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

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.

Download Full-text

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 ◽

10.3390/ani11102976 ◽

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.

Download Full-text

Detecting SARS-CoV-2 variants with SNP genotyping

10.1101/2020.11.18.388140 ◽

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.

Download Full-text

Detecting SARS-CoV-2 variants with SNP genotyping

PLoS ONE ◽

10.1371/journal.pone.0243185 ◽

2021 ◽

Vol 16 (2) ◽

pp. e0243185 ◽

Cited By ~ 2

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.

Download Full-text

Kinect V2 Performance Assessment in Daily-Life Gestures: Cohort Study on Healthy Subjects for a Reference Database for Automated Instrumental Evaluations on Neurological Patients

Applied Bionics and Biomechanics ◽

10.1155/2017/8567084 ◽

2017 ◽

Vol 2017 ◽

pp. 1-16 ◽

Cited By ~ 7

Author(s):

Alessandro Scano ◽

Andrea Chiavenna ◽

Matteo Malosio ◽

Lorenzo Molinari Tosatti

Keyword(s):

Healthy Subjects ◽

Daily Life ◽

Low Cost ◽

Cost Effective ◽

Major Effect ◽

Healthy People ◽

Reference Database ◽

Kinect V2 ◽

Effective Strategies ◽

Neurological Patients

Background. The increase of sanitary costs related to poststroke rehabilitation requires new sustainable and cost-effective strategies for promoting autonomous and dehospitalized motor training. In the Riprendo@Home and Future Home for Future Communities research projects, the promising approach of introducing low-cost technologies that promote home rehabilitation is exploited. In order to provide reliable evaluation of patients, a reference database of healthy people’s performances is required and should consider variability related to healthy people performances.Methods.78 healthy subjects performed several repetitions of daily-life gestures, the reaching movement (RM) and hand-to-mouth (HtMM) movement with both the dominant and nondominant upper limbs. Movements were recorded with a Kinect V2. A synthetic biomechanical protocol based on kinematical, dynamical, and motor control parameters was used to assess motor performance of the healthy people. The investigation was conducted by clustering participants depending on their limb dominancy (right/left), gender (male/female), and age (young/middle/senior) as sources of variability.Results.Results showed that limb dominancy has minor relevance in affecting RM and HtMM; gender has relevance in affecting the HtMM; age has major effect in affecting RM and HtMM.Conclusions.An investigation of healthy subjects’ upper limb performances during daily-life gestures was performed with the Kinect V2 sensor. Findings will be the basis for a database of normative data for neurological patients’ motor evaluation.

Download Full-text

MinION barcodes: biodiversity discovery and identification by everyone, for everyone

10.1101/2021.03.09.434692 ◽

2021 ◽

Author(s):

Amrita Srivathsan ◽

Leshon Lee ◽

Kazutaka Katoh ◽

Emily Hartop ◽

Sujatha Narayanan Kutty ◽

...

Keyword(s):

Real Time ◽

Cost Effective ◽

Biodiversity Loss ◽

Flow Cell ◽

Dna Barcodes ◽

Flow Cells ◽

Sequencing Errors ◽

Sequencing Cost ◽

Science Labs ◽

Barcode Sequencing

AbstractDNA barcodes are a useful tool for discovering, understanding, and monitoring biodiversity. This is critical at a time when biodiversity loss is a major problem for many countries. However, widespread adoption of barcoding programs requires the process to be cost-effective and simple to apply. We here present a workflow that satisfies these conditions. It was developed via “innovation through subtraction” and thus requires minimal lab equipment, can be learned within days, reduces the barcode sequencing cost to <10 cents, and allows fast turnaround from specimen to sequence by using the real-time sequencer MinION. We first describe cost-effective and rapid procedures in a comprehensive workflow for obtaining tagged amplicons. We then demonstrate how a portable MinION device can be used for real-time sequencing of tagged amplicons in many settings (field stations, biodiversity labs, citizen science labs, schools). Small projects can use the flow cell dongle (“Flongle”) while large projects can rely on MinION flow cells that can be stopped and re-used after collecting sufficient data for a given project. We also provide amplicon coverage recommendations that are based on several runs of MinION flow cells (R10.3) involving >24,000 specimen barcodes, which suggest that each run can generate >10,000 barcodes. Additionally, we present a novel software, ONTbarcoder, that overcomes the bioinformatics challenges posed by the sequencing errors of MinION reads. This software is compatible with Windows10, Macintosh, and Linux, has a graphical user interface (GUI), and can generate thousands of barcodes on a standard laptop within hours based on two input files (FASTQ, demultiplexing file). Next, we document that MinION barcodes are virtually identical to Sanger and Illumina barcodes for the same specimens (>99.99%). Lastly, we demonstrate how rapidly MinION data have improved by comparing the performance of sequential flow cell generations. We overall assert that barcoding with MinION is the way forward for government agencies, universities, museums, and schools because it combines low consumable and capital cost with scalability. Biodiversity loss is threatening the planet and the use of MinION barcodes will help with enabling an army of researchers and citizen scientists, which is necessary for effective biodiversity discovery and monitoring.

Download Full-text

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

10.1101/2020.05.30.20117721 ◽

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.

Download Full-text

ONTbarcoder and MinION barcodes aid biodiversity discovery and identification by everyone, for everyone

BMC Biology ◽

10.1186/s12915-021-01141-x ◽

2021 ◽

Vol 19 (1) ◽

Cited By ~ 1

Author(s):

Amrita Srivathsan ◽

Leshon Lee ◽

Kazutaka Katoh ◽

Emily Hartop ◽

Sujatha Narayanan Kutty ◽

...

Keyword(s):

Graphical User Interface ◽

Cost Effective ◽

Biodiversity Loss ◽

Flow Cell ◽

Government Agencies ◽

Dna Barcodes ◽

Flow Cells ◽

Sequencing Cost ◽

Science Labs ◽

Barcode Sequencing

Abstract Background DNA barcodes are a useful tool for discovering, understanding, and monitoring biodiversity which are critical tasks at a time of rapid biodiversity loss. However, widespread adoption of barcodes requires cost-effective and simple barcoding methods. We here present a workflow that satisfies these conditions. It was developed via “innovation through subtraction” and thus requires minimal lab equipment, can be learned within days, reduces the barcode sequencing cost to < 10 cents, and allows fast turnaround from specimen to sequence by using the portable MinION sequencer. Results We describe how tagged amplicons can be obtained and sequenced with the real-time MinION sequencer in many settings (field stations, biodiversity labs, citizen science labs, schools). We also provide amplicon coverage recommendations that are based on several runs of the latest generation of MinION flow cells (“R10.3”) which suggest that each run can generate barcodes for > 10,000 specimens. Next, we present a novel software, ONTbarcoder, which overcomes the bioinformatics challenges posed by MinION reads. The software is compatible with Windows 10, Macintosh, and Linux, has a graphical user interface (GUI), and can generate thousands of barcodes on a standard laptop within hours based on only two input files (FASTQ, demultiplexing file). We document that MinION barcodes are virtually identical to Sanger and Illumina barcodes for the same specimens (> 99.99%) and provide evidence that MinION flow cells and reads have improved rapidly since 2018. Conclusions We propose that barcoding with MinION is the way forward for government agencies, universities, museums, and schools because it combines low consumable and capital cost with scalability. Small projects can use the flow cell dongle (“Flongle”) while large projects can rely on MinION flow cells that can be stopped and re-used after collecting sufficient data for a given project.

Download Full-text

Evaluation Of SYBR Green Real Time PCR For Detecting SARS-CoV-2 From Clinical Samples

10.1101/2020.05.13.093609 ◽

2020 ◽

Author(s):

Álvaro Fajardo ◽

Marianoel Pereira-Gómez ◽

Natalia Echeverría ◽

Fernando López-Tort ◽

Paula Perbolianachis ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Pcr Amplification ◽

Cost Effective ◽

Laboratory Diagnosis ◽

Sybr Green ◽

World Health ◽

Diagnostic Tools ◽

Clinical Samples ◽

One Step

ABSTRACTThe pandemic caused by SARS-CoV-2 has triggered an extraordinary collapse of healthcare systems and hundred thousand of deaths worldwide. Following the declaration of the outbreak as a Public Health Emergency of International Concern by the World Health Organization (WHO) on January 30th, 2020, it has become imperative to develop diagnostic tools to reliably detect the virus in infected patients. Several methods based on real time reverse transcription polymerase chain reaction (RT-qPCR) for the detection of SARS-CoV-2 genomic RNA have been developed. In addition, these methods have been recommended by the WHO for laboratory diagnosis. Since all these protocols are based on the use of fluorogenic probes and one-step reagents (cDNA synthesis followed by PCR amplification in the same tube), these techniques can be difficult to perform given the limited supply of reagents in low and middle income countries. In the interest of economy, time and availability of chemicals and consumables, the SYBR Green-based detection was implemented to establish a convenient assay. Therefore, we adapted one of WHO recommended Taqman-based one-step real time PCR protocols (from the University of Hong Kong) to SYBR Green. Our results suggest that SYBR-Green detection represents a reliable cost-effective alternative to increase the testing capacity.

Download Full-text

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

International Journal of Medical and Biomedical Studies ◽

10.32553/ijmbs.v4i2.1185 ◽

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.

Download Full-text

Janus – A New Approach to Air Combat Pilot Training

Transactions on Aerospace Research ◽

10.2478/tar-2019-0019 ◽

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”.

Download Full-text