Cindy Boer

The Gut Microbiome of Childen and Adults | 207 5.1 Upon arrival at Erasmus MC, samples were recorded and stored at −20 °C. The only modification in the collection protocol for the GenR cohort (as compared to RS) was that, in case of delay, the samples were stored by participants at 4 °C (home fridge) be- fore mailing to Erasmus MC. This modification allowed to better preserve samples that were produced in the evening or during the weekend. DNA isolation Stool samples from the two cohorts were randomly taken from the −20 °C freezer and allowed to thaw for 10minutes at room temperature prior to DNA isolation. Samples with inconsistent or lack of information on sample production and samples in which mold growth was observed were excluded ( Supplementary Figure 1 ). An aliquot of approximately 300mg was homogenized in stool stabilizing buffer according to the manufacturer’s protocol (Arrow Stool DNA; Isogen Life Science, De Meern, The Neth- erlands). Homogenized samples were bead beated in Lysing Matrix B tubes contain- ing 0.1mm silica beads (MP Biomedicals, LLC, Bio Connect Life Sciences BV, Huissen, The Netherlands) using the MagNA Lyser instrument (Roche Diagnostics, Almere, The Netherlands) at 7,000 rpm for 45 seconds. Samples were then centrifuged at 6,000×g for 5min and 0.5ml of supernatant was subjected to automated DNA isolation (Arrow; DiaSorin S.P.A., Saluggia, Italy) according to the manufacturer’s protocol using setting ‘Stool DNA 2.0’ in batches of 12 samples per run. DNA concentration was measured us- ing Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher Scientific, Waltham, MA) and DNA was stored at −20 °C. 16S rRNA gene sequencing The V3 and V4 variable regions of the 16S rRNA gene were amplified using the 309F-806R primer pair and dual indexing (12 base pairs (bp) each on the forward and reverse primers) as previously described[26]. Amplicons were normalized using the SequalPrep Normalization Plate kit (Thermo Fischer Scientific) and pooled. The pools were purified prior to sequencing using Agencourt AMPure XP (Beckman Coulter Life Science, Indianapolis, IN) and the amplicon size and quantity of the pools were assessed on the LabChip GX (PerkinElmer Inc., Groningen, The Netherlands). PhiX Control v3 library (Illumina Inc., San Diego, CA) was spiked into (~10%) the pooled amplicon li- braries and each pool was sequenced on an Illumina MiSeq sequencer (MiSeq Reagent Kit v3, 2×300bp) at an average depth of 50,000 read-pairs per sample.