Survey of SARS-CoV-2 genetic diversity in two major Brazilian cities using a fast and affordable Sanger sequencing strategy

Dorlass, Erick Gustavo; Lourenço, Karine Lima; Magalhães, Rubens Daniel Miserani; Sato, Hugo Itaru; Fiorini, Alex; Peixoto, Renata Barbosa; Coelho, Helena Perez; Telezynski, Bruna Larotonda; Scagion, Guilherme Pereira; Ometto, Tatiana; Thomazelli, Luciano Matsumiya; Oliveira, Danielle Bruna Leal; Fernandes, Ana Paula; Durigon, Edison Luiz; Fonseca, Flávio Guimarães da; Teixeira, Santuza Maria Ribeiro

doi:10.1016/j.ygeno.2021.10.015

Cited by 12 publications

(11 citation statements)

References 34 publications

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“…After that, the number of cases related to the variant continued to rise, and 8 weeks later, VOC Delta was already responsible for more than 70 per cent of cases in the state. The rapid replacement of variants by Delta has already been described in other countries and states in Brazil ( Dorlass et al. 2021 ; Kupferschmidt and Wadman 2021 ; Lamarca et al.…”

Section: Discussionmentioning

confidence: 64%

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Tracking the turnover of SARS-CoV-2 VOCs Gamma to Delta in a Brazilian state (Minas Gerais) with a high-vaccination status

et al. 2022

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The emergence and global dissemination of SARS-CoV-2 variants of concern (VOC) have been described as the main factor driving the COVID-19 pandemic. In Brazil, the Gamma variant dominated the epidemiological scenario during the first period of 2021. Many Brazilian regions detected the Delta variant after its first description and documented its spread. To monitor the introduction and spread of VOC Delta, we performed genotyping-PCR and genome sequencing in ten regional sentinel units from June to October 2021 in the State of Minas Gerais (MG). We documented the introduction and spread of Delta, comprising 70% of the cases eight weeks later. Comparing the viral loads of the Gamma and Delta dominance periods, we provide additional evidence that the latter is more transmissible. The spread and dominance of Delta did not culminate in the increase in cases and deaths, suggesting that the vaccination may have restrained the epidemic growth. Analysis of 224 novel Delta genomes revealed that Rio de Janeiro state was the primary source for disseminating this variant in the state of MG. We present the establishment of Delta, providing evidence of its enhanced transmissibility and showing that this variant shift did not aggravate the epidemiological scenario in a high immunity setting.

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Section: Discussionmentioning

confidence: 64%

“…Samples with wild-type profiles at Positions K417, E484, and N501 and mutant at Positions L452R and P681R were classified as Delta and randomly selected for whole-genome sequencing. Further genotyping was done through partial nucleotide sequencing of the S-coding gene through Sanger sequencing ( Dorlass et al. 2021 ).…”

Section: Methodsmentioning

confidence: 99%

Tracking the turnover of SARS-CoV-2 VOCs Gamma to Delta in a Brazilian state (Minas Gerais) with a high-vaccination status

et al. 2022

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“…Reverse transcription Polymerase chain reaction (RT-PCR) assays have been used to identify specific characteristics that are unique in specific variants, such as S-gene target failure (SGTF) in the Alpha and Omicron variant, and genotyping assays have also been applied for this purpose ( 33 ). Sanger sequencing is also a methodology that allows monitoring SARS-CoV-2 variants with a rapid response ( 34 – 36 ). Recombination can be challenging to detect by classification methods, as the recombinant sequences have high similarity to their shared ancestor ( 28 , 32 ).…”

Section: Introductionmentioning

confidence: 99%

Molecular characterization of a new SARS-CoV-2 recombinant cluster XAG identified in Brazil

et al. 2022

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Recombination events have been described in the Coronaviridae family. Since the beginning of the SARS-CoV-2 pandemic, a variable degree of selection pressure has acted upon the virus, generating new strains with increased fitness in terms of viral transmission and antibody scape. Most of the SC2 variants of concern (VOC) detected so far carry a combination of key amino acid changes and indels. Recombination may also reshuffle existing genetic profiles of distinct strains, potentially giving origin to recombinant strains with altered phenotypes. However, co-infection and recombination events are challenging to detect and require in-depth curation of assembled genomes and sequencing reds. Here, we present the molecular characterization of a new SARS-CoV-2 recombinant between BA.1.1 and BA.2.23 Omicron lineages identified in Brazil. We characterized four mutations that had not been previously described in any of the recombinants already identified worldwide and described the likely breaking points. Moreover, through phylogenetic analysis, we showed that the newly named XAG lineage groups in a highly supported monophyletic clade confirmed its common evolutionary history from parental Omicron lineages and other recombinants already described. These observations were only possible thanks to the joint effort of bioinformatics tools auxiliary in genomic surveillance and the manual curation of experienced personnel, demonstrating the importance of genetic, and bioinformatic knowledge in genomics.

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“…The predicted PCR amplicon with these primers is a 1006 bp product flanking the RBD region of the spike protein of the SARS-CoV-2 virus. The PCR was prepared as described by Dorlas et al [ 11 ], and the products were analyzed in 1% agarose gel electrophoresis (40 min at 110 v). The products were purified with ExoSAP-IT TM PCR Product Cleanup (Affymetrix, Santa Clara, CA, USA) according to the manufacturer’s protocol.…”

Section: Methodsmentioning

confidence: 99%

“…The Sanger sequencing technique is considered the standard method for short nucleotide sequence determination, it is available to many labs, it is less expensive and faster than NGS, and it can run samples individually [ 10 ]. These features allow Sanger sequencing to be used as a screening method for detecting SARS-CoV-2 mutations and generating data of importance for public health and surveillance systems [ 10 , 11 ].…”

Section: Introductionmentioning

confidence: 99%

SARS-CoV-2 Variants of Concern: Presumptive Identification via Sanger Sequencing Analysis of the Receptor Binding Domain (RBD) Region of the S Gene

et al. 2023

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Variants of concern (VOCs) of SARS-CoV-2 are viral strains that have mutations associated with increased transmissibility and/or increased virulence, and their main mutations are in the receptor binding domain (RBD) region of the viral spike. This study aimed to characterize SARS-CoV-2 VOCs via Sanger sequencing of the RBD region and compare the results with data obtained via whole genome sequencing (WGS). Clinical samples (oro/nasopharyngeal) with positive RT-qPCR results for SARS-CoV-2 were used in this study. The viral RNA from SARS-CoV-2 was extracted and a PCR fragment of 1006 base pairs was submitted for Sanger sequencing. The results of the Sanger sequencing were compared to the lineage assigned by WGS using next-generation sequencing (NGS) techniques. A total of 37 specimens were sequenced via WGS, and classified as: VOC gamma (8); delta (7); omicron (10), with 3 omicron specimens classified as the BQ.1 subvariant and 12 specimens classified as non-VOC variants. The results of the partial Sanger sequencing presented as 100% in agreement with the WGS. The Sanger protocol made it possible to characterize the main SARS-CoV-2 VOCs currently circulating in Brazil through partial Sanger sequencing of the RBD region of the viral spike. Therefore, the sequencing of the RBD region is a fast and cost-effective laboratory tool for clinical and epidemiological use in the genomic surveillance of SARS-CoV-2.

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Survey of SARS-CoV-2 genetic diversity in two major Brazilian cities using a fast and affordable Sanger sequencing strategy

Cited by 12 publications

References 34 publications

Tracking the turnover of SARS-CoV-2 VOCs Gamma to Delta in a Brazilian state (Minas Gerais) with a high-vaccination status

Tracking the turnover of SARS-CoV-2 VOCs Gamma to Delta in a Brazilian state (Minas Gerais) with a high-vaccination status

Molecular characterization of a new SARS-CoV-2 recombinant cluster XAG identified in Brazil

SARS-CoV-2 Variants of Concern: Presumptive Identification via Sanger Sequencing Analysis of the Receptor Binding Domain (RBD) Region of the S Gene

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