Towards low-cost and PCR free field-based community metabarcoding

1. 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. 2. To overcome this, we designed a comprehensive workflow allowing rapid community metabarcoding with minimum, self-manufacturable equipment in the field. We combine cellulose-based fast DNA extraction, optimized for environmental samples, with isothermal recombinase polymerase amplification (RPA). We demonstrate that stepwise RPA-based reamplification allows the introduction of non-homologous sequence adapters and index sequences, allowing the complete isothermal preparation of libraries for high-throughput sequencing. 3. Compared to laboratory-dependent approaches, the novel workflow halves costs and reduces hands on time more than fourfold. Our workflow makes metabarcoding broadly accessible for practitioners, conservationists and researchers without extensive laboratory equipment. Using environmental DNA from fish mock communities and a riverine ecotone, we demonstrate the sensitivity of the workflow and its interconnectivity with existing approaches. 4. Making metabarcoding available to researchers around the globe, our approach signifies a critical contribution to the immense task of characterizing and protecting earth’s biodiversity.