Computational protocol: Comparative Analysis of Genome Wide DNA Methylation Profiles for the Genic Male Sterile Cabbage Line 01-20S and Its Maintainer Line

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Protocol publication

[…] The raw reads produced from BS-Seq were filtered to generate high-quality clean reads. DNA methylation is strand specific, resulting in the noncomplementarity of the two DNA strands. To build pre-converted forms of the reference genome, cytosine residues were converted to thymidine residues in the sense strand, whereas guanine residues were converted to adenosine residues in the antisense strand. The reference genome of ‘02-12’ cabbage is available at the B. oleracea Genomics Project []. The high-quality clean reads were then aligned to the prepared reference genome using Bismark []. Unique matches were filtered for further methylation analyses.The individual methylated C sites were identified using an R package methylKit []. The DMRs between the two samples were screened according to the method of Li et al. []. To retrieve the DMRs, each certain (algorithm-specified) methylated genomic region needed to contain at least three C sites, with at least one differentially methylated C site (Q-value < 0.01). Those algorithm-specified regions with an absolute mean methylation difference greater than 10% between F and S were determined as DMRs. The two-sample t-test was performed to measure significant differences between F (01-20F) and S (01-20S) in terms of CG, CHG, CHH using SPSS 17.0 (IBM Corp., Armonk, NY, USA). […]

Pipeline specifications

Software tools Bismark, methylKit
Application BS-seq analysis
Organisms Oryza sativa, Triticum aestivum
Chemicals Cytosine