From single-molecule detection to next-generation sequencing: microfluidic droplets for high-throughput nucleic acid analysis

ABSTRACT The Centers for Disease Control and Prevention recently emphasized the need for enhanced technologies to use in investigations of outbreaks of foodborne illnesses. To address this need, e-probe diagnostic nucleic acid analysis (EDNA) was adapted and validated as a tool for the rapid, effective identification and characterization of multiple pathogens in a food matrix. In EDNA, unassembled next generation sequencing data sets from food sample metagenomes are queried using pathogen-specific sequences known as electronic probes (e-probes). In this study, the query of mock sequence databases demonstrated the potential of EDNA for the detection of foodborne pathogens. The method was then validated using next generation sequencing data sets created by sequencing the metagenome of alfalfa sprouts inoculated with Escherichia coli O157:H7. Nonspecific hits in the negative control sample indicated the need for additional filtration of the e-probes to enhance specificity. There was no significant difference in the ability of an e-probe to detect the target pathogen based upon the length of the probe set oligonucleotides. The results from the queries of the sample database using E. coli e-probe sets were significantly different from those obtained using random decoy probe sets and exhibited 100% precision. The results support the use of EDNA as a rapid response methodology in foodborne outbreaks and investigations for establishing comprehensive microbial profiles of complex food samples.

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E-probe Diagnostic Nucleic acid Analysis (EDNA): A theoretical approach for handling of next generation sequencing data for diagnostics

Journal of Microbiological Methods ◽

10.1016/j.mimet.2013.07.002 ◽

2013 ◽

Vol 94 (3) ◽

pp. 356-366 ◽

Cited By ~ 26

Author(s):

Anthony H. Stobbe ◽

Jon Daniels ◽

Andres S. Espindola ◽

Ruchi Verma ◽

Ulrich Melcher ◽

...

Keyword(s):

Next Generation Sequencing ◽

Nucleic Acid ◽

Theoretical Approach ◽

Next Generation Sequencing Data ◽

Next Generation ◽

Sequencing Data ◽

Nucleic Acid Analysis ◽

Generation Sequencing

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Laboratory implementation of Next Generation Sequencing for medium and high throughput whole genome analysis of clinical M. tuberculosis complex strains

10.26226/morressier.5991c409d462b80292388d15 ◽

2017 ◽

Author(s):

Christian Utpatel

Keyword(s):

Next Generation Sequencing ◽

High Throughput ◽

Genome Analysis ◽

Whole Genome ◽

Next Generation ◽

Tuberculosis Complex ◽

Whole Genome Analysis ◽

Generation Sequencing

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Next-Generation Sequencing: An Emerging Tool for Drug Designing

Current Pharmaceutical Design ◽

10.2174/1381612825666190911155508 ◽

2019 ◽

Vol 25 (31) ◽

pp. 3350-3357 ◽

Cited By ~ 1

Author(s):

Pooja Tripathi ◽

Jyotsna Singh ◽

Jonathan A. Lal ◽

Vijay Tripathi

Keyword(s):

Next Generation Sequencing ◽

High Throughput ◽

Large Scale ◽

Massively Parallel Sequencing ◽

Genomic Research ◽

Biological Research ◽

Next Generation ◽

Human Welfare ◽

Drug Designing ◽

Generation Sequencing

Background: With the outbreak of high throughput next-generation sequencing (NGS), the biological research of drug discovery has been directed towards the oncology and infectious disease therapeutic areas, with extensive use in biopharmaceutical development and vaccine production. Method: In this review, an effort was made to address the basic background of NGS technologies, potential applications of NGS in drug designing. Our purpose is also to provide a brief introduction of various Nextgeneration sequencing techniques. Discussions: The high-throughput methods execute Large-scale Unbiased Sequencing (LUS) which comprises of Massively Parallel Sequencing (MPS) or NGS technologies. The Next geneinvolved necessarily executes Largescale Unbiased Sequencing (LUS) which comprises of MPS or NGS technologies. These are related terms that describe a DNA sequencing technology which has revolutionized genomic research. Using NGS, an entire human genome can be sequenced within a single day. Conclusion: Analysis of NGS data unravels important clues in the quest for the treatment of various lifethreatening diseases and other related scientific problems related to human welfare.

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Next-Generation Sequencing: From Basic Research to Diagnostics

Clinical Chemistry ◽

10.1373/clinchem.2008.112789 ◽

2009 ◽

Vol 55 (4) ◽

pp. 641-658 ◽

Cited By ~ 459

Author(s):

Karl V Voelkerding ◽

Shale A Dames ◽

Jacob D Durtschi

Keyword(s):

Next Generation Sequencing ◽

Dna Sequencing ◽

Single Molecule ◽

Basic Research ◽

Clinical Diagnostics ◽

Massively Parallel ◽

Next Generation ◽

New Paradigm ◽

The Impact ◽

Generation Sequencing

Abstract Background: For the past 30 years, the Sanger method has been the dominant approach and gold standard for DNA sequencing. The commercial launch of the first massively parallel pyrosequencing platform in 2005 ushered in the new era of high-throughput genomic analysis now referred to as next-generation sequencing (NGS). Content: This review describes fundamental principles of commercially available NGS platforms. Although the platforms differ in their engineering configurations and sequencing chemistries, they share a technical paradigm in that sequencing of spatially separated, clonally amplified DNA templates or single DNA molecules is performed in a flow cell in a massively parallel manner. Through iterative cycles of polymerase-mediated nucleotide extensions or, in one approach, through successive oligonucleotide ligations, sequence outputs in the range of hundreds of megabases to gigabases are now obtained routinely. Highlighted in this review are the impact of NGS on basic research, bioinformatics considerations, and translation of this technology into clinical diagnostics. Also presented is a view into future technologies, including real-time single-molecule DNA sequencing and nanopore-based sequencing. Summary: In the relatively short time frame since 2005, NGS has fundamentally altered genomics research and allowed investigators to conduct experiments that were previously not technically feasible or affordable. The various technologies that constitute this new paradigm continue to evolve, and further improvements in technology robustness and process streamlining will pave the path for translation into clinical diagnostics.

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Advances in genetics and molecular breeding of three legume crops of semi-arid tropics using next-generation sequencing and high-throughput genotyping technologies

Journal of Biosciences ◽

10.1007/s12038-012-9228-0 ◽

2012 ◽

Vol 37 (5) ◽

pp. 811-820 ◽

Cited By ~ 44

Author(s):

Rajeev K Varshney ◽

Himabindu Kudapa ◽

Manish Roorkiwal ◽

Mahendar Thudi ◽

Manish K Pandey ◽

...

Keyword(s):

Next Generation Sequencing ◽

High Throughput ◽

Molecular Breeding ◽

Next Generation ◽

Legume Crops ◽

Generation Sequencing ◽

Semi Arid

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Metagenomic Next-Generation Sequencing of Bloodstream Microbial Cell-Free Nucleic Acid in Children With Suspected Sepsis in Pediatric Intensive Care Unit

Frontiers in Cellular and Infection Microbiology ◽

10.3389/fcimb.2021.665226 ◽

2021 ◽

Vol 11 ◽

Author(s):

Gangfeng Yan ◽

Jing Liu ◽

Weiming Chen ◽

Yang Chen ◽

Ye Cheng ◽

...

Keyword(s):

Intensive Care Unit ◽

Intensive Care ◽

Next Generation Sequencing ◽

Nucleic Acid ◽

Pediatric Intensive Care Unit ◽

Pediatric Intensive Care ◽

Microbial Cell ◽

Next Generation ◽

Suspected Sepsis ◽

Generation Sequencing

Bloodstream infection is a life-threatening complication in critically ill patients. Multi-drug resistant bacteria or fungi may increase the risk of invasive infections in hospitalized children and are difficult to treat in intensive care units. The purpose of this study was to use metagenomic next-generation sequencing (mNGS) to understand the bloodstream microbiomes of children with suspected sepsis in a pediatric intensive care unit (PICU). mNGS were performed on microbial cell-free nucleic acid from 34 children admitted to PICU, and potentially pathogenic microbes were identified. The associations of serological inflammation indicators, lymphocyte subpopulations, and other clinical phenotypes were also examined. mNGS of blood samples from children in PICU revealed potential eukaryotic microbial pathogens. The abundance of Pneumocystis jirovecii was positively correlated with a decrease in total white blood cell count and immunodeficiency. Hospital-acquired pneumonia patients showed a significant increase in blood bacterial species richness compared with community-acquired pneumonia children. The abundance of bloodstream bacteria was positively correlated with serum procalcitonin level. Microbial genome sequences from potential pathogens were detected in the bloodstream of children with suspected sepsis in PICU, suggesting the presence of bloodstream infections in these children.

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Robust Sub-nanomolar Library Preparation for High Throughput Next Generation Sequencing

BMC Genomics ◽

10.1186/s12864-018-4677-y ◽

2018 ◽

Vol 19 (1) ◽

Cited By ~ 7

Author(s):

Wells W. Wu ◽

Je-Nie Phue ◽

Chun-Ting Lee ◽

Changyi Lin ◽

Lai Xu ◽

...

Keyword(s):

Next Generation Sequencing ◽

High Throughput ◽

Library Preparation ◽

Next Generation ◽

Generation Sequencing

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Evaluation of TypeSeq, a Novel High-Throughput, Low-Cost, Next-Generation Sequencing-Based Assay for Detection of 51 Human Papillomavirus Genotypes

The Journal of Infectious Diseases ◽

10.1093/infdis/jiz324 ◽

2019 ◽

Vol 220 (10) ◽

pp. 1609-1619 ◽

Cited By ~ 4

Author(s):

Sarah Wagner ◽

David Roberson ◽

Joseph Boland ◽

Aimée R Kreimer ◽

Meredith Yeager ◽

...

Keyword(s):

Human Papillomavirus ◽

Next Generation Sequencing ◽

High Throughput ◽

Vaccine Efficacy ◽

Linear Array ◽

Human Papillomaviruses ◽

Next Generation ◽

Hpv Genotyping ◽

Positive Agreement ◽

Generation Sequencing

AbstractBackgroundHuman papillomaviruses (HPV) cause over 500 000 cervical cancers each year, most of which occur in low-resource settings. Human papillomavirus genotyping is important to study natural history and vaccine efficacy. We evaluated TypeSeq, a novel, next-generation, sequencing-based assay that detects 51 HPV genotypes, in 2 large international epidemiologic studies.MethodsTypeSeq was evaluated in 2804 cervical specimens from the Study to Understand Cervical Cancer Endpoints and Early Determinants (SUCCEED) and in 2357 specimens from the Costa Rica Vaccine Trial (CVT). Positive agreement and risks of precancer for individual genotypes were calculated for TypeSeq in comparison to Linear Array (SUCCEED). In CVT, positive agreement and vaccine efficacy were calculated for TypeSeq and SPF10-LiPA.ResultsWe observed high overall and positive agreement for most genotypes between TypeSeq and Linear Array in SUCCEED and SPF10-LiPA in CVT. There was no significant difference in risk of precancer between TypeSeq and Linear Array in SUCCEED or in estimates of vaccine efficacy between TypeSeq and SPF10-LiPA in CVT.ConclusionsThe agreement of TypeSeq with Linear Array and SPF10-LiPA, 2 well established standards for HPV genotyping, demonstrates its high accuracy. TypeSeq provides high-throughput, affordable HPV genotyping for world-wide studies of cervical precancer risk and of HPV vaccine efficacy.

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