Long-Range Targeted Nanopore Ligation Sequencing Workflow for Targeted Amplicon Sequencing of Secondary Metabolite Gene.

This chapter presents protocols for advanced genotyping and genetic analysis in plant science, utilizing DNA extracted from FTA samples and leaf tissue. Targeted long range PCR is employed to uncover allelic and structural variations within genes of interest, while targeted long-read nanopore sequencing offers a comprehensive view of gene targets in their native and enriched forms. The protocol for nanopore sequencing encompasses DNA preparation, barcoding of up to 96 samples, adapter ligation, purification, and sequencing, culminating in the generation of raw data files (pod5 or fastq) suitable for further analysis. Together, these methods provide a powerful toolkit for plant diagnostics and breeding.