Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Genotyping amplicons from SARS-CoV-2 viruses with or without mutations to the N.iORF3 transcription regulatory sequence to determine relative viral fitness in A549-ACE2-TMPRSS2 cells

ABSTRACT: We have demonstrated that a newly evolved TRS within the Nucleocapsid gene of SARS-CoV-2 (termed N.iORF3) leads to the expression of a novel subgenomic mRNA encoding a truncated C-terminal portion of Nucleocapsid, which is an antagonist of type I interferon production. Using reverse genetics-derived viruses we show N.iORF3 contributes to viral fitness during infection and observe distinct phenotypes when the Nucleocapsid coding sequence is mutated compared to when the TRS alone is ablated. Competition assays were performed to determine the relative fitness of different virus mutants and amplicons were analysed to quantify the proportions of different viruses in tissue culture.

INSTRUMENT(S): Illumina MiSeq

ORGANISM(S): Severe acute respiratory syndrome-related coronavirus

SUBMITTER: Harriet Mears

PROVIDER: E-MTAB-14680 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:BACKGROUND: Microarray comparative genomic hybridization (CGH) is currently one of the most powerful techniques to measure DNA copy number in large genomes. In humans, microarray CGH is widely used to assess copy number variants in healthy individuals and copy number aberrations associated with various diseases, syndromes and disease susceptibility. In model organisms such as Caenorhabditis elegans (C. elegans) the technique has been applied to detect mutations, primarily deletions, in strains of interest. Although various constraints on oligonucleotide properties have been suggested to minimize non-specific hybridization and improve the data quality, there have been few experimental validations for CGH experiments. For genomic regions where strict design filters would limit the coverage it would also be useful to quantify the expected loss in data quality associated with relaxed design criteria. RESULTS: We have quantified the effects of filtering various oligonucleotide properties by measuring the resolving power for detecting deletions in the human and C. elegans genomes using NimbleGen microarrays. Approximately twice as many oligonucleotides are typically required to be affected by a deletion in human DNA samples in order to achieve the same statistical confidence as one would observe for a deletion in C. elegans. Surprisingly, the ability to detect deletions strongly depends on the oligonucleotide 15-mer count, which is defined as the sum of the genomic frequency of all the constituent 15-mers within the oligonucleotide. A similarity level above 80% to non-target sequences over the length of the probe produces significant cross-hybridization. We recommend the use of a fairly large melting temperature window of up to 10 C, the elimination of repeat sequences, the elimination of homopolymers longer than 5 nucleotides, and a threshold of -1 kcal/mol on the oligonucleotide self-folding energy. We observed very little difference in data quality when varying the oligonucleotide length between 50 and 70, and even when using an isothermal design strategy. CONCLUSIONS: We have determined experimentally the effects of varying several key oligonucleotide microarray design criteria for detection of deletions in C. elegans and humans with NimbleGen's CGH technology. Our oligonucleotide design recommendations should be applicable for CGH analysis in most species. One-copy deletions were studied in both the C. elegans (2 samples) and human (1 sample) genomes. There were no replicate or dye-swap hybridizations.

Dataset Information

Genotyping amplicons from SARS-CoV-2 viruses with or without mutations to the N.iORF3 transcription regulatory sequence to determine relative viral fitness in A549-ACE2-TMPRSS2 cells

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