## 2 Materials and Methods
### Metagenomic Sequencing and Analysis of Data  
The amplification of PCR products for the 16S region with 16 SV34 primers was successful for the five samples. The amplicon was sequenced on the Illumina paired-end platform to generate 250 bp paired-end raw reads (Raw PE), and then merged and pretreated to obtain Clean Tags [14]. The chimeric sequences in the Clean Tags were detected and removed to get the Effective Tags, which can be used for subsequent analysis, as was published by [14; 15].  
Metagenomic sequencing was carried out at Novogene (Cambridge, UK). Library preparation was performed with a Nextera DNA Flex kit (Illumina), following standard procedure. The amplicon was sequenced on the Illumina paired-end platform to generate 377 bp paired-end raw reads (Raw PE), and then merged and pretreated to obtain clean tags. The data were processed by QIIME software, v.1.9.1 ([[http://qime.org/](http://qime.org/)]([http://qime.org/](http://qime.org/)) accessed on 15 February 2014), as published by [16]. The first stage of the 16S rRNA gene analysis involved quality control of the sequences to exclude from the analysis those with a length of fewer than 200 nucleotides, with a quality score of less than 25, with misread sequences of primers and multiplex identifiers, extensive homopolymer repeats (more than 8 nucleotides), and unidentified nucleotides. The chimeric sequences in the clean tags were removed to obtain effective tags, which were used for subsequent analysis [17; 18; 19]. The OTUs (operational taxonomic unit) were selected at more than 97% similarity. ACE and Chao indices and Shannon and Simpson diversity indices were calculated using the QIIME v. 1.9.1 [20]. Alpha diversity metrics summarize the structure of an ecological community by measuring the number of taxonomic groups together with the abundances of the groups, as was published by [PERSON] (2019) [21]. Alpha diversity was analysed by the utilization of six indices, including Observed-species and Shannon indices, as calculated using QIIME (Version 1.9.1) and displayed with R software (Version 2.15.3) [16; 22]. The heatmap based on the Weighted Unifrac and Unweighted Unifrac distances was analysed with R software (Version 2.15.3). The same R software was used to find the differences in the dominant taxa between the three groups of samples at each taxonomic rank; the top 10 taxa with the average abundance of the three groups of samples at each taxonomic rank were selected to generate a ternary plot. OTU comparisons were performed using the Venn diagram package (R software v.4.0.3) [22]. In our 16S information analysis process, the input file is the beta diversity distance matrix, which is the difference value matrix composed of the OTU abundance of two samples. After the statistical processing of the data, \(p\) values < 0.05 were considered statistically significant [23].  
The BioProject accession number provided for these SRA data is PRJNA1106797 with submission ID: SUB14407308, submitted on 28-04-2024 The BioSample accessions are SAMN41150171, SAMN41150172, SAMN41150173, SAMN41150174, and SAMN41150175. SRA records will be accessible with the following link: [[https://www.ncbi.nlm.nih.gov/sra/PRJNA1106797](https://www.ncbi.nlm.nih.gov/sra/PRJNA1106797)]([https://www.ncbi.nlm.nih.gov/sra/PRJNA1106797](https://www.ncbi.nlm.nih.gov/sra/PRJNA1106797)) released on 1 May 2024.  
Beta diversity, a comparative analysis of the microbial community composition, was carried out according to the taxonomy annotation results of all the samples and the abundance information of feature sequences [24]. The differences between different samples (groups) were found through Beta diversity index inter-group difference analysis, via the utilisation of the multivariate statistical method of Non-Metric Multidimensional Scaling (NMDS) [25]. For this analysis, samples with the closest distance are clustered together to form a new node. The average distance between the most abundant and other samples is calculated as the two closest samples.