scholarly journals Complete Genome Sequencing of Field Isolates of Peste des Petits Ruminants Virus from Tanzania Revealed a High Nucleotide Identity with Lineage III PPR Viruses

Animals ◽  
2021 ◽  
Vol 11 (10) ◽  
pp. 2976
Author(s):  
Edson Kinimi ◽  
Mana Mahapatra ◽  
Tebogo Kgotlele ◽  
Mariam R. Makange ◽  
Chandana Tennakoon ◽  
...  
Keyword(s):  
Low Cost ◽  
Viral Evolution ◽  
Cost Effective ◽  
Nucleotide Identity ◽  
Clinical Samples ◽  
Depth Information ◽  
Peste Des Petits Ruminants ◽  
Sequencing Technology ◽  
Oxford Nanopore ◽  
Complete Genomes

Peste des petits ruminants virus (PPRV) causes a highly devastating disease of sheep and goats that threatens food security, small ruminant production and susceptible endangered wild ruminants. With policy directed towards achieving global PPR eradication, the establishment of cost-effective genomic surveillance tools is critical where PPR is endemic. Genomic data can provide sufficient in-depth information to identify the pockets of endemicity responsible for PPRV persistence and viral evolution, and direct an appropriate vaccination response. Yet, access to the required sequencing technology is low in resource-limited settings and is compounded by the difficulty of transporting clinical samples from wildlife across international borders due to the Convention on International Trade in Endangered Species (CITES) of Wild Fauna and Flora, and Nagoya Protocol regulations. Oxford nanopore MinION sequencing technology has recently demonstrated an extraordinary performance in the sequencing of PPRV due to its rapidity, utility in endemic countries and comparatively low cost per sample when compared to other whole-genome (WGS) sequencing platforms. In the present study, Oxford nanopore MinION sequencing was utilised to generate complete genomes of PPRV isolates collected from infected goats in Ngorongoro and Momba districts in the northern and southern highlands of Tanzania during 2016 and 2018, respectively. The tiling multiplex polymerase chain reaction (PCR) was carried out with twenty-five pairs of long-read primers. The resulting PCR amplicons were used for nanopore library preparation and sequencing. The analysis of output data was complete genomes of PPRV, produced within four hours of sequencing (accession numbers: MW960272 and MZ322753). Phylogenetic analysis of the complete genomes revealed a high nucleotide identity, between 96.19 and 99.24% with lineage III PPRV currently circulating in East Africa, indicating a common origin. The Oxford nanopore MinION sequencer can be deployed to overcome diagnostic and surveillance challenges in the PPR Global Control and Eradication program. However, the coverage depth was uneven across the genome and amplicon dropout was observed mainly in the GC-rich region between the matrix (M) and fusion (F) genes of PPRV. Thus, larger field studies are needed to allow the collection of sufficient data to assess the robustness of nanopore sequencing technology.

scholarly journals Detecting SARS-CoV-2 variants with SNP genotyping

2020 ◽  
Author(s):  
Helen Harper ◽  
Amanda J. Burridge ◽  
Mark Winfield ◽  
Adam Finn ◽  
Andrew D. Davidson ◽  
...  
Keyword(s):  
High Throughput ◽  
Low Cost ◽  
Cost Effective ◽  
Snp Genotyping ◽  
Clinical Samples ◽  
Marker Panel ◽  
Qrt Pcr ◽  
Crucial Information ◽  
The Uk ◽  
Genotyping Panel

AbstractTracking genetic variations from positive SARS-CoV-2 samples yields crucial information about the number of variants circulating in an outbreak and the possible lines of transmission but sequencing every positive SARS-CoV-2 sample would be prohibitively costly for population-scale test and trace operations. Genotyping is a rapid, high-throughput and low-cost alternative for screening positive SARS-CoV-2 samples in many settings. We have designed a SNP identification pipeline to identify genetic variation using sequenced SARS-CoV-2 samples. Our pipeline identifies a minimal marker panel that can define distinct genotypes. To evaluate the system we developed a genotyping panel to detect variants-identified from SARS-CoV-2 sequences surveyed between March and May 2020- and tested this on 50 stored qRT-PCR positive SARS-CoV-2 clinical samples that had been collected across the South West of the UK in April 2020. The 50 samples split into 15 distinct genotypes and there was a 76% probability that any two randomly chosen samples from our set of 50 would have a distinct genotype. In a high throughput laboratory, qRT-PCR positive samples pooled into 384-well plates could be screened with our marker panel at a cost of < £1.50 per sample. Our results demonstrate the usefulness of a SNP genotyping panel to provide a rapid, cost-effective, and reliable way to monitor SARS-CoV-2 variants circulating in an outbreak. Our analysis pipeline is publicly available and will allow for marker panels to be updated periodically as viral genotypes arise or disappear from circulation.

scholarly journals Detecting SARS-CoV-2 variants with SNP genotyping

PLoS ONE ◽  
2021 ◽  
Vol 16 (2) ◽  
pp. e0243185 ◽  
Author(s):  
Helen Harper ◽  
Amanda Burridge ◽  
Mark Winfield ◽  
Adam Finn ◽  
Andrew Davidson ◽  
...  
Keyword(s):  
High Throughput ◽  
Low Cost ◽  
Cost Effective ◽  
Snp Genotyping ◽  
Clinical Samples ◽  
Marker Panel ◽  
Qrt Pcr ◽  
Crucial Information ◽  
The Uk ◽  
Genotyping Panel

Tracking genetic variations from positive SARS-CoV-2 samples yields crucial information about the number of variants circulating in an outbreak and the possible lines of transmission but sequencing every positive SARS-CoV-2 sample would be prohibitively costly for population-scale test and trace operations. Genotyping is a rapid, high-throughput and low-cost alternative for screening positive SARS-CoV-2 samples in many settings. We have designed a SNP identification pipeline to identify genetic variation using sequenced SARS-CoV-2 samples. Our pipeline identifies a minimal marker panel that can define distinct genotypes. To evaluate the system, we developed a genotyping panel to detect variants-identified from SARS-CoV-2 sequences surveyed between March and May 2020 and tested this on 50 stored qRT-PCR positive SARS-CoV-2 clinical samples that had been collected across the South West of the UK in April 2020. The 50 samples split into 15 distinct genotypes and there was a 61.9% probability that any two randomly chosen samples from our set of 50 would have a distinct genotype. In a high throughput laboratory, qRT-PCR positive samples pooled into 384-well plates could be screened with a marker panel at a cost of < £1.50 per sample. Our results demonstrate the usefulness of a SNP genotyping panel to provide a rapid, cost-effective, and reliable way to monitor SARS-CoV-2 variants circulating in an outbreak. Our analysis pipeline is publicly available and will allow for marker panels to be updated periodically as viral genotypes arise or disappear from circulation.

scholarly journals DNA Thermo-Protection Facilitates Whole Genome Sequencing of Mycobacteria Direct from Clinical Samples by the Nanopore Platform

2020 ◽  
Author(s):  
Sophie George ◽  
Yifei Xu ◽  
Gillian Rodger ◽  
Marcus Morgan ◽  
Nicholas D. Sanderson ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Low Cost ◽  
Clinical Samples ◽  
Heat Inactivation ◽  
Whole Genome ◽  
Genome Coverage ◽  
Human Dna ◽  
Oxford Nanopore ◽  
Oxford Nanopore Technologies

ABSTRACTMycobacterium tuberculosis (MTB) is the leading cause of death from bacterial infection. Improved rapid diagnosis and antimicrobial resistance determination, such as by whole genome sequencing, are required. Our aim was to develop a simple, low-cost method of preparing DNA for Oxford Nanopore Technologies (ONT) sequencing direct from MTB positive clinical samples (without culture). Simultaneous sputum liquefaction, bacteria heat-inactivation (99°C/30min) and enrichment for Mycobacteria DNA was achieved using an equal volume of thermo-protection buffer (4M KCl, 0.05M HEPES buffer pH7.5, 0.1% DTT). The buffer emulated intracellular conditions found in hyperthermophiles, thus protecting DNA from rapid thermo-degradation, which renders it a poor template for sequencing. Initial validation employed Mycobacteria DNA (extracted or intracellular). Next, mock clinical samples (infection-negative human sputum spiked 0-105 BCG cells/ml) underwent liquefaction in thermo-protection buffer and heat-inactivation. DNA was extracted and sequenced. Human DNA degraded faster than Mycobacteria DNA, resulting in target enrichment. Four replicate experiments each demonstrated detection at 101 BCG cells/ml, with 31-59 MTB complex reads. Maximal genome coverage (>97% at 5x-depth) was achieved at 104 BCG cells/ml; >91% coverage (1x depth) at 103 BCG cells/ml. Final validation employed MTB positive clinical samples (n=20), revealed initial sample volumes ≥1ml typically yielded higher mean depth of MTB genome coverage, the overall range 0.55-81.02. A mean depth of 3 gave >96% one-fold TB genome coverage (in 15/20 clinical samples). A mean depth of 15 achieved >99% five-fold genome coverage (in 9/20 clinical samples). In summary, direct-from-sample sequencing of MTB genomes was facilitated by a low cost thermo-protection buffer.

scholarly journals Genome-guided discovery of natural products through multiplexed low coverage whole-genome sequencing of soil Actinomycetes on Oxford Nanopore Flongle

2021 ◽  
Author(s):  
Rahim Rajwani ◽  
Shannon I Ohlemacher ◽  
Gengxiang Zhao ◽  
Hong-Bing Liu ◽  
Carole A. Bewley
Keyword(s):  
Natural Products ◽  
High Resolution Mass Spectrometry ◽  
Low Cost ◽  
Genome Mining ◽  
Cost Effective ◽  
Gene Clusters ◽  
Mass Spectrometry Data ◽  
Human Pathogens ◽  
Oxford Nanopore ◽  
Long Read

Genome-mining is an important tool for discovery of new natural products; however, the number of publicly available genomes for natural product-rich microbes such as Actinomycetes, relative to human pathogens with smaller genomes, is small. To obtain contiguous DNA assemblies and identify large (ca. 10 to greater than 100 Kb) biosynthetic gene clusters (BGCs) with high-GC (>70%) and -repeat content, it is necessary to use long-read sequencing methods when sequencing Actinomycete genomes. One of the hurdles to long-read sequencing is the higher cost. In the current study, we assessed Flongle, a recently launched platform by Oxford Nanopore Technologies, as a low-cost DNA sequencing option to obtain contiguous DNA assemblies and analyze BGCs. To make the workflow more cost-effective, we multiplexed up to four samples in a single Flongle sequencing experiment while expecting low-sequencing coverage per sample. We hypothesized that contiguous DNA assemblies might enable analysis of BGCs even at low sequencing depth. To assess the value of these assemblies, we collected high-resolution mass-spectrometry data and conducted a multi-omics analysis to connect BGCs to secondary metabolites. In total, we assembled genomes for 20 distinct strains across seven sequencing experiments. In each experiment, 50% of the bases were in reads longer than 10 Kb, which facilitated the assembly of reads into contigs with an average N50 value of 3.5 Mb. The programs antiSMASH and PRISM predicted 629 and 295 BGCs, respectively. We connected BGCs to metabolites for N,N-dimethyl cyclic-ditryptophan, a novel lassopeptide and three known Actinomycete-associated siderophores, namely mirubactin, heterobactin and salinichelin.

scholarly journals A comparative evaluation of a dye-based and probe-based RT-qPCR assay for the screening of SARS-CoV-2 using individual and pooled-sample testing

2020 ◽  
Author(s):  
Claudio Verdugo ◽  
Anita Plaza ◽  
Gerardo Acosta-Jamett ◽  
Natalia Castro ◽  
Josefina Gutiérrez ◽  
...  
Keyword(s):  
Low Cost ◽  
Rna Extraction ◽  
Cost Effective ◽  
Clinical Samples ◽  
Analytical Sensitivity ◽  
Detection Rates ◽  
Efficient System ◽  
Population Sampling ◽  
Effective Interventions ◽  
Surveillance Programs

ABSTRACTEffective interventions are mandatory to control the transmission and spread of SARS-CoV-2, a highly contagious virus causing devastating effects worldwide. Cost-effective approaches are pivotal tools required to increase the detection rates and escalate further in massive surveillance programs, especially in countries with limited resources that most of the efforts have focused on symptomatic cases only. Here, we compared the performance of the RT-qPCR using an intercalating dye with the probe-based assay. Then, we tested and compared these two RT-qPCR chemistries in different pooling systems: after RNA extraction (post-RNA extraction) and before RNA extraction (pre-RNA extraction) optimizing by pool size and template volume. We evaluated these approaches in 610 clinical samples. Our results show that the dye-based technique has a high analytical sensitivity similar to the probe-based detection assay used worldwide. Further, this assay may also be applicable in testing by pool systems post-RNA extraction up to 20 samples. However, the most efficient system for massive surveillance, the pre-RNA extraction pooling approach, was obtained with the probe-based assay in test up to 10 samples adding 13.5 µL of RNA template. The low cost and the potential use in pre-RNA extraction pool systems, place of this assays as a valuable resource for scalable sampling to larger populations. Implementing a pool system for population sampling results in an important savings of laboratory resources and time, which are two key factors during an epidemic outbreak. Using the pooling approaches evaluated here, we are confident that it can be used as a valid alternative assay for the detection of SARS-CoV-2 in human samples.

scholarly journals Rapid and Cost-Efficient Enterovirus Genotyping from Clinical Samples Using Flongle Flow Cells

Genes ◽  
2019 ◽  
Vol 10 (9) ◽  
pp. 659 ◽  
Author(s):  
Carole Grädel ◽  
Miguel Angel Terrazos Miani ◽  
Maria Teresa Barbani ◽  
Stephen L Leib ◽  
Franziska Suter-Riniker ◽  
...  
Keyword(s):  
Low Cost ◽  
Clinical Importance ◽  
Pcr Amplification ◽  
Cost Effective ◽  
Flow Cell ◽  
Turnover Time ◽  
Clinical Samples ◽  
Reference Database ◽  
Flow Cells ◽  
Consensus Sequences

Enteroviruses affect millions of people worldwide and are of significant clinical importance. The standard method for enterovirus identification and genotyping still relies on Sanger sequencing of short diagnostic amplicons. In this study, we assessed the feasibility of nanopore sequencing using the new flow cell “Flongle” for fast, cost-effective, and accurate genotyping of human enteroviruses from clinical samples. PCR amplification of partial VP1 gene was performed from multiple patient samples, which were multiplexed together after barcoding PCR and sequenced multiple times on Flongle flow cells. The nanopore consensus sequences obtained from mapping reads to a reference database were compared to their Sanger sequence counterparts. Using clinical specimens sampled over different years, we were able to correctly identify enterovirus species and genotypes for all tested samples, even when doubling the number of barcoded samples on one flow cell. Average sequence identity across sequencing runs was >99.7%. Phylogenetic analysis showed that the consensus sequences achieved with Flongle delivered accurate genotyping. We conclude that the new Flongle-based assay with its fast turnover time, low cost investment, and low cost per sample represents an accurate, reproducible, and cost-effective platform for enterovirus identification and genotyping.

ASSESSMENT OF THE PLATELET COUNT IN THE PREGNANT WOMEN IN IGIMS, PATNA, BIHAR

2020 ◽  
Vol 4 (2) ◽  
Author(s):  
Tanwi Singh ◽  
Anshuman Sinha
Keyword(s):  
Platelet Count ◽  
Pregnant Women ◽  
Screening Test ◽  
Low Cost ◽  
Medical Science ◽  
Cost Effective ◽  
Platelet Indices ◽  
Increased Risk ◽  
Low Platelet Count ◽  
Severity Of The Disease

The major risk associated with low platelet count in pregnancy is the increased risk of bleeding during the childbirth or post that. There is an increased blood supply to the uterus during pregnancy and the surgical procedure requires cutting of major blood vessels. Women with thrombocytopenia are at increased risk of losing excessive blood. The risk is more in case of caesarean delivery as compared to vaginal delivery. Hence based on above findings the present study was planned for Assessment of the Platelet Count in the Pregnant Women in IGIMS, Patna, Bihar. The present study was planned in Department of Pathology, Indira Gandhi Institute of Medical Science, Patna, Bihar, India. The present study was planned from duration of January 2019 to June 2019. In the present study 200 pregnant females samples received for the platelet estimation were enrolled in the present study. Clinically platelet indices can be a useful screening test for early identification of preeclampsia and eclampsia. Also platelet indices can assess the prognosis of this disease in pregnant women and can be used as an effective prognostic marker because it correlates with severity of the disease. Platelet count is a simple, low cost, and rapid routine screening test. Hence the data generated from the present study concludes that platelet count can be used as a simple and cost effective tool to monitor the progression of preeclampsia, thereby preventing complications to develop during the gestational period. Keywords: Platelet Count, Pregnant Women, IGIMS, Patna, Bihar, etc.

Janus – A New Approach to Air Combat Pilot Training

2019 ◽  
Vol 2019 (4) ◽  
pp. 7-22
Author(s):  
Georges Bridel ◽  
Zdobyslaw Goraj ◽  
Lukasz Kiszkowiak ◽  
Jean-Georges Brévot ◽  
Jean-Pierre Devaux ◽  
...  
Keyword(s):  
Low Cost ◽  
Cost Effective ◽  
High Energy ◽  
Training System ◽  
Embedded Sensors ◽  
Pilot Training ◽  
New Approach ◽  
Combat Aircraft ◽  
Air Combat ◽  
The Cost

Abstract Advanced jet training still relies on old concepts and solutions that are no longer efficient when considering the current and forthcoming changes in air combat. The cost of those old solutions to develop and maintain combat pilot skills are important, adding even more constraints to the training limitations. The requirement of having a trainer aircraft able to perform also light combat aircraft operational mission is adding unnecessary complexity and cost without any real operational advantages to air combat mission training. Thanks to emerging technologies, the JANUS project will study the feasibility of a brand-new concept of agile manoeuvrable training aircraft and an integrated training system, able to provide a live, virtual and constructive environment. The JANUS concept is based on a lightweight, low-cost, high energy aircraft associated to a ground based Integrated Training System providing simulated and emulated signals, simulated and real opponents, combined with real-time feedback on pilot’s physiological characteristics: traditionally embedded sensors are replaced with emulated signals, simulated opponents are proposed to the pilot, enabling out of sight engagement. JANUS is also providing new cost effective and more realistic solutions for “Red air aircraft” missions, organised in so-called “Aggressor Squadrons”.

Motivating Compliance: Firm Response to Mandatory Disclosure Policies

2018 ◽  
Vol 32 (2) ◽  
pp. 103-119
Author(s):  
Colleen M. Boland ◽  
Chris E. Hogan ◽  
Marilyn F. Johnson
Keyword(s):  
Conflict Of Interest ◽  
Low Cost ◽  
Cost Effective ◽  
Data Availability ◽  
Policy Adoption ◽  
Future Research ◽  
Regulated Firms ◽  
Firm Response ◽  
Disclosure Rules ◽  
The Impact

SYNOPSIS Mandatory existence disclosure rules require an organization to disclose a policy's existence, but not its content. We examine policy adoption frequencies in the year immediately after the IRS required mandatory existence disclosure by nonprofits of various governance policies. We also examine adoption frequencies in the year of the subsequent change from mandatory existence disclosure to a disclose-and-explain regime that required supplemental disclosures about the content and implementation of conflict of interest policies. Our results suggest that in areas where there is unclear regulatory authority, mandatory existence disclosure is an effective and low cost regulatory device for encouraging the adoption of policies desired by regulators, provided those policies are cost-effective for regulated firms to implement. In addition, we find that disclose-and-explain regulatory regimes provide stronger incentives for policy adoption than do mandatory existence disclosure regimes and also discourage “check the box” behavior. Future research should examine the impact of mandatory existence disclosure rules in the year that the regulation is implemented. Data Availability: Data are available from sources cited in the text.

scholarly journals Computer-supported Detection of M-Components and Evaluation of Immunoglobulins after Capillary Electrophoresis

2001 ◽  
Vol 47 (1) ◽  
pp. 110-117 ◽  
Author(s):  
Magnus Jonsson ◽  
Joyce Carlson ◽  
Jan-Olof Jeppsson ◽  
Per Simonsson
Keyword(s):  
Capillary Electrophoresis ◽  
Decision Support ◽  
Protein Separation ◽  
Serum Proteins ◽  
Cost Effective ◽  
Clinical Samples ◽  
Serum Samples ◽  
Computerized Decision Support ◽  
Cost Effective Method ◽  
Mathematical Algorithms

Abstract Background: Electrophoresis of serum samples allows detection of monoclonal gammopathies indicative of multiple myeloma, Waldenström macroglobulinemia, monoclonal gammopathy of undetermined significance, and amyloidosis. Present methods of high-resolution agarose gel electrophoresis (HRAGE) and immunofixation electrophoresis (IFE) are manual and labor-intensive. Capillary zone electrophoresis (CZE) allows rapid automated protein separation and produces digital absorbance data, appropriate as input for a computerized decision support system. Methods: Using the Beckman Paragon CZE 2000 instrument, we analyzed 711 routine clinical samples, including 95 monoclonal components (MCs) and 9 cases of Bence Jones myeloma, in both the CZE and HRAGE systems. Mathematical algorithms developed for the detection of monoclonal immunoglobulins (MCs) in the γ- and β-regions of the electropherogram were tested on the entire material. Additional algorithms evaluating oligoclonality and polyclonal concentrations of immunoglobulins were also tested. Results: CZE electropherograms corresponded well with HRAGE. Only one IgG MC of 1 g/L, visible on HRAGE, was not visible after CZE. Algorithms detected 94 of 95 MCs (98.9%) and 100% of those visible after CZE. Of 607 samples lacking an MC on HRAGE, only 3 were identified by the algorithms (specificity, 99%). Algorithms evaluating total gammaglobulinemia and oligoclonality also identified several cases of Bence Jones myeloma. Conclusions: The use of capillary electrophoresis provides a modern, rapid, and cost-effective method of analyzing serum proteins. The additional option of computerized decision support, which provides rapid and standardized interpretations, should increase the clinical availability and usefulness of protein analyses in the future.

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