Multiplexed direct detection of barcoded protein reporters on a nanopore array

AbstractGenetically encoded reporter proteins are a cornerstone of molecular biology. While they are widely used to measure many biological activities, the current number of uniquely addressable reporters that can be used together for one-pot multiplexed tracking is small due to overlapping detection channels such as fluorescence. To address this, we built an expanded library of orthogonally-barcoded Nanopore-addressable protein Tags Engineered as Reporters (NanoporeTERs), which can be read and demuxed by nanopore sensors at the single-molecule level. By adapting a commercially available nanopore sensor array platform typically used for real-time DNA and RNA sequencing (Oxford Nanopore Technologies’ MinION), we show direct detection of NanoporeTER expression levels from unprocessed bacterial culture with no specialized sample preparation. These results lay the foundations for a new class of reporter proteins to enable multiplexed, real-time tracking of gene expression with nascent nanopore sensor technology.

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A nanopore interface for high bandwidth DNA computing

10.1101/2021.12.30.474555 ◽

2021 ◽

Author(s):

Karen Zhang ◽

Yuan-Jyue Chen ◽

Kathryn Doroschak ◽

Karin Strauss ◽

Luis Ceze ◽

...

Keyword(s):

Fluorescence Spectroscopy ◽

Single Molecule ◽

Dna Computing ◽

Direct Detection ◽

New Paradigm ◽

Single Molecule Level ◽

Oxford Nanopore ◽

Dna Strand Displacement ◽

Order Of Magnitude ◽

Dna Strand

DNA has emerged as a powerful substrate for programming information processing machines at the nanoscale. Among the DNA computing primitives used today, DNA strand displacement (DSD) is arguably the most popular, with DSD-based circuit applications ranging from disease diagnostics to molecular artificial neural networks. The outputs of DSD circuits are generally read using fluorescence spectroscopy. However, due to the spectral overlap of typical small-molecule fluorescent reporters, the number of unique outputs that can be detected in parallel is limited, requiring complex optical setups or spatial isolation of reactions to make output bandwidths scalable. Here, we present a multiplexable sequencing-free readout method that enables real-time, kinetic measurement of DSD circuit activity through highly parallel, direct detection of barcoded output strands using nanopore sensor array technology (Oxford Nanopore Technologies' MinION device). We show that engineered reporter probes can be detected and classified with high accuracy at the single-molecule level directly from raw nanopore signals using deep learning. We then demonstrate this method's utility in multiplexed detection of clinically relevant microRNA sequences. These results increase DSD output bandwidth by an order of magnitude over what is possible with fluorescence spectroscopy, laying the foundations for a new paradigm in DNA circuit readout and programmable multiplexed molecular diagnostics using portable nanopore devices.

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Faculty Opinions recommendation of Direct detection of DNA methylation during single-molecule, real-time sequencing.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.3246956.793481773 ◽

2013 ◽

Author(s):

Hans Lehrach

Keyword(s):

Dna Methylation ◽

Real Time ◽

Single Molecule ◽

Direct Detection

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Real-Time 3D Imaging at the Single Molecule Level of Signal Transduction in Saccharomyces CerevisiÆ Responding to Environmental Changes

Biophysical Journal ◽

10.1016/j.bpj.2015.11.823 ◽

2016 ◽

Vol 110 (3) ◽

pp. 145a

Author(s):

Erik G. Hedlund ◽

Sviatlana Shashkova ◽

Adam J.M. Wollman ◽

Stefan Hohmann ◽

Mark C. Leake

Keyword(s):

Saccharomyces Cerevisiae ◽

Signal Transduction ◽

Real Time ◽

Single Molecule ◽

3D Imaging ◽

Environmental Changes ◽

Single Molecule Level

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Direct detection of DNA methylation during single-molecule, real-time sequencing

Nature Methods ◽

10.1038/nmeth.1459 ◽

2010 ◽

Vol 7 (6) ◽

pp. 461-465 ◽

Cited By ~ 826

Author(s):

Benjamin A Flusberg ◽

Dale R Webster ◽

Jessica H Lee ◽

Kevin J Travers ◽

Eric C Olivares ◽

...

Keyword(s):

Dna Methylation ◽

Real Time ◽

Single Molecule ◽

Direct Detection

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Oxford Nanopore sequencing-based protocol to detect CpG methylation in human mitochondrial DNA

10.1101/2021.02.20.432086 ◽

2021 ◽

Author(s):

Iacopo Bicci ◽

Claudia Calabrese ◽

Zoe J. Golder ◽

Aurora Gomez-Duran ◽

Patrick F Chinnery

Keyword(s):

Mitochondrial Dna ◽

Single Molecule ◽

Bisulfite Sequencing ◽

Nuclear Dna ◽

Cpg Methylation ◽

Nanopore Sequencing ◽

Single Molecule Level ◽

Oxford Nanopore ◽

Genome Bisulfite Sequencing ◽

Technical Issues

SummaryMethylation on CpG residues is one of the most important epigenetic modifications of nuclear DNA, regulating gene expression. Methylation of mitochondrial DNA (mtDNA) has been studied using whole genome bisulfite sequencing (WGBS), but recent evidence has uncovered major technical issues which introduce a potential bias during methylation quantification. Here, we validate the technical concerns with WGBS, and then develop and assess the accuracy of a protocol for variant-specific methylation identification using long-read Oxford Nanopore Sequencing. Our approach circumvents mtDNA-specific confounders, while enriching for native full-length molecules over nuclear DNA. Variant calling analysis against Illumina deep re-sequencing showed that all expected mtDNA variants can be reliably identified. Methylation calling revealed negligible mtDNA methylation levels in multiple human primary and cancer cell lines. In conclusion, our protocol enables the reliable analysis of epigenetic modifications of mtDNA at single-molecule level at single base resolution, with potential applications beyond methylation.MotivationAlthough whole genome bisulfite sequencing (WGBS) is the gold-standard approach to determine base-level CpG methylation in the nuclear genome, emerging technical issues raise questions about its reliability for evaluating mitochondrial DNA (mtDNA) methylation. Concerns include mtDNA strand asymmetry rendering the C-rich light strand disproportionately vulnerable the chemical modifications introduced with WGBS. Also, short-read sequencing can result in a co-amplification of nuclear sequences originating from ancestral mtDNA with a high nucleotide similarity. Lastly, calling mtDNA alleles with varying proportions (heteroplasmy) is complicated by the C-to-T conversion introduced by WGBS on unmethylated CpGs. Here, we propose an alternative protocol to quantify methyl-CpGs in mtDNA, at single-molecule level, using Oxford Nanopore Sequencing (ONS). By optimizing the standard ONS library preparation, we achieved selective enrichment of native mtDNA and accurate single nucleotide variant and CpG methylation calling, thus overcoming previous limitations.

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FluoSim: simulator of single molecule dynamics for fluorescence live-cell and super-resolution imaging of membrane proteins

10.1101/2020.02.06.937045 ◽

2020 ◽

Cited By ~ 3

Author(s):

Matthieu Lagardère ◽

Ingrid Chamma ◽

Emmanuel Bouilhol ◽

Macha Nikolski ◽

Olivier Thoumine

Keyword(s):

Real Time ◽

Protein Dynamics ◽

Single Molecule ◽

Imaging Techniques ◽

Simulated Data ◽

Super Resolution ◽

Molecular Complexes ◽

Live Cell ◽

Single Molecule Level ◽

Resolution Imaging

AbstractFluorescence live-cell and super-resolution microscopy methods have considerably advanced our understanding of the dynamics and mesoscale organization of macro-molecular complexes that drive cellular functions. However, different imaging techniques can provide quite disparate information about protein motion and organization, owing to their respective experimental ranges and limitations. To address these limitations, we present here a unified computer program that allows one to model and predict membrane protein dynamics at the ensemble and single molecule level, so as to reconcile imaging paradigms and quantitatively characterize protein behavior in complex cellular environments. FluoSim is an interactive real-time simulator of protein dynamics for live-cell imaging methods including SPT, FRAP, PAF, and FCS, and super-resolution imaging techniques such as PALM, dSTORM, and uPAINT. The software, thoroughly validated against experimental data on the canonical neurexin-neuroligin adhesion complex, integrates diffusion coefficients, binding rates, and fluorophore photo-physics to calculate in real time the distribution of thousands of independent molecules in 2D cellular geometries, providing simulated data of protein dynamics and localization directly comparable to actual experiments.

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NanoR: a user-friendly R package to analyze and compare nanopore sequencing data

10.1101/514232 ◽

2019 ◽

Author(s):

Davide Bolognini ◽

Niccolò Bartalucci ◽

Alessandra Mingrino ◽

Alessandro Maria Vannucchi ◽

Alberto Magi

Keyword(s):

Real Time ◽

Low Cost ◽

R Package ◽

Sequencing Data ◽

High Performing ◽

Dna And Rna ◽

Oxford Nanopore ◽

The One ◽

User Friendly ◽

Oxford Nanopore Technologies

AbstractMinION and GridION X5 from Oxford Nanopore Technologies are devices for real-time DNA and RNA sequencing. On the one hand, MinION is the only real-time, low cost and portable sequencing device and, thanks to its unique properties, is becoming more and more popular among biologists; on the other, GridION X5, mainly for its costs, is less widespread but highly suitable for researchers with large sequencing projects. Despite the fact that Oxford Nanopore Technologies’ devices have been increasingly used in the last few years, there is a lack of high-performing and user-friendly tools to handle the data outputted by both MinION and GridION X5 platforms. Here we present NanoR, a cross-platform R package designed with the purpose to simplify and improve nanopore data visualization. Indeed, NanoR is built on few functions but overcomes the capabilities of existing tools to extract meaningful informations from MinION sequencing data; in addition, as exclusive features, NanoR can deal with GridION X5 sequencing outputs and allows comparison of both MinION and GridION X5 sequencing data in one command. NanoR is released as free package for R at https://github.com/davidebolo1993/NanoR.

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Enabling metagenomic surveillance for bacterial tick-borne pathogens using nanopore sequencing with adaptive sampling

10.1101/2021.08.17.456696 ◽

2021 ◽

Author(s):

Evan J. Kipp ◽

Laramie L. Lindsey ◽

Benedict S. Khoo ◽

Christopher Faulk ◽

Jonathan D. Oliver ◽

...

Keyword(s):

Real Time ◽

Single Molecule ◽

Adaptive Sampling ◽

Sequence Data ◽

Ixodes Scapularis ◽

Fold Increase ◽

Borrelia Miyamotoi ◽

Nucleotide Sequence Data ◽

Oxford Nanopore ◽

Sequencing Platforms

Technological and computational advancements in the fields of genomics and bioinformatics are providing exciting new opportunities for pathogen discovery and surveillance. In particular, single-molecule nucleotide sequence data originating from Oxford Nanopore Technologies (ONT) sequencing platforms can be bioinformatically leveraged, in real-time, for enhanced biosurveillance of a vast array of zoonoses. The recently released nanopore adaptive sampling (NAS) pipeline facilitates immediate mapping of individual nucleotide molecules (i.e., DNA, cDNA, and RNA) to a given reference as each molecule is sequenced. User-defined thresholds then allow for the retention or rejection of specific molecules, informed by the real-time reference mapping results, as they are physically passing through a given sequencing nanopore. Here, we show how NAS can be used to selectively sequence entire genomes of bacterial tick-borne pathogens circulating in wild populations of the blacklegged tick vector, Ixodes scapularis. The NAS method provided a two-fold increase in targeted pathogen sequences, successfully enriching for Borrelia (Borreliella) burgdorferi s.s.; Borrelia (Borrelia) miyamotoi; Anaplasma phagocytophilum; and Ehrlichia muris eauclairensis genomic DNA within our I. scapularis samples. Our results indicate that NAS has strong potential for real-time sequence-based pathogen surveillance.

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