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ADMIRE: analysis and visualization of differential methylation in genomic regions using the Infinium HumanMethylation450 Assay.

Preussner J, Bayer J, Kuenne C, Looso M - Epigenetics Chromatin (2015)

Bottom Line: Cytosine bases of the DNA are converted to 5-methylcytosine by the methyltransferase enzyme, acting as a reversible regulator of gene expression.Publication-ready graphics, genome browser tracks, and table outputs include summary data and statistics, permitting instant comparison of methylation profiles between sample groups and the exploration of methylation patterns along the whole genome.The web-based version of ADMIRE provides a simple interface to researchers with limited programming skills, whereas the offline version is suitable for integration into custom pipelines.

View Article: PubMed Central - PubMed

Affiliation: Bioinformatics Group, Max Planck Institute for Heart and Lung Research, Ludwigstrasse 43, 61231 Bad Nauheim, Germany.

ABSTRACT

Background: DNA methylation at cytosine nucleotides constitutes epigenetic gene regulation impacting cellular development and a wide range of diseases. Cytosine bases of the DNA are converted to 5-methylcytosine by the methyltransferase enzyme, acting as a reversible regulator of gene expression. Due to its outstanding importance in the epigenetic field, a number of lab techniques were developed to interrogate DNA methylation on a global range. Besides whole-genome bisulfite sequencing, the Infinium HumanMethylation450 Assay represents a versatile and cost-effective tool to investigate genome-wide changes of methylation patterns.

Results: Analysis of DNA Methylation In genomic REgions (ADMIRE) is an open source, semi-automatic analysis pipeline and visualization tool for Infinium HumanMethylation450 Assays with a special focus on ease of use. It features flexible experimental settings, quality control, automatic filtering, normalization, multiple testing, and differential analyses on arbitrary genomic regions. Publication-ready graphics, genome browser tracks, and table outputs include summary data and statistics, permitting instant comparison of methylation profiles between sample groups and the exploration of methylation patterns along the whole genome. ADMIREs statistical approach permits simultaneous large-scale analyses of hundreds of assays with little impact on algorithm runtimes.

Conclusions: The web-based version of ADMIRE provides a simple interface to researchers with limited programming skills, whereas the offline version is suitable for integration into custom pipelines. ADMIRE may be used via our freely available web service at https://bioinformatics.mpi-bn.mpg.de without any limitations concerning the size of a project. An offline version for local execution is available from our website or GitHub (https://github.molgen.mpg.de/loosolab/admire).

No MeSH data available.


IGV screenshots showing methylation across several genomic locations and boxplots for all significant sites. a–f Tracks shown are as follows: I. Methylation sites present on the HumanMethylation450 K Chip, II.Color-coded methylation values from control samples, III. Color-coded methylation values from AF samples, IV. Differentially methylated 10-kbp tiling regions called by ADMIRE, V. Differentially methylated 5-kbp tiling regions called by ADMIRE. The color bar encodes the m value, with blue indicating low methylation values and red indicating high methylation values. The absolute scale is created indvidually for each bar. Track IV and V are only used if the search with the corresponding input (5- or 10-kB tiling size) resulted in a significant region. a A 5-kbp region from chr17 called to be differentially methylated by RnBeads with an adjusted p value of 0.00008. b–f Top 5 differentially methylated regions from Admire with q values between 0.0004 and 0.003. gBoxplots for 20 significantly changed protein coding genes (higher in AF sample) identified by ADMIRE. Each box illustrates the distribution of absolute differences of the methylation values in the respective significantly changed region (see also Additional file 4). The cutoff at median methylation value of 5 % is shown as red dashedline
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Fig4: IGV screenshots showing methylation across several genomic locations and boxplots for all significant sites. a–f Tracks shown are as follows: I. Methylation sites present on the HumanMethylation450 K Chip, II.Color-coded methylation values from control samples, III. Color-coded methylation values from AF samples, IV. Differentially methylated 10-kbp tiling regions called by ADMIRE, V. Differentially methylated 5-kbp tiling regions called by ADMIRE. The color bar encodes the m value, with blue indicating low methylation values and red indicating high methylation values. The absolute scale is created indvidually for each bar. Track IV and V are only used if the search with the corresponding input (5- or 10-kB tiling size) resulted in a significant region. a A 5-kbp region from chr17 called to be differentially methylated by RnBeads with an adjusted p value of 0.00008. b–f Top 5 differentially methylated regions from Admire with q values between 0.0004 and 0.003. gBoxplots for 20 significantly changed protein coding genes (higher in AF sample) identified by ADMIRE. Each box illustrates the distribution of absolute differences of the methylation values in the respective significantly changed region (see also Additional file 4). The cutoff at median methylation value of 5 % is shown as red dashedline

Mentions: In order to compare ADMIRE to RnBeads, the current gold standard for HumanMethylation450 Assay analysis, we used an additional dataset of smaller size since the RnBeads [16] web interface is restricted to 24 samples. Our test dataset contains 11 samples from a study analyzing permanent atrial fibrillation (GEO GSE62727). This dataset was analyzed by RnBeads using default parameters (5-kB pre-calculated tiling regions) as well as the ADMIRE pipeline. To match the output from RnBeads and enable a direct comparison, we selected all 5-kB tiling regions as input for ADMIRE (see “Methods”). Our tool found twenty 5-kB regions corresponding to protein coding genes to be higher methylated in fibrillating atria (see Additional file 4) with a median methylation change of up to 12 %. Next, we carried out a second run with ADMIRE using 10-kB tiling regions as input to test for reproducibility of statistically significantly changed regions. Besides nine genes present in both result files, another 14 genes were identified from 10-kB regions only, with a median methylation change up to 45 % (see Additional file 5). RnBeads identified only one region to be higher methylated in fibrillating atria. This genomic location was not reported by ADMIRE. Some representative significant regions found by ADMIRE and the single region found by RnBeads are shown in Fig. 4a–f. We chose an indirect way to evaluate specificity and significance of regions reported by ADMIRE but not by RnBeads. To evaluate the latter, we visualized the homogeneity of the methylation change over all 5-kB tiling regions detected by ADMIRE in Fig. 4g. The boxplots represent all single methylation sites, combined in accordance to the tiling region. Their level and spread present a global overview in order to investigate the magnitude of the methylation changes. The user can interpret this information to select an appropriate threshold. To evaluate the specificity of our findings, we performed a functional analysis. This showed an enrichment of transcriptional regulation, driven by transcription factors such as HOX A, TBX5, and PITX2 (Additional file 6). This is remarkable, as initial GWAS studies identified a major risk region where the presence of a variant increased the risk of AF up to 65 %. Located proximally to the variant, PITX2 is a transcription factor import for cardiogenesis, especially for left–right signaling and L/R atrial identity. Knockout of PITX2 lead to a shortened atrial action potential in haploinsufficient mice and increased the susceptibility to AF [17]. Expression analysis identified the Sinoatrial node (SAN) specific genes Shox2, Tbx3, and Hcn4 as upregulated in PITX2 -mutant embryos [18]. A recent study additionally identified two microRNAs miR-17-92 and miR-106b-25 as direct targets of PITX2 that can repress Shox2 and Tbx3 upon transcription [19] and promote the expression of Cx43, a connexin protein forming gap junctions that allow the interchange of charged ions between adjacent cells [20]. Another GWAS study linked TBX5 to AF [21]. The homeobox transcription factor may play a role in heart development and specification of limb identity [22]. Interestingly, TBX5 was identified as interactor of Tbx3, a regulator of the SAN gene program [23]. Hoxa3 is another important gene in heart chamber morphogenesis, since Hoxa3-expressing progenitor cells in the second heart field give rise to the atria and parts of the outflow tract [24].Fig. 4


ADMIRE: analysis and visualization of differential methylation in genomic regions using the Infinium HumanMethylation450 Assay.

Preussner J, Bayer J, Kuenne C, Looso M - Epigenetics Chromatin (2015)

IGV screenshots showing methylation across several genomic locations and boxplots for all significant sites. a–f Tracks shown are as follows: I. Methylation sites present on the HumanMethylation450 K Chip, II.Color-coded methylation values from control samples, III. Color-coded methylation values from AF samples, IV. Differentially methylated 10-kbp tiling regions called by ADMIRE, V. Differentially methylated 5-kbp tiling regions called by ADMIRE. The color bar encodes the m value, with blue indicating low methylation values and red indicating high methylation values. The absolute scale is created indvidually for each bar. Track IV and V are only used if the search with the corresponding input (5- or 10-kB tiling size) resulted in a significant region. a A 5-kbp region from chr17 called to be differentially methylated by RnBeads with an adjusted p value of 0.00008. b–f Top 5 differentially methylated regions from Admire with q values between 0.0004 and 0.003. gBoxplots for 20 significantly changed protein coding genes (higher in AF sample) identified by ADMIRE. Each box illustrates the distribution of absolute differences of the methylation values in the respective significantly changed region (see also Additional file 4). The cutoff at median methylation value of 5 % is shown as red dashedline
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4666223&req=5

Fig4: IGV screenshots showing methylation across several genomic locations and boxplots for all significant sites. a–f Tracks shown are as follows: I. Methylation sites present on the HumanMethylation450 K Chip, II.Color-coded methylation values from control samples, III. Color-coded methylation values from AF samples, IV. Differentially methylated 10-kbp tiling regions called by ADMIRE, V. Differentially methylated 5-kbp tiling regions called by ADMIRE. The color bar encodes the m value, with blue indicating low methylation values and red indicating high methylation values. The absolute scale is created indvidually for each bar. Track IV and V are only used if the search with the corresponding input (5- or 10-kB tiling size) resulted in a significant region. a A 5-kbp region from chr17 called to be differentially methylated by RnBeads with an adjusted p value of 0.00008. b–f Top 5 differentially methylated regions from Admire with q values between 0.0004 and 0.003. gBoxplots for 20 significantly changed protein coding genes (higher in AF sample) identified by ADMIRE. Each box illustrates the distribution of absolute differences of the methylation values in the respective significantly changed region (see also Additional file 4). The cutoff at median methylation value of 5 % is shown as red dashedline
Mentions: In order to compare ADMIRE to RnBeads, the current gold standard for HumanMethylation450 Assay analysis, we used an additional dataset of smaller size since the RnBeads [16] web interface is restricted to 24 samples. Our test dataset contains 11 samples from a study analyzing permanent atrial fibrillation (GEO GSE62727). This dataset was analyzed by RnBeads using default parameters (5-kB pre-calculated tiling regions) as well as the ADMIRE pipeline. To match the output from RnBeads and enable a direct comparison, we selected all 5-kB tiling regions as input for ADMIRE (see “Methods”). Our tool found twenty 5-kB regions corresponding to protein coding genes to be higher methylated in fibrillating atria (see Additional file 4) with a median methylation change of up to 12 %. Next, we carried out a second run with ADMIRE using 10-kB tiling regions as input to test for reproducibility of statistically significantly changed regions. Besides nine genes present in both result files, another 14 genes were identified from 10-kB regions only, with a median methylation change up to 45 % (see Additional file 5). RnBeads identified only one region to be higher methylated in fibrillating atria. This genomic location was not reported by ADMIRE. Some representative significant regions found by ADMIRE and the single region found by RnBeads are shown in Fig. 4a–f. We chose an indirect way to evaluate specificity and significance of regions reported by ADMIRE but not by RnBeads. To evaluate the latter, we visualized the homogeneity of the methylation change over all 5-kB tiling regions detected by ADMIRE in Fig. 4g. The boxplots represent all single methylation sites, combined in accordance to the tiling region. Their level and spread present a global overview in order to investigate the magnitude of the methylation changes. The user can interpret this information to select an appropriate threshold. To evaluate the specificity of our findings, we performed a functional analysis. This showed an enrichment of transcriptional regulation, driven by transcription factors such as HOX A, TBX5, and PITX2 (Additional file 6). This is remarkable, as initial GWAS studies identified a major risk region where the presence of a variant increased the risk of AF up to 65 %. Located proximally to the variant, PITX2 is a transcription factor import for cardiogenesis, especially for left–right signaling and L/R atrial identity. Knockout of PITX2 lead to a shortened atrial action potential in haploinsufficient mice and increased the susceptibility to AF [17]. Expression analysis identified the Sinoatrial node (SAN) specific genes Shox2, Tbx3, and Hcn4 as upregulated in PITX2 -mutant embryos [18]. A recent study additionally identified two microRNAs miR-17-92 and miR-106b-25 as direct targets of PITX2 that can repress Shox2 and Tbx3 upon transcription [19] and promote the expression of Cx43, a connexin protein forming gap junctions that allow the interchange of charged ions between adjacent cells [20]. Another GWAS study linked TBX5 to AF [21]. The homeobox transcription factor may play a role in heart development and specification of limb identity [22]. Interestingly, TBX5 was identified as interactor of Tbx3, a regulator of the SAN gene program [23]. Hoxa3 is another important gene in heart chamber morphogenesis, since Hoxa3-expressing progenitor cells in the second heart field give rise to the atria and parts of the outflow tract [24].Fig. 4

Bottom Line: Cytosine bases of the DNA are converted to 5-methylcytosine by the methyltransferase enzyme, acting as a reversible regulator of gene expression.Publication-ready graphics, genome browser tracks, and table outputs include summary data and statistics, permitting instant comparison of methylation profiles between sample groups and the exploration of methylation patterns along the whole genome.The web-based version of ADMIRE provides a simple interface to researchers with limited programming skills, whereas the offline version is suitable for integration into custom pipelines.

View Article: PubMed Central - PubMed

Affiliation: Bioinformatics Group, Max Planck Institute for Heart and Lung Research, Ludwigstrasse 43, 61231 Bad Nauheim, Germany.

ABSTRACT

Background: DNA methylation at cytosine nucleotides constitutes epigenetic gene regulation impacting cellular development and a wide range of diseases. Cytosine bases of the DNA are converted to 5-methylcytosine by the methyltransferase enzyme, acting as a reversible regulator of gene expression. Due to its outstanding importance in the epigenetic field, a number of lab techniques were developed to interrogate DNA methylation on a global range. Besides whole-genome bisulfite sequencing, the Infinium HumanMethylation450 Assay represents a versatile and cost-effective tool to investigate genome-wide changes of methylation patterns.

Results: Analysis of DNA Methylation In genomic REgions (ADMIRE) is an open source, semi-automatic analysis pipeline and visualization tool for Infinium HumanMethylation450 Assays with a special focus on ease of use. It features flexible experimental settings, quality control, automatic filtering, normalization, multiple testing, and differential analyses on arbitrary genomic regions. Publication-ready graphics, genome browser tracks, and table outputs include summary data and statistics, permitting instant comparison of methylation profiles between sample groups and the exploration of methylation patterns along the whole genome. ADMIREs statistical approach permits simultaneous large-scale analyses of hundreds of assays with little impact on algorithm runtimes.

Conclusions: The web-based version of ADMIRE provides a simple interface to researchers with limited programming skills, whereas the offline version is suitable for integration into custom pipelines. ADMIRE may be used via our freely available web service at https://bioinformatics.mpi-bn.mpg.de without any limitations concerning the size of a project. An offline version for local execution is available from our website or GitHub (https://github.molgen.mpg.de/loosolab/admire).

No MeSH data available.