title: Targeted capture and sequencing of gene-sized DNA molecules.
abstract: 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.
authors.affiliation: Earlham Institute (EI), Norwich Research Park, Norwich, UK.
authors.forename: Michael
authors.initials: M
authors.lastname: Giolai
authors.affiliation: Earlham Institute (EI), Norwich Research Park, Norwich, UK.
authors.forename: Pirita
authors.initials: P
authors.lastname: Paajanen
authors.affiliation: Earlham Institute (EI), Norwich Research Park, Norwich, UK.
authors.forename: Walter
authors.initials: W
authors.lastname: Verweij
authors.affiliation: Earlham Institute (EI), Norwich Research Park, Norwich, UK.
authors.forename: Lawrence
authors.initials: L
authors.lastname: Percival-Alwyn
authors.affiliation: Earlham Institute (EI), Norwich Research Park, Norwich, UK.
authors.forename: David
authors.initials: D
authors.lastname: Baker
authors.affiliation: The Sainsbury Laboratory, Norwich Research Park, Norwich, UK.
authors.forename: Kamil
authors.initials: K
authors.lastname: Witek
authors.affiliation: The Sainsbury Laboratory, Norwich Research Park, Norwich, UK.|Plant Biology Laboratory, Salk Institute for Biological Studies, La Jolla, CA.
authors.forename: Florian
authors.initials: F
authors.lastname: Jupe
authors.affiliation: The James Hutton Institute, Dundee, UK.
authors.forename: Glenn
authors.initials: G
authors.lastname: Bryan
authors.affiliation: The James Hutton Institute, Dundee, UK.
authors.forename: Ingo
authors.initials: I
authors.lastname: Hein
authors.affiliation: The Sainsbury Laboratory, Norwich Research Park, Norwich, UK.
authors.forename: Jonathan D G
authors.initials: JD
authors.lastname: Jones
authors.affiliation: Earlham Institute (EI), Norwich Research Park, Norwich, UK.|School of Environmental Sciences, University of East Anglia, Norwich Research Park, Norwich, UK.
authors.forename: Matthew D
authors.initials: MD
authors.lastname: Clark
mesh_terms: D004247:DNA / Q000032:analysis / Q000235:genetics* / Q000302:isolation & purification*; D005091:Exons / Q000235:genetics; D015723:Gene Library; D017343:Genes, Plant / Q000235:genetics; D023281:Genomics / Q000379:methods*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D008957:Models, Genetic; D017422:Sequence Analysis, DNA / Q000379:methods*; D032322:Solanum / Q000235:genetics
chemical_list: D004247:DNA
keywords: NB-LRR gene; PacBio; RenSeq; gene enrichment; resistance gene; targeted capture