Whole-genome MinION sequencing of EcN-GFP harboring plasmid: pZE21 replicate: 6
Genomic DNA from 30 mL of overnight culture of control EcN was extracted using a Genomic DNA Buffer Set (19060, Qiagen) with the Qiagen protocol for Gram-negative bacteria (Qiagen Genomic DNA Handbook 06/2015) and the Genomic-tip 500/G (10262, Qiagen). Purified genomic DNA was sheared to a target fragment size of 10 kilobases using the Covaris g-TUBE. All following steps were carried out in Eppendorf DNA LoBind tubes. Sheared DNA was repaired using the NEBNext FFPE Repair Mix (M6630), and repaired DNA purified from the reaction using Agencourt AMPure XP beads. DNA was then end-repaired and dA-tailed using the NEBNext End repair / dA-tailing Module (E7546) and again purified with AMPure XP beads. DNA was then prepared for MinION sequencing using the MinION Ligation Sequencing Kit 1D (SQK-LSK108). Briefly, adapters were ligated to the DNA using the MinION Adapter Mix (AMX1D) and NEB Blunt/TA Ligase Master Mix (M0367). Then the DNA product was purified using AMPure XP beads and the Minion Adapter Bead Binding Buffer (ABB). 350 ng of DNA product was then sequenced on a MinION R9.4 flow cell for 48 hours using the Running Buffer with Fuel Mix (RBF) and the Library Loading Bead Kit (EXP-LLB001). Average read lengths were 7 kilobases and the genome was sequenced with an average coverage of 50 reads.