scholarly journals Single nucleotide detection using bilayer MoS2 nanopores with high efficiency

RSC Advances ◽  
2021 ◽  
Vol 11 (11) ◽  
pp. 6114-6123
Author(s):  
Payel Sen ◽  
Manisha Gupta
Keyword(s):  
Dna Sequencing ◽  
Dwell Time ◽  
High Efficiency ◽  
Single Nucleotide

Bilayer MoS2 nanopores are suitable for fast and high-efficiency single nucleotide detection and DNA sequencing due to fast analyte capture and improved dwell time.

scholarly journals Highly multiplexed, fast and accurate nanopore sequencing for verification of synthetic DNA constructs and sequence libraries

2019 ◽  
Vol 4 (1) ◽  
Author(s):  
Andrew Currin ◽  
Neil Swainston ◽  
Mark S Dunstan ◽  
Adrian J Jervis ◽  
Paul Mulherin ◽  
...  
Keyword(s):  
Synthetic Biology ◽  
Dna Sequencing ◽  
Cost Effective ◽  
Polymorphism Analysis ◽  
Sequencing Data ◽  
Single Nucleotide Variants ◽  
Single Nucleotide ◽  
Synthetic Dna ◽  
Design Build ◽  
Hardware Costs

Abstract Synthetic biology utilizes the Design–Build–Test–Learn pipeline for the engineering of biological systems. Typically, this requires the construction of specifically designed, large and complex DNA assemblies. The availability of cheap DNA synthesis and automation enables high-throughput assembly approaches, which generates a heavy demand for DNA sequencing to verify correctly assembled constructs. Next-generation sequencing is ideally positioned to perform this task, however with expensive hardware costs and bespoke data analysis requirements few laboratories utilize this technology in-house. Here a workflow for highly multiplexed sequencing is presented, capable of fast and accurate sequence verification of DNA assemblies using nanopore technology. A novel sample barcoding system using polymerase chain reaction is introduced, and sequencing data are analyzed through a bespoke analysis algorithm. Crucially, this algorithm overcomes the problem of high-error rate nanopore data (which typically prevents identification of single nucleotide variants) through statistical analysis of strand bias, permitting accurate sequence analysis with single-base resolution. As an example, 576 constructs (6 × 96 well plates) were processed in a single workflow in 72 h (from Escherichia coli colonies to analyzed data). Given our procedure’s low hardware costs and highly multiplexed capability, this provides cost-effective access to powerful DNA sequencing for any laboratory, with applications beyond synthetic biology including directed evolution, single nucleotide polymorphism analysis and gene synthesis.

scholarly journals Single-molecule detection of deoxyribonucleoside triphosphates in microdroplets

2019 ◽  
Vol 47 (17) ◽  
pp. e101-e101 ◽  
Author(s):  
Boris Breiner ◽  
Kerr Johnson ◽  
Magdalena Stolarek ◽  
Ana-Luisa Silva ◽  
Aurel Negrea ◽  
...  
Keyword(s):  
Dna Sequencing ◽  
Single Molecule ◽  
Dna Polymerases ◽  
Single Molecule Detection ◽  
Synthesis Methods ◽  
Single Nucleotide ◽  
New Approach ◽  
Fluorescent Signal ◽  
Single Molecule Level ◽  
Current Sequence

AbstractA new approach to single-molecule DNA sequencing in which dNTPs, released by pyrophosphorolysis from the strand to be sequenced, are captured in microdroplets and read directly could have substantial advantages over current sequence-by-synthesis methods; however, there is no existing method sensitive enough to detect a single nucleotide in a microdroplet. We have developed a method for dNTP detection based on an enzymatic two-stage reaction which produces a robust fluorescent signal that is easy to detect and process. By taking advantage of the inherent specificity of DNA polymerases and ligases, coupled with volume restriction in microdroplets, this method allows us to simultaneously detect the presence of and distinguish between, the four natural dNTPs at the single-molecule level, with negligible cross-talk.

scholarly journals Homogeneous Real-Time Detection of Single-Nucleotide Polymorphisms by Strand Displacement Amplification on the BD ProbeTec ET System

2003 ◽  
Vol 49 (10) ◽  
pp. 1599-1607 ◽  
Author(s):  
Sha-Sha Wang ◽  
Keith Thornton ◽  
Andrew M Kuhn ◽  
James G Nadeau ◽  
Tobin J Hellyer
Keyword(s):  
Single Nucleotide Polymorphisms ◽  
Dna Sequencing ◽  
Real Time ◽  
Whole Blood ◽  
Nucleotide Polymorphisms ◽  
Strand Displacement ◽  
Strand Displacement Amplification ◽  
Single Nucleotide ◽  
Buccal Swabs ◽  
Real Time Detection

Abstract Background: The BD ProbeTec™ ET System is based on isothermal strand displacement amplification (SDA) of target nucleic acid coupled with homogeneous real-time detection using fluorescent probes. We have developed a novel, rapid method using this platform that incorporates a universal detection format for identification of single-nucleotide polymorphisms (SNPs) and other genotypic variations. Method: The system uses a common pair of fluorescent Detector Probes in conjunction with unlabeled allele-specific Adapter Primers and a universal buffer chemistry to permit analysis of multiple SNP loci under generic assay conditions. We used Detector Probes labeled with different dyes to facilitate differentiation of two alternative alleles in a single reaction with no postamplification manipulation. We analyzed six SNPs within the human β2-adrenergic receptor (β2AR) gene, using whole blood, buccal swabs, and urine samples, and compared results with those obtained by DNA sequencing. Results: Unprocessed whole blood was successfully genotyped with as little as 0.1–1 μL of sample per reaction. All six β2AR assays were able to accommodate ≥20 μL of unprocessed whole blood. For the 14 individuals tested, genotypes determined with the six β2AR assays agreed with DNA sequencing results. Conclusion: SDA-based allelic differentiation on the BD ProbeTec ET System can detect SNPs rapidly, using whole blood, buccal swabs, or urine.

scholarly journals Discovery of Single Nucleotide Polymorphisms and Mutations by Pyrosequencing

2002 ◽  
Vol 3 (1) ◽  
pp. 51-56 ◽  
Author(s):  
Mostafa Ronaghi ◽  
Elahe Elahi
Keyword(s):  
Comparative Genomics ◽  
Dna Sequencing ◽  
Large Scale ◽  
Nucleotide Polymorphisms ◽  
Single Nucleotide ◽  
Mutation Scanning ◽  
Large Scale Analysis ◽  
Polymorphism Discovery ◽  
Sequencing Method ◽  
The Cost

Comparative genomics, analyzing variation among individual genomes, is an area of intense investigation. DNA sequencing is usually employed to look for polymorphisms and mutations. Pyrosequencing, a real-time DNA sequencing method, is emerging as a popular platform for comparative genomics. Here we review the use of this technology for mutation scanning, polymorphism discovery and chemical haplotyping. We describe the methodology and accuracy of this technique and discuss how to reduce the cost for large-scale analysis.

scholarly journals Elucidation of Japanese pepper (Zanthoxylum piperitum De Candolle) domestication using RAD-Seq

2020 ◽  
Author(s):  
Maddumage Dona Ginushika Priyadarsh Premarathne ◽  
Nami Fukutome ◽  
Kazuaki Yamasaki ◽  
Fumiyo Hayakawa ◽  
Atsushi J. Nagano ◽  
...  
Keyword(s):  
Dna Sequencing ◽  
Restriction Site ◽  
Early Stage ◽  
Agricultural Fields ◽  
Single Nucleotide Variants ◽  
Single Nucleotide ◽  
Second Stage ◽  
Zanthoxylum Piperitum ◽  
Production Area

AbstractJapanese pepper, Zanthoxylum piperitum, is native to Japan and has four famous varieties: Asakura, Takahara, Budou, and Arima, named after their production area or morphology. Restriction-site associated DNA sequencing (RAD-Seq) was used to analyse 93 accessions from various areas, including these four varieties. The analysis of the single nucleotide variants was used to classify the plants into eight groups: each of the Asakura and Arima varieties has two groups, each of the Takahara and Budou varieties has one group, and two additional groups are present. In one group of the Asakura varieties and two groups of the Arima varieties, the plants were present in agricultural fields and mountains, representing the early stage of domestication of the Japanese pepper. The second group of the Asakura varieties was of genetically close plants present in various areas, which represents the second stage of domestication of this plant because, after early domestication, genetically related varieties of excellent traits spread to the periphery. These results demonstrate that the domestication of the Japanese pepper is ongoing. In addition, this study shows that spineless plants are polyphyletic, despite the spineless variety being considered a subspecies of the Japanese pepper.

scholarly journals MQuad enables clonal substructure discovery using single cell mitochondrial variants

2021 ◽  
Author(s):  
Aaron Wing Cheung Kwok ◽  
Chen Qiao ◽  
Rongting Huang ◽  
Mai-Har Sham ◽  
Joshua W. K. Ho ◽  
...  
Keyword(s):  
Dna Sequencing ◽  
Single Cell ◽  
Single Cells ◽  
High Sensitivity ◽  
Copy Number Variations ◽  
Sequencing Data ◽  
Single Nucleotide ◽  
Single Cell Sequencing ◽  
Mtdna Variants ◽  
Python Package

AbstractMitochondrial mutations are increasingly recognised as informative endogenous genetic markers that can be used to reconstruct cellular clonal structure using single-cell RNA or DNA sequencing data. However, there is a lack of effective computational methods to identify informative mtDNA variants in noisy and sparse single-cell sequencing data. Here we present an open source computational tool MQuad that accurately calls clonally informative mtDNA variants in a population of single cells, and an analysis suite for complete clonality inference, based on single cell RNA or DNA sequencing data. Through a variety of simulated and experimental single cell sequencing data, we showed that MQuad can identify mitochondrial variants with both high sensitivity and specificity, outperforming existing methods by a large extent. Furthermore, we demonstrated its wide applicability in different single cell sequencing protocols, particularly in complementing single-nucleotide and copy-number variations to extract finer clonal resolution. MQuad is a Python package available via https://github.com/single-cell-genetics/MQuad.

scholarly journals Epidemic Territorial Spread of IncP-2-Type VIM-2 Carbapenemase-Encoding Megaplasmids in Nosocomial Pseudomonas aeruginosa Populations

2021 ◽  
Vol 65 (4) ◽  
Author(s):  
Paweł Urbanowicz ◽  
Ibrahim Bitar ◽  
Radosław Izdebski ◽  
Anna Baraniak ◽  
Elżbieta Literacka ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Large Scale ◽  
High Efficiency ◽  
Donor Cell ◽  
Stress Factors ◽  
Single Nucleotide ◽  
S1 Nuclease ◽  
Content Type ◽  
Short Read Sequence ◽  
Sequence Types

ABSTRACT In 2003 to 2004, the first five VIM-2 metallo-β-lactamase (MBL)-producing Pseudomonas aeruginosa (MPPA) isolates with an In4-like integron, In461 (aadB-blaVIM-2-aadA6), on conjugative plasmids were identified in three hospitals in Poland. In 2005 to 2015, MPPA expanded much in the country, and as many as 80 isolates in a collection of 454 MPPA (∼18%) had In461, one of the two most common MBL-encoding integrons. The organisms occurred in 49 hospitals in 33 cities of 11/16 main administrative regions. Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) classified them into 55 pulsotypes and 35 sequence types (STs), respectively, revealing their remarkable genetic diversity overall, with only a few small clonal clusters. S1 nuclease/hybridization assays and mating of 63 representative isolates showed that ∼85% of these had large In461-carrying plasmids, ∼350 to 550 kb, usually self-transmitting with high efficiency (∼10−1 to 10−2 per donor cell). The plasmids from 19 isolates were sequenced and subjected to structural and single-nucleotide-polymorphism (SNP)-based phylogenetic analysis. These formed a subgroup within a family of IncP-2-type megaplasmids, observed worldwide in pseudomonads from various environments and conferring resistance/tolerance to multiple stress factors, including antibiotics. Their microdiversity in Poland arose mainly from acquisition of different accessory fragments, as well as new resistance genes and multiplication of these. Short-read sequence and/or PCR mapping confirmed the In461-carrying plasmids in the remaining isolates to be the IncP-2 types. The study demonstrated a large-scale epidemic spread of multidrug resistance plasmids in P. aeruginosa populations, creating an epidemiological threat. It contributes to the knowledge on IncP-2 types, which are interesting research objects in resistance epidemiology, environmental microbiology, and biotechnology.

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