Non-C methylation in Bismark .cov files

bedward1 · 02-03-2015, 01:31 PM

Hi
I am analyzing RRBS using TrimGalore/Bismark and the methylation extractor, and am noticing quite a few non-C (T, A, and G) methylation signals (maybe 20% compared to C, by eye). Is this a normal technical artifact that has been observed?
Thanks

dpryan · 02-03-2015, 02:17 PM

In the case of G, that is a C, just on the other strand. In the case of T or A, that's either an off by 1 error or the sequence you're looking at is different from the sequence against which the aligning was done.

bedward1 · 02-03-2015, 02:37 PM

Thanks for the replay Devon.
Is the 'off by 1 error' common? What would cause this?

dpryan · 02-04-2015, 12:56 AM

Off by 1 errors are quite common (common enough that it has its own wikipedia article). The likely cause is the file using 1-based coordinates while whatever you're using to visualize things assumes it's using 0-based coordinates. I remember this being an issue with some of bismark's output at one point, though I don't know what the status of that is. If you look at those and there's an appropriate C (or G, depending on how things were done) just to the right, then that's what's going on.

fkrueger · 02-04-2015, 03:19 AM

Wouldn't off-by-1 errors more result in 50/50 values than 20%? I'd be happy to have a closer look at whatever problems you are running into, just drop me a line.

dpryan · 02-04-2015, 04:42 AM

Given the non-random distribution of bases in the genome, probably not.

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02-03-2015, 01:31 PM	#1
bedward1 Junior Member Location: San Diego Join Date: Aug 2013 Posts: 2	Non-C methylation in Bismark .cov files Hi I am analyzing RRBS using TrimGalore/Bismark and the methylation extractor, and am noticing quite a few non-C (T, A, and G) methylation signals (maybe 20% compared to C, by eye). Is this a normal technical artifact that has been observed? Thanks

02-03-2015, 02:17 PM	#2
dpryan Devon Ryan Location: Freiburg, Germany Join Date: Jul 2011 Posts: 3,480	In the case of G, that is a C, just on the other strand. In the case of T or A, that's either an off by 1 error or the sequence you're looking at is different from the sequence against which the aligning was done.

02-03-2015, 02:37 PM	#3
bedward1 Junior Member Location: San Diego Join Date: Aug 2013 Posts: 2	Thanks for the replay Devon. Is the 'off by 1 error' common? What would cause this?

02-04-2015, 12:56 AM	#4
dpryan Devon Ryan Location: Freiburg, Germany Join Date: Jul 2011 Posts: 3,480	Off by 1 errors are quite common (common enough that it has its own wikipedia article). The likely cause is the file using 1-based coordinates while whatever you're using to visualize things assumes it's using 0-based coordinates. I remember this being an issue with some of bismark's output at one point, though I don't know what the status of that is. If you look at those and there's an appropriate C (or G, depending on how things were done) just to the right, then that's what's going on.

02-04-2015, 03:19 AM	#5
fkrueger Senior Member Location: Cambridge, UK Join Date: Sep 2009 Posts: 625	Wouldn't off-by-1 errors more result in 50/50 values than 20%? I'd be happy to have a closer look at whatever problems you are running into, just drop me a line.

02-04-2015, 04:42 AM	#6
dpryan Devon Ryan Location: Freiburg, Germany Join Date: Jul 2011 Posts: 3,480	Given the non-random distribution of bases in the genome, probably not.