An efficient and robust laboratory workflow and tetrapod database for larger scale environmental DNA studies

BACKGROUND: The use of environmental DNA for species detection via metabarcoding is growing rapidly. We present a co-designed lab workflow and bioinformatic pipeline to mitigate the 2 most important risks of environmental DNA use: sample contamination and taxonomic misassignment. These risks arise from the need for polymerase chain reaction (PCR) amplification to detect the trace amounts of DNA combined with the necessity of using short target regions due to DNA degradation. FINDINGS: Our high-throughput workflow minimizes these risks via a 4-step strategy: (i) technical replication with 2 PCR replicates and 2 extraction replicates; (ii) using multi-markers (12S,16S,CytB); (iii) a "twin-tagging," 2-step PCR protocol; and (iv) use of the probabilistic taxonomic assignment method PROTAX, which can account for incomplete reference databases. Because annotation errors in the reference sequences can result in taxonomic misassignment, we supply a protocol for curating sequence datasets. For some taxonomic groups and some markers, curation resulted in >50% of sequences being deleted from public reference databases, owing to (i) limited overlap between our target amplicon and reference sequences, (ii) mislabelling of reference sequences, and (iii) redundancy. Finally, we provide a bioinformatic pipeline to process amplicons and conduct PROTAX assignment and tested it on an invertebrate-derived DNA dataset from 1,532 leeches from Sabah, Malaysia. Twin-tagging allowed us to detect and exclude sequences with non-matching tags. The smallest DNA fragment (16S) amplified most frequently for all samples but was less powerful for discriminating at species rank. Using a stringent and lax acceptance criterion we found 162 (stringent) and 190 (lax) vertebrate detections of 95 (stringent) and 109 (lax) leech samples. CONCLUSIONS: Our metabarcoding workflow should help research groups increase the robustness of their results and therefore facilitate wider use of environmental and invertebrate-derived DNA, which is turning into a valuable source of ecological and conservation information on tetrapods

Shifting up a gear with iDNA: From mammal detection events to standardised surveys

Invertebrate-derived DNA (iDNA), in combination with high throughput sequencing, has been proposed as a cost-efficient and powerful tool to survey vertebrate species. Previous studies, however, have only provided evidence that vertebrates can be detected using iDNA, but have not taken the next step of placing these detection events within a statistical framework that allows for robust biodiversity assessments. Here, we compare concurrent iDNA and camera-trap surveys. Leeches were repeatedly collected in close vicinity to 64 camera-trap stations in Sabah, Malaysian Borneo. We analyse iDNA-derived mammalian detection events in a modern occupancy model that accounts for imperfect detection and compare the results with those from occupancy models parameterised with camera-trap-derived detection events. We also combine leech-iDNA and camera-trap data in a single occupancy model. We found consistent estimates of occupancy probabilities produced by our camera-trap and leech datasets. This indicates that the metabarcoding of leech-iDNA method provides reasonable estimates of occupancy and may be a suitable method for studying and monitoring mammal species in tropical rainforests. However, we also show that a more extensive collection of leeches would be needed to assess mammal biodiversity with a robustness similar to that of camera traps. As certain taxa were only detected in leeches, we see great potential in complementing camera-trap studies with the iDNA approach, as long as the collection of leeches follows a robust and standardised sampling scheme. Synthesis and applications. Here, we describe an approach to analyse detection records of mammals derived from leech samples using an occupancy framework that accounts for leech-specific factors influencing the detection probability. We further combined camera trap and leech data, which lead to increased confidence in occupancy estimates. Our approach is not restricted to the processing of leech samples, but can be used for the analysis of other invertebrate DNA and environmental DNA data. Our study is the first step to shift the application of invertebrate DNA studies from opportunistic ad-hoc collections to the systematic surveys required for long-term management of wildlife populations

Supporting data for "An efficient and improved laboratory workflow and tetrapod database for larger scale eDNA studies"

The use of environmental DNA, eDNA, for species detection via metabarcoding is growing rapidly and now, even terrestrial mammals can be monitored via invertebrate-derived DNA or iDNA from hematophagous invertebrates. We present a co-designed lab workflow and bioinformatic pipeline to mitigate the two most important risks of e/iDNA: sample contamination and taxonomic mis-assignment. These risks arise from the need for amplification to detect the trace amounts of DNA and the necessity of using short target regions due to DNA degradation. Here we present a high-throughput laboratory workflow that minimises these risks via a three-step strategy: (1) each sample is sequenced for two PCR replicates from each of two extraction replicates; (2) we use a ‘twin-tagging,’ two-step PCR protocol; (3) and a multi-marker approach targeting three mitochondrial loci: 12S, 16S and CytB. As a test, 1532 leeches were analysed from Sabah, Malaysian Borneo. Twin-tagging allowed us to detect and exclude chimeric sequences. The smallest DNA fragment (16S) amplified best for all samples but often at lower taxonomic resolution. We only accepted assignments that were found in both extraction replicates, totalling 174 assignments for 96 samples. To avoid false taxonomic assignments, we also present an approach to create curated reference databases that can be used with the powerful taxonomic-assignment method PROTAX. For some taxonomic groups and some markers, curation resulted in over 50% of sequences being deleted from public reference databases, due mainly to: (1) limited overlap between our target amplicon and available reference sequences; (2) apparent mislabelling of reference sequences; (3) redundancy. A provided bioinformatics pipeline processes amplicons and conducts the PROTAX taxonomic assignment. Our metabarcoding workflow should help research groups to increase the robustness of their results and therefore facilitate wider usage of e/iDNA, which is turning into a valuable source of ecological and conservation information on tetrapods