2.5. Lytic Phage Genome Sequencing and Assembly

Because the classification of the isolated phage was unknown, we performed de novo genome sequencing. In brief, phage DNA samples were sent for de novo sequencing to GENEWIZ Biological Technology (Suzhou, China) and a library of these DNA samples that had passed the inspection was constructed for cluster preparation and sequencing. Bcl2fastq (v.2.17.1.14) was used to perform image-based recognition (Base Calling) on the original image data. After quality analysis, the raw sequencing data were obtained. Trimmomatic-0.36 was used to trim and remove adaptors and low-quality nucleotides from the original readings to obtain clean data for subsequent information analysis [41]. Spades Genome Assembler 3.8.1-Linux version was used to assemble the post-processed readings into contig sequences [42]. SSPACE (v.3.0) was used to further assemble contig sequences into scaffold sequences and GapFiller (v.1.10) was used to complement and extend the scaffold sequence to obtain the final scaffold sequence of the whole phage genome.

Three online websites, Open Reading Frame Finder (www.ncbi.nlm.nih.gov/orffinder/, accessed on 29 February 2020, RASTtk (https://rast.nmpdr.org/, accessed on 29 February 2020) and GeneMarks (http://topaz.gatech.edu/GeneMark/, accessed on 29 February 2020), were jointly used to predict all genes in the genome [43,44,45]. After comparisons with protein sequences of known function for similarity and identity, the best hit was selected for the annotation of the gene products. The manual search method was also used to locate ARGs and virulence genes in the genome using the online websites VFDB and ARDB. Finally, CGview (v.1.0) was used to draw the whole genome map of the phage.

All uploaded phage genome sequences in NCBI were downloaded to compare with the isolated phage genome sequences and amino acid sequences by local BLAST+ (v.2.7.1). The genome alignment map of the isolated phage was drawn in Easy-fig; the gene encoding products with different functions are indicated with different colors.

The evolutionary relationships between phages were analyzed using phylogenetic trees based on the amino acid sequences of the terminase large subunits of different Escherichia phages. The phage terminase is a unique protein of the dsDNA phage and possesses highly conserved functions to drive packaging by energizing the DNA [46]. Terminase of the double-stranded DNA phage can be divided into a large subunit and a small subunit according to their molecular weight [47]. The large subunit has endonucleolytic and ATPase activities and is generally composed of 400 to 750 amino acids [48]. After aligning the sequences of the terminase large subunit, similar sequences were downloaded from the National Center for Biotechnology (NCBI). Phylogenetic analysis was performed with MEGA7 by the neighbor-joining method (Bootstrap: 1000 with the Poisson Model) [49]. Alignment analysis was based on the ClustalW alignment of amino acid sequences by MEGA7 (parameter setting: Gap Opening Penalty: 10, Gap Extension Penalty: 0.2) [50].