title: Shotgun sequencing of humanM stool
design: The DNA was extracted from each sample using the ZymoBIOMICS DNA extraction protocol (Zymo Research, USA). The DNA purity was quantified using a Nanodrop Spectrophotometer (NanoDrop Technology, USA) by measuring the A260/280 and A260/230 ratios. The DNA concentration was quantified using a Qubit 3.0 Fluorometer (Invitrogen, USA). The integrity of the extracted DNA was evaluated with agarose gel electrophoresis (Thermo Scientific, USA). Extracted DNA samples were stored at 4 C for 24 hours before sequencing. Each extracted genomic DNA sample was adjusted to 0.2 ng/L and then prepared for sequencing with  a Nextera XT DNA Sample Prep Kit (Illumina Inc., USA). In total, 1 ng of each sample was used for tagmentation, as directed by the manufacturer. After tagmentation, PCR amplification was done using a unique combination of barcode primers for each of the 6 samples. Then, each DNA library was purified using AMPure XP beads (Beckman Coulter, USA) and normalized with Library Normalization beads/additives. Normalized libraries were pooled, and pair-end sequencing was performed using the MiSeq Reagent Kit v3 (300 cycles) through the Illumina MiSeq platform.