We have mapped sequence-directed nucleosome positioning on genomic DNA molecules 
using high-throughput sequencing. Chromatins, prepared by reconstitution with 
either chicken or frog histones, were separately digested to mononucleosomes 
using either micrococcal nuclease (MNase) or caspase-activated DNase (CAD). Both 
enzymes preferentially cleave internucleosomal (linker) DNA, although they do so 
by markedly different mechanisms. MNase has hitherto been very widely used to 
map nucleosomes, although concerns have been raised over its potential to 
introduce bias. Having identified the locations and quantified the strength of 
both the chicken or frog histone octamer binding sites on each DNA, the results 
obtained with the two enzymes were compared using a variety of criteria. Both 
enzymes displayed sequence specificity in their preferred cleavage sites, 
although the nature of this selectivity was distinct for the two enzymes. In 
addition, nucleosomes produced by CAD nuclease are 8-10 bp longer than those 
produced with MNase, with the CAD cleavage sites tending to be 4-5 bp further 
out from the nucleosomal dyad than the corresponding MNase cleavage sites. 
Despite these notable differences in cleavage behaviour, the two nucleases 
identified essentially equivalent patterns of nucleosome positioning sites on 
each of the DNAs tested, an observation that was independent of the histone 
type. These results indicate that biases in nucleosome positioning data 
collected using MNase are, under our conditions, not significant.