Comparative analysis of environmental DNA extraction and purification methods from different humic acid-rich soils

Lakay, Francisco; Botha, Alfred; Prior, Bernard A.

doi:10.1111/j.1365-2672.2006.03052.x

Cited by 172 publications

(106 citation statements)

References 36 publications

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“…The numbers of tested soils in various studies were, for example, one soil type (27), three soils (28), four soils (9), six soils (5), and eight soils (29). Vegetation cover, one of the most important factors influencing PCR performance in our study, was not considered in many studies (4,7,10,15) and only generally described in others (5,12,27,29). The other important factor, pH, was more often included in the studies, but in some cases studied soils were in narrow pH ranges, such as pH 4.3 to 5.8 (10), pH 5.9 to 7.1 (28), pH 6.2 to 6.3 (15), and pH 5.7 to 6.5 (17), or included just one sample of extremely low pH, such as pH 4.8 (29) or pH 4 (27).…”

Section: Discussionmentioning

confidence: 99%

Innovative Methods for Soil DNA Purification Tested in Soils with Widely Differing Characteristics

Sagova‐Mareckova

Čermák

Novotná

et al. 2008

Appl Environ Microbiol

216

113

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). This study demonstrated significant differences between the tested methods in terms of DNA yield, PCR performance, and recovered bacterial diversity. The differences in DNA yields were correlated to vegetation cover, soil pH, and clay content. The differences in PCR performances were correlated to vegetation cover and soil pH. The innovative methods improved PCR performance in our set of soils, in particular for forest acidic soils. PCR was successful in 95% of cases by the method using CaCl 2 purification and in 93% of cases by the method based on CaCO 3 pretreatment, but only in 79% by Mo Bio PowerSoil, for our range of soils. Also, the innovative methods recovered a higher percentage of actinomycete diversity from a subset of three soils. Recommendations include the assessment of soil characteristics prior to selecting the optimal protocol for soil DNA extraction and purification.

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Section: Discussionmentioning

confidence: 99%

Innovative Methods for Soil DNA Purification Tested in Soils with Widely Differing Characteristics

Sagova‐Mareckova

Čermák

Novotná

et al. 2008

Appl Environ Microbiol

216

113

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“…DNA extraction from soil is a key step in the metagenomic approach (3,12,21). Two different methods are routinely used.…”

Section: Methodsmentioning

confidence: 99%

Accessing the Soil Metagenome for Studies of Microbial Diversity

Delmont

Robe²,

Cecillon

et al. 2011

Appl Environ Microbiol

260

185

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Soil microbial communities contain the highest level of prokaryotic diversity of any environment, and metagenomic approaches involving the extraction of DNA from soil can improve our access to these communities. Most analyses of soil biodiversity and function assume that the DNA extracted represents the microbial community in the soil, but subsequent interpretations are limited by the DNA recovered from the soil. Unfortunately, extraction methods do not provide a uniform and unbiased subsample of metagenomic DNA, and as a consequence, accurate species distributions cannot be determined. Moreover, any bias will propagate errors in estimations of overall microbial diversity and may exclude some microbial classes from study and exploitation. To improve metagenomic approaches, investigate DNA extraction biases, and provide tools for assessing the relative abundances of different groups, we explored the biodiversity of the accessible community DNA by fractioning the metagenomic DNA as a function of (i) vertical soil sampling, (ii) density gradients (cell separation), (iii) cell lysis stringency, and (iv) DNA fragment size distribution. Each fraction had a unique genetic diversity, with different predominant and rare species (based on ribosomal intergenic spacer analysis [RISA] fingerprinting and phylochips). All fractions contributed to the number of bacterial groups uncovered in the metagenome, thus increasing the DNA pool for further applications. Indeed, we were able to access a more genetically diverse proportion of the metagenome (a gain of more than 80% compared to the best single extraction method), limit the predominance of a few genomes, and increase the species richness per sequencing effort. This work stresses the difference between extracted DNA pools and the currently inaccessible complete soil metagenome.

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“…To be sure that the genetic information obtained is representative of the whole microbiome, mDNA preparation requires specific protocols to preserve, as much as possible, the quality and the amount of the nucleic acids to guarantee best metagenomic library and the subsequent sequencing approach. mDNA extraction from geothermal environments can be performed by following two general strategies sharing the critical removal of humic acid, a major soil component made of phenolic moieties covalently bound to DNA (Lakay et al 2007), which inhibits restriction enzymes and PCR amplification (Tebbe and Vahjen 1993). The first method is the ''direct mDNA extraction'' and consists in cell lysis directly followed by the nucleic acids separation from soil particles within the sample, generally providing quickly high DNA amounts.…”

Section: Crisprmentioning

confidence: 99%

Metagenomics of microbial and viral life in terrestrial geothermal environments

Strazzulli

Fusco

Cobucci‐Ponzano

et al. 2017

Rev Environ Sci Biotechnol

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Geothermally heated regions of Earth, such as terrestrial volcanic areas (fumaroles, hot springs, and geysers) and deep-sea hydrothermal vents, represent a variety of different environments populated by extremophilic archaeal and bacterial microorganisms. Since most of these microbes thriving in such harsh biotopes, they are often recalcitrant to cultivation; therefore, ecological, physiological and phylogenetic studies of these microbial populations have been hampered for a long time. More recently, culture-independent methodologies coupled with the fast development of next generation sequencing technologies as well as with the continuous advances in computational biology, have allowed the production of large amounts of metagenomic data. Specifically, these approaches have assessed the phylogenetic composition and functional potential of microbial consortia thriving within these habitats, shedding light on how extreme physico-chemical conditions and biological interactions have shaped such microbial communities. Metagenomics allowed to better understand that the exposure to an extreme range of selective pressures in such severe environments, accounts for genomic flexibility and metabolic versatility of microbial and viral communities, and makes extreme-and hyper-thermophiles suitable for bioprospecting purposes, representing an interesting source for novel thermostable proteins that can be potentially used in several industrial processes.Keywords Hyperthermophiles Á Geothermal environments Á Microbial and viral metagenomics Á CRISPR Á Enzyme discovery BackgroundHot springs populated by extreme thermophiles (T opt : 65-79°C) and hyperthermophiles (T opt [ 80°C) (DeCastro et al. 2016) are very diverse and some of them show combinations of extreme chemo-physical conditions, such as temperature, acidic or alkaline pHs, high pressure, and high concentrations of salts and heavy metals (López-López et al. 2013). As with all studies of environmental microbiology, our understanding of the composition, functional and physiological dynamics, and evolution of extreme-and hyper-thermophilic microbial consortia has lagged A. Strazzulli Á M. Moracci Á P. Contursi

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Comparative analysis of environmental DNA extraction and purification methods from different humic acid-rich soils

Cited by 172 publications

References 36 publications

Innovative Methods for Soil DNA Purification Tested in Soils with Widely Differing Characteristics

Innovative Methods for Soil DNA Purification Tested in Soils with Widely Differing Characteristics

Accessing the Soil Metagenome for Studies of Microbial Diversity

Metagenomics of microbial and viral life in terrestrial geothermal environments

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