DNA Extraction for Streamlined Metagenomics of Diverse Environmental Samples

Marotz, Clarisse; Almasi‐Hashiani, Amir; Humphrey, Greg; Gaffney, James; Gogul, Grant; Knight, Rob

doi:10.2144/000114559

Cited by 162 publications

(148 citation statements)

References 9 publications

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“…In our previous study on comparison of DNA extraction methods [6] we assembled a set of 96 samples spanning a broad range of environments, including 48 fecal samples, 12 soil samples, 12 marine sediment samples, six seawater samples, five skin samples, five oral samples and six mattress dust samples. We used the DNA from this previous study, extracted using the Earth Microbiome Project (EMP) protocol [7] on the Kingfisher instrument, for this study.…”

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confidence: 99%

Triplicate PCR Reactions for 16S rRNA Gene Amplicon Sequencing are Unnecessary

et al. 2019

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Conventional wisdom holds that PCR amplification for sequencing should employ pooled replicate reactions to reduce bias due to jackpot effects and chimera formation. However, modern amplicon data analysis employs methods that may be less sensitive to such artifacts. Here we directly compare results from single versus triplicate reactions for 16S amplicon sequencing and find no significant impact of adopting a less labor-intensive single-reaction protocol.

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confidence: 99%

Triplicate PCR Reactions for 16S rRNA Gene Amplicon Sequencing are Unnecessary

et al. 2019

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“…16S rRNA sequences were available for a subset of 436 isolates cultivated from dust samples collected from athletic facilities in the Pacific Northwest. DNA extraction, library preparation, 16S rRNA amplicon sequencing, and taxonomic identification (Data S1) were conducted following the Earth Microbiome Project, targeting the V4 region of the 16S SSU rRNA …”

Section: Methodsmentioning

confidence: 99%

“…DNA extraction, library preparation, 16S rRNA amplicon sequencing, and taxonomic identification (Data S1) were conducted following the Earth Microbiome Project, targeting the V4 region of the 16S SSU rRNA. [27][28][29]…”

Section: Whole-genome Sequencingmentioning

confidence: 99%

“…M.oleivoranson average represented less than 0.05% of the total population ( Figure 5B). In subsequent samples, M oleivorans was TA B L E 2 Genus-level abundance comparison among 16S rRNA gene sequences, [27][28][29] metagenomics, 32 To determine whether the pH of the different paints was contributing to selection within the microbial community, we tested the tolerance of each individual community member to variations in pH in liquid media. Specifically, we tested pH 4 and 10, which correspond to the pH of latex and clay surfaces, respectively ( Figure S2).…”

Section: Microbial Composition and Viability Reduction In Relationmentioning

confidence: 99%

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Impacts of indoor surface finishes on bacterial viability

Maamar

Glawe

et al. 2019

Indoor Air

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Microbes in indoor environments are constantly being exposed to antimicrobial surface finishes. Many are rendered non‐viable after spending extended periods of time under low‐moisture, low‐nutrient surface conditions, regardless of whether those surfaces have been amended with antimicrobial chemicals. However, some microorganisms remain viable even after prolonged exposure to these hostile conditions. Work with specific model pathogens makes it difficult to draw general conclusions about how chemical and physical properties of surfaces affect microbes. Here, we explore the survival of a synthetic community of non‐model microorganisms isolated from built environments following exposure to three chemically and physically distinct surface finishes. Our findings demonstrated the differences in bacterial survival associated with three chemically and physically distinct materials. Alkaline clay surfaces select for an alkaliphilic bacterium, Kocuria rosea, whereas acidic mold‐resistant paint favors Bacillus timonensis, a Gram‐negative spore‐forming bacterium that also survives on antimicrobial surfaces after 24 hours of exposure. Additionally, antibiotic‐resistant Pantoea allii did not exhibit prolonged retention on antimicrobial surfaces. Our controlled microcosm experiment integrates measurement of indoor chemistry and microbiology to elucidate the complex biochemical interactions that influence the indoor microbiome.

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“…The first is the degraded nature of ancient DNA (aDNA) itself, and the second is that reconstruction of a complex microbial community, such as the gut microbiome, can be influenced by DNA extraction methods (Wesolowska-Andersen et al, 2014). For example, both the Earth Microbiome Project (Marotz et al, 2017;Thompson et al, 2017) and the Human Microbiome Project (HMP) (Aagaard et al, 2013) have recommended using the Qiagen PowerSoil (formerly MoBio PowerSoil) DNA extraction kit, and consequently, this kit has become relatively standard in modern microbiome research. On the other hand, modern microbiome reconstruction depends on the efficiency with which bacterial lysis occurs during DNA extraction, which is influenced by differences in cell wall structure, spore formation, and other factors.…”

Section: Introductionmentioning

confidence: 99%

Comparison of extraction methods for recovering ancient microbial DNA from paleofeces

Hagan

Hofman

Hübner

et al. 2019

American J Phys Anthropol

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Objectives: Paleofeces are valuable to archeologists and evolutionary biologists for their potential to yield health, dietary, and host information. As a rich source of preserved biomolecules from host-associated microorganisms, they can also provide insights into the recent evolution and changing ecology of the gut microbiome. However, there is currently no standard method for DNA extraction from paleofeces, which combine the dual challenges of complex biological composition and degraded DNA. Due to the scarcity and relatively poor preservation of paleofeces when compared with other archeological remains, it is important to use efficient methods that maximize ancient DNA (aDNA) recovery while also minimizing downstream taxonomic biases. Methods:In this study, we use shotgun metagenomics to systematically compare the performance of five DNA extraction methods on a set of well-preserved human and dog paleofeces from Mexico (~1,300 BP). Results:Our results show that all tested DNA extraction methods yield a consistent microbial taxonomic profile, but that methods optimized for ancient samples recover significantly more DNA.Conclusions: These results show promise for future studies that seek to explore the evolution of the human gut microbiome by comparing aDNA data with those generated in modern studies. K E Y W O R D S

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DNA Extraction for Streamlined Metagenomics of Diverse Environmental Samples

Cited by 162 publications

References 9 publications

Triplicate PCR Reactions for 16S rRNA Gene Amplicon Sequencing are Unnecessary

Triplicate PCR Reactions for 16S rRNA Gene Amplicon Sequencing are Unnecessary

Impacts of indoor surface finishes on bacterial viability

Comparison of extraction methods for recovering ancient microbial DNA from paleofeces

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