Extended Data Fig 3: SSDS signal at hotspots is narrower in ovaries than in spermatocytes.

a, SSDS coverage is a measure of DMC1-bound ssDNA either side of each meiotic DSB. In a population of meiocytes, DSBs will occur in a several hundred nucleotide window around the hotspot center (orange rectangle). To assess coverage, we first convert the position of each SSDS fragment into the distance along ssDNA from the hotspot center. Merging the top and bottom strand fragments in this way increases coverage two-fold and minimizes the influence of asymmetric gaps and fluctuations in coverage. Coverage at each hotspot was normalized by the maximum value at the hotspot to prevent strong hotspots from dominating the average profile. The average normalized coverage across all hotspots was then calculated. DSB hotspots identified in b, females (ovary sample O1) and c, males (testis sample T1) were each split into three bins by strength. Coverage was calculated for all nine male and two female samples for each set. The SSDS signal is narrower for all female samples compared to male samples. The difference is particularly pronounced at stronger hotspots, where coverage estimates are most accurate. At the widest point, the mean male and female profiles diverge by ~0.4 Kb. d, We also examined the MACS-determined hotspot boundaries to further negate the possibility that the average profiles in B-C are not a reflection of the population. By this metric, the mean hotspot width estimated from male samples (1,759 ± 73 bp; mean (solid blue line) ± SEM (dashed blue lines); n = 9) is significantly wider the mean width of hotspots in female samples (1,490 ± 89 bp; mean (solid pink line) ± SEM (dashed pink lines); n = 2) (t-test; P = 0.0007). Sequencing quality and sample SPoT can affect width estimates, therefore, we processed each sample as follows; we reduced the SPoT of each sample to that of the lowest quality sample (O2; see Methods), considering only uniquely mapping and high quality (Q >30) ssDNA type 1 fragments. We then reduced all samples to have the same number of fragments as the smallest. On these datasets, we performed peak calling and retained only DSB hotspots that were called in all samples (N = 1,975). e, Potential mechanistic explanations for the difference in SSDS signal between males and females. These differences may manifest in all meiocytes or in sub-populations. Importantly, we see no evidence of shape differences at hotspots in sub-populations of spermatocytes (data not shown).