Same-day tagmentation PCR-based whole genome sequencing of bacteriophage genomes from a single plaque without DNA...

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 bacteriophage genomes via tagmentation and PCR amplification of crude plaque material, bypassing classical phage amplification, filtration and DNA extraction. When applied to our phage collection, this technique provided 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. This PCR-based approach is however not suitable for studying phage DNA modifications, akin to sequencing by synthesis, although developed for Oxford Nanopore Technologies sequencing which traditionally allows for interrogation modified bases. The optimization of bacteriophage identification by the technique of tagmentation directly to isolated plaques can enable same-day plaque-to-sequence workflows for novel phages, as demonstrated here in a proof-of-concept evaluation of 14 plaque genomes.