whole genome sequencing of E. coli isolated from the chicken gut
From selective MacConkey agar showing colonies of interest (tetracycline-resistant E. coli), a single colony (one isolate per bird per necropsy; n = 36) was picked and subcultured overnight in LB broth at 37C and used for DNA extraction using the PureLink Genomic DNA kit (Invitrogen, Carlsbad, CA) following the manufacturers instructions. DNA concentrations were determined using a Qubit dsDNA Broad Range Assay with the Qubit Fluorimeter (Qiagen), and normalized to 400 ng input DNA in 7.5 l final volume for sequencing using an Oxford Nanopore MinION standard flow cell; and 200 ng DNA in 3.75 l final volume for sequencing using an Oxford Nanopore MinION Flongle flow cell. DNA libraries were generated with the rapid barcoding kit (SQK-RBK004; Oxford Nanopore, Oxford, United Kingdom) according to the manufacturers instructions. Genome sequencing was performed at USDA, NADC, in Ames, IA on a MinION instrument (Oxford Nanopore) using a standard flow cell (FLO-MIN106 R9.4.1) or a Flongle flow cell (FLO-FLG106). The MinION flow cell was run for 4872h, and were base called with Guppy (GPU) version 5.0.11+2b6dbff (Wick et al., 2019).