Unbiased mosaic variant assessment in sperm: a cohort study to test predictability of transmission

Essential revisions:

1) In Figure 1b – Individual F03 has more than twice as many mosaic variants as F01 or F02. Is there a difference in sequence depth, age, or another explanation? Is the difference statistically significant?

In our previous work, we found that—on average—a male harbors around 30 mosaic variants in sperm. We did, however, observe a range from 10 to more than 50, and all three individuals would fall within this range (even though F01 and F02 would show numbers at the lower end of the spectrum). We do not believe that this is due to sequencing depth, as F03 is sequenced at a depth between F01 and F02; likewise, we do not believe it to be an age effect: while F03 is older than F01 and F02 (50 years, compared to 36 and 38), the difference is much less than between our young and aged cohorts in our previous publication (~20 years vs. 48+ years). Therefore, we believe these differences to be within the expected biological variation range and not due to a systematic issue. Prompted by this comment we have made two changes to the manuscript:

1) We have now added the ages of the three sperm donors to the Methods section for clarity.

Additional comments/suggestions for authors:

1) The manuscript is well-written and the figures are generally clear. However, this is a specialized field and some of the genetic concepts are quite complex and may be of limited accessibility to the general reader. The authors might consider trying to provide a more general and accessible summary of the work and findings and their implications, including the limitation to only a relatively small subset of de novo genetic mutations.