Segmentation of Noisy Signals Generated By a Nanopore

2015 ◽  
Author(s):  
Jacob Matthew Schreiber ◽  
Kevin Karplus
Keyword(s):  
Real Time ◽  
Single Molecule ◽  
Ionic Current ◽  
Divide And Conquer ◽  
Single Molecule Sequencing ◽  
Automated Method ◽  
Noisy Signals ◽  
Low Pass ◽  
Current Steps ◽  
Pass Through

Nanopore-based single-molecule sequencing techniques exploit ionic current steps produced as biomolecules pass through a pore to reconstruct properties of the sequence. A key task in analyzing complex nanopore data is discovering the boundaries between these steps, which has traditionally been done in research labs by hand. We present an automated method of analyzing nanopore data, by detecting regions of ionic current corresponding to the translocation of a biomolecule, and then segmenting the region. The segmenter uses a divide-and-conquer method to recursively discover boundary points, with an implementation that works several times faster than real time and that can handle low-pass filtered signals.

A review of DNA sequencing techniques

2002 ◽  
Vol 35 (2) ◽  
pp. 169-200 ◽  
Author(s):  
Lilian T. C. França ◽  
Emanuel Carrilho ◽  
Tarso B. L. Kist
Keyword(s):  
Dna Sequencing ◽  
Real Time ◽  
Single Molecule ◽  
Fluorescent Dyes ◽  
Read Length ◽  
Chemical Methods ◽  
Single Molecule Sequencing ◽  
Exonuclease Digestion ◽  
Speed Accuracy ◽  
Sanger Method

1. Summary 1692. Introduction 1703. Sanger's method and other enzymic methods 1703.1 Random approach 1713.2 Direct approach 1713.3 Enzyme technology 1753.4 Sample preparation 1753.5 Labels and DNA labelling 1763.5.1 Radioisotopes 1763.5.2 Chemiluminescent detection 1763.5.3 Fluorescent dyes 1773.6 Fragment separation and analysis 1803.6.1 Electrophoresis 1803.6.2 Mass spectrometry – an alternative 1824. Maxam & Gilbert and other chemical methods 1835. Pyrosequencing – DNA sequencing in real time by the detection of released PPi 1876. Single molecule sequencing with exonuclease 1907. Conclusion 1928. Acknowledgements 1929. References 193The four best known DNA sequencing techniques are reviewed. Important practical issues covered are read-length, speed, accuracy, throughput, cost, as well as the automation of sample handling and preparation. The methods reviewed are: (i) the Sanger method and its most important variants (enzymic methods); (ii) the Maxam & Gilbert method and other chemical methods; (iii) the PyrosequencingTM method – DNA sequencing in real time by the detection of released pyrophosphate (PPi); and (iv) single molecule sequencing with exonuclease (exonuclease digestion of a single molecule composed of a single strand of fluorescently labelled deoxynucleotides). Each method is briefly described, the current literature is covered, advantages, disadvantages, and the most suitable applications of each method are discussed.

scholarly journals The Nanopore-Tweezing-Based, Targeted Detection of Nucleobases on Short Functionalized Peptide-Nucleic Acid Sequences

2021 ◽  
Author(s):  
Isabela Dragomir ◽  
Alina Asandei ◽  
Irina Schiopu ◽  
Ioana Bucataru ◽  
Loredana Mereuta ◽  
...  
Keyword(s):  
Single Molecule ◽  
Peptide Nucleic Acid ◽  
Peptide Nucleic Acids ◽  
Conformational Dynamics ◽  
Ionic Current ◽  
Single Molecule Sequencing ◽  
The Past ◽  
Stochastic Fluctuations ◽  
Targeted Detection ◽  
Recognition Capability

Quantum leaps advances in the single-molecule investigative science have been made possible over the past decades through the implication of nanopores, as versatile components on dedicated biosensors. Here, we employed the nanopore-tweezing technique to capture amino acid-functionalized, peptide-nucleic acids (PNA) with -hemolysin-based nanopores, and correlate the ensuing stochastic fluctuations of the ionic current through the nanopore with the composition and order of bases in the PNAs primary structure. We demonstrate that while the system enables detection of distinct bases on homopolymeric PNA or triplet bases on heteropolymeric strands, it also reveals rich insights into the conformational dynamics of the entrapped PNA within the nanopore, relevant for perfecting the recognition capability single-molecule sequencing.

scholarly journals Massively multiplex single-molecule oligonucleosome footprinting

2020 ◽  
Author(s):  
Nour J Abdulhay ◽  
Colin P McNally ◽  
Laura J Hsieh ◽  
Sivakanthan Kasinathan ◽  
Aidan Keith ◽  
...  
Keyword(s):  
Real Time ◽  
High Throughput ◽  
Single Molecule ◽  
Nucleosome Occupancy ◽  
Binding Motifs ◽  
Single Molecule Sequencing ◽  
Genome Wide ◽  
Sequencing Method ◽  
Transcription Factor Binding Motifs

ABSTRACTOur understanding of the beads-on-a-string arrangement of nucleosomes has been built largely on high-resolution sequence-agnostic imaging methods and sequence-resolved bulk biochemical techniques. To bridge the divide between these approaches, we present the single-molecule adenine methylated oligonucleosome sequencing assay (SAMOSA). SAMOSA is a high-throughput single-molecule sequencing method that combines adenine methyltransferase footprinting and single-molecule real-time DNA sequencing to natively and nondestructively measure nucleosome positions on individual chromatin fibres. SAMOSA data allows unbiased classification of single-molecular ‘states’ of nucleosome occupancy on individual chromatin fibres. We leverage this to estimate nucleosome regularity and spacing on single chromatin fibres genome-wide, at predicted transcription factor binding motifs, and across both active and silent human epigenomic domains. Our analyses suggest that chromatin is comprised of a diverse array of both regular and irregular single-molecular oligonucleosome patterns that differ subtly in their relative abundance across epigenomic domains. This irregularity is particularly striking in constitutive heterochromatin, which has typically been viewed as a conformationally static entity. Our proof-of-concept study provides a powerful new methodology for studying nucleosome organization at a previously intractable resolution, and offers up new avenues for modeling and visualizing higher-order chromatin structure.1-sentence summaryHigh-throughput single-molecule real-time footprinting of chromatin arrays reveals heterogeneous patterns of oligonucleosome occupancy.

scholarly journals The Nanopore-Tweezing-Based, Targeted Detection of Nucleobases on Short Functionalized Peptide Nucleic Acid Sequences

Polymers ◽  
2021 ◽  
Vol 13 (8) ◽  
pp. 1210
Author(s):  
Isabela S. Dragomir ◽  
Alina Asandei ◽  
Irina Schiopu ◽  
Ioana C. Bucataru ◽  
Loredana Mereuta ◽  
...  
Keyword(s):  
Single Molecule ◽  
Peptide Nucleic Acid ◽  
Conformational Dynamics ◽  
Ionic Current ◽  
Molecular Medicine ◽  
Single Molecule Sequencing ◽  
Stochastic Fluctuations ◽  
Wide Range ◽  
Targeted Detection ◽  
Recognition Capability

The implication of nanopores as versatile components in dedicated biosensors, nanoreactors, or miniaturized sequencers has considerably advanced single-molecule investigative science in a wide range of disciplines, ranging from molecular medicine and nanoscale chemistry to biophysics and ecology. Here, we employed the nanopore tweezing technique to capture amino acid-functionalized peptide nucleic acids (PNAs) with α-hemolysin-based nanopores and correlated the ensuing stochastic fluctuations of the ionic current through the nanopore with the composition and order of bases in the PNAs primary structure. We demonstrated that while the system enables the detection of distinct bases on homopolymeric PNA or triplet bases on heteropolymeric strands, it also reveals rich insights into the conformational dynamics of the entrapped PNA within the nanopore, relevant for perfecting the recognition capability of single-molecule sequencing.

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