An efficient and robust laboratory workflow and tetrapod database for larger scale environmental DNA studies
Research article published in GigaScience (2019)
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.
Abstract sourced from PubMed (NCBI) for the cited record. See the original publication for the authoritative version.
Resumen
The use of environmental DNA for species detection via metabarcoding is growing rapidly.
Por qué esto importa para la hirudoterapia
Este estudio presenta un flujo de trabajo de laboratorio y un pipeline bioinformático para metabarcoding de ADN ambiental diseñado para mitigar la contaminación de las muestras y la asignación taxonómica incorrecta mediante replicación técnica, enfoques multimarcador (12S, 16S, CytB), PCR con doble etiquetado y asignación taxonómica probabilística a través de PROTAX. El pipeline se probó con ADN derivado de invertebrados procedente de 1.532 sanguijuelas de Sabah, Malasia, obteniendo 162 detecciones de vertebrados bajo criterios estrictos (a partir de 95 muestras de sanguijuelas) y 190 bajo criterios laxos (a partir de 109 muestras de sanguijuelas). Las sanguijuelas actúan aquí como recolectores pasivos de ADN de vertebrados para monitorización ecológica y de conservación, no como objeto de investigación terapéutica ni de investigación sobre el secretoma. Este trabajo no tiene relevancia directa para la hirudoterapia ni para el ámbito de ASH.
Citación
An efficient and robust laboratory workflow and tetrapod database for larger scale environmental DNA studies
Axtner J et al. · GigaScience, 2019
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Añadido a la biblioteca ASH: May 27, 2026 · Última actualización del sitio: 18 de junio de 2026