scholarly journals INSIGHT: A population-scale COVID-19 testing strategy combining point-of-care diagnosis with centralized high-throughput sequencing

2021 ◽  
Vol 7 (7) ◽  
pp. eabe5054
Author(s):  
Qianxin Wu ◽  
Chenqu Suo ◽  
Tom Brown ◽  
Tengyao Wang ◽  
Sarah A. Teichmann ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Limit Of Detection ◽  
Point Of Care ◽  
Population Level ◽  
Reaction Products ◽  
Testing Strategy ◽  
Asymptomatic Patients ◽  
Testing Stage ◽  
Population Scale

We present INSIGHT [isothermal NASBA (nucleic acid sequence–based amplification) sequencing–based high-throughput test], a two-stage coronavirus disease 2019 testing strategy, using a barcoded isothermal NASBA reaction. It combines point-of-care diagnosis with next-generation sequencing, aiming to achieve population-scale testing. Stage 1 allows a quick decentralized readout for early isolation of presymptomatic or asymptomatic patients. It gives results within 1 to 2 hours, using either fluorescence detection or a lateral flow readout, while simultaneously incorporating sample-specific barcodes. The same reaction products from potentially hundreds of thousands of samples can then be pooled and used in a highly multiplexed sequencing–based assay in stage 2. This second stage confirms the near-patient testing results and facilitates centralized data collection. The 95% limit of detection is <50 copies of viral RNA per reaction. INSIGHT is suitable for further development into a rapid home-based, point-of-care assay and is potentially scalable to the population level.

scholarly journals INSIGHT: a population scale COVID-19 testing strategy combining point-of-care diagnosis with centralised high-throughput sequencing

2020 ◽  
Author(s):  
Qianxin Wu ◽  
Chenqu Suo ◽  
Tom Brown ◽  
Tengyao Wang ◽  
Sarah A. Teichmann ◽  
...  
Keyword(s):  
High Throughput Sequencing ◽  
Limit Of Detection ◽  
Point Of Care ◽  
Viral Rna ◽  
Testing Strategy ◽  
Two Stage ◽  
Asymptomatic Patients ◽  
Testing Stage ◽  
Population Scale ◽  
Stage 1

AbstractWe present INSIGHT (Isothermal NASBA-Sequencing-based hIGH-througput Test): a two-stage COVID-19 testing strategy, using a barcoded isothermal NASBA reaction that combines point-of-care diagnosis with next generation sequencing, aiming to achieve population-scale COVID-19 testing. INSIGHT combines the advantages of near-patient with centralised testing. Stage 1 allows a quick decentralised readout for early isolation of pre-symptomatic or asymptomatic patients. The same reaction products can then be used in a highly multiplexed sequencing-based assay in Stage 2, confirming the near-patient testing results and facilitating centralised data collection. Based on experiments using commercially acquired human saliva with spiked-in viral RNA as input, the INSIGHT platform gives Stage 1 results within one to two hours, using either fluorescence detection or a lateral flow (dipstick) readout, whilst simultaneously incorporating sample-specific barcodes into the amplification product. INSIGHT Stage 2 can be performed by directly pooling and sequencing all post-amplification barcoded Stage 1 products from hundreds of thousands of samples with minimal sample preparation steps. The 95% limit of detection (LoD-95) for INSIGHT is estimated to be below 50 copies of viral RNA per 20 μl of reaction. Our two-stage testing strategy is suitable for further development into a rapid home-based and point-of-care assay, and is potentially scalable to the population level.

scholarly journals Selection and Identification of Novel Aptamers Specific for Clenbuterol Based on ssDNA Library Immobilized SELEX and Gold Nanoparticles Biosensor

Molecules ◽  
2018 ◽  
Vol 23 (9) ◽  
pp. 2337 ◽  
Author(s):  
Xixia Liu ◽  
Qi Lu ◽  
Sirui Chen ◽  
Fang Wang ◽  
Jianjun Hou ◽  
...  
Keyword(s):  
Gold Nanoparticles ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Limit Of Detection ◽  
Retention Rate ◽  
Pcr Amplification ◽  
Cross Reactivity ◽  
Enriched Library ◽  
Amplification Curve ◽  
Specificity And Sensitivity

We describe a multiple combined strategy to discover novel aptamers specific for clenbuterol (CBL). An immobilized ssDNA library was used for the selection of specific aptamers using the systematic evolution of ligands by exponential enrichment (SELEX). Progress was monitored using real-time quantitative PCR (Q-PCR), and the enriched library was sequenced by high-throughput sequencing. Candidate aptamers were picked and preliminarily identified using a gold nanoparticles (AuNPs) biosensor. Bioactive aptamers were characterized for affinity, circular dichroism (CD), specificity and sensitivity. The Q-PCR amplification curve increased and the retention rate was about 1% at the eighth round. Use of the AuNPs biosensor and CD analyses determined that six aptamers had binding activity. Affinity analysis showed that aptamer 47 had the highest affinity (Kd = 42.17 ± 8.98 nM) with no cross reactivity to CBL analogs. Indirect competitive enzyme linked aptamer assay (IC-ELAA) based on a 5′-biotin aptamer 47 indicated the limit of detection (LOD) was 0.18 ± 0.02 ng/L (n = 3), and it was used to detect pork samples with a mean recovery of 83.33–97.03%. This is the first report of a universal strategy including library fixation, Q-PCR monitoring, high-throughput sequencing, and AuNPs biosensor identification to select aptamers specific for small molecules.

scholarly journals Molecular beacon assay development for Severe Acute Respiratory Syndrome Coronavirus 2 detection

2021 ◽  
Author(s):  
Josué Carvalho ◽  
Jéssica Lopes Nunes ◽  
Joana Figueiredo ◽  
Tiago Santos ◽  
André Miranda ◽  
...  
Keyword(s):  
Molecular Beacon ◽  
Point Of Care ◽  
Total Duration ◽  
Fluorescence Analysis ◽  
Population Level ◽  
Clinical Samples ◽  
Test Duration ◽  
Upper Respiratory Tract ◽  
Testing Strategy ◽  
Two Stage

The fast spread of SARS-CoV-2 has led to a global pandemic, calling for fast and accurate assays to allow infection diagnosis and prevention of transmission. We aimed to develop a molecular beacon (MB)-based detection assay for SARS-CoV-2, designed to, detect the ORF1ab and S genes, proposing a two-stage COVID-19 testing strategy, using MBs to detect the presence of target amplicons by fluorescence analysis. Two MBs were designed, optimized in terms of concentration, fluorescence plateaus of hybridization, reaction temperature and best real-time results. A total of 450 nasopharyngel and throat swab samples (418 positive and 32 negative) were tested with the MB assay and the fluorescence levels compared with the cycle threshold (Ct) values obtained from a commercial RT-PCR test in terms of test duration, sensitivity and specificity. Our results show that the samples with higher fluorescence levels correspond to those with low Ct values, suggesting a correlation between viral load and increased MB fluorescence. The proposed assay represents a fast (total duration of 2 h 20 min including amplification and fluorescence reading stages) and simple way of detecting SARS-CoV-2 in clinical samples from the upper respiratory tract. Our two-stage testing strategy is suitable for further development into a point-of-care assay and potentially scalable to population level.

Profiling population-level diversity and dynamics of Accumulibacter via high throughput sequencing of ppk1

2019 ◽  
Vol 103 (23-24) ◽  
pp. 9711-9722 ◽  
Author(s):  
Wei Song ◽  
Min Jia Zheng ◽  
Hao Li ◽  
Wei Zheng ◽  
Feng Guo
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Population Level

scholarly journals A comparison of DNA/RNA extraction protocols for high-throughput sequencing of microbial communities

2020 ◽  
Author(s):  
Justin P. Shaffer ◽  
Clarisse Marotz ◽  
Pedro Belda-Ferre ◽  
Cameron Martino ◽  
Stephen Wandro ◽  
...  
Keyword(s):  
Microbial Community ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Limit Of Detection ◽  
Sequence Data ◽  
Rna Virus ◽  
Microbial Community Composition ◽  
Rna Extraction ◽  
Acid Extraction ◽  
Dna And Rna

AbstractOne goal among microbial ecology researchers is to capture the maximum amount of information from all organisms in a sample. The recent COVID-19 pandemic, caused by the RNA virus SARS-CoV-2, has highlighted a gap in traditional DNA-based protocols, including the high-throughput methods we previously established as field standards. To enable simultaneous SARS-CoV-2 and microbial community profiling, we compare the relative performance of two total nucleic acid extraction protocols and our previously benchmarked protocol. We included a diverse panel of environmental and host-associated sample types, including body sites commonly swabbed for COVID-19 testing. Here we present results comparing the cost, processing time, DNA and RNA yield, microbial community composition, limit of detection, and well-to-well contamination, between these protocols.Accession numbersRaw sequence data were deposited at the European Nucleotide Archive (accession#: ERP124610) and raw and processed data are available at Qiita (Study ID: 12201). All processing and analysis code is available on GitHub (github.com/justinshaffer/Extraction_test_MagMAX).Methods summaryTo allow for downstream applications involving RNA-based organisms such as SARS-CoV-2, we compared the two extraction protocols designed to extract DNA and RNA against our previously established protocol for extracting only DNA for microbial community analyses. Across 10 diverse sample types, one of the two protocols was equivalent or better than our established DNA-based protocol. Our conclusion is based on per-sample comparisons of DNA and RNA yield, the number of quality sequences generated, microbial community alpha- and beta-diversity and taxonomic composition, the limit of detection, and extent of well-to-well contamination.

scholarly journals Towards routine employment of computational tools for antimicrobial resistance determination via high-throughput sequencing

2021 ◽  
Author(s):  
Simone Marini ◽  
Rodrigo Mora ◽  
Christina Boucher ◽  
Noelle Noyes ◽  
Mattia Prosperi
Keyword(s):  
Antimicrobial Resistance ◽  
High Throughput ◽  
Bacterial Infections ◽  
High Throughput Sequencing ◽  
Markov Models ◽  
Point Of Care ◽  
Economic Loss ◽  
Clinical Samples ◽  
Sequencing Data ◽  
Data Repositories

Antimicrobial resistance (AMR) is a growing threat to public health and farming at large. Without appropriate interventions, it can lead to millions of deaths per year and substantial economic loss worldwide. In clinical and veterinary practice, a timely characterization of the antibiotic susceptibility profile of bacterial infections is a crucial step in optimizing treatment. Fast turnaround of AMR testing is also needed in food safety and infection control surveillance (e.g., contamination of healthcare or long-term nursing facilities). High-throughput sequencing is a promising option for clinical point-of-care and ecological surveillance, opening the opportunity to develop genotyping-based AMR determination as a possibly faster alternative to phenotypic testing. In the present work, we compare the performance of state-of-the-art methods for detection of AMR from high-throughput sequencing data in healthcare settings. We consider five complementary computational approaches --alignment (AMRPlusPlus), deep learning (DeepARG), k-mer genomic signatures (KARGA, ResFinder), and hidden Markov models (Meta-MARC). We use an extensive collection of clinical studies never employed for model training. To do so, we assemble data from multiple, independent AMR high-throughput sequencing experiments collected in a variety of hospital settings, comprising of 585 isolates with a available AMR resistance profiles determined by phenotypic tests across nine antibiotic classes. We show how the prediction landscape of AMR classifiers is highly heterogeneous, with balanced accuracy varying from 0.4 to 0.92. Although some algorithms---ResFinder, KARGA, and AMRPlusPlus-- exhibit overall better balanced accuracy than others, the high per-AMR-class variance and related findings suggest that: (1) all algorithms might be subject to sampling bias present both in data repositories used for training and experimental/clinical settings; and (2) a portion of clinical samples might contain uncharacterized AMR genes that the algorithms---mostly trained on known AMR genes---fail to generalize upon. These results lead us to formulate practical advice for software configuration and application, as well as give suggestions for future study design to further develop AMR prediction tools from proof-of-concept to bedside.

scholarly journals A 'Variant Viewer' for investigating population-level variation in Arabidopsis thaliana

2021 ◽  
Author(s):  
Matthew Cumming ◽  
Eddi Esteban ◽  
Vincent Lau ◽  
Asher Pasha ◽  
Nicholas J. Provart
Keyword(s):  
Arabidopsis Thaliana ◽  
Genetic Variation ◽  
High Throughput ◽  
Web Application ◽  
High Throughput Sequencing ◽  
Population Level ◽  
Gene Families ◽  
Level Variation ◽  
Splice Isoforms ◽  
Large Populations

High throughput sequencing has opened the doors for investigators to probe genetic variation present in large populations of organisms. In plants, the 1001 Genomes Project (1001genomes.org) is one such effort that sought to characterize the extant worldwide variation in Arabidopsis thaliana for future analyses to compare and draw upon. We developed a web application that accesses the 1001 Genomes database called The Variant Viewer, for investigators to view variants in any A. thaliana gene and within gene families. These variants may be visualized in the context of alignments of queried genes, across splice isoforms of these genes and in relation to conserved domains.

scholarly journals A comparison of DNA/RNA extraction protocols for high-throughput sequencing of microbial communities

BioTechniques ◽  
2021 ◽  
Author(s):  
Justin P Shaffer ◽  
Clarisse Marotz ◽  
Pedro Belda-Ferre ◽  
Cameron Martino ◽  
Stephen Wandro ◽  
...  
Keyword(s):  
Microbial Community ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Limit Of Detection ◽  
Rna Virus ◽  
Microbial Community Composition ◽  
Rna Extraction ◽  
Acid Extraction ◽  
Dna And Rna ◽  
Community Profiling

One goal of microbial ecology researchers is to capture the maximum amount of information from all organisms in a sample. The recent COVID-19 pandemic, caused by the RNA virus SARS-CoV-2, has highlighted a gap in traditional DNA-based protocols, including the high-throughput methods the authors previously established as field standards. To enable simultaneous SARS-CoV-2 and microbial community profiling, the authors compared the relative performance of two total nucleic acid extraction protocols with the authors' previously benchmarked protocol. The authors included a diverse panel of environmental and host-associated sample types, including body sites commonly swabbed for COVID-19 testing. Here the authors present results comparing the cost, processing time, DNA and RNA yield, microbial community composition, limit of detection and well-to-well contamination between these protocols.

scholarly journals Novel Insights for Biosurveillance of Bat-Borne Viruses

Proceedings ◽  
2020 ◽  
Vol 50 (1) ◽  
pp. 47
Author(s):  
Adrian C. Paskey ◽  
Justin H. J. Ng ◽  
Gregory K. Rice ◽  
Wan Ni Chia ◽  
Casandra W. Philipson ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Temporal Patterns ◽  
Population Level ◽  
The Body ◽  
Population Persistence ◽  
Target Enrichment ◽  
Species Barrier ◽  
Future Directions ◽  
New Knowledge

Bats are rich reservoirs of viruses, including several high consequence zoonoses. In this study, high throughput sequencing was used to characterize the virome through a longitudinal study of a captive colony of lesser dawn bats, species Eonycteris spelaea, in Singapore. This study utilized viral RNA extracted from swabs of four body sites per bat per timepoint. Swabs of the exterior of the bat (head and body) were used to evaluate virus populations and demonstrate utility as a sample site for future surveillance to extrapolate population-level infection. Through unbiased shotgun and target-enrichment sequencing, we identified both the known and previously unknown viruses of zoonotic relevance and defined the population persistence and temporal patterns of viruses from families that have the capacity to jump the species barrier. We observed the population persistence of three zoonotic-related viral families that are known to be associated with spillover from bats to humans: Paramyxoviridae, Reoviridae, and Coronaviridae. To our knowledge, this is the first study that combines probe-based viral enrichment with high-throughput sequencing or that creates a viral profile from multiple swab sites on individual bats and their cohorts. This work demonstrates temporal patterns of the lesser dawn bat virome, including several novel viruses. Noninvasive surveillance methods that target the body of bats not only detect viruses shed within the colony but can also represent viral populations dispersed throughout the entire colony. New knowledge of persistent viral families should inform future directions for the biosurveillance of viruses that have the potential to cross the species barrier from bats to humans or other amplifying hosts.

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