Cindy Boer

244 | Chapter 5.2 for an all-female random subset of the Rotterdam Study III cohort (RSIII), by knee MRIs using a 1.5T MRI scanner (General Electric Healthcare, Milwaukee, Wisconsin, USA). For further detail of our used MRI protocol see ref. [51]. All MRI images were scored by a trained reader (blinded for clinical, radiographic and genetic data). Joint effusion was determined in the tibiofemoral joint (TFJ) and in the patellofemoral joint (PFJ) togeth- er (grade 0–3: 0=no joint effusion, 1= small joint effusion, 2=moderate joint effusion, and 3= severe joint effusion). The scores of the left and the right knee were summed, resulting in a score from 0 to 6, where higher scores represent more severe knee joint effusion. For all phenotypes, if data on either the left or right knee joint was missing, individuals were excluded. Oral medication usage such as, proton pump inhibitors (PPIs) and non-steroidal anti-inflammatory drugs (NSAIDs), were determined by ques- tionnaire at the same time point as WOMAC scores, knee X-rays, knee MRIs, and stool sample collection. Lifelines-DEEP replication cohort The LifeLines-DEEP (LLD) cohort is a subcohort of the LifeLines cohort (167,729 partic- ipants) that employs a broad range of investigative procedures to assess the biomedical, socio-demographic, behavioral, physical and psychological factors that contribute to health and disease in the general Dutch population[52]. A subset of approximately 1500 participants was included in LLD subcohort. For these participants, additional biologi- cal materials were collected, including analysis of the gut-microbiome composition. The collection, phenotyping, and processing of LLD have been described in detail[27,53]. Briefly, microbiome data was generated for 1179 LLD samples. Fecal samples were col- lected at home within two weeks of blood sample collection and stored immediately at 20 °C. After transport on dry ice, fecal samples were stored at −80 °C. Aliquots were made and DNA was isolated with the AllPrep DNA/RNA Mini Kit (Qiagen; cat. #80204). The 16S rRNA gene of the isolated DNA was sequenced at the Broad Institute, Boston, using Illumina MiSeq pair-ends. Hypervariable region V4 was selected using forward primer 515F (GTGCCAGCMGCCGCGGTAA) and reverse primer 806R (GGACTACHVGG- GTWTCTAAT) (Supplementary Table 9)[54]. Closed-reference OUT picking has been done with 97% similarity cutoff using UCLUST[55] program and GreenGenes 13.5 reference database[56] from QIIME[46] software. Overall, for 878 samples, both WO- MAC scores and microbiome information was available. The library size of microbial sequencing was rarefied to 10,000 read-depth using the rarefy function in R package vegan (version 2.5–2). At this depth, 11 subjects were excluded. After the exclusion step, we had 867 samples (362 men and 505 women) remained for the final analysis. Their characteristics are summarized in Supplementary Table 7. The LifeLines-DEEP study has been approved by the medical ethical committee of the University Medical Center Groningen, The Netherlands.