design: DNA ExtractionStool samples collected from subjects and donors were stored in 2 mL cryovials and frozen at -80C. A single fecal pellet is deposited into a Qiagen PowerBead glass 0.1 mm tube (13118-50). Using a Promega Maxwell RSC PureFood GMO and Authentication Kit (AS1600), 1mL of CTAB buffer & 20l of RNAse A Solution is added to the PowerBead tube containing the sample. The sample/buffer is mixed for 10 seconds on a Vortex Genie2 and then incubated at 95C for 5 minutes on an Eppendorf ThermoMixer F2.0, shaking at 1500 rpm. The tube is removed and clipped to a horizontal microtube attachment on a Vortex Genie2 (SI-H524) and vortexed at high-speed for 20 minutes. The sample is removed from the Vortex and centrifuged on an Eppendorf Centrifuge 5430R at 40C, 12700 rpm for 10 minutes. Upon completion, the sample is centrifuged again for an ADDITIONAL 10 minutes to eliminate foam. The sample is checked for foam and particulates (they will clog the robotic instrument tips). If foam or particulates are found in the sample, they are carefully removed using P1000 pipette. DNA extraction is then processed via the Promega MaxPrep Liquid Handler and Promega Maxwell RSC 48 instruments. DNA is quantified using Quant-iT dsDNA High Sensitivity Assay Kit using Promega GloMax plate reader on a microplate (655087).Metagenomic Shotgun Sequencing Library GenerationLibrary generation follows the Illumina Nextera XT DNA Library Prep Kit Reference Guide:https://support.illumina.com/content/dam/illumina-support/documents/documentation/chemistry_documentation/samplepreps_nextera/nextera-xt/nextera-xt-library-prep-reference-guide-15031942-05.pdfLibrary Verification, Quality Check, PoolingDNA sequencing libraries are washed using Beckman Coulter AMPure XP magnetic beads. Library quality & size verification is performed using PerkinElmer LabChip GXII instrument with DNA 1K Reagent Kit (CLS760673). Library concentrations are quantified using Quant-iT dsDNA High Sensitivity Assay Kit using Promega GloMax plate reader on a microplate (655087). Library molarity is calculated based on library peak size & concentration. Libraries are normalized to 2nM using the PerkinElmer Zephyr G3 NGS Workstation (133750) and pooled together using the same volume across all normalized libraries into a 1.5ml Eppendorf DNA tube (022431021).SequencingPooled libraries are sequenced on the Illumina HiSeqX instrument at loading concentration of 10pM, paired-end 150bp.