Baiye Sun

Amplicon sequencing provides a suitable approach for microbiome profiling, supported by a variety of R-based and web-based tools. In this review, we systematically evaluated eight freely accessible web-based tools suitable for users without scripting experience, comparing their performance across modules including alpha and beta diversity, taxonomic composition, differential comparison, network and correlation analysis, functional profiling, machine learning, tree-plot and user experience. While all tools exhibit limited data filtering and normalization options, performance varied considerably across modules. Mian and MicrobiomeAnalyst 2.0 excelled in alpha diversity analysis and taxonomic composition analysis, METAGENassist outperformed others in beta diversity, and MicrobiomeAnalyst 2.0 achieved the highest score in differential comparison and functional analysis. Namco and Mian outperform in network analysis and correlation analysis, respectively. Machine-learning functions were comparable across animalcules, MicrobiomeAnalyst 2.0 and METAGENassist, with the best treeplot visualization in animalcules and MicrobiomeAnalyst 2.0. And, user experience was highest for animalcules and Mian. Overall, MicrobiomeAnalyst 2.0 achieving the highest overall performance, followed by Mian and Namco. Several limitations among evaluated tools include inconsistent accessibility, diverse input data formats, restricted feature sets, and incomplete retention of key information in exported figures. Future development should integrate preprocessing, interactive visualization and figure export, alongside advanced statistical methods, multi-omics integration and meta-analytical capabilities, to enhance flexibility, reproducibility and interpretability. This comprehensive assessment provides a practical reference for researchers in selecting the most suitable web-based tools for specific microbiome analysis tasks, highlighting the importance of both module-specific performance and overall tool capabilities.