Abstract
Soil-borne microbes are major ecological players in terrestrial environments since they cycle organic matter, channel nutrients across trophic levels and influence plant growth and health. Therefore, the identification, taxonomic characterization and determination of the ecological role of members of soil microbial communities have become major topics of interest. The development and continuous improvement of high-throughput sequencing platforms have further stimulated the study of complex microbiota in soils and plants. The most frequently used approach to study microbiota composition, diversity and dynamics is polymerase chain reaction (PCR), amplifying specific taxonomically informative gene markers with the subsequent sequencing of the amplicons. This methodological approach is called DNA metabarcoding. Over the last decade, DNA metabarcoding has rapidly emerged as a powerful and cost-effective method for the description of microbiota in environmental samples. However, this approach involves several processing steps, each of which might introduce significant biases that can considerably compromise the reliability of the metabarcoding output. The aim of this review is to provide state-of-the-art background knowledge needed to make appropriate decisions at each step of a DNA metabarcoding workflow, highlighting crucial steps that, if considered, ensures an accurate and standardized characterization of microbiota in environmental studies.
Highlights
Soil microorganisms have been recognized as an integral part of terrestrial ecosystems because they play a central role in nutrient transformation and in plant community productivity, composition and diversity [1]
Based on literature review and experience, we provide a comprehensive overview of the positive and negative aspects related to each step of the metabarcoding workflow for microbiota studies on samples associated with terrestrial ecosystems (Figure 1)
The A260/A230 ratio is a metric for DNA quality, and it is best if it is greater than 1.5. If these ratios are appreciably lower in either case, it may indicate the presence of protein, phenol or other contaminants that may be introduced by extraction procedures and can act as polymerase chain reaction (PCR) inhibitors
Summary
Soil microorganisms have been recognized as an integral part of terrestrial ecosystems because they play a central role in nutrient transformation and in plant community productivity, composition and diversity [1]. The relative short length of these markers does not always allow a resolution to species level, so alternative approaches like single-cell metagenomics or isolation via cultivation are needed to fully discriminate microbial species Despite this limitation, DNA metabarcoding has rapidly emerged as a powerful, repeatable and cost-effective method for characterizing microbial communities in small and large-scale studies. DNA metabarcoding has rapidly emerged as a powerful, repeatable and cost-effective method for characterizing microbial communities in small and large-scale studies This comprehensive approach has enabled soil microbiologists to explore important ecological aspects related to soil–plant–microbe systems, such as the identification of microbial taxa that are (i) dominant or low in abundance across different terrestrial ecosystems; (ii) involved in specific processes (e.g., litter decomposition, nitrogen cycling, degradation of toxic compounds and many more); (iii) more sensitive to abiotic and biotic factors. Since sampling procedures for soil- and plant-associated microbiome were already covered in
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