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Pipeline publication

[…] Bisulfite conversion of 500 ng of genomic DNA was performed using EZ DNA methylation kit (Zymo Research) following manufacturer’s instructions. 200 ng of bisulfite converted DNA were used for hybridization on the HumanMethylation450 BeadChip (Illumina). Briefly, samples were whole genome amplified followed by an enzymatic end-point fragmentation, precipitation and resuspension. The resuspended samples were hybridized onto the beadchip for 16 h at 48°C and washed. A single nucleotide extension with labeled dideoxy-nucleotides was performed and repeated rounds of staining were applied with a combination of labeled antibodies differentiating between biotin and DNP., Data were normalized using GenomeStudio V2010.3 (Illumina). The DNA methylation level is displayed as β-values ranging from 0 to 1. Methylation level (β-value) for each of the 485,577 CpG sites were calculated as the ratio of methylated signal divided by the sum of methylated and unmethylated signals plus 100. After normalization step, probes related to X and Y chromosomes were removed as well as those containing a SNPs with a frequency > 1% (Caucasian population; 1000 Genome project) in the probe sequence or interrogated CpG site. Samples were clustered by hierarchical clustering using Manhattan distances., Transcription factor enrichment analysis was performed using PSCAN. Transcription factor binding annotations were extracted from TRANSFAC. The gene promoter region was defined as -1,000 and +0 bp to the transcription start site. Z-test p-values were corrected for multiple hypotheses testing using the Bonferroni method., Gene Ontology (GO) enrichment analysis was performed using the Database for Annotation, Visualization and Integrated Discovery (DAVID; v6.7).,, The research leading to these results has received funding from the European Research Council (ERC) grant EPINORC under the agreement n° 268626, the MICINN Project – SAF2011-22803, the Cellex Foundation, the European Community's Seventh Framework Programme (FP7/2007-2013) by the grant HEALTH-F5-2011-282510 - BLUEPRINT and the Health and Science Departments of the Generalitat de Catalunya. M.E. is an ICREA Research Professor., Previously published online:, No potential conflicts of interest were disclosed.] […]

Pipeline specifications

Software tools GenomeStudio, Pscan, DAVID