McCarthy Ann, Chiang Edna, Schmidt Marian L, Denef Vincent J
Department of Ecology and Evolutionary Biology, University of Michigan, Ann Arbor, MI, United States of America.
PLoS One. 2015 Mar 23;10(3):e0121659. doi: 10.1371/journal.pone.0121659. eCollection 2015.
Bias is a pervasive problem when characterizing microbial communities. An important source is the difference in lysis efficiencies of different populations, which vary depending on the extraction protocol used. To avoid such biases impacting comparisons between gene and transcript abundances in the environment, the use of one protocol that simultaneously extracts both types of nucleic acids from microbial community samples has gained popularity. However, knowledge regarding tradeoffs to combined nucleic acid extraction protocols is limited, particularly regarding yield and biases in the observed community composition. Here, we evaluated a commercially available protocol for simultaneous extraction of DNA and RNA, which we adapted for freshwater microbial community samples that were collected on filters. DNA and RNA yields were comparable to other commonly used, but independent DNA and RNA extraction protocols. RNA protection agents benefited RNA quality, but decreased DNA yields significantly. Choice of extraction protocol influenced the perceived bacterial community composition, with strong method-dependent biases observed for specific phyla such as the Verrucomicrobia. The combined DNA/RNA extraction protocol detected significantly higher levels of Verrucomicrobia than the other protocols, and those higher numbers were confirmed by microscopic analysis. Use of RNA protection agents as well as independent sequencing runs caused a significant shift in community composition as well, albeit smaller than the shift caused by using different extraction protocols. Despite methodological biases, sample origin was the strongest determinant of community composition. However, when the abundance of specific phylogenetic groups is of interest, researchers need to be aware of the biases their methods introduce. This is particularly relevant if different methods are used for DNA and RNA extraction, in addition to using RNA protection agents only for RNA samples.
在描述微生物群落时，偏差是一个普遍存在的问题。一个重要来源是不同种群的裂解效率差异，这取决于所使用的提取方案。为避免此类偏差影响环境中基因丰度与转录本丰度之间的比较，使用一种能同时从微生物群落样本中提取这两种核酸的方案已受到广泛欢迎。然而，关于组合核酸提取方案的权衡取舍的知识有限，特别是在产量和观察到的群落组成偏差方面。在这里，我们评估了一种用于同时提取DNA和RNA的市售方案，并将其应用于在滤膜上收集的淡水微生物群落样本。DNA和RNA产量与其他常用的但独立的DNA和RNA提取方案相当。RNA保护剂有利于RNA质量，但显著降低了DNA产量。提取方案的选择影响了所感知的细菌群落组成，对于特定门类如疣微菌门，观察到强烈的方法依赖性偏差。与其他方案相比，DNA/RNA联合提取方案检测到的疣微菌水平显著更高，并且通过显微镜分析证实了这些更高的数量。使用RNA保护剂以及独立的测序运行也导致了群落组成的显著变化，尽管比使用不同提取方案导致的变化小。尽管存在方法偏差，但样本来源是群落组成的最强决定因素。然而，当关注特定系统发育类群的丰度时，研究人员需要意识到他们的方法所引入的偏差。如果除了仅对RNA样本使用RNA保护剂之外，还对DNA和RNA提取使用不同方法，这一点尤其重要。
