An isothermal workflow for low‐cost and PCR‐free field‐based community metabarcoding
Abstract DNA metabarcoding has revolutionized our ability to monitor ecosystems. However, the method is still rarely used in developing countries where resources are limited and fieldwork is challenging. To overcome this limitation, we designed a comprehensive workflow allowing rapid community metab...
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| Hauptverfasser: | , , |
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| Format: | Artikel |
| Sprache: | Englisch |
| Veröffentlicht: |
Wiley
2025-07-01
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| Schriftenreihe: | Methods in Ecology and Evolution |
| Schlagworte: | |
| Online-Zugang: | https://doi.org/10.1111/2041-210X.70069 |
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| Zusammenfassung: | Abstract DNA metabarcoding has revolutionized our ability to monitor ecosystems. However, the method is still rarely used in developing countries where resources are limited and fieldwork is challenging. To overcome this limitation, we designed a comprehensive workflow allowing rapid community metabarcoding with minimum, self‐manufacturable equipment in the field. The workflow combines fast DNA extraction with isothermal recombinase polymerase amplification (RPA). Fast DNA extraction takes about 2 min and is optimized for environmental samples, employing cellulose columns to capture DNA. RPA‐based preparation of libraries for high‐throughput sequencing is completely isothermal and requires stepwise reamplification (3X for MiSeq and 2X for MinION libraries). We used environmental DNA from fish mock communities to demonstrate the sensitivity of the novel workflow and subsequently tested it on‐site with European fish in a riverine ecotone. Fast DNA extraction yielded reduced DNA mass when compared with kit‐based extraction, yet recovered complete community composition. RPA‐based sequencing libraries equally detected complete composition and structure of communities when compared to PCR‐based approaches. Combined with simplified, self‐manufacturable field laboratories and MinION sequencing, the workflow is completely field‐deployable while providing alternatives to remain interconnective with established lab‐based protocols such as MiSeq sequencing. Compared to laboratory‐dependent approaches, the novel workflow halves costs and reduces hands‐on time more than fourfold. The protocol can be widely adapted for any taxonomic group with the design of RPA‐compatible primer sets. Making metabarcoding available to practitioners and researchers around the globe, our approach signifies a critical contribution to the immense task of characterizing and protecting the earth's biodiversity. |
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| ISSN: | 2041-210X |