Keeping reads at CG SNPs using Bismark

[felixkrueger]

Keeping reads at CG SNPs using Bismark

[FelixKrueger]

The reason for entirely removing C/T SNP positions in a bisulfite experiment is obviously that you cannot trust the result at all.
Let's assume genome 1 would normally be a C, but due to its methylation state you might see it as either a C (methylated) or T (unmethylated) after bisulfite treatment. Genome 2 would always have a T at that position in any case.
While a C in a read would have to be a (methylated) genome 1 read, there is no way to discriminate between a genome 2 or unmethylated genome 1 read, unless the read contains additional informative SNPs at the same time. In other words, the methylation state might not be a real methylation state but could be confounding signal coming simply from the underlying genomic sequence (that just looks like non-methylation).

If you still wanted to include reads covering those C/T positions (I am not sure you should be wanting that...) and I am not mistaken, you should be able to pre-filter your list of SNPs to exclude C/T SNPs, and then run the N-masking and indexing again. This way you wouldn't N-mask and thus include all C/T positions, and therefore also get 'methylation calls'. You should not observe any mapping bias in this case because Cs are in-silico C-to-T converted to Ts for the mapping procedure anyway. Since those positions were not N-masked they would also not participate in allele (or haplotype) assignment, but please be aware that the 'methylation' you observe at these positions might be confounded by genetics, i.e. the T SNP.

Do I make sense?

[jrmerritt6]

Yes, I do see your point about the confound of looking at methylation at C/T SNPs. The good thing is that I'm studying F1 hybrids, and we have a very good list of non-CG fixed SNPs to assign reads to haplotype, so dissecting out methylation vs. SNP is possible with my particular system. The approach you suggested, excluding C/T SNPs, worked for me. Thank you!