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Commun Biol ; 4(1): 1215, 2021 10 22.
Article in English | MEDLINE | ID: covidwho-1479821


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 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 RNA samples from anonymized SARS-CoV-2 positive subjects and we observed a recurrent and characteristic pattern of sgmRNAs expression in relation to the total viral RNA content. Additionally, expression profiles of sgmRNAs, as determined by meta-transcriptomics sequencing of a subset of 110 RNA samples, were highly correlated with those obtained by ddPCR. By providing a comprehensive and dynamic snapshot of the levels of SARS-CoV-2 sgmRNAs in infected individuals, our results may contribute a better understanding of the dynamics of transcription and expression of the genome of SARS-CoV-2 and facilitate the development of more accurate molecular diagnostic tools for the stratification of COVID-19 patients.

COVID-19 Nucleic Acid Testing/methods , COVID-19/genetics , COVID-19/metabolism , Coronavirus Nucleocapsid Proteins , Polymerase Chain Reaction/methods , RNA, Viral/metabolism , SARS-CoV-2 , Spike Glycoprotein, Coronavirus , Transcriptome , Computational Biology , Humans , Limit of Detection , Open Reading Frames , Phosphoproteins , RNA, Messenger/metabolism , Real-Time Polymerase Chain Reaction , Reproducibility of Results
Brief Bioinform ; 22(2): 616-630, 2021 03 22.
Article in English | MEDLINE | ID: covidwho-1343634


Various next generation sequencing (NGS) based strategies have been successfully used in the recent past for tracing origins and understanding the evolution of infectious agents, investigating the spread and transmission chains of outbreaks, as well as facilitating the development of effective and rapid molecular diagnostic tests and contributing to the hunt for treatments and vaccines. The ongoing COVID-19 pandemic poses one of the greatest global threats in modern history and has already caused severe social and economic costs. The development of efficient and rapid sequencing methods to reconstruct the genomic sequence of SARS-CoV-2, the etiological agent of COVID-19, has been fundamental for the design of diagnostic molecular tests and to devise effective measures and strategies to mitigate the diffusion of the pandemic. Diverse approaches and sequencing methods can, as testified by the number of available sequences, be applied to SARS-CoV-2 genomes. However, each technology and sequencing approach has its own advantages and limitations. In the current review, we will provide a brief, but hopefully comprehensive, account of currently available platforms and methodological approaches for the sequencing of SARS-CoV-2 genomes. We also present an outline of current repositories and databases that provide access to SARS-CoV-2 genomic data and associated metadata. Finally, we offer general advice and guidelines for the appropriate sharing and deposition of SARS-CoV-2 data and metadata, and suggest that more efficient and standardized integration of current and future SARS-CoV-2-related data would greatly facilitate the struggle against this new pathogen. We hope that our 'vademecum' for the production and handling of SARS-CoV-2-related sequencing data, will contribute to this objective.

COVID-19/virology , Genome, Viral , High-Throughput Nucleotide Sequencing/methods , SARS-CoV-2/genetics , COVID-19/epidemiology , Humans , Pandemics