Journal: Epigenetics

Article Title: Semiconductor-based sequencing of genome-wide DNA methylation states

doi: 10.1080/15592294.2014.1003747

Figure Lengend Snippet: Workflow of Ion Torrent-compatible MeDIP-Seq protocol. Schematic representation of the MeDIP-Seq protocol developed for the Ion Torrent platform to profile DNA methylation genome wide. (a) Genomic DNA (1 μg) is isolated from a sample of interest and sonicated for 300 s on a Covaris Focused-ultrasonicator (Peak intensity power: 50, Duty: 20%, Cycles: 200, Temp: 20°C) to a mean fragment size of 300 bp. (b) Fragmented DNA is nick repaired and ligated with Ion Torrent sequencing adapters. (c) Ligated DNA is enriched for either 5mC (data shown) or 5hmC variants of methylation. Efficiency of MeDIP reaction is determined by using spiked in methylated and unmethylated DNA and compared to a 10% input DNA control. (d) Fragments of immunoprecipitated DNA are separated on a 2% agarose gel and size selected from 200–400 bp. (e) Size-selected fragmented DNA library is amplified for 18 cycles. (f) Amplified MeDIP library is cleaned up and size selected again on a 2% E-gel. (g) MeDIP library (1 μl) is used on a bioanalyzer instrument to assess quality and determine concentration. Figure displays bioanalyzer traces for WT (top) and DKO (bottom) 5mC MeDIP libraries. (h) High quality MeDIP library is templated on ISP beads appropriate to the capacity of sequencing (Life Technologies). (i) Templated MeDIP library is loaded onto an Ion Torrent semi-conductor sequencing chip and sequenced for 500 flows (Life Technologies). An example of the Ion Torrent 318 chip is shown in the figure using scanning electron microscopy to visualize the wells of a semi-conductor sequencing chip. (j) Sequencing data is processed on the Ion Torrent software (Life Technologies). An example of a loaded P1 chip after an Ion Proton sequencing run is shown. WT, HCT116-WT; DKO, HCT116-DKO.

Article Snippet: After the adapter ligation reaction, we immunoprecipitated methylated DNA using a previously validated magnetic capture-based MeDIP kit (Diagenode; cat. no. mc-magme-A10) that included an antibody with high affinity and specificity for 5-methylcytosine.

Techniques: Methylated DNA Immunoprecipitation, DNA Methylation Assay, Genome Wide, Isolation, Sonication, Sequencing, Methylation, Immunoprecipitation, Agarose Gel Electrophoresis, Amplification, Concentration Assay, Electron Microscopy, Software

Journal: Epigenetics

Article Title: Semiconductor-based sequencing of genome-wide DNA methylation states

doi: 10.1080/15592294.2014.1003747

Figure Lengend Snippet: Scalability of Ion Torrent-compatible MeDIP-Seq protocol. (a) Global 5-methylcytosine (5mC) content measured as a percentage of the genome in DNA from WT and DKO cells determined by an ELISA assay. (b) Number of peaks identified by MACS v2.1.0 software from MeDIP-Seq data in WT and DKO cells. (c) Sequence coverage of genome-wide CpGs for WT and DKO cells on the Ion Torrent Proton sequencer calculated using MEDIPS v1.14.0 software. Pie charts illustrate the fraction of CpGs covered by the indicated reads according to their fold-coverage in relation to the total genomic CpG content from MeDIP-Seq libraries of the indicated sample sequenced on the Proton with the total number of non-redundant mapped reads in parentheses. (d) Sequence coverage of genome-wide CpGs for WT and DKO cells in MeDIP-Seq on the Ion Torrent PGM sequencer. Data displayed as in c . (e) Scaled chromosomal view of the 5mC MeDIP-Seq enrichment of methylation over the MX1 gene in WT and DKO cells sequenced on the 2 different Ion Torrent sequencers as indicated. MeDIP-Seq data are displayed as RPM (Reads Per Million mapped reads) below the RefSeq annotation of the gene (blue lines). Methylation enrichment is displayed in gray, with red vertical lines corresponding to areas containing CpGs. Chromosome position and gene orientation are displayed.

Article Snippet: After the adapter ligation reaction, we immunoprecipitated methylated DNA using a previously validated magnetic capture-based MeDIP kit (Diagenode; cat. no. mc-magme-A10) that included an antibody with high affinity and specificity for 5-methylcytosine.

Techniques: Methylated DNA Immunoprecipitation, Enzyme-linked Immunosorbent Assay, Software, Sequencing, Genome Wide, Methylation

Journal: Epigenetics

Article Title: Semiconductor-based sequencing of genome-wide DNA methylation states

doi: 10.1080/15592294.2014.1003747

Figure Lengend Snippet: Ion Torrent-compatible MeDIP-Seq detects expected differences in DNA methylation between DNMT -proficient and - deficient cells. Distribution of 5mC methylation over the indicated bp window of all RefSeq annotated TSS ( a ), gene bodies ( b ), CpG islands ( c ), and exons ( d ). Methylation of WT (green line) and DKO (orange line) HCT116 cells are displayed as mean RPM values with s.e.m. indicated as a semi-transparent shade around the mean curve. Yellow shaded areas highlight regions of significant difference between WT and DKO methylation calculated using a one-sided KS test ( P -values shown). In c and d , a KS test was performed using data over the non-shaded areas as well ( P -values shown).

Article Snippet: After the adapter ligation reaction, we immunoprecipitated methylated DNA using a previously validated magnetic capture-based MeDIP kit (Diagenode; cat. no. mc-magme-A10) that included an antibody with high affinity and specificity for 5-methylcytosine.

Techniques: Methylated DNA Immunoprecipitation, DNA Methylation Assay, Methylation

Journal: Epigenetics

Article Title: Semiconductor-based sequencing of genome-wide DNA methylation states

doi: 10.1080/15592294.2014.1003747

Figure Lengend Snippet: Validation of Ion Torrent-compatible MeDIP-Seq data by integration with the single-nucleotide resolution 450K array. ( a ) Histogram of the frequency and distribution of CpG cytosine methylation levels of probes on the 450K array in HCT116-WT (green) and -DKO (orange) cells stratified into categories based on their percentage of methylation. ( b ) Percentage of CpGs on the 450K array (black) overlapping with those underlying MeDIP-Seq peaks (white) of WT cells. ( c ) Distribution of methylation levels measured in WT cells of all probes on the 450K array that integrate with MeDIP-Seq peaks. ( d ) Distribution of methylation levels measured in DKO cells of all probes on the 450K array that integrate with MeDIP-Seq peaks in WT cells. ( e ) Scaled chromosomal view of the MeDIP-Seq enrichment of methylation over the ICR of the SNRPN gene. MeDIP-Seq from WT and DKO cells are displayed as indicated below the RefSeq annotation of SNRPN/SNURF (blue lines) and a CpG island (green). CpG methylation data of each 450K array probe over this region from WT and DKO cells are shown to scale as colored bars representing the degree of methylation (blue, low-; violet, intermediate-; and orange, high-levels of methylation). Percent methylation (calculated from β-values) of these CpGs is shown under each bar. The expanded region below these values shows a scaled depiction of CpG methylation of alleles sequenced after bisulfite-PCR in WT and DKO cells as indicated. Each circle represents a CpG cytosine in the amplicon sequenced, the color of which depicts its methylation state (white, unmethylated; black, methylated). *, the CpG on the 450K array also covered by the bisulfite-PCR amplicon. ( f ) The average expression of SNURF is significantly elevated in DKO compared to WT cells. Average expression values and s.e.m. from triplicate qPCR are shown. ( g ) DNA methylation over the SNURF promoter region is significantly higher in WT compared to DKO cells. Using bisulfite-PCR sequencing data described in e , percent methylation was calculated from the sum of methylated CpGs over the total CpGs evaluated. Average methylation values and s.e.m from individual clones are shown. P -values shown in f and g were calculated using 2-sided Student's t -test.

Article Snippet: After the adapter ligation reaction, we immunoprecipitated methylated DNA using a previously validated magnetic capture-based MeDIP kit (Diagenode; cat. no. mc-magme-A10) that included an antibody with high affinity and specificity for 5-methylcytosine.

Techniques: Methylated DNA Immunoprecipitation, Methylation, CpG Methylation Assay, Amplification, Expressing, DNA Methylation Assay, Sequencing, Clone Assay

Journal: Epigenetics

Article Title: Semiconductor-based sequencing of genome-wide DNA methylation states

doi: 10.1080/15592294.2014.1003747

Figure Lengend Snippet: Ion Torrent-compatible MeDIP-Seq confirms a role for DNA methylation in alternative splicing. (a) Distribution of 5mC methylation over alternatively spliced exons (ASE) flanked by constitutively spliced exons (5’ or 3’ exons) in WT and DKO cells. Mean RPM values are displayed over ± 500 bp windows relative to the splice acceptor and donor sites at each of the exons as indicated, with s.e.m. depicted by shaded areas for WT (green) and DKO (orange) profiles. A schematic representation of cassette exons is shown, with the number of exons aberrantly included (blue) and excluded (gray) in WT vs. DKO cells indicated. P- values shown were calculated using one-sided KS test. (b) The ASE of the FAM204A gene (exon 2) is aberrantly excluded in DKO compared to WT cells. Schematic shows the 2 known isoforms for the FAM204A gene (blue bars depict the first 3 exons of the gene in the orientation indicated), under which a Sashimi plot generated by MISO analysis of RNA-Seq data measured as RPKM in WT and DKO cells illustrates the number of exon-exon junction reads as indicated to infer isoform expression. The left graphs show the MISO calculated distribution of a percent exon inclusion score (Psi-value; 95% confidence intervals in brackets) for the FAM204A ASE from WT (top) and DKO (bottom) RNA-Seq data. (c) Demethylation of the FAM204A ASE correlates with its aberrant exclusion. Scaled chromosomal view at the FAM204A gene region (ASE highlighted in yellow) of the indicated WT and DKO distribution of RNA-Seq (top pair) and DNA methylation (bottom pair) data displayed as RPKM. The first 3 exons of the gene are represented (blue bars) with a CpG island in the promoter region indicated (green bar). In the MeDIP-Seq data, red vertical lines correspond to areas containing CpGs.

Article Snippet: After the adapter ligation reaction, we immunoprecipitated methylated DNA using a previously validated magnetic capture-based MeDIP kit (Diagenode; cat. no. mc-magme-A10) that included an antibody with high affinity and specificity for 5-methylcytosine.

Techniques: Methylated DNA Immunoprecipitation, DNA Methylation Assay, Methylation, Generated, RNA Sequencing Assay, Expressing

Journal: Epigenetics

Article Title: Semiconductor-based sequencing of genome-wide DNA methylation states

doi: 10.1080/15592294.2014.1003747

Figure Lengend Snippet: Ion Torrent-compatible MeDIP-Seq accurately distinguishes between variant DNA modifications. (a) Global 5mC (blue bar) content in bulk DNA from a human brain tissue is significantly higher than that of 5hmC (red bar). Percent methylation of the indicated variant modification was measured using an ELISA assay. Mean values of triplicate experiments are shown, with the P -value calculated by 2-sided Student's t -test. (b) Number of peaks identified by MACS v2.1.0 software for 5mC and 5hmC variants of DNA methylation in brain. (c) Venn diagram illustrating the indicated total number of CpGs contained in the peaks identified for 5mC and 5hmC, showing the degree of overlap of CpGs (as a percentage of the total in 5mC and 5hmC) between the 2 libraries. Distribution of 5mC and 5hmC methylation in brain over indicated bp window of all RefSeq annotated TSS (d) , gene bodies (e) , CpG islands (f) , and exons (g) . For d-g , all mean values from brain 5mC (blue line) and 5hmC (red line) are shown as RPM with s.e.m. displayed as a semi-transparent shade around the mean curve. Yellow shaded areas highlight regions of significant difference between 5mC and 5hmC calculated using a one-sided KS test ( P -values shown).

Article Snippet: After the adapter ligation reaction, we immunoprecipitated methylated DNA using a previously validated magnetic capture-based MeDIP kit (Diagenode; cat. no. mc-magme-A10) that included an antibody with high affinity and specificity for 5-methylcytosine.

Techniques: Methylated DNA Immunoprecipitation, Variant Assay, Methylation, Modification, Enzyme-linked Immunosorbent Assay, Software, DNA Methylation Assay