Bisulfite-seq paired end

[yaping liu]

In Bisulfite-seq, the second end read is from the complementary of original +/- strand, But it is still the directional protocol. Here is a graph I found from a book chapter to describe paired-end bisulfite-sequecing (Johnson, et al. 2012. Current protocol in molecular biology)

e.g. if in a particular position, C are in the + strand, 

in the 1st end reads, all the unmethylated Cs are converted to Ts in + strand, while in - strand, all Gs are still kept to be G since the directional protocol only convert one strand.

But in the 2nd end reads, in - strand, all the Gs are converted to As (represent the complementary + strand's unmethylated Cs to Ts), while + strand, all Cs are still kept to be C.

1). You can use the latest IGV browser, which contains a bisulfite mode (developed by Dr.Ben Berman in our group),  to view the raw methylation status inside BAM file.

2).You can also extract 1st end reads only or 2nd reads only from the original BAM file by using command: "samtools view -bh sample.bam -f 64 > sample.1st.bam" and "samtools view -bh sample.bam -f 128> sample.2nd.bam"

Then you will see that for 1st end, C/(C+T) represent the methylation value, the other strand's G not changed (if no SNP or sequencing error)

for 2nd end, G/(G+A) represent the methylation value, the other strand's C not changed.

3). BisSNP could automate detect the reads status(1st end or 2nd end, + strand or - strand) from the flag field inside BAM file, and then using our probability model to tell you the genotype status and methylation status.