Robust, Open-Source and Automation-Friendly DNA Extraction Protocol for Hologenomic Research.

Global efforts to standardise methodologies benefit greatly from open-source procedures that enable the generation of comparable data. Here, we present a modular, high-throughput nucleic acid extraction protocol standardised within the Earth Hologenome Initiative to generate both genomic and microbial metagenomic data from faecal samples of vertebrates. The procedure enables the purification of either RNA and DNA in separate fractions (DREX1) or as total nucleic acids (DREX2). We demonstrate their effectiveness across faecal samples from amphibians, reptiles and mammals, with reduced performance observed on bird guano. Despite some variation in laboratory performance metrics, both DREX1 and DREX2 yielded highly similar microbial community profiles, as well as comparable depth and breadth of host genome coverages. Benchmarking against a commercial kit widely used in microbiome research showed comparable recovery of host genomic data and microbial community complexity. Our open-source method offers a robust, cost-effective, scalable and automation-friendly nucleic acid extraction procedure to generate high-quality hologenomic data across vertebrate taxa. The method enhances research comparability and reproducibility by providing standardised, high-throughput, open-access protocols with fully transparent reagents. It is designed to integrate automatised pipelines, and its modular structure also supports continuous development and improvement.