Cindy Boer

242 | Chapter 5.2 Methods Rotterdam study cohort The Rotterdam study (RS) is a large population-based prospective study population (14,926 participants) ongoing since 1990 to study determinants of chronic disabling diseases. The first cohort RS-I, started in 1990 and includes individuals of 55 years and older living in the Ommoord district of Rotterdam in the Netherlands. In 2000, a second cohort was started RS-II with individuals who had become 55 years of age or moved into the study district since the start of the study. In 2006, the third cohort was initiat- ed, RSIII, of individuals aged>45, living in the Ommoord district. The cohorts are pre- dominantly Caucasian and a further detailed description of the design and rationale of the Rotterdam Study has been published elsewhere[44]. The Rotterdam Study has been approved by the Medical Ethical Committee of the Erasmus MC, University Medical Cen- ter Rotterdam, the Netherlands (MEC 02.1015). All subjects provided written consent prior to participation in the Rotterdam Study. Stool sample collection started in 2012 during the second visit of the Rotterdam Study III population (RSIII-2). A random group of 2440 participants was invited to provide a stool sample. In total 1691 (response rate=69%) stool samples were received via mail at the Erasmus MC for analysis of stool microbiome composition as a marker for gastrointestinal-microbiome composition. Af- ter quality control 1,427 samples remained for further analysis. Taxonomic profiling of gastrointestinal microbiota in RS Sample collection: Stool samples were collected at home by the participant using a Commode Specimen Collection System (Covidien, Mansfield, MA) and ~1g aliquot was transferred to a 25×76mm feces collection tube, which was sent via the regular mail to the Erasmus MC. Participants also filled out a short questionnaire addressing date and time of defecation, current or recent antibiotics use, current probiotics use, and recent travel activities. Upon arrival samples were stored (−20 °C), samples taking longer than 3 days to arrive at the Erasmus MC were excluded from further analysis, for the remain- ing samples TimeInMail (in days) was registered as a technical covariate. DNA isolation: For each participant, frozen stool samples were allowed to thaw for 10min at room temperature prior to DNA isolation. An Aliquot of ~300mg of stool was homogenized using 0.1mm silica beads (MP Biomedicals, LLC, Bio Connect Life Sciences BV, Huissen, The Netherlands) and DNA was isolated from the samples using the Arrow stool DNA kit according to the manufacturers’ protocol (Arrow Stool DNA; Isogen Life Science, de Meern, The Netherlands). 16S rRNA gene sequencing: The V3 and V4 hypervariable regions of the 16S rRNA gene were amplified using the 319F (ACTCCTACGGGAGGCAG-