Long-read shotgun sequencing
We applied long-read sequencing to faecal samples obtained at the 0- and 3-month timepoints from each of the 15 upper-quartile WLZ responders in the MDCF-2 treatment group. Aliquots containing 400–1,000 ng of DNA from each biospecimen were transferred to a 96-well, 0.8 ml, deep-well plate (Nunc, Thermo Fisher Scientific) and prepared for long-read sequencing using the SMRTbell Express Template Prep Kit 2.0 (PacBio). All subsequent DNA handling and transfer steps were performed with wide-bore, genomic DNA pipette tips (ART, Thermo Fisher Scientific). Barcoded adapters were ligated to A-tailed DNA fragments by overnight incubation at 20 °C. Adapter-ligated fragments were then treated using the SMRTbell Enzyme Cleanup Kit to remove damaged or partial SMRTbell templates. A high molecular mass DNA fraction was purified using AMPure beads (ratio of 0.45× well-mixed AMPure bead volume to sample) and eluted in 12 µl of PacBio elution buffer. DNA libraries were sequenced on the Sequel System (Pacific Biosciences) using the Sequel Binding Kit 3.0 and Sequencing Primer v4 with 24 h of data collection. A total of 3.0 × 10Hybrid assembly of short- and long-read data was performed using OPERA-MS