Computational protocol: Exploring genome wide bisulfite sequencing for DNA methylation analysis in livestock: a technical assessment

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Protocol publication

[…] Quality control of data was undertaken using FastQC software (Babraham Bioinformatics, UK). Quality and adapter trimming for all samples was carried out using Trim Galore software, which was run in -RRBS mode for the RRBS samples. A Phred score of 20 was used as the quality cut-off value as this is the community accepted value and relates to a 1/100 chance of the assigned nucleotide being in correct, this provided a useful balance between using only high quality DNA without discarding too much sequence. To analyze the relationship between sequencing depth and CpG coverage within promoters, genes and CpG islands, sequence data generated from both RRBS and WGBS strategies was sampled at random from the fastq file using a script developed in house. This sampling created smaller datasets originating from the same library and sequence file. Sequences were mapped using paired end mapping to sheep genome assembly OARv3 using Bismark software (Babraham Bioinformatics, UK) which utilizes the Bowtie short read aligner (). After considerable optimisation, a seed length of 50 bp was chosen and only one mismatch was tolerated. Only sequences in which both ends mapped uniquely with an appropriate insert size in the correct orientation were used for subsequent calculation of DNA methylation levels. Sequencing read counts and levels of methylation were calculated and visualized using Seqmonk software (Babraham Bioinformatics, UK). Analysis of the CpG site coverage across the whole genome, as well as within genes and promoter regions was performed using Seqmonk. The number of CpG sites within these regions that were represented by 1x and ≥10x coverage was identified. Promoter regions were defined as 2 kb upstream of the transcription start site. […]

Pipeline specifications

Software tools FastQC, Trim Galore!, Bismark, Bowtie, SeqMonk
Application BS-seq analysis
Organisms Homo sapiens, Mus musculus, Ovis aries