Targeted capture and sequencing of gene-sized DNA molecules

Michael Giolai, Pirita Paajanen, Walter Verweij, Lawrence Percival-Alwyn, David Baker, Kamil Witek, Florian Jupe, Glenn Bryan, Ingo Hein, Jonathan D. G. Jones, Matthew D. Clark (Lead / Corresponding author)

Research output: Contribution to journalArticle

12 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)315-322
Number of pages8
JournalBioTechniques
Volume61
Issue number6
DOIs
Publication statusPublished - Dec 2016

Keywords

  • Targeted capture
  • Gene enrichment
  • PacBio
  • RenSeq
  • NB-LRR gene
  • Resistance gene

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    Giolai, M., Paajanen, P., Verweij, W., Percival-Alwyn, L., Baker, D., Witek, K., Jupe, F., Bryan, G., Hein, I., Jones, J. D. G., & Clark, M. D. (2016). Targeted capture and sequencing of gene-sized DNA molecules. BioTechniques, 61(6), 315-322. https://doi.org/10.2144/000114484