From COI to Complete Mitogenomes on NGS Platforms
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DNA Barcodes 2015; 3: 170–186 Research Article Open Access Damien D. Hinsinger, Regis Debruyne, Maeva Thomas, Gaël P. J. Denys, Marion Mennesson, Jose Utge, Agnes Dettai Fishing for barcodes in the Torrent: from COI to complete mitogenomes on NGS platforms DOI 10.1515/dna-2015-0019 samples, adaptable on any desktop NGS platform. It enables Received March 31, 2015; accepted September 8, 2015 to extend from the prevalent barcoding approach by shifting Abstract: The adoption of Next-Generation Sequencing from the single COI to complete mitogenome sequencing. (NGS) by the field of DNA barcoding of Metazoa has been hindered by the fit between the classical COI Keywords: DNA barcoding; mitogenome assembly; Next- barcode and the Sanger-based sequencing method. Generation Sequencing; sample multiplexing; sequence Here we describe a framework for the sequencing post-processing and multiplexing of mitogenomes on NGS platforms that implements (I) a universal long-range PCR-based amplification technique, (II) a two-level multiplexing approach (i.e. divergence-based and specific tag indexing), 1 Introduction and (III) a dedicated demultiplexing and assembling For over a decade now, the DNA-barcoding effort (sensu [1] script from an Ion Torrent sequencing platform. for Actinopterygian fish species has focused on extensively We provide a case study of mitogenomes obtained for two sequencing the barcode region of the mitochondrial vouchered individuals of daces Leuciscus burdigalensis Cytochrome Oxidase 1 gene (COI) via a classical sanger- and L. oxyrrhis and show that this workflow enables to based approach [2,3]. This identification and preliminary recover over 100 mitogenomes per sequencing chip on a analysis tool for characterizing fish diversity has been PGM sequencer, bringing the individual cost down below very successful thanks to the variability within the COI 7,50€ per mitogenome (as of current 2015 sequencing costs). barcode sequence coupled with taxonomically efficient The use of several kilobases for identification purposes, as PCR primers (e.g. [4], and the availability of a dedicated, involved in the improved DNA-barcode we propose, stress easy to use database to store and analyze the reference the need for data reliability, especially through metadata. sequences: the Barcode of Life Database (BOLD; [5]. It Based on both scientific and economic considerations, this has also brought methodological improvements through framework presents a relevant approach for multiplexing the encouraged use of shared, standardized markers and systematic vouchering of specimens attached to *Corresponding author: Agnes Dettai, Institut de Systématique, the sequences [1,6,7]. The appropriateness of COI as a Évolution, Biodiversité ISYEB, UMR 7205 CNRS, MNHN, UPMC, EPHE marker for fish identification has been questioned, and Muséum national d’Histoire naturelle, Sorbonne Universités. 57 rue studies have used other mitochondrial markers instead Cuvier, CP30, 75005 Paris, France, E-mail: [email protected] (for instance [8–10], leading to some dispersion of efforts Damien D. Hinsinger, Institut de Systématique, Évolution, Biodiver- sité ISYEB, UMR 7205 CNRS, MNHN, UPMC, EPHE Muséum national in the search for a more variable and obtainable marker. d’Histoire naturelle, Sorbonne Universités. 57 rue Cuvier, CP30, While there are limitations to the current COI barcoding 75005 Paris, France strategy within metazoans, we consider that over the last Jose Utage, Regis Debruyne, Outils et Méthodes de la Systématique twelve years its benefits have been noticeable. Although Intégrative, UMS 2700, MNHN, CNRS, Muséum national d’Histoire not perfect, it has provided a clear and unified framework naturelle, Sorbonne Universités. 57 rue Cuvier, CP26, 75005 Paris, France for biodiversity analysis, especially in Actinopterygians Maeva Thomas, Gaël P. J. Denys, Marion Mennesson, Unité Biologie where COI has proven appropriate [2,3], although COI des organismes et écosystèmes aquatiques (BOREA, UMR 7208), lacks variability in some groups [11]. However, the rise of Sorbonne Universités, Muséum national d’Histoire naturelle, Uni- the second-generation sequencing techniques with their versité Pierre et Marie Curie, Université de Caen Basse-Normandie, ability to generate large amounts of genomic data has CNRS, IRD, 57 rue Cuvier, CP26, 75005 Paris, France increasingly challenged this framework [12], to the point © 2015 Damien D. Hinsinger et al. licensee De Gruyter Open. This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivs 3.0 License. A multiplex approach for sequencing mitogenomes of fishes 171 where some authors consider it nothing but outdated and second best represented vertebrate group after mammals irrelevant [13,14]. in mitogenome sequence number in NCBI Nucleotide, and The current second-generation sequencing platforms the first in number of represented species (figure 1). Since were originally designed to deep sequence individual the COI barcode belongs to the mitochondrial genome, whole-genome DNA libraries. Thus, they are not directly it exhibits the same evolutionary characteristics. Thus adapted to the multiplexed targeted sequencing of short both have the same advantages: unambiguous orthology genomic fragments like COI barcodes. As some other and uniparental inheritance facilitating their use within authors point out [15], this contrasts with the simplicity bifurcating phylogenetic approaches [29,31,32], as well and immediate efficacy of COI barcode sequencing. One as high levels of phylogenetic content with somewhat of the greatest difficulties on those platforms pertains to uniform evolutionary rates [33]. But they also have the attaching sequence reads/consensuses to actual specimen same limitations: potential species paraphyly, lineage- vouchers while sequencing a cost-effective number of specific rates of evolution and base compositions [34]. samples, without multiplying sequence tags and their We promote here an affordable and easy approach added cost and labwork [16–18]. Methods implementing to develop actinopterygian mitogenomics still further, by known indexed libraries in order to demultiplex sequences sequencing complete or large fragments of mitogenomes from individual organisms have indeed proven work- using next generation sequencing technologies and two- intensive (when PCR-based; [19,20] and/or expensive level multiplexing. In the line of Timmermans et al. [16], (when ligation-based) and, despite being productive Kane et al. [35], Dettai et al. [17] and Strohm et al. [7], [21], lack the simplicity and appeal of the Sanger-based we discuss how this could represent an extension of the sequencing of individual PCRs. original barcode marker for more precise identification To overcome this problem for the Next Generation of vouchers and metagenomics, while remaining fully Sequencing methods (NGS; [22], several pragmatic compatible with the previously obtained mitochondrial approaches intend to improve multiplexing by considering datasets. We describe briefly the mitogenomes derived from an implicit tagging of the samples based on their sequence the presented workflow for two specimens from species divergence, as originally proposed by Pollock et al. [23] of daces endemic to the South-West of France, Leuciscus in the pre-NGS era. Both PCR-based [16] and PCR-free burdigalensis Valenciennes 1844 and Leuciscus oxyrrhis [17,24] approaches have thus been described. They all (La Blanchère 1873), as an example of improvements for make use of the throughput of current NGS platforms barcoding, phylogeographic and taxonomic studies. We to sequence not only the COI barcode sequence but the also evaluate the presence of sequences of mitogenomic entire mitogenome, with high multiplexing of individuals. origin in 11 complete fish nuclear genomes available in In these new NGS frameworks, currently available COI the ENSEMBL database to assess the risk of sequencing barcode reference data have proved very useful to guide NUMTs instead of the desired mitogenomes. the a priori taxon-multiplexing strategy [17] as well as for a posteriori assignation of assembled mitogenomes to the samples. The COI and other mitochondrial sequences are 2 Methods used as ‘baits’ [16,25], and thus the necessary traceability to specimen vouchers is maintained. 2.1 Dedicated Teleost long-range PCR ampli- In recent years, the number of mitogenome fication of mitogenomes sequence releases in GenBank has considerably risen thanks to increased throughput in new sequencing We set out to amplify the complete mitochondrial genome platforms [7,17,26]. Furthermore, the number of available for organisms of various groups of local research interest mitogenomes, and their usefulness for phylogeny at largely spread within teleosts: Cyprinidae, Esocidae, different scales has been particularly well explored Cottidae, Nototheniidae, Gobiidae, Eleotridae, Lampridae, in Actinopterygians since the description of the carp and Zoarcidae. We downloaded complete mitochondrial mitogenome in 1994 [27]. This is largely due the effort of sequences from NCBI Nucleotide for species from these one team over the last 15 years which is responsible for the groups and aligned them using ClustalW (with default publication of more than 1340 fish mitogenomes and the settings; [36]). Conserved sequence regions (identified corresponding publications (from [28] to [29] This includes by eye) were subsequently imported into Oligo 4.1 Primer the creation of Mitofish and MitoAnnotator, a dedicated