Comparative analysis of fish environmental DNA reveals higher sensitivity achieved through targeted sequence-based metabarcoding.

Environmental DNA (eDNA)-based methods of species detection are enabling various applications in ecology and conservation including large-scale biomonitoring efforts. qPCR is widely used as the standard approach for species-specific detection, often targeting a fish species of interest from aquatic eDNA. However, DNA metabarcoding has the potential to displace qPCR in certain eDNA applications. In this study, we compare the sensitivity of the latest Illumina NovaSeq 6000 NGS platform to qPCR TaqMan assays by measuring limits of detection and by analysing eDNA from water samples collected from Churchill River and Lake Melville, NL, Canada. Species-specific, targeted next generation sequencing (NGS) assays had significantly higher sensitivity than qPCR, with limits of detection 14- to 29-fold lower. For example, when analysing eDNA, qPCR detected Gadus ogac (Greenland cod) in 21% of samples, but targeted NGS detected this species in 29% of samples. General NGS assays were as sensitive as qPCR, while simultaneously detecting 15 fish species from eDNA samples. With over 34,000 fish species on the planet, parallel and sensitive methods such as NGS will be required to support effective biomonitoring at both regional and global scales.

Large-Scale Monitoring of Plants through Environmental DNA Metabarcoding of Soil: Recovery, Resolution, and Annotation of Four DNA Markers.

In a rapidly changing world we need methods to efficiently assess biodiversity in order to monitor ecosystem trends. Ecological monitoring often uses plant community composition to infer quality of sites but conventional aboveground surveys only capture a snapshot of the actively growing plant diversity. Environmental DNA (eDNA) extracted from soil samples, however, can include taxa represented by both active and dormant tissues, seeds, pollen, and detritus. Analysis of this eDNA through DNA metabarcoding provides a more comprehensive view of plant diversity at a site from a single assessment but it is not clear which DNA markers are best used to capture this diversity. Sequence recovery, annotation, and sequence resolution among taxa were evaluated for four established DNA markers (matK, rbcL, ITS2, and the trnL P6 loop) in silico using database sequences and in situ using high throughput sequencing of 35 soil samples from a remote boreal wetland. Overall, ITS2 and rbcL are recommended for DNA metabarcoding of vascular plants from eDNA when not using customized or geographically restricted reference databases. We describe a new framework for evaluating DNA metabarcodes and, contrary to existing assumptions, we found that full length DNA barcode regions could outperform shorter markers for surveying plant diversity from soil samples. By using current DNA barcoding markers rbcL and ITS2 for plant metabarcoding, we can take advantage of existing resources such as the growing DNA barcode database. Our work establishes the value of standard DNA barcodes for soil plant eDNA analysis in ecological investigations and biomonitoring programs and supports the collaborative development of DNA barcoding and metabarcoding.

A new way to contemplate Darwin's tangled bank: how DNA barcodes are reconnecting biodiversity science and biomonitoring.

Encompassing the breadth of biodiversity in biomonitoring programmes has been frustrated by an inability to simultaneously identify large numbers of species accurately and in a timely fashion. Biomonitoring infers the state of an ecosystem from samples collected and identified using the best available taxonomic knowledge. The advent of DNA barcoding has now given way to the extraction of bulk DNA from mixed samples of organisms in environmental samples through the development of high-throughput sequencing (HTS). This DNA metabarcoding approach allows an unprecedented view of the true breadth and depth of biodiversity, but its adoption poses two important challenges. First, bioinformatics techniques must simultaneously perform complex analyses of large datasets and translate the results of these analyses to a range of users. Second, the insights gained from HTS need to be amalgamated with concepts such as Linnaean taxonomy and indicator species, which are less comprehensive but more intuitive. It is clear that we are moving beyond proof-of-concept studies to address the challenge of implementation of this new approach for environmental monitoring and regulation. Interpreting Darwin's 'tangled bank' through a DNA lens is now a reality, but the question remains: how can this information be generated and used reliably, and how does it relate to accepted norms in ecosystem study?This article is part of the themed issue 'From DNA barcodes to biomes'.