TAXON 63 (4) • August 2014: 899–910 Wysocki & al. • Full plastome sequence assembly methods in grasses METHODS AND TECHNIQUES A multi-step comparison of short-read full plastome sequence assembly methods in grasses William P. Wysocki,1 Lynn G. Clark,2 Scot A. Kelchner,3 Sean V. Burke,1 J. Chris Pires,4 Patrick P. Edger,5 Dustin R. Mayfield,4 Jimmy K. Triplett,6 J. Travis Columbus,7 Amanda L. Ingram8 & Melvin R. Duvall1 1 Biological Sciences, Northern Illinois University, 1425 W. Lincoln Hwy, DeKalb, Illinois 60115-2861, U.S.A. 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 Division of Biological Sciences, University of Missouri, 1201 Rollins St, Columbia, Missouri 65211, U.S.A. 5 Department of Plant and Microbial Biology, University of California – Berkeley, Berkeley, California 94720, U.S.A. 6 Department of Biology, Jacksonville State University, 144B Martin Hall, Jacksonville, Alabama 36265, U.S.A. 7 Rancho Santa Ana Botanic Garden & Claremont Graduate University, 1500 North College Avenue, Claremont, California 91711, U.S.A. 8 Department of Biology, Wabash College, P.O. Box 352, Crawfordsville, Indiana 47933, U.S.A. Author for correspondence: William P. Wysocki, [email protected] ORCID: J.C.P., http://www.orcid.org/ J. Chris Pires: 0000-0001-9682-2639 DOI http://dx.doi.org/10.12705/634.5 Abstract 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. Keywords ACRE; de novo assembly; next-generation sequencing; plastome; Poaceae Supplementary Material The Electronic Supplement (Tables S1–S2; Appendix S1: Guide to short-read plastome assembly) is available in the Supplementary Data section of the online version of this article at http://www.ingentaconnect.com/ content/iapt/tax INTRODUCTION and their complex evolutionary history. These phylogenomic studies—which are genome-scale phylogenetic studies—have Systematic studies of land plants have advanced in the been shown to offer improved resolution, stronger support, context of molecular phylogenetic research. Access to next- and allow for more confident estimates of divergence dates. generation sequencing (NGS) methods has given plant sys- In this context, complete chloroplast genomes (plastomes) are tematists the ability to analyze genome-scale data for large tools of demonstrated phylogenetic utility for studies at differ- numbers of terminal taxa to investigate evolutionary relation- ent taxonomic levels. Intrageneric phylogenomic studies show ships. Taxa within the grass family (Poaceae) are of particu- fine-scale relationships, document microstructural events, and lar interest due to the economic importance of cereal grains, offer explanations for historical biogeographic patterns (Cronn their use in functional genomic studies (Botiri & al., 2008) & al., 2008; Parks & al., 2009, 2012; Burke & al., 2012, 2014). Received: 22 Jan 2014 | returned for first revision: 3 May 2014 | last revision received: 27 May 2014 | accepted: 27 May 2014 | published online ahead of inclusion in print and online issues: 5 Aug 2014 || © International Association for Plant Taxonomy (IAPT) 2014 Version of Record (identical to print version). 899 Wysocki & al. • Full plastome sequence assembly methods in grasses TAXON 63 (4) • August 2014: 899–910 Intergeneric studies show broader evolutionary patterns even A robust test for accuracy of plastome assembly would be in taxa with slowly evolving plastomes, indicate patterns of to compare duplicated sequences from the same plant using genome evolution, and show lineage-specific rate variations two different methods, such as Sanger and Illumina. To this (Zhang, Y.J. & al., 2011; Hand & al., 2013). Studies within fami- end a Sanger-sequenced plastome of Neyraudia reynaudiana lies have resolved formerly intractable phylogenetic issues and (Kunth) Keng ex Hitchc. along with NGS sequenced plastome revealed readily interpretable patterns of molecular evolution of the same individual was used to test their accuracy. Addi- (Leseberg & Duvall, 2009; Duvall & al., 2010; Wu & Ge, 2012). tionally we used Sanger-sequenced plastomes of two other spe- Growing use of complete plastomes by plant systematists cies (Arundinaria gigantea (Walt.) Muhl., Pharus latifolius L.) does not reflect uniform methodological choices for determin- along with NGS sequenced plastomes from closely related con- ing and assembling these data. In part, this is due to the rapid geners of these species to perform a somewhat less stringent changes of the NGS technologies, with periodic advances often test, similar to the strategy employed by Steele & al. (2012). accompanied by increases in read length, which directly impact This is justified because of the high identities of plastomes the efficiency and accuracy of assembly. The development of between grass and other monocot congeners (see above). new software tools for assembly is another factor. However, The study species represent three major lineages of these do not entirely account for the diversity of methods of grasses. From the PACMAD clade (acronym abbreviates the plastome assembly employed in published reports. Although subfamilial membership for Panicoideae, Arundinoideae, Chlo- there are numerous comparative studies of different methods ridoideae, Micrairoideae, Aristidoideae, and Danthonioideae) of assembling large genomes (Lin & al., 2011; Zhang, W. & al., the chloridoid Neyraudia reynaudiana was selected and com- 2011; Liu & al., 2012; and many others), we find a single such pared against itself for the two sequencing methods. From the study for assembling complete plastomes (Steele & al., 2012). BEP clade (acronym for: Bambusoideae, Ehrhartoideae, and Plastome assembly presents challenges such as a large inverted Pooideae) the New World bambusoid species (following Clark repeat region, mitogenomes with similar sequences that are & Triplett, 2007) Arundinaria gigantea and both of its conge- likely intermingled in the same pool of reads, and AT-richness, ners, A. tecta (Walt.) Muhl. and A. appalachiana Triplett & al., which introduces periodic regions of low sequence complex- were selected. Finally, from one lineage of the deeply diverging ity. Plastomes also present unique opportunities for phyloge- grade of grasses Pharus latifolius and the congeneric species nomic analysis, foremost of which are their highly conserved P. lappulaceus Aubl. were selected. By assessing the accuracy, sequences and structures. Major structural changes have been completeness, and efficiency of these assembly methods we occasionally well documented at intergeneric or interfamilial compared different approaches to assembly and established levels (e.g., Doyle & al., 1992; Cosner & al., 2004). However, guidelines for full plastome determination of grasses using plastomes from monocot congeners such as Acorus america- short reads of approximately 100 base pairs (bp) produced from nus (Raf.) Raf. (NC010093) and A. calamus L. (NC007407) single-read libraries. and those from Oryza sativa L. “Japonica Group” (NC001320) and “Indica Group” (NC008155), O. meridionalis N.Q.Ng (NC016927) and O. nivara S.D.Shartma & Shastry (NC005973) MATERIALS AND METHODS show 99.54% and 99.49% nucleotide identities in alignments of these plastomes respectively (Wysocki, unpub. comparisons). DNA extraction and Sanger sequencing. — Silica- In existing studies of complete plastomes, different com- dried leaf samples were obtained from five species: Arundi- binations of assembly methods have been used. Prior to assem- naria appalachiana, U.S.A., J. Triplett JT099 (ISC); A. tecta, bly, NGS reads may be trimmed based on sequence quality