scholarly journals A Multiplex Pharmacogenetics Assay using the MinION Nanopore Sequencing Device

2019 ◽  
Author(s):  
Yusmiati Liau ◽  
Simone L. Cree ◽  
Simran Maggo ◽  
Allison L. Miller ◽  
John F. Pearson ◽  
...  

AbstractAimThe MinION nanopore sequencing device opens the opportunity to cost-effective and point-of-care DNA sequencing. We developed a multiplex assay targeting pharmacogenetic variants related to clopidogrel and warfarin, two commonly used drugs that show response variability due to genetic polymorphisms.Materials & MethodsSix reference and 78 clinical DNA samples were amplified by PCR to generate 15 amplicons targeting key variants. These products were then barcoded to enable sample multiplexing. Three variant calling tools were used to compare genotyping accuracy.Results and ConclusionsAll but three samples were successfully sequenced and genotyped. Nanopolish software achieved accuracy > 90 % for all except one variant. While minor mis-genotyping issues exist, this work demonstrates that drug-specific or broad pharmacogenetic screening assays are possible on the MinION sequencing device.

2021 ◽  
Vol 9 (12) ◽  
pp. 2598
Author(s):  
Anton Pembaur ◽  
Erwan Sallard ◽  
Patrick Philipp Weil ◽  
Jennifer Ortelt ◽  
Parviz Ahmad-Nejad ◽  
...  

The scale of the ongoing SARS-CoV-2 pandemic warrants the urgent establishment of a global decentralized surveillance system to recognize local outbreaks and the emergence of novel variants of concern. Among available deep-sequencing technologies, nanopore-sequencing could be an important cornerstone, as it is mobile, scalable, and cost-effective. Therefore, streamlined nanopore-sequencing protocols need to be developed and optimized for SARS-CoV-2 variants identification. We adapted and simplified existing workflows using the ‘midnight’ 1200 bp amplicon split primer sets for PCR, which produce tiled overlapping amplicons covering almost the entire SARS-CoV-2 genome. Subsequently, we applied Oxford Nanopore Rapid Barcoding and the portable MinION Mk1C sequencer combined with the interARTIC bioinformatics pipeline. We tested a simplified and less time-consuming workflow using SARS-CoV-2-positive specimens from clinical routine and identified the CT value as a useful pre-analytical parameter, which may help to decrease sequencing failures rates. Complete pipeline duration was approx. 7 h for one specimen and approx. 11 h for 12 multiplexed barcoded specimens. The adapted protocol contains fewer processing steps and can be completely conducted within one working day. Diagnostic CT values deduced from qPCR standardization experiments can act as principal criteria for specimen selection. As a guideline, SARS-CoV-2 genome copy numbers lower than 4 × 106 were associated with a coverage threshold below 20-fold and incompletely assembled SARS-CoV-2 genomes. Thus, based on the described thermocycler/chemistry combination, we recommend CT values of ~26 or lower to achieve full and high-quality SARS-CoV-2 (+)RNA genome coverage.


Diagnostics ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 1352
Author(s):  
Darius Riziki Martin ◽  
Nicole Remaliah Sibuyi ◽  
Phumuzile Dube ◽  
Adewale Oluwaseun Fadaka ◽  
Ruben Cloete ◽  
...  

The transmission of Tuberculosis (TB) is very rapid and the burden it places on health care systems is felt globally. The effective management and prevention of this disease requires that it is detected early. Current TB diagnostic approaches, such as the culture, sputum smear, skin tuberculin, and molecular tests are time-consuming, and some are unaffordable for low-income countries. Rapid tests for disease biomarker detection are mostly based on immunological assays that use antibodies which are costly to produce, have low sensitivity and stability. Aptamers can replace antibodies in these diagnostic tests for the development of new rapid tests that are more cost effective; more stable at high temperatures and therefore have a better shelf life; do not have batch-to-batch variations, and thus more consistently bind to a specific target with similar or higher specificity and selectivity and are therefore more reliable. Advancements in TB research, in particular the application of proteomics to identify TB specific biomarkers, led to the identification of a number of biomarker proteins, that can be used to develop aptamer-based diagnostic assays able to screen individuals at the point-of-care (POC) more efficiently in resource-limited settings.


2021 ◽  
Vol 13 (1) ◽  
Author(s):  
Andrew W. Kirkpatrick ◽  
Jessica L. McKee ◽  
John M. Conly

AbstractCOVID-19 has impacted human life globally and threatens to overwhelm health-care resources. Infection rates are rapidly rising almost everywhere, and new approaches are required to both prevent transmission, but to also monitor and rescue infected and at-risk patients from severe complications. Point-of-care lung ultrasound has received intense attention as a cost-effective technology that can aid early diagnosis, triage, and longitudinal follow-up of lung health. Detecting pleural abnormalities in previously healthy lungs reveal the beginning of lung inflammation eventually requiring mechanical ventilation with sensitivities superior to chest radiographs or oxygen saturation monitoring. Using a paradigm first developed for space-medicine known as Remotely Telementored Self-Performed Ultrasound (RTSPUS), motivated patients with portable smartphone support ultrasound probes can be guided completely remotely by a remote lung imaging expert to longitudinally follow the health of their own lungs. Ultrasound probes can be couriered or even delivered by drone and can be easily sterilized or dedicated to one or a commonly exposed cohort of individuals. Using medical outreach supported by remote vital signs monitoring and lung ultrasound health surveillance would allow clinicians to follow and virtually lay hands upon many at-risk paucisymptomatic patients. Our initial experiences with such patients are presented, and we believe present a paradigm for an evolution in rich home-monitoring of the many patients expected to become infected and who threaten to overwhelm resources if they must all be assessed in person by at-risk care providers.


The Analyst ◽  
2021 ◽  
Author(s):  
Diwakar M. Awate ◽  
Cicero C. Pola ◽  
Erica Shumaker ◽  
Carmen L Gomes ◽  
Jaime Javier Juarez

Despite having widespread application in the biomedical sciences, flow cytometers have several limitations that prevent their application to point-of-care (POC) diagnostics in resource-limited environments. 3D printing provides a cost-effective approach...


2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Shan Wei ◽  
Esther Kohl ◽  
Alexandre Djandji ◽  
Stephanie Morgan ◽  
Susan Whittier ◽  
...  

AbstractThe COVID-19 pandemic has resulted in an urgent need for a rapid, point of care diagnostic testing that could be rapidly scaled on a worldwide level. We developed and tested a highly sensitive and robust assay based on reverse transcription loop mediated isothermal amplification (RT-LAMP) that uses readily available reagents and a simple heat block using contrived spike-in and actual clinical samples. RT-LAMP testing on RNA-spiked samples showed a limit of detection (LoD) of 2.5 copies/μl of viral transport media. RT-LAMP testing directly on clinical nasopharyngeal swab samples in viral transport media had an 85% positive percentage agreement (PPA) (17/20), and 100% negative percentage agreement (NPV) and delivered results in 30 min. Our optimized RT-LAMP based testing method is a scalable system that is sufficiently sensitive and robust to test for SARS-CoV-2 directly on clinical nasopharyngeal swab samples in viral transport media in 30 min at the point of care without the need for specialized or proprietary equipment or reagents. This cost-effective and efficient one-step testing method can be readily available for COVID-19 testing world-wide, especially in resource poor settings.


2015 ◽  
Vol 69 (1) ◽  
Author(s):  
Pawan Jolly ◽  
Nello Formisano ◽  
Pedro Estrela

AbstractThe use of aptamers in biosensing has attracted considerable attention as an alternative to antibodies because of their unique properties such as long-term stability, cost-effectiveness and adjustability to various applications. Among cancers, the early diagnosis of prostate cancer (PCa) is one of the greatest concerns for ageing men worldwide. One of the most commonly used biomarkers for PCa is prostate-specific antigen (PSA), which can be found in elevated levels in patients with cancer. This review presents the gradual transition of research from antibody-based to aptamerbased biosensors, specifically for PSA. A brief description on aptamer-based biosensing for other PCa biomarkers is also presented. Special attention is given to electrochemical methods as analytical techniques for the development of simple, sensitive and cost-effective biosensors. The review also focuses on the different surface chemistries exploited for fabrication and their applications in clinical samples. The use of aptamers represents a promising tool for the development of point-ofcare biosensors for the early detection of prostate cancer. In view of the unmatched upper hand of aptamers, future prospects are also discussed, not only in the point-of-care format but also in other novel applications.


Antibiotics ◽  
2018 ◽  
Vol 7 (4) ◽  
pp. 106 ◽  
Author(s):  
Emily Holmes ◽  
Sharman Harris ◽  
Alison Hughes ◽  
Noel Craine ◽  
Dyfrig Hughes

More appropriate and measured use of antibiotics may be achieved using point-of-care (POC) C-reactive protein (CRP) testing, but there is limited evidence of cost-effectiveness in routine practice. A decision analytic model was developed to estimate the cost-effectiveness of testing, compared with standard care, in adults presenting in primary care with symptoms of acute respiratory tract infection (ARTI). Analyses considered (1) pragmatic use of testing, reflective of routine clinical practice, and (2) testing according to clinical guidelines. Threshold and scenario analysis were performed to identify cost-effective scenarios. In patients with symptoms of ARTI and based on routine practice, the incremental cost-effectiveness ratios of CRP testing were £19,705 per quality-adjusted-life-year (QALY) gained and £16.07 per antibiotic prescription avoided. Following clinical guideline, CRP testing in patients with lower respiratory tract infections (LRTIs) cost £4390 per QALY gained and £9.31 per antibiotic prescription avoided. At a threshold of £20,000 per QALY, the probabilities of POC CRP testing being cost-effective were 0.49 (ARTI) and 0.84 (LRTI). POC CRP testing as implemented in routine practice is appreciably less cost-effective than when adhering to clinical guidelines. The implications for antibiotic resistance and Clostridium difficile infection warrant further investigation.


2017 ◽  
Vol 99 (6) ◽  
pp. 490-496 ◽  
Author(s):  
KSS Dayananda ◽  
VY Kong ◽  
JL Bruce ◽  
GV Oosthuizen ◽  
GL Laing ◽  
...  

INTRODUCTION Selective non-operative management (SNOM) of abdominal stab wounds is well established in South Africa. SNOM reduces the morbidity associated with negative laparotomies while being safe. Despite steady advances in technology (including laparoscopy, computed tomography [CT] and point-of-care sonography), our approach has remained clinically driven. Assessments of financial implications are limited in the literature. The aim of this study was to review isolated penetrating abdominal trauma and analyse associated incurred expenses. METHODS Patients data across the Pietermaritzburg Metropolitan Trauma Service (PMTS) are captured prospectively into the regional electronic trauma registry. A bottom-up microcosting technique produced estimated average costs for our defined clinical protocols. RESULTS Between January 2012 and April 2015, 501 patients were treated for an isolated abdominal stab wound. Over one third (38%) were managed successfully with SNOM, 5% underwent a negative laparotomy and over half (57%) required a therapeutic laparotomy. Over five years, the PMTS can expect to spend a minimum of ZAR 20,479,800 (GBP 1,246,840) for isolated penetrating abdominal stab wounds alone. CONCLUSIONS Provided a stringent policy is followed, in carefully selected patients, SNOM is effective in detecting those who require further intervention. It minimises the risks associated with unnecessary surgical interventions. SNOM will continue to be clinically driven and promulgated in our environment.


2019 ◽  
Author(s):  
Hyou-Arm Joung ◽  
Zachary S. Ballard ◽  
Jing Wu ◽  
Derek K. Tseng ◽  
Hailemariam Teshome ◽  
...  

ABSTRACTCaused by the tick-borne spirochete, Borrelia burgdorferi, Lyme disease (LD) is the most common vector-borne infectious disease in North America and Europe. Though timely diagnosis and treatment are effective in preventing disease progression, current tests are insensitive in early-stage LD, with a sensitivity <50%. Additionally, the serological testing currently recommended by the US Center for Disease Control has high costs (>$400/test) and extended sample-to-answer timelines (>24 hours). To address these challenges, we created a cost-effective and rapid point-of-care (POC) test for early-stage LD that assays for antibodies specific to seven Borrelia antigens and a synthetic peptide in a paper-based multiplexed vertical flow assay (xVFA). We trained a deep learning-based diagnostic algorithm to select an optimal subset of antigen/peptide targets, and then blindly-tested our xVFA using human samples (N(+) = 42, N(−)= 54), achieving an area-under-the-curve (AUC), sensitivity, and specificity of 0.950, 90.5%, and 87.0% respectively, outperforming previous LD POC tests. With batch-specific standardization and threshold tuning, the specificity of our blind-testing performance improved to 96.3%, with an AUC and sensitivity of 0.963 and 85.7%, respectively.


2020 ◽  
Author(s):  
Timour Baslan ◽  
Sam Kovaka ◽  
Fritz J. Sedlazeck ◽  
Yanming Zhang ◽  
Robert Wappel ◽  
...  

ABSTRACTGenome copy number is an important source of genetic variation in health and disease. In cancer, clinically actionable Copy Number Alterations (CNAs) can be inferred from short-read sequencing data, enabling genomics-based precision oncology. Emerging Nanopore sequencing technologies offer the potential for broader clinical utility, for example in smaller hospitals, due to lower instrument cost, higher portability, and ease of use. Nonetheless, Nanopore sequencing devices are limited in terms of the number of retrievable sequencing reads/molecules compared to short-read sequencing platforms. This represents a challenge for applications that require high read counts such as CNA inference. To address this limitation, we targeted the sequencing of short-length DNA molecules loaded at optimized concentration in an effort to increase sequence read/molecule yield from a single nanopore run. We show that sequencing short DNA molecules reproducibly returns high read counts and allows high quality CNA inference. We demonstrate the clinical relevance of this approach by accurately inferring CNAs in acute myeloid leukemia samples. The data shows that, compared to traditional approaches such as chromosome analysis/cytogenetics, short molecule nanopore sequencing returns more sensitive, accurate copy number information in a cost effective and expeditious manner, including for multiplex samples. Our results provide a framework for the sequencing of relatively short DNA molecules on nanopore devices with applications in research and medicine, that include but are not limited to, CNAs.


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