Dataset Information

Calibrating ChIP-Seq with Nucleosomal Internal Standards to Measure Histone Modification Density Genome Wide.

ABSTRACT: Chromatin immunoprecipitation (ChIP) serves as a central experimental technique in epigenetics research, yet there are serious drawbacks: it is a relative measurement, which untethered to any external scale obscures fair comparison among experiments; it employs antibody reagents that have differing affinities and specificities for target epitopes that vary in abundance; and it is frequently not reproducible. To address these problems, we developed Internal Standard Calibrated ChIP (ICeChIP), wherein a native chromatin sample is spiked with nucleosomes reconstituted from recombinant and semisynthetic histones on barcoded DNA prior to immunoprecipitation. ICeChIP measures local histone modification densities on a biologically meaningful scale, enabling unbiased trans-experimental comparisons, and reveals unique insight into the nature of bivalent domains. This technology provides in situ assessment of the immunoprecipitation step, accommodating for many experimental pitfalls as well as providing a critical examination of untested assumptions inherent to conventional ChIP.

SUBMITTER: Grzybowski AT

PROVIDER: S-EPMC4458216 | biostudies-literature | 2015 Jun

REPOSITORIES: biostudies-literature

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Calibrating ChIP-Seq with Nucleosomal Internal Standards to Measure Histone Modification Density Genome Wide.

Grzybowski Adrian T AT Chen Zhonglei Z Ruthenburg Alexander J AJ

Molecular cell 20150521 5

Chromatin immunoprecipitation (ChIP) serves as a central experimental technique in epigenetics research, yet there are serious drawbacks: it is a relative measurement, which untethered to any external scale obscures fair comparison among experiments; it employs antibody reagents that have differing affinities and specificities for target epitopes that vary in abundance; and it is frequently not reproducible. To address these problems, we developed Internal Standard Calibrated ChIP (ICeChIP), whe ...[more]

PMID: 26004229

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Project description:BackgroundThe advent of ChIP-seq technology has made the investigation of epigenetic regulatory networks a computationally tractable problem. Several groups have applied statistical computing methods to ChIP-seq datasets to gain insight into the epigenetic regulation of transcription. However, methods for estimating enrichment levels in ChIP-seq data for these computational studies are understudied and variable. Since the conclusions drawn from these data mining and machine learning applications strongly depend on the enrichment level inputs, a comparison of estimation methods with respect to the performance of statistical models should be made.ResultsVarious methods were used to estimate the gene-wise ChIP-seq enrichment levels for 20 histone methylations and the histone variant H2A.Z. The Multivariate Adaptive Regression Splines (MARS) algorithm was applied for each estimation method using the estimation of enrichment levels as predictors and gene expression levels as responses. The methods used to estimate enrichment levels included tag counting and model-based methods that were applied to whole genes and specific gene regions. These methods were also applied to various sizes of estimation windows. The MARS model performance was assessed with the Generalized Cross-Validation Score (GCV). We determined that model-based methods of enrichment estimation that spatially weight enrichment based on average patterns provided an improvement over tag counting methods. Also, methods that included information across the entire gene body provided improvement over methods that focus on a specific sub-region of the gene (e.g., the 5' or 3' region).ConclusionThe performance of data mining and machine learning methods when applied to histone modification ChIP-seq data can be improved by using data across the entire gene body, and incorporating the spatial distribution of enrichment. Refinement of enrichment estimation ultimately improved accuracy of model predictions.

Dataset Information

Calibrating ChIP-Seq with Nucleosomal Internal Standards to Measure Histone Modification Density Genome Wide.

Publications

Calibrating ChIP-Seq with Nucleosomal Internal Standards to Measure Histone Modification Density Genome Wide.

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