Amplicon-Based Detection and Sequencing of SARS-CoV-2 in Nasopharyngeal Swabs from Patients With COVID-19 and Identification of Deletions in the Viral Genome That Encode Proteins Involved in Interferon Antagonism
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Abstract
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19). Sequencing the viral genome as the outbreak progresses is important, particularly in the identification of emerging isolates with different pathogenic potential and to identify whether nucleotide changes in the genome will impair clinical diagnostic tools such as real-time PCR assays. Although single nucleotide polymorphisms and point mutations occur during the replication of coronaviruses, one of the biggest drivers in genetic change is recombination. This can manifest itself in insertions and/or deletions in the viral genome. Therefore, sequencing strategies that underpin molecular epidemiology and inform virus biology in patients should take these factors into account. A long amplicon/read length-based RT-PCR sequencing approach focused on the Oxford Nanopore MinION/GridION platforms was developed to identify and sequence the SARS-CoV-2 genome in samples from patients with or suspected of COVID-19. The protocol, termed Rapid Sequencing Long Amplicons (RSLAs) used random primers to generate cDNA from RNA purified from a sample from a patient, followed by single or multiplex PCRs to generate longer amplicons of the viral genome. The base protocol was used to identify SARS-CoV-2 in a variety of clinical samples and proved sensitive in identifying viral RNA in samples from patients that had been declared negative using other nucleic acid-based assays (false negative). Sequencing the amplicons revealed that a number of patients had a proportion of viral genomes with deletions.
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SciScore for 10.1101/2020.03.05.20032011: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement Consent: Ethics and clinical information: Patients used in this study gave informed consent and were recruited under the International Severe Acute Respiratory and emerging Infection Consortium ( Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Experimental Models: Cell Lines Sentences Resources Total RNA was purified from SARS-CoV-2 infected Vero cells using the Qiagen RNA minikit following AVL inactivation. Verosuggested: CLS Cat# 605372/p622_VERO, RRID:CVCL_0059)Software and Algorithms Sentences Resources EPI2ME (WIMP): Fast5s generated by the … SciScore for 10.1101/2020.03.05.20032011: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement Consent: Ethics and clinical information: Patients used in this study gave informed consent and were recruited under the International Severe Acute Respiratory and emerging Infection Consortium ( Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Experimental Models: Cell Lines Sentences Resources Total RNA was purified from SARS-CoV-2 infected Vero cells using the Qiagen RNA minikit following AVL inactivation. Verosuggested: CLS Cat# 605372/p622_VERO, RRID:CVCL_0059)Software and Algorithms Sentences Resources EPI2ME (WIMP): Fast5s generated by the MinION sequencer were base called into fastqs by Guppy. MinIONsuggested: (MinION, RRID:SCR_017985)Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:One of the limitations is working at higher containment. We would note that our base calling used Epi2ME and artefacts are often observed by Kraken. BLAST analysis provides greater accuracy but takes longer. Sequencing of viral genomes during outbreaks provides much needed information in terms of viral adaptation [4] and informs molecular epidemiological studies [11]. Due to recombination in coronaviruses (e.g. [12, 13]) current diagnostics may not remain fit for purpose and therefore metagenomic approaches provide independent verification of the presence of viral genomes as well information on the underlying microbiome – which may contribute to severe disease in COVID-19. One limitation of the metagenomic approach is the limit of detection and in this study, not all of the SARS-CoV-2 genome was sequenced using the SISPA approach. For diagnostic purposes, RT-qPCR generally is more sensitive, provided that the primer binding sites remain conserved in the pathogen being tested. In this case, RT-qPCR diagnostic reagents can be revaluated based upon using sequencing as sentinel for these events. Determining the background microbiome in near real time can inform potential treatment strategies in the event specific co-infections are identified. Future efforts will quantify the limits of detection using genome sequencing by these approaches.
Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
Results from JetFighter: We did not find any issues relating to colormaps.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
- Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
- Thank you for including a protocol registration statement.
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Excerpt
Using Amplicon-based Nanopore sequencing, the authors of this paper were able to detect SARS-CoV-2 from lung tissue samples of 2 patients.
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