Generating amplicon reads for microbial community assessment with next‐generation sequencing

Gołębiewski, Marcin; Tretyn, Andrzej

doi:10.1111/jam.14380

Cited by 70 publications

(50 citation statements)

References 161 publications

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“…Several studies have shown that larger PCR cycle numbers cause chimera generation and interfere with bacterial community structure analysis 39 , 40 . Hence, minimizing the number of PCR cycles by optimizing the starting template conditions and concentrations is important 26 . However, in our situation, reducing the number of PCR cycles to less than 35 decreased the number of environmental DNA samples that were amplified.…”

Section: Discussionmentioning

confidence: 99%

Evaluation of PCR conditions for characterizing bacterial communities with full-length 16S rRNA genes using a portable nanopore sequencer

Fujiyoshi

Muto-Fujita

Maruyama

2020

Sci Rep

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MinION (Oxford Nanopore Technologies), a portable nanopore sequencer, was introduced in 2014 as a new DNA sequencing technology. MinION is now widely used because of its low initial start-up costs relative to existing DNA sequencers, good portability, easy-handling, real-time analysis and long-read output. However, differences in the experimental conditions used for 16S rRNA-based PCR can bias bacterial community assessments in samples. Therefore, basic knowledge about reliable experimental conditions is needed to ensure the appropriate use of this technology. Our study concerns the reliability of techniques for obtaining accurate and quantitative full-length 16S rRNA amplicon sequencing data for bacterial community structure assessment using MinION. We compared five PCR conditions using three independent mock microbial community standard DNAs and established appropriate, standardized, better PCR conditions among the trials. We then sequenced two mock communities and six environmental samples using Illumina MiSeq for comparison. Modifying the PCR conditions improved the sequencing quality; the optimized conditions were 35 cycles of 95 °C for 1 min, 60 °C for 1 min and 68 °C for 3 min. Our results provide important information for researchers to determine bacterial community using MinION accurately.

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Section: Discussionmentioning

confidence: 99%

Evaluation of PCR conditions for characterizing bacterial communities with full-length 16S rRNA genes using a portable nanopore sequencer

Fujiyoshi

Muto-Fujita

Maruyama

2020

Sci Rep

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“…The identification of the microbial communities using modern genomic tools has enabled the detection of distinctive microorganisms that were not approachable by culture-based techniques. Gene amplification (using PCR) and sequencing techniques have proven exceptionally useful in evaluating the microbial community (Malik et al, 2008;Rani et al, 2008;Gołebiewski and Tretyn, 2020).…”

Section: Genomics and 16s Rrna For Bioremediationmentioning

confidence: 99%

Microbial Biodiversity and Bioremediation Assessment Through Omics Approaches

Chandran

Meena

Sharma

2020

Front. Environ. Chem.

131

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Industrialization, population burst, and changing lifestyles have resulted in the genesis of non-degradable pollutants languishing the environment and human health. Biological approaches using microorganisms are gaining importance as an eco-friendly and cost-effective substitute to mitigate the pollution load. Microorganisms can survive in a divergent environment and produce metabolites that can degrade and transform pollutants making it possible to revive contaminated sites naturally. Modern omics technologies like metagenomics, transcriptomics, proteomics, etc. have been used nowadays design strategies to study ecology and diversity of microorganisms and their application in environmental monitoring and bioremediation. The present article will focus on the omics techniques reportedly used in environmental monitoring to tackle the pollution load.

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“…Nonetheless, the application of these techniques is not free from difficulties, due to (among the others) the semiquantitative nature of data, the short DNA reads obtained by the most common HTS techniques, the variability in the copy number per cell of the most common taxonomic markers used (i.e. 16S and 18S rDNA), the incompleteness of genetic databases, which are still fed by information obtained by the isolation and cultivation approaches (Gołębiewski & Tretyn, 2020;Salmaso et al, 2020). Despite these constraints, the use of HTS techniques in the study of phytoplankton, which is just at the beginning, is contributing to revolutionize the approach we are using in the assessment of aquatic biodiversity in freshwater environments, opening the way to a next generation of investigations in phytoplankton ecology and a new improved understanding of plankton ecology.…”

Section: Culture Independent Approaches-metagenomicsmentioning

confidence: 99%

Freshwater phytoplankton diversity: models, drivers and implications for ecosystem properties

et al. 2020

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Our understanding on phytoplankton diversity has largely been progressing since the publication of Hutchinson on the paradox of the plankton. In this paper, we summarise some major steps in phytoplankton ecology in the context of mechanisms underlying phytoplankton diversity. Here, we provide a framework for phytoplankton community assembly and an overview of measures on taxonomic and functional diversity. We show how ecological theories on species competition together with modelling approaches and laboratory experiments helped understand species coexistence and maintenance of diversity in phytoplankton. The nonequilibrium nature of phytoplankton and the role of disturbances in shaping diversity are also discussed. Furthermore, we discuss the role of water body size, productivity of habitats and temperature on phytoplankton species richness, and how diversity may affect the functioning of lake ecosystems. At last, we give an insight into molecular tools that have emerged in the last decades and argue how it has broadened our perspective on microbial diversity. Besides historical backgrounds, some critical comments have also been made.

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Generating amplicon reads for microbial community assessment with next‐generation sequencing

Cited by 70 publications

References 161 publications

Evaluation of PCR conditions for characterizing bacterial communities with full-length 16S rRNA genes using a portable nanopore sequencer

Evaluation of PCR conditions for characterizing bacterial communities with full-length 16S rRNA genes using a portable nanopore sequencer

Microbial Biodiversity and Bioremediation Assessment Through Omics Approaches

Freshwater phytoplankton diversity: models, drivers and implications for ecosystem properties

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