Abstract：
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 surveyed the available literature for environmental aDNA projects—from sample collection to data analysis—and 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 (<100 bp) than a commercial kit or a phenol−chloroform 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.
摘要：
高通量DNA测序（HTS）彻底改变了古微生物学领域，导致微生物古DNA（aDNA）研究的爆炸性增长，尤其是环境样本。然而,检查环境微生物的aDNA研究通常无法验证aDNA，检查实验室和环境污染，和控制样品处理过程中引入的偏差。这里,我们调查了环境aDNA项目的现有文献-从样本收集到数据分析-并评估了已发表的微生物aDNA研究中使用的先前方法和方法。然后我们将这些概念整合到案例研究中，使用鸟枪宏基因组学来检查方法学，技术,以及对土壤微生物的环境aDNA研究中的分析偏差。具体来说,我们比较了五种DNA提取方法和八种生物信息学管道对极端环境中土壤核心微生物aDNA信息回收的影响。我们的结果表明，与商业试剂盒或苯酚-氯仿方法相比，为aDNA研究优化的基于二氧化硅的方法恢复了明显更多的受损和更短的读数（＆lt;100bp）。此外,我们描述了一个严格的数据预处理管道，有效地减少我们的数据集和下游分析中的低复杂度和重复读数的表示，减少分类学分类中的分析偏差。
