Single-nucleotide variants (SNVs) in segmental duplications (SDs) have not been systematically assessed because of the limitations of mapping short-read sequencing data^1,2. Here we constructed 1:1 unambiguous alignments spanning high-identity SDs across 102 human haplotypes and compared the pattern of SNVs between unique and duplicated regions^3,4. We find that human SNVs are elevated 60% in SDs compared to unique regions and estimate that at least 23% of this increase is due to interlocus gene conversion (IGC) with up to 4.3 megabase pairs of SD sequence converted on average per human haplotype. We develop a genome-wide map of IGC donors and acceptors, including 498 acceptor and 454 donor hotspots affecting the exons of about 800 protein-coding genes. These include 171 genes that have 'relocated' on average 1.61 megabase pairs in a subset of human haplotypes. Using a coalescent framework, we show that SD regions are slightly evolutionarily older when compared to unique sequences, probably owing to IGC. SNVs in SDs, however, show a distinct mutational spectrum: a 27.1% increase in transversions that convert cytosine to guanine or the reverse across all triplet contexts and a 7.6% reduction in the frequency of CpG-associated mutations when compared to unique DNA. We reason that these distinct mutational properties help to maintain an overall higher GC content of SD DNA compared to that of unique DNA, probably driven by GC-biased conversion between paralogous sequences^5,6.

There has been tremendous progress in phased genome assembly production by combining long-read data with parental information or linked-read data. Nevertheless, a typical phased genome assembly generated by trio-hifiasm still generates more than 140 gaps. We perform a detailed analysis of gaps, assembly breaks, and misorientations from 182 haploid assemblies obtained from a diversity panel of 77 unique human samples. Although trio-based approaches using HiFi are the current gold standard, chromosome-wide phasing accuracy is comparable when using Strand-seq instead of parental data. Importantly, the majority of assembly gaps cluster near the largest and most identical repeats (including segmental duplications [35.4%], satellite DNA [22.3%], or regions enriched in GA/AT-rich DNA [27.4%]). Consequently, 1513 protein-coding genes overlap assembly gaps in at least one haplotype, and 231 are recurrently disrupted or missing from five or more haplotypes. Furthermore, we estimate that 6-7 Mbp of DNA are misorientated per haplotype irrespective of whether trio-free or trio-based approaches are used. Of these misorientations, 81% correspond to bona fide large inversion polymorphisms in the human species, most of which are flanked by large segmental duplications. We also identify large-scale alignment discontinuities consistent with 11.9 Mbp of deletions and 161.4 Mbp of insertions per haploid genome. Although 99% of this variation corresponds to satellite DNA, we identify 230 regions of euchromatic DNA with frequent expansions and contractions, nearly half of which overlap with 197 protein-coding genes. Such variable and incompletely assembled regions are important targets for future algorithmic development and pangenome representation.