Neb quick t4 dna ligase

A total of 1.5 μg of gDNA was end-repaired (NEBnext ultra II end repair kit, New England Biolabs, MA, USA) and purified using 1 × AmPure beads (Beckmann Coulter, USA). Adapter ligation (AMX) was performed at RT (20 °C) for 20 min using NEB Quick T4 DNA Ligase (New England Biolabs, MA, USA). The reaction mixture was purified using 0.6 × AmPure beads (Beckmann Coulter, USA) and sequencing library was eluted in 15 μl of elution buffer provided in the ligation sequencing kit (SQK-LSK109) from Oxford Nanopore Technology (ONT). Sequencing was performed on GridION X5 (Oxford Nanopore Technologies, Oxford, UK) using SpotON flow cell R9.4 (FLO-MIN106) in 48 h sequencing protocol on MinKNOW (version 1.1.20, ONT) with Albacore (v1.1.2)³⁷ live base calling enabled with default parameters.

For long-read sequencing, the genomic DNA was prepared using the ligation sequencing kit SQK-LSK112 (Oxford Nanopore Technologies, Oxford, United Kingdom) according to the manufacturer’s guidelines. Briefly, genomic DNA (1020 ng) was subjected to end repair, 5’ phosphorylation and dA-tailing by NEBNext FFPE DNA Repair and NEBNext Ultra II End prep modules (New England Biolabs) and purified with AMPure XP (Beckman Coulter, Pasadena, CA, USA) magnetic beads. The sequencing adaptors were ligated using the NEB Quick T4 DNA Ligase (New England Biolabs) in combination with ligation buffer (LNB) from the SQK-LSK112 kit. The library was finally cleaned up using the long fragment buffer (LFB) and purified with AMPure XP magnetic beads. For sequencing, 12 µL of library (~ 400 ng) were loaded onto a primed FLO-MIN112 (ID: FAT29688) flow cell on a MinION device for a 42-h run. Data acquisition was carried out with MinKNOW software v22.05.5 (Oxford Nanopore Technologies).