Bioinformatics Analyses for T4 Genome, Expression Vectors, and ECOR Reference Library

The T4 phage genome was isolated using the manufacturer-suggested extended protocol from the Norgen Biotek Phage Genome Isolation kit (Norgen Biotek, Thorold, ON, Canada). Genome isolation for each strain of the ECOR Library was accomplished using the DNeasy Blood and Tissue DNA extraction kit (Qiagen, Hilden, Germany) and then prepared for WGS. WGS analysis was performed as a service (Animal Health Diagnostic Center, College of Veterinary Medicine, Cornell University, Ithaca, NY, United States), using the Illumina MiSeq NGS platform, a Nextera XT DNA Library Kit, and data collection with QC analysis performed in BaseSpace Sequence Hub (Illumina, San Diego, CA, United States). Raw sequence reads were assembled and analyzed using the Geneious Prime 2020.0.3 software¹. Sanger Sequencing was performed (Biotechnology Resource Center, Cornell University, Ithaca, NY, United States) to confirm proper assembly of all constructed plasmids (Supplementary Table 2). For all relevant T4 genes (g37, g38, and g57), alignments between our assembled T4 genome and NCBI’s published T4 genome ({"type":"entrez-nucleotide","attrs":{"text":"NC_000866","term_id":"29366675","term_text":"NC_000866"}}NC_000866) were performed at both the nucleotide and amino acid level. For WGS, all returned contigs were evaluated to ensure the quality of the reads were >85%. T4 genome was assembled against the annotated T4 genome from NCBI. Contigs from each ECOR strain were trimmed, filtered, normalized, and error-corrected before undergoing de novo assembly using E. coli K-12 substr. MG1655 (GenBank: {"type":"entrez-nucleotide","attrs":{"text":"U00096.2","term_id":"48994873","term_text":"U00096.2"}}U00096.2) as a reference. The raw contigs from sequenced ECOR strains have been deposited in a NCBI BioProject (#PRJNA579348) for open access by future researchers. Amino acid sequences of OmpC porin proteins from E. coli K-12, E. coli O157:H7 (GenBank: {"type":"entrez-nucleotide","attrs":{"text":"BA000007.2","term_id":"47118301","term_text":"BA000007.2"}}BA000007.2), and E. coli B (GenBank: {"type":"entrez-nucleotide","attrs":{"text":"CP000819.1","term_id":"253972022","term_text":"CP000819.1"}}CP000819.1) were used as reference strains to align assembled OmpC sequences from the assembled ECOR strains. The “EMBOSS Protein” plug-in allowed secondary structures to be predicted for comparisons between all ECOR strain OmpC sequences.