Journal article
A Targeted Enrichment Strategy for Massively Parallel Sequencing of Angiosperm Plastid Genomes
Applications in Plant Sciences, Vol.1(2), pp.1200497-n/a
02/2013
DOI: 10.3732/apps.1200497
PMCID: PMC4105372
PMID: 25202518
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.
Details
- Title: Subtitle
- A Targeted Enrichment Strategy for Massively Parallel Sequencing of Angiosperm Plastid Genomes
- Creators
- Gregory W. Stull - Florida Museum of Natural HistoryMichael J. Moore - Oberlin CollegeVenkata S. Mandala - Oberlin CollegeNorman A. Douglas - Oberlin CollegeHeather Rose Kates - University of FloridaXinshuai Qi - Zhejiang UniversitySamuel F. Brockington - University of CambridgePamela S. Soltis - Florida Museum of Natural HistoryDouglas E. Soltis - University of FloridaMatthew A. Gitzendanner - Florida Museum of Natural History
- Resource Type
- Journal article
- Publication Details
- Applications in Plant Sciences, Vol.1(2), pp.1200497-n/a
- DOI
- 10.3732/apps.1200497
- PMID
- 25202518
- PMCID
- PMC4105372
- NLM abbreviation
- Appl Plant Sci
- ISSN
- 2168-0450
- eISSN
- 2168-0450
- Grant note
- 0735191; 0431266 / National Science Foundation (http://data.elsevier.com/vocabulary/SciValFunders/100000001)
- Language
- English
- Date published
- 02/2013
- Academic Unit
- Biochemistry and Molecular Biology
- Record Identifier
- 9985113008202771
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