Introduction to ChIP-Seq

definition

    ChIP-Seq, aiming to identify genome-wide transcription factor binding sites and histone-modificatin enriched regions, comprises two parts:  ChIP experiments and sequencing.  Main steps of ChIP consist of cross-link proteins to DNA, fragment (sonication), IP with antibody,reverse crosslink, size selection and PCR.

figure 1. ChIP experiment steps

System error       

     Not all DNA fragments enriched in the regions binding to a protein of interest are actual signals. The proportion of DNA fragments containing the actual binding site of the protein depends on the number of active binding sites, the number of starting genomes, and the efficiency of the IP. Some DNA fragments appears enriched due to the following reasons:

     * open chromatin regions are fragmented more easily than closed regions

     * repetitive sequences might seem to be enriched(copy number inaccuracies in genome assembly)

     * Uneven distribution of sequence reads across the genome

Control

        There are two kinds of controls for ChIP-Seq: lgG control and input control. IgG control is DNA resulting from a "mock" ChIP with Immunoglobulin G (IgG) antibody, which binds to non-nuclear antigen;input control is DNA purified from cells that are cross-linked,fragmented, but without adding any antibody for enrichment. If too little DNA is recovered in IgG control, sequencing library will be of low complexity and binding sites identified using this control could be biased. Input control is ideal in most of the cases. It represents all the chromatin that was available for IP.