Flow cell adapters and indexing barcodes

Immediately upon receipt, the fecal samples were homogenized, aliquoted, and stored at −80°C until the day of analysis. Total DNA was extracted from fecal samples using the Qiagen Fast QiaAmp DNA Stool Mini Kit (QIAGEN, Valencia, CA, USA) according to the manufacturer’s protocol with the inclusion of a physical lysis step. The sample was lysed in FastPrep-24 (MPBio, Derby, UK) with lysis matrix E tubes (MPBio, Derby, UK) twice at 6.5 m/s for 45 seconds. The hypervariable V4 region of the 16s rRNA gene was amplified by polymerase chain reaction with 515-806 primers tailed with sequences to incorporate Illumina flow cell adapters and indexing barcodes (Illumina, San Diego, USA).
Primer dimers and low-molecular-weight products were removed using Agencourt Ampure Beads (Beckman Coulter, Spain) and samples were quantified and quality checked for amplicon size using the 4200 TapeStation (Agilent Technologies, Santa Clara, CA, USA). Amplicons were pooled in equimolar amounts and sequenced (2 × 250) on an Illumina Miseq system (Illumina, San Diego, USA) according to standard protocols.

Immediately upon receipt, the faecal samples were homogenized, aliquoted and stored at −80°C until the day of analysis. Total DNA was extracted from faecal samples using the Qiagen Fast QiaAmp DNA stool mini kit (QIAGEN, Valencia, CA, USA) according to the manufacturer’s protocol with the inclusion of a physical lysis step. The sample was lysed twice at 6.5m/s for 45 seconds in FastPrep-24 (MPBio, Derby, UK) with lysis matrix tubes E (MPBio, Derby, UK). The hypervariable V4 region of the 16S rRNA gene was amplified by PCR, with 515-806 primers (515:GTGCCAGCMGCCGCGGTAA, 806:GGACTACHVGGGTWTCTAAT) tailed with sequences to incorporate Illumina flow cell adapters and indexing barcodes (Illumina, San Diego, CA, USA). PCR amplification program was 1 cycle of 98°C 30 seconds; 25 cycles of 98°C 10 seconds, 60°C 20 seconds, 72°C 20 seconds; 1 cycle of 72°C 2 minutes. Q5® High-Fidelity 2X Master Mix (New England Biolabs, Ipswich, MA, USA)
Primer dimers and low-molecular-weight products were removed using Agencourt Ampure Beads (Beckman Coulter, Spain). Samples were quantified and quality was checked for amplicon size using the 4200 TapeStation (Agilent Technologies, Santa Clara, CA, USA). Amplicons were equimolar pooled and sequenced (2 × 250) on an Illumina Miseq platform (Illumina, San Diego, CA, USA) according to standard protocols.