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In genotyping from NGS data, what is the best way to account for bias (sequencing, mapping, etc) when identifying a locus as homozygous vs heterozygous?
Thanks for any help!
Thanks for any help!
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Not quite sure what you mean by locus. Do you mean specific SNP calls? If so there are many programs out there to call SNPs and will attempt to correctly determine if they are homozygous or heterozygous.
As for bias, it's good to make sure to remove any PCR duplicates. -
Yes, exactly. I was wondering what those programs do to determine if a locus is homozygous or heterozygous.Comment
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SAMtools mpileup is pretty good. It's the only common one I'm really familiar with so I don't want to try to compare it to others in any way, but I know it's used fairly commonly.Comment
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The programs that I know (samtools mpileup + bcf tools and GATK unified genotyper), basically count the number of reads in your experiment supporting one or another allele at a SNP position, and then they estimate whether the ratio of 1st allele to 2nd allele is 50 : 50 or 100 : 0 (usually one of the alleles corresponds to the reference sequence, and the other to the alternative sequence).Originally posted by andy11 View Post
The field "AF" (allele frequency) in the vcf-format file gives you this information.
Hope that helps.
Cheers,
SophiaComment
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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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