Microfluidic PCR-based target enrichment： A case study in two rapid radiations of Commiphora （Burseraceae） from Madagascar  

作　　者：Morgan R. Gostel Kiera A. Coy Andrea Weeks 

机构地区：[1]Department of Environmental Science and Policy, George Mason University, MSN 5F2, 4400 University Drive, Fairfax, Virginia 22030, USA [2]Smithsonian Conservation Biology Institute, 3001 Connecticut Ave NW, Washington, D.C. 20008, USA [3]Department of Biology and Ted R. Bradley Herbarium, George Mason University, MSN 3E1, 4400 University Drive, Fairfax, Virginia 22030, USA

出　　处：《Journal of Systematics and Evolution》2015年第5期411-431,共21页植物分类学报（英文版）

基　　金：Research was funded by the National Science Foundation （NSF） #0919179 （AW）, NSF #14o315o （AW and MRG）, the American Society of Plant Taxonomists （Graduate Research Grant to MRG）, the George Mason University Department of Environmental Science and Policy （Graduate Research Fellow- ship to MRG）, and the George Mason University Office of Student Scholarship, Creative Activities, and Research （OSCAR Undergraduate Research Scholars Program award to KAC and MRG）. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the NSF or our other sponsors. Joe Boland （NIH AGI）, John Lynch and Lena Emadi （Fluidigm Corporation）, Simon Uribe-Convers （University of Idaho）, Doug Soltis （University of Florida）, Eric Carpenter （OneKP project）, and the Beijing Genomics Institute （OneKP） provided invaluable technical advice as well as generous material support and guidance. Lastly, we thank herbaria and other institutions that were integral to our conducting this research： Parc Botanique et Zoologique de Tsimbazaza, Madagascar （TAN）; Centre National de la Recherche Appliquee au Developement Rural, Madagascar （TEF）; Mis- souri Botanical Garden; and the Museum National d＇Histoire Naturelle, Paris.

摘　　要：Developing effective and cost-efficient multilocus nuclear datasets for angiosperm species is a continuing challenge to the systematics community. Here we describe the development and validation of a novel set of 91 nuclear markers for PCR-based target enrichment. Using microfluidic PCR and Illumina MiSeq, we generated nuclear, subgenomic libraries for 96 species simultaneously and sequenced them for a total cost of ca. S6000 USD. Approximately half of these costs include reusable reagents （primers, barcodes, and custom sequencing primers） and taxon sampling could be increased by an order of magnitude to maximize sequencing depth efficiency. The principle benefit of microfluidic PCR over alternative target enrichment strategies is that it bypasses costly library preparation. After sequencing, we evaluated the ability of the loci to resolve species level relationships within two recently radiated lineages of endemic Madagascan Commiphora Jacq. （Burseraceae） species. Our results demonstrate that （i） effective nuclear markers can be designed for non-model angiosperm taxa from these publicly available datasets; （ii） that microfluidic PCP, amplification followed by high throughput sequencing can produce highly complete taxon by locus sequence data matrices with minimal resource investment; and （iii） that these numerous nuclear phylogenomic markers can improve our understanding of phylogenetic relationships within Commiphora. We provide a synopsis of ongoing activities to enhance this microfluidic PCR-based target enrichment strategy through broader primer assays, multiplexing, and increased efficiency of sequencing depth.Developing effective and cost-efficient multilocus nuclear datasets for angiosperm species is a continuing challenge to the systematics community. Here we describe the development and validation of a novel set of 91 nuclear markers for PCR-based target enrichment. Using microfluidic PCR and Illumina MiSeq, we generated nuclear, subgenomic libraries for 96 species simultaneously and sequenced them for a total cost of ca. S6000 USD. Approximately half of these costs include reusable reagents （primers, barcodes, and custom sequencing primers） and taxon sampling could be increased by an order of magnitude to maximize sequencing depth efficiency. The principle benefit of microfluidic PCR over alternative target enrichment strategies is that it bypasses costly library preparation. After sequencing, we evaluated the ability of the loci to resolve species level relationships within two recently radiated lineages of endemic Madagascan Commiphora Jacq. （Burseraceae） species. Our results demonstrate that （i） effective nuclear markers can be designed for non-model angiosperm taxa from these publicly available datasets; （ii） that microfluidic PCP, amplification followed by high throughput sequencing can produce highly complete taxon by locus sequence data matrices with minimal resource investment; and （iii） that these numerous nuclear phylogenomic markers can improve our understanding of phylogenetic relationships within Commiphora. We provide a synopsis of ongoing activities to enhance this microfluidic PCR-based target enrichment strategy through broader primer assays, multiplexing, and increased efficiency of sequencing depth.

关 键 词：genome-tagged amplification lllumina MiSeq microfluidic PCR PHYLOGENOMICS targeted amplicon sequencing. 

分 类 号：TP211.3[自动化与计算机技术—检测技术与自动化装置] Q78[自动化与计算机技术—控制科学与工程]