Data on the KIT mutation rate in skin melanoma in the central European region is missing. Accordingly, in a cohort of 79 double wild type (BRAF/NRAS) skin melanoma KIT mutation was assessed by Sanger sequencing of exons 9,11,13,17 and 18. In this skin melanoma cohort KIT mutation frequency was found to be 34/79 (43%) with a significantly higher rate in acrolentiginous melanoma (ALM) as compared to UV-induced common variants (20/34, 58.8% versus 14/45, 31.1%, p=0.014). Exon 11 was the most frequent mutation site (44.7%) followed by exon 9 (21.1%) equally characterizing UV-induced common histotypes and ALM tumors. KIT mutation hotspots were identified in exon 9 (c491/492), in exon 11 (c559,c572, c570), in exon 13 (c642), in exon 17 (c822) and in exon 18 (c853). The relatively high KIT mutation rate in skin melanoma in this central-European cohort justifies regular testing of this molecular target.

PIK3CA is a gene usually mutated in breast cancer and has an important role in tumor progression and treatment. Therefore, there is required a technique to detect low-rate PIK3CA mutations improving the clinical conduct. This study aimed to compare chip-based dPCR and Sanger sequencing to detect PIK3CA mutations in breast cancer patients. Fifty-seven tumor samples from breast cancer patients were collected and analyzed by Sanger sequencing and dPCR for PIK3CA mutations (E545K, H1047R, and H1047L). Digital PCR sensitivity, specificity, and overall performance were estimated by contingency tables, receptor operator characteristic (ROC), and area under the curve (AUC). Sanger sequencing identified PIK3CA mutations in six patients (10.5%), two with H1047R, and four with E545K. Digital PCR confirmed those mutations and identified 19 additional patients with at least one mutation. Comparison between dPCR and Sanger sequencing showed a sensitivity of 100% (95% CI 53-100%), and a specificity of 84.2% (95% CI 83 - 84.2%). Besides, H1047R mutation showed a significant association with breast cancer phenotype (p =0.019) and lymphatic node infiltration (p =0.046). Digital PCR showed a high sensitivity to detect mutations in tumor samples and it might be capable to detect low-rate mutations and tumor subpopulations not detected by Sanger sequencing.

The world has now entered into a replacement era of genomics due to the continued advancements within the next generation high throughput sequencing technologies, which incorporates sequencing by synthesis-fluorescent in place sequencing (FISSEQ), pyrosequencing, sequencing by ligation using polony amplification, supported oligonucleotide detection (SOLiD), sequencing by hybridization alongside sequencing by ligation, and nanopore technology. Great impacts of those methods are often seen for solving the genome related problems of plant and Animalia which will open the door of a replacement era of genomics. This might ultimately overcome the Sanger sequencing that ruled for 30 years. NGS is predicted to advance and make the drug discovery process more rapid.

DNA sequencing methods were first developed more than 20 years ago with the publication of two approaches to sequencing methodology that became known as Sanger sequencing (1), based on enzymatic synthesis from a single-stranded DNA template with chain termination using dideoxynucleotides (ddNTPs) and Maxim-Gilbert sequencing (2),which involved chemical degradation ofend-radio-labeled DNA fragments. Both methods relied on four-lane,high resolution polyacrylamide gel electrophoresis to separate the labeled fragment and allow the base sequence to be read in a staggered ladder-like fashion. Sanger sequencing was technically easier and faster, and thus became the main DNA sequencing method for the vast majority of applications.

Aim: Despite the endemic nature of contagious ecthyma in Nigeria, there is limited report on the molecular characterization of the isolates responsible for disease outbreaks. The aim of this study was to molecularly characterize ORFV isolated from clinical infections in goats in Sokoto metropolis. Materials and Methods: Seronegative embryonated chicken eggs were used to isolate ORFV via the chorio allantoic membrane (CAM) route according to the established protocol. Viral DNA was extracted from infected CAM and the full coding region of B2L gene was amplified by PCR and subsequently sequenced by Sanger’s method. The nucleotide sequence results were blasted for identification and phylogenetically analyzed using MEGA and Bioedit softwares. Results and Discussion: The results showed that B2L gene sequences of the ORFV UDUS/01/19/More strain showed slight variability (96- 98.7%) with the reference sequences. Our isolate clustered within the same clade with Korean strain signifying a close genetic relationship. Unique amino acid substitutions were noted in our isolate when compared with other references. This is arguably the first genetic characterisation of B2L gene of ORFV circulating in Nigeria. Conclusion: Our study has provided in sight into the genetic diversity of ORFV in the study area. This is crucial for the design of effective vaccines against the disease which are currently lacking in the country.

Despite several decades’ effort to detect and identify phytoplasmas (Tenericutes: Mollicutes) using PCR and Sanger sequencing focusing on diseased plants, knowledge of phytoplasma biodiversity and vector associations remains highly incomplete. To improve protocols for documenting phyto-plasma diversity and ecology, we used DNA extracted from phloem-feeding insects and com-pared traditional Sanger sequencing with a next-generation sequencing method, Anchored Hy-brid Enrichment (AHE) for detecting and characterizing phytoplasmas. Among 22 of 180 leaf-hopper samples that initially tested positive for phytoplasmas using qPCR, AHE yielded phyto-plasma 16Sr sequences for 20 (19 complete and 1 partial sequence) while Sanger sequencing yield-ed sequences for 16 (11 complete and 5 partial). AHE yielded phytoplasma sequences for an addi-tional 7 samples (3 complete and 4 partial) that did not meet the qPCR threshold for phytoplasma positivity or yielded non-phytoplasma sequences using Sanger sequencing. This suggests that AHE is more efficient for obtaining phytoplasma sequences. Twenty-three samples with sufficient data were classified into eight 16Sr subgroups (16SrI-B, I-F, I-AO, III-U, V-C, IX-J, XI-C, XXXVII-A), three new subgroups (designated as 16SrVI-L, XV-D, XI-G) and three possible new groups. Our results suggest that screening phloem-feeding insects using qPCR and AHE sequencing may be the most efficient method for discovering new phytoplasmas.

Fusarium spp. are commonly associated with the root rot complex of soybean (Glycine max). Previous surveys identified six common Fusarium species from Manitoba, including F. oxysporum, F. redolens, F. graminearum, F. solani, F. avenaceum and F. acuminatum. This study aimed to determine their pathogenicity, assess host resistance and evaluate the genetic diversity of Fusarium spp. isolated from Canada. Pathogenicity was tested on two cultivars, ‘Akras’ (moderate resistance) and ‘B150Y1’ (susceptible), under greenhouse conditions. The virulence of the fungal isolates varied, with root rot severities ranging from 1.5 to 3.3 on a 0-4 scale. The six species were used to screen a panel of 20 Canadian soybean cultivars for resistance in a greenhouse. Cluster and principal component analyses were conducted based on the same traits as for the pathogenicity study. Two of the cultivars, ‘P15T46R2’ and ‘B150Y1’, were consistently found to be tolerant to F. oxysporum, F. redolens, F. graminearum and F. solani. To investigate the incidence and prevalence of Fusarium spp. in Canada, fungi were isolated from 106 soybean fields surveyed across Manitoba, Saskatchewan, Ontario and Quebec. Two-hundred twenty-one Fusarium isolates were identified, with phylogenetic analyses indicating diversity of the isolates in the major soybean production regions of Canada.

Infant mortality remains a major global concern. Sudden unexpected death in infancy (SUDI) is reported globally and in the Western Cape of South Africa it accounted for 40% of all infant deaths between 2012 and 2016. Research highlighting molecular typing of respiratory viruses in SUDI cases is lacking. A total of 116 PCR-positive human rhinovirus (HRV) and respiratory syncytial virus (RSV) swab samples collected from the lungs and trachea of SUDI cases admitted to Tygerberg Medico-legal Mortuary in Cape Town between 2015 and 2019 were included and underwent sequencing and phylogenetic analysis. Three distinct HRV species were identified; HRV-A (n=28), HRV-C (n=11), and HRV-B (n=4), including eight HRV-A, seven HRV-C and one HRV-B genotypes. For RSV, both RSV-A (n=5) and -B (n=5) were detected, but no RSV-A. After amino acid alignment indicating 20 amino acid duplication and nine substitutions, two BA9 genotypes were confirmed. This study describes the first molecular and phylogenetic characterisation of respiratory viruses in SUDI cases in South Africa. The rapid decline in viral viability in post-mortem samples and the retrospective study design employed for the current study did not allow for any correlations between viral genotypes and cause of death and future prospective studies should investigate temporality and specific viral strains associated with clinical disease severity.

Background: SARS-CoV-2 Omicron JN.1 has the highest number of amino acid mutations in its receptor binding domain (RBD) and has acquired a hallmark L455S mutation. The immune evasion capability of JN.1 is a subject of scientific investigation. The US CDC used RT-qPCR SGTF as the proxy indicator of JN.1 infections for evaluation of the effectiveness of updated monovalent XBB.1.5 COVID-19 vaccines against JN.1 and recommended that all persons aged ≥6 months should receive an updated COVID-19 vaccine dose. Objective: Recommend Sanger sequencing for diagnosis of JN.1 infections to generate the data based on which guidelines are made to direct vaccination policies. Methods: The RNA in nasopharyngeal swab specimens from patients with clinical respiratory infection was subjected to nested RT-PCR, targeting a 398-base segment of the N-gene and a 445-base segment of the RBD of SARS-CoV-2 for amplification. The DNA sequences were analyzed for amino acid mutations. Results: The N-gene sequence showed Q229K, usually associated with Omicron BA.2.86 (+JN.1). The RBD sequence showed 24 amino acid mutations, including the hallmark L455S mutation for JN.1 and the V483del for BA.2.86 lineage. Conclusions: The CDC should consider Sanger sequencing of the RBD to diagnose JN.1 infections for statistical analysis in making vaccination policies.

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.

As SARS-CoV-2 continues to spread among human populations, genetic changes occur and accumulate in the circulating virus. Some of these genetic changes have caused amino acid mutations, including deletions, which may have potential impact on critical SARS-CoV-2 countermeasures, including vaccines, therapeutics, and diagnostics. Considerable efforts have been made to categorize the amino acid mutations of the angiotensin-converting enzyme 2 (ACE2) receptor binding domain (RBD) of the spike (S) protein along with certain mutations in other regions within the S protein as specific variants in an attempt to study the relationship between these mutations and the biological behavior of the virus. However, the currently used whole genome sequencing surveillance technologies can test only a small fraction of the positive specimens with high viral loads and often generate uncertainties in nucleic acid sequencing that needs additional verification for precision determination of mutations. This article introduces a generic protocol to routinely sequence a 437-bp nested RT-PCR cDNA amplicon of the ACE2 RBD and a 490-bp nested RT-PCR cDNA amplicon of the N-terminal domain (NTD) of the S gene for detection of the amino acid mutations needed for accurate determination of all variants of concern and variants of interest according to the definitions published by the U.S. Centers for Disease Control and Prevention. This protocol was able to amplify both nucleic acid targets into cDNA amplicons to be used as templates for Sanger sequencing on all 16 clinical specimens that were positive for SARS-CoV-2.