1d genomic dna ligation sequencing kit sqk lsk109

For nanopore sequencing via the Oxford Nanopore technology method, purified PCR amplified sequences containing encoded data were subjected to library preparation using the 1D Genomic DNA ligation sequencing kit (SQK-LSK109) from Oxford Nanopore Technologies following the manufacturer’s protocols. Briefly, 0.5 pmol of the double-stranded DNA strands were used as starting material. The DNA was repaired and end-prepped using NEBNext FFPE DNA repair mix (NEB, M6630S) and NEBNext Ultra II End Repair/dA-Tailing Module (NEB, E7546) followed by bead purification using Agencourt AMPure XP beads (Beckman Coulter, A63880) at 1:2 sample to bead ratio. Adapters were then ligated to the end-prepped samples using the NEBNext Quick T4 DNA ligase (NEB, E6056S). The flow cells (R4.2.1) were primed, the sample was loaded onto the priming port of the flow cell and sequenced on the MinION, that generated ~500 K reads/hr. Sequencing was performed using the MinKNOW software (version 18.3.1, Oxford Nanopore Technologies) that converted raw data (in the form of fast5 files) into FastQ files which were used for downstream analysis.

Genomic DNA of L. curvatus KP 3-4 was extracted using Wizard^® Genomic DNA Purification Kit (A1620; Promega, Madison, WI, USA) following the manufacturer’s instructions. Here, 1 μg DNA was subjected to library construction using the 1D Genomic DNA Ligation Sequencing Kit (SQK-LSK 109, Oxford Nanopore Technologies, Oxford, UK) and sequenced on the MinION sequencer (Mk1B, Oxford Nanopore Technologies) according to the manufacturer’s protocol. The resulting ONT long reads were corrected with Canu ver 1.9 [30 (link)] using default configuration with the option of a total contig size of 3 Mb. The raw ONT reads were registered in DDBJ (accession number: DRA013072).