Bacterial Lysis Observation Over Time Assay (BLOTS) for High-Throughput Isolation, Sequencing, and Characterization of Bacteriophages.

Phages remain severely under-sampled, largely due to the limitations of current isolation methods, which are labor-intensive, low-throughput, and often restricted to a narrow set of bacterial hosts. To address this gap, we developed a robust and scalable field-to-genome workflow that leverages laboratory automation to screen environmental samples against a comprehensive 188-strain bacterial host library using a 384-well plate format. We demonstrate the workflow using Acinetobacter baumannii as the primary model host. Phage activity is detected through Bacterial Lysis Observation over Time assay (BLOTS) using spectrophotometry. Subsequently, isolation and subculture of the phages are done with a single layer agar spot assay. Phage genomes are then sequenced using long-read technology and annotated. This enables systematic and reproducible phage detection and enrichment from diverse sample types. The use of large numbers of host strains streamlines phage discovery and substantially increases the chances of isolating novel phages. The BLOTS high-throughput approach can also be used for screening phage collections against specified clinical hosts of interest for phage research and candidate selection for phage therapy applications. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Preparation of host libraries and multi-host primary enrichment Basic Protocol 2: Preparation of the BLOTS screening plate using an automated liquid handler Alternate Protocol 1: Manual phage screening without laboratory automation Basic Protocol 3: Phage screening using BLOTS Basic Protocol 4: Phage validation using the single-layer agar spot assay Basic Protocol 5: Phage amplification for downstream applications Basic Protocol 6: Phage concentration using PEG-8000 precipitation Alternate Protocol 2: Phage concentration using centrifugal filtration Basic Protocol 7: Automated DNA extraction with magnetic beads Basic Protocol 8: Whole-genome sequencing using long reads Basic Protocol 9: De novo genome assembly using the Phage Galaxy platform Basic Protocol 10: Annotation and characterization of phage genome Basic Protocol 11: Host range testing and characterization of isolated phages using BLOTS.