Journal
BIOTECHNIQUES
Volume 61, Issue 6, Pages 315-322Publisher
BIOTECHNIQUES OFFICE
DOI: 10.2144/000114484
Keywords
targeted capture; gene enrichment; PacBio; RenSeq; NB-LRR gene; resistance gene
Funding
- BBSRC, Institute Strategic Programme Grant at the Earlham Institute [BB/J004669/1]
- BBSRC Grant Controlling important diseases in potato by cloning functional NB-LRR-type resistance genes [BB/L009757/1]
- BBSRC [BB/K018299/1, BB/K019090/1, BBS/E/T/000PR6193, BB/H019820/1, BB/L009293/1, BB/L009757/1, BB/L008025/1, BBS/E/T/000PR5885] Funding Source: UKRI
- Biotechnology and Biological Sciences Research Council [BB/K018299/1, BB/L009757/1, BB/K019090/1, BB/L009293/1, BB/H019820/1, BBS/E/T/000PR6193, BB/L008025/1, BBS/E/T/000PR5885] Funding Source: researchfish
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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.
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