2.4. Fecal Microbiota
LYSIS
During pre- and post-intervention study days, participants were asked to collect fecal samples using a stool sampling collection kit. Samples were taken using an EasySampler Stool Collector and a sample tube with a spoon lid. The sample was protected by being placed in yet another tube and stored immediately in the freezer (−20 °C). The samples were transported in a cooling box with an ice pack to the clinic within 72 h after the sample was collected. At the clinic, the sample is transferred to −80 °C within 24 h. The samples were analyzed with the 16S rRNA gene amplicon sequencing method, where DNA was extracted and purified from fecal samples using the QIAamp FAST DNA Stool mini kit from Qiagen, Venlo, The Netherlands. The DNA extraction followed the protocol from the manufacturer with one exception, where lysis of the bacterial cell walls used a mechanical lysis step (bead beating 2 × 1 min in a Precellys Evolution using 0.1 mm zirconium/silica beads). Once the DNA was extracted and purified from the sample, polymerase chain reaction (PCR) was used to amplify the V3-V4 region of the gene encoding 16S rRNA. This gene exists in all bacteria and is normally used for the taxonomic classification of bacteria since parts of the 16S gene vary in sequence composition between different bacteria. Sample-specific barcodes and Illumina adapters were then attached to the PCR amplicons to enable the pooling of samples. The library of PCR amplicons was then sequenced on the Illumina NovaSeq 6000 platform at Novogene, Singapore. The bioinformatics analysis to handle the generated sequence data used QIIME2 via the dada2 pipeline. The sequences were first demultiplexed, i.e., separated according to the sample-specific barcode in the specific sample. Then, quality control and filtration of the sequence data was performed to remove sequences with poor quality. Finally, a taxonomic classification of the sequences was performed. The gut microbiota analysis and subsequent data processing and analysis were described in detail by Iversen and Dicksved [Species that were known from the literature to associate with glucose regulation were selected to assess association with response clusters. The selected species were “