C40B 50/06 - Biochemical methods, e.g. using enzymes or whole viable microorganisms
C12N 15/10 - Processes for the isolation, preparation or purification of DNA or RNA
C12Q 1/68 - Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
C12Q 1/6881 - Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
C12Q 1/6886 - Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
2.
STRUCTURE TO PREVENT THREADING OF NUCLEIC ACID TEMPLATES THROUGH A NANOPORE DURING SEQUENCING
The invention related to forming nucleic add templates including control templates for sequencing using a nanopore-based method, wherein the templates of the novel structure disclosed herein are limited or prevented from threading into the nanopore during sequencing.
C40B 50/06 - Biochemical methods, e.g. using enzymes or whole viable microorganisms
C12N 15/10 - Processes for the isolation, preparation or purification of DNA or RNA
C12Q 1/68 - Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
C12Q 1/6881 - Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
C12Q 1/6886 - Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
4.
Method for labeling ligation products with cell-specific barcodes I
A method of barcoding is provided. The method comprises: providing a population of fixed cells or cell organelles in a first reaction volume, hybridizing oligonucleotide probes to target molecules that are in or on the cells or cell organelles in the first reaction volume, splitting the population of cells or cell organelles into a plurality of second reactions volumes, wherein at least some of the second reaction volumes receive a single fixed cell or cell organelle from the population of fixed cells or fixed cell organelles, and adding cell-specific nucleic acid barcodes onto: the oligonucleotide probes, ligation products comprising the oligonucleotide probes, or complements of the oligonucleotide probes or ligation products, in the plurality of second reaction volumes.
A method of barcoding is provided. The method comprises performing a ligation assay on target nucleic acid molecules that are in or on cells or cell organelles to produce ligation products and adding cell-origination barcodes onto the ligation products or complements thereof by a split-pool barcoding process.
Devices for sequencing linear biomolecules (e.g., DNA, RNA, polypeptides, proteins, and the like) using quantum tunneling effects, and methods of making and using such devices, are provided. A nanofabricated device can include a small gap formed by depositing a thin film between two electrodes, and subsequently removing the film using an etching process. The width of the resulting gap can correspond with the size of a linear biomolecule such that when a part of the biomolecule (e.g., a nucleobase or amino acid) is present in the gap, a change in tunneling current, voltage, or impedance can be measured and the part of the biomolecule identified. The gap dimensions can be precisely controlled at the atomic-scale by, for example, atomic layer deposition (ALD) of the sacrificial film. The device can be made using existing integrated circuit fabrication equipment and facilities, and multiple devices can be formed on a single chip.
The present disclosure is directed to automated systems including an electrophoretic device including one or more separation conduits. In some embodiments, the automated systems are suitable for use in sample cleanup and/or target enrichment processes, such as sample cleanup and/or target enrichment processes conducted prior to sequencing, e.g., next generation sequencing.
C40B 50/06 - Biochemical methods, e.g. using enzymes or whole viable microorganisms
C12Q 1/68 - Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
C12Q 1/6881 - Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
C12N 15/10 - Processes for the isolation, preparation or purification of DNA or RNA
C12Q 1/6886 - Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
The present disclosure provides 3′ protected nucleotides, including those 3′ protected nucleotides having a detectable tag. Systems and methods of sequencing nucleic acids using the 3′ protected nucleotides are also disclosed, such as the sequencing of a nucleic acid using a nanopore or the sequencing of a nucleic acid via sequencing-by-synthesis.
The present disclosure provides variant OmpG polypeptides, compositions comprising the OmpG variant polypeptides, and methods for using the variant OmpG polypeptides as nanopores for determining the sequence of single stranded nucleic acids. The variant OmpG nanopores reduce the ionic current noise versus the parental OmpG polypeptide from which they are derived and thereby enable sequencing of polynucleotides with single nucleotide resolution. The reduced ionic current noise also provides for the use of these OmpG nanopore variants in other single molecule sensing applications, e.g., protein sequencing.
A method of forming a nanopore in a lipid bilayer is disclosed. A nanopore forming solution is deposited over a lipid bilayer. The nanopore forming solution has a concentration level and a corresponding activity level of pore molecules such that nanopores are substantially not formed un-stimulated in the lipid bilayer. Formation of a nanopore in the lipid bilayer is initiated by applying an agitation stimulus level to the lipid bilayer. In some embodiments, the concentration level and the corresponding activity level of pore molecules are at levels such that less than 30 percent of a plurality of available lipid bilayers have nanopores formed un-stimulated therein.
Disclosed herein are embodiments of single-molecule array sequencing (SMAS) devices and systems. Each sensor of an array of sensors of the SMAS device is capable of detecting labels attached to nucleotides incorporated into a single nucleic acid strand bound to a respective binding site. Each sensor can detect a single label (e.g., fluorescent, magnetic, organometallic, charged molecule, etc.) attached to the incorporated nucleotide. Also disclosed are methods of using SMAS devices and systems for highly-scalable nucleic acid (e.g., DNA) sequencing based on sequencing by synthesis (SBS) of multiple instances of clonally amplified DNA immobilized on such SMAS devices. Also disclosed are error correction methods that mitigate errors (e.g., errant label detections or non-detections) made in sequencing individual nucleic acid strands.
The invention is a method of single cell transcriptome analysis. The method comprises detecting multiple transcripts in each individual cell of the plurality of cells by barcoding the transcripts with a cell-specific compound barcode formed using a DNA polymerase and a terminal transferase, optionally in a single enzyme such as a reverse transcriptase.
This disclosure provides a biochip comprising a plurality of wells. The biochip includes a membrane that is disposed in or adjacent to an individual well of the plurality of wells. The membrane comprises a nanopore, and the individual well comprises an electrode that detects a signal upon ionic flow through the pore in response to a species passing through or adjacent to the nanopore. The electrode can be a non-sacrificial electrode. A lipid bilayer can be formed over the plurality of wells using a bubble.
A method for adding cell origination barcodes onto beads is provided. The method comprises: splitting a pool of beads into a plurality of reaction volumes, appending pre-made oligonucleotides onto the beads in the reaction volumes, wherein at least some of the reaction volumes each receive an oligonucleotide that contains a sequence that is different from the other oligonucleotides added to the reaction volumes, pooling the beads and repeating the splitting, appending and pooling steps one or more times to produce a pool of beads that comprise the cell origination barcodes. In the one or more repeats the oligonucleotides that are appended are added to previously appended oligonucleotides to form the cell origination barcodes.
A kit for split-pool barcoding is provided. The kit comprises: a binding agent that binds to a target molecule that is in or on cells or cell organelles and at least two sets of assayable polymer subunit (APS) oligonucleotides. In the kit each set comprises at least 10 unique APS oligonucleotides, the APS oligonucleotides in a set each comprise a sequence that distinguishes the APS oligonucleotides from one another, and the APS oligonucleotides from different sets are configured to link together in an ordered fashion to form all or part of a cell or organelle origination barcode.
Ecole Polytechnique Federale De Lausanne (EPFL) (Switzerland)
Roche Sequencing Solutions, Inc. (USA)
Inventor
Feng, Jiandong
Liu, Ke
Radenovic, Aleksandra
Astier, Yann
Abstract
The invention relates to a method for making nanopores in thin layers or monolayers of transition metal dichalcogenides that enables accurate and controllable formation of pore within those thin layer(s) with sub-nanometer precision.
The present disclosure generally relates to devices and methods for effecting epitachophoresis in order to isolate/purify analytes from urine samples or other samples comprising high salt concentrations, e.g., sodium or potassium salts. Epitachophoresis may be used to effect sample analysis, such as by selective separation, detection, extraction, and/or pre-concentration of target analytes such as, for example, DNA, RNA, and/or other biological molecules. Said target analytes may be collected following epitachophoresis and used for desired downstream applications and further analysis.
The present invention is a method and compositions for primer extension target enrichment of nucleic acids and improvements thereto including simultaneously enriching for RNA and DNA and optionally sequencing the enriched products.
The present disclosure generally relates to devices and methods for effecting epitachophoresis. Epitachophoresis may be used to effect sample analysis, such as by selective separation, detection, extraction, and/or pre-concentration of target analytes such as, for example, DNA, RNA, and/or other biological molecules. Said target analytes may be collected following epitachophoresis and used for desired downstream applications and further analysis.
Embodiments of the present technology may allow for the analysis of molecules by tunneling recognition at a tunneling junction. A tunneling junction of the present technology can include an insulating layer between two electrodes. A voltage may be applied to the electrodes. When a molecule makes contact with both electrodes, the molecule allows current to tunnel through the molecule. The characteristics of the current may aid in identifying a portion of the molecule, for example, a particular nucleotide or base present in a nucleic acid molecule. Methods and systems for analysis of molecules are described.
The present disclosure generally relates to devices and methods for effecting epitachophoresis. Epitachophoresis may be used to effect sample analysis, such as by selective separation, detection, extraction, and/or pre-concentration of target analytes such as, for example, DNA, RNA, and/or other biological molecules. Said target analytes may be collected following epitachophoresis and used for desired downstream applications and further analysis.
Disclosed herein are apparatuses for nucleic acid sequencing using magnetic labels (e.g., magnetic particles) and magnetic sensors. Also disclosed are methods of making and using such apparatuses. An apparatus for nucleic acid sequencing comprises a plurality of magnetic sensors, a plurality of binding areas disposed above the plurality of magnetic sensors, each of the binding areas for holding fluid, and at least one line for detecting a characteristic of at least a first magnetic sensor of the plurality of magnetic sensors, the characteristic indicating presence or absence of one or more magnetic nanoparticles coupled to a first binding area associated with the first magnetic sensor.
The invention provides methods and compositions for analysis of single cell in tissue sample allowing for simultaneous detection and localization of multiple targets in the cells.
Described herein are variants of alpha-hemolysin having at least one amino acid substitution at H35G, E111N, M113A, and/or K147N in the mature, wild-type alpha-hemolysin amino acid sequence. In certain examples, the variant may have a substitution at E111S, M113S, T145S, K147S, or L135I in the mature alpha-hemolysin amino acid sequence. The α-hemolysin variants may also include a substitution at H144A and/or a series of glycine residues spanning residues 127 to 131 of the mature, wild-type alpha hemolysin. Also provided are nanopore assemblies including the alpha-hemolysin variants, the assembly having an increased nanopore lifetime. Further, provided are variants that, in addition to providing increased lifetime, provide a decreased time-to-thread. Hence, the variants provided herein both increase nanopore lifetime and improve efficiency and accuracy of DNA sequencing reactions using nanopores comprising the variants.
C07K 14/31 - Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F) from Staphylococcus (G)
A61K 38/02 - Peptides of undefined number of amino acids; Derivatives thereof
C12N 15/01 - Preparation of mutants without inserting foreign genetic material therein; Screening processes therefor
Molecules may be analyzed (e.g., sequencing of nucleic acid molecules) by tunneling recognition at a tunneling junction. Embodiments of the present invention may allow detecting individual nucleotides and the sequencing of a nucleic acid molecule using a tunneling junction. By labeling a specific nucleotide with a moiety, tunneling junctions may generate a signal with a suitable signal-to-noise ratio. The tunneling recognition uses a tunneling current that is mostly through the moiety rather than mostly through the nucleotide or a portion of the molecule of interest. Because a single nucleotide can be detected with a signal with a suitable signal-to-noise ratio resulting from the tunneling current passing through the moiety, embodiments of the present invention may allow for fast detection of nucleotides using a tunneling current.
Recombinant DPO4-type DNA polymerase variants with amino acid substitutions that confer modified properties upon the polymerase for improved single molecule sequencing applications are provided. Such properties may include enhanced binding and accurate incorporation of bulky nucleotide analog substrates into daughter strands and the like. Also provided are compositions comprising such DPO4 variants and nucleotide analogs, as well as nucleic acids which encode the polymerases with the aforementioned phenotypes.
The invention provides methods, compositions, kits and devices for the detection of target molecules. In some embodiments, the invention allows for multiplexed target molecule detection.
The present disclosure relates to a method for detection reaction volume deviations in a digital polymerase chain reaction (dPCR) and to a method for determining the amount or concentration of a nucleic acid of interest in a sample with dPCR.
Techniques described herein can apply AC signals with different phases to different groups of nanopore cells in a nanopore sensor chip. When a first group of nanopore cells is in a dark period and is not sampled or minimally sampled by an analog-to-digital converter (ADC) to capture useful data, a second group of nanopore cells is in a bright period during which output signals from the second group of nanopore cells are sampled by the analog-to-digital converter. The reference level setting of the ADC is dynamically changed based on the applied AC signals to fully utilize the dynamic range of the ADC.
Some embodiments relate to methods, systems, uses, or software for generating a consensus sequence of a particular molecule. A set of sequences of the particular molecule can be accessed, each having been generated independently from other sequences in the set of sequences and each including an ordered set of bases. An alignment process may be performed using the set of sequences to generate an alignment result associating, for each base of the ordered sets of bases of the sets of sequences. The base may have a reference position. For each reference position of a set of reference positions, a feature vector for the reference position may be generated that represents each base from the ordered sets of bases aligned to the reference position. The feature vectors for the set of references positions may be processed using a machine learning model to generate the consensus sequence for the particular molecule.
G06N 3/0442 - Recurrent networks, e.g. Hopfield networks characterised by memory or gating, e.g. long short-term memory [LSTM] or gated recurrent units [GRU]
Described herein are variants of alpha-hemolysin having at least one mutation selected from T12R, T12K, N17R, N17K or combinations of T12 and N17 mutations. The variants in some embodiments may further comprise H144A. The α-hemolysin variants have a decreased time to thread.
C07K 14/31 - Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F) from Staphylococcus (G)
Disclosed are compositions, kits, and methods for detecting gene fusions involving an unknown fusion partner using locked nucleic acid primers. In some embodiments, the compositions include a compound including at least two nucleotide sequences which are joined, directly or indirectly, through a 5′ to 5′ linkage. In some embodiments, the compound further includes a spacer moiety and/or a cleavage moiety.
C12Q 1/6886 - Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
C12N 15/10 - Processes for the isolation, preparation or purification of DNA or RNA
A nanopore based sequencing system includes a plurality of nanopore sensors. Each nanopore sensor has a portion for receiving a fluid. The nanopore based sequencing system includes a fluid chamber configured to guide the fluid over the plurality of nanopore sensors and an inlet configured to deliver the fluid into the fluid chamber. At least a portion of the fluid chamber is made of a material that has been molded around at least a portion of an electrode.
A method of using a sequencing cell includes applying voltage across the sequencing cell, acquiring one or more signal values from the sequencing cell, and acquiring one or more correlated signal values that are correlated with respective values of the plurality of acquired signal values thereby forming a plurality of two-dimensional data points. The plurality of two-dimensional data points comprise values in a first dimension that equal the plurality of acquired signal value and values in a second dimension that equal the plurality of correlated signal values. The method can further include computing a plurality of transformed signal values by applying a two-dimensional transformation to the plurality of two-dimensional data points.
Disclosed herein are devices, systems, and methods for monitoring single-molecule biological processes using magnetic sensors and magnetic particles (MNP). A MNP is attached to a biopolymer (e.g., a nucleic acid, protein, etc.), and motion of the MNP is detected and/or monitored using a magnetic sensor. Because the MNP is small (e.g., its size is comparable to the size of the molecule being monitored) and is tethered to a biopolymer, changes in the volume of Brownian motion of the MNP in a solution can be monitored to monitor the movement of the MNP and, by inference, the tethered biopolymer. The magnetic sensor is small (e.g., nanoscale or having a size on the order of the sizes of the MNP and the biopolymer) and can be used to detect even small changes in the position of the MNP within the sensing region of the magnetic sensor.
Methods for analyzing a nucleic acid molecule are described. Methods may include attaching the nucleic acid molecule to a particle having a first characteristic dimension. In addition, methods may include applying an electric field through an aperture to move the particle to the aperture. Also, methods may include applying a voltage across a first electrode and a second electrode. Further, methods may include contacting a portion of the nucleic acid molecule to both the first electrode and the second electrode within the aperture, where the portion may include a nucleotide. In addition, methods may include measuring a current through the first electrode, the portion of the nucleic acid molecule, and the second electrode, where the measured current runs in a direction parallel to a longitudinal axis of the aperture. Also, methods may include identifying the nucleotide of the portion of the nucleic acid molecule based on the current.
Described herein are systems and methods for dividing a population of particles into two or more subpopulations, reacting each formed subpopulation of particles with a different reagent, pooling the reacted subpopulations of particles back together.
In one aspect of the present disclosure is a targeted sequencing workflow where an input sample comprising a sufficient quantity of genomic material is provided such minimal or no amplification cycles are utilized prior to sequencing.
The invention provides methods and compositions for removal of undesired or excess oligonucleotides from reaction mixtures using a double hairpin nucleic acid comprising a single nucleic acid strand having: i. a first hairpin at the 5′-end; ii. a second hairpin at the 3′-end; and iii. a single-stranded region between the 5′-end and the 3′-end, wherein the single-stranded region comprises a sequence capable of hybridizing to the oligonucleotide to be removed, e.g, excess primers, subcodes or adaptor molecules.
Disclosed are methods and compositions for detecting structural rearrangements in a genome using rearrangement-specific enrichment probes or rearrangement- specific amplification primers.
C12Q 1/6874 - Methods for sequencing involving nucleic acid arrays, e.g. sequencing by hybridisation [SBH]
C12Q 1/6883 - Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
45.
Generation of single-stranded circular DNA templates for single molecule sequencing
The invention is a novel method of separately sequencing each strand of a nucleic acid involving the use of an adaptor comprising a strand cleavage site or a strand synthesis termination site. The adaptor may also be self-priming at the strand cleavage site.
The invention provides methods, compositions, kits and devices for the detection of target molecules. In some embodiments, the invention allows for multiplexed target molecule detection.
This application discloses compositions comprising primer compounds that reduce or block deleterious threading into a nanopore of nucleic acid strands displaced by a nanopore-linked polymerase, for example during the use of a nanopore device for nucleic acid sequencing. Also disclosed are methods for using the compositions to reduce deleterious threading events during nanopore-based nucleic acid detection techniques, such as nanopore sequencing.
The present invention provides a new building block for peptide synthesis, which introduces a cleavage site that can be used to generate cleavable fragments subsequent to a peptide sequence.
C07K 1/04 - General processes for the preparation of peptides on carriers
C07C 271/22 - Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms to carbon atoms of hydrocarbon radicals substituted by carboxyl groups
C07K 14/00 - Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
The invention provides methods, compositions, kits and devices for the detection of target molecules. In some embodiments, the invention allows for multiplexed target molecule detection.
The invention provides methods, compositions, kits and devices for the detection of target molecules. In some embodiments, the invention allows for multiplexed target molecule detection.
A method of detecting a lipid bilayer formed in a cell of a nanopore based sequencing chip is disclosed. An integrating capacitor is coupled with a lipid membrane, wherein the lipid membrane is between a working electrode and a counter electrode. An alternating current (AC) voltage is applied to the counter electrode. A voltage across the integrating capacitor is periodically sampled by an analog-to-digital converter (ADC). A change in the sampled voltage across the integrating capacitor in response to a change in the AC voltage is determined. Whether the lipid membrane comprises a lipid bilayer is detected based on the determined change in the sampled voltage across the integrating capacitor in response to the change in the AC voltage.
Systems and methods for inserting a single pore into a membrane under faradaic conditions are described herein. A stepped or ramped voltage waveform can be applied across the membranes of the cells of an array, where the voltage waveform starts at first voltage and increases in magnitude over a period of time to a second voltage. The voltage waveform has a polarity that maintains a first species of a redox couple in its current oxidation state. The first voltage is selected to be low enough to reduce the risk of damaging the membrane, while the rate of voltage increase is selected to provide sufficient time for the pores to insert into the membranes. Once a pore is inserted into the membrane, the voltage across the membrane rapidly drops, thereby reducing the risk of damaging the membrane even if the applied voltage between the electrodes is further increased.
A nanopore cell may include a well having a seamless porous electrode and hydrophobic sidewalls. The seamless porous electrode may be formed by depositing porous electrode material on a planar electrode support layer formed by a conductive layer island and a dielectric layer. The porous electrode material may form uniform seamless columns and may be protected during manufacturing by depositing a selectably removable protective layer thereon. The well may be formed by forming and then patterning hydrophobic cladding over the protective layer. The protective layer may be removed to expose the seamless porous electrode at the bottom of the well.
G01N 27/12 - Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating impedance by investigating resistance of a solid body in dependence upon reaction with a fluid
56.
SEQUENCE ALIGNMENT SYSTEMS AND METHODS TO IDENTIFY SHORT MOTIFS IN HIGH-ERROR SINGLE-MOLECULE READS
Described herein is a novel alignment method which leverages multi-stage secondary analysis, with each stage progressively reducing the amount of data to be analyzed in the next stage(s), but increasing exhaustiveness of the search on the remaining data received from previous stage(s). This way, less noisy alignments can be quickly identified from the initially large data-pools in early stage(s), while very noisy alignments can be identified equally fast from smaller data-pools in latter stage(s) of computation, thus maintaining target sensitivity while reducing overall compute times.
The invention is a method of predicting response to therapy in a colorectal cancer patient, the method comprising analysis of circulating tumor DNA from a patient's sample.
C12Q 1/6886 - Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
G16B 20/20 - Allele or variant detection, e.g. single nucleotide polymorphism [SNP] detection
The present disclosure provides a kit for preparing a library of nucleic acids. The kit includes first and second oligonucleotide, each having a tail sequence, a common sequence, and at least one of a unique identifier sequence, and a variable length punctuation mark. The kit further includes a first primer having a first sample identifier sequence and a first priming sequence at a 3′ end of the first primer. The first priming sequence includes the tail sequence of the first oligonucleotide. The kit further includes a second primer having a second sample identifier sequence and a second priming sequence at a 3′ end of the second primer. The second priming sequence is complimentary to the second tail sequence of the second oligonucleotide.
Sequencing adaptors and methods are provided for preparation of polynucleotides for sequencing. The sequencing adaptors contain a portion of a recognition sequence for a methyl-dependent endonuclease. Unwanted adaptor dimers that form during ligation of adaptors to target polynucleotides produce a complete restriction sequence and are cleaved by the endonuclease, followed by exonuclease digestion, thereby removing the dimers.
C12Q 1/6806 - Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
C12Q 1/6848 - Nucleic acid amplification reactions characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
61.
METHODS OF SEQUENCING BY SYNTHESIS USING A CONSECUTIVE LABELING SCHEME
The present disclosure provides a method for sequencing target polynucleotide molecules. In some embodiments, the present disclosure provides a method of sequencing by synthesis where different subsets of nucleotide-conjugate complexes are sequentially formed and detected during each iterative extension of a plurality of nascent nucleic acid copy strands, where each nascent nucleic acid copy strand is complementary to one of a plurality of target polynucleotide molecules. In some embodiments, the plurality of target polynucleotide molecules are arrayed on a solid support.
A method for identifying a sub-population within a mixed population of cells is disclosed. The method involves contacting the mixed population of cells with at least one unique binding agent, wherein the at least one unique binding agent is designed to bind to a target molecule present in the sub-population, and wherein the at least one unique binding agent is attached to an epitope specific barcode that represents the identity of the target molecule. The method further involves sequentially attaching two or more assayable polymer subunits to the epitope specific barcode to create unique cell origination barcodes that represent the identities of individual cells to which the at least one unique binding agent has bound; and decoding the epitope specific barcode and cell origination barcodes, thereby identifying the sub-population within the mixed population of cells.
C12Q 1/70 - Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
The invention provides methods, compositions, kits and devices for the detection of target molecules. In some embodiments, the invention allows for multiplexed target molecule detection.
The invention provides methods, compositions, kits and devices for the detection of target molecules. In some embodiments, the invention allows for multiplexed target molecule detection.
The invention provides methods, compositions, kits and devices for the detection of target molecules. In some embodiments, the invention allows for multiplexed target molecule detection.
The present disclosure is directed to automated systems including a microfluidic chip having one or more independently operable processing conduits. In some embodiments, the automated systems are suitable for use in sample cleanup and/or target enrichment processes, such as sample cleanup and/or target enrichment processes conducted prior to sequencing.
The Trustees of Columbia University in the City of New York (USA)
Roche Sequencing Solutions, Inc. (USA)
Inventor
Fuller, Carl W.
Kumar, Shiv
Ju, Jingyue
Davis, Randall
Chen, Roger
Abstract
This disclosure provides systems and methods for attaching nanopore-detectable tags to nucleotides. The disclosure also provides methods for sequencing nucleic acids using the disclosed tagged nucleotides.
Phosphoramidate-based monomers are provided for use in the synthesis of expandable polymers for nanopore-based sensing. Such monomers comprising a reporter construct that contain a first reporter code, a symmetrical chemical brancher bearing a translocation control element, and a second reporter code, wherein the ends of the reporter construct are attached to phosphoramidate-nucleoside. Related methods and products are also provided.
The present invention relates to diagnostic test and technology. In particular, it relates to a method for determining an analyte suspected to be present in a sample comprising contacting said sample with at least one sensor element comprising at least one binding agent which is capable of specifically binding to the analyte and which comprises at least one magnetic label; and in functional proximity thereto a magnetic tunnel junction generating a signal which is altered upon binding of the analyte to the binding agent for a time and under conditions which allow for specific binding of the analyte suspected to be present in the sample to the at least one binding agent, measuring an altered signal generated by the magnetic tunnel junction upon analyte binding to the at least one binding agent comprising the at least one magnetic label, and determining the analyte based on the altered signal which is generated by the magnetic tunnel junction. The present invention further relates to a device for determining an analyte suspected to be present in a sample and for using such a device. Moreover, the present invention furthermore relates to an aptamer which is capable of specifically binding to an analyte and which comprises at least one magnetic label and a method for identifying such an aptamer. Finally, the invention relates to a kit for determining an analyte suspected to be present in a sample.
Here we describe a statistical approach based on beta mixture modelling to detect contamination and report contamination levels as both point estimates and confidence intervals in liquid biopsy samples. We validate our method with both in silico simulation and in vitro contamination spiked samples. Although we focus on liquid biopsy samples, the same strategy is applicable to any generic NGS application with minor modifications. For example, tissue samples from a biopsy can be used according to the systems and methods described herein.
G16H 20/10 - ICT specially adapted for therapies or health-improving plans, e.g. for handling prescriptions, for steering therapy or for monitoring patient compliance relating to drugs or medications, e.g. for ensuring correct administration to patients
Ecole Polytechnique Federale De Lausanne (EPFL) (Switzerland)
Roche Sequencing Solutions, Inc. (USA)
Inventor
Feng, Jiandong
Liu, Ke
Radenovic, Aleksandra
Astier, Yann
Abstract
The invention relates to a method for making nanopores in thin layers or monolayers of transition metal dichalcogenides that enables accurate and controllable formation of pore within those thin layer(s) with sub-nanometer precision.
The invention provides methods, compositions, kits and devices for the detection of target molecules. In some embodiments, the invention allows for multiplexed target molecule detection.
The invention is a method of assessing a mammalian immune repertoire by primer extension target enrichment (PETE). Methods and compositions for assessing an immune repertoire and the status of additional genetic markers are disclosed.
The invention provides methods, compositions, kits and devices for the detection of target molecules. In some embodiments, the invention allows for multiplexed target molecule detection.
The present disclosure provides 3′ protected nucleotides, including those 3′ protected nucleotides having a detectable tag. Systems and methods of sequencing nucleic acids using the 3′ protected nucleotides are also disclosed, such as the sequencing of a nucleic acid using a nanopore or the sequencing of a nucleic acid via sequencing-by-synthesis.
The present disclosure provides biochips and methods for making biochips. A biochip can comprise a nanopore in a membrane (e.g., lipid bilayer) adjacent or in proximity to an electrode. Methods are described for forming the membrane and inserting the nanopore into the membrane. The biochips and methods can be used for nucleic acid (e.g., DNA) sequencing. The present disclosure also describes methods for detecting, sorting, and binning molecules (e.g., proteins) using biochips.
The present disclosure generally relates to devices and methods for effecting epitachophoresis. Epitachophoresis may be used to effect sample analysis, such as by selective separation, detection, extraction, and/or pre-concentration of target analytes such as, for example, DNA, RNA, and/or other biological molecules. Said target analytes may be collected following epitachophoresis and used for desired downstream applications and further analysis.
C12N 15/10 - Processes for the isolation, preparation or purification of DNA or RNA
C12Q 1/6806 - Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
C12Q 1/6827 - Hybridisation assays for detection of mutation or polymorphism
C12Q 1/6883 - Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
C12Q 1/70 - Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
78.
And methods for measuring analytes using nanofabricated device
Devices for sequencing linear biomolecules (e.g., DNA, RNA, polypeptides, proteins, and the like) using quantum tunneling effects, and methods of making and using such devices, are provided. A nanofabricated device can include a small gap formed by depositing a thin film between two electrodes, and subsequently removing the film using an etching process. The width of the resulting gap can correspond with the size of a linear biomolecule such that when a part of the biomolecule (e.g., a nucleobase or amino acid) is present in the gap, a change in tunneling current, voltage, or impedance can be measured and the part of the biomolecule identified. The gap dimensions can be precisely controlled at the atomic-scale by, for example, atomic layer deposition (ALD) of the sacrificial film. The device can be made using existing integrated circuit fabrication equipment and facilities, and multiple devices can be formed on a single chip.
The present disclosure provides, in some embodiments, a computing device comprising an improved user interface. In some embodiments, the improved user interface enables the visualization of clinically relevant information pertaining to interacting gene variants, including therapeutic recommendations and longitudinal data visualization. In some embodiments, the improved user interface facilitates the contemporaneous visualization of clinically relevant information pertaining to individual gene variants and the visualization of clinically relevant information pertaining to an interaction between gene variants, including therapeutic recommendations, over time. In some embodiments, the visualization(s), through the improved user interface, facilitates the rapid interpretation of clinically relevant information by a medical professional such that decisions regarding patient care may be made accurately and efficiently.
G16B 45/00 - ICT specially adapted for bioinformatics-related data visualisation, e.g. displaying of maps or networks
G16B 20/20 - Allele or variant detection, e.g. single nucleotide polymorphism [SNP] detection
G16H 10/40 - ICT specially adapted for the handling or processing of patient-related medical or healthcare data for data related to laboratory analysis, e.g. patient specimen analysis
G16H 40/67 - ICT specially adapted for the management or administration of healthcare resources or facilities; ICT specially adapted for the management or operation of medical equipment or devices for the operation of medical equipment or devices for remote operation
G16H 10/60 - ICT specially adapted for the handling or processing of patient-related medical or healthcare data for patient-specific data, e.g. for electronic patient records
80.
Concentrating a target molecule for sensing by a nanopore
Methods and related products are disclosed that improve the probability of interaction between a target molecule and a nanopore by capturing the target molecule on a surface comprising the nanopore. The captured target molecule, the nanopore, or both, are able to move relative to each other along the surface. When the leader of the target molecule is in proximity with the nanopore, interaction of the target portion of the target molecule with the nanopore occurs, thereby permitting sensing of the target portion. Confining the target molecule and nanopore in this manner leads to significantly enhanced interaction with the nanopore.
A system includes a plurality of nanopore cells. Data corresponding to nanopore states of the plurality of nanopore cells is received. The data is analyzed to determine a compressed output size of the data given at least one compression technique. It is determined whether the compressed output size exceeds a data budget. In the event it is determined that the compressed output size exceeds the data budget, the data is modified. The modified data is outputted.
The present disclosure provides, in some embodiments, a computing device comprising an improved user interface. In some embodiments, the improved user interface enables the visualization of clinically relevant information pertaining to interacting gene variants, including therapeutic recommendations. In some embodiments, the improved user interface facilitates the contemporaneous visualization of clinically relevant information pertaining to individual gene variants and the visualization of clinically relevant information pertaining to an interaction between gene variants, including therapeutic recommendations. In some embodiments, the visualization(s), through the improved user interface, facilitates the rapid interpretation of clinically relevant information by a medical professional such that decisions regarding patient care may be made accurately and efficiently.
G16H 15/00 - ICT specially adapted for medical reports, e.g. generation or transmission thereof
G16H 10/60 - ICT specially adapted for the handling or processing of patient-related medical or healthcare data for patient-specific data, e.g. for electronic patient records
G16H 20/10 - ICT specially adapted for therapies or health-improving plans, e.g. for handling prescriptions, for steering therapy or for monitoring patient compliance relating to drugs or medications, e.g. for ensuring correct administration to patients
G16B 45/00 - ICT specially adapted for bioinformatics-related data visualisation, e.g. displaying of maps or networks
G16B 50/00 - ICT programming tools or database systems specially adapted for bioinformatics
A consumable device used in a nanopore based sequencing system can include a nanopore chip, a flow cell with one or more flow channels, and a flow cell cover. A fluidic interface can be used to deliver fluid to the flow cell. The fluid interface can include a flow cell boss and the flow cell cover can include a receptacle for receiving the flow cell boss. A dispense tip can be used to introduce fluid into the flow cell through the flow cell boss.
A nanopore cell includes a conductive layer and a working electrode disposed above the conductive layer and at the bottom of a well into which an electrolyte may be contained, such that at least a portion of a top base surface area of the working electrode is exposed to the electrolyte. The nanopore cell further includes a first insulating wall disposed above the working electrode and surrounding a lower section of a well, and a second insulating wall disposed above the first insulating wall and surrounding an upper section of the well, forming an overhang above the lower section of the well. The upper section of the well includes an opening that a membrane may span across, and wherein a base surface area of the opening is smaller than the at least a portion of the top base surface area of the working electrode that is exposed to the electrolyte.
Techniques for measuring sequences of nucleic acids are provided. Time-based measurements (e.g., forming a histogram) particular to a given sequencing cell can be used to generate a tailored model. The model can include probability functions, each corresponding to different states (e.g., different states of a nanopore). Such probability functions can be fit to a histogram of measurements obtained for that cell. The probability functions can be updated over a sequencing run of the nucleic acid so that drifts in physical properties of the sequencing cell can be compensated. A hidden Markov model can use such probability functions as emission probabilities for determining the most likely nucleotide states over time. For sequencing cells involving a polymerase, a 2-state classification between bound and unbound states of the polymerase can be performed. The bound regions can be further analyzed by a second classifier to distinguish between states corresponding to different bound nucleotides.
G16B 40/00 - ICT specially adapted for biostatistics; ICT specially adapted for bioinformatics-related machine learning or data mining, e.g. knowledge discovery or pattern finding
G16B 45/00 - ICT specially adapted for bioinformatics-related data visualisation, e.g. displaying of maps or networks
The present invention relates generally to classification of biological samples, and more specifically to cell of original classification. In particular, some embodiments of the invention relate to diffuse large B cell lymphoma cell of origin classification using machine learning models. The machine learning models can be based on decision trees such as a random forest algorithm or a gradient boosted decision tree. Features for the models can be determined through analysis of variant data from plasma or blood samples from a plurality of subjects with the disease.
C12Q 1/6886 - Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
G16B 20/20 - Allele or variant detection, e.g. single nucleotide polymorphism [SNP] detection
Methods for the rapid detection of the presence or absence of Epstein Barr Virus (EBV) in a biological or non-biological sample are described. The methods can include performing an amplifying step, a hybridizing step, and a detecting step. Furthermore, primers and probes targeting EBV, and kits are provided that are designed for the detection of target regions of EBV. Also described are kits, reaction mixtures, and oligonucleotides (e.g., primer and probe) for the amplification and detection of EBV. Also described are primers and probes that detect different regions of EBV, and can be employed in a dual target assay for simultaneously detecting two different and non-overlapping target regions of EBV.
C12Q 1/70 - Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
Embodiments may include a nucleic acid molecule system. The system may include a nucleic acid polymerase attached to a tether compound. The polymerase may be configured to elongate a nascent strand. The system may also include a nucleotide attached to a label compound. The label compound may include a moiety. The system may further include a transistor in electrical communication with a power supply. The polymer may be attached to the transistor. In addition, the system may include a meter device configured to measure an electrical characteristic of the transistor from the moiety after the label compound is cleaved from the nucleotide by the nucleic acid polymerase.
Methods and compositions for the manufacture and use of a detection apparatus based on one or more native biological nanopores are provided. Uses include, but are not limited to, detection and sequencing of nucleic acids.
A nanopore measurement circuit includes a first analog memory configured to store a first electrical value corresponding to a first measurement sample of a nanopore and a second analog memory configured to store a second electrical value corresponding to a second measurement sample of the nanopore. The nanopore measurement circuit also includes a measurement circuitry configured to provide an output indicating a difference between the first electrical value of the first analog memory and the second electrical value of the second analog memory.
The present technology provides an approach to designing libraries of peptide sequences for discovery and testing of significantly more motifs than would be otherwise available in a given fixed library format. The technology includes a plurality of x-mers embedded in N-mer peptides sequences, where N and x are integers and where N is greater than x. This approach provides for the representation of multiple unique x-mer peptides in a single N-mer peptide feature.
Recombinant DPO4-type DNA polymerase variants with amino acid substitutions that confer modified properties upon the polymerase for improved single molecule sequencing applications are provided. Such properties may include enhanced binding and incorporation of bulky nucleotide analog substrates into daughter strands and the like. Also provided are compositions comprising such DPO4 variants and nucleotide analogs, as well as nucleic acids which encode the polymerases with the aforementioned phenotypes.
C07H 21/04 - Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
The invention is a method of single cell transcriptome analysis. The method comprises detecting multiple transcripts in each individual cell of the plurality of cells by barcoding the transcripts with a cell-specific compound barcode formed using a DNA polymerase and a terminal transferase, optionally in a single enzyme such as a reverse transcriptase.
The present invention is a method and compositions for forming a library for nucleic acids sequencing simultaneously from DNA and RNA present in a sample.
A device for controlling, detecting, and measuring a molecular complex is disclosed. The device comprises a common electrode. The device further comprises a plurality of measurement cells. Each measurement cell includes a cell electrode and an integrator electronically coupled to the cell electrode. The integrator measures the current flowing between the common electrode and the cell electrode. The device further comprises a plurality of analog-to-digital converters, wherein an integrator from the plurality of measurement cells is electrically coupled to one analog-to-digital converter of the plurality of analog-to-digital converters.
The invention is an improved method of target enrichment and target depletion where probe binding is facilitated by the FANCA protein. Improved workflows for next regeneration sequencing and related methods involving nucleic acid probe hybridization are improved by the use of FANCA protein.
The present disclosure provides systems and methods of classifying and/or identifying a cancer subtype. The present disclosure also provides methods of enhancing the prediction of a tumor mutational burden by using both synonymous and non-synonymous somatic mutations in the computation method. It is believed that by increasing the number of mutations in the computation of the tumor mutational burden, a comparatively more consistent tumor mutational burden may be derived, especially for targeted-panel sequencing. It is believed that the consistent computation of the tumor mutational burden from targeted panels allows for computationally quicker and less costly analysis of sequencing data as compared with a tumor mutational burden computed from whole exome sequencing data.
G16H 50/30 - ICT specially adapted for medical diagnosis, medical simulation or medical data mining; ICT specially adapted for detecting, monitoring or modelling epidemics or pandemics for individual health risk assessment
G16B 20/00 - ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
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