FelixKrueger
commented
4 years ago Hi @soleilll
I assume the oligo sequence you have is an oligo you spiked into your experiment before doing the bisulfite conversion, e.g. the 5mC/5hmC oligos CEGX used to distribute?
Generally, if you use Bismark in end-to-end mode, which is the default, then you can only align to a 60bp oligo sequence if the read length in your experiment was 58bp or shorter. Longer reads can by definition not map fully. You have a few options here:
-
you could and (probably should) pad the oligo sequence with
NNon either side, so that a full match to the sequence doesn't get removed because the sequence is too short to extract the cytosine context of the first two and last two base pairs -
in case your read-length exceeds 60bp, you could hard-trim the sequences to 60bp (e.g. with Trim Galore, option
--hardtrim5 60) -
instead of hard-trimming you could also try to align the reads in
--localmode to allow softclipping. Like hard-trimming, this will most ikely also require the padding withNson either side
You don't have to repeat the alignments to the 'genome+oligo' reference, but you can just index the oligo sequence itself. Alignments to just this sequence should be lightning fast.
Let me know how you get on.
soleilll
Hello！ I have some problem when i calculate the bs rate.I combined the genome.fa and oligo.fa.Then make index and bismark mapped.I try to extract the reads mapped to the oligo.fa,but there were no reads succeeded.So i can't get the bs rate(C/C+T). Is the oligo 60bp too short to map? Can you help me with the problem?Thank you very much!