title: Comparison of DNA extraction protocols for the analysis of gut microbiota in fishes.
abstract: This study investigated the impacts of bacterial DNA extraction methodology on downstream analysis of fish gut microbiota. Feces and intestine samples were taken from three sympatric freshwater fish species with varying diets. Samples were processed immediately (approximately 4 h after capture; fresh), stored at -20 °C for 15 days or preserved in RNAlater® reagent for 15 days. DNA was then extracted using two commercial kits: one designed for animal tissues and one specifically formulated for stool samples. Microbial community fingerprints were generated using ribosomal intergenic spacer analysis. Factors including diversity as depicted by band number, band intensity, repeatability and practicalities such as cost and time were considered. Despite significant differences in microbiota structure, results were similar between feces and intestine samples. Frozen samples were consistently outperformed by other storage methods and the stool kit typically outperformed the tissue kit. Overall, we recommend extraction of bacterial DNA from fresh samples using the stool kit for both sample types. If samples cannot be processed immediately, preservation in RNAlater® is preferred to freezing. Choice of DNA extraction method significantly influences the results of downstream microbial community analysis and thus should be taken into consideration for metadata analysis.
authors.affiliation: Aquatic Microbiology Laboratory, School of Fisheries, Aquaculture, and Aquatic Sciences, Auburn University, Auburn, AL 36849, USA.
authors.forename: Andrea M
authors.initials: AM
authors.lastname: Larsen
authors.affiliation: Aquatic Microbiology Laboratory, School of Fisheries, Aquaculture, and Aquatic Sciences, Auburn University, Auburn, AL 36849, USA.
authors.forename: Haitham H
authors.initials: HH
authors.lastname: Mohammed
authors.affiliation: Aquatic Microbiology Laboratory, School of Fisheries, Aquaculture, and Aquatic Sciences, Auburn University, Auburn, AL 36849, USA ariascr@auburn.edu.
authors.forename: Covadonga R
authors.initials: CR
authors.lastname: Arias
mesh_terms: D000818:Animals; D004269:DNA, Bacterial / Q000302:isolation & purification*; D005243:Feces / Q000382:microbiology*; D005399:Fishes / Q000382:microbiology*; D007422:Intestines / Q000382:microbiology*; D064307:Microbiota; D016133:Polymerase Chain Reaction; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D017422:Sequence Analysis, DNA
chemical_list: D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S
keywords: RISA; fish gut; microbial community