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A multi-step comparison of short-read full plastome sequence assembly methods in grasses

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Technological advances have allowed phylogenomic studies of plants, such as full chloroplast genome (plastome) analysis, to become increasingly popular and economically feasible. Although next-generation short-read sequencing allows for full plastomes to be sequenced relatively rapidly, it requires additional attention using software to assemble these reads into comprehensive sequences. Here we compare the use of three de novo assemblers combined with three contig assembly methods. Seven plastome sequences were analyzed. Three of these were Sanger-sequenced. The other four were assembled from short, single-end read files generated from next-generation libraries. These plastomes represented a total of six grass species (Poaceae), one of which was sequenced in duplicate by the two methods to allow direct comparisons for accuracy. Enumeration of missing sequence and ambiguities allowed for assessments of completeness and efficiency. All methods that used de Bruijn-based de novo assemblers were shown to produce assemblies comparable to the Sanger-sequenced plastomes but were not equally efficient. Contig assembly methods that utilized automatable and repeatable processes were generally more efficient and advantageous when applied to larger scale projects with many full plastomes. However, contig assembly methods that were less automatable and required more manual attention did show utility in determining plastomes with lower read depth that were not able to be assembled when automatable procedures were implemented. Although the methods here were used exclusively to generate grass plastomes, these could be applied to other taxonomic groups if previously sequenced plastomes were available. In addition to comparing sequencing methods, a supplemental guide for short-read plastome assembly and applicable scripts were generated for this study.
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Keywords: ACRE; DE NOVO ASSEMBLY; NEXT-GENERATION SEQUENCING; PLASTOME; POACEAE

Document Type: Research Article

Affiliations: 1: Biological Sciences, Northern Illinois University, 1425 W. Lincoln Hwy, DeKalb, Illinois 60115-2861, U.S.A.;, Email: [email protected] 2: Ecology, Evolution and Organismal Biology, Iowa State University, 353 Bessey Hall, Ames, Iowa 50011-1020, U.S.A. 3: Biological Sciences, Idaho State University, 921 S. 8th Ave, Pocatello, Idaho 83209-8007, U.S.A. 4: Biological Sciences, Northern Illinois University, 1425 W. Lincoln Hwy, DeKalb, Illinois 60115-2861, U.S.A. 5: Division of Biological Sciences, University of Missouri, 1201 Rollins St, Columbia, Missouri 65211, U.S.A. 6: Department of Plant and Microbial Biology, University of California – Berkeley, Berkeley, California 94720, U.S.A. 7: Department of Biology, Jacksonville State University, 144B Martin Hall, Jacksonville, Alabama 36265, U.S.A. 8: Rancho Santa Ana Botanic Garden & Claremont Graduate University, 1500 North College Avenue, Claremont, California 91711, U.S.A. 9: Department of Biology, Wabash College, P.O. Box 352, Crawfordsville, Indiana 47933, U.S.A.

Publication date: 28 August 2014

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