72 hours (37uC, 5% CO2). Colcemid was then added (final concentration 0.05 ug/ml) and cells were harvested after a 1 hour incubation. Cells were spun down by centrifugation, the media was discarded and the pellet was resuspended in 8 ml of hypotonic solution. After incubating for 20 minutes, the standard fixative solution (1 part Acetic Acid, 3 parts Methanol) was added and cells were centrifuged at 2500 rpm for 5 minutes. The pellet was washed with fixative solution and cells were kept at 4uC overnight. DNA from BACs was extracted using PureLink Miniprep kit (Invitrogen, Cat#K2100-10). FISH experiments were performed essentially as described by Lichter et al. [41]. BACs were labeled either with Cy3-dUTP or FITC-dUTP by standard nick- translation assay. Images were acquired using Nikon 80i microscope, equipped with CCD camera Cool Snap HQ2 (Photometrics) and software Nis Elements Br (NIKON). Elabora- tion of the images was done using Photoshop. Bisulfite allelic sequencing of Alu elements Primers for 14 Alus (Table S5) were designed using ‘‘MethPri- mer’’ (http://www.urogene.org/methprimer/) [42] making sure to target unique sequences flanking the Alu. Out of the 14 Alus, 8 were near the breakpoints (,150 bp); as our goal was to amplify Alus orthologous in human and gibbon, we had to take into account the synteny between human and gibbon and had to eliminate all the cases where the Alus were located across the breakpoint. The remaining Alus were located randomly in the gibbon genome but had to have a CpG content high enough to allow us to make a statistic. The genomic DNA from whole-blood from gibbon and human was bisulfite converted using EpiTect Bisulfite kit (Qiagen, Cat.# 59104PCR) and the amplification was performed using the FastStart Taq DNA Polymerase (Roche, Cat#12032929001). PCR products were purified and cloned using TA-cloning procedures (Qiagen PCR cloning Kit, Cat.# 231124). We sequenced 12 clones for each Alu in order to have fair representation of all the alleles. Supporting Information Figure S1 Random sampling simulations for human and gibbon segmental duplications. Random sampling simulations were carried on as described in Materials and Methods. Histograms were obtained for human SD (A) and the in silico set of gibbon SD (B). We also tested the overlap with a ‘‘stringent’’ sample (lighter color) where all the BP that in gibbon overlap with centromeres were removed. Even in this case it is evident that the overlap of the gibbon sample with both classes of SDs is significant. Found at: doi:10.1371/journal.pgen.1000538.s001 (1.42 MB TIF) Figure S2 dN/dS ratios for gibbon and macaque genes. The ratio of the average dN/dS compute for gibbons (vs. human) and macaques (vs. human) for all genes found within the fully sequenced BACs (Total), for genes found within the NISC database (NISC), for genes located within 50 kb (,50 kb) from the breakpoint found within the BAC sequences and genes located further than 50 kb (.50 kb) from the breakpoint found within the BAC sequences. p values were calculated using the nonparametric Mann-Whitney test. Found at: doi:10.1371/journal.pgen.1000538.s002 (8.62 MB TIF) Figure S3 Random sampling simulations for Alu and Line 1 elements. Random sampling simulations were carried on as described in Materials and Methods. The two charts show the histogram resulted from counting the overlap between random regions of the human genome and Alu (A) and Line (B). The Evolutionary Breakpoints in Gibbon random sampling was repeated 5,000 times in both cases. The corresponding value for the gibbon dataset is indicated by the blue arrow. Found at: doi:10.1371/journal.pgen.1000538.s003 (1.51 MB TIF) Table S1 Sequenced Gibbon BACs. The fully sequenced and assembled 23 gibbon BACs from the genomic BAC library CHORI-271 (http://bacpac.chori.org/library.php?id=228) are reported here with the corresponding accession numbers. Found at: doi:10.1371/journal.pgen.1000538.s004 (0.04 MB DOC) Table S2 Gibbon breakpoints mapping information and anno- tations. We report here the sequencing status and annotations for 80 BACs used in this study. All the BACs were End-sequenced and mapped on the human assembly (hg18). The breakpoint sequence was obtained for 46 BACs whereas for 7 clones we identify Trace Archives mate pairs whose mapping indicated the presence of the breakpoint but no breakpoint sequence was found. For 24 clones the breakpoints could not be narrowed down at a resolution higher than a BAC clone. For each breakpoint sequence repeat, segmental duplications and gene contents are annotated. Abbre- viations: gSD = gibbon segmental duplication, hSD = human segmental duplication. Found at: doi:10.1371/journal.pgen.1000538.s005 (0.11 MB PDF) Table S3 Estimates of dN/dS for each gene within the BAC sequences. The tables report the estimates of dN/dS for genes within the NISC BACs (left-most table) and for the fully sequenced gibbon BACs (center table). The small table on the right illustrates the averaged estimates of dN/dS for genes where human, gibbon and macaque sequences were available. (*) Hypothetical genes were removed to provide a more stringent analysis and genes found within BAC CH271-262e11 were omitted because they belong to a gene cluster family with high sequence identity. This sequence identity along with their position upstream and downstream of the BP leads to uncertainty in alignment data and gene coordinates. Found at: doi:10.1371/journal.pgen.1000538.s006 (0.04 MB XLS) Table S4 Estimates of dN/dS for genes located within specified distances from the breakpoint tested in the study. Estimates of dN/ dS for each gene found within 400–600 kb from the BP where human, gibbon and macaque sequences were available (left-most table). Estimates of dN/dS for each gene found within 0.9–1.1 Mb from the BP where human, gibbon, and macaque sequences were available (center table). The small table on the right side summarizes the average dN/dS values at various distances from the BP tested in the study. Distances were determined by calculating the minimum distance from either the start or end of the gene to the BP. Found at: doi:10.1371/journal.pgen.1000538.s007 (0.03 MB XLS) Table S5 Primers used for Bisulfite Allelic sequencing. The table lists the primers that have been used to amplify the 14 Alus and carry on allelic bisulfite sequencing. Found at: doi:10.1371/journal.pgen.1000538.s008 (0.03 MB XLS) Protocol S1 Breakpoints mapping strategy. Found at: doi:10.1371/journal.pgen.1000538.s009 (0.03 DOC) Protocol S2 dN/dS analysis. Found at: doi:10.1371/journal.pgen.1000538.s010 (0.03 DOC) MB MB PLoS Genetics | www.plosgenetics.org 9 June 2009 | Volume 5 | Issue 6 | e1000538