Preservation of nucleic acids by freeze-drying for next generation sequencing analyses of soil microbial communities

Structure and diversity of microbial communities are an important research topic in biology, since microbes play essential roles in the ecology of various environments. Different DNA isolation protocols can lead to data bias and can affect results of next-generation sequencing. To evaluate the impact of protocols for DNA isolation from soil samples and also the influence of individual handling of samples, we compared results obtained by two researchers (R and T) using two different DNA extraction kits: (1) MO BIO PowerSoil® DNA Isolation kit (MO_R and MO_T) and (2) NucleoSpin® Soil kit (MN_R and MN_T). Samples were collected from six different sites on Okinawa Island, Japan. For all sites, differences in the results of microbial composition analyses (bacteria, archaea, fungi, and other eukaryotes), obtained by the two researchers using the two kits, were analyzed. For both researchers, the MN kit gave significantly higher yields of genomic DNA at all sites compared to the MO kit (ANOVA; P <0.006). In addition, operational taxonomic units for some phyla and classes were missed in some cases: Micrarchaea were detected only in the MN_T and MO_R analyses; the bacterial phylum Armatimonadetes was detected only in MO_R and MO_T; and WIM5 of the phylum Amoebozoa of eukaryotes was found only in the MO_T analysis. Our results suggest the possibility of handling bias; therefore, it is crucial that replicated DNA extraction be performed by at least two technicians for thorough microbial analyses and to obtain accurate estimates of microbial diversity.

Download Full-text

Profiling soil microbial communities with next-generation sequencing: the influence of DNA kit selection and technician technical expertise

10.7287/peerj.preprints.3073v1 ◽

2017 ◽

Author(s):

Taha Soliman ◽

Sung-Yin Yang ◽

Tomoko Yamazaki ◽

Holger Jenke-Kodama

Keyword(s):

Next Generation Sequencing ◽

Microbial Communities ◽

Dna Extraction ◽

Dna Isolation ◽

Soil Microbial Communities ◽

Next Generation ◽

Microbial Composition ◽

Okinawa Island ◽

The Impact ◽

Generation Sequencing

Structure and diversity of microbial communities are an important research topic in biology, since microbes play essential roles in the ecology of various environments. Different DNA isolation protocols can lead to data bias and can affect results of next-generation sequencing. To evaluate the impact of protocols for DNA isolation from soil samples and also the influence of individual handling of samples, we compared results obtained by two researchers (R and T) using two different DNA extraction kits: (1) MO BIO PowerSoil® DNA Isolation kit (MO_R and MO_T) and (2) NucleoSpin® Soil kit (MN_R and MN_T). Samples were collected from six different sites on Okinawa Island, Japan. For all sites, differences in the results of microbial composition analyses (bacteria, archaea, fungi, and other eukaryotes), obtained by the two researchers using the two kits, were analyzed. For both researchers, the MN kit gave significantly higher yields of genomic DNA at all sites compared to the MO kit (ANOVA; P <0.006). In addition, operational taxonomic units for some phyla and classes were missed in some cases: Micrarchaea were detected only in the MN_T and MO_R analyses; the bacterial phylum Armatimonadetes was detected only in MO_R and MO_T; and WIM5 of the phylum Amoebozoa of eukaryotes was found only in the MO_T analysis. Our results suggest the possibility of handling bias; therefore, it is crucial that replicated DNA extraction be performed by at least two technicians for thorough microbial analyses and to obtain accurate estimates of microbial diversity.

Download Full-text

Profiling soil microbial communities with next-generation sequencing: the influence of DNA kit selection and technician technical expertise

PeerJ ◽

10.7717/peerj.4178 ◽

2017 ◽

Vol 5 ◽

pp. e4178 ◽

Cited By ~ 17

Author(s):

Taha Soliman ◽

Sung-Yin Yang ◽

Tomoko Yamazaki ◽

Holger Jenke-Kodama

Keyword(s):

Next Generation Sequencing ◽

Microbial Communities ◽

Dna Extraction ◽

Dna Isolation ◽

Soil Microbial Communities ◽

Next Generation ◽

Microbial Composition ◽

Okinawa Island ◽

The Impact ◽

Generation Sequencing

Structure and diversity of microbial communities are an important research topic in biology, since microbes play essential roles in the ecology of various environments. Different DNA isolation protocols can lead to data bias and can affect results of next-generation sequencing. To evaluate the impact of protocols for DNA isolation from soil samples and also the influence of individual handling of samples, we compared results obtained by two researchers (R and T) using two different DNA extraction kits: (1) MO BIO PowerSoil®DNA Isolation kit (MO_R and MO_T) and (2) NucleoSpin®Soil kit (MN_R and MN_T). Samples were collected from six different sites on Okinawa Island, Japan. For all sites, differences in the results of microbial composition analyses (bacteria, archaea, fungi, and other eukaryotes), obtained by the two researchers using the two kits, were analyzed. For both researchers, the MN kit gave significantly higher yields of genomic DNA at all sites compared to the MO kit (ANOVA;P < 0.006). In addition, operational taxonomic units for some phyla and classes were missed in some cases: Micrarchaea were detected only in the MN_T and MO_R analyses; the bacterial phylum Armatimonadetes was detected only in MO_R and MO_T; and WIM5 of the phylum Amoebozoa of eukaryotes was found only in the MO_T analysis. Our results suggest the possibility of handling bias; therefore, it is crucial that replicated DNA extraction be performed by at least two technicians for thorough microbial analyses and to obtain accurate estimates of microbial diversity.

Download Full-text

Rhizosphere Microbial Community Dynamics in Glyphosate-Treated Susceptible and Resistant Biotypes of Giant Ragweed (Ambrosia trifida)

Weed Science ◽

10.1614/ws-d-13-00164.1 ◽

2014 ◽

Vol 62 (2) ◽

pp. 370-381 ◽

Cited By ~ 8

Author(s):

Jessica R. Schafer ◽

Steven G. Hallett ◽

William G. Johnson

Keyword(s):

Microbial Community ◽

Next Generation Sequencing ◽

Microbial Communities ◽

Soil Microbial Communities ◽

Microbial Interactions ◽

Field Soil ◽

Next Generation ◽

Giant Ragweed ◽

Generation Sequencing ◽

Rhizosphere Microbial Community

In a previous study, glyphosate-susceptible and -resistant giant ragweed biotypes grown in sterile field soil survived a higher rate of glyphosate than those grown in unsterile field soil, and the roots of the susceptible biotype were colonized by a larger number of soil microorganisms than those of the resistant biotype when treated with 1.6 kg ae ha−1glyphosate. Thus, we concluded that soil-borne microbes play a role in glyphosate activity and now hypothesize that the ability of the resistant biotype to tolerate glyphosate may involve microbial interactions in the rhizosphere. The objective of this study was to evaluate differences in the rhizosphere microbial communities of glyphosate-susceptible and -resistant giant ragweed biotypes 3 d after a glyphosate treatment. Giant ragweed biotypes were grown in the greenhouse in unsterile field soil and glyphosate was applied at either 0 or 1.6 kg ha−1. Rhizosphere soil was sampled 3 d after the glyphosate treatment, and DNA was extracted, purified, and sequenced with the use of Illumina Genome Analyzer next-generation sequencing. The taxonomic distribution of the microbial community, diversity, genera abundance, and community structure within the rhizosphere of the two giant ragweed biotypes in response to a glyphosate application was evaluated by metagenomics analysis. Bacteria comprised approximately 96% of the total microbial community in both biotypes, and differences in the distribution of some microbes at the phyla level were observed. Select soil-borne plant pathogens (VerticilliumandXanthomonas) and plant-growth–promoting rhizobacteria (Burkholderia) present in the rhizosphere were influenced by either biotype or glyphosate application. We did not, however, observe large differences in the diversity or structure of soil microbial communities among our treatments. The results of this study indicate that challenging giant ragweed biotypes with glyphosate causes perturbations in rhizosphere microbial communities and that the perturbations differ between the susceptible and resistant biotypes. However, biological relevance of the rhizosphere microbial community data that we obtained by next-generation sequencing remains unclear.

Download Full-text

Soil microbiota manipulation and its role in suppressing soil-borne plant pathogens in organic farming systems under the light of microbiome-assisted strategies

Chemical and Biological Technologies in Agriculture ◽

10.1186/s40538-020-00183-7 ◽

2020 ◽

Vol 7 (1) ◽

Cited By ~ 2

Author(s):

Ugo De Corato

Keyword(s):

Next Generation Sequencing ◽

Microbial Communities ◽

Organic Farming ◽

Plant Pathogens ◽

Soil Microbial Communities ◽

Farming Systems ◽

Soil Microbiota ◽

Soil Microbial ◽

Organic Farming Systems ◽

Generation Sequencing

Abstract Soil microbiota plays a key role in suppressing soil-borne plant pathogens improving the natural soil suppressiveness. Microbiome disturbance triggers specific perturbation to change and shape the soil microbial communities’ network for increasing suppression against phytopathogens and related diseases. Very important goals have been reached in manipulation of soil microbiota through agronomical practices based on soil pre-fumigation, organic amendment, crop rotation and intercropping. Nevertheless, to limit inconsistencies, drawbacks and failures related to soil microbiota disturbance, a detailed understanding of the microbiome shifts during its manipulation is needed under the light of the microbiome-assisted strategies. Next-generation sequencing often offers a better overview of the soil microbial communities during microbiomes manipulation, but sometime it does not provide information related to the highest taxonomic resolution of the soil microbial communities. This review work reports and discusses the most reliable findings in relation to a comprehensive understanding of soil microbiota and how its manipulation can improve suppression against soil-borne diseases in organic farming systems. Role and functionality of the soil microbiota in suppressing soil-borne pathogens affecting crops have been basically described in the first section of the paper. Characterization of the soil microbiomes network by high-throughput sequencing has been introduced in the second section. Some relevant findings by which soil microbiota manipulation can address the design of novel sustainable cropping systems to sustain crops’ health without use (or reduced use) of synthetic fungicides and fumigants have been extensively presented and discussed in the third and fourth sections, respectively, under the light of the new microbiome-assisted strategies. Critical comparisons on the next-generation sequencing have been provided in the fifth section. Concluding remarks have been drawn in the last section.

Download Full-text