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Old 03-02-2017, 04:24 PM   #4
nucacidhunter
Jafar Jabbari
 
Location: Melbourne

Join Date: Jan 2013
Posts: 1,226
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Generally there are three WGBS library prep methods:
1- Post-ligation bisulfite conversion: DNA fragmentation and standard library preparation with methylated adapters followed by bisulfite conversion and amplification
2- Post-bisulfite conversion library preparation by second strand synthesis of converted ssDNA followed by standard end repair, A tailing and adapter ligation and PCR amplification of double stranded DNA.
3- Post-bisulfite conversion library preparation by synthesise of second strand with random primers appended with one partial Illumina adapter sequence and tagging the 3’ end of new strand with Terminal Tagging Oligo appended with other partial Illumina adapter followed by PCR amplification.

I assume your library was prepared with method 1. Peak size of 200 on average would have insert size of 75 nt so I would expect that large number of reads have been trimmed at 5’ end.

It would be interesting to see the FastQC “per base sequence content” plot for reads and that should show similar portion of converted Cs. For an example see following plots for low diversity RRBS library that shows low %C in R1 and correspondingly low %G in R2. If your plots show similar C and G then issue could be analysis step.

RRBS.pdf
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