Sanger Sequencing Of Samples Research Articles

Mutagenic primer-based novel multiplex PCR-RFLP technique to genotype BECN1 SNPs rs10512488 and rs11552192.

Single Nucleotide Polymorphisms (SNPs) in candidate autophagy gene BECN1 could influence its functions thereby autophagy process. BECN1 noncoding SNPs were found to be significantly associated with neurodegenerative disease and type 2 diabetes mellitus. This study aimed to develop a simultaneous genotyping technique for two BECN1 SNPs (rs10512488 and rs11552192). A mutagenic primer-based approach was used to introduce a NdeI restriction site to genotype rs10512488 by Artificial-Restriction Fragment Length Polymorphism (A-RFLP) along with rs11552192 by Polymerase Chain Reaction (PCR)-RFLP. Multiplexing PCR and restriction digestion reactions were set up for simultaneous genotyping of both SNPs in 100 healthy individuals. Genotypic and allele frequencies were manually calculated, and the Hardy-Weinberg Equilibrium was assessed using the chi-square test. We successfully developed PCR and RFLP conditions for the amplification and restriction digestion of both SNPs within the same tube for genotyping. The results of genotyping by newly developed multiplexing PCR-RFLP technique were concordant with the genotypes obtained by Sanger sequencing of samples. Allelic frequencies of rs10512488 obtained were 0.15 (A) and 0.85 (G), whereas allelic frequencies of rs11552192 were 0.16 (T) and 0.84 (A). The newly developed technique is rapid, cost-effective and time-saving for large-scale applications compared to sequencing methods and would play an important role in low-income settings. For the first time, allelic frequencies of rs10512488 and rs11552192 were reported among the North Indian population.

Resurgence of SARS-CoV-2 Delta after Omicron variant superinfection in an immunocompromised pediatric patient

BackgroundPersistent SARS-CoV-2 infection in immunocompromised hosts is thought to contribute to viral evolution by facilitating long-term natural selection and viral recombination in cases of viral co-infection or superinfection. However, there are limited data on the longitudinal intra-host population dynamics of SARS-CoV-2 co-infection/superinfection, especially in pediatric populations. Here, we report a case of Delta-Omicron superinfection in a hospitalized, immunocompromised pediatric patient.MethodsWe conducted Illumina whole genome sequencing (WGS) for longitudinal specimens to investigate intra-host dynamics of SARS-CoV-2 strains. Topoisomerase PCR cloning of Spike open-reading frame and Sanger sequencing of samples was performed for four specimens to validate the findings. Analysis of publicly available SARS-CoV-2 sequence data was performed to investigate the co-circulation and persistence of SARS-CoV-2 variants.ResultsResults of WGS indicate the patient was initially infected with the SARS-CoV-2 Delta variant before developing a SARS-CoV-2 Omicron variant superinfection, which became predominant. Shortly thereafter, viral loads decreased below the level of detection before resurgence of the original Delta variant with no residual trace of Omicron. After 54 days of persistent infection, the patient tested negative for SARS-CoV-2 but ultimately succumbed to a COVID-19-related death. Despite protracted treatment with remdesivir, no antiviral resistance mutations emerged. These results indicate a unique case of persistent SARS-CoV-2 infection with the Delta variant interposed by a transient superinfection with the Omicron variant. Analysis of publicly available sequence data suggests the persistence and ongoing evolution of Delta subvariants despite the global predominance of Omicron, potentially indicative of continued transmission in an unknown population or niche.ConclusionA better understanding of SARS-CoV-2 intra-host population dynamics, persistence, and evolution during co-infections and/or superinfections will be required to continue optimizing patient care and to better predict the emergence of new variants of concern.

Rare Variants Residing in Novel Cis-Acting Element in Visual System Homeobox 1 and Their Contribution in the Pathogenesis of Keratoconus

(1) Background: The visual system homeobox 1 (VSX1) may contribute to the incidence of keratoconus (KC) in different populations. The present study investigated the role of VSX1 in autosomal recessive Pakistani families and sporadic KC patients using in silico analysis of the rare variants for the identification of the cis-acting elements in VSX1; (2) Methods: Mutation analysis of VSX1 was undertaken using Sanger sequencing of samples from seven KC families and 100 sporadic patients. In silico analysis of the rare variants and identification of cis-acting elements was determined using Human Splicing Finder (HSF), ESE finder, RESCUE-ESE and through Exon- Identity Element (EIEs) prediction software suits, combined with various algorithms to identify the effect of variations in splicing motifs; (3) Results: Screening of VSX1 did not reveal any novel mutation in the KC panel, but a synonymous polymorphism rs12480307 (c.546A>G; p.Ala182Ala) in exon three and 3′UTR rs76499395 (c.*496A>G) were observed in two separate probands. These polymorphisms were not found in any of the sporadic KC cases or 100 ethnically matched control samples. The analysis of these rare variants revealed a plausible role for these two single nucleotide polymorphisms (SNPs) in KC development through the identification of novel cis-acting elements, an exonic splicing enhancer element (ESE) and binding motifs for two micro RNAs, miRNA-203 binding and hsa-miR-3938, in the VSX1 gene structure; (4) Conclusions: Rare genetic variations in the VSX1 were found to have a potential contribution to KC development.

Next generation sequencing reveals past and current widespread occurrence of maize yellow mosaic virus in South Africa

Maize yellow mosaic virus (MaYMV) is a single-stranded RNA polerovirus first identified in China. MaYMV was recently reported from West and East Africa, however it had not yet been reported from southern Africa. RNA-seq data from South African field-grown fungal-infected maize was mined for viral sequences by de novo assembly of reads that did not map to the maize or fungal genomes. Predicted proteins from the de novo-assembled unmapped reads matched MaYMV proteins with regions of 96–100% identity. MaYMV was detected in maize RNAseq data from 2009, 2012 and 2013. Complete South African MaYMV genome sequences (5642 nt) were determined by RT-PCR and Sanger sequencing of samples from two different maize genotypes, years, and sites. Phylogenetic analysis confirmed the species identity as MaYMV, and showed separate clustering of isolates between Africa, Asia and South America. Some MaYMV positive samples had reads matching Potyviridae (Johnson grass mosaic virus and Sugarcane mosaic virus), and mycoviruses (Setosphaeria turcica hypovirus 1, Bipolaris maydis partitivirus 1, and Pleospora typhicola fusarivirus 1). A 2016/2017 RT-PCR survey of maize plants exhibiting virus-like symptoms, such as yellowing and streaking patterns, revealed MaYMV in 39 samples from six provinces in South Africa. This report documents the earliest known MaYMV infection world-wide, and indicates that the virus is widespread throughout Africa.

CHCHD2 mutations in autosomal dominant late-onset Parkinson's disease: a genome-wide linkage and sequencing study

Identification of causative genes in mendelian forms of Parkinson's disease is valuable for understanding the cause of the disease. We did genetic studies in a Japanese family with autosomal dominant Parkinson's disease to identify novel causative genes. We did a genome-wide linkage analysis on eight affected and five unaffected individuals from a family with autosomal dominant Parkinson's disease (family A). Subsequently, we did exome sequencing on three patients and whole-genome sequencing on one patient in family A. Variants were validated by Sanger sequencing in samples from patients with autosomal dominant Parkinson's disease, patients with sporadic Parkinson's disease, and controls. Participants were identified from the DNA bank of the Comprehensive Genetic Study on Parkinson's Disease and Related Disorders (Juntendo University School of Medicine, Tokyo, Japan) and were classified according to clinical information obtained by neurologists. Splicing abnormalities of CHCHD2 mutants were analysed in SH-SY5Y cells. We used the Fisher's exact test to calculate the significance of allele frequencies between patients with sporadic Parkinson's disease and unaffected controls, and we calculated odds ratios and 95% CIs of minor alleles. We identified a missense mutation (CHCHD2, 182C>T, Thr61Ile) in family A by next-generation sequencing. We obtained samples from a further 340 index patients with autosomal dominant Parkinson's disease, 517 patients with sporadic Parkinson's disease, and 559 controls. Three CHCHD2 mutations in four of 341 index cases from independent families with autosomal dominant Parkinson's disease were detected by CHCHD2 mutation screening: 182C>T (Thr61Ile), 434G>A (Arg145Gln), and 300+5G>A. Two single nucleotide variants (-9T>G and 5C>T) in CHCHD2 were confirmed to have different frequencies between sporadic Parkinson's disease and controls, with odds ratios of 2·51 (95% CI 1·48-4·24; p=0·0004) and 4·69 (1·59-13·83, p=0·0025), respectively. One single nucleotide polymorphism (rs816411) was found in CHCHD2 from a previously reported genome-wide association study; however, there was no significant difference in its frequency between patients with Parkinson's disease and controls in a previously reported genome-wide association study (odds ratio 1·17, 95% CI 0·96-1·19; p=0·22). In SH-SY5Y cells, the 300+5G>A mutation but not the other two mutations caused exon 2 skipping. CHCHD2 mutations are associated with, and might be a cause of, autosomal dominant Parkinson's disease. Further genetic studies in other populations are needed to confirm the pathogenicity of CHCHD2 mutations in autosomal dominant Parkinson's disease and susceptibility for sporadic Parkinson's disease, and further functional studies are needed to understand how mutant CHCHD2 might play a part in the pathophysiology of Parkinson's disease. Japan Society for the Promotion of Science; Japanese Ministry of Education, Culture, Sports, Science and Technology; Japanese Ministry of Health, Labour and Welfare; Takeda Scientific Foundation; Cell Science Research Foundation; and Nakajima Foundation.