Help!!!! masc2, paired-end(BAMPE)

[wendy]

Dear All,

Could any one offer me some suggestions?

I am using paired-end ChIP-seq data for transcription factors, without control/input.

Previously I run macs2 by using command: 

macs2 callpeak -t treat.bam -f BAMPE -g mm -n output -q 0.05

I saw suggestions saying that it is better to use --nolambda when you have no control/input sample and this could give you more real peaks. Thus, I added --nolambda to my command line and it turned out that only 2 peaks left instead of 1300 peaks I had before.

My questions are:

(1) What's the best parameter settings to deal with "paired-end" and "without control (input)" data? Is my command line fine?

(2) Should I use --nolambda when I have no control/input ? And why does this give me so few peaks? Is it strange that there are a little bit more peaks when I make the p value lower (more stringent)?

(3) In theory, --nolambda should generate more peaks or fewer peaks comparing with not using it? 

(4) In the case of not using --nolambda, by checking *treat_pileup.bdg and *summits.bed, I saw some smaller peaks were called, but at the same time the higher ones were not called. How to explain this?

Wish to get your reply. Thanks a lot!

Best wishes,

Wendy

[Ian Donaldson]

Apologies for not answering your questions directly, I do not know about the effect of --nolambda.  However, if you do not have a control then I would definately filter the reads using a blacklist file from ENCODE for your genome, as there is no means to remove non-specific binding events:

https://sites.google.com/site/anshulkundaje/projects/blacklists

Depending on the cell-line/tissue you could try to find an appropriate input sample in GEO/ArrayExpress, which although not ideal for publication would at least give you an idea.

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