Duplication rate for whole genome bisulfite sequencing

weigrc · 09-07-2015, 08:02 PM

Hi folks,

For WGBS, we've experienced with human gDNA using Illumina EpiGnome/TruSeq DNA Methylation kit and HiSeq sequencing.

The sequencing duplication rate is dynamic, from <5% to >20%; the more sequencing output we request, the higher duplication rate we get. Is it normal to get 5%-20% of duplication rate?

Can you share your experience on WGBS, which kit you've tried, how much sequencing depth you've requested and how much duplication rate you've got?

Besides, have you seen such "dynamic" duplication rate with different sequencing output?

Thanks,
Wei

nucacidhunter · 09-07-2015, 09:18 PM

In any PCR amplified library increasing sequencing depth will increase duplication rate as the diversity (# of unique fragments) is limited and sequencing more fragments will result in reads originating from the same initial fragment.
What you see is normal and I have seen around 10% dup rate using the same kit when around 40M reads/library was obtained. The rate was similar between bisulphite converted and non-converted libraries indicating the limits of kit and chemistry employed.

avilella · 09-09-2015, 10:54 AM

As an additional note to the previous answer, the duplicate rate will increase the deeper you sequence a given sample.

The curve of total reads and total distinct reads will depend on the diversity, as depicted here:

http://smithlabresearch.org/software/preseq/challenge/

There are ways to improve the duplicate rate of WGBS by using more recent commercial conversion kits from other vendors, generally categorized as post-BS kits.

weigrc · 09-16-2015, 05:52 PM

Dear Nucacidhunter and Avilella,

Thanks for your information. They help!!

BTW, rather than EpiGenome and TruSeq DNA Methylation kits, I read there is a new kit for methylation study as Accel-NGS® Methyl-Seq DNA Library Kit, which claims a better performance than former two kits. Has anyone tried out yet?

Accel-NGS® Methyl-Seq DNA Library Kit: http://www.swiftbiosci.com/LP/methyl-seq

Thanks in advance for the sharing.

Regards,
Wei

shldbcrzy · 04-18-2017, 01:47 PM

Weigrc, what has been your experience with the swift accel NGS kit?

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09-07-2015, 08:02 PM	#1
weigrc Member Location: Hong Kong Join Date: Oct 2011 Posts: 46	Duplication rate for whole genome bisulfite sequencing Hi folks, For WGBS, we've experienced with human gDNA using Illumina EpiGnome/TruSeq DNA Methylation kit and HiSeq sequencing. The sequencing duplication rate is dynamic, from <5% to >20%; the more sequencing output we request, the higher duplication rate we get. Is it normal to get 5%-20% of duplication rate? Can you share your experience on WGBS, which kit you've tried, how much sequencing depth you've requested and how much duplication rate you've got? Besides, have you seen such "dynamic" duplication rate with different sequencing output? Thanks, Wei Last edited by weigrc; 09-07-2015 at 08:04 PM.

09-09-2015, 10:54 AM	#3
avilella Member Location: uk Join Date: Mar 2009 Posts: 34	As an additional note to the previous answer, the duplicate rate will increase the deeper you sequence a given sample. The curve of total reads and total distinct reads will depend on the diversity, as depicted here: http://smithlabresearch.org/software/preseq/challenge/ There are ways to improve the duplicate rate of WGBS by using more recent commercial conversion kits from other vendors, generally categorized as post-BS kits. Last edited by avilella; 09-13-2015 at 03:19 AM.

09-16-2015, 05:52 PM	#4
weigrc Member Location: Hong Kong Join Date: Oct 2011 Posts: 46	Dear Nucacidhunter and Avilella, Thanks for your information. They help!! BTW, rather than EpiGenome and TruSeq DNA Methylation kits, I read there is a new kit for methylation study as Accel-NGS® Methyl-Seq DNA Library Kit, which claims a better performance than former two kits. Has anyone tried out yet? Accel-NGS® Methyl-Seq DNA Library Kit: http://www.swiftbiosci.com/LP/methyl-seq Thanks in advance for the sharing. Regards, Wei Last edited by weigrc; 09-17-2015 at 08:58 PM.

09-07-2015, 09:18 PM	#2
nucacidhunter Jafar Jabbari Location: Melbourne Join Date: Jan 2013 Posts: 1,238	In any PCR amplified library increasing sequencing depth will increase duplication rate as the diversity (# of unique fragments) is limited and sequencing more fragments will result in reads originating from the same initial fragment. What you see is normal and I have seen around 10% dup rate using the same kit when around 40M reads/library was obtained. The rate was similar between bisulphite converted and non-converted libraries indicating the limits of kit and chemistry employed.

04-18-2017, 01:47 PM	#5
shldbcrzy Junior Member Location: NYC Join Date: Sep 2011 Posts: 1	Weigrc, what has been your experience with the swift accel NGS kit?