Sonawala Unnati, Derevnina Lida, Eves-van den Akker Sebastian
The Crop Science Centre, Department of Plant Sciences, University of Cambridge, CB2 3EA Cambridge, UK.
The Crop Science Centre, Department of Plant Sciences, University of Cambridge, CB2 3EA Cambridge, UK.
STAR Protoc. 2024 Dec 20;5(4):103427. doi: 10.1016/j.xpro.2024.103427. Epub 2024 Nov 1.
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..
我们提出了一种方案，通过基于Cas9的纳米孔富集方法，在不进行扩增的情况下，实现对马铃薯胞囊线虫感兴趣区域的长读长测序的更高深度。我们描述了设计与Cas9核酸酶一起使用的高保真引导RNA、从线虫中提取高分子量DNA以及对基因组DNA末端进行去磷酸化的步骤。然后，我们详细介绍了使用Cas9-引导RNA复合物对感兴趣区域进行靶向切割，随后进行纳米孔文库制备的程序。有关此方案的使用和执行的完整详细信息，请参考索纳瓦拉等人的研究。
