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Freg 07-25-2018 03:43 AM

Newbie questions about Methyl-Seq (Bismark)
2 Attachment(s)

I'm quite new to epigenetics and DNA methylation data analysis, so many excuses in advance if my questions seem too naive or have already been answered elsewhere.

I was recently charged with analysis of Targeted bisulfite sequencing data of human patients.

Patients were sequenced on 3 different runs.
They used Illumina's TruSeq MethylCapture EPIC Library prep kit (107 Mb, 3,340,894 CpG sites) and the sequencing was performed on a NextSeq 500. The data is paired-end (fastq R1 + fastq R2).

After the initial QC (fastQC) and adapter trimming (Trim Galore!), I aligned my fastqs on a reference genome (UCSC hg19). I used Bismark tool (0.19.0) for Alignment, Deduplication and Methylation calling.
All patients were analysed with the same workflow.

What concerns me is a big difference of bismark reports between the runs (PDFs attached), especially the deduplication rate (75% for run1!) and CHG/CHH methylation (nothing for run2).

I don't really know what to make of this, and how much it will affect the downstream analysis. I'm very new to Methyl-Seq but the ultimate goal is to perform a case-control study to identify Differentially Methylated CpG Sites (I was thinking of using methylkit).

Am I doing something wrong or is it the problem with the initial data?

Any insight will be appreciated.

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