Abstract:The SARS-CoV-2 spike (S) protein, the viral mediator for binding and entry into the host cell, has sparked great interest as a target for vaccine development and treatments with neutralizing antibodies. Initial data suggest that the virus has low mutation rates, but its large genome could facilitate recombination, insertions, and deletions, as has been described in other coronaviruses. Here, we deep-sequenced the complete SARS-CoV-2
S
gene from 18 patients (10 with mild and 8 with severe… Show more
“…Even if several studies report variants found at frequencies below 0.1 (25,27,37-39) only a few evaluated the confidence of such calls (26,40). We found that the number of variants with VAF>0.1 detected in samples with at least 1000 g.c.p.r is similar between contemporaneous samples and increases with time since the start of the outbreak, as expected.…”
Background: SARS-CoV-2 genotyping has been instrumental to monitor virus evolution and transmission during the pandemic. The reliability of the information extracted from the genotyping efforts depends on a number of aspects, including the quality of the input material, applied technology and potential laboratory-specific biases. These variables must be monitored to ensure genotype reliability. The current lack of guidelines for SARS-CoV-2 genotyping leads to inclusion of error-containing genome sequences in studies of viral spread and evolution. Results: We used clinical samples and synthetic viral genomes to evaluate the impact of experimental factors, including viral load and sequencing depth, on correct sequence determination using an amplicon-based approach. We found that at least 1000 viral genomes are necessary to confidently detect variants in the genome at frequencies of 10% or higher. The broad applicability of our recommendations was validated in >200 clinical samples from six independent laboratories. The genotypes of clinical isolates with viral load above the recommended threshold cluster by sampling location and period. Our analysis also supports the rise in frequency of 20A.EU1 and 20A.EU2, two recently reported European strains whose dissemination was favoured by travelling during the summer 2020. Conclusions: We present much-needed recommendations for reliable determination of SARS-CoV-2 genome sequence and demonstrate their broad applicability in a large cohort of clinical samples.
“…Even if several studies report variants found at frequencies below 0.1 (25,27,37-39) only a few evaluated the confidence of such calls (26,40). We found that the number of variants with VAF>0.1 detected in samples with at least 1000 g.c.p.r is similar between contemporaneous samples and increases with time since the start of the outbreak, as expected.…”
Background: SARS-CoV-2 genotyping has been instrumental to monitor virus evolution and transmission during the pandemic. The reliability of the information extracted from the genotyping efforts depends on a number of aspects, including the quality of the input material, applied technology and potential laboratory-specific biases. These variables must be monitored to ensure genotype reliability. The current lack of guidelines for SARS-CoV-2 genotyping leads to inclusion of error-containing genome sequences in studies of viral spread and evolution. Results: We used clinical samples and synthetic viral genomes to evaluate the impact of experimental factors, including viral load and sequencing depth, on correct sequence determination using an amplicon-based approach. We found that at least 1000 viral genomes are necessary to confidently detect variants in the genome at frequencies of 10% or higher. The broad applicability of our recommendations was validated in >200 clinical samples from six independent laboratories. The genotypes of clinical isolates with viral load above the recommended threshold cluster by sampling location and period. Our analysis also supports the rise in frequency of 20A.EU1 and 20A.EU2, two recently reported European strains whose dissemination was favoured by travelling during the summer 2020. Conclusions: We present much-needed recommendations for reliable determination of SARS-CoV-2 genome sequence and demonstrate their broad applicability in a large cohort of clinical samples.
“…Sequencing technical replicates of multiple cDNAs generated from the same sample has been proposed as a mitigation strategy to identify iSNVs more reliably [ 23 ]. However, whereas amplicon-based sequencing has been widely used for the analysis of low-frequency variants [ [20] , [21] , [22] , 37 , 38 ] only a few studies thus far have evaluated the confidence of such calls and have implemented the sequencing of cDNA replicates to ensure accuracy [ 23 ]. False positives have also been reported among high-frequency variants supported by good sequencing depth, indicating that the risks of inaccurate sequencing are not limited to suboptimal samples [ 39 ].…”
Sequencing the SARS-CoV-2 genome from clinical samples can be challenging, especially in specimens with low viral titer. Here we report Accurate SARS-CoV-2 genome Reconstruction (ACoRE), an amplicon-based viral genome sequencing workflow for the complete and accurate reconstruction of SARS-CoV-2 sequences from clinical samples, including suboptimal ones that would usually be excluded even if unique and irreplaceable. The protocol was optimized to improve flexibility and the combination of technical replicates was established as the central strategy to achieve accurate analysis of low-titer/suboptimal samples. We demonstrated the utility of the approach by achieving complete genome reconstruction and the identification of false-positive variants in >170 clinical samples, thus avoiding the generation of inaccurate and/or incomplete sequences. Most importantly, ACoRE was crucial to identify the correct viral strain responsible of a relapse case, that would be otherwise mis-classified as a re-infection due to missing or incorrect variant identification by a standard workflow.
“…ACE2-binding affinity of the S protein is one of the most important determinants of SARS-CoV-2 infectivity and disease severity. While some of the S protein mutations enhance ACE2 binding, resulting in increased susceptibility and severity of COVID-19, others can inhibit S protein binding or result in a truncated S1 protein, conferring a high degree of resistance to infection or reducing the severity of it [ [116] , [117] , [118] ]. Fig.…”
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