Peer review of: Germline de novo mutation rates on exons versus introns in humans (2019)

Reviewers' comments: 

Reviewer #1 (Remarks to the Author): 

This paper describes an analysis of published de novo mutation datasets to test the hypothesis that 
the exonic mutation rate is different from the intronic mutation rate in the germline. This hypothesis 
was first posited by Frigola et al in a paper in 2017, following observation of this effect in the somatic 
cells of a cohort of colorectal tumours. It is an important hypothesis to test because, if true, it would 
undermine many evolutionary genetics models that assume that mutation rate is independent of 
coding status of the DNA sequence, so that any difference in diversity can be interpreted as 
differences in the action of population genetic processes following mutation, such as selection. 

The authors here assembled a large amount of data and tested this hypothesis rigorously. I have very 
few comments on the science – they have approached the topic logically and have explored it 
thoroughly. The paper is clearly written. The result is clearly in line with previous expectations and the 
hypothesis proposed by Frigola is not only not supported, but is effectively refuted by the data and the 
analyses. This is a very important result for molecular evolutionary and population genetics. 

I have some minor comments. 
1. Page 2 para 3. POLE should be defined and explained at the first mention. 
2. Page 2 para 3. “mutation rates are surprisingly lower”. Surprisingly should be removed or be placed 
at the start of the sentence (replacing “Recently”) – at the moment the placing of “surprisingly” 
suggests that the magnitude of the drop in exonic mutation rate is surprising, “surprisingly lower”, 
rather than the actual fact of the mutation rate being lower at all being surprising. 
3. Page 3 para 1. “Differential” should be “different”. 
4. Page 3 para 3. After “In contrast” it should read “In contrast, in our study, the observed 
germline...” to prevent ambiguity and emphasise that you are referring to this study not the study by 
Frigola et al discussed in the previous sentence. 
5. Page 11 para 1. Coordinates of exonic and intronic boundaries after mappability filtering should 
either be available as a supplementary txt file hosted by the journal, or hosted by figshare or similar 
stable open access repository. 
5. Page 11 Code availability. These scripts should be made openly accessible on Github or Figshare or 
a suitable open access repository. 

Reviewer #2 (Remarks to the Author): 

Rodriguez-Galindo et al. investigated whether there is an elevated mutation rate in exons compared 
with that in introns. This study was conducted after Frigola et al 2017 showed that there is a reduced 
mutation rate in exons compared with introns in somatic mutations in tumors, which the authors 
showed that it would be due to enhanced MMR activities in exons. In this study, the authors showed 
that there is no evidence of higher mutation rates in exons, and that in mixed disease cohorts, there is 
an increased mutation rate in nonsynonymous mutation rates compared with the intron mutation rate. 

This study is particularly crucial because evolutionary models are based on the assumption that the 
base mutation rate is the same between the two, except for those variants that are subject to positive 
or negative selection. The authors gave strong evidence that the assumption holds in germline 
mutations. The article is well written and easy to follow. While I have some concerns about the 
differences in the results in the different cohorts, I would recommend this manuscript to be published 
with some additional analysis. 

Firstly, as the authors showed in Table 1, the point estimates for exon excess compared with intron 
are mostly positive, although most of them are insignificant. They showed that when they stratify the 

exonic mutations by synonymous and nonsynonymous, only nonsynonymous mutations showed 
excess mutations compared with expectation. They further showed that this is the case in the Autism 
probands but not the "healthy" probands. 
The authors hypothesize that it could be due to ascertainment bias from disease probands being more 
likely to carry functional impactful variants (Table S3). I think that the disease cohort may exhibit a 
slight increase in the number of functional variants, but 10.5% excess seems pretty substantial. Can 
the authors also look into the largest single cohort, Halldorsson dataset, which consists of multiple 
diseases, does it also exhibit such trend in non-synonymous and synonymous mutations? 

Secondly, in Frigola et al. 2017, after filtering and removing cancer driving genes, there are 12,104 
genes left (including X, Y); in this manuscript, after filtering (and removing X, Y), there are 12,754 
genes left. Assuming the way of constructing the meta-exon is the same (which brings me to the final 
point that data and scripts need to be shared), could the difference in the observation partially be 
explained by the differences in the genes selected? 1) Can the authors take the intersect between the 
two sets of genes and repeat the analysis? 2) show the distribution of exon vs. intron mutation rate to 
see if the excess mutations in exons is driven by a few genes. 

Finally, the authors claimed that custom scripts for data analysis are available upon request from the 
authors. I think the key scripts and processed data need to be released. 

Minor comments: 

On page 33, Table S3. the calling of Complete Genomics variants in the Yuen dataset does not involve 
GATK, but uses a heuristic method described in 
Yuen RKC, Thiruvahindrapuram B, Merico D, Walker S, Tammimies K, Hoang N, et al. Whole-genome 
sequencing of quartet families with autism spectrum disorder. Nat Med. 2015;21: 185–191.