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However, aDNA studies that examine environmental microbes routinely fail to authenticate aDNA, examine laboratory and environmental contamination, and control for biases introduced during sample processing. Here, we surveyed the available literature for environmental aDNA projects\u2014from sample collection to data analysis\u2014and assessed previous methodologies and approaches used in the published microbial aDNA studies. We then integrated these concepts into a case study, using shotgun metagenomics to examine methodological, technical, and analytical biases during an environmental aDNA study of soil microbes. Specifically, we compared the impact of five DNA extraction methods and eight bioinformatic pipelines on the recovery of microbial aDNA information in soil cores from extreme environments. Our results show that silica-based methods optimized for aDNA research recovered significantly more damaged and shorter reads (&lt;100 bp) than a commercial kit or a phenol\u2013chloroform method. Additionally, we described a stringent pipeline for data preprocessing, efficiently decreasing the representation of low-complexity and duplicated reads in our datasets and downstream analyses, reducing analytical biases in taxonomic classification.<\/jats:p>","DOI":"10.3390\/microorganisms10081623","type":"journal-article","created":{"date-parts":[[2022,8,10]],"date-time":"2022-08-10T09:42:56Z","timestamp":1660124576000},"page":"1623","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":16,"title":["A Case Study for the Recovery of Authentic Microbial Ancient DNA from Soil Samples"],"prefix":"10.3390","volume":"10","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-7996-2189","authenticated-orcid":false,"given":"Vilma","family":"P\u00e9rez","sequence":"first","affiliation":[{"name":"Australian Centre for Ancient DNA (ACAD), School of Biological Sciences, University of Adelaide, Adelaide, SA 5005, Australia"},{"name":"ARC Centre of Excellence for Australian Biodiversity and Heritage (CABAH), School of Biological Sciences, University of Adelaide, Adelaide, SA 5005, Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yichen","family":"Liu","sequence":"additional","affiliation":[{"name":"Key Laboratory of Vertebrate Evolution and Human Origins, Institute of Vertebrate Paleontology and Paleoanthropology, Center for Excellence in Life and Paleoenvironment, Chinese Academy of Sciences, Beijing 100044, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Martha B.","family":"Hengst","sequence":"additional","affiliation":[{"name":"Laboratorio de Ecolog\u00eda Molecular y Microbiolog\u00eda Aplicada, Departamento de Ciencias Farmac\u00e9uticas, Facultad de Ciencias, Universidad Cat\u00f3lica del Norte, Antofagasta 1270300, Chile"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Laura S.","family":"Weyrich","sequence":"additional","affiliation":[{"name":"ARC Centre of Excellence for Australian Biodiversity and Heritage (CABAH), School of Biological Sciences, University of Adelaide, Adelaide, SA 5005, Australia"},{"name":"Department of Anthropology and Huck Institutes of the Life Sciences, The Pennsylvania State University, State College, PA 16802, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2022,8,10]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"289","DOI":"10.1007\/978-1-4939-3369-3_17","article-title":"Analysis of Ancient DNA in Microbial Ecology","volume":"1399","author":"Bennett","year":"2016","journal-title":"Methods Mol. 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