Be aware of the allele-specific bias and compositional effects in multi-template PCR

High-throughput sequencing of amplicon libraries is the most widespread and one of the most effective ways to study the taxonomic structure of microbial communities, even despite growing accessibility of whole metagenome sequencing. Due to the targeted amplification, the method provides unparalleled resolution of communities, but at the same time perturbs initial community structure thereby reducing data robustness and compromising downstream analyses. Experimental research of the perturbations is largely limited to comparative studies on different PCR protocols without considering other sources of experimental variation related to characteristics of the initial microbial composition itself. Here we analyse these sources and demonstrate how dramatically they effect the relative abundances of taxa during the PCR cycles. We developed the mathematical model of the PCR amplification assuming the heterogeneity of amplification efficiencies and considering the compositional nature of data. We designed the experiment—five consecutive amplicon cycles (22–26) with 12 replicates for one real human stool microbial sample—and estimated the dynamics of the microbial community in line with the model. We found the high heterogeneity in amplicon efficiencies of taxa that leads to the non-linear and substantial (up to fivefold) changes in relative abundances during PCR. The analysis of possible sources of heterogeneity revealed the significant association between amplicon efficiencies and the energy of secondary structures of the DNA templates. The result of our work highlights non-trivial changes in the dynamics of real-life microbial communities due to their compositional nature. Obtained effects are specific not only for amplicon libraries, but also for any studies of metagenome dynamics

Microbiomes of the initial soils of mining areas of Yakutsk City (Eastern Siberia, Russia)

The microbiome of initial soils formed at the heaps and bottoms of surface sediment quarries in the surroundings of Yakutsk City(Eastern Siberia,Russia) has been characterized for the first time. In the initial Entisols, we detected Alphaproteobacteria (represented mainly by the family Rizobiales), Gammaproteobacteria, Actinobacteria, Acidobacteria, Bacteroidetes (mostly Chitinophagales), Deltaproteobacteria, and Chloroflexi. The lower soil horizons had a more homogenous species diversity taxonomy that was dominated by Gammaproteobacteria. The morphologically different horizons did not differ microbiologically. This is caused by the limited soil development and relatively slow rate of revegetation of the spoil banks and heaps of the quarries under the severe climatic conditions of the Eastern part of Russian Arctic zone. Based on our findings, we propose that the soil microbiomes in such recently abandoned quarries are characterized by low diversity, which is a characteristic feature of the polar soils surrounding Yakutsk. Data obtained can be used for elaboration of reclamation strategies with taking into account the information about key microbial drivers of soil processes

Diversity Indices of Plant Communities and Their Rhizosphere Microbiomes: An Attempt to Find the Connection

The rhizosphere community represents an “ecological interface” between plant and soil, providing the plant with a number of advantages. Despite close connection and mutual influence in this system, the knowledge about the connection of plant and rhizosphere diversity is still controversial. One of the most valuable factors of this uncertainty is a rough estimation of plant diversity. NGS sequencing can make the estimations of the plant community more precise than classical geobotanical methods. We investigate fallow and crop sites, which are similar in terms of environmental conditions and soil legacy, yet at the same time are significantly different in terms of plant diversity. We explored amplicons of both the plant root mass (ITS1 DNA) and the microbial communities (16S rDNA); determined alpha- and beta-diversity indices and their correlation, and performed differential abundance analysis. In the analysis, there is no correlation between the alpha-diversity indices of plants and the rhizosphere microbial communities. The beta-diversity between rhizosphere microbial communities and plant communities is highly correlated (R = 0.866, p = 0.01). ITS1 sequencing is effective for the description of plant root communities. There is a connection between rhizosphere communities and the composition of plants, but on the alpha-diversity level we found no correlation. In the future, the connection of alpha-diversities should be explored using ITS1 sequencing, even in more similar plant communities—for example, in different synusia

Microbiome composition of disturbed soils from sandy-gravel mining complexes with different reclamation approach

Activities connected to mineral mining disrupt the soil layer and bring up to surface parent rock material. As a result, they leave behind vast areas of disturbed lands, that are difficult to restore due to altered environmental conditions. Returning these lands to the natural ecosystems is an important contemporary challenge. Soil microbiome composition reflects changes happening to disturbed lands, its analysis helps to evaluate disturbance degree and estimate the effect of implementation of remediation techniques. Also, factors connected to the characteristics of a particular geographical region have a certain impact and should be taken into account. We focus on microbiomes of disturbed lands from two sandy-gravel mining complexes in mountainous areas with moderate continental climate of Central Caucasus. These quarries share the same parent rock material but differ in benchmark soil type and presence of remediation practices. Comparative analysis of microbiome composition based on sequencing of 16S rRNA gene libraries showed that region and disturbance are the key factors explaining microbiome variation, which surpass the influence of local vegetation factors. However, application of remediation techniques greatly reduces dissimilarity of soil microbiomes caused by disturbance. Linking of soil agrochemical parameters to microbiome composition showed that disturbance factor correlates with a lack of organic carbon. Other agrochemical parameters, like pH, ammonium, nitrates and total carbon explain variation of microbiomes on a smaller scale between sampling sites. Thus, while regional and disturbance factors reflected differentiation of soil microbiomes, soil agrochemical parameters explained local variation of certain groups of microorganisms

Microbiome composition of disturbed soils from sandy-gravel mining complexes with different reclamation approaches

Activities connected to mineral mining disrupt the soil layer and bring parent rock material to the surface. It leads to altering the environmental conditions and leaves behind vast areas of disturbed lands. Returning these lands to natural ecosystems is an important contemporary challenge, which can be acquired by reclamation practices. Soil microbiome composition reflects changes happening to disturbed lands; thus, its analysis is a powerful tool for evaluating the disturbance degree and estimating the effect of the implementation of reclamation techniques. Additionally, factors connected to the characteristics of a particular geographical region have a certain impact on the microbiome and should be taken into account. Thereby, studies of soil microbiomes of disturbed soils of different origins are essential in understanding the dynamics of soil restoration. Here, we focus on soil microbiomes from two sandy-gravel mining complexes in mountainous areas with a moderate continental climate of the Central Caucasus. These quarries share the same parent rock material, but differ in benchmark soil type and reclamation approach - one was left for passive recovery and the other was technically reclaimed with overburden material. Comparative analysis of microbiome composition, based on sequencing of 16S rRNA gene libraries, showed that region and disturbance are the key factors explaining microbiome variation, which surpass the influence of local factors. However, the application of reclamation techniques greatly reduces the dissimilarity of soil microbiomes caused by disturbance. Linking of soil chemical parameters to microbiome composition showed that the disturbance factor correlates with a lack of organic carbon. Other chemical parameters, like pH, ammonium, nitrates and total carbon explain microbiome variability on a smaller scale between sampling sites. Thus, while regional and disturbance factors reflected differentiation of soil microbiomes, soil chemical parameters explained local variation of certain groups of microorganisms

Additional file 1 of Microbial features of mature and abandoned soils in refractory clay deposits

Additional file 1: Supplementary Table 1. Agrochemical analysis of soils

RIAM: A Universal Accessible Protocol for the Isolation of High Purity DNA from Various Soils and Other Humic Substances

A single universal open protocol RIAM (named after Research Institute for Agricultural Microbiology) for the isolation of high purity DNA from different types of soils and other substrates (high and low in humic, clay content, organic fertilizer, etc.) is proposed. The main features of the RIAM protocol are the absence of the sorption–desorption stage on silica columns, the use of high concentrations of phosphate in buffers, which prevents DNA sorption on minerals, and DNA precipitation using CTAB. The performance of RIAM was compared with a reference commercial kit and showed very good results in relation to the purity and quantity of DNA, as well as the absence of inhibitory activity on PCR. In all cases, the RIAM ensured the isolation of DNA in quantities much greater than the commercial kit without the effect of PCR inhibition up to 50 ng DNA per reaction in a volume of 15 µL. The latter circumstance along with the ability of the protocol to extract low molecular weight DNA fractions makes the method especially suitable for those cases where quantitative assessments, detection of minor components of soil microbiota, and completeness of isolation of all DNA fractions are required

Microbial Composition on Abandoned and Reclaimed Mining Sites in the Komi Republic (North Russia)

Restoration of anthropogenically disturbed soils is an urgent problem in modern ecology and soil biology. Restoration processes in northern environments are especially important, due to the small amounts of fertile land and low levels of natural succession. We analyzed the soil microbiota, which is one of the indicators of the succession process is the soil. Samples were obtained from three disturbed soils (self-overgrown and reclaimed quarries), and two undisturbed soils (primary and secondary forests). Primary Forest soil had a well-developed soil profile, and a low pH and TOC (total organic carbon) amount. The microbial community of this soil had low richness, formed a clear remote cluster in the beta-diversity analysis, and showed an overrepresentation of Geobacter (Desulfobacteriota). Soil formation in clay and limestone abandoned quarries was at the initial stage, and was caused by both a low rate of mineral profile formation and severe climatic conditions in the region. Microbial communities of these soils did not have specific abundant taxa, and included a high amount of sparse taxa. Differences in taxa composition were correlated with abiotic factors (ammonium concentration), which, in turn, can be explained by the parent rock properties. Limestone quarry reclaimed by topsoil coverage resulted in an adaptation of the top soil microbiota to a novel parent rock. According to the CCA analysis, the microbial composition of samples was connected with pH, TOC and ammonium nitrogen concentration. Changes in pH and TOC were connected with ASVs from Chloroflexota, Gemmatimonadota and Patescibacteria. ASVs from Gemmatimonadota also were correlated with a high ammonium concentration

Water Stress, Cadmium, and Plant Genotype Modulate the Rhizosphere Microbiome of Pisum sativum L.

Drought and heavy metals seriously affect plant growth and the biodiversity of the associated rhizosphere microbiomes, which, in turn, could be involved in the adaptation of plants to these environmental stresses. Rhizosphere soil was collected from a three-factor pot experiment, where pea line SGE and its Cd-tolerant mutant SGECdt were cultivated under both optimal and limited water conditions and treated with a toxic Cd concentration. The taxonomic structure of the prokaryotic rhizosphere microbiome was analyzed with the high-throughput sequencing of 16S rRNA amplicon libraries. A permutation test demonstrated statistically significant effects of Cd and water stress but not of pea genotype on the rhizosphere microbiome structure. Phylogenetic isometric log-ratio data transformation identified the taxonomic balances that were affected by abiotic factors and pea genotypes. A small number of significant (log ratio [−3.0:+3.0]) and phylogenetically deep balances characterized water stress, while a larger number of weak (log ratio [−0.8:+0.8]) phylogenetically lower balances described the influence of the plant genotype. Stress caused by cadmium took on an intermediate position. The main conclusion of the study is that the most powerful factor affecting the rhizosphere microbiome was water stress, and the weakest factor was plant genotype since it demonstrated a very weak transformation of the taxonomic structure of rhizosphere microbiomes in terms of alpha diversity indices, beta diversity, and the log ratio values of taxonomic balances