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Advancements in NGS technologies have significantly expanded the scope and efficiency of metagenomics, surpassing traditional culture-based methods (Fig. 2).These innovations enable multi-domain microbial analysis through advanced bioinformatics tools, long-read sequencing, single-cell omics, and enhanced culture-based techniques, facilitating deeper insights into microbial diversity, function, and host interactions.The integration of cutting-edge computational tools and experimental methodologies has enhanced microbiome research, enabling the discovery of novel microbes, metabolic pathways, and therapeutic targets. Multi-domain microbial analysis and advanced bioinformatics tools.Recent metagenomic research has expanded beyond bacterial communities to encompass archaea, fungi, and viruses, fostering a more holistic understanding of the microbiome.Specialized databases such as IMG/VR for viruses 64 and fungiDB for fungal communities 65 facilitate comprehensive taxonomic and functional analyses.Advanced bioinformatics tools further enhance the ability to predict microbial functions and uncover disease-associated elements.For taxonomic classification, 16S rRNA reference databases, such as SILVA, Greengenes2 (GG2), and the Ribosomal Database Project (RDP) improve the accuracy of amplicon sequencing annotations 666768.Functional prediction tools, including PICRUSt/PICRUSt2, Tax4Fun, and FARPROTAX, provide expanded insights into microbial functions by leveraging enhanced genome reference databases 69707172.Metagenomic shotgun sequencing tools such as MetaPhlAn 3, Kraken2, and HUMAnN 3 allow for high-resolution taxonomic
Yang et al. (Wed,) studied this question.