Journal ArticleWet Lab Protocols Matter: Choice of DNA Extraction and Library Preparation Protocols Bias Ancient Oral Microbiome RecoveryWright, Sterling L [Center for Health Through Microbiomes, The Biodesign Institute Arizona State University Tempe Arizona USA] (ORCID:0000000305269132); Abdul‐Aziz, Muslih [Australian Centre for Ancient DNA, School of Biological Sciences and the Environment Institute, and Centre of Excellence for Australian Biodiversity and Heritage The University of Adelaide Adelaide Australia]; Blaha, Grace N [Department of Anthropology The Pennsylvania State University University Park Pennsylvania USA]; Ta, Christine K [One Health Microbiome Center The Pennsylvania State University University Park Pennsylvania USA]; Gancz, Abigail [One Health Microbiome Center The Pennsylvania State University University Park Pennsylvania USA]; Ademola‐Popoola, Iyunoluwa J [One Health Microbiome Center The Pennsylvania State University University Park Pennsylvania USA]; Szécsényi‐Nagy, Anna [ELTE Research Centre for the Humanities Institute of Archaeogenomics, MTA‐BTK Lendület 'Momentum' Research Group Budapest Hungary]; Sereno, Paul C [Institut de Recherches en Sciences Humains Université Abdou Moumouni de Niamey Niamey Niger République]; Weyrich, Laura S [Australian Centre for Ancient DNA, School of Biological Sciences and the Environment Institute, and Centre of Excellence for Australian Biodiversity and Heritage The University of Adelaide Adelaide Australia]<title>ABSTRACT</title> <p>Ancient DNA (aDNA) analysis of archaeological dental calculus has provided a wealth of insights into ancient health, demography and lifestyles. However, the workflow for ancient metagenomics is still evolving, raising concerns about reproducibility. Few systematic investigations have examined how DNA extraction methods and library preparation protocols influence ancient oral microbiome recovery, despite evidence from modern populations suggesting that they do. This leaves a gap in our understanding of how wet‐lab protocols impact aDNA recovery from dental calculus. In this study, we apply two DNA extraction and two library preparation methods in the aDNA field on dental calculus samples from Hungary and Niger. Samples from each context have similar chronological ages, but differences in their levels of aDNA preservation are notable, providing additional insights into how the efficacy of wet‐lab protocols is impacted by sample preservation. Several metrics were employed to assess intra‐ and inter‐sample variability, such as DNA fragment length recovery, GC content, clonality, endogenous content, DNA deamination and microbial composition. Our findings indicate that both DNA extraction and library preparation protocols can considerably impact ancient DNA recovery from archaeological dental calculus. Furthermore, no single protocol consistently outperformed the others across all assessments, and the effectiveness of specific protocol combinations depended on the preservation of the sample. These findings highlight the challenges of meta‐analyses and underscore the need to account for technical variability. Lastly, our study raises the question of whether the field should strive to standardise methods for comparability or optimise protocols based on sample preservation and specific research objectives.</p>Molecular Ecology Resources2025-11-0110667397Molecular Ecology Resources2581755-098Xhttps://doi.org/10.1111/1755-0998.700542235545National Science Foundation