Phusion (NEB).

Any advice will be appreciated. Also as an aside, how much of a GC% difference can current GC-normalization algorithms solve? How much is a complete write-off?
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Old 01-07-2016, 02:42 PM   #2
DJ Flowcell
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Default

check out "Analyzing and minimizing PCR amplification bias
in Illumina sequencing libraries" by Aird et. al. Phusion sucks for AT rich...there's a lot of tweaking that can be done in you amplification step to help get representation from extreme base composition sequences.
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Old 01-08-2016, 06:18 AM   #3
zaratieg
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Thanks!! It's an interesting read. We'll try other Taqs.
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chip-seq, gc bias, library construction, sonication

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