Direct identification of de novo mobile element insertions from single molecule sequencing of human sperm

Mobile element insertions (MEIs) are a significant source of human genetic variation, yet the rates and properties of de novo MEIs are poorly characterized due to technical limitations in sequencing technology. Here, we directly sequenced individual gametes from sperm samples of 14 donors (aged 28-62) using highly accurate PacBio long-read sequencing to identify de novo retrotransposition events without familial inference. We developed a “self-alignment” strategy using personal genome assemblies that enables high-precision, single-read detection of de novo MEIs. Using this method, we identified 51 de novo Alu insertions, revealing 7-fold variation in Alu retrotransposition rates between individuals (0.023 to 0.17 insertions/gamete). We found a significant increase in Alu activity with paternal age, yielding an additional 0.003 insertions/gamete/year, of additional paternal age, representing a direct observation of age-associated increases in structural variant (SV) mutation rates. These active Alu elements are predominantly comprised of the evolutionarily young AluYa5 and AluYb8 subfamilies and bear characteristic molecular signatures of target-primed reverse transcription (TPRT). Our population-averaged rate of 7.4 insertions per 100 gametes aligns well with previous population genetic estimates, validating both direct observation and population approaches for estimating de novo MEI rates. These results establish direct gamete sequencing as a powerful method for characterizing germline mutation processes and reveal age as a significant determinant of de novo retrotransposition in the male germline.