title: A targeted enrichment strategy for massively parallel sequencing of angiosperm plastid genomes.
abstract: OBJECTIVE
  We explored a targeted enrichment strategy to facilitate rapid and low-cost next-generation sequencing (NGS) of numerous complete plastid genomes from across the phylogenetic breadth of angiosperms. •
  
  
  RESULTS
  A custom RNA probe set including the complete sequences of 22 previously sequenced eudicot plastomes was designed to facilitate hybridization-based targeted enrichment of eudicot plastid genomes. Using this probe set and an Agilent SureSelect targeted enrichment kit, we conducted an enrichment experiment including 24 angiosperms (22 eudicots, two monocots), which were subsequently sequenced on a single lane of the Illumina GAIIx with single-end, 100-bp reads. This approach yielded nearly complete to complete plastid genomes with exceptionally high coverage (mean coverage: 717×), even for the two monocots. •
  
  
  CONCLUSIONS
  Our enrichment experiment was highly successful even though many aspects of the capture process employed were suboptimal. Hence, significant improvements to this methodology are feasible. With this general approach and probe set, it should be possible to sequence more than 300 essentially complete plastid genomes in a single Illumina GAIIx lane (achieving ∼50× mean coverage). However, given the complications of pooling numerous samples for multiplex sequencing and the limited number of barcodes (e.g., 96) available in commercial kits, we recommend 96 samples as a current practical maximum for multiplex plastome sequencing. This high-throughput approach should facilitate large-scale plastid genome sequencing at any level of phylogenetic diversity in angiosperms.
authors.affiliation: Department of Biology, University of Florida, Gainesville, Florida 32611-8525 USA ; Florida Museum of Natural History, University of Florida, Gainesville, Florida 32611-7800 USA.
authors.forename: Gregory W
authors.initials: GW
authors.lastname: Stull
authors.affiliation: Department of Biology, Oberlin College, Oberlin, Ohio 44074-1097 USA.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Moore
authors.affiliation: Department of Biology, Oberlin College, Oberlin, Ohio 44074-1097 USA.
authors.forename: Venkata S
authors.initials: VS
authors.lastname: Mandala
authors.affiliation: Department of Biology, Oberlin College, Oberlin, Ohio 44074-1097 USA.
authors.forename: Norman A
authors.initials: NA
authors.lastname: Douglas
authors.affiliation: Florida Museum of Natural History, University of Florida, Gainesville, Florida 32611-7800 USA ; Genetics Institute, University of Florida, Gainesville, Florida 32611 USA.
authors.forename: Heather-Rose
authors.initials: HR
authors.lastname: Kates
authors.affiliation: Key Laboratory of Conservation Biology for Endangered Wildlife of the Ministry of Education, and Laboratory of Systematic and Evolutionary Botany and Biodiversity, College of Life Sciences, Zhejiang University, Hangzhou 310058 People's Republic of China.
authors.forename: Xinshuai
authors.initials: X
authors.lastname: Qi
authors.affiliation: Department of Plant Sciences, University of Cambridge, Cambridge CB13EA United Kingdom.
authors.forename: Samuel F
authors.initials: SF
authors.lastname: Brockington
authors.affiliation: Florida Museum of Natural History, University of Florida, Gainesville, Florida 32611-7800 USA ; Genetics Institute, University of Florida, Gainesville, Florida 32611 USA.
authors.forename: Pamela S
authors.initials: PS
authors.lastname: Soltis
authors.affiliation: Department of Biology, University of Florida, Gainesville, Florida 32611-8525 USA ; Florida Museum of Natural History, University of Florida, Gainesville, Florida 32611-7800 USA ; Genetics Institute, University of Florida, Gainesville, Florida 32611 USA.
authors.forename: Douglas E
authors.initials: DE
authors.lastname: Soltis
authors.affiliation: Department of Biology, University of Florida, Gainesville, Florida 32611-8525 USA ; Florida Museum of Natural History, University of Florida, Gainesville, Florida 32611-7800 USA ; Genetics Institute, University of Florida, Gainesville, Florida 32611 USA.
authors.forename: Matthew A
authors.initials: MA
authors.lastname: Gitzendanner
keywords: next-generation sequencing; phylogenomics; plastid genomes