Performance of 16S rRNA Taxonomic Classification Tools in Metagenomic Data From a Beetle's Gut Microbiome

Understanding symbiosis between insects and their gut microbiota is critical for uncovering ecological and evolutionary processes. Taxonomic classification of microbial communities using the 16S rRNA gene is an initial standard approach, yet the accuracy and limitations of bioinformatic tools for this purpose, particularly in non-model organisms like beetles, are not well-studied. This study provides a novel benchmarking of three widely used classification tools-QIIME2, Mothur, and VSEARCH-on beetle gut metagenomic data, revealing new insights into their performance. Our results show that all tools achieved an ASV retrieval accuracy above 99 % with consistent taxonomic assignments. Based on these findings, all three tools are suitable for analyzing 16S data from insect gut samples, with the choice depending on specific research objectives or computational limitations. We emphasize the need for careful selection of reference databases to maximize taxonomic accuracy, contributing valuable guidelines for future studies involving complex microbial communities in underexplored hosts.