Broad range kit

The enriched raw milk (1 ml) was centrifuged and the raw milk fat layer was removed before the pellet was washed with 1× PBS as described above for raw milk screening. DNA extraction and purification was performed using the MasterPure complete DNA and RNA extraction and purification kit (Lucigen), following the manufacturer’s instructions and including an RNase A treatment for 30 min (Qiagen; 100 mg ml⁻¹) [26 (link)]. Genomic DNA was quantified using a Qubit 3.0 fluorometer and the broad range kit (Agilent), and quality control of the DNA extracts was assessed using the Nanodrop 1.0 or 2.0 spectrophotometer (Agilent).

DNA was quality checked using Qubit (Thermo Fisher) using the Broad Range kit and the Agilent TapeStation, which registered a peak fragment size of 58 kb. Oxford nanopore sequencing was carried out using flow cell Flo-MIN107 (R9.5 nanopore) and the rapid barcoding kit SQK-RBK001, with the barcode NB01 chosen for this isolate. Barcoding was used to enable exclusion of reads from this first isolate in subsequent data, in the event that this flow cell was reused for other isolates at a later date. MinKNOW version 1.6.11 was used with local 1D base calling enabled. The sequencing run was stopped after 23 hours as sufficient data had been obtained.