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Old 02-03-2015, 01:31 PM   #1
bedward1
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Default 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
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Old 02-03-2015, 02:17 PM   #2
dpryan
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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.
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Old 02-03-2015, 02:37 PM   #3
bedward1
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Thanks for the replay Devon.
Is the 'off by 1 error' common? What would cause this?
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Old 02-04-2015, 12:56 AM   #4
dpryan
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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.
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Old 02-04-2015, 03:19 AM   #5
fkrueger
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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.
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Old 02-04-2015, 04:42 AM   #6
dpryan
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Given the non-random distribution of bases in the genome, probably not.
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bismark, bs-seq, cytosine, methylation

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