Dehydroepiandrosterone (DHEA) is considered an endogenous steroid hormone precursor, and 17-ß Estradiol (E2) is one of the estrogen steroid hormones. Of the thirteen known human cytosolic sulfotransferases (SULTs), SULT2B1a has been shown to be expressed in steroid hormone-responsive tissues such as the prostate, ovary, and placenta, as well as the fetal brain. Previous studies have demonstrated that SULT2B1a is capable of sulfating 3β-hydroxysteroids such as DHEA and pregnenolone. The present study aimed to investigate the effects of human SULT2B1 SNPs on the enzymatic characteristics of SULT2B1a allozymes in mediating the sulfation of DHEA and E2. To inspect the effects of single nucleotide polymorphisms of the SULT2B1 gene on the sulfation of DHEA and E2 by SULT2B1a allozymes, 13 recombinant SULT2B1a allozymes were produced, expressed, and purified using established procedures. 13 SULT 2B1a nonsynonymous missense coding SNPs (cSNPs) were selected among numerous identified human SULT 2B1a SNPs by a comprehensive database search. The corresponding cDNAs, packaged in pGEX-2TK expression vector, and encoding the selected 13 SULT2B1a allozymes, have been generated by performing site-directed mutagenesis. These were then bacterially expressed in BL21 E. coli cells and purified using glutathione-Sepharose affinity chromatography. The purified allozymes were tested for their ability to sulfonate DHEA and E2. In terms of the kinetic parameters, the wild-type SULT2B1a exhibited higher enzyme affinity towards DHEA than with E2. In comparison with the wild-type SULT2B1a, the purified allozymes displayed differential sulfating activities towards DHEA and E2. Accordingly, these findings indicate an apparent effect of SULT2B1 cSNPs on the sulfating activities of SULT2B1a allozymes toward DHEA and E2, and may provide for a better understanding of the pharmacokinetics of DHEA and E2 in individuals with differing SULT2B1a genotypes.

Background/Objectives: Lung cancer remains a global health concern, with substantial variation in patient survival. Despite advances in detection and treatment, the genetic basis for the divergent outcomes is not understood. We investigated germline polymorphisms that modulate overall survival in 1,464 surgically resected lung adenocarcinoma patients. Methods: A multivariable Cox proportional hazard model was used to assess the association of more than 7 million polymorphisms with overall survival at the 60-month follow-up, considering age, sex, pathological stage, decade of surgery and principal components as covariates. Genes in which variants were identified were studied in silico to investigate functional roles. Results: Six germline variants passed the genome-wide significance threshold. These single nucleotide polymorphisms mapped to non-coding (intronic) regions on chromosomes 2, 3, and 5. The minor alleles of rs13000315, rs151212827, and rs190923216 (chr. 2, 3 and 5, respectively) were found to be independent negative prognostic factors. All six variants have been reported to regulate the expression of nine genes, seven of which are protein-coding, in different tissues. Survival-associated variants on chromosomes 2 and 3 were already reported to regulate the expression of NT5DC2 and NAGK, with high expression associated with the minor alleles. High NT5DC2 and NAGK expression in lung adenocarcinoma tissue were already shown to correlate with poor overall survival. Conclusions: This study highlights a potential regulatory role of the identified polymorphisms in influencing outcome and suggests a mechanistic link between these variants, gene expression regulation, and lung adenocarcinoma prognosis. Validation and functional studies are warranted to elucidate the mechanisms underlying these associations.

Anti-Müllerian hormone (AMH) has been proposed as biomarker for fertility in cattle, yet this associative relationship appears to be influenced by heat stress. Objective was to test serum AMH and AMH-related single nucleotide polymorphisms (SNPs) as markers potentially predictive of reproductive traits in heat-stressed dairy cows. The study included 300 Holstein cows that were genotyped using BovineSNP50 (54,000 SNP) after blood samples were collected and DNA extracted. A genome wide association study was then executed. Nine intragenic SNPs within the pathways that influence the AMH gene were found important with multiple comparisons adjustment test (p<1.09x10-6). A further validation study was performed in an independent Holstein cattle population, which was divided into moderate (MH; n=152) and severe heat-stressed (SH; n=128) groups, and subjected to a summer reproductive management program. Serum AMH was confirmed as a predictor of fertility measures (p<0.05) in MH but not in SH group. Cows were genotyped using the TaqMan allelic discrimination method and qPCR, which revealed four SNPs as predictive markers for serum AMH (p<0.01), reproductive traits (p<0.01) and physiological variables (p<0.05). These SNPs were in the genes AMH, IGFBP1, LGR5 and TLR4. In conclusion, both serum AMH concentrations and AMH-related SNPs appear to be associated with fertility traits in dairy cows managed during summer.

Vitamin D receptor (VDR) is an important candidate gene in musculoskeletal phenotypes. Polymorphisms in VDR have been previously associated with several pathologies and muscular strength in athletes and elderly people, however literature reported contradictory results. The object of this research is to verify the association between the most studied VDR variants (rs2228570, rs7975232, and rs1544410) and the increase of muscle mass in elite young soccer players. A sample of 55 soccer players (15-18 years old) form a professional team was selected for this study. DNA was extracted by salting out method and polymorphisms were genotyped by PCR-RFLP, followed by 2% agarose gel electrophoresis. To test the effect of the three SNPs, logistic regression analysis was applied. Body composition was carried out through skinfold thickness method, and muscular area of arm and lower limb were calculated using Frisancho formulas. All three polymorphisms met Hardy-Weinberg equilibrium (p > 0.05) and their frequencies fell within the worldwide variability. A significant correlation between rs1544410 and increase of calf muscle mass was observed. Individuals carrying A allele showed higher calf muscular mass than those carrying the G allele (p = 0.034). Moreover, haplotype analysis applied to the two SNPs in linkage disequilibrium (rs7975232 and rs1544410), showed that AG haplotype resulted negatively correlated to calf muscle area. In conclusion, we confirm an association between VDR polymorphisms and muscular mass that could encourage the genetic screening of VDR gene to identify a potential risk of injury and for individual nutritional interventions.

Single Nucleotide Polymorphisms (SNPs) are the most common type of genetic variation found in individual’s DNA sequences. SNPs can occur in both coding and non-coding regions of the genome and can affect gene expression, protein function, and disease susceptibility. In this systematic review we evaluate the influence of SNPs as a biomarker in the assessment of oral mucositis (OM) in head and neck cancer (HNC) patients. The study selection process involved screening 66 articles from different platforms, and after removing duplicates and excluding irrelevant articles, 23 articles were included for full-text evaluation. The main characteristics and findings of the selected articles are presented and discussed. We conclude that SNPs can be used as a reliable biomarker for the assessment of OM in HNC patients, and further research is needed to explore the potential of SNPs in personalized medicine for HNC treatment.

Rapeseed (Brassica napus L.) is one of the most oil crop, and its commercial value is contingent upon its agronomic characteristics and oil quality. In this study, 73,226 single nucleotide polymorphisms (SNPs) across 95 rapeseed mutant lines derived from gamma rays and their original cultivar (‘Tamra’) obtained from genotyping-by-sequencing (GBS) was investigated gene ontology (GO) analysis and genome-wide association study (GWAS). GWAS was conducted on agronomic (plant height, ear length, thousand seed weight and seed yield) and oil (fatty acid and crude fat) traits. GO analysis showed that many genes displaying SNPs were involved in cellular processes, intracellular anatomical structures and organic cyclic compound binding. A total of 149 significant SNPs were associated with the agronomic traits (76 SNPs) and oil traits (73 SNPs). Bna.A05p02350D (SFGH) and Bna.C02p22490D (MDN1) were selected as novel candid genes for thousand seed weight. In addition, Bna.C03p14350D (EXO70) and Bna.A09p05630D (PI4Kα1) were selected as novel candidate genes for the erucic acid and crude fat content, respectively. These findings could facilitate the identification of optimal genotypes for breeding new cultivars, and association studies represent an economically efficient tool for mutant screening and the selection of elite rapeseed-breeding lines.

Mycobacterium tuberculosis variant bovis (MBO) has one of the widest known mammalian host ranges, including humans. Despite characterization of this pathogen in the 1800s, and whole genome sequencing of a UK strain (AF2122) nearly two decades ago, the basis of its host specificity and pathogenicity remains poorly understood. Recent experimental calf infection studies show that MBO strain Ravenel (MBO Ravenel) is attenuated in the cattle host. In the present study, experimental infections were performed to define attenuation and whole genome sequencing completed to identify regions of differences (RD) and single nucleotide polymorphisms (SNPs) to explain the observed attenuation. Comparative genomic analysis of MBO Ravenel against three pathogenic strains of MBO (strains AF2122-97, 10-7428 and 95-1315) was performed. Experimental infection studies on 5 calves each, with either MBO Ravenel or 95-1315, revealed no visible lesions in all 5 animals in the Ravenel group despite robust IFN-γ responses. Out of 486 polymorphisms in the present analysis, 173 were unique to MBO Ravenel among the strains compared. A high-confidence subset of 9 unique SNPs were missense mutations in genes with annotated functions impacting 2 major MBO survival and virulence pathways: 1) Cell wall synthesis & transport [espH (A103T), mmpL8 (V888I), aftB (H484Y), eccC5 (T507M), rpfB (E263G)], and 2) Lipid metabolism & respiration [mycP1(T125I), pks5 (G455S), fadD29 (N231S), fadE29 (V360G)]. These substitutions likely contribute to the observed attenuation. Results from experimental calf infections and the functional attributions of polymorphic loci on the genome of MBO Ravenel provide new insights into the strain’s genotype-disease phenotype associations.

Understanding the function of a locus is a challenge in molecular biology. Although numerous molecular data have been generated in the last decades, it remains difficult to grasp, how these data are related at a locus? In this study, we describe an analytical workflow that can solve this problem using the knowledge available at single-nucleotide polymorphisms (SNPs) level. The underlying algorithm uses SNPs as connectors to link omics data and identify correlation between them through a joint bioinformatical/statistical approach. We describe its application in finding the mechanism whereby a mutation causes a phenotype and in revealing the path whereby a gene is being regulated and impacts the phenotypes. We translated our workflow into freely available shell scripts that carry out the analyses. Our approach provides a basic framework to solve the information overload problem in biology.

Sesame seeds and their edible oil are highly nutritious and rich in mono- and polyunsaturated fatty acids. Bioactive compounds such as sterols, tocopherols, and sesamol provide significant medicinal benefits. The high oil content (50%) and favorable mono-and polyunsaturated fatty acids balance, as well as sesame's resilience to water stress makes, make it a promising candidate for global agricultural expansion. However, sesame production faces challenges such as low yields, poor response to agricultural inputs, and losses due to capsule dehiscence. To enhance yield, traits like determinate growth, dwarfism, high harvest index, non-shattering capsules, and photoperiod sensitivity are needed. These traits can be achieved through variation or induced mutation breeding. Traditional breeding methods often result in unwanted genetic changes. The CRISPR/Cas9 technology has the potential to suppress detrimental alleles and improve the fatty acid profile by inhibiting polyunsaturated fatty acid biosynthesis. Even though sesame is an orphan crop, it has entered the genomic era, with available sequences assisting molecular breeding efforts. This progress aids in associating single nucleotide polymorphisms (SNPs) and simple sequence repeats (SSRs) with key economic traits, as well as identifying genes related to adaptability, oil production, fatty acid synthesis, and photosynthesis. Additionally, transcriptomic studies can reveal genes involved in abiotic stress responses and adaptation to diverse climates. Quantitative trait loci (QTL) mapping can identify loci linked to crucial traits such as capsule size, seed count per capsule, and capsule number per plant. This paper reviews recent advances in sesame breeding, discusses ongoing challenges, and explores potential strategies for future improvement. Hence, integrating advanced genomic tools and breeding strategies provides promising ways to enhance sesame production to meet global demands.

Abnormalities in sperm nuclei and chromatin can interfere with normal fertilization, embryonic development, implantation, and pregnancy. We aimed in this study to determine the impact of H2BFWT gene variants in sperm DNA on ICSI outcomes in couples undergoing ART treatment. One hundred and nineteen partners were divided into pregnant (G1) and non-pregnant (G2) groups. After semen analysis, complete DNA was extracted from purified sperm samples. The sequence of the H2BFWT gene was amplified by PCR and then subjected to Sanger sequencing. The results showed that there are three mutations in this gene: rs7885967, rs553509 and rs578953. rs553509 and rs578953 showed significant differences in the distribution of alternative and reference alleles between G1 and G2 (P = 0.0004 and P = 0.0020, respectively). However, there was no association between these SNPs and the studied parameters. This study is the first to shed light on the connection between H2BFWT gene variants in sperm DNA and pregnancy after ICSI therapy. This is a pilot study, so further investigations about these gene variants at the transcriptional and translational levels, in a larger cohort of patients, are needed to determine its functional consequences and to understand the mechanism of how the pregnancy is affected by sperm DNA.

The dengue virus (DENV) is an arbovirus belonging to the Flaviviridae family; the species comprises four antigenically distinct serotypes (DENV–1, DENV–2, DENV–3, and DENV–4), which are further subdivided into genotypes. The virus is transmitted to humans through mosquito bites, primarily from the Aedes spp. genus. Dengue is endemic in various parts of the world, including tropical and subtropical regions of Asia, Latin America, Africa, and Oceania. In Brazil, the state of Tocantins, located in north-central Brazil, has experienced a significant number of arboviral disease cases, particularly dengue. This study aimed to monitor DENV circulation within the state by conducting full genome sequencing of viral genomes recovered from 61 patients between June 2021 and July 2022. During this period, both DENV-1 and DENV-2 serotypes were identified. Our findings confirm the circulation of DENV serotypes 1 and 2 in Tocantins, affecting males and females equally, with younger age groups (4 to 43 years old) being the most susceptible. Phylogenetic analysis revealed that the circulating viruses belong to DENV–1 genotype V American and DENV–2 genotype III Southeast Asian/American. The Bayesian analysis of DENV-1 Genotype V sequenced here is closely related to genomes previously sequenced in the state of São Paulo. Regarding DENV-2 genotype III genomes, these are clustered in a distinct, well-supported subclade, along with previously reported isolates from the states of Goiás and São Paulo. In both cases, our results suggest that multiple introductions of these genotypes occurred in the Tocantins state. This observation highlights the significant impact of major population centers in Brazil on virus dispersion, including other Latin-American countries and the USA. In the SNP analysis, DENV-1 displayed 122 distinct missense mutations, while DENV-2 had 44, with significant mutations predominantly occurring in the envelope and NS5 proteins. The analyses performed here reveal the concomitant circulation of distinct DENV-1 and -2 genotypes in some Brazilian states, underscoring the dynamic evolution of the DENV and the ongoing significance of surveillance efforts in supporting public health policies.

Background: The myeloid-epithelial-reproductive tyrosine kinase (MERTK) is involved in hepatic steatosis, inflammation and liver fibrosis. Here we evaluated the association between the MERTK rs4374383 single nucleotide polymorphism (SNP) and liver fibrosis progression in hepatitis C virus (HCV)-infected patients. Methods: We performed a retrospective study (repeated measures design) in 208 patients who had liver stiffness measurement (LSM), which was assessed by transient elastography No patient had cirrhosis at baseline (LSM≥12.5 kPa). Results: At baseline, 53.8% were male, the median age was 47.1 years, 13.5% reported a high intake of alcohol, 10.1% were prior injection drug users, 85.3% were infected by HCV genotype 1, and 22.6% had previously failed antiviral therapy (pegylated-interferon-alpha/ribavirin). During a median follow-up of 46.6 months, 26 patients developed cirrhosis. The rs4374383 G carriers had a higher risk of increasing LSM (adjusted arithmetic mean ratio (aAMR)=1.14; p=0.006) and a higher likelihood of having an increase in LSM greater than 5 kPa (ΔLSM≥5 kPa) [adjusted odds ratio (aOR)=2.37; p=0.029], and greater than 7 kPa (ΔLSM≥7 kPa) [aOR=3.24; p=0.032], after controlling for confounding. The SNP’s association with cirrhosis progression was close to statistical significance (aOR=2.18; p=0.070). Conclusions: MERTK rs4374383 A carriers had a lower risk of liver fibrosis progression than G carriers, supporting the hypothesis that this SNP seems to have a critical role in the pathogenesis of liver disease in HCV-infected patients.

Dipeptidyl peptidase (DPP) 9, DPP8, DPP4 and fibroblast activation protein (FAP) are the four enzymatically active members of the S9b protease family. Associations of DPP9 with human liver cancer, exonic single nucleotide polymorphisms (SNPs) in DPP9 and loss of function (LoF) variants have not been explored. Human genomic databases including The Cancer Genome Atlas (TCGA) were interrogated to identify DPP9 LoF variants and associated cancers. Survival and gene signature analyses were performed on hepatocellular carcinoma (HCC) data. We found that DPP9 and DPP8 are intolerant to LoF variants. DPP9 LoF variants were most often associated with uterine carcinoma. Two DPP9 intronic SNPs that have been associated with lung fibrosis and COVID-19 were not associated with liver fibrosis or cancer. All four DPP4-like genes were overexpressed in liver tumours and their joint high expression was associated with poor survival in HCC. Increased DPP9 expression was associated with obesity in HCC patients.. High expression of genes that positively correlated with overexpression of DPP4, DPP8, and DPP9 were associated with very poor survival in HCC. Enriched pathways analysis of these positively correlated genes featured Toll-like receptor and SUMOylation pathways. This comprehensive data mining suggests that DPP9 is essential for human survival and the DPP4 protease family is important in cancer pathogenesis.

The suitability of an animal model to study human diseases heavily relies on the similarity between the two species at the genetic, epigenetic, gene expression and metabolic levels. However, consistent data from different animal models at each level to evaluate this suitability is lacking. With the availability of genome sequences for many mammalian species it is now possible to compare animal models based on the genomic similarities. Here, we compare the coding sequences (CDS) of five mammalian models that include rhesus macaque, marmoset, pig, mouse and rat with those from human. We identified 10,316 conserved CDS across the five organisms and human based on the sequence similarity. Mapping the human disease-associated single nucleotide polymorphisms (SNPs) from these conserved CDS in each species has identified species-specific association with various human diseases. While associations for a disease such as colon cancer were prevalent in multiple model species, pig showed the highest number (117 diseases) of model-specific human disease associations. Based on the percentage of disease-associated SNP-containing genes, marmoset models are well suited to study many human ailments including behavioral and gastrointestinal diseases. Comparison of gene expression levels of the conserved CDS from colon, heart, kidney, lung, skeletal muscle, and spleen tissues in these models showed correlations with human expression levels in a tissue-specific manner. In the gastrointestinal tissues (colon, spleen), the pig showed the highest correlation while mouse displayed a better correlation in the heart and kidney. This study demonstrated genomic as well as tissue-specific expression-based similarity evaluation of five animal models against human that could help investigators select a suitable animal model to study their targeted disease.

The generation of forensic DNA profiles consisting of single nucleotide polymorphisms (SNPs) is now being facilitated by wider adoption of next-generation sequencing (NGS) methods in casework laboratories. At the same time, and in part because of this advance, there is an intense focus on the generation of SNP profiles from evidentiary specimens for so-called forensic or investigative genetic genealogy (FGG or IGG) applications. However, FGG methods are constrained by the algorithms for genealogical database searches, which were designed for use with single-source profiles, and the fact that many forensic samples are mixtures. To enable the use of two-person mixtures for FGG, we developed a workflow, MixDeR, for the deconvolution of mixed SNP profiles. MixDeR, a flexible and easy to use R package and Shiny app, processes ForenSeq Kintelligence® (QIAGEN, Inc.) SNP genotyping results and directs deconvolution of the profiles in EuroForMix (EFM). MixDeR then filters the EFM outputs to produce inferred single-source genotypes in reports formatted for use with GEDmatch® PRO. An optional MixDeR output includes metrics that assist with testing and validation of the workflow. As the Shiny app provides a graphical user interface and the software is designed to be run offline, MixDeR should be suitable for use by any laboratory developing FGG capabilities, no matter their bioinformatic resources or expertise.

Temperature is one of the most important range-limiting factors for many seaweeds. Driven by the recent climatic changes, rapid northward shifts of species’ distribution ranges can potentially modify the phylogeographic signature of Last Glacial Maximum such as increased genetic diversity at lower latitudes. To explore this question in detail in the kelp Saccharina latissima, we used microsatellites and double digest RAD-sequencing derived SNPs on S. latissima sporophytes sampled within 11 sites spanning the entire European Atlantic latitudinal range of this species. In addition, we checked for statistical correlation between genetic marker allele frequencies and three environmental proxies (sea surface temperature, salinity, and water turbidity). Our findings revealed that genetic diversity was significantly higher for the northern localities compared to the southern ones in contrast to the expected phylogeographic pattern. This suggests that the southernmost S. latissima populations are negatively affected by the recent climatic changes but also that the recolonization of S. latissima range following the LGM may have occurred from northerly refugia. Seven SNPs and 12 microsatellite alleles were found to be significantly associated with at least one of the three environmental variables. We discuss the potential adaptive functions of the genes associated with the outlier markers and the importance of these markers for successful conservation and aquaculture strategies for S. latissima in this age of rapid global change.

Chicory, a horticultural crop cultivated worldwide, presents many botanical varieties and local biotypes. Among these, Italian radicchio group cultivars of pure Cichorium intybus L. and interspecific hybrids with Cichorium endivia L., as the “Red of Chioggia” biotype which in turn includes several phenotypes. This study deals with a pipeline for the marker-assisted breeding of F1 hybrids: it presents the genotyping-by-sequencing results of four elite inbred lines using a RADseq approach and an original molecular assay based on CAPS markers for screening mutants with nuclear male-sterility in radicchio of Chioggia. Two thousand nine hundred fifty-three SNP-carrying RADtags were identified and used to compute the actual estimates of homozygosity, and overall genetic similarity and uniformity of the populations, as well as to determine their genetic distinctiveness and differentiation. Molecular data were further used to investigate the genomic distribution of the RADtags among the two Cichorium species, allowing their mapping in 1,131 and 1,071 coding sequences in chicory and endive, respectively. In parallel, an assay to screen the genotype at the male-sterility locus Cims-1 was developed to discriminate wild-type and mutant alleles of the causative gene myb80-like. Moreover, a RADtag mapped close to this genomic region proved the potential application of this method for future marker-assisted selection tools. Finally, combining the genotype information of the core collection, the best ten individuals from each inbred line were selected to compute the observed genetic similarity as a measure of uniformity as well as the expected homozygosity and heterozygosity estimates scorable by the putative progenies derived from selfing (pollen parent) and full-sibling (seed parent) or pair-wise crossing (F1 hybrids). This predictive approach was conducted as a pilot study for understanding the potential application of RADseq in improving molecular breeding strategies aimed at the development of inbred lines and F1 hybrids in leaf chicory.

Wheat dwarf virus (WDV) causes an important vector transmitted virus disease, which leads to significant yield losses in barley production. Due to the fact, that at the moment no plant protection products are approved to combat the vector Psammotettix alienus and this disease cannot be controlled by chemical means, therefore the use of WDV resistant or tolerant genotypes is the most efficient method to control and reduce negative effects of WDV on barley growth and production. In this study, a set of 480 barley genotypes were screened to identify genotypic differences in response to WDV and five traits were assessed under infected and non-infected conditions. In total, 32 genotypes showed resistance or tolerance against WDV. Subsequently, the phenotypic data of 191 out of 480 genotypes combined with 34,408 single nucleotide polymorphisms (SNPs) in a genome wide association study to identify quantitative trait loci (QTLs) and markers linked to resistance/tolerance to WDV, using Tassel, GAPIT, and FARM CPU. In total, 1, 3, 2, 2 and 1 significantly associated markers on chromosomes 3H, 4H, 5H and 7H identified by all three methods for ELISA-60, relative performance of total grain weight, plant height, number of ears per plant and thousand grain weight, respectively.

Large population passages of the SARS-CoV-2 in the past two and a half years have allowed the circulating virus to accumulate an increasing number of mutations in its genome. The most recently emerging Omicron subvariants have the highest number of mutations in the Spike (S) protein gene and these mutations mainly occur in the receptor-binding domain (RBD) and the N-terminal domain (NTD) of the S gene. The eCDC and the WHO recommend partial Sanger sequencing of the SARS-CoV-2 S gene RBD and NTD on the PCR-positive samples in diagnostic laboratories as a practical means of determining the variants of concern to monitor a possible increased transmissibility, increased virulence, or reduced effectiveness of vaccines against them. The author’s diagnostic laboratory has implemented the eCDC/WHO recommendation by sequencing a 398-base segment of the N gene for the definitive detection of SARS-CoV-2 in clinical samples, and sequencing a 445-base segment of the RBD and a 490-509-base segment of the NTD for variant determination. This paper presents 5 selective cases to illustrate the challenges of using Sanger sequencing to diagnose Omicron subvariant when the samples harbor a high level of co-existing minor subvariant sequences with multi-allelic single nucleotide polymorphisms (SNPs) or possible recombinant Omicron subvariants containing a BA.1 NTD and a BA.2 RBD, which can only be detected by using specially designed PCR primers. The current large-scale surveillance programs using next-generation sequencing (NGS) do not face similar problems because NGS focuses on deriving consensus sequence.

Both SARS-CoV-2 and SARS-CoV initially appeared in China and spread to other parts of the world. SARS-CoV-2 has generated a COVID-19 pandemic causing more than 6 million human deaths worldwide while the SARS outbreak quickly ended in six months with a global total of 774 reported deaths. One of the factors contributing to this stunning difference in the outcome between these two outbreaks is the inaccuracy of the RT-qPCR tests for SARS-CoV-2, which generated a large number of false-negative and false-positive test results that have misled patient management and public health policy-makers. This article presented Sanger sequencing evidence to show that the RT-PCR diagnostic protocol established in 2003 for SARS-CoV can in fact detect SARS-CoV-2 accurately due to the well-known nonspecific PCR amplification of DNAs with similar sequences. Using nested RT-PCR followed by Sanger sequencing to retest 50 patient samples collected in January, 2022 and sold as RT-qPCR positive reference confirmed 21 (42%) were false-positive. Although the other 29 positive isolates were categorized as Omicron variant by partial sequencing of the N gene, and the RBD and the NTD of the S gene, 9 (31%) showed focal to complete sequencing failure in the S gene segments due to multi-allelic SNPs. During the course of the study, an Omicron variant isolate containing a BA.1 NTD and a BA.2 RBD in its S gene was also detected. Routine partial S gene sequencing of all PCR-positive samples can timely discover multi-allelic SNPs and viral recombination in the circulating variants for investigation of their impacts on vaccine efficacies, therapeutics and diagnostics.