High duplication percentage

klebsiella · 01-09-2013, 02:47 AM

Dear all,

I know that this thread was already asked before, however I wonder if someone has a new/better explanation/suggestions.

I am encountering a high duplication level in my chip-seq libraries. almost 99% of the reads are eliminated when considering only one read to map with Bowtie.

I am starting my libraries with around 2/3 ng of IP materials. I am using the NEB kit for the library prep. The library concentration is not that high too.
I am doing 18 cycles. I am multiplexing 10 samples at a time.
Inputs sequencing seems better with around 40% of duplication.

Any suggestion is the most welcome,

Best,

huguesparri · 01-17-2013, 05:31 AM

You can try to size select after the PCR enrichment rather than before. It tends to increase the diversity in the final libraries.

klebsiella · 01-17-2013, 05:45 AM

Dear Huguesparri,

Many thanks for your reply,
Indeed, I will try now either 1) avoiding completely the first size selection and put everything into the PCR or 2) making a really big size selection first (i.e 200-800 bp) and go with that to the PCR.
I think that I will go for the second option. I believe that in both cases this will help to increase the PCR complexity and get better results (less duplication).

PS: Also I will also try to start with more materials at the beginning,

Best

Similar Threads
Thread	Thread Starter	Forum	Replies	Last Post
High duplication levels in FASTQC	flobpf	Bioinformatics	3	11-27-2013 01:28 PM
Concern about short fragment size and high duplication rate in paired-end ChIP-Seq	biznatch	Epigenetics	9	08-13-2013 11:51 PM
High percentage of N calls in the library	apredeus	Bioinformatics	2	12-08-2012 04:27 PM
high sequence duplication levels for Illumina RNA-Seq meta-transcriptomics	Marcus	RNA Sequencing	12	07-20-2012 07:41 AM
Very high duplication of sequences in ChIP-Seq sequencing results	OptimusBrien	Epigenetics	8	09-15-2011 09:23 AM

01-09-2013, 02:47 AM	#1
klebsiella Member Location: Heidelberg Join Date: Jul 2010 Posts: 10	High duplication percentage Dear all, I know that this thread was already asked before, however I wonder if someone has a new/better explanation/suggestions. I am encountering a high duplication level in my chip-seq libraries. almost 99% of the reads are eliminated when considering only one read to map with Bowtie. I am starting my libraries with around 2/3 ng of IP materials. I am using the NEB kit for the library prep. The library concentration is not that high too. I am doing 18 cycles. I am multiplexing 10 samples at a time. Inputs sequencing seems better with around 40% of duplication. Any suggestion is the most welcome, Best,

01-17-2013, 05:31 AM	#2
huguesparri Member Location: Montpellier (France) Join Date: May 2008 Posts: 93	You can try to size select after the PCR enrichment rather than before. It tends to increase the diversity in the final libraries.

01-17-2013, 05:45 AM	#3
klebsiella Member Location: Heidelberg Join Date: Jul 2010 Posts: 10	Dear Huguesparri, Many thanks for your reply, Indeed, I will try now either 1) avoiding completely the first size selection and put everything into the PCR or 2) making a really big size selection first (i.e 200-800 bp) and go with that to the PCR. I think that I will go for the second option. I believe that in both cases this will help to increase the PCR complexity and get better results (less duplication). PS: Also I will also try to start with more materials at the beginning, Best