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When using bismark, if we set -I(min insert size) as 20, will sam file exclude overlapped reads? In my case 70% of pe reads in sam file are overlapped, though I set -I 20.
Is this normal?
Is this normal?
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The latest version of bismark uses bowtie2, which has options for dealing with overlapping reads and by default can map mates that overlap. I am not sure if bismark uses the default for this or not, or whether or not this is one of the options available to adjust in Bismark. -
A following question is If I use bismark methylation call extractor for the overlapped pair end reads(count the overlapped CpG twice), will the same reads actually report two CpG calling in the same site?(I want to take advantage of this to check the accuracy of methylation calling)Originally posted by chadn737 View PostComment
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See the --no_overlap option. I recall that, by default, it will report both. With the --no_overlap option, I recall it reporting the call from the first read in a pair. I wrote my own version that checks to see if the calls in overlapping reads are equivalent and found that they differ in ~0.01% of cases in a dataset that I checked. BTW, the accuracy of methylation calls in this case will be purely a function of the sequencing accuracy.Originally posted by serenaliao View PostComment
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Love this - good data definitely starts from good input, and poor input can only give relatively poor data. I particularly like the mention of Nanodrop/absorbance based methods for quantification. It's such a toss up if you'll get an accurate reading or what amounts to a randomly generated number, and a lot of library/sequencing related issues can be traced back to poor quant.07-01-2026, 11:43 AM
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I’m not a sequencing expert. I’m a purification scientist who uses NGS to evaluate workflows my group develops. With this perspective, we think about the sample first and the NGS workflow second. The sequencer is an exceptionally honest reporter, but it can only report on what you give it, so whether you get clean, interpretable data from an NGS workflow is largely determined before you begin.
Here are nine questions we think about, in roughly the order they matter, before...
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