Questions about Bismark and bisulfite-seq

[sero]

Hello everyone,

I am new to bis-seq data analysis and really feel confused about the 'directional' mapping.

In my paired-end sequencing data, I noticed there were very few 'C's in the first read file while very few 'G's in the second. (about 2%)

Is this the 'directional libraries'? When Bismark maps the reads, will it (or Bowtie) first turn the reads in the second read file into reverse complements?

I think in my data, there are only 'C' to 'T' in the first read file and 'G' to 'A' in the second, but I am not sure how to use this information in Bismark.

Thank you.

[fkrueger]

Hi Sero,

By the sounds of it you do indeed have directional libraries. Bismark will C-to-T convert read 1 and G-to-A convert read 2 before starting the alignments. So you don't need to worry about anything, it should all be taken care of.

If you are just beginning to understand BS-Seq may I suggest you read the following protocol.

[sero]

Thank you for your reply and the link!

I just felt unsure whether I was doing the right thing ...

Because Bismark says the directional mapping will only try the OT and OB strands ... so I worried whether I needed to reverse complement the Read 2 ...

Thanks a lot!

[fkrueger]

For paired-end alignments this only means that read 1 of the pair will only align to the OT or OB strands. By definition, read 2 has to be the reverse complement or read 1, and if you would align read 2 in single-end mode they would indeed align to the CTOT and CTOB strands only. But again, don't worry, Bismark will handle it just fine.

[sero]

Thank you!