Dataset Information

Effect of storage and DNA extraction method on 16S rRNA-profiled fecal microbiota in Japanese adults.

ABSTRACT: The effect of two factors, storage and the bacterial DNA extraction method, that potentially affect the 16S rRNA-based profiling of the microbiota in the feces of Japanese adults, were evaluated. Profiles of the microbiota in feces stored in DESS (DMSO-EDTA-salt solution) for 1, 2 and 3 weeks at room temperature, and for 3 weeks at 4°C were compared with those in fresh feces and feces stored in guanidine thiocyanate solution for 3 weeks at 4°C. None of the storage variables (preservation solution, temperature and duration) considerably affected ?- and ?-diversity of the fecal microbiota and OTU profiles. Regarding the bacterial DNA extraction methods, four were evaluated; A) silica membrane DNA purification combined with bead-beating bacterial disruption, B) magnetic bead DNA purification combined with bead-beating bacterial disruption, C) manual DNA purification using phenol-chloroform and ethanol precipitation combined with enzymatic bacterial lysis, and D) DNA extraction by a commercially available DNA stool kit. While methods A, B, and C did not markedly affect ?- and ?-diversity of the fecal microbiota and the OTU profiles, method D noticeably altered both ?- and ?-diversity. In addition, method D caused significant changes in the abundance of two predominant genera; Bacteroides and Bifidobacterium.

SUBMITTER: Kawada Y

PROVIDER: S-EPMC6436037 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

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Effect of storage and DNA extraction method on 16S rRNA-profiled fecal microbiota in Japanese adults.

Kawada Yuki Y Naito Yuji Y Andoh Akira A Ozeki Motoyuki M Inoue Ryo R

Journal of clinical biochemistry and nutrition 20181213 2

The effect of two factors, storage and the bacterial DNA extraction method, that potentially affect the 16S rRNA-based profiling of the microbiota in the feces of Japanese adults, were evaluated. Profiles of the microbiota in feces stored in DESS (DMSO-EDTA-salt solution) for 1, 2 and 3 weeks at room temperature, and for 3 weeks at 4°C were compared with those in fresh feces and feces stored in guanidine thiocyanate solution for 3 weeks at 4°C. None of the storage variables (preservation solutio ...[more]

PMID: 30936622

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Project description:Proper preservation of stool samples to minimize microbial community shifts and inactivate infectious agents is important for self-collected specimens requiring shipment to laboratories when cold chain transport is not feasible. In this study, we evaluated the performance of six preservation solutions (Norgen, OMNI, RNAlater, CURNA, HEMA, and Shield) for these aspects. Following storage of human stool samples with these preservatives at room temperature for 7 days, three hypervariable regions of the bacterial 16S rRNA gene (V1-V2, V3-V4, and V4) were amplicon sequenced. We found that samples collected in two preservatives, Norgen and OMNI, showed the least shift in community composition relative to -80°C standards compared with other storage conditions, and both efficiently inhibited the growth of aerobic and anaerobic bacteria. RNAlater did not prevent bacterial activity and exhibited relatively larger community shift. Although the effect of preservation solution was small compared to intersubject variation, notable changes in microbiota composition were observed, which could create biases in downstream data analysis. When community profiles inferred from different 16S rRNA gene hypervariable regions were compared, we found differential sensitivity of primer sets in identifying overall microbial community and certain bacterial taxa. For example, reads generated by the V4 primer pair showed a higher alpha diversity of the gut microbial community. The degenerate 27f-YM primer failed to detect the majority of Bifidobacteriales. Our data indicate that choice of preservation solution and 16S rRNA gene primer pair are critical determinants affecting gut microbiota profiling. IMPORTANCE Large-scale human microbiota studies require specimens collected from multiple sites and/or time points to maximize detection of the small effects in microbe-host interactions. However, batch biases caused by experimental protocols, such as sample collection, massively parallel sequencing, and bioinformatics analyses, remain critical and should be minimized. This work evaluated the effects of preservation solutions and bacterial 16S rRNA gene primer pairs in revealing human gut microbiota composition. Since notable changes in detecting bacterial composition and abundance were observed among choice of preservatives and primer pairs, a consistent methodology is essential in minimizing their effects to facilitate comparisons between data sets.

Dataset Information

Effect of storage and DNA extraction method on 16S rRNA-profiled fecal microbiota in Japanese adults.

Publications

Effect of storage and DNA extraction method on 16S rRNA-profiled fecal microbiota in Japanese adults.

Similar Datasets

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