BactoFlow: An Automated and Reproducible Pipeline for High-Resolution Bacterial Genomics

Next-generation sequencing has transformed bacterial genomics, but fragmented bioinformatics tools restrict accessibility. BactoFlow is a modular, automated, end-to-end pipeline, integrating quality control, mapping, assembly, annotation, resistance and virulence screening, antiviral defense detection and publication-ready visualization. Conda-managed environments and checkpoint-based execution for reproducible, scalable and fault-tolerant genomic analyses. We used BactoFlow to characterize a clinical Pseudomonas aeruginosa isolate (SRR33893847) and assigned it by MLST to sequence type ST308, a globally disseminated, high-risk clone with a well-documented association with multidrug resistance. Quality trimming with Trimmomatic retained 92.4% of 1,661,330 raw reads. Subsequent alignment via BWA-MEM enabled FreeBayes to identify 56,587 high-confidence variants, reflecting substantial genomic divergence from the reference strain. De novo assembly with SPAdes produced a 6.92 Mb draft genome distributed across 73 contigs, with an N50 of 412,000 bp and a GC content of 66.11%. Prokka annotation identified 6,380 coding sequences along with 68 tRNA genes and 3 rRNA features. Multi-database functional screening filled out a detailed resistance and virulence profile: CARD detected 51 antimicrobial resistance genes, VFDB flagged 290 virulence-associated factors, and PADLOC identified 12 antiviral defense systems, among them Gabija, RosmerTA, and zorya_type_I, alongside 3 integrated prophage regions. Overall, these findings validate a genomically well-equipped and clinically significant isolate. BactoFlow offers a scalable, accessible solution for researchers seeking high-resolution bacterial genomics without the overhead of assembling and managing separate tools.