Methods to validate samples

Frozen stool samples were thawed and approximately 10 mg was used for DNA extraction using a QIAamp Fast DNA Stool Mini Kit (Qiagen, Hilden, Germany) using the manufacturer’s instruction with the following modifications. Contents were incubated for 45 min at 37 °C in lysozyme-mutanolysin buffer (22 mg/ml lysozyme, 0.1 U/ml mutanolysin, 20 mM TrisHCl, 2 mM EDTA, 1.2% Triton-x, pH 8.0), before homogenization for 150 s with 0.1 mm zirconia beads. Samples were then incubated at 95 °C for 5 min with InhibitEX Buffer and incubated at 70 °C for 10 min with Proteinase K and Buffer AL. Following this step, the QIAamp Fast DNA Stool Mini Kit isolation protocol was followed, beginning with the ethanol step. DNA was quantified with the Qubit 2.0 Fluorimeter (Life Technologies, Carlsbad, CA) using the dsDNA Broad Range Assay Kit.

Here we described briefly the validation analysis for detecting short chain fatty acid (SCFA) measurement in stools. Details including the chemicals needed, SCFA derivatization and sample preparation, and validation analysis are included in the Supplemental Appendix.

The SCFAs are generally not easy to be detected via Liquid Chromatography – Mass Spectrometry (LC-MS) system due to their volatility. Therefore, a chemical derivatization method is needed to increase their detectability as previously reported [27]. Feces were prepared with a 2:1 propanol ratio (w/w), and ¹³C₄-sodium butyrate was spiked into these samples and served as internal standards. Then samples were frozen at -80^oC until analysis. Before Ultrahigh Pressure Liquid Chromatography – High Resolution Mass Spectrometer (UPLC-HRMS) analysis, 60 µL of fecal sample solution was added 200 µL 50% acetonitrile for SCFAs extraction. After 2 min vortex, samples were centrifuged at 23,748 × g for 10 min and 40 µL supernatant was used for derivatization following the procedures outlined in the supplemental appendix. A Thermo Scientific Vanquish Flex UPLC coupled Q Exactive (QE) system was used to analyze derivatized SCFAs. According to the FDA guideline for bioanalytical method validation [28], the calibration curve, accuracy, precision, recovery and stability of targeted SCFAs were validated as described in our earlier study [29].