Zhong Nan Da Xue Xue Bao Yi Xue Ban. 2025 Dec 28;50(12):2312-2324. doi: 10.11817/j.issn.1672-7347.2025.240565.
ABSTRACT
OBJECTIVES: 16S rRNA gene sequencing is an important method for studying microbial structure in samples. However, whether selecting different hypervariable regions for sequencing in the same sample affects the results remains unclear. This study aims to compare the sequencing results of 16S rRNA gene hypervariable regions V3 to V4 and V4 in children with obesity-related non-alcoholic fatty liver disease (NAFLD), and to provide evidence for scientifically evaluating gut microbiota detection results in obese children with NAFLD.
METHODS: Obese children with NAFLD and children with simple obesity who visited Hunan Children’s Hospital between January 2019 and September 2021 were selected as study subjects. Fecal samples were collected, and total DNA was extracted. After PCR amplification of the gut microbiota V3 to V4 region and V4 region, sequencing was performed. α-diversity, β-diversity, and microbial community structure differences between the 2 hypervariable regions were compared. Seven samples were selected for metagenomic sequencing as the gold standard to evaluate the performance of V3 to V4 and V4 region sequencing.
RESULTS: A total of 145 participants were included, including 92 in the case group and 53 in the control group. The number of operational taxonomic units (OTUs) obtained by V3 to V4 sequencing (16 977) was higher than that obtained by V4 sequencing (3 362). α-diversity analysis showed that in the overall population, the Shannon index (5.49±1.11) and Chao1 index (1 843.04±580.78) in the V3 to V4 region were higher than the Shannon index (4.98±0.65) and Chao1 index (379.59±47.27) in the V4 region (all P<0.001). β-diversity analysis showed overall differences in microbial community structure between the V3 to V4 and V4 regions, and the intergroup differences were greater than the intragroup differences (P<0.05). Welch’s t-test results showed that in the overall population, the numbers of differential taxa detected by V3 to V4 and V4 sequencing at the phylum, class, order, family, and genus levels were 2, 9, 35, 33, and 72, respectively; in the case group, the numbers were 1, 9, 32, 35, and 66; and in the control group, the numbers were 0, 7, 27, 21, and 0. Linear discriminant analysis effect size (LEfSe) analysis showed that V3 to V4 sequencing identified 29 differential taxa between the case group and control group, whereas V4 sequencing identified 7 differential taxa. Sensitivity analysis showed that the Shannon index obtained by V3 to V4 sequencing (5.41±1.62) was not significantly different from that of metagenomic sequencing (6.39±0.42) (P=0.169), while the Chao1 index (1 889.92±781.73) was lower than that of metagenomic sequencing (3 092.71±505.89), with a statistically significant difference (P<0.01). The Shannon index and Chao1 index obtained by V4 sequencing were both lower than those of metagenomic sequencing, with statistically significant differences (4.89±0.94 vs 6.39±0.42, 362.41±35.22 vs 3 092.71±505.89, respectively, both P<0.01).
CONCLUSIONS: Sequencing of the V3 to V4 and V4 regions of the 16S rRNA gene affects the results of gut microbiota structure analysis in obese children. The V3 to V4 region is more likely to detect differential taxa between case and control groups and provides a more accurate estimation of α-diversity. It may therefore be considered a preferred region for gut microbiota sequencing in children with NAFLD. However, there is currently no unified standard for selecting V regions in 16S rRNA gene sequencing, and the detection region and method should be selected comprehensively according to research objectives and sample characteristics.
PMID:42032992 | DOI:10.11817/j.issn.1672-7347.2025.240565
