methylation values. To exclude unreliable data, only methylation signals identified by a number of reads $10 were used for further analyses. After this filtering, we computed, for each CpG the mean value between two replicas, obtaining methylation values of genomic CpGs per cell line in the range (5–8) 105 . We next selected methylation values of CpG dinucleotides in CGIs, filtering them by the CpG Islands track of UCSC-GB. The final CGI methylation value was obtained by calculating the mean methylation of all CpGs contained in the CGI. Integrated haplotype score (iHS). The normalized iHS scores were obtained from UCSC Genome Browser ‘‘HGDP iHS’’ track. The per-continent integrated haplotype score (iHS) [23] is a measure of recent positive selection. The scores present in the UCSC Genome Browser were calculated using SNPs genotyped in 53 populations worldwide by the Human Genome Diversity Project in collaboration with the Centre d’Etude du Polymor- phisme Humain (HGDP-CEPH). Samples from 1,043 individuals from different geographical regions were genotyped for 657,000 SNPs at Stanford. The 53 populations were divided into seven continental groups: Africa (Bantu populations only), Middle East, Europe, South Asia, East Asia, Oceania and the Americas. iHS was calculated for each population group and then normalizing the resulting unstandardized iHS scores in derived allele frequency bins as described in [23]. Per-SNP iHS scores were smoothed in windows of 31 SNPs, centered on each SNP. The final score is 2log10 of the proportion of smoothed scores higher than each SNP’s smoothed score. We converted genome coordinates from assembly NCBI36/ hg18 to assembly GRCh37/hg19 by using Batch Coordinate Conversion (liftOver) utility (UCSC Genome Browser). We scanned normalized iHS scores across the whole genome and selected the genomic intervals where iHS values $2. Once detected such compact regions, we extended their boundaries to the nearest loci where iHS was exactly vanishing. Selective Sweep Scan: 5% Smallest S scores. Green et al. [25] identified polymorphic sites among five modern human genomes and determined ancestral or derived state of each single SNP. The human allele states were used to estimate an expected number of derived alleles in Neanderthal in the 100,000-base window around each SNP. The measure called S score compare the observed number of Neanderthal alleles in each window to the expected number. An S score significantly less than zero indicates an increase of human-derived alleles not found in Neanderthal, suggesting positive selection in the human lineage since divergence from Neanderthals. Regions with S scores in the lowest 5% (strongest negative scores, ‘‘5% Lowest S’’ track of UCSC Genome Browser) were used in our analyses. Conserved Elements. Conserved elements were down- loaded from the UCSC GB Conservation (cons46way) Track. In this track conserved elements were predicted using the methods phastCons and phyloP. Both phastCons and phyloP are phylogenetic methods that rely on a tree model containing the tree topology, branch lengths representing evolutionary distance at neutrally evolving sites, the background distribution of nucleotides, and a substitution rate matrix. Pairwise alignments with the human genome were generated for each species using blastz from repeat-masked genomic sequence. The conserved elements were predicted using 10 primate species. Primate species used are :Homo Sapiens (reference species), Pan troglodytes, Gorilla gorilla gorilla, Pongo pygmaeus abelii, Macaca mulatta, Papio hamadryas, Callithrix jacchus, Tarsier syrichta, Microcebus murinus, Otolemur garnettii. CpG Islands Methylation and Selective Pressure Statistical analysis In order to test the hypothesis that two distributions have the same means we use a ‘‘bootstrapping approach’’. In particular we take the mean of the smaller sample, hereafter denoted by m, and compare this value with the probability distribution of mean, p(m), obtained from a large number (104) of randomly sampled cohorts of the same size taken from the larger sample. Type I error to reject the hypothesis even if it is true, denoted as ‘‘Bootstrap p-value’’ of the test, by definition is the sum of p(m) for m$m. Since we have 104 cohorts of the larger sample the precision of our ‘‘Bootstrap p-value’’ is 1024, which is however small enough since we have fixed the threshold of statistical significance at 1023. All statistical analyses were carried out with R ver. 2.10.1 [42] Supporting Information Figure S1 Histogram of CGIs mean methylation values (y-axis) and their Standard Errors for each cell line group: Cancer Transformed (CT), EBV transformed (EBV), and Normal Untransformed (NU). (TIF) Figure S2 Histogram of the percentages of methylation of HIR+CE CGIs (open bars) compared to CE CGIs (closed bars) for each cell line. Error bars represent standard errors. (TIF) Figure S3 Histogram of the percentages of methylation of 5SLR+CE CGIs (open bars) compared to CE CGIs (closed bars) for each cell line. Error bars represent standard errors. (TIF) Table S1 Complete list of the cell used in this study, with their characteristics. (DOC) Table S2 Lists, for each cell type, the number of CpG analyzed, the number of CpGs inside CGIs, the number of CGIs for which we were able to estimate methylation, the number of CpG analyzed per CGI and the mean value of CGI methylation. (DOC) Table S3 Lists, for each HIR identified, the chromosome, the start position, the end position, the total length and the human population in which it has been detected. Genomic coordinates refer to assembly GRCh37/hg19. (DOC) Table S4 Lists, for each cell type, the mean methylation of CGIs inside HIRs (with its standard error), the mean methylation of CGIs localized outside these regions (with its standard error), the number of CGIs inside HIRs, the number of CGIs localized outside HIRs and the Bootstrap p-values. (DOC) Table S5 Lists, for each 5SLR identified, the chromosome, the start position, the end position and the total length. Genomic coordinates refer to assembly GRCh37/hg19. (DOC) Table S6 Lists, for each cell type, the mean methylation of CGIs inside 5SLRs (with its standard error), the mean methylation of CGIs localized outside these regions (with its standard error), the number of CGIs inside 5SLRs s, the number of CGIs localized outside 5SLRs and the Bootstrap p-values. (DOC) Table S7 Lists, for each cell type, the mean methylation of CGIs containing CEs (with its standard error), the mean methylation of PLoS ONE | www.plosone.org 8 August 2011 | Volume 6 | Issue 8 | e23156