A BEST PRACTICE GUIDE TO MICROBIAL ANCIENT DNA ANALYSIS OF SOIL

High Throughput DNA Sequencing (HTS) revolutionized the field of paleomicrobiology, leading to an explosive growth of microbial ancient DNA (aDNA) studies, especially from environmental samples. 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 survey available literature for environmental aDNA projects, from sample collection to data analysis, and assess previous methodologies and approaches used in published aDNA microbial studies. We then integrate these concepts into a case study, where we use shotgun metagenomics to examine methodological, technical, and analytical biases during an environmental aDNA study of microbes. Specifically, we compare the impact of five DNA extraction methods and eight bioinformatic pipelines on the recovery of ancient microbial DNA information in soil cores from extreme environments. Our results show that silica-based methods optimized for ancient DNA research recovered significantly more damaged and short reads ( less than 100 bp) compared to a commercial kit or a phenol-chloroform method. Additionally, we describe 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.