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

Abstract

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.

Host selection of hematophagous leeches (Haemadipsa japonica): Implications for iDNA studies

“…Data on the life history and behavior of leeches would also be needed to interpret the results, such as the time interval between feeding and sampling (Schnell et al, ). However, the studies in Vietnam, Bangladesh and Borneo indicated that the hosts were taxonomically variable (Axtner et al, ; Schnell et al, ; Weiskopf et al, ), so other Haemadipsa leeches might be suitable for making a species inventory.…”

“…Another relatively unexplored topic is on how different analysis method affects the results. Next generation sequencing (NGS) technology has been applied in the iDNA studies (Abrams et al, 2019;Axtner et al, 2019;Calvignac-Spencer, Merkel, et al, 2013b). One obvious advantage of NGS is that it can detect the DNA of multiple species collected from one individual of an invertebrate.…”

“…Aside from studies using hematophagous invertebrates (Kocher et al, ) and flies (Hoffmann, Stockhausen, Merkel, Calvignac‐Spencer, & Leendertz, ; Hoffmann et al, ) to study diseases across landscapes and using flies to study diseases within primate social groups (Gogarten et al, ), iDNA has mostly been used to study the presence of a particular species or biodiversity (Axtner et al, ; Calvignac‐Spencer et al, ; Drinkwater et al, ; Lee, Gan, Clements, & Wilson, ; Perez‐Flores, Rueda‐Calderon, Kvist, Siddall, & Oceguera‐Figueroa, ; Rodgers et al, ; Schnell et al, , ; Schubert et al, ; Tessler et al, ). For example, Schnell et al () analyzed iDNA from 25 individuals of hematophagous leeches in Viet Nam and detected six species of mammals, including two recently described species of rare muntjac and rabbit.…”

Non-invasive surveys of mammalian viruses using environmental DNA

“…Samples were stored in RNA fixating saturated ammonium sulfate solution and exported under the permit 'JKM/MBS.1000-2/3 JLD.2 (8)' issued by the Sabah Biodiversity Council. A total of 68 pools (L1-L68) were selected for viral capture to maximize representation of host wildlife species identified from bloodmeals [31].…”

“…Host identification of leeches followed an eDNA/iDNA workflow recently published [31]. In summary, leech samples were digested and short fragments of the mitochondrial markers 12S, 16S and cytochrome B were amplified in four PCR replicates each resulting in 12 PCR replicates per sample.…”