DOI: 10.5061/dryad.8pk0p2njx
title: Pedigree-based and phylogenetic methods support surprising patterns of mutation rate and spectrum in the gray mouse lemur

description: Mutations are the raw material on which evolution acts, and knowledge of
 their frequency and genomic distribution is crucial for understanding how
 evolution operates at both long and short timescales. At present, the rate
 and spectrum of de novo mutations have been directly characterized in
 relatively few lineages. Our study provides the first direct mutation rate
 estimate for a strepsirrhine (i.e., the lemurs and lorises), which
 comprise nearly half of the primate clade. Using high-coverage linked-read
 sequencing for a focal quartet of gray mouse lemurs (Microcebus murinus),
 we estimated the mutation rate to be 1.52 × 10–8 (95% credible interval:
 1.28 × 10−8 to 1.78 × 10−8) mutations/site/generation, a rate among the
 highest calculated for a mammal. Further, we found an unexpectedly low
 count of paternal mutations, and only a modest overrepresentation of
 mutations at CpG-sites. Despite the surprising nature of these results, we
 found both the rate and spectrum to be robust to the manipulation of a
 wide range of computational filtering criteria. We also sequenced a
 technical replicate to estimate a false negative and false positive rate
 for our data and show that any point estimate of a de novo mutation rate
 should be considered with a large degree of uncertainty. To validate these
 observations, we conducted an independent analysis of context-dependent
 substitution types for gray mouse lemur and five additional primate
 species for which de novo mutation rates have also been estimated. These
 comparisons revealed general consistency of the mutation spectrum between
 the pedigree-based and the substitution rate analyses for all species
 compared.
description: Mutation rate estimation was done with a single pedigree using
 10x linked read data. Alignment to the Mmur3.0 reference genome was done
 with LongRanger with initial variant calling by GATK. Mutations were
 called with two software 1) DeNovoGear and 2) VarScan2. VCFs produced by
 both methods are provided. Context-dependent
 substitution rate analyses were done by MULTIDIVTIME, based on stochastic
 mappings from PhyloBayes. Analyses were based on whole-genome alignments
 from Ensembl, which included the Mmur3.0 reference genome. A Concatenated
 whole-genome alignment was used to produce 10 randomly selected alignments
 that were 1Mb in length. Scripts for analysing data with MCMCTREE and the
 resulting variance-covariance matrices from stochastic mappings (https://github.com/HuiJieLee/ParsePhyloBayes) with MULTIDIVTIME are provided.
description: There are three folders available which have readme files
 describing their contents: 1) VCF - VCF files resulting
 from mutation-calling software 2) SubstitutionRates -
 Contains folders for analyses with MCMCTREE and MULTIDIVTIME. Includes
 alignments used for analyses of context-dependent substitution
 rates. 3) MutationList - Contains a simple csv file
 with all of the final mutations analyzed in the manuscript and a readme
 describing the fields. 10x reads are deposited in
 NCBI's SRA database as bam files with
 identifiers SRR10130788-SRR10130796. Additional metadata and pedigree
 information are available in the supplementary material.