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Author Soltis, Pamela S. ♦ Soltis, Douglas E. ♦ Stull, Gregory W. ♦ Qi, Xinshuai ♦ Douglas, Norman A. ♦ Mandala, Venkata S. ♦ Kates, Heatherrose ♦ Moore, Michael J. ♦ Brockington, Samuel F. ♦ Gitzendanner, Matthew A.
Source Directory of Open Access Journals (DOAJ)
Content type Text
Publisher Botanical Society of America
File Format HTM / HTML
Date Created 2014-05-01
Copyright Year ©2013
Language English
Subject Domain (in LCC) Q ♦ QH301-705.5
Subject Keyword Biology ♦ Next-generation sequencing ♦ Science ♦ Plastid genomes ♦ Phylogenomics
Abstract Premise of the study: 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. Methods and 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.
ISSN 21680450
Age Range 18 to 22 years ♦ above 22 year
Educational Use Research
Education Level UG and PG ♦ Career/Technical Study
Learning Resource Type Article
Publisher Date 2013-02-01
e-ISSN 21680450
Journal Applications in Plant Sciences
Volume Number 1
Issue Number 2
Starting Page 1200497

Source: Directory of Open Access Journals (DOAJ)