Stool sample collection, DNA extraction, and metagenomic sequencing
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To assess taxonomic and functional changes in fecal samples collected we used shotgun sequencing of stool DNA for whole metagenome analysis. Patients collected their fresh stool samples at home, which were then immediately frozen in their home freezer at -20°C. Frozen samples were delivered to the hospital within two days using insulating polystyrene foam containers and were kept at -80°C until analysis. DNA extraction was performed using the QIAamp DNA stool kit (Qiagen, Hilden, Germany). DNA quantification was performed using a Qubit 3.0 Fluorometer (Thermo Fisher Scientific, Carlsbad, CA), and one ng of each sample (0.2 ng/μL) was used for shotgun library preparation using the Nextera XT DNA Library Preparation Kit (Illumina, Inc., San Diego, CA). Sequencing was carried out on a NextSeq 500 sequencer (Illumina) with 150-bp paired-end chemistry at the Sequencing and Bioinformatic Service of FISABIO (Valencia, Spain).Taxonomic assignments of total DNA metagenomic sequencing were carried out through Kaiju (