NIH, National Cancer Institute, Division of Cancer Treatment and Diagnosis (DCTD) NIH - National Institutes of Health National Cancer Institute DCTD - Division of Cancer Treatment and Diagnosis

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

Author(s): Kawada Y, Naito Y, Andoh A, Ozeki M, Inoue R

Publication: J Clin Biochem Nutr, 2019, Vol. 64, Page 106-111

PubMed ID: 30936622 PubMed Review Paper? No

Purpose of Paper

This paper compared the microbial biodiversity of fresh fecal specimens and fecal specimens stored in a DMSO EDTA salt solution (DESS) for 1, 2, or 3 weeks at room temperature; in DESS at 4°C for 3 weeks; in guanidine thiocyanate solution for 3 weeks at 4°C; and among specimens extracted using four different methods.

Conclusion of Paper

Regardless of storage solution, duration, and temperature, specimens clustered by patient source and had similar α-diversity. Although the intra-individual operational taxonomic unit (OTU) profiles were very strongly correlated, the inter-individual correlation was modest. Nevertheless, some differences in the microbial profile were noted after storage at the phylum (Bacteroidetes and Firmicutes) and the genus levels (Ochrobactrum and Peptoniphilus).

While specimens generally clustered by patient source rather than by DNA extraction method, specimens extracted using the QIAamp Stool Mini Kit were less tightly clustered than specimens that were extracted with the other three methods. The Shannon α-diversity index was significantly lower when DNA extraction was with QIAamp DNA Stool Mini Kit compared to the other methods, but the Chao1 index was comparable among specimens extracted with all four methods. More differences in the microbial profile at both the phylum (Actinobacteria Firmicutes, and Bacteroidetes) and genus level (Bacteroides and Bifidobacterium) were noted when extraction was with the QIAamp DNA Stool Mini Kit compared to the other three methods. Feces extracted with phenol-chloroform had less Bacteroidetes and Bacteroides compared to when DNA extraction was with the QuickGene DNA Tissue Kit or the MaxWell Extraction Kit.

Studies

  1. Study Purpose

    This study compared the microbial biodiversity of fresh fecal specimens and fecal specimens stored in a DMSO EDTA salt solution (DESS) for 1, 2, or 3 weeks at room temperature; in DESS at 4°C for 3 weeks; in guanidine thiocyanate solution for 3 weeks at 4°C; and among specimens extracted using four different methods. Fecal specimens were collected using a stool collection brush from three healthy men and two healthy women into tubes containing QuickGene lysis buffer (fresh, 1 specimen per volunteer), guanidine thiocyanate solution (one specimen per volunteer), and DESS (eight specimens per volunteer). The specimen in guanidine thiocyanate solution and one specimen in DESS were then stored at 4°C for 3 weeks while three of the remaining specimens in DESS were stored at room temperature for 1, 2, and 3 weeks, respectively, before DNA extraction. DNA was extracted from all specimens using the QuickGene DNA Tissue Kit. To test the effect of extraction method, the remaining four specimens in DESS from each volunteer underwent DNA extraction using: 1) the QuickGene DNA Tissue Kit, 2) bead-beating bacterial disruption and the Maxwell RSC Blood DNA Kit for purification, 3) enzyme-based lysing and phenol-chloroform extraction, and 4) bead-beating bacterial disruption with the QIAamp DNA Stool Mini Kit. 16S rRNA sequencing libraries were prepared and sequenced using a MiSeq instrument. The R phyloseq package was used to calculate the Chao1 and Shannon α-Diversity indices and β-Diversity was estimated based on the Bray-Curtis distances and principal coordinate analysis (PCoA) using phyloseq.

    Summary of Findings:

    Regardless of storage solution, duration and temperature specimens clustered by patient source with no clear effects of storage solution or duration and no effect on α-diversity. The Pearson’s correlation between the OTU profile of fresh and stored specimens from the same individual was generally > 0.9, but the interindividual correlation was only 0.62. Nevertheless, at the phylum level, stored specimens (regardless of method) had approximately 10% higher abundance of Bacteroidetes (P<0.05) and specimens stored in DESS for 2 weeks at room temperature or guanidine thiocyanate solution for 3 weeks at 4°C had lower abundance of Firmicutes (P<0.05). At the genus level, feces stored in DESS for 2 weeks at room temperature had higher abundance of Ochrobactrum and specimens stored in guanidine thiocyanate for 3 weeks at 4°Chad higher abundance of Peptoniphilus.

    Overall, fecal specimens clustered by patient source rather than by DNA extraction method, but specimens extracted using the QIAamp Kit were less tightly clustered than the specimens extracted with the other three methods. The Pearsons correlation was highest when specimens extracted with QuickGene Kit and the Maxwell Kit (r=0.97) were compared and lowest when specimens that were extracted using phenol chloroform and the QIAamp Kit (r=0.69) were compared. At the phylum level, extraction with the QIAamp Kit resulted in lower abundance of Actinobacteria and Firmicutes and higher abundance of Bacteroidetes compared to when extraction was by any of the other three methods (P<0.05, all); levels of Bacteroidetes were also lower when extraction was with phenol chloroform rather than the QuickGene Kit or the MaxWell Kit (P<0.05, both). At the genus level, Bacteroides were more abundant and Bifidobacterium less abundant when extraction was with QIAamp Kit compared to any of the other three methods (P<0.05), and Bacteroides were less abundant when extraction was with phenol-chloroform rather than any other method (P<0.05). Further, the Shannon α-diversity index was significantly lower when DNA extraction was with QIAamp Kit than the other methods evaluated (P<0.05), but the Chao1 index was comparable among fecal specimens extracted with all four methods.

    Biospecimens
    Preservative Types
    • Other Preservative
    • None (Fresh)
    Diagnoses:
    • Normal
    Platform:
    AnalyteTechnology Platform
    DNA Next generation sequencing
    Pre-analytical Factors:
    ClassificationPre-analytical FactorValue(s)
    Storage Storage duration 0 weeks
    1 week
    2 weeks
    3 weeks
    Analyte Extraction and Purification Analyte isolation method QuickGene DNA tissue kit
    Maxwell RSC blood DNA kit for purification
    Phenol-chloroform extraction
    QIAamp DNA Stool Mini kit
    Biospecimen Preservation Type of fixation/preservation Dimethyl sulfoxide
    Guanidine thiocyanate
    None (fresh)
    Refrigeration
    EDTA
    Storage Storage temperature 4°C
    Room temperature

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