Advancements and challenges in bioinformatics tools for microbial genomics in the last decade: Toward the smart...

Over the past decade, microbial genomics has been transformed by advances in sequencing technologies and bioinformatics, enabling the transition from targeted gene markers to complete genome assemblies and ecological scale metagenomic surveys. This review presents a comprehensive overview of the bioinformatics pipelines that structure this field, from sample preparation, PCR amplification, and next-generation sequencing (NGS) to read preprocessing, genome assembly, polishing, structural and functional annotation, and submission to public databases. We highlight the major tools that have become standards at each stage, including FastQC, SPAdes, Prokka, Bakta, CARD, GTDB-Tk, QIIME 2, and Kraken2, while also emphasizing recent innovations such as hybrid assemblers, ontology-driven annotation frameworks, and automated workflows (nf-core, Bactopia). Applications extend across microbiology, from antimicrobial resistance surveillance and phylogenetic classification to ecological studies, exemplified here by three case studies: termite gut microbiota profiling by 16S metabarcoding, the description of new Bartonella species from bats, and the genomic characterization of rare Salmonella enterica serovars from primates. Despite these advances, persistent challenges remain, including incomplete and biased reference databases, computational bottlenecks, and economic disparities in sequencing and storage capacities. In response, international initiatives increasingly promote open, interoperable, and reusable bioinformatics infrastructures. Conforming to the Findable, Accessible, Interoperable, Reusable (FAIR) principles and global frameworks such as Global Alliance for Genomics and Health (GA4GH), these efforts are driving greater standardization, transparency, and data sharing across the microbial genomics community. Future perspectives point toward the integration of artificial intelligence, long-read and telomere-to-telomere (T2T) sequencing, cloud-native infrastructures, and even quantum computing, paving the way for a predictive, reproducible, and globally inclusive microbial genomics.