scholarly journals Single-Molecule Analysis Methods Using Nanogap Electrodes and Their Application to DNA Sequencing Technologies

2017 ◽  
Vol 90 (11) ◽  
pp. 1189-1210 ◽  
Author(s):  
Masateru Taniguchi
Keyword(s):  
Dna Sequencing ◽  
Single Molecule ◽  
Sequencing Technologies ◽  
Analysis Methods ◽  
Nanogap Electrodes ◽  
Single Molecule Analysis

Perspectives and Challenges of Emerging Single-Molecule DNA Sequencing Technologies

Small ◽  
2009 ◽  
Vol 5 (23) ◽  
pp. 2638-2649 ◽  
Author(s):  
Mingsheng Xu ◽  
Daisuke Fujita ◽  
Nobutaka Hanagata
Keyword(s):  
Dna Sequencing ◽  
Single Molecule ◽  
Sequencing Technologies

scholarly journals Single-molecule DNA sequencing of widely varying GC-content using nucleotide release, capture and detection in microdroplets

2020 ◽  
Vol 48 (22) ◽  
pp. e132-e132
Author(s):  
Tim J Puchtler ◽  
Kerr Johnson ◽  
Rebecca N Palmer ◽  
Emma L Talbot ◽  
Lindsey A Ibbotson ◽  
...  
Keyword(s):  
Dna Sequencing ◽  
Single Molecule ◽  
Direct Detection ◽  
Gc Content ◽  
Cost Effective ◽  
Epigenetic Modifications ◽  
Fluorescence Signal ◽  
Sequencing Platform ◽  
Sequencing Technologies ◽  
Lower Accuracy

Abstract Despite remarkable progress in DNA sequencing technologies there remains a trade-off between short-read platforms, having limited ability to sequence homopolymers, repeated motifs or long-range structural variation, and long-read platforms, which tend to have lower accuracy and/or throughput. Moreover, current methods do not allow direct readout of epigenetic modifications from a single read. With the aim of addressing these limitations, we have developed an optical electrowetting sequencing platform that uses step-wise nucleotide triphosphate (dNTP) release, capture and detection in microdroplets from single DNA molecules. Each microdroplet serves as a reaction vessel that identifies an individual dNTP based on a robust fluorescence signal, with the detection chemistry extended to enable detection of 5-methylcytosine. Our platform uses small reagent volumes and inexpensive equipment, paving the way to cost-effective single-molecule DNA sequencing, capable of handling widely varying GC-bias, and demonstrating direct detection of epigenetic modifications.

scholarly journals Implementation and Data Analysis of Tn-seq, Whole-Genome Resequencing, and Single-Molecule Real-Time Sequencing for Bacterial Genetics

2016 ◽  
Vol 199 (1) ◽  
Author(s):  
Peter E. Burby ◽  
Taylor M. Nye ◽  
Jeremy W. Schroeder ◽  
Lyle A. Simmons
Keyword(s):  
Dna Sequencing ◽  
Real Time ◽  
Single Molecule ◽  
De Novo ◽  
Large Data ◽  
Data Sets ◽  
Bacterial Genetics ◽  
Sequencing Technologies ◽  
Bioinformatics Tools ◽  
Whole Genome Resequencing

ABSTRACT Few discoveries have been more transformative to the biological sciences than the development of DNA sequencing technologies. The rapid advancement of sequencing and bioinformatics tools has revolutionized bacterial genetics, deepening our understanding of model and clinically relevant organisms. Although application of newer sequencing technologies to studies in bacterial genetics is increasing, the implementation of DNA sequencing technologies and development of the bioinformatics tools required for analyzing the large data sets generated remain a challenge for many. In this minireview, we have chosen to summarize three sequencing approaches that are particularly useful for bacterial genetics. We provide resources for scientists new to and interested in their application. Here, we discuss the analysis of data from transposon mutagenesis followed by deep sequencing (Tn-seq) to determine gene disruptions differentially represented in a mutant population and Illumina sequencing for identification of suppressor or other mutations, and we summarize single-molecule real-time (SMRT) sequencing for de novo genome assembly and the use of the output data for detection of DNA base modifications.

Direct Read and Quantify Damage Nucleotide from an Oligonucleotide Using a Single Molecule Interface

2019 ◽  
Author(s):  
Jiajun Wang ◽  
Meng-Yin Li ◽  
Jie Yang ◽  
Ya-Qian Wang ◽  
Xue-Yuan Wu ◽  
...  
Keyword(s):  
Dna Sequencing ◽  
Single Molecule ◽  
Genetic Diseases ◽  
High Sensitivity ◽  
Dna Lesions ◽  
Lesion Detection ◽  
The Novel ◽  
Structural Variations ◽  
Dna Lesion ◽  
Base Lesion

DNA lesion such as metholcytosine(<sup>m</sup>C), 8-OXO-guanine(<sup>O</sup>G), inosine(I) <i>etc</i> could cause the genetic diseases. Identification of the varieties of lesion bases are usually beyond the capability of conventional DNA sequencing which is mainly designed to discriminate four bases only. Therefore, lesion detection remain challenge due to the massive varieties and less distinguishable readouts for minor structural variations. Moreover, standard amplification and labelling hardly works in DNA lesions detection. Herein, we designed a single molecule interface from the mutant K238Q Aerolysin, whose confined sensing region shows the high compatible to capture and then directly convert each base lesion into distinguishable current readouts. Compared with previous single molecule sensing interface, the resolution of the K238Q Aerolysin nanopore is enhanced by 2-order. The novel K238Q could direct discriminate at least 3 types (<sup>m</sup>C, <sup>O</sup>G, I) lesions without lableing and quantify modification sites under mixed hetero-composition condition of oligonucleotide. Such nanopore could be further applied to diagnose genetic diseases at high sensitivity.

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