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Old 06-25-2012, 04:20 AM   #1
hui_shi
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Default Strand bias on estimation of DNA methylation level

Hi Guys,

I was just wondering how you might deal with strand bias in DNA methylation calling using RRBS (reduced representation bisulphite sequencing). I know that strand bias is often an indication of erroneous alignment in NGS, so I am just not quite sure if I should filter out regions with disproportional number of plus and minus strands in estimating DNA methylation levels at CpG sites.

Thanks,

Hui

Last edited by hui_shi; 06-25-2012 at 09:13 AM.
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Old 04-22-2014, 01:48 PM   #2
raphael123
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I like this question ... And I am interested !
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Old 04-22-2014, 03:35 PM   #3
dpryan
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It depends on how bad it is. I've seen cases where the bias is more in read#2 versus read#1, which isn't surprising. Regardless, the methylation extractor that I wrote for bison can exclude biased regions according to both read (1 vs. 2) and strand. I would expect at least some of the other methylation extractors (at least those packaged with programs that will make the methylation bias graphs as well) to have similar functionality.

Last edited by dpryan; 04-22-2014 at 03:36 PM. Reason: swapped the reads!
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Old 04-23-2014, 11:06 AM   #4
raphael123
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Quote:
Originally Posted by dpryan View Post
It depends on how bad it is. I've seen cases where the bias is more in read#2 versus read#1, which isn't surprising.
I guess you talk about pair end sequencing, we are talking about a atrand bias, a difference of methylation between FORWARD and REVERSE strand ...
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Old 04-23-2014, 01:57 PM   #5
dpryan
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Quote:
Originally Posted by raphael123 View Post
I guess you talk about pair end sequencing, we are talking about a atrand bias, a difference of methylation between FORWARD and REVERSE strand ...
I understand that. I mentioned that due to it being the biggest source of bias. I've only ever seen very small differences due to strand, but, as I mentioned, programs like the bison_methylation_extractor can trivially deal with cases of strand-bias in methylation. Presumably at least some others can as well.
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