Single-Run Consolidation of Targeted Next-Generation Sequencing Assays for Pathogen Identification, Genotyping, and Antimicrobial Resistance Profiling on the DNBSEQ-E25 Platform: A Proof-of-Concept Study

Background/Objectives: Routine diagnostic microbiology relies on separate workflows for pathogen identification, resistance genotyping, and molecular surveillance. This proof-of-concept study evaluated the feasibility of consolidating targeted assays for bacterial pathogens and DNA and RNA viruses within a single DNBSEQ-E25 sequencing run coupled with automated bioinformatic interpretation. Methods: Twenty-five libraries generated from 21 unique external quality assessment materials, reference strains, and anonymized residual clinical specimens were analysed. Targeted assays covered Mycobacterium tuberculosis resistance-associated regions, human immunodeficiency virus type 1 (HIV-1), cytomegalovirus (CMV), full-length bacterial 16S rRNA, and influenza A/B. Outcomes included on-target sequencing yield, coverage, and pathogen-specific resistance and typing results. Results: The 25 libraries generated 39,962,528 reads, with 82.6% mapping to the intended targets. Both tuberculosis materials yielded resistance profiles concordant with the expected results. HIV-1 pol libraries achieved complete target coverage, whereas whole-genome coverage reached 89.4–91.1%. Both cytomegalovirus formats achieved complete target coverage except for UL54 in the multiplex format, reflecting substantial UL54 depth imbalance. Bacterial 16S results were consistent with the reference organisms, and influenza sequencing enabled subtype and clade assignment. Conclusions: These findings support the technical feasibility of consolidating the evaluated targeted assays within a single DNBSEQ-E25 run. The limited sample set and absence of replicate runs preclude conclusions regarding reproducibility or clinical performance.