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Data Exploration, Quality Control and Statistical Analysis of ChIP-exo/nexus Experiments

ABSTRACT: ChIP-exo/nexus experiments present modifications on the commonly used ChIP-seq protocol for high resolution mapping of transcription factor binding sites. Although many aspects of the ChIP-exo data analysis are similar to those of ChIP-seq, these high throughput experiments pose a number of unique quality control and analysis challenges. We develop a statistical quality control pipeline and accompanying R package, ChIPexoQual, to enable exploration and analysis of ChIP-exo and related experiments. ChIPexoQual evaluates a number of key issues including strand imbalance, library complexity, and signal enrichment of data. Assessment of these features are facilitated through diagnostic plots and summary statistics calculated over regions of the genome with varying levels of coverage. We evaluated our QC pipeline with both large collections of public ChIP-exo/nexus data and multiple, new ChIP-exo datasets from E. coli. ChIPexoQual analysis of these datasets resulted in guidelines for using these QC metrics across a wide range of sequencing depths and provided further insights for modelling ChIP-exo data. Finally, although ChIP-exo experiments have been compared to ChIP-seq experiments with single-end (SE) sequencing, we provide, for the first time, comparisons with paired-end (PE) ChIP-seq experiments. We illustrate that, at fixed sequencing depths, ChIP-exo provides higher sensitivity, specificity, and spatial resolution than PE ChIP-seq and both significantly outperform their SE ChIP-seq counterpart.

ORGANISM(S): Escherichia coli

PROVIDER: GSE84830 | GEO | 2017/03/01

SECONDARY ACCESSION(S): PRJNA338159

REPOSITORIES: GEO

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Project description:We have established a certification system for antibodies to be used in chromatin immunoprecipitation assays coupled to massive parallel sequencing (ChIP-seq). This certification comprises a standardized ChIP procedure and the attribution of a numerical quality control indicator (QCi) to biological replicate experiments. The QCi computation is based on a universally applicable quality assessment that quantitates the global deviation of randomly sampled subsets of ChIP-seq dataset with the original genome-aligned sequence reads. Comparison with a QCi database for >28,000 ChIP-seq assays were used to attribute quality grades (ranging from âAAAâ to âDDDâ) to a given dataset. In the present report we used the numerical QC system to assess the factors influencing the quality of ChIP-seq assays, including the nature of the target, the sequencing depth and the commercial source of the antibody. We have used this approach specifically to certify mono and polyclonal antibodies obtained from Active Motif directed against the histone modification marks H3K4me3, H3K27ac and H3K9ac for ChIP-seq. The antibodies received the grades AAA to BBA (www.ngs-qc.org). We propose to attribute such quantitative grading of all antibodies attributed with the label âChIP-seq gradeâ. The NGS-QC team has setup a certification procedure for Antibodies dedicated to ChIP-seq applications based on (1)a highly reproducible pipeline for performing ChIP-seq assays including the use of an automated liquid handling platform (TECAN EVO75) for ChIP and sequencing library preparation steps and (2) the use of the NGS-QC Generator tool for providing a ChIP-seq quality grade and to evaluate other parameters like the Optimal sequencing depth, the degree of reproducibility among biological replicates and the comparison of the quality grades of the certified antibody with datasets retrieved in the public domain. This quality certification structure makes possible to assign a measured Quality Stamp to ChIP-seq grade Antibodies.

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Data Exploration, Quality Control and Statistical Analysis of ChIP-exo/nexus Experiments

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