Sperm sequencing reveals extensive positive selection in the male germline

Mutations that occur in the cell lineages of sperm or eggs can be transmitted to offspring. In humans, positive selection of driver mutations during spermatogenesis is known to increase the birth prevalence of certain developmental disorders. Until recently, characterising the extent of this selection in sperm has been limited by the error rates of sequencing technologies. Using the duplex sequencing method NanoSeq, we sequenced 81 bulk sperm samples from individuals aged 24 to 75 years. Our findings revealed a linear accumulation of 1.67 (95% CI = 1.41-1.92) mutations per year per haploid genome, driven by two mutational signatures associated with human ageing. Deep targeted and exome NanoSeq of sperm samples identified over 35,000 germline coding mutations. We detected 40 genes (31 novel) under significant positive selection in the male germline, implicating both activating and loss-of-function mechanisms and diverse cellular pathways. Most positively selected genes are associated with developmental or cancer predisposition disorders in children, while four genes that exhibit elevated frequencies of protein-truncating variants in healthy populations. We find that positive selection during spermatogenesis drives a 2-3 fold elevated risk of known disease-causing mutations in sperm, resulting in 3-5% of sperm from middle-aged to elderly individuals carrying a pathogenic mutation across the exome. These findings shed light on the dynamics of germline mutations and highlight a broader increased disease risk for children born to fathers of advanced age than previously appreciated.