Protocol for Cas9-targeted long-read sequencing in Globodera pallida and Globodera rostochiensis

Summary: We present a protocol to achieve a higher depth of long-read sequencing of region(s) of interest in potato cyst nematodes without amplification using a Cas9-based Nanopore enrichment approach. We describe steps for designing high-fidelity guide RNAs to be used with Cas9 nuclease, extracting high-molecular-weight DNA from the nematodes, and dephosphorylating genomic DNA ends. We then detail procedures for using Cas9-guide RNA complex to make targeted cleavage of the region of interest followed by a Nanopore library preparation.For complete details on the use and execution of this protocol, please refer to Sonawala et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.