Plaque-2-seq

Plaque-2-seq

Plaque-2-Seq is a sequencing and analysis framework developed to enable rapid, low-cost generation of high-quality bacteriophage genomes directly from individual plaques. Traditional phage genome sequencing often requires large amounts of DNA, extensive culturing, or costly workflows, which can limit throughput and scalability. Plaque-2-Seq addresses these challenges by optimising both laboratory and computational steps for low-input phage material.

The approach combines transposase-based library preparation, amplification, and Oxford Nanopore long-read sequencing, alongside a tailored bioinformatics workflow designed to minimise common artefacts such as chimeric reads and fragmented assemblies. This allows reliable recovery of complete or near-complete phage genomes from single plaques with minimal hands-on time.

Plaque-2-Seq was evaluated across phages infecting a wide range of bacterial hosts, including Escherichia, Pseudomonas, Klebsiella, Enterococcus, and Synechococcus. The resulting genome assemblies were assessed using standard quality metrics and benchmarking tools, demonstrating that the method produces accurate, high-quality genomes suitable for comparative genomics, taxonomy, and downstream functional analysis.

By substantially reducing cost and turnaround time compared with conventional short-read sequencing approaches, Plaque-2-Seq enables high-throughput phage genome generation at scale. This makes it particularly well suited for large isolation campaigns, phage biobank development, and studies of phage diversity in both clinical and environmental contexts.

The full methodology, benchmarking, and validation are described in our preprint on bioRxiv, and the complete, reproducible pipeline is openly available on GitHub.