Abstract
Chondrichthyes - sharks, rays, skates, and chimeras, are among the most threatened and data deficient vertebrate species. Global demand for shark and ray derived products, drives unregulated and exploitative fishing practices, which are in turn facilitated by the lack of ecological data required for effective conservation of these species. Here, we describe a Next Generation Sequencing method (using the MinION, a hand-held portable sequencing device from Oxford Nanopore Technologies), and analyses pipeline for molecular ecological studies in Chondrichthyes. Using this method, the complete mitochondrial genome and nuclear intergenic and protein-coding sequences were obtained by direct sequencing of genomic DNA obtained from shark fin tissue. Recovered loci include mitochondrial barcode sequences- Cytochrome oxidase I, NADH2, 16S rRNA and 12S rRNA- and nuclear genetic loci such as 5.8S rRNA, Internal Transcribed Spacer 2, and 28S rRNA regions, which are commonly used for taxonomic identification. Other loci recovered were the nuclear protein-coding genes for antithrombin or SerpinC, Immunoglobulin lambda light chain, Preprogehrelin, selenium binding protein 1(SBP1), Interleukin-1 beta (IL-1β) and Recombination-Activating Gene 1 (RAG1). The median coverage across all genetic loci was 20x and sequence accuracy was ≥99.8% compared to reference sequences. Analyses of the nuclear ITS2 region and the mitochondrial protein-encoding loci allowed accurate taxonomic identification of the shark specimen as Carcharhinus falciformis, a CITES Appendix II species. MinION sequencing provided 1,152,211 bp of new shark genome, increasing the number of sequenced shark genomes to five. Phylogenetic analyses using both mitochondrial and nuclear loci provided evidence that Prionace glauca is nested within Carcharhinus, suggesting the need for taxonomic reassignment of P. glauca. We increased genomic information about a shark species for ecological and population genetic studies, enabled accurate identification of the shark tissue for biodiversity indexing and resolved phylogenetic relationships among multiple taxa. The method was independent of amplification bias, and adaptable for field assessments of other Chondrichthyes and wildlife species in the future.
Highlights
Chondrichthyes are among the oldest extant vertebrates
Molecular taxonomy is conducted by PCR amplification and sequencing of DNA barcodes such as Cytochrome oxidase 1(COI)[6,7,8] NADH29,10 16S rRNA and 12S rRNA11,12, of the mitochondrial genome and the Recombination-Activating Gene 1(RAG1)[13] and Internal transcribed spacer 2 (ITS)[14] regions of the nuclear genome
Sequencing was done on the MinION portable sequencer from Oxford Nanopore Technologies (ONT) which requires library preparation and loading times of as little as thirty minutes, allows sequence acquisition as early as 2 hours into sequencing, and allows local sequencing and analyses of genomic DNA with a user-friendly software interface installed on a portable computer
Summary
Chondrichthyes (sharks, rays, skates, and chimaeras) are among the oldest extant vertebrates. Adopting a genomics approach could allow assessment of multiple genomic loci without amplification bias and without the need for a priori sequence information Such an approach would be ideal to increase ecological and evolutionary investigations in Chondrichthyes, similar to those of other commercial fisheries[25]. Increasing capacity for use of genome-wide data addresses taxonomic inadequacies and enables complex ecological and evolutionary questions to be answered by providing information about population size, structure, biogeography and priority conservation units. These data together will address chondrichthyan data deficiencies and assist conservation efforts. Our sequencing and analyses methods increase the available genomic information about C. falciformis and provide an accessible tool for genome-wide studies across Chondrichthyes
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