Targeted capture and sequencing of gene-sized DNA molecules

BioTechniques

Matthew D. Clark¹  Michael Giolai¹  Pirita Paajanen¹  Lawrence Percival-Alwyn¹  Walter Verweij¹  David Baker¹  Florian Jupe²  Jonathan D. G. Jones²  Kamil Witek²  Ingo Hein³  Glenn Bryan³ 

[1] 1Earlham Institute (EI), Norwich Research Park, Norwich, UK;2The Sainsbury Laboratory, Norwich Research Park, Norwich, UK;4The James Hutton Institute, Dundee, UK;

关键词: targeted capture;    gene enrichment;    PacBio;    RenSeq;    NB-LRR gene;    resistance gene;   

DOI  :  10.2144/000114484

来源: DOAJ

【 摘 要 】

Targeted capture provides an efficient and sensitive means for sequencing specific genomic regions in a high-throughput manner. To date, this method has mostly been used to capture exons from the genome (the exome) using short insert libraries and short-read sequencing technology, enabling the identification of genetic variants or new members of large gene families. Sequencing larger molecules results in the capture of whole genes, including intronic and intergenic sequences that are typically more polymorphic and allow the resolution of the gene structure of homologous genes, which are often clustered together on the chromosome. Here, we describe an improved method for the capture and single-molecule sequencing of DNA molecules as large as 7 kb by means of size selection and optimized PCR conditions. Our approach can be used to capture, sequence, and distinguish between similar members of the NB-LRR gene family—key genes in plant immune systems.

【 授权许可】

Unknown