title: Nanopore DNA-Seq of Pseudomonas fluorescens phage: UNO-G1W1
design: A high titer stock was prepared and genomic DNA was isolated with standard phenol-chloroform extraction for sequencing. The gDNA library was prepared using 640 ng of input gDNA with the ONT Ligation Sequencing Kit (SQK-LSK109) and NEBNext Companion Module for Oxford Nanopore Techniologies Ligation Sequencing (NEB, E7180S). The Ligation Sequencing gDNA protocol (Version: GDE_9063_v109_revAP_25May2022) was followed, unless otherwise stated. The optional 10 ng of DNA Control sample (DNA CS), a 3.6 kb amplicon mapping to the 3 end of the Lambda genome, was included in the library preparation as a positive quality control for library prep and sequencing performance. Agencourt AMPure XP beads (Beckman Coulter, A63881) were used for all required and optional clean-up steps throughout the entirety of library prep. All quantifications of gDNA were quantified using the Qubit dsDNA BR Assay Kit (Invitrogen, Q32850) and Qubit 4 Fluorometer (Invitrogen, Q33238), following the manufacturers protocol (MAN0002325, Revisions B.0). To enrich for and improve recovery of long fragments following adapter ligation, Long Fragment Buffer (LFB) was used for washes and incubation with Elution Buffer (EB)  was carried out at 37C. The prepared DNA library was eluted and stored on ice before loading the recommended amount onto a FLO-MIN106D (R9.4.1) flow cell and sequencing was performed on the MinION Mk1B device. MinKNOW software (ver. 5.8.6) was used and live base calling was performed with Dorado (ver. 7.2.13) with the Super High Accuracy mode enabled and filtering on a Q-score threshold of 10. The entire sequencing effort, irrespective of Q score, yielded 7.4 Gb and 451,617 reads. The passed reads (Q>10) were composed of 4.09 Gb (230,557 reads) achieving a coverage of approximately 41,735x, based on a predicted genome size of 98 kb.