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Old 05-01-2010, 01:26 PM   #1
flobpf
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Question ChIP-Seq and Bisulfite sequencing coverage

Hello all,

I plan to perform some ChIP-Seq and Bisulfite sequencing. Does anyone have any idea how much coverage is "good" or "acceptable" for each of these methods and how many replicates are required?

My issue is my organism's genome is over 200 Mb. If I want just 1X coverage using GAIIx, it would itself require 1.2 entire flow-cells under ideal conditions. Then there need to be replicates and controls and the cost just explodes.

I know some groups have started to use multiplexing for genotyping and ChIP-Seq. But that is at the cost of coverage. So, something ~ 0.1X coverage in bisulfite sequencing or ChIP-Seq...is that good?

Any thoughts welcome

Thanks,
Flobpf
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Old 05-02-2010, 12:16 AM   #2
simonandrews
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I presume you meant your genome is 200Gbp rather than Mbp?

I suppose the amount of data you need to collect will depend on what you want to measure. If you're doing bisulphite seq to get an idea of the overall level of methylation in your genome or to do a very low density map then you won't need all that much data.

If you want to be able to look in detail at specific regions then effectively you need the same amount of data as for resequencing the whole genome since you need to observe everything to measure it. Arguably you need even more than that since the top and bottom strands could have different methylation patterns.

If you're only interested in CpG islands then reduced representation bisulphite seq can reduce the amount of data you need to collect by a factor of 4 or 5.

Our experience has been that within a sample the technical reproducibility of bs-seq has been very good so the increased amount of sequence is mostly just to provide coverage rather than repeated measures. To look at biological variability you'll need to do replicates though but the number of these required will depend on what you're trying to measure. It may be more cost effective to do a single replicate and follow up specific regions with other techniques - at the cost of missing some valid targets.
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Old 05-03-2010, 01:29 PM   #3
flobpf
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Simon, Thanks for the reply. I did not know about RRBS and that indeed could be used for my stuff

Thanks,
Flobpf
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