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Pipeline publication

[…] land Biolabs, USA). For the MspI digested segments, the sticky ends were filled with CG nucleotides and 3′ A overhangs were added. Secondly, methylated Illumina sequencing adapters with 3′ T overhangs were ligated to the digested segments, and the products obtained were purified. Then 110–220 bp fragments were selected and converted by bisulfite using an EZ DNA Methylation Gold Kit (Zymo Research, USA). Lastly, libraries of 110–220 bp fragments were PCR amplified and each library was sequenced using one lane of an Illumina HiSeq 2500 and 100 bp paired-end reads. The first two nucleotides were trimmed from all the second read sequences to blunt-end the MspI site. All reads were trimmed using Trim Galore (v0.4.0) software (Babraham Bioinformatics, http://www.bioinformatics.babraham.ac.uk/projects/trim_galore/) and a Phred quality score of 20 as the minimum. The adaptor pollution reads and multiple N reads (where N >10% of one read) were removed to generate the clean reads. The quality control checks were performed by FastQC (v0.11.3) software (Babraham Bioinformatics). The clean reads were mapped to the porcine reference genome (Sscrofa 10.2, downloaded from Ensembl, http://www.ensembl.org/Sus_scrofa/Info/Index), and then call the DNA methylation by Bismark (v0.14.5) with default parameters. For the overlapped reads, only the methylation calls of read 1 were used for in the process by Bismark with the option “—no_overlap”, in order to avoid scoring the overlapping methylation calls twice. The bisulfite conversion rates were calculated as the number of covered CpHs, which were unconverted, was divided by the total number of covered CpHs. The bisulfite conversion efficiencies of these nine libraries were 99.23%, 99.15%, 99.17%, 99.47%, 97.57%, 99.41%, 99.47%, 99.45%, 99.40% for hypothalamus 1, hypothalamus 2, hypothalamus 3, pituitary 1, pituitary 2, pituitary 3, ovary 1, ovary 2, ovary 3. Uniquely mapped reads were retained for further a […]

Pipeline specifications

Software tools Trim Galore!, FastQC, Bismark