Faculty Opinions recommendation of Real-time selective sequencing using nanopore technology.

AbstractThe Oxford MinION, the first commercial nanopore sequencer, is also the first to implement molecule-by-molecule real-time selective sequencing or “Read Until”. As DNA transits a MinION nanopore, real-time pore current data can be accessed and analyzed to provide active feedback to that pore. Fragments of interest are sequenced by default, while DNA deemed non-informative is rejected by reversing the pore bias to eject the strand, providing a novel means of background depletion and/or target enrichment. In contrast to the previously published pattern-matching Read Until approach, our RUBRIC method is the first example of real-time selective sequencing where on-line basecalling enables alignment against conventional nucleic acid references to provide the basis for sequence/reject decisions. We evaluate RUBRIC performance across a range of optimizable parameters, apply it to mixed human/bacteria and CRISPR/Cas9-cut samples, and present a generalized model for estimating real-time selection performance as a function of sample composition and computing configuration.

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BOSS-RUNS: a flexible and practical dynamic read sampling framework for nanopore sequencing

10.1101/2020.02.07.938670 ◽

2020 ◽

Author(s):

Nicola De Maio ◽

Charlotte Manser ◽

Rory Munro ◽

Ewan Birney ◽

Matthew Loose ◽

...

Keyword(s):

Mathematical Model ◽

Real Time ◽

A Priori ◽

Dna Fragments ◽

Decision Strategy ◽

Oxford Nanopore ◽

A Genome ◽

Optimal Dynamic ◽

Genomic Regions ◽

Selective Sequencing

AbstractReal-time selective sequencing of individual DNA fragments, or ‘Read Until’, allows the focusing of Oxford Nanopore Technology sequencing on pre-selected genomic regions. This can lead to large improvements in DNA sequencing performance in many scenarios where only part of the DNA content of a sample is of interest. This approach is based on the idea of deciding whether to sequence a fragment completely after having sequenced only a small initial part of it. If, based on this small part, the fragment is not deemed of (sufficient) interest it is rejected and sequencing is continued on a new fragment. To date, only simple decision strategies based on location within a genome have been proposed to determine what fragments are of interest. We present a new mathematical model and algorithm for the real-time assessment of the value of prospective fragments. Our decision framework is based not only on which genomic regions are a priori interesting, but also on which fragments have so far been sequenced, and so on the current information available regarding the genome being sequenced. As such, our strategy can adapt dynamically during each run, focusing sequencing efforts in areas of highest uncertainty (typically areas currently low coverage). We show that our approach can lead to considerable savings of time and materials, providing high-confidence genome reconstruction sooner than a standard sequencing run, and resulting in more homogeneous coverage across the genome, even when entire genomes are of interest.Author SummaryAn existing technique called ‘Read Until’ allows selective sequencing of DNA fragments with an Oxford Nanopore Technology (ONT) sequencer. With Read Until it is possible to enrich coverage of areas of interest within a sequenced genome. We propose a new use of this technique: combining a mathematical model of read utility and an algorithm to select an optimal dynamic decision strategy (i.e. one that can be updated in real time, and so react to the data generated so far in an experiment), we show that it possible to improve the efficiency of a sequencing run by focusing effort on areas of highest uncertainty.

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Real-time selective sequencing using nanopores and deep learning

10.21203/rs.3.rs-540693/v1 ◽

2021 ◽

Author(s):

Artem Danilevsky ◽

Avital Luba Polsky ◽

Noam Shomron

Keyword(s):

Deep Learning ◽

Real Time ◽

Human Cell Line ◽

Genomic Research ◽

Nanopore Sequencing ◽

Sequencing Data ◽

Total Input ◽

Unique Method ◽

Cell Line Sample ◽

Selective Sequencing

Abstract Nanopore sequencing is an emerging technology that utilizes a unique method of reading nucleic acid sequences and, at the same time, it detects various chemical modifications. Deep learning has increased in popularity as a useful technique to solve many complex computational tasks. Selective sequencing has been widely used in genomic research; although it introduces several caveats to the process of sequencing, its advantages supersede them. In this study we demonstrate an alternative method of software-based selective sequencing that is performed in real time by combining nanopore sequencing and deep learning. Our results show the feasibility of using deep learning for classifying signals from only the first 200 nucleotides in a raw nanopore sequencing signal format. Using custom deep learning models and a script utilizing "Read-Until" framework to target mitochondrial molecules in real time from a human cell line sample, we achieved a significant separation and enrichment ability of more than 2-fold. In a series of very short sequencing runs (10, 30, and 120 minutes), we identified genomic and mitochondrial reads with accuracy above 90%, although mitochondrial DNA comprises only 0.1% of the total input material. We believe that our results will lay the foundation for rapid and selective sequencing using nanopore technology and will pave the way for future clinical applications using nanopore sequencing data.

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Real-Time Selective Sequencing with RUBRIC: Read Until with Basecall and Reference-Informed Criteria

Scientific Reports ◽

10.1038/s41598-019-47857-3 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 7

Author(s):

Harrison S. Edwards ◽

Raga Krishnakumar ◽

Anupama Sinha ◽

Sara W. Bird ◽

Kamlesh D. Patel ◽

...

Keyword(s):

Real Time ◽

Selective Sequencing

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Real-time selective sequencing using nanopore technology

Nature Methods ◽

10.1038/nmeth.3930 ◽

2016 ◽

Vol 13 (9) ◽

pp. 751-754 ◽

Cited By ~ 110

Author(s):

Matthew Loose ◽

Sunir Malla ◽

Michael Stout

Keyword(s):

Real Time ◽

Selective Sequencing

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Some Recent Results in Quiescent Prominence Spectrometry

International Astronomical Union Colloquium ◽

10.1017/s0252921100065179 ◽

1979 ◽

Vol 44 ◽

pp. 41-47

Author(s):

Donald A. Landman

Keyword(s):

Real Time ◽

Computer System ◽

Spectral Response ◽

Absolute Intensity ◽

Solar Observatory ◽

Target Size ◽

Small Target ◽

Signal Averaging ◽

Quiescent Prominence

This paper describes some recent results of our quiescent prominence spectrometry program at the Mees Solar Observatory on Haleakala. The observations were made with the 25 cm coronagraph/coudé spectrograph system using a silicon vidicon detector. This detector consists of 500 contiguous channels covering approximately 6 or 80 Å, depending on the grating used. The instrument is interfaced to the Observatory’s PDP 11/45 computer system, and has the important advantages of wide spectral response, linearity and signal-averaging with real-time display. Its principal drawback is the relatively small target size. For the present work, the aperture was about 3″ × 5″. Absolute intensity calibrations were made by measuring quiet regions near sun center.

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Video real-time imaging of artificial membranes during freeze-drying by cryo-electron microscopy

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s042482010010216x ◽

1988 ◽

Vol 46 ◽

pp. 16-17

Author(s):

Alan S. Rudolph ◽

Ronald R. Price

Keyword(s):

Real Time ◽

Self Assembly ◽

Freeze Drying ◽

Video Capture ◽

Drying Process ◽

Artificial Membranes ◽

The Past ◽

Cryo Electron Microscopy ◽

Spherical Structures ◽

Capture Techniques

We have employed cryoelectron microscopy to visualize events that occur during the freeze-drying of artificial membranes by employing real time video capture techniques. Artificial membranes or liposomes which are spherical structures within internal aqueous space are stabilized by water which provides the driving force for spontaneous self-assembly of these structures. Previous assays of damage to these structures which are induced by freeze drying reveal that the two principal deleterious events that occur are 1) fusion of liposomes and 2) leakage of contents trapped within the liposome [1]. In the past the only way to access these events was to examine the liposomes following the dehydration event. This technique allows the event to be monitored in real time as the liposomes destabilize and as water is sublimed at cryo temperatures in the vacuum of the microscope. The method by which liposomes are compromised by freeze-drying are largely unknown. This technique has shown that cryo-protectants such as glycerol and carbohydrates are able to maintain liposomal structure throughout the drying process.

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An Interactive Minicomputer Program for the Indexing and Simulation of Electron Diffraction Patterns

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100092670 ◽

1976 ◽

Vol 34 ◽

pp. 542-543

Author(s):

R.P. Goehner ◽

W.T. Hatfield ◽

Prakash Rao

Keyword(s):

Electron Diffraction ◽

Real Time ◽

Pattern Analysis ◽

Flow Diagram ◽

Hard Copy ◽

Time Analysis ◽

Real Time Analysis ◽

Transmission Electron ◽

One Step ◽

Diffraction Patterns

Computer programs are now available in various laboratories for the indexing and simulation of transmission electron diffraction patterns. Although these programs address themselves to the solution of various aspects of the indexing and simulation process, the ultimate goal is to perform real time diffraction pattern analysis directly off of the imaging screen of the transmission electron microscope. The program to be described in this paper represents one step prior to real time analysis. It involves the combination of two programs, described in an earlier paper(l), into a single program for use on an interactive basis with a minicomputer. In our case, the minicomputer is an INTERDATA 70 equipped with a Tektronix 4010-1 graphical display terminal and hard copy unit.A simplified flow diagram of the combined program, written in Fortran IV, is shown in Figure 1. It consists of two programs INDEX and TEDP which index and simulate electron diffraction patterns respectively. The user has the option of choosing either the indexing or simulating aspects of the combined program.

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Microstructural investigation of surface roughness in MBE-grown AlAs

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100138646 ◽

1995 ◽

Vol 53 ◽

pp. 454-455

Author(s):

R. Rajesh ◽

R. Droopad ◽

C. H. Kuo ◽

R. W. Carpenter ◽

G. N. Maracas

Keyword(s):

Thin Film ◽

Real Time ◽

Growth Control ◽

Native Oxide ◽

Effusion Cell ◽

Surface Oxide Layer ◽

Microstructural Investigation ◽

Mbe Growth ◽

Film Substrate ◽

And Control

Knowledge of material pseudodielectric functions at MBE growth temperatures is essential for achieving in-situ, real time growth control. This allows us to accurately monitor and control thicknesses of the layers during growth. Undesired effusion cell temperature fluctuations during growth can thus be compensated for in real-time by spectroscopic ellipsometry. The accuracy in determining pseudodielectric functions is increased if one does not require applying a structure model to correct for the presence of an unknown surface layer such as a native oxide. Performing these measurements in an MBE reactor on as-grown material gives us this advantage. Thus, a simple three phase model (vacuum/thin film/substrate) can be used to obtain thin film data without uncertainties arising from a surface oxide layer of unknown composition and temperature dependence.In this study, we obtain the pseudodielectric functions of MBE-grown AlAs from growth temperature (650°C) to room temperature (30°C). The profile of the wavelength-dependent function from the ellipsometry data indicated a rough surface after growth of 0.5 μm of AlAs at a substrate temperature of 600°C, which is typical for MBE-growth of GaAs.

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