Metagenomic Analysis of Gut Microbiome

DNA was extracted from ∼0.1 g aliquots of the fecal samples using the NucleoSpin 96 Soil (Macherey-Nagel) kit. Fragmented DNA was used for library construction using a NEBNext Ultra Library Prep Kit for Illumina (New England Biolabs). Quantitative real-time PCR (qPCR) was used to determine the concentration of the final library before sequencing. The library was sequenced using 2 × 150-bp paired-end sequencing on the Illumina platform. The metagenomic analysis was performed using the metagenomic species (MGS) concept (17 (link)) and the Clinical Microbiomics human gut MGS database. For taxonomic abundance profiling, we used the Clinical Microbiomics HGMGS version HG4.D.1 set of 2095 metagenomic species (MGS), each represented by a set of genes with highly coherent abundance profiles and base compositions in the 12,170 metagenomes, which includes 481 from infants, 9,428 publicly available metagenomes (18 (link)), and 3,567 publicly available genome assemblies from isolated microbial strains. Individual high-quality non-host reads were mapped to a gene if the mapping quality (MAPQ) was ≥ 20, and the reads aligned with ≥ 95% identity over ≥ 100 base pairs.