SEQanswers

Go Back   SEQanswers > Bioinformatics > Bioinformatics



Similar Threads
Thread Thread Starter Forum Replies Last Post
High duplication rate for INPUT (ChIP-seq) vadoue Bioinformatics 0 11-06-2014 06:58 AM
High duplication levels in FASTQC flobpf Bioinformatics 3 11-27-2013 12:28 PM
Concern about short fragment size and high duplication rate in paired-end ChIP-Seq biznatch Epigenetics 9 08-13-2013 10:51 PM
high sequence duplication levels for Illumina RNA-Seq meta-transcriptomics Marcus RNA Sequencing 12 07-20-2012 06:41 AM
Very high duplication of sequences in ChIP-Seq sequencing results OptimusBrien Epigenetics 8 09-15-2011 08:23 AM

Reply
 
Thread Tools
Old 09-07-2016, 01:41 PM   #1
fh331
Member
 
Location: UK

Join Date: Apr 2016
Posts: 19
Default ChIP-Seq high degree duplication levels

Hi all,

I am a newbie to bioinformatics. I've got ChIP-Seq data for 3 different proteins and 2 chromatin modification markers for two cancer cell lines. One library was prepared for each sample and run two different lanes (HiSeqV4). I thus have two files for each sample (one from each lane). The sequencing facility gives aligned data as CRAM files. I have converted CRAM into BAM and merged them using different tools: Picard, samtools, bamtools to see if it makes any difference. (Apparently, it doesn't). After merging, I have at least over 80 million reads for each sample. Next, using 'samtools view -b -f 2 -F4 -q 1' I filter the data to remove reads not in pairs and to select uniquely mapped reads. This filtering step reduces the number of reads by ~10 million. Next, I check the stats of the filtered reads for each sample using bamtools stats functions. It finds a lot of PCR duplication in almost all samples. The level of duplication ranges from 43% to over 80% in the samples. The Input samples (genomic DNA with no IP) has very little duplication (below 1%). I am assuming something has gone wrong either with library prepartion or at another step. Is this data too bad to work with? Is anyone able to suggest where the problem might be? If I deduplicate the data using picardtools, I am left with as little as 3 million reads. Can I peak call on this little reads?

Any input on this will be highly appreciated!
fh331 is offline   Reply With Quote
Old 09-07-2016, 02:36 PM   #2
mastal
Senior Member
 
Location: uk

Join Date: Mar 2009
Posts: 667
Default

I think your data sounds like what you would expect from a ChIP-Seq experiment.

You expect to see peaks of reads where your proteins of interest are binding to the DNA, and you don't see these in your input DNA control.

Here is a link to a previous thread where this has been discussed:

http://seqanswers.com/forums/showthread.php?t=40440

Last edited by mastal; 09-07-2016 at 02:40 PM.
mastal is offline   Reply With Quote
Reply

Tags
high duplication chip-seq

Thread Tools

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off




All times are GMT -8. The time now is 09:13 PM.


Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2020, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO