BackgroundSmall RNAs (19-24 nt) are key regulators of gene expression that guide both transcriptional and post-transcriptional silencing mechanisms in eukaryotes. Current studies have demonstrated that microRNAs (miRNAs) act in several plant pathways associated with tissue proliferation, differentiation, and development and in response to abiotic and biotic stresses. In order to identify new miRNAs in soybean and to verify those that are possibly water deficit and rust-stress regulated, eight libraries of small RNAs were constructed and submitted to Solexa sequencing.ResultsThe libraries were developed from drought-sensitive and tolerant seedlings and rust-susceptible and resistant soybeans with or without stressors. Sequencing the library and subsequent analyses detected 256 miRNAs. From this total, we identified 24 families of novel miRNAs that had not been reported before, six families of conserved miRNAs that exist in other plants species, and 22 families previously reported in soybean. We also observed the presence of several isomiRNAs during our analyses. To validate novel miRNAs, we performed RT-qPCR across the eight different libraries. Among the 11 miRNAs analyzed, all showed different expression profiles during biotic and abiotic stresses to soybean. The majority of miRNAs were up-regulated during water deficit stress in the sensitive plants. However, for the tolerant genotype, most of the miRNAs were down regulated. The pattern of miRNAs expression was also different for the distinct genotypes submitted to the pathogen stress. Most miRNAs were down regulated during the fungus infection in the susceptible genotype; however, in the resistant genotype, most miRNAs did not vary during rust attack. A prediction of the putative targets was carried out for conserved and novel miRNAs families.ConclusionsValidation of our results with quantitative RT-qPCR revealed that Solexa sequencing is a powerful tool for miRNA discovery. The identification of differentially expressed plant miRNAs provides molecular evidence for the possible involvement of miRNAs in the process of water deficit- and rust-stress responses.

Microorganisms are widely distributed all over the Earth, inhabiting very diverse natural ecosystems, from the human body to inanimate indoor environments. Until recently, the methods most commonly used to study microbes have been culture-dependent approaches relying on the phenotypic evaluation of isolates that can grow in laboratory conditions. Given the advances in molecular biology and high-throughput DNA sequencing methodologies, scientists could expand their microbiome knowledge to microorganisms that do not grow well in the laboratory or have been considered too difficult and laborious to be cultivated. Culture-independent methods such as direct DNA sequencing can be performed for many samples at once, revealing the entire microbial profile of the samples and making possible the rapid characterization of the whole environmental microbiome. Investigating the microbiome profile of indoor environments such as hospitals, houses, offices and other buildings is of major concern because it could include a number of opportunistic, pathogenic or nosocomial microbes. Additionally, these environments could serve as reservoirs of virulence or antimicrobial resistance, which could be spread by humans or other vectors. High-throughput DNA sequencing has enabled large-scale microbiome screening for multiple indoor areas in a single analysis. Using this approach, we can easily track microorganisms in the environment and monitor microbiome composition related to hygiene processes or environment quality. Gaining such information and resolution regarding indoor microbiome analysis can lend very important assistance for epidemiological surveillance. Impact statement Research concerning the microbiome of indoor environments like hospitals, houses or buildings could have several implications for human health. Today, there is an ongoing shift in the paradigm of microbial analysis, from single isolated bacterial samples to entire microbiome profiles using high-throughput DNA sequencing methods. The use of sequencing methods in several studies has revealed an unprecedented microbial diversity in indoor environments, leading to a larger comprehension of the entire microbiome context. Here, we present a review of these microbiome studies using high-throughput DNA sequencing, including some new approaches and ideas that can be broadly applied in microbial tracking and epidemiological surveillance of indoor environments.