MOSAIC: A Modular Single-Molecule Analysis Interface for Decoding Multistate Nanopore Data

Forstater, Jacob H.; Briggs, Kyle; Robertson, Joseph W. F.; Ettedgui, Jessica; Marie-Rose, Olivier; Vaz, Canute; Kasianowicz, John J.; Tabard‐Cossa, Vincent; Balijepalli, Arvind

doi:10.1021/acs.analchem.6b03725

Cited by 97 publications

(94 citation statements)

References 58 publications

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“…Data is post-filtered for analysis at 900 kHz using a digital low-pass Bessel filter, and analyzed to extract passage times and sub-level structure using both the adept2state module of MOSAIC 51,52 (https://pages.nist.gov/mosaic/) for 100 bp molecules which do not fold, and a custom implementation of the CUSUM+ algorithm 52,53 for the rest of the events (https://github.com/shadowk29/CUSUM). Both analysis programs are available freely online.…”

Section: Methodsmentioning

confidence: 99%

DNA Translocations through Nanopores under Nanoscale Preconfinement

et al. 2017

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To reduce unwanted variation in the passage speed of DNA through solid-state nanopores, we demonstrate nanoscale pre-confinement of translocating molecules using an ultra-thin nanoporous silicon nitride membrane separated from a single sensing nanopore by a nanoscale cavity. We present comprehensive experimental and simulation results demonstrating that the presence of an integrated nanofilter within nanoscale distances of the sensing pore eliminates the dependence of molecular passage time distributions on pore size, revealing a global minimum in the coefficient of variation of the passage time. These results provide experimental verification that the inter- and intra-molecular passage time variation depends on the conformational entropy of each molecule prior to translocation. Furthermore, we show that the observed consistently narrower passage time distributions enables a more reliable DNA length separation independent of pore size and stability. We also demonstrate that the composite nanofilter/nanopore devices can be configured to suppress the frequency of folded translocations, ensuring single-file passage of captured DNA molecules. By greatly increasing the rate at which usable data can be collected, these unique attributes will offer significant practical advantages to many solid-state nanopore-based sensing schemes, including sequencing, genomic mapping, and barcoded target detection.

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Section: Methodsmentioning

confidence: 99%

DNA Translocations through Nanopores under Nanoscale Preconfinement

et al. 2017

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“…Data were filtered at 10 kHz and acquired at 250 kHz using the DigiData 1200 digitizer with a custom National Instruments LabVIEW program. Data was processed and events were detected using MOSAIC 101 as follows. The values for the open pore current ( I o ) and the standard deviation of the noise ( σ ) was extracted (108 and 8.3 pA, respectively, for β -CD, Figure S13).…”

Section: Methodsmentioning

confidence: 99%

Porphyrin-Assisted Docking of a Thermophage Portal Protein into Lipid Bilayers: Nanopore Engineering and Characterization

Cressiot¹,

Greive

et al. 2017

ACS Nano

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Nanopore-based sensors for nucleic acid sequencing and single-molecule detection typically employ pore-forming membrane proteins with hydrophobic external surfaces, suitable for insertion into a lipid bilayer. In contrast, hydrophilic pore-containing molecules, such as DNA origami, have been shown to require chemical modification to favor insertion into a lipid environment. In this work, we describe a strategy for inserting polar proteins with an inner pore into lipid membranes, focusing here on a circular 12-subunit assembly of the thermophage G20c portal protein. X-ray crystallography, electron microscopy, molecular dynamics, and thermal/chaotrope denaturation experiments all find the G20c portal protein to have a highly stable structure, favorable for nanopore sensing applications. Porphyrin conjugation to a cysteine mutant in the protein facilitates the protein’s insertion into lipid bilayers, allowing us to probe ion transport through the pore. Finally, we probed the portal interior size and shape using a series of cyclodextrins of varying sizes, revealing asymmetric transport that possibly originates from the portal’s DNA-ratchet function.

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“…Data was recorded at 1 K Hz bandwidth with a sampling frequency 20 KHz. The Clampex 10 (Molecular Devices), Clampfit 10 (Molecular Devices), MOSAIC [50] and Origin 8 (Origin Lab Corporation) were used to collect and analyze data. The events that last <0.4 ms were excluded for translocation data analysis.…”

Section: Methodsmentioning

confidence: 99%

Nano-channel of viral DNA packaging motor as single pore to differentiate peptides with single amino acid difference

Kang

Wang

et al. 2018

Biomaterials

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Detection, differentiation, mapping, and sequencing of proteins are important in proteomics for the assessment of cell development such as protein methylation or phosphorylation as well as the diagnosis of diseases including metabolic disorder, mental illness, immunological ailments, and malignant cancers. Nanopore technology has demonstrated the potential for the sequencing or sensing of DNA, RNA, chemicals, or other macromolecules. Due to the diversity of protein in shape, structure and charge and the composition versatility of 20 amino acids, the sequencing of proteins remains challenging. Herein, we report the application of the channel of bacteriophage T7 DNA packaging motor for the differentiation of an assortment of peptides of a single amino acid difference. Explicit fingerprints or signatures were obtained based on current blockage and dwell time of individual peptide. Data from the clear mapping of small proteins after protease digestion suggests the potential of using T7 motor channel for proteomics including protein sequencing.

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MOSAIC: A Modular Single-Molecule Analysis Interface for Decoding Multistate Nanopore Data

Cited by 97 publications

References 58 publications

DNA Translocations through Nanopores under Nanoscale Preconfinement

DNA Translocations through Nanopores under Nanoscale Preconfinement

Porphyrin-Assisted Docking of a Thermophage Portal Protein into Lipid Bilayers: Nanopore Engineering and Characterization

Nano-channel of viral DNA packaging motor as single pore to differentiate peptides with single amino acid difference

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