Metagenomic DNA extraction for shotgun metagenomic sequencing.
The QIAamp Fast DNA stool minikit (Qiagen GmbH, Hilden, Germany) was used to extract metagenomic DNA from fecal samples. The DNA concentration and quality were checked by using a Nanodrop spectrophotometer and the Qubit double-stranded DNA (dsDNA) assay kit in combination with a Qubit 2.0 fluorometer (Life Technologies, CA, USA), and 1% agarose gel electrophoresis was used to evaluate DNA integrity. Shotgun metagenomic sequencing was performed on all samples using an Illumina NovaSeq instrument, and the NEBNext Ultra DNA library prep kit (New England BioLabs [NEB]) was used to construct the library. All operations were done according to the manufacturers’ instructions. A total of 96 stool samples were subjected to shotgun sequencing (probiotics group, 