technical replicates uncorrelate with each other

[tujchl]

Hi:

I do chip-seq aganist gammaH2AX, one of histone modification, which indicates DNA damage and repair.
At the beginning I just have about 7M reads (chip) and 4.7M reads (input) and using SICER to get enrishment profile on genome (human hg18).
Then I send the exactly the same sample to sequencing (chip sample) and getting 11M reads and I don`t sequence input sample this time and use the same input to get enrishment profile.
My question is I find those two profiles have large differences.

can you give me some advice?
thanks in advance

[mudshark]

Hi
- did you confirm the SICER peaks by qPCR on unprocessed ChIP samples or any other technology?
- are there positive/negative regions already documented and do your sequencing results conform to those?
- my initial guess is that you a) do not have enough reads for a robust analysis b) differences in sequencing depth will be the main source for inconsistency. c) most of your SICER peaks are false positives

[tujchl]

thanks mudshark

1. I have ordered primers to do the verification via qPCR
2. what do you mean by positive/negative regative?
3. I hope the variability occurs just beacuse I don`t have enough reads.

[ETHANol]

When you say you sequenced exactly the same sample does that mean that you:
1) Made a new library from the same ChIP or
2) Made a single library from one ChIP and sequenced it twice?

[tujchl]

Thanks ETHANol

The exactly the same sample means just one library and I sequence it twice