Comparison of PCA and Welch’s t-test analysis results at the phylum, class, order, and family levels of gut microbiota in obese children with NAFLD using 16S rRNA gene V3-V4, V4 regions, and metagenomic sequencing

Select obese children aged 6-18 who visited the outpatient departments of the Children's Health Center and Liver Disease Center at Hunan Children's Hospital from January 2020 to September 2021 as the sample source. The case group consisted of obese children with NAFLD, while the control group consisted of simply obese children. Collect fecal samples, extract total DNA from the samples, perform PCR amplification and sequencing on the V3-V4 and V4 regions of the gut microbiota, and compare the differences in alpha and beta diversity and microbiota structure between the two highly variable regions after amplification. Select 7 samples for metagenomic sequencing as the gold standard. A total of 145 research subjects were included, with 92 in the case group and 53 in the control group. SPSS 26.0, STAMP 2.1.3 statistical software, and OmicShare platform were used for data analysis. Compare the Shannon index and Chao 1 index between the V3-V4 and V4 regions using a two sample t-test. PCA and PCoA analysis were used to evaluate the β diversity of microbial communities at the phylum, class, order, family, and genus levels in different populations using V3-V4 and V4 sequencing, and to observe the overall differences in microbial communities between different groups. Welch's t-test was used to analyze and compare the differential microbial communities at different levels among different populations using V3-V4 and V4 sequencing, and multiple corrections were performed using the Benjamini Hochberg FDR method. Figure 1 shows the PCA analysis of different taxonomic levels of microbial communities obtained from the V3-V4 and V4 regions of the total population, including (A) phyla, (B) classes, (C) orders, and (D) families. Figure 2 shows PCA analysis of different taxonomic levels of microbiota obtained from the V3-V4 and V4 regions of the case group and control group, (A) phylum, (B) class, (C) order, and (D). Figure 3-6 shows the differential phyla, classes, orders, and families obtained from sequencing the V3-V4 and V4 regions of the total population sample. Figure 7-10 shows the differential phyla, classes, orders, and families obtained from sequencing the V3-V4 and V4 regions of the case group samples. Figure 11-13 shows the differential bacterial classes, orders, and families obtained by sequencing the V3-V4 and V4 regions of the control group samples. Figure 14-15 shows the comparison of metagene sequencing with Shannon index and Chao1 index in V4 and V3-V4 regions (* * represents P<0.01). Figure 16 shows the PCA analysis of microbial communities obtained from metagenomic sequencing and V3-V4 and V4 regions at different taxonomic levels, including phylum (A), class (B), order (C), family (D), and genus (E). Table 1 shows the inter group differences in Anosim levels of microbial communities between metagene sequencing and V3-V4 and V4 region sequencing. Figure 17-19 shows the differential bacterial orders, families, and genera obtained from metagene and V3-V4 region sequencing. Figures 20-21 show the differential bacterial families and genera obtained from metagene and V4 region sequencing.