Dataset Information

Nanopore sequencing of long ribosomal DNA amplicons enables portable and simple biodiversity assessments with high phylogenetic resolution across broad taxonomic scale.

ABSTRACT: BACKGROUND:In light of the current biodiversity crisis, DNA barcoding is developing into an essential tool to quantify state shifts in global ecosystems. Current barcoding protocols often rely on short amplicon sequences, which yield accurate identification of biological entities in a community but provide limited phylogenetic resolution across broad taxonomic scales. However, the phylogenetic structure of communities is an essential component of biodiversity. Consequently, a barcoding approach is required that unites robust taxonomic assignment power and high phylogenetic utility. A possible solution is offered by sequencing long ribosomal DNA (rDNA) amplicons on the MinION platform (Oxford Nanopore Technologies). FINDINGS:Using a dataset of various animal and plant species, with a focus on arthropods, we assemble a pipeline for long rDNA barcode analysis and introduce a new software (MiniBar) to demultiplex dual indexed Nanopore reads. We find excellent phylogenetic and taxonomic resolution offered by long rDNA sequences across broad taxonomic scales. We highlight the simplicity of our approach by field barcoding with a miniaturized, mobile laboratory in a remote rainforest. We also test the utility of long rDNA amplicons for analysis of community diversity through metabarcoding and find that they recover highly skewed diversity estimates. CONCLUSIONS:Sequencing dual indexed, long rDNA amplicons on the MinION platform is a straightforward, cost-effective, portable, and universal approach for eukaryote DNA barcoding. Although bulk community analyses using long-amplicon approaches may introduce biases, the long rDNA amplicons approach signifies a powerful tool for enabling the accurate recovery of taxonomic and phylogenetic diversity across biological communities.

SUBMITTER: Krehenwinkel H

PROVIDER: S-EPMC6503943 | biostudies-literature | 2019 May

REPOSITORIES: biostudies-literature

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Nanopore sequencing of long ribosomal DNA amplicons enables portable and simple biodiversity assessments with high phylogenetic resolution across broad taxonomic scale.

Krehenwinkel Henrik H Pomerantz Aaron A Henderson James B JB Kennedy Susan R SR Lim Jun Ying JY Swamy Varun V Shoobridge Juan Diego JD Graham Natalie N Patel Nipam H NH Gillespie Rosemary G RG Prost Stefan S

GigaScience 20190501 5

<h4>Background</h4>In light of the current biodiversity crisis, DNA barcoding is developing into an essential tool to quantify state shifts in global ecosystems. Current barcoding protocols often rely on short amplicon sequences, which yield accurate identification of biological entities in a community but provide limited phylogenetic resolution across broad taxonomic scales. However, the phylogenetic structure of communities is an essential component of biodiversity. Consequently, a barcoding a ...[more]

PMID: 30824940

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Project description:Finding standard cost-effective methods for monitoring biodiversity is challenging due to trade-offs between survey costs (including expertise), specificity, and range of applicability. These trade-offs cause a lack of comparability among datasets collected by ecologists and conservationists, which is most regrettable in taxonomically demanding work on megadiverse inconspicuous taxon groups. We have developed a site-scale survey method for diverse sessile land organisms, which can be analyzed over multiple scales and linked with ecological insights and management. The core idea is that field experts can effectively allocate observation effort when the time, area, and priority sequence of tasks are fixed. We present the protocol, explain its specifications (taxon group; expert qualification; plot size; effort) and applications based on >800 original surveys of four taxon groups; and we analyze its effectiveness using data on polypores in hemiboreal and tropical forests. We demonstrate consistent effort-species richness curves and among-survey variation in contrasting ecosystems, and high effectiveness compared with casual observations both at local and regional scales. Bias related to observer experience appeared negligible compared with typical assemblage variation. Being flexible in terms of sampling design, the method has enabled us to compile data from various projects to assess conservation status and habitat requirements of most species (specifically rarities and including discovery of new species); also, when linked with site descriptions, to complete environmental assessments and select indicator species for management. We conclude that simple rules can significantly improve expert-based biodiversity surveys. Ideally, define (i) a common plot size that addresses multiple taxon groups and management goals; (ii) taxon groups based on field expertise and feasible number of species; (iii) sufficient and practical search time; (iv) a procedure for recording within-plot heterogeneity. Such a framework, combined with freedom to allocate effort on-site, helps utilizing full expertise of observers without losing technical rigor.

Project description:Background:Advancements in portable scientific instruments provide promising avenues to expedite field work in order to understand the diverse array of organisms that inhabit our planet. Here, we tested the feasibility for in situ molecular analyses of endemic fauna using a portable laboratory fitting within a single backpack in one of the world's most imperiled biodiversity hotspots, the Ecuadorian Chocó rainforest. We used portable equipment, including the MinION nanopore sequencer (Oxford Nanopore Technologies) and the miniPCR (miniPCR), to perform DNA extraction, polymerase chain reaction amplification, and real-time DNA barcoding of reptile specimens in the field. Findings:We demonstrate that nanopore sequencing can be implemented in a remote tropical forest to quickly and accurately identify species using DNA barcoding, as we generated consensus sequences for species resolution with an accuracy of >99% in less than 24 hours after collecting specimens. The flexibility of our mobile laboratory further allowed us to generate sequence information at the Universidad Tecnológica Indoamérica in Quito for rare, endangered, and undescribed species. This includes the recently rediscovered Jambato toad, which was thought to be extinct for 28 years. Sequences generated on the MinION required as few as 30 reads to achieve high accuracy relative to Sanger sequencing, and with further multiplexing of samples, nanopore sequencing can become a cost-effective approach for rapid and portable DNA barcoding. Conclusions:Overall, we establish how mobile laboratories and nanopore sequencing can help to accelerate species identification in remote areas to aid in conservation efforts and be applied to research facilities in developing countries. This opens up possibilities for biodiversity studies by promoting local research capacity building, teaching nonspecialists and students about the environment, tackling wildlife crime, and promoting conservation via research-focused ecotourism.

Dataset Information

Nanopore sequencing of long ribosomal DNA amplicons enables portable and simple biodiversity assessments with high phylogenetic resolution across broad taxonomic scale.

Publications

Nanopore sequencing of long ribosomal DNA amplicons enables portable and simple biodiversity assessments with high phylogenetic resolution across broad taxonomic scale.

Similar Datasets

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