A hybrid and poly-polish workflow for the complete and accurate assembly of phage genomes: a case study of ten przondoviruses

Bacteriophages (phages) within the Przondovirus genus are T7-like podoviruses belonging to the Studiervirinae subfamily, within the Autographiviridae family and have a highly conserved genome organisation. The genome size of these phages ranges from 37 kb to 42 kb, encode 50-60 genes and are characterised by the presence of direct terminal repeats (DTRs) flanking the linear chromosome. These DTRs are often deleted during short-read-only and hybrid assemblies. Moreover, long-read-only assemblies are often littered with sequencing and/or assembly errors and require additional curation. Here, we present the isolation and characterisation of ten novel przondoviruses targeting Klebsiella spp. We describe HYPPA – a HYbrid and Poly-polish Phage Assembly workflow, which utilises long-read assemblies in combination with short-read sequencing to resolve phage DTRs and correcting errors, negating the need for laborious primer walking and Sanger sequencing validation. Our data demonstrate the importance of careful curation of phage assemblies before publication, and prior to using them for comparative genomics. IMPACT STATEMENT The current workflows employed for phage genome assembly are often error-prone and can lead to many incomplete phage genomes being deposited within databases. This can create challenges when performing comparative genomics, and may also lead to incorrect taxonomic assignment. To overcome these challenges we proposed HYPPA, a workflow that can produce complete and high-quality phage genomes without the need for laborious lab-based validation. DATA SUMMARY Phage raw reads are available from the National Centre for Biotechnology Information Sequence Read Archive (NCBI-SRA) under the BioProject number PRJNA914245. Phage annotated genomes have been deposited at GenBank under the accessions [OQ579023][1]-[OQ579032][2] ( [Table 1][3] ). Bacterial WGS data for clinical preterm infant samples have been deposited at GenBank under BioProject accession PRJNA471164 ( Table S1 ). Bacterial raw reads for food samples are available from NCBI-SRA with individual accessions (SAMN33593347-SAMN33593351), and can be found under the BioProject number PRJNA941224 ( Table S1 ). Strain-specific details for bacteria and publicly-available phages used in these analyses, along with accessions for the latter can be found in Table S1 and Table S6 , respectively. The CL1-CL8 clinical Klebsiella strains ( Table S1 ) were under a Materials Transfer Agreement, for which sequencing data and strain information is not available. ### Competing Interest Statement The authors have declared no competing interest. * BHI : brain heart infusion CPS : capsular polysaccharide DNAP : DNA polymerase DTR : direct terminal repeat HYPPA : hybrid and poly-polish phage assembly ICTV : International Committee on Taxonomy of Viruses LPS : lipopolysaccharide NCBI : National Centre for Biotechnology Information MLST : multilocus sequence typing ONT : Oxford Nanopore Technologies PEG : polyethylene glycol QC : quality control QIB : Quadram Institute Bioscience RNAP : RNA polymerase [1]: /lookup/external-ref?link_type=GEN&access_num=OQ579023&atom=%2Fbiorxiv%2Fearly%2F2023%2F03%2F09%2F2023.03.09.531871.atom [2]: /lookup/external-ref?link_type=GEN&access_num=OQ579032&atom=%2Fbiorxiv%2Fearly%2F2023%2F03%2F09%2F2023.03.09.531871.atom [3]: #T1