title: MinION sequencing; Urine MinION sequencing, R7.0 chemistry, SQK-MAP-002, Metrichor (R7.0)
design: Methodology was refined during the project. In its final iteration, urines (4–10 mL) were centrifuged at 300 g for 2 min to deplete human cells. The supernatant was collected and re-centrifuged at 12 300 g for 5 min, with the resulting bacterial pellet resuspended in 1 mL of PBS and processed with a MolYsis Basic 5 Kit (MolYsis Life Science, Bremen, Germany) to lyse residual human cells and to remove their DNA. Bacterial Lysis Buffer (Roche, Basel, Switzerland) and proteinase K (14–22 mg/mL) (Roche) were added and, after incubation for 10 min at 65°C, DNA was purified using the MagNA Pure Compact Nucleic Acid Isolation Kit (Roche) and DNA Bacteria v3_2 protocol. Variations, in early iterations, were (i) the initial centrifugation was omitted and no human DNA depletion performed (CU1), and (ii) a NEBNext® Microbiome DNA Enrichment kit (New England BioLabs, Hitchin, UK) was used to remove human DNA instead of MolYsis (CU2–4).  To generate a library with an average fragment size of ∼8 kb, up to 2 μg of DNA was fragmented by centrifugation at 7200 rpm (3600 g) in a G-tube (Covaris, Brighton, UK), used according to the manufacturer's instructions, then end-repaired (New England BioLabs), cleaned with Agencourt AMPure XP Beads (Beckman Coulter, High Wycombe, UK) and dA-tailed (New England BioLabs). The product was re-cleaned and eluted in 31 μL of Tris-HCl (pH 8.5). The library was then prepared according to the SQK-MAP-002 Genomic Sequencing Kit protocol provided by ONT.  MinION sequencing was performed using R7.0 flow cells.  ONT MinKNOW software (version 0.45.2.6) was used to collect raw electronic signal data, which were base-called using Metrichor™ software (R7.0)
library_construction_protocol: SQK-MAP_002