2.5. Gut Microbiota Data
LYSIS
In total, 621 feces samples were analyzed. Three-level constrained randomization assured that the participant order was randomized, and that samples from the same participant ID were analyzed next to each other in the same batch, that the order of the interventions for the participant ID were randomized, and that the order of intervention and preceding run-in/washout week was also randomized. DNA was isolated from the feces samples with QIAamp DNA Stool Mini kit (Qiagen, Hilden, Germany) with an additional bead-beating step to increase efficiency in the lysis of bacterial cell walls. The bead beating was carried out with 0.1-mm Silica/Zirconia beads for 2 × 45 s using a Precellys homogenizer (Bertin Technologies, Montigny-le-Bretonneux, France). Purified DNA was stored at −20 °C until analysis. The 16S RNA gene amplicon libraries were generated by amplifying the V3-V4 region of 16S rRNA genes with the primers (341F 5′-CCTACGGGAGGCAGCAG-3′ and 806R 5′-GGACTACNNGGGTATCTAAT-3′). PCR results were purified with GeneJET Gel Extraction Kit (Thermo Fisher Scientific, Waltham, MA, USA). Sequencing libraries were generated using NEB NextThe raw demultiplexed reads from the sequencing were processed using DADA2 to denoise (filterAndTrim parameters: maxN = 0, maxEE = c (2,2), truncQ = 2, rm.phix = TRUE, compress = TRUE), dereplicate reads, merge paired-end reads and remove chimeras [