Cindy Boer

Genetics of Osteoarthritis Consortium GWAS Meta-Analyses | 185 4.1 site wasn’t included in this analysis as it wasn’t clear which osteoarthritis subpheno- type was leading the signal. Each of the 100 independent genome wide significant SNVs was assigned to the above groups only if it wasn’t nominally significant (p-value>0.05) for any of the other phenotypes in the other classification groups, resulting in 86 SNVs to be further analysed. Fine mapping For each independent signal we included all variants within 1Mb of the index variant. In situations where there was more than one independent signal in the region we used the conditional summary statistics of the meta-analysis conditioned on all other signals. We calculated Wakefield’s asymptotic Bayes factors [85] and we determined the posterior probability of each variant being causal. To produce a 95% credible set of variants we ranked according to posterior probability and included those variants with the highest probability of being causal until the shared probability was at least 95%. Some regions were large therefore we considered only variants in the 95% credible with a posterior probability of causality > 3% ( Supplementary Table 7) . RNA sequencing analysis of the RAAK cohort Preserved and lesioned cartilage and subchondral bone samples from the same donor were obtained from the Research in Articular osteoArthritis Cartilage (RAAK) study consisting of patients with osteoarthritis who underwent joint replacement surgery due to an end-stage disease [86-88]. Total RNA from articular cartilage and subchon- dral bone was isolated using Qiagen RNeasy Mini Kit (Qiagen, GmbH, Hilden, Germany). Paired-end 2×100 bp RNA-sequencing (Illumina TruSeq RNA Library Prep Kit, Illumina HiSeq2000 and Illumina HiSeq4000) was performed. Strand specific RNA-Seq librar- ies were generated which yielded a mean of 20 million reads per sample. More details on mapping and quality control (QC) from cartilage are previously described (PMID: 30504444, 30298554). Methods of subchondral bones RNA sequencing have been pre- viously described[89]. After QC, 35 paired cartilage samples (N = 70) and 24 paired subchondral bone samples (18 paired knee and 6 paired hip samples) remained for further differential expression analysis. Normalization and statistical framework was performed using the DESeq2 v1.20 R package. A general linear model (GLM) assuming a negative binomial distribution was applied followed by a paired Wald-test between preserved and lesioned osteoarthritis cartilage and subchondral bone samples. Benja- mini-Hochberg multiple testing corrected p-values with significance cut-off of 0.05 are reported as False Discovery Rate (FDR).