indicate weak (black) and prominent (red) Rec12 peaks identified by PeakFinder.
We extended this analysis genome-wide, except for the telomeres and centromeres, which are considered separately in the Discussion. Rec12-DNA linkage positions were identified as the maxima of Rec12 peaks using the PeakFinder program . Using the mean of the haploid 5-h data, this program identified 353 such positions genome-wide. The 149, 124, and 80 positions seen on Chromosomes I, II, and III respectively (Figure S7) are in good agreement with the relative sizes of the chromosomes (5.6, 4.5, and 2.5 Mb, not including the rDNA at the ends of Chromosome III). Of the 353 Rec12 peak positions from the haploid data, 340 (96%) were also identified in the diploid data. Using a cutoff of p < 10−8, a cutoff that excludes all but 18 (12%) of the false-positive peaks identified in the 0-h data, the ChIPOTle program  independently identified peak regions in the haploid data that included all of the positions identified by PeakFinder, plus an additional 16 (4%). We used the peak positions identified by PeakFinder from the haploid data for the analysis below.
As mentioned above, we estimated the relative frequency of Rec12-DNA linkage at each hotspot by integrating the IP:WCE ratios across each peak and classified the peaks as “prominent” (≥0.5% estimated DSBs) or “weak” (<0.5%) based on the relation in Figure 4. Among the 353 peaks identified by PeakFinder, 194 are prominent hotspots. Based on the sum of the integrals, these prominent peaks account for 88% of all detectable Rec12-DNA linkages (DSBs).
Our analysis shows that DSB hotspots are over-represented in large IGRs and under-represented in coding DNA. Figure 6A shows the proportion of all genomic DNA and the proportion of all prominent and weak Rec12 peaks found in coding sequences and in IGRs of different sizes. The distributions of all DNA and Rec12 peaks are clearly distinct (for prominent peaks p < 0.0001 and for weak peaks p < 0.005; two-tailed contingency chi-squared test). IGRs >2 kb comprise only 13% of the genome, but they contain 62% of all prominent Rec12 peaks and 37% of the weak Rec12 peaks. Similarly, IGRs >3 kb comprise only 8% of the genome but contain 47% of the prominent Rec12 peaks and 19% of the weak Rec12 peaks. Thus, prominent peaks are more strongly enriched in large IGRs than are weak peaks. In fact, among prominent peaks, the probability of a peak lying in an IGR >3 kb rises with peak integral so that the very strongest peaks are even more highly associated with large IGRs (Figure S8). The Rec12 peak integrals, a measure of Rec12-DNA linkages and hence DSBs, are similarly skewed toward large IGRs: 65% of all Rec12-DNA linkages are in IGRs >2 kb, and 52% are in IGRs >3 kb. In addition, fewer prominent hotspots than weak hotspots (19% versus 38%) occur in coding DNA, which accounts for 61% of the genome.
Frequency distributions of DNA (green bars), weak Rec12-DNA linkage sites (gray bars), and prominent Rec12-DNA linkage sites (red bars) are shown for the classes of DNA sequence indicated. Analysis was carried out using the whole genome excluding the centromeres and 50 kb at the end of each chromosomal contig (to exclude telomeric regions).
(A) Number distribution. Peaks of Rec12-DNA linkage (DSB hotspots) identified from the mean of the haploid 5-h data were classified according to their position in coding sequences (CDS) or in IGRs. A total of 62% of prominent Rec12 peaks occur in IGRs >2 kb.
(B) Probability of a prominent Rec12 peak occurring within an IGR increases with IGR size.
(C) Density of prominent Rec12 peaks (number of peaks per kb) increases with IGR size.
(D) DSB density (Rec12 peak integrals per kb) is greatest in large IGRs.
Prominent DSB hotspots, as indicated by sites of Rec12-DNA linkage, are therefore particularly strongly associated with large IGRs. In addition, large IGRs are highly predictive of DSB hotspots, since 44% of the IGRs >3 kb long have prominent Rec12 peaks (Figure 6B) and an additional 12% have weak Rec12 peaks. Furthermore, prominent Rec12