Assessing Hardy-Weinberg equilibrium in T2T-aligned 1000 genomes project

Quality control of markers in genome-wide association studies often includes testing for Hardy-Weinberg equilibrium (HWE). However, this is usually implemented in a homogeneous population without stratifying by sex. Previous work indicates sex-based selection at numerous autosomal loci in cohorts with active recruitment. Sex chromosome sequences can also interfere with autosomal SNPs. These motivate a re-examination of HWE in sex-aware analyses. Using the telomere-to-telomere (T2Tv2)-aligned high-coverage whole genome sequencing data from 2,490 individuals in the 1000 Genomes Project, we examined genome-wide sex-specific deviations from HWE across five super-populations. Our analyses were restricted to bi-allelic SNPs with non-missing genotypes and minor allele frequency (MAF) ≥5% in both sexes of the five super-populations. We applied an allele-based framework to quantify both the magnitude and direction of Hardy–Weinberg disequilibrium (HWD), followed by a second-order omnibus meta-analysis that combined HWD results across populations and sexes. At a genome-wide significance threshold of p < 5e-8, 0.9% of autosomal SNPs exhibited significant deviations from HWE. The majority of these deviations were associated with genomic features indicative of poor sequence quality. Restricting the analysis to reliable genomic regions substantially reduced the number of signals, yielding 255 autosomal SNPs and one non-pseudoautosomal chromosome X SNP. Among these, 140 autosomal SNPs displayed significant heterogeneity across populations but not across sexes. Notably, eight SNPs within a 15-bp region on chromosome 14q31.3 showed excess heterozygosity in both sexes of the African super-population (AFR). Finally, we developed a multivariate predictor of HWD based on sequence features, providing a practical tool that can be integrated into existing quality control pipelines for whole genome sequencing studies.