Question: duplicated read in ChIP seq
0
gravatar for op263
2.4 years ago by
op2630
op2630 wrote:

Hello,

I just received sequences from the first ChIP seq experiment done in my lab. I run triplicates for the samples and used input as a control. I started the analysis with UseGalaxy and already have some problems after FastQC step!! I found high level of duplicated read in my samples (input are fines) with only 4% of seqs remaining if deduplicated.

Is it worth making the analysis after removing the duplicates? I was considering removing the duplicated read and combining single reads from the triplicates.

many thanks for any help!

Olivier

chip-seq • 2.2k views
ADD COMMENTlink modified 2.4 years ago by harold.smith.tarheel4.3k • written 2.4 years ago by op2630
4
gravatar for harold.smith.tarheel
2.4 years ago by
United States
harold.smith.tarheel4.3k wrote:

Duplication is expected in ChIP-Seq, but 96% duplication is not unless 1) you depth of coverage is massively excessive, or 2) your binding factor interacts with very few sites. A much more common explanation is that your IP failed and/or the amount of IPed chromatin was too low for efficient library construction, which results in a huge amount of PCR duplication. You can discriminate via genome browser of your non-deduplicated data. Bona fide peaks will have multiple overlapping reads with offsets, while samples with only PCR duplicates will stack up perfectly without offsets.

ADD COMMENTlink modified 2.4 years ago • written 2.4 years ago by harold.smith.tarheel4.3k
1
gravatar for mastal511
2.4 years ago by
mastal5112.0k
mastal5112.0k wrote:

You would expect to find duplicated sequences in Chip-Seq data, because you are only sequencing the parts of the genome pulled down by the IP procedure. Your data is probably fine, so don't remove the duplicates.

ADD COMMENTlink modified 2.4 years ago • written 2.4 years ago by mastal5112.0k
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