Extracting data from the muck: deriving biological insight from complex microbial communities and non-model organisms with next 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

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

Harnessing the Power of PCR Molecular Fingerprinting Methods and Next Generation Sequencing for Understanding Structure and Function in Microbial Communities

Methods in Molecular Biology - PCR ◽

10.1007/978-1-4939-7060-5_16 ◽

2017 ◽

pp. 225-248 ◽

Cited By ~ 1

Author(s):

Sujal Phadke ◽

Andreia Filipa Salvador ◽

Joana Isabel Alves ◽

Orianna Bretschger ◽

Maria Madalena Alves ◽

...

Keyword(s):

Next Generation Sequencing ◽

Microbial Communities ◽

Structure And Function ◽

Molecular Fingerprinting ◽

Next Generation ◽

And Function ◽

Generation Sequencing ◽

Fingerprinting Methods

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

Microsatellite loci discovery from next-generation sequencing data and loci characterization in the epizoic barnacleChelonibia testudinaria(Linnaeus, 1758)

PeerJ ◽

10.7717/peerj.2019 ◽

2016 ◽

Vol 4 ◽

pp. e2019 ◽

Cited By ~ 5

Author(s):

Christine Ewers-Saucedo ◽

John D. Zardus ◽

John P. Wares

Keyword(s):

Next Generation Sequencing ◽

Microsatellite Markers ◽

Microsatellite Loci ◽

Next Generation Sequencing Data ◽

Model Organisms ◽

Next Generation ◽

Sequencing Data ◽

Genetic Studies ◽

Evolutionary Features ◽

Generation Sequencing

Microsatellite markers remain an important tool for ecological and evolutionary research, but are unavailable for many non-model organisms. One such organism with rare ecological and evolutionary features is the epizoic barnacleChelonibia testudinaria(Linnaeus, 1758).Chelonibia testudinariaappears to be a host generalist, and has an unusual sexual system, androdioecy. Genetic studies on host specificity and mating behavior are impeded by the lack of fine-scale, highly variable markers, such as microsatellite markers. In the present study, we discovered thousands of new microsatellite loci from next-generation sequencing data, and characterized 12 loci thoroughly. We conclude that 11 of these loci will be useful markers in future ecological and evolutionary studies onC. testudinaria.

Download Full-text

Genomic characterization of fecal microbial communities in severely sick humans and broiler chickens using next generation sequencing

10.21203/rs.2.22699/v1 ◽

2020 ◽

Author(s):

Ome Kalsoom Afridi ◽

Johar Ali ◽

Jeong Ho Chang

Keyword(s):

Next Generation Sequencing ◽

Microbial Communities ◽

Broiler Chickens ◽

Human Subjects ◽

Fecal Microbiota ◽

Next Generation ◽

Microbial Association ◽

Taxonomic Profiling ◽

Microbial Dysbiosis ◽

Generation Sequencing

Abstract Background: Microbiota plays an important role in food safety and its alteration poses a serious threat to humans. Comparative microbiome profiling using next-generation sequencing (NGS) enabled the understanding of microbial diversity and similarity between different species. In this study, we used NGS to profile the fecal microbiota of sick human and broiler chickens. A total of 26 fecal samples were collected from severely sick human subjects (n= 13) and broiler chickens (n=13) with similar symptoms. Results: The total number of microbial species detected in broiler chickens fecal microbiota was higher than that of humans. Phylum Proteobacteria was the most abundant in both human and broiler chickens fecal microbiota while Tenericutes was found to be least abundant in both species. Phylum Actinobacteria was found only in the human fecal microbiota. In both humans and broiler chickens, E.coli was found to be phylogenetically related suggesting a microbial association between both species. Conclusion: NGS based taxonomic profiling revealed the association of microbial dysbiosis with extreme sickness in both humans and broiler chickens. The dominance of phylum Proteobacteria in both the species ascertains their altered gut microbiota. Both human and broiler chickens microbial communities were found to be genetically related indicating horizontal transfer of microbes between the two species.

Download Full-text