Each of the chromatin accessibility assays discussed here has inherent limitations in identifying regions of enrichment, based on the fragmentation method used and the involvement of any size selection steps. MNase-seq, DNase-seq and ATAC-seq are all based on the double enzymatic cleavage of DNA fragments and are sensitive to the excision-ability of a fragment. As shown in MNase-seq and ATAC-seq experiments, this sensitivity represents an issue only when mapping larger fragments (>100 bp) because the data is heavily biased by the overall nucleosome configuration at the region [55, 103]. In MNase-seq experiments, it was specifically shown that nucleosomes flanked by hypersensitive sites or long linkers are excised easier at low enzymatic concentrations and exhibit artificially higher nucleosome occupancy compared to nucleosomes without these characteristics, thus leading to biased results [55].
