SARS-CoV-2 Genomes From Oklahoma, United States

Narayanan, Sai; Ritchey, John C.; Patil, Girish; Narasaraju, Teluguakula; More, Sunil S.; Malayer, Jerry R.; Saliki, Jeremiah T.; Kaul, Anil; Agarwal, Pratul K.; Ramachandran, Akhilesh

doi:10.3389/fgene.2020.612571

Cited by 7 publications

(3 citation statements)

References 51 publications

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“…However, some SARS-CoV-2 isolates with an N gene harboring P13L, Δ31-33, R203K and G204R mutations may not have a demonstrable RBD mutation profile to support an Omicron variant diagnosis as shown in the GenBank sequences ID# OL898842, OL901854, OL902308 and OL920485 even when the NTD of the S gene in these isolates has been sequenced to show the presence of A67V, Δ69-70, T95I, G142D and Δ143-145 mutations, as shown in Figure 31. The N gene R203K and G204R mutations are not reliable for Omicron variant diagnosis because they were already found in the SARS-CoV-2 strains circulating in early 2020 [40] long before the Omicron variant emerged. In the current series, 2 (M22-44 and M22-68) of 29 positive samples did not yield an RBD sequence for a definitive diagnosis of an Omicron variant.…”

Section: The N Gene Is a More Reliable Target For Rt-pcr Detection Wh...mentioning

confidence: 99%

Evidence-Based Evaluation of PCR Diagnostics for SARS-Cov-2 and The Omicron Variants by Sanger Sequencing

Lee¹

2022

Preprint

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Both SARS-CoV-2 and SARS-CoV initially appeared in China and spread to other parts of the world. SARS-CoV-2 has generated a COVID-19 pandemic causing more than 6 million human deaths worldwide while the SARS outbreak quickly ended in six months with a global total of 774 reported deaths. One of the factors contributing to this stunning difference in the outcome between these two outbreaks is the inaccuracy of the RT-qPCR tests for SARS-CoV-2, which generated a large number of false-negative and false-positive test results that have misled patient management and public health policy-makers. This article presented Sanger sequencing evidence to show that the RT-PCR diagnostic protocol established in 2003 for SARS-CoV can in fact detect SARS-CoV-2 accurately due to the well-known nonspecific PCR amplification of DNAs with similar sequences. Using nested RT-PCR followed by Sanger sequencing to retest 50 patient samples collected in January, 2022 and sold as RT-qPCR positive reference confirmed 21 (42%) were false-positive. Although the other 29 positive isolates were categorized as Omicron variant by partial sequencing of the N gene, and the RBD and the NTD of the S gene, 9 (31%) showed focal to complete sequencing failure in the S gene segments due to multi-allelic SNPs. During the course of the study, an Omicron variant isolate containing a BA.1 NTD and a BA.2 RBD in its S gene was also detected. Routine partial S gene sequencing of all PCR-positive samples can timely discover multi-allelic SNPs and viral recombination in the circulating variants for investigation of their impacts on vaccine efficacies, therapeutics and diagnostics.

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Section: The N Gene Is a More Reliable Target For Rt-pcr Detection Wh...mentioning

confidence: 99%

Evidence-Based Evaluation of PCR Diagnostics for SARS-Cov-2 and The Omicron Variants by Sanger Sequencing

Lee¹

2022

Preprint

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“…Sequencing of the S gene can be a valuable tool for confirmation of known variants following screening by PCR as described above. However, the gold standard for the identification of new variants remains sequencing of the entire viral genome by NGS, since mutations outside the S gene, able to change the infectivity of the virus and the prognosis of the infection, have also been reported [ 11 , 12 , 13 ]. Indeed, mutations associated with prognosis changes have been described in the ORF8 , ORF3a , ORF6 , and N genes; for example, deletion of 382 nucleotides (D382) within ORF8 has been associated with a milder infection and a lower concentration of proinflammatory cytokines [ 14 ].…”

Section: Discussionmentioning

confidence: 99%

Different Strategies for the Identification of SARS-CoV-2 Variants in the Laboratory Practice

Anaclerio

Mandatori

Antonucci

et al. 2021

Genes

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A considerable effort has been devoted in all countries to react to the COVID-19 pandemic by tracing infected individuals, containing the spread of the disease, identifying therapies, and producing and distributing vaccines. Currently, a significant concern is the appearance of variants of the virus that may frustrate these efforts by showing increased transmissibility, increased disease severity, reduced response to therapy or vaccines, and ability to escape diagnosis. All countries have therefore devoted a massive attempt to the identification and tracking of these variants, which requires a vast technological effort to sequence a large number of viral genomes. In this paper, we report our experience as one of the Italian laboratories involved in SARS-CoV-2 variant tracing. We summarize the different approaches used, and outline a potential model combining several techniques to increase tracing ability while at the same time minimizing costs.

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“…The N gene of the Omicron variant strains invariably harbors the R203K and G204R mutations. But these two mutations were already sporadically reported in nasopharyngeal swab specimens collected in the early part of 2020 [42] long before the appearance of the Omicron variant. Therefore, variant determination based on N gene sequencing alone is not reliable.…”

Section: The N Gene Is the More Reliable Target Than The S Gene For A...mentioning

confidence: 99%

Implementation of the eCDC/WHO Recommendation for Molecular Diagnosis of SARS-CoV-2 Omicron Subvariants and Its Challenges

Lee¹

2022

Preprint

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Large population passages of the SARS-CoV-2 in the past two and a half years have allowed the circulating virus to accumulate an increasing number of mutations in its genome. The most recently emerging Omicron subvariants have the highest number of mutations in the Spike (S) protein gene and these mutations mainly occur in the receptor-binding domain (RBD) and the N-terminal domain (NTD) of the S gene. The eCDC and the WHO recommend partial Sanger sequencing of the SARS-CoV-2 S gene RBD and NTD on the PCR-positive samples in diagnostic laboratories as a practical means of determining the variants of concern to monitor a possible increased transmissibility, increased virulence, or reduced effectiveness of vaccines against them. The author’s diagnostic laboratory has implemented the eCDC/WHO recommendation by sequencing a 398-base segment of the N gene for the definitive detection of SARS-CoV-2 in clinical samples, and sequencing a 445-base segment of the RBD and a 490-509-base segment of the NTD for variant determination. This paper presents 5 selective cases to illustrate the challenges of using Sanger sequencing to diagnose Omicron subvariant when the samples harbor a high level of co-existing minor subvariant sequences with multi-allelic single nucleotide polymorphisms (SNPs) or possible recombinant Omicron subvariants containing a BA.1 NTD and a BA.2 RBD, which can only be detected by using specially designed PCR primers. The current large-scale surveillance programs using next-generation sequencing (NGS) do not face similar problems because NGS focuses on deriving consensus sequence.

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SARS-CoV-2 Genomes From Oklahoma, United States

Cited by 7 publications

References 51 publications

Evidence-Based Evaluation of PCR Diagnostics for SARS-Cov-2 and The Omicron Variants by Sanger Sequencing

Evidence-Based Evaluation of PCR Diagnostics for SARS-Cov-2 and The Omicron Variants by Sanger Sequencing

Different Strategies for the Identification of SARS-CoV-2 Variants in the Laboratory Practice

Implementation of the eCDC/WHO Recommendation for Molecular Diagnosis of SARS-CoV-2 Omicron Subvariants and Its Challenges

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