Figure 4. The impact of Fragment Length and Complex Peak Structures in ChIP-seq.

(a) The average DNA fragment length can affect resolution with respect to binding site determination. A ChIP-seq experiment yields distributions for tags sequenced from the forward and reverse strands, the maxima of which should be separated by the average fragment length. In real experimental data, an overlap of the two distributions is often observed. If the average fragment length is much longer than the width of the strand distributions, the binding site will fall in between the two distributions. Tag locations are shifted toward the middle will result in a single summit (top illustration). Intermediate fragment lengths yield a single broadened peak in the unshifted aggregate distribution, and tag shifting may improve resolution a small amount by more precisely locating the summit (middle illustration). Very short fragments, such that the strand-specific densities are separated by a distance significantly less than the width of the individual distributions, can yield good binding site resolution without tag shifting tag. (b) Overlapping tag distributions are observed for clusters of nearby peaks such as the pictured double for a CTCF peak region in human⁷. Motif mapping reveals two CTCF binding sites (in red), though ChIP-seq signal suggests a single binding site call lying between the two motifs. As an example, the ERANGE region call (orange) is shown to cover both motifs. The problem of reliably discriminating multiple binding sites with very closely overlapping signals is an ongoing area of research.