SARS-CoV-2 replication requires the synthesis of a set of structural proteins expressed through discontinuous transcription of ten subgenomic mRNAs (sgmRNAs). Here, we have fine-tuned a droplet digital PCR (ddPCR) assays to accurately detect and quantify SARS-CoV-2 genomic ORF1ab and sgmRNAs for the nucleocapsid (N) and spike (S) proteins. We analyzed 166 RNAs from anonymized COVID-19 positive subjects and we found a recurrent and characteristic pattern of sgmRNAs expression in relation to the total viral RNA content. Further, we observed that expression profiles of sgmRNAs analyzed in a subset of 110 samples subjected to meta-transcriptomics sequencing were highly correlated with those obtained by ddPCR. Our results, providing a comprehensive and dynamic snapshot of SARS-CoV-2 sgmRNAs expression and replication, may contribute to provide a better understanding of SARS-CoV-2 transcription and expression mechanisms, and support the development of more accurate molecular diagnostic tools and for the stratification of COVID-19 patients.

Background: In nature, microbial communities undergo changes in composition threatening their resilient behaviour. Here, we interrogated sourdough, a natural cereal-fermenting metacommunity, as challenging ecosystem, where players undergo continuous environmental and spatiotemporal stimuli. Results: Inspecting meta-genomes and -transcriptomes, spontaneous sourdoughs harboured dominant, sub-dominant and satellite players differently engaged in functional pathways. The highest microbial richness bared the highest number of gene copies per pathway. Merging meta-24 omics data or using the same microbiota of a spontaneous sourdough, we de novo reconstructed two synthetic microbial communities, respectively, SDG and SMC-SD43. In vitro, the KEGG number of dominant players in SDG was not affected by single player depletion; that of sub-dominants and satellites fluctuated but showed unique contributions. Compared to SMC-SD43, SDG ensured broader transcriptome redundancy. After in situ long-time back slopping, SDG kept stable, with constant volatilome. SMC-SD43 lost many members. Dominant, sub-dominant and satellite players altogether ensured gene and transcript redundancy. Conclusions: Our study demonstrates how starting from spontaneous sourdoughs and by reconstructing the synthetic community it was possible to unravel the metabolic contribution of individual players. To be resilient and performing, the sourdough metacommunity had to comprise dominant, sub-dominant and satellite players, which, only altogether, ensured gene and transcript redundancy. Overall, our study changed the paradigm and introduced theoretical foundations for steering food fermentations.

As human activities on our planet persist, causing widespread and irreversible environmental degradation, the need to biomonitor ecosystems has never been more pressing. These circumstances have required a renewal in monitoring techniques, encouraged by necessity to develop more rapid and accurate tools which will support timely observations of ecosystem structure and function. The World Exposition (from now 'EXPO2015') hosted in Milan from May to October 2015 was a global event that could be categorized as a mega-event, which can be defined as an acute environmental stressor, possibly generating biodiversity alteration and disturbance. During the six months of EXPO2015, exhibitors from more than 135 countries and 22 million visitors insisted on a 1.1 million square meters area. Faced with such a massive event, we explore the potential of DNA metabarcoding using three molecular markers to improve the understanding of anthropogenic impacts in the area, both considering air and water monitoring. Furthermore, we explore the effectiveness of the taxonomy assignment phase considering different taxonomic levels of analysis and the use of data mining approaches to predict sample origin. Unless the degree of taxa identification still remains open, our results showed that DNA metabarcoding is a powerful genomic-based tool to monitor biodiversity at the microscale, allowing us to capture exact fingerprints of specific event sites and to explore in a comprehensive manner the eukaryotic community alteration. With this work, we aim to disentangle and overcome the crucial issues related to the generalization of DNA metabarcoding in order to support future applications.