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

PloS One
Ann McCarthyVincent J Denef

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 signifi...Continue Reading

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Citations

Dec 17, 2016·Nucleic Acids Research·Dieter M TourlousseYuji Sekiguchi
Feb 28, 2020·FEMS Microbiology Ecology·Marian L SchmidtVincent J Denef
Jul 5, 2017·The Journal of Antimicrobial Chemotherapy·Johan Bengtsson-PalmeErik Kristiansson
Apr 10, 2021·Folia Microbiologica·Paul Iturbe-EspinozaRob J M van Spanning
Jan 25, 2019·Journal of Microbiological Methods·Athenia L OldhamKathleen E Duncan

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Datasets Mentioned

BETA
SRP051811

Methods Mentioned

BETA
PCR
amplicon sequencing
nucleic acid extraction

Software Mentioned

MiSeq
AllPrep
AMOVA
mothur
SILVA
RTA
Metastats
MCS

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