Criteria for assessing the quality of a ChIP-seq experiment. (A) Library complexity. Individual reads mapping to the plus (red) or minus strand (blue) are represented. (B) Distribution of functional regulatory elements with respect to the strength of the ChIP-seq signal. ChIP-seq was performed against myogenin, a major regulator of muscle differentiation, in differentiated mouse myocytes. While many extensively characterized muscle regulatory elements exhibit strong myogenin binding, a large number of known functional sites are at the low end of the binding strength continuum. (C) Number of called peaks vs. ChIP enrichment. Except in special cases, successful experiments identify thousands to tens of thousands of peaks for most TFs and, depending on the peak finder used, numbers in the hundreds or low thousands indicate a failure. Peaks were called using MACS with default thresholds. (D) Generation of a cross-correlation plot. Reads are shifted in the direction of the strand they map to by an increasing number of base pairs and the Pearson correlation between the per-position read count vectors for each strand is calculated. Read coverage as wigglegram is represented, not to the same scale in the top and bottom panels.) (E) Two cross-correlation peaks are usually observed in a ChIP experiment, one corresponding to the read length (“phantom” peak) and one to the average fragment length of the library. (F) Correlation between the fraction of reads within called regions and the relative cross-correlation coefficient for 1052 human ChIP-seq experiments. (G) The absolute and relative height of the two peaks are useful determinants of the success of a ChIP-seq experiment. A high-quality IP is characterized by a ChIP peak that is much higher than the “phantom” peak, while often very small or no such peak is seen in failed experiments.
