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Abstract
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.
Highlights
Molecular analyses performed directly on nucleic acid extracts from environmental samples eliminate the biases associated with culture-dependent approaches [1], but introduce a variety of other biases that create differences between real and perceived community composition [2,3]
As samples taken for DNA extraction tend to be stored differently than those taken for RNA extraction, we evaluated the effect of different preservation methods on yield, RNA quality, and bacterial community composition
We compared the combined Qiagen AllPrep DNA/RNA/miRNA protocol (AP), which has been used for extraction of aquatic microbial community samples [28,32,33], to two DNA-only protocols (enzymatic (Enz) and bead-beating (Bead)) and one RNA-only protocol (Mirvana) that have been commonly used for the extraction of nucleic acids from aquatic samples [25,34,35]
Summary
Molecular analyses performed directly on nucleic acid extracts from environmental samples eliminate the biases associated with culture-dependent approaches [1], but introduce a variety of other biases that create differences between real and perceived community composition [2,3]. Previous studies have shown differences in observed community composition due to sample storage conditions [6], extraction method [7,8,9,10,11,12,13], sequencing protocol and user bias [5,14,15,16,17], and sequence analysis approach [5,18,19]. Another potential source of bias is differential treatment of DNA and RNA extracts. In addition to extraction method biases, it has been argued that the heterogeneity of natural environments necessitates the extraction of all biomolecules from the same sample, using one lysis method to break open the cells prior to separating DNA, RNA, and other molecules of interest [29]
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