Shotgun metagenome analysis
Three representative samples were selected from the C2, M1, and M3 groups based on the criteria that the samples showed the highest differences in the unweighted pair group method with an arithmetic mean tree comparing samples of 16S rRNA sequencing data (data not shown)). Sequencing libraries were prepared according to the manufacturer’s instructions using the TruSeq Nano DNA High-Throughput Library Prep Kit (Illumina). Briefly, 100 ng of genomic DNA was sheared using adaptive focused acoustic technology (Covaris, Woburn, MA, USA), and the fragmented DNA was end-repaired to create 5’-phosphorylated, blunt-ended dsDNA molecules. Following end-repair, the DNA was size-selected using a bead-based method. These DNA fragments went through the addition of a single ‘A’ base and ligation of the TruSeq DNA UD Indexing adapters. The products were purified and enriched by PCR to create the final DNA library. The libraries were quantified using qPCR according to the qPCR Quantification Protocol Guide (KAPA Library Quantification kits for Illumina Sequencing platforms) and qualified using the TapeStation D1000 ScreenTape (Agilent Technologies). We then sequenced the data using NovaSeq (Illumina).After sequencing was completed, the raw data were demultiplexed using index sequences, and paired-end FASTQ files were generated for each sample. Adapter sequences and data with an average phred quality score < 20 were removed using trimmomatic (version 0.39)