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Ethics approval for this study was obtained from the North West–Liverpool Central Research Ethics Committee, with additional earlier approvals from the St Thomas’ Hospital and London–Westminster committees.
Semen samples were gathered from 75 participants within the TwinsUK cohort, some of which were archival. Additionally, whole-blood samples were collected from 67 of these men. In total, 104 semen samples were collected from individuals aged 24 to 75 years, including 29 men who had samples taken at two different times, averaging about 12 years apart. Blood samples ranged from 22 to 83 years old and were taken across one to four time points, with an average interval between samples being 8.1 years. The cohort included nine pairs of monozygotic twins and three pairs of dizygotic twins, with successful sequencing counts summarized in Supplementary Table 1.
Regarding metadata, self-reported data on age, height, weight, and other lifestyle factors like smoking and alcohol consumption were sourced from questionnaires. Notably, all individuals reporting ethnicity identified as white. BMI was calculated based on weight divided by height squared, while smoking was quantified in pack-years. Alcohol consumption was calculated from average weekly intake extrapolated to the participant’s adult life span.
For DNA extraction, sperm samples underwent a specific protocol using a Qiagen kit, with minor revisions to the elution process. Whole blood DNA extraction followed the Gentra Puregene protocol.
The study utilized three different targeted gene panels designed by Twist Bioscience for targeted NanoSeq sequencing, including a custom pilot panel of 210 genes and a more extensive panel of 263 genes. Coverage from 84 total samples was achieved, with different sequencing methodologies applied to the panels.
Sequencing libraries were prepared according to established methods and were subsequently sequenced on NovaSeq platforms. Each sample was aligned to the human reference genome, and the calling process utilized specific parameters to identify variants.
To assess DNA contamination, the highly accurate duplex sequencing method revealed challenges regarding contamination from non-target cell types. A specific analysis tool, verifyBamID, helped determine the presence and extent of foreign DNA contamination, leading to the exclusion of some samples exceeding the set thresholds.
The mutation burden calculations considered variations in trinucleotide sequences, correcting for differences across targeted genomic regions. The study compared mutation burdens from different sequencing methodologies and examined the influence of age on these rates through regression analyses.
Mutational signatures were extracted using a hierarchical process, revealing important data on mutations in sperm and their relation to cancer. Positively selected genes were annotated to assess their potential impact on diseases, focusing particularly on those related to sperm and cancers.
Visualizations of gene mutations were created to illustrate significant findings, incorporating variant annotations stemming from various mutation analysis tools. Associations with mutations and lifestyle factors like BMI were analyzed through statistical methods, examining various mutation outcome variables related to both sperm and blood samples.
Further details regarding the research can be found in the linked Nature Portfolio Reporting Summary.
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