A Comparative Analysis of Microbial DNA Preparation Methods for Use With Massive and Branching Coral Growth Forms.

Alejandra Hernandez-Agreda,Tracy D Ainsworth,William Leggat

doi:10.3389/fmicb.2018.02146

Abstract

In the last two decades, over 100 studies have investigated the structure of the coral microbiome. However, as yet there are no standardized methods applied to sample preservation and preparation, with different studies using distinct methods. There have also been several comparisons made of microbiome data generated across different studies, which have not addressed the influence of the methodology employed over each of the microbiome datasets. Here, we assess three different preservation methods; salt saturated dimethyl sulfoxide (DMSO) – EDTA, snap freezing with liquid nitrogen and 4% paraformaldehyde solution, and two different preparation methodologies; bead beating and crushing, that have been applied to study the coral microbiome. We compare the resultant bacterial assemblage data for two coral growth forms, the massive coral Goniastrea edwardsi and the branching coral Isopora palifera. We show that microbiome datasets generated from differing preservation and processing protocols are comparable in composition (presence/absence). Significant discrepancies between preservation and homogenization methods are observed in structure (relative abundance), and in the occurrence and dominance of taxa, with rare (low abundance and low occurrence) phylotypes being the most variable fraction of the microbial community. Finally, we provide evidence to support chemical preservation with DMSO as effective as snap freezing samples for generating reliable and robust microbiome datasets. In conclusion, we recommend where possible a standardized preservation and extraction method be taken up by the field to provide the best possible practices for detailed assessments of symbiotic and conserved bacterial associations.

Highlights

Sequencing of the gene 16S rRNA is by far the most common technique used to study the microbiome (Shokralla et al, 2012; D’Amore et al, 2016; Lear et al, 2018)
Samples were preserved using three reagents: two samples were snap frozen in liquid nitrogen and stored at −80◦C, two samples were preserved in salt-saturated 20% dimethyl sulfoxide (DMSO) – 0.5 M EDTA and stored at 4◦C, and one sample was fixed in 4% PFA solution and stored at 4◦C
Richness (d), diversity (H ), and evenness (J ) of the G. edwardsi microbiome were similar for the combination preservation (DMSO and liquid nitrogen) and homogenization method (Figures 2A,C,E)

Summary

Introduction

Sequencing of the gene 16S rRNA is by far the most common technique used to study the microbiome (Shokralla et al, 2012; D’Amore et al, 2016; Lear et al, 2018). Coral Microbiome DNA Preparation Methods with the initial sampling protocol, through to the analysis (Lear et al, 2018). Snap freezing has become widely used as the sample is preserved immediately upon collection with minimal handling and exposure of the sample to preservation artifacts (Fouhy et al, 2015; Vandeputte et al, 2017). This method has been limited by the capacity to transport and store liquid nitrogen or dry ice in remote areas. Like DMSO, sample preservation in PFA provides an transportable and widely applicable preservation system; but it has not yet been widely taken up in environmental microbiome studies, and the impact of histological preparation on coral microbiome has not been evaluated

Objectives

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Results

Discussion

Conclusion