The effects of incomplete bisulfite conversion upon mapping efficiency This question has also been posted on Biostars

I have sequenced numerous multiplexed pools of BS amplicon-seq libraries derived from human samples on a MiSeq over the past few weeks. I have been utilising trim-galore and Bismark for alignment and am finding the mapping efficiency to be really low for two pools (55% &amp; 30% respectively) both of which had a cytosine per base sequence of around 10-20% throughout the entire read in their fastqc files.

The high C content visible in the fastqc files makes me think the poor mapping efficiency is due to poor bisulfite conversion, as this would be expected to be close to zero is bisulfite conversion had actually took place.

I am new to the field so any help would be greatly appreciated.
Bisulfite conversion efficiency has no effect on the mapping rate in bismark and similar tools. The reason is that the reads are fully bisulfite converted in silico before alignment to minimize mapping bias.

I would suggest that you play around with the settings handed to bowtie2, such as using local alignment and modifying the --score-min option to allow more mismatches. Alternatively, you might try a different aligner like bwameth, which will always do local alignment.