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Pipeline publication

[…] CA) according to manufacturer’s instructions. During the bisulfite conversion, 5 ng of unmethylated lambda DNA per microgram of DNA sample was added to assess the bisulfite conversion error rate. Ultra-high-throughput pair-end sequencing for all samples was carried out using the Illumina HiSeq-2000 (Illumina) according to the manufacturer’s instructions. Raw sequencing data were processed by the Illumina 1.5 base-calling pipeline, resulting in 90 bp reads. The bisulfite-treated sequence data have been deposited to NCBI GEO under reference GSE60475 while the other sequence data have been deposited to NCBI SRA under reference PRJNA281096., Overall quality of the reads was evaluated using the FastQC software (Babraham Institute, Cambridge, UK). Reads containing >5% N bases were omitted. The remaining reads were dynamically trimmed to the longest stretch of bases which had a Phred score higher or equal to 30 (i.e., ∼99.9% base-call accuracy) using Trim Galore! 0.3.2 software (Babraham Institute) with standard settings. In addition to removal of poor-quality bases, adaptor sequences were trimmed from the reads. For bisulfite-treated samples, trimmed reads were subsequently transformed into fully bisulfite-converted forward (C -> T conversion) and reverse read (G -> A conversion of the forward strand) versions, before being mapped to similarly converted versions of the genome (also C -> T and G -> A converted) using Bowtie2 v.2.1.0 () while setting the scoring function as −score_min L, 0, −0.6. These four mapping processes were run in parallel and only the unique best mapping of each read was withheld. Reads from the nonbisulfite-treated samples did not need conversion and were mapped to the nonconverted version of the genome using the same scoring function. Nonuniquely mapping reads were discarded for further analysis. For bisulfite-treated samples, reads that might have occurred as PCR duplicates were removed using the Bismark deduplicate script (). The D. pulex filtered reference genome assembly with ∼5,000 scaffolds (Dappu1; ) was obtained from the DOE Joint Genome Institute (JGI) Genome Portal. The D. magna reference genome assembly v2.4, which was based on the exact same isolate, was used for mapping the D. magna data (, last accessed April 4, 2016). The above-described procedure was applied to each biological sample separately., The conversion error rate (supplementary table S3, Supplementary Material online) was defined as the percentage of reads mapping to the unmethylated lambda phage control DNA and which yielded a methylation call […]

Pipeline specifications

Software tools FastQC, Trim Galore!, Bowtie2, Bismark