Kingfisher flex automated purification system

Fecal samples were collected by the participants at home and frozen immediately. They were transported to the laboratory frozen and stored at − 80 °C until processing.
Bacterial DNA was extracted using a previously described repeated bead-beating method^{35 (link)} with the following modifications for automated DNA purification: ca. 125 mg of fecal material were suspended in 1 ml of sterile ice-cold PBS, and 175 μl of fecal suspension was combined with 235 μl of RBB lysis buffer (500 mM NaCl, 50 mM Tris–HCl (pH 8.0), 50 mM EDTA, 4% SDS) in a bead-beating tube from the Ambion Magmax™ Total Nucleic Acid Isolation Kit (Life Technologies, Carlsbad, CA, USA). After repeated bead-beating, 200 μl of the supernatant was used for DNA extraction with a KingFisher™ Flex automated purification system (ThermoFisher Scientific, Waltham, MA, USA) using a MagMAX™ Pathogen High Vol. Duo program. DNA was quantified using a Quanti-iT™ Pico Green dsDNA assay (Invitrogen, San Diego, CA, USA) and 1 ng was used for V3–V4-region amplicon PCR of the 16S rRNA gene as previously described^{36 (link)}. Sequencing was carried out with Illumina HiSeq 2500 equipment in Rapid Run mode.

Genomic DNA was isolated from the subclones by magnetic nanoparticles in 96-well format using NAxtra™ nucleic acids extraction kit (Lybe Scientific) and the KingFisher™ Flex automated purification system (Thermo Scientific). Barcoded amplicons for next generation (NG) sequencing were generated in two steps. The first PCR reactions were performed with genomic DNA using primers and conditions given in Supplementary Table S1 and S3, which are compatible with our in-house 96-well 6-nt-tag barcoding system. Second PCR reactions (same program) were performed with diluted PCR products as template (1 μl 1:10 diluted) and barcoded primer pars specific for each well in a 96-well plate. All PCR steps were performed with Q5^® Hot Start High-Fidelity 2X Master Mix (NEB), according to the manufacturer's instructions. All amplicons (∼200 bp) from each 96-well plate were pooled and purified by Quiaquick PCR purification kit (Quiagen), ∼1 μg DNA from each plate was pooled and sent to Eurofins Genomics for multiplexed amplicon sequencing (150 bp PE). Sequencing data were analysed in-house implementing CRISPResso, a computational pipeline for the analysis of CRISPR/Cas9 genome editing outcomes from deep sequencing data (25 (link)). Unique combinations of forward and reverse barcoded primers were used to demultiplex the sequencing data.

Bacterial DNA was extracted from the swabs using a previously described bead beating method^{25 (link)} with the following modifications: The swabs were vortexed in 0.5 ml of sterile ice-cold PBS, of which 175 μL was combined with 235 μL of RBB lysis buffer (500 mM NaCl, 50 mM Tris-HCl (pH 8.0), 50 mM EDTA, 4% SDS) in a bead beating tube. The samples were bead beaten using a FastPrep-24 instrument at 5.5 m/s (MP Biomedicals, Inc., USA) with 0.1 mm zirconium-silica beads (Biospec Products, Bartlesville, OK, USA) for 1 min. Samples were then heated at +95 °C for 15 min with shaking 400 rpm and centrifuged at room temperature for 5 min at 13 000 rpm. The supernatant (200 μL) was used for DNA extraction with KingFisher Flex automated purification system (ThermoFisher Scientific, USA) and Ambion Magma Total Nucleic Acid Isolation Kit (Life Technologies, USA) using MagMAX Pathogen High Vol Duo program. DNA was quantified using Quanti-iT Pico Green dsDNA Assay (Invitrogen, San Diego, CA, USA). An aliquot of the DNA extract was sent to Karolinska Institutet, Sweden for Human papillomavirus (HPV) genotyping^{49 (link)}.