Zirconia bead mix

DNA was extracted using the QiAmp Fast DNA Stool Mini kit (Qiagen Ltd, West Sussex, UK). Collected samples were individually placed in 2-mL tubes prefilled with 0.1 mm silica and zirconia bead mix (Benchmark Scientific, Edison, USA), dissolved in 1 mL InhibitEX buffer (Qiagen Ltd, West Sussex, UK) and vortexed until homogenized. A bead-beating step (Beadbug microcentrifuge homogenizer, Benchmark Scientific, USA) was applied for 3 x 60 s at 5 m/s with 5-min rest in between. Total genomic DNA was eluted in sterile microcentrifuge tubes and quantified by Qubit Fluorimetric Quantitation (ThermoFisher Scientific Ltd, UK) as per manufacturer’s instructions. Metataxonomic sequencing (16S rRNA gene sequencing) was performed at Research & Testing RTL Genomics (Lubbock, TX, USA), using primers detecting the V1-V2 regions of the 16S rRNA gene plus bifidobacteria regions to generate 10,000 paired-ends reads on a Illumina MiSeq (Illumina, San Diego, USA.

A total of 29 scraped intestinal samples and 96 fecal pellets were individually placed in 2-mL tubes prefilled with 0.1 mm silica and zirconia bead mix (Benchmark Scientific, Edison, USA), dissolved in 1 mL InhibitEX buffer (Qiagen Ltd., West Sussex, UK) and vortexed until homogenized. A bead-beating step (Beadbug microcentrifuge homogenizer, Benchmark Scientific, USA) was applied for 3 × 60 s at 5 m/s with 5 min rest in-between. The DNA extraction has been performed with QiAmp Fast DNA Stool Mini kit (Qiagen Ltd., UK), following the manufacturer’s instruction. Total genomic DNA was eluted in sterile microcentrifuge tubes and quantified by Qubit Fluorimetric Quantitation (ThermoFisher Scientific Ltd., UK), following the manufacturer’s instructions. DNA aliquots were kept at − 20 °C until used. Sequencing of the variable regions of the 16S rRNA gene was performed at Research and Testing Laboratory LLC (Lubbock, Texas, USA). Primers used to amplify the V1–V2 regions of 16S rRNA gene were 28F (5′-GAGTTTGATCNTGGCTCAG-3′) and 388R (5′-TGCTGCCTCCCGTAGGAGT-3′). Sequencing was performed using an Illumina Miseq (Illumina, San Diego, USA), with 10K paired-end sequencing protocol.