Loss-of-Function Variant in PPP1R12A-Related Urogenital and/or Brain Malformation Syndrome: Expanded Phenotype of Sex Reversal.

Differences of sex development (DSDs) are a heterogeneous group of congenital conditions in which chromosomal, gonadal, or anatomical sex does not match. The broad spectrum of phenotypes associated with DSDs requires accurate diagnosis, which influences the care and quality of life of affected patients. The decreasing costs of next-generation sequencing (NGS) and international research collaborations in rare diseases have allowed the identification of new genes associated with DSDs. Recently, Hughes et al. in 2020 reported the association of loss-of-function (LoF) variants in PPP1R12A with morphological anomalies of the midline, including holoprosencephaly and urogenital malformations, also known as genitourinary and/or brain malformation syndrome (OMIM #618820). In this report, we describe a Mexican individual with hypertelorism, multiple skin hemangiomas, testicular atrophy, and sex reversal, in whom a c.1880delC frameshift variant in PPP1R12A was detected by exome sequencing. Segregation analysis confirmed it as a de novo variant through Sanger sequencing. The main objective of this report is to expand PPP1R12A-related urogenital and/or brain malformation syndrome.

The novel HLA allele, HLA-DQB1*02:211, identified in a Brazilian bone marrow donor.

HLA-DQB1*02:211 allele differs from DQB1*02:02:01:02 by change of C → A in exon 2.

16S rDNA Sequencing for Bacterial Identification in Preterm Infants with Suspected Early-Onset Neonatal Sepsis.

BACKGROUND: The high prevalence of suspected early-onset neonatal sepsis among preterm infants leads to immediate antibiotic administration upon admission. Notably, most blood cultures for suspected early-onset neonatal sepsis do not yield a causative pathogen. This study aimed to assess polymerase chain reaction (PCR) targeting the variable region V4 of the 16S ribosomal gene (16S rDNA) and Sanger sequencing for bacterial identification in preterm infants with suspected early-onset neonatal sepsis. METHODS: Therefore, this prospective study was conducted. Preterm infants with suspected early-onset neonatal sepsis were included in this study. The three groups were formed based on the risk of infection and clinical sepsis. Blood samples were collected upon admission to the neonatal unit for culture and molecular analysis. PCR amplification and subsequent Sanger sequencing of the V4 region of the 16S rDNA were performed. RESULTS: Twenty-eight patients were included in this study. Blood cultures were negative in 100% of the patients. Amplification and sequencing of the V4 region identified bacterial genera in 19 patients across distinct groups. The predominant taxonomically identified genus was Pseudomonas. CONCLUSIONS: Amplifying the 16S rDNA variable region through PCR and subsequent Sanger sequencing in preterm neonates with suspected early-onset neonatal sepsis can enhance the identification of microbial species that cause infection, especially in negative cultures.

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

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.

Comparison of four different human papillomavirus genotyping methods in cervical samples: Addressing method-specific advantages and limitations.

Since human papillomavirus (HPV) is recognized as the causative agent of cervical cancer and associated with anogenital non-cervical and oropharyngeal cancers, the characterization of the HPV types circulating in different geographic regions is an important tool in screening and prevention. In this context, this study compared four methodologies for HPV detection and genotyping: real-time PCR (Cobas® HPV test), nested PCR followed by conventional Sanger sequencing, reverse hybridization (High + Low PapillomaStrip® kit) and next-generation sequencing (NGS) at an Illumina HiSeq2500 platform. Cervical samples from patients followed at the Family Health Strategy from Juiz de Fora, Minas Gerais, Brazil, were collected and subjected to the real-time PCR. Of those, 114 were included in this study according to the results obtained with the real-time PCR, considered herein as the gold standard method. For the 110 samples tested by at least one methodology in addition to real-time PCR, NGS showed the lowest concordance rates of HPV and high-risk HPV identification compared to the other three methods (67-75 %). Real-time PCR and Sanger sequencing showed the highest rates of concordance (97-100 %). All methods differed in their sensitivity and specificity. HPV genotyping contributes to individual risk stratification, therapeutic decisions, epidemiological studies and vaccine development, supporting approaches in prevention, healthcare and management of HPV infection.

Detection, Characterization and Sequencing of BTV Serotypes Circulating in Cuba in 2022.

In Cuba, despite a high sero-prevalence of bluetongue virus (BTV), circulating serotypes remain unknown. The aim of this study was to identify circulating BTV serotypes in farms throughout the western region of Cuba. Blood samples were collected from 200 young cattle and sheep between May and July 2022 for virological analyses (PCR, viral isolation and virus neutralization) and genome sequencing. The results confirmed viral circulation, with viro-prevalence of 25% for BTV. The virus was isolated from 18 blood samples and twelve BTV serotypes were identified by sequencing RT-PCR products targeting the segment 2 of the BTV genome (BTV-1, 2, 3, 6, 10, 12, 13, 17, 18, 19, 22 and 24). Finally, the full genome sequences of 17 Cuban BTV isolates were recovered using a Sequence Independent Single Primer Amplification (SISPA) approach combined to MinION Oxford Nanopore sequencing technology. All together, these results highlight the co-circulation of a wide diversity of BTV serotypes in a quite restricted area and emphasize the need for entomological and livestock surveillance, particularly in light of recent changes in the global distribution and nature of BTV infections.

Molecular landscape of the JAK2 gene in chronic myeloproliferative neoplasm patients from the state of Amazonas, Brazil.

JAK2V617F (dbSNP: rs77375493) is the most frequent and most-studied variant in BCR::ABL1 negative myeloproliferative neoplasms and in the JAK2 gene. The present study aimed to molecularly characterize variants in the complete coding region of the JAK2 gene in patients with BCR::ABL1 negative chronic myeloproliferative neoplasms. The study included 97 patients with BCR::ABL1 negative myeloproliferative neoplasms, including polycythemia vera (n=38), essential thrombocythemia (n=55), and myelofibrosis (n=04). Molecular evaluation was performed using conventional PCR and Sanger sequencing to detect variants in the complete coding region of the JAK2 gene. The presence of missense variants in the JAK2 gene including rs907414891, rs2230723, rs77375493 (JAK2V617F), and rs41316003 were identified. The coexistence of variants was detected in polycythemia vera and essential thrombocythemia. Thus, individuals with high JAK2V617F variant allele frequency (≥50% VAF) presented more thrombo-hemorrhagic events and manifestations of splenomegaly compared with those with low JAK2V617F variant allele frequency (<50% VAF). In conclusion, individuals with BCR::ABL1 negative neoplasms can display >1 variant in the JAK2 gene, especially rs2230722, rs2230724, and rs77375493 variants, and those with high JAK2V617F VAF show alterations in the clinical-laboratory profile compared with those with low JAK2V617F VAF.

A tool for the cheap and rapid screening of SARS-CoV-2 variants of concern (VoCs) by Sanger sequencing.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an emerging virus that, since March 2020, has been responsible for a global and ongoing pandemic. Its rapid spread over the past nearly 3 years has caused novel variants to arise. To monitor the circulation and emergence of SARS-CoV-2 variants, surveillance systems based on nucleotide mutations are required. In this regard, we searched in the spike, ORF8, and nucleocapsid genes to detect variable sites among SARS-CoV-2 variants. We describe polymorphic genetic regions that enable us to differentiate between the Alpha, Beta, Gamma, Delta, and Omicron variants of concern (VoCs). We found 21 relevant mutations, 13 of which are unique for Omicron lineages BA.1/BA.1.1, BA.2, BA.3, BA.4, and BA.5. This genetic profile enables the discrimination between VoCs using only four reverse transcription PCR fragments and Sanger sequencing, offering a cheaper and faster alternative to whole-genome sequencing for SARS-CoV-2 surveillance. IMPORTANCE Our work describes a new (Sanger sequencing-based) screening methodology for SARS-CoV-2, performing PCR amplifications of a few target regions to detect diagnostic mutations between virus variants. Using the methodology developed in this work, we were able to discriminate between the following VoCs: Alpha, Beta, Gamma, Delta, and Omicron (BA.1/BA.1.1, BA.2, BA.3, BA.4, and BA.5). This becomes important, especially in low-income countries where current methodologies like next-generation sequencing have prohibitive costs. Furthermore, rapid detection would allow sanitary authorities to take rapid measures to limit the spread of the virus and therefore reduce the probability of new virus dispersion. With this methodological approach, 13 previously unreported diagnostic mutations among several Omicron lineages were found.

The first report of Dirofilaria repens infection in dogs from Colombia.

Dirofilariasis is a mosquito-borne disease caused by Dirofilaria parasites, affecting both wild and domestic animals, including humans considered as accidental hosts. Dirofilaria repens is the principal causative agent of dirofilariasis in the Old World, with increasing reports of the parasite in countries where it has not been previously identified, due to several factors such as the expansion of mosquito vectors' geographical distribution. By utilizing newly designed primers for molecular detection and confirming through next-generation sequencing, here, we report the first plausible cases of D. repens in dogs from Colombia. Our results support the classification of this species as an emergent pathogen in the Americas. Finally, we encourage an increase in diagnostic and surveillance efforts to prevent and control the current and future dirofilariasis cases in this region.

Comparative analysis of onychomycosis in Puerto Rico using molecular and conventional approaches.

Onychomycosis is the most prevalent nail ailment in adults, accounting for 50% of all nail infections. Dermatophyte fungi are the primary cause, but non-dermatophyte molds (NDM) and yeasts can also cause onychomycosis. It remains important to precisely determine the fungal cause of onychomycosis since the response to current treatments may vary between fungal classes. Real-time polymerase chain reaction (qPCR) has become a widespread tool for detecting fungal organisms for diagnosis due to its sensitivity and ability to detect down to the species level. This retrospective study aims to evaluate the qPCR Onycho+ test for dermatophyte detection using remnants of toenails from a cohort of patients from Puerto Rico.  Two hundred forty-two toenail samples submitted for histological examination via Periodic acid Schiff (PAS) staining for suspected onychomycosis were analyzed by the Onycho+ test and Sanger sequencing of the internal transcribed spacer (ITS-2). Compared to the gold standard Sanger sequencing method, the Onycho+ test reported an agreement of 91.39%, a sensitivity of 100% and a specificity of 84.5% in detecting dermatophytes, superior to the histology method which had a 69.53% agreement, 85.1% sensitivity and 57.1% specificity. The distribution of fungal organisms detected in this cohort shows a dermatophyte majority but a higher-than-expected proportion of NDMs. Nails negative for the Onycho+ test and positive for histology were mostly NDMs. This study demonstrates that the clinical performance of the Onycho+ test is superior to histology in detecting dermatophytes and that a combination of Onycho+ and histology can result in a higher clinical accuracy.

Validation of a new strategy for the identification of SARS-CoV-2 variants by sequencing the spike gene by Sanger.

INTRODUCTION: The emergence of multiple variants of SARS-CoV-2 during the COVID-19 pandemic is of great world concern. Until now, their analysis has mainly focused on next-generation sequencing. However, this technique is expensive and requires sophisticated equipment, long processing times, and highly qualified technical personnel with experience in bioinformatics. To contribute to the analysis of variants of interest and variants of concern, increase the diagnostic capacity, and process samples to carry out genomic surveillance, we propose a quick and easy methodology to apply, based on Sanger sequencing of 3 gene fragments that code for protein spike. METHODS: Fifteen positive samples for SARS-CoV-2 with a cycle threshold below 25 were sequenced by Sanger and next-generation sequencing methodologies. The data obtained were analyzed on the Nextstrain and PANGO Lineages platforms. RESULTS: Both methodologies allowed the identification of the variants of interest reported by the WHO. Two samples were identified as Alpha, 3 Gamma, one Delta, 3 Mu, one Omicron, and 5 strains were close to the initial Wuhan-Hu-1 virus isolate. According to in silico analysis, key mutations can also be detected to identify and classify other variants not evaluated in the study. CONCLUSION: The different SARS-CoV-2 lineages of interest and concern are classified quickly, agilely, and reliably with the Sanger sequencing methodology.

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

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.

[Validation of a new strategy for the identification of SARS-CoV-2 variants by sequencing the spike gene by Sanger]. / Validación de una nueva estrategia para la identificación de variantes de SARS-CoV-2 mediante secuenciación del gen espiga por Sanger.

Introduction: The emergence of multiple variants of SARS-CoV-2 during the COVID-19 pandemic is of great world concern. Until now, their analysis has mainly focused on next-generation sequencing. However, this technique is expensive and requires sophisticated equipment, long processing times, and highly qualified technical personnel with experience in bioinformatics. To contribute to the analysis of variants of interest and variants of concern, increase the diagnostic capacity, and process samples to carry out genomic surveillance, we propose a quick and easy methodology to apply, based on Sanger sequencing of 3 gene fragments that code for protein spike. Methods: Fifteen positive samples for SARS-CoV-2 with a cycle threshold below 25 were sequenced by Sanger and next-generation sequencing methodologies. The data obtained were analyzed on the Nextstrain and PANGO Lineages platforms. Results: Both methodologies allowed the identification of the variants of interest reported by the WHO. Two samples were identified as Alpha, 3 Gamma, one Delta, 3 Mu, one Omicron, and 5 strains were close to the initial Wuhan-Hu-1 virus isolate. According to in silico analysis, key mutations can also be detected to identify and classify other variants not evaluated in the study. Conclusion: The different SARS-CoV-2 lineages of interest and concern are classified quickly, agilely, and reliably with the Sanger sequencing methodology.

An overview of the trypanosomatid (Kinetoplastida: Trypanosomatidae) parasites infecting several mammal species in Colombia.

BACKGROUND: Trypanosomatids are among the most critical parasites for public health due to their impact on human, animal, and plant health. Diseases associated with these pathogens manifest mainly in poor and vulnerable populations, where social, environmental, and biological factors modulate the case incidence and geographical distribution. METHODS: We used Sanger and amplicon-based next-generation sequencing (NGS) in samples from different mammals to identify trypanosomatid infections in several departments in Colombia. A total of 174 DNA samples (18 humans, 83 dogs, and 73 wild mammals) were analyzed by conventional PCR using a fragment of the heat shock protein 70 (Hsp70) gene and Sanger sequenced the positive samples. Twenty-seven samples were sent for amplicon-based NGS using the same gene fragment. Data obtained were used to perform diversity analyses. RESULTS: One hundred and thirteen samples were positive for PCR by Hsp70 fragment; these corresponded to 22.1% Leishmania spp., 18.6% L. amazonensis, 9.7% L. braziliensis, 14.2% L. infantum, 8% L. panamensis, and 27.4% Trypanosoma cruzi. Comparison of the identified species by the two sequencing technologies used resulted in 97% concordance. Alpha and beta diversity indices were significant, mainly for dogs; there was an interesting index of coinfection events in the analyzed samples: different Leishmania species and the simultaneous presence of T. cruzi and even T. rangeli in one of the samples analyzed. Moreover, a low presence of L. braziliensis was observed in samples from wild mammals. Interestingly, to our knowledge, this is the first report of Leishmania detection in Hydrochaeris hydrochaeris (capybara) in Colombia. CONCLUSIONS: The Hsp70 fragment used in this study is an optimal molecular marker for trypanosomatid identification in many hosts and allows the identification of different species in the same sample when amplicon-based sequencing is used. However, the use of this fragment for molecular diagnosis through conventional PCR should be carefully interpreted because of this same capacity to identify several parasites. This point is of pivotal importance in highly endemic countries across South America because of the co-circulation of different genera from the Trypanosomatidae family. The findings show an interesting starting point for One Health approaches in which coevolution and vector-host interactions can be studied.

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

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.

SH2B1 variants as potential causes of non-syndromic monogenic obesity in a Brazilian cohort.

PURPOSE: SH2B1 gene encodes an important adaptor protein to receptor tyrosine kinases or cytokine receptors associated with Janus kinases. This gene has been associated with the structural and functional modulation of neurons and other cells, and impacts on energy and glucose homeostasis. Several studies suggested that alterations in this gene are strong candidates for the development of obesity. However, only a few studies have screened SH2B1 point variants in individuals with obesity. Therefore, the aim of this study was to investigate the prevalence of SH2B1 variants in a Brazilian cohort of patients with severe obesity and candidates to bariatric surgery. METHODS: The cohort comprised 122 individuals with severe obesity, who developed this phenotype during childhood. As controls, 100 normal-weight individuals were included. The coding region of SH2B1 gene was screened by Sanger sequencing. RESULTS: A total of eight variants were identified in SH2B1, of which p.(Val345Met) and p.(Arg630Gln) variants were rare and predicted as potentially pathogenic by the in the silico algorithms used in this study. The p.(Val345Met) was not found in either the control group or in publicly available databases. This variant was identified in a female patient with severe obesity, metabolic syndrome and hyperglycemia. The p.(Arg630Gln) was also absent in our control group, but it was reported in gnomAD with an extremely low frequency. This variant was observed in a female patient with morbid obesity, metabolic syndrome, hypertension and severe binge-eating disorder. CONCLUSION: Our study reported for the first time two rare and potentially pathogenic variants in Brazilian patients with severe obesity. Further functional studies will be necessary to confirm and elucidate the impact of these variants on SH2B1 protein function and stability, and their impact on energetic metabolism. LEVEL OF EVIDENCE: Level V, cross-sectional descriptive study.

Characterization of the Complete Mitochondrial Genome of the Bromeliad Crab Metopaulias depressus (Rathbun, 1896) (Crustacea: Decapoda: Brachyura: Sesarmidae).

Metopaulias depressus is a non-marine crab endemic to Jamaica that dwells in rainforest bromeliads and exhibits elaborate active parental care behavior. Current genomic resources on M. depressus are rare, limiting the understanding of its adaptation to terrestrial life in species that evolved from marine ancestors. This study reports the complete mitochondrial genome of M. depressus assembled using Sanger sequencing. The AT-rich mitochondrial genome of M. depressus is 15,765 bp in length and comprises 13 protein-coding genes (PCGs), 2 ribosomal RNA genes, and 22 transfer RNA genes. A single 691 bp-long intergenic space is assumed to be the control region (CR) or D-loop. A set of selective pressure analyses indicate that the entirety of the PCGs experience purifying selection. Cox1, cox2, nad5, cox3, and atp6 experience strong purifying selection, and atp8 experiences weak purifying selection compared to the rest of the PCGs. The secondary structures of most tRNA genes exhibit a standard 'cloverleaf' structure, with the exception of trnS1, which lacks the dihydroxyuridine (DHU) arm but not the loop, the trnH gene, which lacks the thymine pseudouracil cytosine (T) loop but not the arm, and trnM, which exhibits an overly developed T loop. A maximum likelihood phylogenetic analysis based on all PCGs indicated that M. depressus is more closely related to the genera Clistocoeloma, Nanosesarma, and Parasesarma than to Chiromantes, Geosesarma, and Orisarma. This study contributes to deciphering the phylogenetic relationships within the family Sesarmidae and represents a new genomic resource for this iconic crab species.

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

Genetic variants of SARS-CoV-2 have been emerging and circulating in many places across the world. Rapid detection of these variants is essential since their dissemination can impact transmission rates, diagnostic procedures, disease severity, response to vaccines or patient management. Sanger sequencing has been used as the preferred approach for variant detection among circulating human immunodeficiency and measles virus genotypes. Using primers to amplify a fragment of the SARS-CoV-2 genome encoding part of the Spike protein, we showed that Sanger sequencing allowed us to rapidly detect the introduction and spread of three distinct SARS-CoV-2 variants in two major Brazilian cities. In both cities, after the predominance of variants closely related to the virus first identified in China, the emergence of the P.2 variant was quickly followed by the detection of the P1 variant, which became dominant in less than one month after it was first detected.

Genetic diversity across the mitochondrial genome of eastern oysters (Crassostrea virginica) in the northern Gulf of Mexico.

The eastern oyster, Crassostrea virginica, is divided into four populations along the western North Atlantic, however, the only published mitochondrial genome sequence was assembled using one individual in Delaware. This study aimed to (1) assemble C. virginica mitochondrial genomes from Texas with pooled restriction-site-associated DNA sequencing (ezRAD), (2) evaluate the validity of the mitochondrial genome assemblies including comparison with Sanger sequencing data, and (3) evaluate genetic differentiation both between the Delaware and Texas genomes, as well as among three bays in Texas. The pooled-genome-assembled-genomes (PAGs) from Texas exhibited several characteristics indicating that they were valid, including elevated nucleotide diversity in non-coding and the third position of codons, placement as the sister haplotype of the genome from Delaware in a phylogenetic reconstruction of Crassostrea mitochondrial genomes, and a lack of genetic structure in the ND4 gene among the three Texas bays as was found with Sanger amplicons in samples from the same bays several years prior. In the comparison between the Delaware and Texas genome, 27 of 38 coding regions exhibited variability between the two populations, which were differentiated by 273 mutations, versus 1-13 mutations among the Texas samples. Using the full PAGs, there was no additional evidence for population structure among the three Texas bays. While population genetics is rapidly moving towards larger high-density datasets, studies of mitochondrial DNA (and genomes) can be particularly useful for comparing historic data prior to the modern era of genomics. As such, being able to reliably compile mitochondrial genomes from genomic data can improve the ability to compare results across studies.

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

The 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 has 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 SARS-CoV-2 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). Twelve COVID-19 samples from Brazilian patients were evaluated for both WGS and Sanger sequencing: three P.2, two P.1, six B.1.1 and one B.1.1.117 lineage. All results from the Sanger sequencing method perfectly matched the mutational profile of VOCs and non-VOCs RBD's characterized by WGS. In summary, this approach allows a much broader network of laboratories to perform molecular surveillance of SARS-CoV-2 VOCs and report results within a shorter time frame, which is of utmost importance in the context of rapid public health decisions in a fast evolving worldwide pandemic.