CBI ChIPSeq Analysis

Chromatin immunoprecipitation is frequently used to map the location of specific protein factors or epigenetic chromatin modifications. The Cancer Bioinformatics (CBI) Shared Resource has extensive, historical experience with ChIPSeq and related analyses, including Cut&Run and ATAC-Seq. While single-factor ChIPSeq is straightforward to process, investigators are increasingly use ChIPSeq in quantitative or comparative assays, which require more customized approaches. Talk to a CBI member for advice when starting these experiments.

Experiment Design

We generally recommend three biological replicates per sample (antibody or experimental condition). For preliminary, exploratory ChIPSeq, one replicate could be used, but for publication and quantitative purposes, multiple replicates are required. For vertebrate genomes, 20 million reads should be a minimum target sequencing depth. For small model organism genomes, much less can be used. Either paired-end or single-end sequencing strategies may be used. When at all possible, ChIP should be checked by quantitative PCR at known loci prior to submission.

Multi-Replicate Multi-Sample Pipeline

CBI has assembled a comprehensive multiple-sample, multiple-replicate pipeline that wraps around the MACS peak caller, which is an industry-standard ChIPSeq peak caller. This pipeline was developed explicitly to handle multiple samples so that comparisons between samples may be made as consistently and reliable as possible. This is accomplished through a number of approaches, including masking false-positive regions, normalizing alignment duplication through sub-sampling, and enforcing reproducibility between replicates. The pipeline is considerably customizable and can handle a wide variety of assays, including ChIP-Seq, ATAC-Seq, Cut&Run, and Cut&Tag. An HTML report is generated upon completion with a number of correlation, QC, and comparative plots. Peaks, data tracks, and count tables are provided for high-level evaluation and differential analysis.

To have CBI analyze your chromatin-assay experiments, please submit a help request.

The pipeline is installed on HCI and CHPC compute resources. Advanced users who are interested in running the pipeline themselves can run the command module load multirepchipseq to load the package into their environment. Learn more about our infrastructure.

High-Level Analysis

For quality control comparison, visualization, and high-level analysis, the deepTools package is recommended for many needs. The BioToolBox package is also available for collecting processed data with respect to annotation for graphing purposes. The Homer package is recommended for motif analysis. The Chipseeker package is useful for annotation, and DiffBind for differential analysis.

The HCI interactive Linux servers have these and other software installed.