PCR-Sequencing Approaches to Assess Informative Mutations in SARS-Cov-2 Spike (S) and ORF7, ORF8 and N Genes Characterizing Variants of Concern and Variants of Interest (original) (raw)

A Simplified Sanger Sequencing Method for Detection of Relevant SARS-CoV-2 Variants

Diagnostics

Molecular surveillance of the new coronavirus through new genomic sequencing technologies revealed the circulation of important variants of SARS-CoV-2. Sanger sequencing has been useful in identifying important variants of SARS-CoV-2 without the need for whole-genome sequencing. A sequencing protocol was constructed to cover a region of 1000 base pairs, from a 1120 bp product generated after a two-step RT-PCR assay in samples positive for SARS-CoV-2. Consensus sequence construction and mutation identification were performed. Of all 103 samples sequenced, 69 contained relevant variants represented by 20 BA.1, 13 delta, 22 gamma, and 14 zeta, identified between June 2020 and February 2022. All sequences found were aligned with representative sequences of the variants. Using the Sanger sequencing methodology, we were able to develop a more accessible protocol to assist viral surveillance with a more accessible platform.

A Sanger-based approach for scaling up screening of SARS-CoV-2 variants of interest and concern

ABSTRACTThe global spread of new SARS-CoV-2 variants of concern underscore an urgent need of simple deployed molecular tools that can differentiate these lineages. Several tools and protocols have been shared since the beginning of the COVID-19 pandemic, but they need to be timely adapted to cope with SARS-CoV-2 evolution. Although whole-genome sequencing (WGS) of the virus genetic material have been widely used, it still presents practical difficulties such as high cost, shortage of available reagents in the global market, need of a specialized laboratorial infrastructure and well-trained staff. These limitations result in genomic surveillance blackouts across several countries. Here we propose a rapid and accessible protocol based on Sanger sequencing of a single PCR fragment that is able to identify and discriminate all SARS-CoV-2 variants of concern (VOCs) identified so far, according to each characteristic mutational profile at the Spike-RBD region (K417N/T, E484K, N501Y, A570D...

Implementation of an In-House Platform for Rapid Screening of SARS-CoV-2 Genome Variations

Archives of Iranian Medicine, 2023

Background: Global real-time monitoring of SARS-CoV-2 variants is crucial to controlling the COVID-19 outbreak. The purpose of this study was to set up a Sanger-based platform for massive SARS-CoV-2 variant tracking in laboratories in low-resource settings. Methods: We used nested RT-PCR assay, Sanger sequencing and lineage assignment for 930-bp of the SARS-CoV-2 spike gene, which harbors specific variants of concern (VOCs) mutations. We set up our platform by comparing its results with whole genome sequencing (WGS) data on 137 SARS-CoV-2 positive samples. Then, we applied it on 1028 samples from March-September 2021. Results: In total, 125 out of 137 samples showed 91.24% concordance in mutation detection. In lineage assignment, 123 out of 137 samples demonstrated 89.78% concordance, 65 of which were assigned as VOCs and showed 100% concordance. Of 1028 samples screened by our in-house method, 78 distinct mutations were detected. The most common mutations were: S

Enhancing the epidemiological surveillance of SARS-CoV-2 using Sanger sequencing to identify circulating variants and recombinants

Brazilian journal of microbiology, 2024

Since the emergence of SARS-CoV-2 in December 2019, more than 12,000 mutations in the virus have been identified. These could cause changes in viral characteristics and directly impact global public health. The emergence of variants is a great concern due to the chance of increased transmissibility and infectivity. Sequencing for surveillance and monitoring circulating strains is extremely necessary as the early identification of new variants allows public health agencies to make faster and more effective decisions to contain the spread of the virus. In the present study, we identified circulating variants in samples collected in Belo Horizonte, Brazil, and detected a recombinant lineage using the Sanger method. The identification of lineages was done through gene amplification of SARS-CoV-2 by Reverse Transcription-Polymerase Chain Reaction (RT-PCR). By using these specific fragments, we were able to differentiate one variant of interest and five circulating variants of concern. We were also able to detect recombinants. Randomly selected samples were sequenced by either Sanger or Next Generation Sequencing (NGS). Our findings validate the effectiveness of Sanger sequencing as a powerful tool for monitoring variants. It is easy to perform and allows the analysis of a larger number of samples in countries that cannot afford NGS.

Evaluation of the Sequence Variability within the PCR Primer/Probe Target Regions of the SARS-CoV-2 Genome

BIO-PROTOCOL, 2020

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2; initially named 2019-nCoV) is responsible for the recent coronavirus disease (COVID-19) pandemic, and polymerase chain reaction (PCR) is the current standard method for diagnosis from patient samples. As PCR assays are prone to sequence mismatches due to mutations in the viral genome, it is important to verify the genomic variability at primer/probe binding regions periodically. This step-by-step protocol describes a bioinformatics approach for an extensive evaluation of the sequence variability within the primer/probe target regions of the SARS-CoV-2 genome. The protocol can be applied to any molecular diagnostic assay of choice using freely available software programs and the ready-to-use multiple sequence alignment (MSA) file provided.

Overview of genomic surveillance related to Severe Acute Respiratory Syndrom Coronavirus 2 (SARS- CoV-2)

E3S web of conferences, 2021

Since the start of the Severe Acute Respiratory Syndrom Coronavirus 2 (SARS-CoV-2) pandemic, several thousand of variants circulated and others are emerging. Therefore, genomic surveillance is crucial, which aims to detect the emergence of new variants, in particular Variants of Concern (VOC) and to assess the impact of priority mutations on the transmissibility and lethality of the virus, the performance of viral diagnostic methods and vaccine efficiency. An overview of available papers was performed to understand conduct, tools and utility of genomic sequencing and surveillance related to Covid-19 disease. We also report the experience of Morocco in this filed through available data. A national SARS-Cov-2 genomic consortium has been established in order to continuously inform the health authorities of the genetic evolution of circulating strains in Morocco. Genomic sequencing shows that Moroccan genomes spread did not show a predominant SARS-CoV-2 lineage. Genomes are dispersed across the evolutionary tree of SARS-CoV-2 and held between 4 and 16 mutations. As the pandemic ongoing, continuous genomic surveillance and regular sequencing are fundamental to understand the spread of SARS-CoV-2, to rapidly identify potential global transmission networks and to consolidate response strategies especially targeted Covid-19 vaccination.

Global landscape of SARS-CoV-2 mutations and conserved regions

Journal of Translational Medicine

Background At the end of December 2019, a novel strain of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) disease (COVID-19) has been identified in Wuhan, a central city in China, and then spread to every corner of the globe. As of October 8, 2022, the total number of COVID-19 cases had reached over 621 million worldwide, with more than 6.56 million confirmed deaths. Since SARS-CoV-2 genome sequences change due to mutation and recombination, it is pivotal to surveil emerging variants and monitor changes for improving pandemic management. Methods 10,287,271 SARS-CoV-2 genome sequence samples were downloaded in FASTA format from the GISAID databases from February 24, 2020, to April 2022. Python programming language (version 3.8.0) software was utilized to process FASTA files to identify variants and sequence conservation. The NCBI RefSeq SARS-CoV-2 genome (accession no. NC_045512.2) was considered as the reference sequence. Results Six mutations had more than 50% frequenc...

Characterization of SARS-CoV-2 Mutational Signatures from 1.5+ Million Raw Sequencing Samples

Viruses, 2022

We present a large-scale analysis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) substitutions, considering 1,585,456 high-quality raw sequencing samples, aimed at investigating the existence and quantifying the effect of mutational processes causing mutations in SARS-CoV-2 genomes when interacting with the human host. As a result, we confirmed the presence of three well-differentiated mutational processes likely ruled by reactive oxygen species (ROS), apolipoprotein B editing complex (APOBEC), and adenosine deaminase acting on RNA (ADAR). We then evaluated the activity of these mutational processes in different continental groups, showing that some samples from Africa present a significantly higher number of substitutions, most likely due to higher APOBEC activity. We finally analyzed the activity of mutational processes across different SARS-CoV-2 variants, and we found a significantly lower number of mutations attributable to APOBEC activity in samples assigned to the Omicron variant.