DNA Metabarcoding: A Rapid Method of Biodiversity Assessment
Author: Dr. Suresh Kaushik


Introduction

Managing the health of global ecosystems requires detailed inventories of species and a good understanding of the patterns and trends of biodiversity. Evolutionary and ecological studies often rely on our ability to identify the species involved in the process under investigation or our capacity to provide robust biodiversity estimates. For about three centuries, the acquisition of biodiversity data was based on morphological characterization of plants and animals. The idea of identifying species on the basis of molecular markers emerged soon after the advent of molecular biology. Early methods involved the use of hybridization, restriction enzyme digestion or other molecular probes. DNA-based species identification was introduced by Arnot et al. and further development, standardized and advanced by Hebert et al. The ability to extract and store DNA for prolonged periods of time provides a unique opportunity to assess the evolution of biodiversity over time in relation to global change and to develop concrete measures to reserve these features.

DNA Barcoding

A short DNA fragment as a barcode is used to identify specimen by studying a small piece of the genome as a marker found in a broad range of species. In other words, DNA barcoding is the identification of species using standardized DNA fragments. DNA barcodes are being used to identify specimens and the approach has wide applications in biodiversity conservation, the study of trophic interactions, food safety and environmental management.

DNA Metabarcoding

DNA metabarcoding refers to the automated identification of multiple species from a single bulk sample containing entire organisms or from a single environmental sample containing degraded DNA. This offers unprecedented scientific and operation opportunities in order to understand biodiversity distribution and dynamics in a better way. These opportunities also come with challenges that the scientific community will have to face. Such challenges are related to standardization of methods, methodological development for additional taxonomic groups, development of a common database, clarification of terminology and ways to avoid waste of information and data derived from metabarcoding. The availability of next-generation sequencing platforms and the ecologists' need for high throughput taxon identification have facilitated the emergence of DNA metabarcoding.

Approaches for DNA Metabarcoding

The performance of various DNA barcodes has been one of the main challenges of the early barcoding initiative. The use of the standardized animal barcode, mitochondrial chytochrome c oxidase subunit I (COI), was not practical for many targeted taxonomic group such as fungi and plants. More recently, the technical advancements provided by the genomic revolution have enabled direct evaluation of biodiversity. A rapid method of biodiversity assessment by DNA metabarcoding combines two technologies:

• DNA-based identification
• High-throughput DNA sequencing.

DNA metabarcoding approaches requires the amplification of shorter DNA fragments which are more appropriate for studies of target degraded environmental DNA (eDNA) and well adapted for spanning different taxonomic groups. Hence, the metabarcoding primers have to be versatile enough to amplify equally and exhausting different targeted groups. It uses universal PCR primers to mass-amplify DNA barcodes from mass collections of organisms or from environmental DNA. The PCR produced is sent to a next generation sequencer and the result is a wealth of DNA sequences. This rapidly growing, high-throughput and sensitive method is likely to generate an increase in the speed , accuracy and resolution of species identification.

Two techniques - DNA barcoding and metabarcoding - are similar in that they both use DNA-based identification of species but they have divergent assets that are determined by their distinct sequencing technologies and specific goals. DNA barcoding involves sequencing one well-curated individual at a time while metabarcoding entails massive parallel sequencing of complex bulk samples for which morphological identification and curation is not practical. The metabarcoding technique has the potential to capitalize on the enormous advantage offered by second-generation sequencing technology capable of generating millions of sequences in one run.

Operational Taxonomic Units (OTUs) and Interpretations

Current DNA metabarcoding studies provide biodiversity estimate that are highly dependent on the resolution of the marker used, the quality of the sequenced libraries, bioinformatics and the parameters used. The operational taxonomic units (OTUs) obtained are not easily reconcilable across sites or studies and inferences regarding species distribution are difficult to make. One of the main goals of metabarcoding project is to cluster sequences into OTUs that correspond to ecological species, unique in their particular niche or biological species as unique reproductive pools. The recovered OTUs can be also analysed using traditional phylogenetic interferences to provide taxonomic assignment and phylogentic placement with confidence estimates at each taxonomic level. OTUs generated by metabarcoding pipelines against references sequenced associated with taxonomic information should be validated. Metabarcoding data are similar to species-sample matrices used in ecology. Many existing tools applied to identify correlations and statistically significant patterns are preferable. Comprehensive comparative studies of OTUs generation methods tested on standard set of data set are needed and best practices in quality filtering, clustering and defining OTUs need to be established. But the ability to interpret the results depends not only on further bioinformatical refinements but also on the availability of well-populated databases that contain references sequences of taxa of interest. A large number of dedicated software packages are available to assess with data processing and analyses.

Challenges for DNA metabarcoding approach

The metabarcoding approaches faces challenges that can hinder our ability to produce robust, comparable biodiversity estimates. DNA metabarcoding depend on the intermediate PCR step. This step can generates amplification biases and contributes to errors which can influence biodiversity estimates. The second-generation sequencing platform can further introduce errors. Another set of challenges come from the need to build appropriate bioinformatics tools and the infrastructure to accommodate robust algorithms and efficient pipelines for data analysis.

Conclusions

DNA metabarcoding is emerging as a promising way of scrutinising the health of our ecosystems and advancing biodiversity research. It requires a global, integrative and interdisciplinary programmes for biodiversity research so as to generate reliable, verifiable and easily interpretable biodiversity estimates by robust, well-probed methods and generally accepted standards.

References
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About Author / Additional Info:
Researcher and teacher with Ph.D. Molecular Biology and Biotechnology