To quantify the effects of external branch length heterogeneity on singleton clustering patterns, we used the stdpopsim library95 to simulate variants across chromosome 1 for 2,000 European and 2,000 African haploid samples, using a previously reported demographic model10. Simulations were performed using a per-site, per-generation mutation rate96 of 1.29 × 10−8, and using recombination rates derived from the HapMap genetic map97. Because our aim was to compare these simulated singletons to unphased singletons observed in the TOPMed data, we randomly assigned each of the 2,000 haploid samples from each population into one of 1,000 diploid pairs, and calculated the inter-singleton distances per diploid sample, ignoring the haplotype on which each simulated singleton originated.
