In-House, Rapid, and Low-Cost SARS-CoV-2 Spike Gene Sequencing Protocol to Identify Variants of Concern Using Sanger Sequencing
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Abstract
Background
The need for active genomic sequencing surveillance to rapidly identify circulating SARS-CoV-2 variants of concern (VOCs) is critical. However, increased global demand has led to a shortage of commercial SARS-CoV-2 sequencing kits, and not every country has the technological capability or the funds for high-throughput sequencing platforms. Therefore, this study aimed to develop and validate a rapid, cost-efficient genome sequencing protocol that uses supplies, equipment, and methodologic expertise available in standard molecular or diagnostic laboratories to identify circulating SARS-CoV-2 variants of concern.
Methods
Sets of primers flanking the SARS-CoV-2 spike gene were designed using SARS-CoV-2 genome sequences retrieved from the Global Initiative on Sharing Avian Influenza Data (GISAID) Database and synthesized in-house. Primer specificity and final sequences were verified using online prediction analyses with BLAST. The primers were validated using 282 nasopharyngeal samples collected from patients assessed as positive for SARS-CoV-2 at the diagnostic laboratory of the hospital. The patient samples were subjected to RNA extraction followed by cDNA synthesis, conventional polymerase chain reaction, and Sanger sequencing. Protocol specificity was confirmed by comparing these results with SARS-CoV-2 whole genome sequencing of the same samples.
Results
Sanger sequencing using the newly designed primers and next-generation whole genome sequencing of 282 patient samples indicated identical VOCs results: 123 samples contained the alpha variant (B.1.1.7); 78, beta (B.1.351), 0, gamma (P.1), and 13, delta (B.1.617.2). The remaining samples were not 100% identical to the reference genome; however, 99.97% identity indicated that there was minimal variation as the virus spread throughout the nation. Only four samples had poor sequence quality by Sanger sequencing owing to a low RNA count (Ct value >38). Therefore, mutation calls were >98% accurate.
Conclusions
Sanger sequencing method using in-house primers is an alternative approach that can be used in facilities with existing equipment to mitigate limitations in high throughput supplies required to identify SARS-CoV-2 variants of concern during the COVID-19 pandemic. This protocol is easily adaptable for detection of emerging variants.
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SciScore for 10.1101/2021.08.09.21261723: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics IRB: Sample Collection and Ethical Considerations: This study was performed in compliance with all applicable national and international ethical guidelines for conducting research on human participants, including in accordance with the Code of Ethics of the World Medical Association (Declaration of Helsinki), and was approved by the institutional review board at King Faisal Specialist Hospital and Research Centre (KFSHRC) (IRB #220 0021).
Consent: This board also granted a waiver for obtaining informed consent owing to the use of deidentified samples for this study.Sex as a biological variable not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Table 2: …
SciScore for 10.1101/2021.08.09.21261723: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics IRB: Sample Collection and Ethical Considerations: This study was performed in compliance with all applicable national and international ethical guidelines for conducting research on human participants, including in accordance with the Code of Ethics of the World Medical Association (Declaration of Helsinki), and was approved by the institutional review board at King Faisal Specialist Hospital and Research Centre (KFSHRC) (IRB #220 0021).
Consent: This board also granted a waiver for obtaining informed consent owing to the use of deidentified samples for this study.Sex as a biological variable not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Table 2: Resources
Software and Algorithms Sentences Resources Primer3 online tools (version 4.1.0) (https://primer3.ut.ee/) was used for primer design. https://primer3.ut.ee/suggested: (Primer3, RRID:SCR_003139)The specificity of the primers and final sequences were verified using in silico prediction analyses with the online Basic Local Alignment Search Tool (BLAST) (https://blast.ncbi.nlm.nih.gov/Blast.cgi). BLASTsuggested: (BLASTX, RRID:SCR_001653)https://blast.ncbi.nlm.nih.gov/Blast.cgisuggested: (TBLASTX, RRID:SCR_011823)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:However, major limitations of using conventional PCR and Sanger sequencing are that this approach is not high throughput and only sequence short reads (≤1000 bp) compared with NGS.
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.
- No protocol registration statement was detected.
Results from scite Reference Check: We found no unreliable references.
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