Abstract
BackgroundDNA-based sequencing approaches are commonly used to identify microorganisms and their genes and document trends in microbial community diversity in environmental samples. However, extraction of microbial DNA from complex environmental samples like corals can be technically challenging, and extraction methods may impart biases on microbial community structure. MethodsWe designed a two-phase study in order to propose a comprehensive and efficient method for DNA extraction from microbial cells present in corals and investigate if extraction method influences microbial community composition. During phase I, total DNA was extracted from seven coral species in a replicated experimental design using four different MO BIO Laboratories, Inc., DNA Isolation kits: PowerSoil®, PowerPlant® Pro, PowerBiofilm®, and UltraClean® Tissue & Cells (with three homogenization permutations). Technical performance of the treatments was evaluated using DNA yield and amplification efficiency of small subunit ribosomal RNA (SSU ribosomal RNA (rRNA)) genes. During phase II, potential extraction biases were examined via microbial community analysis of SSU rRNA gene sequences amplified from the most successful DNA extraction treatments.ResultsIn phase I of the study, the PowerSoil® and PowerPlant® Pro extracts contained low DNA concentrations, amplified poorly, and were not investigated further. Extracts from PowerBiofilm® and UltraClean® Tissue and Cells permutations were further investigated in phase II, and analysis of sequences demonstrated that overall microbial community composition was dictated by coral species and not extraction treatment. Finer pairwise comparisons of sequences obtained from Orbicella faveolata, Orbicella annularis, and Acropora humilis corals revealed subtle differences in community composition between the treatments; PowerBiofilm®-associated sequences generally had higher microbial richness and the highest coverage of dominant microbial groups in comparison to the UltraClean® Tissue and Cells treatments, a result likely arising from using a combination of different beads during homogenization.ConclusionsBoth the PowerBiofilm® and UltraClean® Tissue and Cells treatments are appropriate for large-scale analyses of coral microbiota. However, studies interested in detecting cryptic microbial members may benefit from using the PowerBiofilm® DNA treatment because of the likely enhanced lysis efficiency of microbial cells attributed to using a variety of beads during homogenization. Consideration of the methodology involved with microbial DNA extraction is particularly important for studies investigating complex host-associated microbiota.
Highlights
DNA-based sequencing approaches are commonly used to identify microorganisms and their genes and document trends in microbial community diversity in environmental samples
Phase I: DNA yield and polymerase chain reaction (PCR) efficiency DNA concentrations varied among the extraction treatments (PS, PowerPlant® Pro (PP), PB, Vortex Garnet (VG), Powerlyzer Glass (PG), and Vortex Glass (VGl)) with PB yielding the highest average concentration of 12.53 ± 15.73 ng μl−1 (Fig. 2, Table 2) among all seven coral species
Assessment of DNA yields by coral species revealed that PG and VGl P. lobata extracts had significantly lower DNA yields than VG extracts (Table 2; oneway FRMANOVA; Holm-Sidak method, p < 0.05), but this trend was not observed for the other species
Summary
DNA-based sequencing approaches are commonly used to identify microorganisms and their genes and document trends in microbial community diversity in environmental samples. The roles that bacteria and archaea may play in coral health and functioning have encouraged comprehensive investigations into the taxonomic identities and functional genes of microorganisms associated with globally distributed coral species These studies have described widespread as well as health-related and ecologically important coral-microbial associations [10,11,12]. Cultivation-independent methods coupled with nextgeneration sequencing technologies have been increasingly used to examine coral-microbial associations [11,12,13,14,15] These methods rely on the extraction of nucleic acids (DNA and RNA) from environmental samples and are advantageous because they allow for the study of hostmicrobe interactions that are difficult to examine using cultivation-dependent methods (reviewed within [3]). Investigators seeking to understand coral-associated microorganisms need to strive for representative lysis of morphologically diverse prokaryotic cells embedded within coral tissue [3, 13, 15, 17] and elution of high-quality nucleic acids
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