Association of microbial pathways predicted/inferred using 16S and shotgun metagenome with the abundance of faecal metabolome

Faecal metabolomics profiling was carried out at the Parkville node of the Monash Proteomics and Metabolomics Platform. Briefly, the freeze-dried faecal samples were subjected to extraction using a methanol-water mixture (2:1). An internal standard, 3-[(3-Cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS), was added at a concentration of 1 µM. The faecal metabolome was measured using a Q-Exactive Orbitrap mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) coupled with a Dionex Ultimate® 3000 RS high-performance liquid chromatography (HPLC) system (Thermo Scientific, Waltham, MA, USA). Chromatographic separation was achieved with a ZIC-pHILIC column (5 µm, 4.6 × 150 mm, SeQuant®, Merck) (Merck Millipore, Victoria, Australia) equipped with a guard column (ZIC-pHILIC). The mass spectrometer operated in full scan mode with both positive and negative ionization. Retention times for standards from the IROA MSMLS library were extracted using the targeted peak detection module in MZmine 2.32, implemented via the RT Extractor Preprocess 2.1.pl script, and manually verified in MZmine 2.32. LC-MS data processing was carried out using IDEOMv20 with default parameters to identify features and remove artefacts (fragments, adducts, isotopes, background, etc). Metabolites were identified (MSI level 1) based on accurate mass and standard retention time. Putative metabolite annotation of other features was based on accurate mass and predicted retention time.