DNA extraction free whole genome sequencing of bacteriophage genomes from a single plaque

DNA sequencing is at the core of genome characterization, proteomics, and identification of novel organisms. For microorganisms such as bacteriophages, sequencing their DNA can provide key insights into their tropism, infectivity, and virulence. There remains however a critical lack of rapid sequencing techniques with the traditional process of replating and incubating individual plaques, collecting lysate, extracting DNA, preparing the DNA library, and sequencing that is labor intensive. Herein, we demonstrate the use of an adapted Nanopore Rapid PCR Barcoding protocol to sequence the bacteriophage genome directly from individual plaques. This technique provides sequencing genome assemblies with 99.88-100% (mean 99.97%) average nucleotide identity (ANI) scores when compared to the traditional methods involving phage amplification, extraction, and sequencing using Illumina. The optimization of bacteriophage identification by the technique of tagmentation directly to isolated plaques will enable rapid and cost-effective sequencing of novel phages.