71_pea protein diet human fecal metagenome
Fecal samples were homogenized in a Qiagen PowerBead glass 0.1 mm tube (13118-50) using the Promega Maxwell RSC PureFood GMO and Authentication Kit (AS1600), 1mL of CTAB buffer & 20l of RNAse A Solution. The sample/buffer was vortexed, incubated at 95C for 5 minutes while shaking at 1500 rpm, and then vortexed at high speed for 20 minutes. The sample centrifuged at 40C, 12700 rpm for 10 minutes. Using an automated Promega MaxPrep Liquid Handler instrument loaded with proteinase K, lysis buffer, and elution buffer. 300 l of homogenized sample is used for DNA extraction. After DNA elution, DNA is quantified using Quant-iT dsDNA High Sensitivity Assay Kit using Promega GloMax plate reader on a microplate (655087). To generate metagenomic shotgun sequencing libraries, the Illumina Nextera XT DNA Library Prep Kit Reference Guide was used. DNA sequencing libraries were washed using Beckman Coulter AMPure XP magnetic beads. Library quality & size verification was performed using the Revvity Labchip GX Touch HT instrument with DNA 1K Reagent Kit (CLS760673). Library concentrations were quantified using Quant-iT dsDNA High Sensitivity Assay Kit using Promega GloMax plate reader on a microplate (655087). Library molarity was calculated based on library peak size & concentration. Libraries were normalized to 2nM using the Hamilton Microlab NIMBUS instrument and pooled together using the same volume across all normalized libraries into a 1.5ml Eppendorf DNA tube (022431021). Pooled libraries were sequenced on the Illumina NovaSeq X instrument at loading concentration of 0.13nM + 5% PhiX, paired-end 150bp. Quality-based filtering and removal of host sequences and adapter and quality trimming was performed using kneadData (https://huttenhower.sph.harvard.edu/kneaddata/) version 0.10.0, using the hg37 human reference genome. Taxonomic composition was calculated using MetaPhlAn4 (Blanco-Miguez et al. 2023). Abundance of gene families and metabolic pathways were quantified using HUMAnN3 (version 3.6, Beghini et al. 2021).  Gene families and pathways were renormalized to relative abundances after removing unmapped/ungrouped counts. Differential abundance analysis was performed with MaAslin2 (Mallick et al. 2021) and ANCOM-BC2 (Lin & Peddada 2024), using a mixed model with subject as random effect and a fixed effect for the primary grouping variable. Diversity analysis was performed with QIIME2 (Boylen et al. 2019).