Rolling out <i>Plaque-2-seq</i>: a single plaque sequencing approach enabling rapid, low-cost sequencing of phages directly from plaques

Rapid, accurate and scalable sequencing of bacteriophage genomes is critical to advance phage therapy, build phage biobanks and understand phage genomic diversity. Current methods are based on sequencing and assembling complete bacteriophage genomes using short- or long-read technologies. However, current protocols require large DNA input and are cost-prohibitive, which limits their application to phage collections that typically are large and have low biomass. In order to address this, we have developed <i>Plaque-2-seq</i>, a robust and cost-effective workflow for high-throughput phage genome sequencing that will transform the speed and cost of attaining phage genomes. <i>Plaque-2-seq</i> combines low-input transposase-based library preparation, amplification, nanopore sequencing and optimized assembly steps tailored to phage genomes. We applied the method to phages isolated on seven genetically diverse bacterial hosts: <i>Escherichia</i>, <i>Pseudomonas</i>, <i>Synechococcus</i>, <i>Enterococcus</i>, <i>Klebsiella, Serratia</i> and <i>Enterobacter</i>. High-quality genome assemblies were validated using CheckV and benchmarked against previously sequenced phage isolates. Compared to standard Illumina sequencing, <i>Plaque-2-seq</i> offers up to ~5-10-fold savings in sequencing price for individual labs. Furthermore, it substantially decreases the time required to produce a phage genome, once a plaque is obtained. Offering the ability to routinely obtain hundreds of phage genome sequences a week, with minimal hands-on time. <i>Plaque-2-seq</i> enables systematic genomic characterization of phage isolates, facilitating taxonomic classification, for the development of large-scale phage biobanks.