DOI RECORD
Sequencing of Genomic DNA through Oxford Nanopore Technology- Using Native Barcoding Kit v1
Abstract
Whole-genome sequencing provides a complete view of an organism's genetic makeup and is widely used to study genome structure, diversity, and evolution. This protocol describes multiplexed long-read sequencing of genomic DNA using the Oxford Nanopore Technologies (ONT) Ligation Sequencing workflow with the Native Barcoding Kit [SQK-NBD114.24 or SQK-NBD114.96] on a PromethION flow cell. Starting with quality-checked, high-molecular-weight DNA from bacteria, fungi, plants, or animals, it covers DNA repair and end preparation, native barcode ligation, sample pooling, adapter ligation and cleanup, flow cell priming and loading, and sequencing run setup, including basecalling, Q-score filtering, output format, and run duration. Native barcoding allows multiple samples to share a flow cell, reducing per-sample cost. The resulting long reads span repetitive regions, enabling genome assemblies, structural variant detection, and characterisation of plasmids and organellar genomes. Applications include species identification, comparative genomics, clinical and public health genomics, agricultural and environmental research, and biotechnology.