Cindy Boer

Microbiome Composition, Joint Pain and Inflammation | 243 5.2 CAG) −806R (GGACTACHVGGGTWTCTAAT) primer pair and dual indexing[45], a full list of all primers used can be found in Supplementary Table 9. Amplicons were normalized using the SequalPrep Normalization Plate kit (Thermo Fischer Scientific) and pooled. Amplicon pools were purified prior to sequencing (Agencourt AMPure XP, Beckman Coulter Life Science, Indianapolis, IN) and size and quantity was assessed (LabChip GX, PerkinElmer Inc., Groningen, The Netherlands). A Control library was added to ~10% of each amplicon pool as a positive control (PhiX Control v3 library, Illumina Inc., San Diego, CA). The hypervariable V3 and V4 regions of the 16S rRNA gene were sequenced in paired-end mode (2×300bp) using the MiSeq platform (Illumina Inc., San Diego, CA) with an average depth of 50,000 paired reads per sample. Data processing and quali- ty control: Sequence read quality control and taxonomic classification, was done using an in-house pipeline (µRAPtor) based on QIIME version 1.9.0[46] and UPARSE version 8.1[47]. Low-quality, merged, and chimeric reads were excluded. Duplicate samples, samples with <10,000 reads, and samples from participants that have used antibiotics (self-reported) in the 6 months prior to sample production were excluded. The remain- der of the reads (~93%) were normalized using random 10,000 read subsampling (rar- efication). To reconstruct taxonomic composition a direct classification of 16S sequenc- ing reads using RDP classifier (2.12) and the SILVA 16S rRNA database (relase.128)[48]. This was done for each taxonomic level available: domain, phylum, class, order, family, and genus, with binning posterior probability cut-off of 0.8. The microbial Shannon di- versity index was calculated on the taxonomic level of genera, using vegan package in R. Relative abundancies were calculated for each taxonomic level prior to any additional QC (domain, phylum, class, order, family and genus). Unknown or unassigned classifi- cations were excluded from the final dataset (n=69), as these currently cannot be used for clinical or therapeutic applications. In total, we were left with gut-microbiome tax- onomies for 1427 individuals and 596 taxonomic classifications for analysis (Figure 1). Phenotype descriptions Osteoarthritic knee joint pain was determined by the WOMAC questionnaire, which is a disease specific questionnaire to assess the severity of hip and knee OA, consisting of 24 items covering three domains: pain, stiffness, and function[49]. The WOMAC includes five items that measure OA joint specific pain, scored on a 5-point Likert scale, 0–4. The five knee specific WOMAC-pain scores were summed to create the knee WOMAC pain score, ranging from 0 to 20, where higher scores represent worse OA-related knee joint pain. Knee OA severity was determined by the radiographic Kellgren and Law- rence score (KL-score)[50]. Using radiographs of both knee joints, left and right, the KL-score was determined for each joint. Scores were subsequently summed for the left and right knee to form the Knee KLsum score. Knee joint effusion could be determined,