RNA preservation agents and nucleic acid extraction method bias perceived bacterial community composition

Area of Science:

• Microbiology
• Molecular Biology
• Environmental Science

Background:

• Bias in microbial community characterization is often caused by varying nucleic acid extraction efficiencies.
• Simultaneous DNA and RNA extraction protocols are increasingly used to avoid biases in gene and transcript abundance comparisons.

Purpose of the Study:

• To evaluate a commercial simultaneous DNA/RNA extraction protocol for freshwater microbial communities.
• To assess potential tradeoffs in yield and community composition bias associated with this protocol.

Main Methods:

• Adaptation of a commercial simultaneous DNA/RNA extraction kit for filtered freshwater samples.
• Comparison of DNA and RNA yields and microbial community composition against independent extraction protocols.
• Evaluation of the impact of RNA protection agents and separate sequencing runs on results.

Main Results:

• DNA and RNA yields were comparable to independent extraction methods.
• RNA protection agents improved RNA quality but reduced DNA yield.
• Extraction protocol choice significantly influenced bacterial community composition, notably increasing detected Verrucomicrobia levels.
• Microscopic analysis confirmed higher Verrucomicrobia counts with the combined protocol.

Conclusions:

• While sample origin is the primary driver of community composition, extraction methods introduce significant, method-dependent biases.
• Researchers must be aware of these biases, especially when using different protocols for DNA and RNA or employing RNA protection agents, to accurately interpret phylogenetic group abundances.