Yiyuan Li

Ultra-deep sequencing enables high-fidelity recovery of biodiversity for bulk arthropod samples without PCR amplification

BackgroundNext-generation-sequencing (NGS) technologies combined with a classic DNA barcoding approach have enabled fast and credible measurement for biodiversity of mixed environmental samples. However, the PCR amplification involved in nearly all existing NGS protocols inevitably introduces taxonomic biases. In the present study, we developed new Illumina pipelines without PCR amplifications to analyze terrestrial arthropod communities.ResultsMitochondrial enrichment directly followed by Illumina shotgun sequencing, at an ultra-high sequence volume, enabled the recovery of Cytochrome c Oxidase subunit 1 (COI) barcode sequences, which allowed for the estimation of species composition at high fidelity for a terrestrial insect community. With 15.5 Gbp Illumina data, approximately 97% and 92% were detected out of the 37 input Operational Taxonomic Units (OTUs), whether the reference barcode library was used or not, respectively, while only 1 novel OTU was found for the latter. Additionally, relatively strong correlation between the sequencing volume and the total biomass was observed for species from the bulk sample, suggesting a potential solution to reveal relative abundance.ConclusionsThe ability of the new Illumina PCR-free pipeline for DNA metabarcoding to detect small arthropod specimens and its tendency to avoid most, if not all, false positives suggests its great potential in biodiversity-related surveillance, such as in biomonitoring programs. However, further improvement for mitochondrial enrichment is likely needed for the application of the new pipeline in analyzing arthropod communities at higher diversity.

Multiplex sequencing of pooled mitochondrial genomes—a crucial step toward biodiversity analysis using mito-metagenomics

The advent in high-throughput-sequencing (HTS) technologies has revolutionized conventional biodiversity research by enabling parallel capture of DNA sequences possessing species-level diagnosis. However, polymerase chain reaction (PCR)-based implementation is biased by the efficiency of primer binding across lineages of organisms. A PCR-free HTS approach will alleviate this artefact and significantly improve upon the multi-locus method utilizing full mitogenomes. Here we developed a novel multiplex sequencing and assembly pipeline allowing for simultaneous acquisition of full mitogenomes from pooled animals without DNA enrichment or amplification. By concatenating assemblies from three de novo assemblers, we obtained high-quality mitogenomes for all 49 pooled taxa, with 36 species >15 kb and the remaining >10 kb, including 20 complete mitogenomes and nearly all protein coding genes (99.6%). The assembly quality was carefully validated with Sanger sequences, reference genomes and conservativeness of protein coding genes across taxa. The new method was effective even for closely related taxa, e.g. three Drosophila spp., demonstrating its broad utility for biodiversity research and mito-phylogenomics. Finally, the in silico simulation showed that by recruiting multiple mito-loci, taxon detection was improved at a fixed sequencing depth. Combined, these results demonstrate the plausibility of a multi-locus mito-metagenomics approach as the next phase of the current single-locus metabarcoding method.

Quantification of mesocosm fish and amphibian species diversity via environmental DNA metabarcoding.

Freshwater fauna are particularly sensitive to environmental change and disturbance. Management agencies frequently use fish and amphibian biodiversity as indicators of ecosystem health and a way to prioritize and assess management strategies. Traditional aquatic bioassessment that relies on capture of organisms via nets, traps and electrofishing gear typically has low detection probabilities for rare species and can injure individuals of protected species. Our objective was to determine whether environmental DNA (eDNA) sampling and metabarcoding analysis can be used to accurately measure species diversity in aquatic assemblages with differing structures. We manipulated the density and relative abundance of eight fish and one amphibian species in replicated 206‐L mesocosms. Environmental DNA was filtered from water samples, and six mitochondrial gene fragments were Illumina‐sequenced to measure species diversity in each mesocosm. Metabarcoding detected all nine species in all treatment replicates. Additionally, we found a modest, but positive relationship between species abundance and sequencing read abundance. Our results illustrate the potential for eDNA sampling and metabarcoding approaches to improve quantification of aquatic species diversity in natural environments and point the way towards using eDNA metabarcoding as an index of macrofaunal species abundance.

Estimating species richness using environmental DNA

Abstract The foundation for any ecological study and for the effective management of biodiversity in natural systems requires knowing what species are present in an ecosystem. We assessed fish communities in a stream using two methods, depletion‐based electrofishing and environmental DNA metabarcoding (eDNA) from water samples, to test the hypothesis that eDNA provides an alternative means of determining species richness and species identities for a natural ecosystem. In a northern Indiana stream, electrofishing yielded a direct estimate of 12 species and a mean estimated richness (Chao II estimator) of 16.6 species with a 95% confidence interval from 12.8 to 42.2. eDNA sampling detected an additional four species, congruent with the mean Chao II estimate from electrofishing. This increased detection rate for fish species between methods suggests that eDNA sampling can enhance estimation of fish fauna in flowing waters while having minimal sampling impacts on fish and their habitat. Modern genetic approaches therefore have the potential to transform our ability to build a more complete list of species for ecological investigations and inform management of aquatic ecosystems.

Effects of sampling effort on biodiversity patterns estimated from environmental DNA metabarcoding surveys

Environmental DNA (eDNA) metabarcoding can greatly enhance our understanding of global biodiversity and our ability to detect rare or cryptic species. However, sampling effort must be considered when interpreting results from these surveys. We explored how sampling effort influenced biodiversity patterns and nonindigenous species (NIS) detection in an eDNA metabarcoding survey of four commercial ports. Overall, we captured sequences from 18 metazoan phyla with minimal differences in taxonomic coverage between 18 S and COI primer sets. While community dissimilarity patterns were consistent across primers and sampling effort, richness patterns were not, suggesting that richness estimates are extremely sensitive to primer choice and sampling effort. The survey detected 64 potential NIS, with COI identifying more known NIS from port checklists but 18 S identifying more operational taxonomic units shared between three or more ports that represent un-recorded potential NIS. Overall, we conclude that eDNA metabarcoding surveys can reveal global similarity patterns among ports across a broad array of taxa and can also detect potential NIS in these key habitats. However, richness estimates and species assignments require caution. Based on results of this study, we make several recommendations for port eDNA sampling design and suggest several areas for future research.