Prior to imputation PLINK31 was used to remove samples with >5% missing data. We also excluded SNPs characterized by >1% missing values, a Hardy-Weinberg equilibrium P < 0.001 and a minor allele frequency of <5%. These were recoded as vcf files using PLINK1.948 and VCFtools49 before uploading to the Michigan Imputation Server (https://imputationserver.sph.umich.edu/start.html#!pages/home) which uses SHAPEIT50,51 to phase haplotypes, and Minimac352 with the most recent 1000 Genomes reference panel (phase 3, version 5). Imputed genotypes were then filtered and recoded with PLINK1.948 removing samples with >5% missing values, and SNPs with >2 alleles, those indicated as a fail in the FILTER columns using the flag ‘--vcf-filter’, in addition to those characterized by >1% missing values, a Hardy-Weinberg equilibrium P < 0.001, a minor allele frequency of < 5%, or < 5 observations for any genotype group in line with the SNP filtering for the raw genotype groups. This resulted in 5,177,320 variants in the imputed set of genotypes. MatrixEQTL46 was used to test genome-wide mQTLs as previously described, except only mQTL with P < 1×10−8 were recorded.
