[Comparison of 16S rRNA gene hypervariable regions V3-V4 and V4 sequencing results of gut microbiota in obese children with non-alcoholic fatty liver disease].

objectives16S 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.

methodsObese 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.

resultsA 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

conclusionsSequencing 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.