scholarly journals Fast SARS-CoV-2 Variant Detection Using Snapback Primer High-Resolution Melting

Diagnostics ◽  
2021 ◽  
Vol 11 (10) ◽  
pp. 1788
Author(s):  
Joseph C. Lownik ◽  
Jared S. Farrar ◽  
Grayson W. Way ◽  
Angela McKay ◽  
Pavitra Roychoudhury ◽  
...  
Keyword(s):  
High Resolution ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
High Resolution Melting ◽  
Whole Genome ◽  
Current Standard ◽  
The Past ◽  
The World ◽  
Variant Detection ◽  
Sensitive Testing

SARS-CoV-2, the virus responsible for COVID-19, emerged in late 2019 and has since spread throughout the world, infecting over 200 million people. The fast spread of SARS-CoV-2 showcased the need for rapid and sensitive testing methodologies to help track the disease. Over the past 18 months, numerous SARS-CoV-2 variants have emerged. Many of these variants are suggested to be more transmissible as well as less responsive to neutralization by vaccine-induced antibodies. Viral whole-genome sequencing is the current standard for tracking these variants. However, whole-genome sequencing is costly and the technology and expertise are limited to larger reference laboratories. Here, we present the feasibility of a fast, inexpensive methodology using snapback primer-based high-resolution melting to test for >20 high-consequence SARS-CoV-2 spike mutations. This assay can distinguish between multiple variant lineages and be completed in roughly 2 h for less than $10 per sample.

scholarly journals Case report: whole genome sequencing based investigation of maternal-neonatal listeriosis in Sichuan, China

2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Lijuan Luo ◽  
Xi Chen ◽  
Michael Payne ◽  
Xiaolong Cao ◽  
Yan Wang ◽  
...  
Keyword(s):  
High Resolution ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Caesarean Delivery ◽  
Severe Disease ◽  
Metal Resistance ◽  
Meat Sample ◽  
Local Market ◽  
Whole Genome ◽  
Food Isolate

Abstract Background Neonatal listeriosis is a rare but severe disease manifesting as septicemia and central nervous system (CNS) infections with a high fatality rate of around 20 to 30%. Whole genome sequencing (WGS) is a promising technique for pathogen identification and infection source tracing with its high resolution. Case presentation A case of neonatal sepsis with listeriosis was reported with positive blood culture for Listeria monocytogenes. The case was investigated to confirm the vertical transmission of the infection and identify the potential food source of the maternal L. monocytogenes infection using WGS. L. monocytogenes was isolated from the neonate’s blood sample the day after caesarean delivery and from the mother’s genital and pudenda swab samples 5 days and 13 days after caesarean delivery. WGS showed that the isolate from the neonate was identical to the genome type of the isolates from the mother, with only one of the 4 isolates from the mother differing by one single nucleotide polymorphism (SNP). By WGS, one L. monocytogenes isolate from a ready-to-eat (RTE) meat sample in the patients’ community market shared the same sequence type but was ruled out as the cause of infection, with 57 SNP differences to the strain causing the maternal-neonatal infection. The food isolate also carried a novel plasmid pLM1686 that harbored heavy metal resistance genes. After caesarean section, the mother was treated with a third generation cephalosporin which L. monocytogenes is naturally resistant to, which may explain why genital and pudenda swabs were still culture-positive for L. monocytogenes 13 days after delivery. Conclusions Genital swab culture for L. monocytogenes had been informative in the diagnosis of maternal listeriosis in this case. The high resolution of WGS confirmed the maternal-neonatal transmission of L. monocytogenes infection and ruled out the L. monocytogenes contaminated RTE meat from the local market as the direct source of the mother’s infection.

scholarly journals The Emergence and Spread of Novel SARS-CoV-2 Variants

2021 ◽  
Vol 9 ◽  
Author(s):  
Huaimin Yi ◽  
Jin Wang ◽  
Jiong Wang ◽  
Yuying Lu ◽  
Yali Zhang ◽  
...  
Keyword(s):  
Immune Response ◽  
Severe Acute Respiratory Syndrome ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Neutralizing Antibodies ◽  
Whole Genome ◽  
Distribution Characteristics ◽  
S Protein ◽  
The World ◽  
Viral Genes

Since severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) began to spread in late 2019, laboratories around the world have widely used whole genome sequencing (WGS) to continuously monitor the changes in the viral genes and discovered multiple subtypes or branches evolved from SARS-CoV-2. Recently, several novel SARS-CoV-2 variants have been found to be more transmissible. They may affect the immune response caused by vaccines and natural infections and reduce the sensitivity to neutralizing antibodies. We analyze the distribution characteristics of prevalent SARS-CoV-2 variants and the frequency of mutant sites based on the data available from GISAID and PANGO by R 4.0.2 and ArcGIS 10.2. Our analysis suggests that B.1.1.7, B.1.351, and P.1 are more easily spreading than other variants, and the key mutations of S protein, including N501Y, E484K, and K417N/T, have high mutant frequencies, which may have become the main genotypes for the spread of SARS-CoV-2.

scholarly journals Comparative Analysis of Genotyping by Sequencing and Whole-Genome Sequencing Methods in Diversity Studies of Olea europaea L.

Plants ◽  
2021 ◽  
Vol 10 (11) ◽  
pp. 2514
Author(s):  
James Friel ◽  
Aureliano Bombarely ◽  
Carmen Dorca Fornell ◽  
Francisco Luque ◽  
Ana Maria Fernández-Ocaña
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Olea Europaea ◽  
Genetic Relationships ◽  
Germplasm Collection ◽  
Whole Genome ◽  
Population Genetic Analysis ◽  
Olea Europaea L ◽  
The World ◽  
Olea Europaéa

Olive, Olea europaea L., is a tree of great economic and cultural importance in the Mediterranean basin. Thousands of cultivars have been described, of which around 1200 are conserved in the different olive germplasm banks. The genetic characterisation of these cultivars can be performed in different ways. Whole-genome sequencing (WGS) provides more information than the reduced representation methods such as genotype by sequencing (GBS), but at a much higher cost. This may change as the cost of sequencing continues to drop, but, currently, genotyping hundreds of cultivars using WGS is not a realistic goal for most research groups. Our aim is to systematically compare both methodologies applied to olive genotyping and summarise any possible recommendations for the geneticists and molecular breeders of the olive scientific community. In this work, we used a selection of 24 cultivars from an olive core collection from the World Olive Germplasm Collection of the Andalusian Institute of Agricultural and Fisheries Research and Training (WOGBC), which represent the most of the cultivars present in cultivated fields over the world. Our results show that both methodologies deliver similar results in the context of phylogenetic analysis and popular population genetic analysis methods such as clustering. Furthermore, WGS and GBS datasets from different experiments can be merged in a single dataset to perform these analytical methodologies with proper filtering. We also tested the influence of the different olive reference genomes in this type of analysis, finding that they have almost no effect when estimating genetic relationships. This work represents the first comparative study between both sequencing techniques in olive. Our results demonstrate that the use of GBS is a perfectly viable option for replacing WGS and reducing research costs when the goal of the experiment is to characterise the genetic relationship between different accessions. Besides this, we show that it is possible to combine variants from GBS and WGS datasets, allowing the reuse of publicly available data.

scholarly journals 807. Same-day Transmission Analysis of Nosocomial Transmission Using Nanopore Whole Genome Sequencing

2021 ◽  
Vol 8 (Supplement_1) ◽  
pp. S497-S498
Author(s):  
Mohamad Sater ◽  
Remy Schwab ◽  
Ian Herriott ◽  
Tim Farrell ◽  
Miriam Huntley
Keyword(s):  
High Resolution ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Variant Calling ◽  
Cost Effective ◽  
Error Rates ◽  
Sequencing Error ◽  
Snp Analysis ◽  
Whole Genome ◽  
Snp Calling

Abstract Background Healthcare associated infections (HAIs) are a major contributor to patient morbidity and mortality worldwide. HAIs are increasingly important due to the rise of multidrug resistant pathogens which can lead to deadly nosocomial outbreaks. Current methods for investigating transmissions are slow, costly, or have poor detection resolution. A rapid, cost-effective and high-resolution method to identify transmission events is imperative to guide infection control. Whole genome sequencing of infecting pathogens paired with a single nucleotide polymorphism (SNP) analysis can provide high-resolution clonality determination, yet these methods typically have long turnaround times. Here we examined the utility of the Oxford Nanopore Technologies (ONT) platform, a rapid sequencing technology, for whole genome sequencing based transmission analysis. Methods We developed a SNP calling pipeline customized for ONT data, which exhibit higher sequencing error rates and can therefore be challenging for transmission analysis. The pipeline leverages the latest basecalling tools as well as a suite of custom variant calling and filtering algorithms to achieve highest accuracy in clonality calls compared to Illumina-based sequencing. We also capitalize on ONT long reads by assembling outbreak-specific genomes in order to overcome the need for an external reference genome. Results We examined 20 bacterial isolates from 5 HAI investigations previously performed at Day Zero Diagnostics as part of epiXact®, our commercialized Illumina-based HAI sequencing and analysis service. Using the ONT data and pipeline, we achieved greater than 90% SNP-calling sensitivity and precision, allowing 100% accuracy of clonality classification compared to Illumina-based results across common HAI species. We demonstrate the validity and increased resolution of our SNP analysis pipeline using assembled genomes from each outbreak. We also demonstrate that this ONT-based workflow can produce isolate to transmission determination (i.e. including WGS and analysis) in less than 24 hours. SNP calling performance ONT-based SNP calling sensitivity and precision compared to Illumina-based pipeline Conclusion We demonstrate the utility of ONT for HAI investigation, establishing the potential to transform healthcare epidemiology with same-day high-resolution transmission determination. Disclosures Mohamad Sater, PhD, Day Zero Diagnostics (Employee, Shareholder) Remy Schwab, MSc, Day Zero Diagnostics (Employee, Shareholder) Ian Herriott, BS, Day Zero Diagnostics (Employee, Shareholder) Tim Farrell, MS, Day Zero Diagnostics, Inc. (Employee, Shareholder) Miriam Huntley, PhD, Day Zero Diagnostics (Employee, Shareholder)

scholarly journals Evaluation of High-Resolution Melting for Rapid Differentiation of Phytophthora Hybrids and Their Parental Species

Plant Disease ◽  
2019 ◽  
Vol 103 (9) ◽  
pp. 2295-2304
Author(s):  
Maria F. Ratti ◽  
Rhys A. Farrer ◽  
Liliana M. Cano ◽  
Roberto Faedda ◽  
Erica M. Goss
Keyword(s):  
High Resolution ◽  
Genome Sequencing ◽  
High Resolution Melting ◽  
Parental Species ◽  
Phytophthora Species ◽  
Wild Plants ◽  
Whole Genome ◽  
Advance Research ◽  
Species Hybrids ◽  
Rapid Tool

Phytophthora species hybrids have been repeatedly reported as causing damaging diseases to cultivated and wild plants. Two known hybrids, P. andina and P. × pelgrandis, are pathogens of Solanaceae and ornamentals, respectively, although the extent of their host ranges are unknown. P. andina emerged from hybridization of P. infestans and an unidentified related species, whereas P. × pelgrandis emerged from P. nicotianae and P. cactorum. Considering that hybrids and parental species can coexist in the same regions and to distinguish them usually requires cloning or whole genome sequencing, we aimed to develop a rapid tool to distinguish them. Specifically, we used high-resolution melting (HRM) assays to differentiate genotypes based on their amplicon melting profiles. We designed primers for P. × pelgrandis and parental species based on available sequences of P. nicotianae and P. cactorum nuclear genes containing polymorphisms between species. For P. andina, heterozygous sites from Illumina short reads were used for the same purpose. We identified multiple amplicons exhibiting differences in melting curves between parental species and hybrids. We propose HRM as a rapid method for differentiation of P. andina and P. × pelgrandis hybrids from parental species that could be employed to advance research on these pathogens.

scholarly journals Evaluation of whole-genome sequencing of four Chinese crested dogs for variant detection using the ion proton system

2015 ◽  
Vol 2 (1) ◽  
Author(s):  
Agnese Viluma ◽  
Shumaila Sayyab ◽  
Sofia Mikko ◽  
Göran Andersson ◽  
Tomas F. Bergström
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Whole Genome ◽  
Ion Proton ◽  
Variant Detection

scholarly journals Whole genome sequencing reveals high-resolution epidemiological links between clinical and environmental Klebsiella pneumoniae

2017 ◽  
Vol 9 (1) ◽  
Author(s):  
Chakkaphan Runcharoen ◽  
Danesh Moradigaravand ◽  
Beth Blane ◽  
Suporn Paksanont ◽  
Jeeranan Thammachote ◽  
...  
Keyword(s):  
High Resolution ◽  
Klebsiella Pneumoniae ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Whole Genome

scholarly journals Whole genomic sequencing based genotyping reveals a specific X3 sublineage restricted to Mexico and related with multidrug resistance

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Ana Cristina Jiménez-Ruano ◽  
Carlos Francisco Madrazo-Moya ◽  
Irving Cancino-Muñoz ◽  
Paulina M. Mejía-Ponce ◽  
Cuauhtémoc Licona-Cassani ◽  
...  
Keyword(s):  
Multidrug Resistance ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Multidrug Resistant ◽  
Genomic Sequencing ◽  
Whole Genome ◽  
Drug Resistant ◽  
The Past ◽  
High Prevalence ◽  
Genomic Cluster

AbstractWhole genome sequencing (WGS) has been shown to be superior to traditional procedures of genotyping in tuberculosis (TB), nevertheless, reports of its use in drug resistant TB (DR-TB) isolates circulating in Mexico, are practically unknown. Considering the above the main of this work was to identify and characterize the lineages and genomic transmission clusters present in 67 DR-TB isolates circulating in southeastern Mexico. The results show the presence of three major lineages: L1 (3%), L2 (3%) and L4 (94%), the last one included 16 sublineages. Sublineage 4.1.1.3 (X3) was predominant in 18 (27%) of the isolates, including one genomic cluster, formed by eleven multidrug resistant isolates and sharing the SIT 3278, which seems to be restricted to Mexico. By the use of WGS, it was possible to identify the high prevalence of L4 and a high number of sublineages circulating in the region, also was recognized the presence of a novel X3 sublineage, formed exclusively by multidrug resistant isolates and with restrictive circulation in Mexico for at least the past 17 years.

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