Mediterranean diet intervention for intestinal microecology in metabolic-related fatty liver disease

Fastq software processed raw Illumina sequencing data for quality assessment. Sequencing reads were assembled with MEGAHIT. Binning analysis was performed using vamb software and the results were screened against the following criteria: genome integrity >50% and contamination rate less than 10%. Unigenes were aligned with sequences of bacteria, fungi, archaea and viruses extracted from NCBI NR database using DIAMOND(15) software. Alpha diversity was measured to assess species diversity per sample. beta-diversity was estimated using Bray-Curtis distance based on principal coordinate analysis (PCoA), non-metric multidimensional scale analysis (NMDS), and principal component analysis (PCA) as appropriate. Statistical comparisons of bacterial abundance and diversity were conducted using the Wilcoxon rank-sum test and Welch's t-test. Heatmaps visualizing phylum- and genus-level taxonomic profiles were generated based on non-parametric Wilcoxon test results (p < 0.05). Taxa with significant abundance differences were assessed using LEfSe (LDA effect size) analysis. Inter-group differential analysis of gut microbiota was performed using STAMP software. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis (KOBAS) with FDR correction (Benjamini-Hochberg, p < 0.05) identified significantly enriched pathways in differentially expressed genes.