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Old 04-18-2010, 07:53 PM   #1
Hao
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Location: New York

Join Date: Dec 2008
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Default How much DNA does the sequencer take in and how are peak heights determined

I am going to do my first ChIP-seq, and I wonder if any of you could help me with a technical question that is critical to my specific ChIP-seq application: How much DNA from the amplified library does the Illumina GA sequencer take in for analysis? Does it always take in a fixed amount of DNA (say, 100ng as determined by Bioanalyzer) or does it take a fixed proportion (say 10% of total libary DNA) of DNA? I am asking this because I am trying to compare two ChIP samples: A is control, B is treatment which results in genome-wide reduction of a specific histone methylation (that is, around three fold decrease on every gene locus). My goal is to be able to see the drop of methylation on every gene locus in B by ChIP-seq when compared to A. But if the sequencer takes in equal ng of DNA for A and B, I would not see a difference for the number of reads for each gene locus; if it takes in equal percent of DNA for A and B, I would see three fold lower number of reads in B (however, if the peak height is represented by the ratio of the number of reads for a gene over the total number of detected reads, then we still won't see a differenence, as the whole genome is affected evenly). or is it just difficult for ChIP-seq to make quantitative comparison across different samples? I guess I am don't know how the peak heights, representing the binding strength, are generated, and how they are normalized between two different samples. I wonder whether people just normalize to total read number, which is usually assumed not changed biologically, but does drop for the treated samples (around three fold) in my specific case.

Sorry for this long question, but any input will be greatly appreciated!
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