To mock or not: a comprehensive comparison of mock IP and DNA input for ChIP-seq

Chromatin immunoprecipitation (IP) followed by sequencing (ChIP-seq) is the gold standard to detect genome-wide DNA-protein binding. The binding sites of transcription factors facilitate many biological studies. Of emerging concern is the abundance of spurious sites in ChIP-seq, which are mainly caused by uneven genomic sonication and nonspecific interactions between chromatin and antibody. A "mock" IP is designed to correct for both factors, whereas a DNA input control corrects only for uneven sonication. However, a mock IP is more susceptible to technical noise than a DNA input, and empirically, these two controls perform similarly for ChIP-seq. Therefore, DNA input is currently being used almost exclusively. With a large dataset, we demonstrate that using a DNA input control results in a definable set of spurious sites, and their abundance is tightly associated with the intrinsic properties of the ChIP-seq sample. For example, compared to human cell lines, samples such as human tissues and whole worm and fly have more accessible genomes, and thus have more spurious sites. The large and varying abundance of spurious sites may impede comparative studies across multiple samples. In contrast, using a mock IP as control substantially removes these spurious sites, resulting in high-quality binding sites and facilitating their comparability across samples. Although outperformed by mock IP, DNA input is still informative and has unique advantages. Therefore, we have developed a method to use both controls in combination to further improve binding site detection.