Methods for amplifying a template/target nucleic acid, where the original template/target nucleic acid has short primer-binding sites (e.g., ≤ 15 nucleotides), by employing long primers are described. Long forward primers and long reverse primers have two regions: (1) a region for annealing/hybridizing to a region of the template/target nucleic acid; and (2) a region that does not anneal/hybridize to a region of the template/target nucleic acid region. Put another way, the long primers are longer than the length of the primer-binding site (i.e., the site upon which the long primers are to anneal/hybridize). Use of these long primers results overcomes the problems and obstacles of short primer-binding sequences.
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.
A61K 38/02 - Peptides à nombre indéterminé d'amino-acidesLeurs dérivés
C07K 14/00 - Peptides ayant plus de 20 amino-acidesGastrinesSomatostatinesMélanotropinesLeurs dérivés
C07K 14/31 - Peptides ayant plus de 20 amino-acidesGastrinesSomatostatinesMélanotropinesLeurs dérivés provenant de bactéries provenant de Micrococcaceae (F) provenant de Staphylococcus (G)
C12N 15/01 - Préparation de mutants sans introduction de matériel génétique étrangerProcédés de criblage à cet effet
This disclosure provides systems and methods for molecular identification and polymer (e.g., nucleic acid) sequencing using nanopores. The polymer may be passed through or in proximity to the nanopore and various subunits of the polymer may affect the current flowing through the nanopore. The various subunits may be identified by measuring the current at a plurality of voltages applied across the nanopore and/or membrane. In some cases, the polymerization of tagged nucleotides presents tag molecules to the nanopore that can be identified by measuring the current at a plurality of voltages applied across the nanopore and/or membrane. Also provided herein are systems and methods for sequencing both the sense and anti-sense strand of a double stranded nucleic acid molecule with a nanopore and methods for using ribonucleic acid (RNA) speed bump molecules to slow the passage of a nucleic acid molecule through or in proximity to a nanopore.
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 insert-ing 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.
C12N 15/11 - Fragments d'ADN ou d'ARNLeurs formes modifiées
G01N 33/487 - Analyse physique de matériau biologique de matériau biologique liquide
G01N 33/543 - Tests immunologiquesTests faisant intervenir la formation de liaisons biospécifiquesMatériaux à cet effet avec un support insoluble pour l'immobilisation de composés immunochimiques
A method of detecting a state of a lipid membrane in a cell of a nanopore based sequencing chip is disclosed. A lipid membrane is coupled with an integrating capacitor, 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 an intermediate change in the AC voltage is determined. A state of the lipid membrane is determined based on the determined change in the sampled voltage across the integrating capacitor in response to the intermediate change in the AC voltage.
C12Q 1/68 - Procédés de mesure ou de test faisant intervenir des enzymes, des acides nucléiques ou des micro-organismesCompositions à cet effetProcédés pour préparer ces compositions faisant intervenir des acides nucléiques
G01N 33/487 - Analyse physique de matériau biologique de matériau biologique liquide
G01R 27/26 - Mesure de l'inductance ou de la capacitanceMesure du facteur de qualité, p. ex. en utilisant la méthode par résonanceMesure de facteur de pertesMesure des constantes diélectriques
6.
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.
G01N 33/543 - Tests immunologiquesTests faisant intervenir la formation de liaisons biospécifiquesMatériaux à cet effet avec un support insoluble pour l'immobilisation de composés immunochimiques
7.
SYSTEMS AND METHODS FOR SELF-LIMITING PROTEIN PORE INSERTION IN A MEMBRANE
Systems and methods for inserting a single pore into a membrane 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 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.
Disclosed herein are compositions for use in preparing target nucleic acid molecules including one or more 5-formyl cytosine bases or adducts of 5-formyl cytosine. Also disclosed herein are methods of efficiently synthesizing nucleic acid molecules including one or more 5-formyl cytosine bases from target nucleic acid molecules which include one or more 5-hydroxymethyl cytosine bases. The present disclosure also provides for methods of detecting epigenetic modifications in a target nucleic acid molecule, such as those epigenetic modifications characterized by methylation of cytosine at the 5-position position (e.g., 5-methyl cytosine; 5-hydroxymethyl cytosine).
C07H 1/00 - Procédés de préparation des dérivés du sucre
C07H 21/04 - Composés contenant au moins deux unités mononucléotide comportant chacune des groupes phosphate ou polyphosphate distincts liés aux radicaux saccharide des groupes nucléoside, p. ex. acides nucléiques avec le désoxyribosyle comme radical saccharide
C12Q 1/6806 - Préparation d’acides nucléiques pour analyse, p. ex. pour test de réaction en chaîne par polymérase [PCR]
9.
ENHANCEMENT OF NUCLEIC ACID POLYMERIZATION BY MINOR GROOVE BINDING MOIETIES
The invention relates to methods and compositions for improving on nucleic acid polymerization, including DNA replication by in vitro primer extension to generate, for example, polymers for nanopore-based single molecule sequencing of a DNA template. A nucleic acid polymerase reaction composition is provided with polymerization enhancement moieties, which allows enhanced DNA polymerase activity with nucleotide analogs, resulting in improved length of primer extension products for sequencing applications.
C12Q 1/6848 - Réactions d’amplification d’acides nucléiques caracterisées par les moyens d’empêcher la contamination ou d’augmenter la spécificité ou la sensibilité d’une réaction d’amplification
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.
C12Q 1/68 - Procédés de mesure ou de test faisant intervenir des enzymes, des acides nucléiques ou des micro-organismesCompositions à cet effetProcédés pour préparer ces compositions faisant intervenir des acides nucléiques
C12Q 1/6806 - Préparation d’acides nucléiques pour analyse, p. ex. pour test de réaction en chaîne par polymérase [PCR]
C12Q 1/6816 - Tests d’hybridation caractérisés par les moyens de détection
C12Q 1/686 - Réaction en chaine par polymérase [PCR]
11.
ALPHA-HEMOLYSIN VARIANTS WITH ALTERED CHARACTERISTICS
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 ayant plus de 20 amino-acidesGastrinesSomatostatinesMélanotropinesLeurs dérivés provenant de bactéries provenant de Micrococcaceae (F) provenant de Staphylococcus (G)
C40B 50/06 - Procédés biochimiques, p. ex. utilisant des enzymes ou des micro-organismes viables entiers
C12N 15/10 - Procédés pour l'isolement, la préparation ou la purification d'ADN ou d'ARN
C12Q 1/68 - Procédés de mesure ou de test faisant intervenir des enzymes, des acides nucléiques ou des micro-organismesCompositions à cet effetProcédés pour préparer ces compositions faisant intervenir des acides nucléiques
C12Q 1/6881 - Produits d’acides nucléiques utilisés dans l’analyse d’acides nucléiques, p. ex. amorces ou sondes pour le typage de tissu ou de cellule, p. ex. sondes d’antigène leucocytaire humain [HLA]
C12Q 1/6886 - Produits d’acides nucléiques utilisés dans l’analyse d’acides nucléiques, p. ex. amorces ou sondes pour les maladies provoquées par des altérations du matériel génétique pour le cancer
13.
PHOSPHOROAMIDATE ESTERS, AND USE AND SYNTHESIS THEREOF
Phosphoramidate esters and related nucleotide analogs useful in polynucleotide sequencing techniques, and synthetic methods for preparing those compounds, are disclosed. These compounds include nucleotide phosphoramidates analogs that are modified on the alpha-phosphate to enable attachment of a variety of application-specific substituents such as tether molecules.
Described herein are methods, systems, and programming for determining a tumor immunophenotype of an image of a tumor. Some embodiments include dividing an image into tiles depicting tumor epithelium and/or tumor stroma. For each tile, an epithelium-immune cell density and a stroma-immune cell density may be calculated based on a number of immune cells identified in the tumor epithelium and the tumor stroma, respectively. Based on the epithelium-immune cell density and the stroma-immune cell density, an inflammation type of the type may be determined, and a tumor immunophenotype may be determined based on each tile's inflammation type.
KAPA BIOSYSTEMS, INC., SOUTH AFRICA (Afrique du Sud)
Inventeur(s)
Klass, Daniel
Chang, Shwu Shin
Graf Grachet, Nathalia
Garcia-Montoya, Gladys
Saelee, Seng Lor
Ristow, Peter
Abrégé
The present disclosure relates, in general, to the enzymatic conversion of methylated nucleic acids in order to distinguish between methylated and unmethylated cytosines in DNA and, more particularly, to improved methods and compositions for enzymatic methylation sequencing. In one aspect, diverse compositions and methods are provided for improved recovery of methylation signal. The methods include, one or more of: a nick repair step, restoration of methylation signal, use of modified methylcytosine nucleic acid adaptors, and use of helicase, ssDNA binding proteins, engineered DNA ligase, or a combination thereof.
A method of forming a plurality of lipid bilayers over an array of cells in a nanopore based sequencing chip is disclosed. Each of the cells comprises a well. A first salt buffer solution with a first osmolarity is flowed over a cell in the nanopore based sequencing chip to substantially fill a well in the cell with the first salt buffer solution. A lipid and solvent mixture is flowed over the cell to deposit a lipid membrane over the well that encloses the first salt buffer solution in the well. A second salt buffer solution with a second osmolarity is flowed above the well to reduce the thickness of the lipid membrane, wherein the second osmolarity is a lower osmolarity than the first osmolarity such that an osmotic imbalance is created between a first volume inside the well and a second volume outside the well.
G01N 15/12 - Recherche de particules individuelles en mesurant des effets électriques ou magnétiques en observant des changements de résistance ou d’impédance à travers des fentes traversées par des particules individuelles, p. ex. en utilisant le principe de Coulter
17.
FORMATION AND CALIBRATION OF NANOPORE SEQUENCING CELLS
Improved multi-cell nanopore-based sequencing chips and methods can employ formation, characterization, calibration, and/or normalization techniques. For example, various methods may include one or more steps of performing physical checks of cell circuitry, forming and characterizing a lipid layer on the cells, performing a zero point calibration of the cells, forming and characterizing nanopores on the lipid layers of each cell, performing a sequencing operation to accumulate sequencing signals from the cells, normalizing those sequencing signals, and determining bases based on the normalized sequencing signals.
G01N 33/487 - Analyse physique de matériau biologique de matériau biologique liquide
B82Y 5/00 - Nanobiotechnologie ou nanomédecine, p. ex. génie protéique ou administration de médicaments
B82Y 15/00 - Nanotechnologie pour l’interaction, la détection ou l'actionnement, p. ex. points quantiques comme marqueurs en dosages protéiques ou moteurs moléculaires
This application discloses triblock copolymers (TBC) molecules with modified chemical headgroup moieties. The triblock copolymers are poly(2-methyl-2-oxazoline)-poly(dimethylsiloxane)-poly(2-methyl-2-oxazoline) (PMOXA-PDMS-PMOXA) copolymers. The headgroup moieties comprise azide or triazole. The TBC molecules are useful as components in polymersome, vesicle, and membrane compositions, such as synthetic membranes used in nanopore sequencing devices. The application also discloses methods of preparing the modified TBC molecules and methods of use.
A61K 9/127 - Vecteurs à bicouches synthétiques, p. ex. liposomes ou liposomes comportant du cholestérol en tant qu’unique agent tensioactif non phosphatidylique
C08G 77/452 - Polymères séquencés ou greffés contenant des segments de polysiloxanes contenant des segments de polymères contenant de l'azote
19.
HYBRID TRIBLOCK COPOLYMER MEMBRANE COMPOSITIONS AND METHODS FOR NANOPORE SEQUENCING
This application discloses hybrid lipid bilayer compositions that include a phospholipid, a triblock copolymer, and a molecule with a pore connecting the two sides of the bilayer, and the use of these lipid bilayer compositions in electrochemical cells for nanopore-based nucleic acid detection techniques, such as nanopore Sequencing-by-Expansion (Nano-SBX) and nanopore Sequencing-by-Synthesis (Nano-SBS) methods.
Provided herein is a method for barcoding, comprising: (a) obtaining multiple populations of cells or cell organelles in a plurality of first volumes, wherein: i. within each first volume the cells or cell organelles comprise nucleic acid molecules that are associated with a first subcode of a set of first subcodes, and ii. in different first volumes the nucleic acid molecules are associated with different first subcodes of the set of first subcodes, (b) pooling the cells or cell organelles, (c) separating the pooled cells or cell organelles into a plurality of second volumes, and (d) associating the nucleic acid molecules with a set of second subcodes in the second volumes, wherein a plurality of the second volumes each receive a different second subcode. This method produces at least some nucleic acid molecules that comprise a first subcode and a second subcode.
C12Q 1/68 - Procédés de mesure ou de test faisant intervenir des enzymes, des acides nucléiques ou des micro-organismesCompositions à cet effetProcédés pour préparer ces compositions faisant intervenir des acides nucléiques
C12Q 1/6806 - Préparation d’acides nucléiques pour analyse, p. ex. pour test de réaction en chaîne par polymérase [PCR]
C12Q 1/6816 - Tests d’hybridation caractérisés par les moyens de détection
C12Q 1/686 - Réaction en chaine par polymérase [PCR]
21.
METHOD FOR INCREASING THROUGHPUT OF SINGLE MOLECULE SEQUENCING BY CONCATENATING SHORT DNA FRAGMENTS
The invention comprises a method and compositions for sequencing library preparation, which increases the throughput of single-molecule sequencing (SMS) platforms by generating long concatenated templates from pools of short DNA molecules.
C12Q 1/686 - Réaction en chaine par polymérase [PCR]
C12Q 1/6876 - Produits d’acides nucléiques utilisés dans l’analyse d’acides nucléiques, p. ex. amorces ou sondes
C40B 40/06 - Bibliothèques comprenant des nucléotides ou des polynucléotides ou leurs dérivés
C40B 50/08 - Synthèse en phase liquide, c.-à-d. dans laquelle tous les blocs servant à créer la bibliothèque sont en phase liquide ou en solution au cours de la création de la bibliothèqueProcédés particuliers de clivage à partir du support liquide
C40B 80/00 - Groupes de liaison ("linkers") ou bras-espaceurs ("spacers") spécialement adaptés à la chimie combinatoire ou aux chimiothèques, p. ex. "linkers" de type "traceless" ou "safety-catch"
22.
METHODS AND COMPOSITIONS FOR DNA LIBRARY PREPARATION AND ANALYSIS
Provided are DNA library preparation methods and compositions that duplicate a target nucleic acid sequence. A target DNA template including the target sequence is circularized via an end adapter to form a circular construct, which is bidirectionally extended by a polymerase-mediated extension that is initiated at nick sites of the end adapter. Following polymerase-mediated extension, a double-length DNA template is formed that includes two copies of the target DNA template (and hence two copies of the target sequence). Each strand of the double-length DNA template includes a parental polynucleotide strand joined to a newly synthesized daughter strand copy of the parental polynucleotide strand. Predetermined sequences can be included in the double-length DNA template, such a primer sequences, unique molecule identifiers, and sequence indexes. Sequencing of the double-length DNA template can reveal genetic/epigenetic information associated with the target sequence. Also provided are methods to create asymmetric and multi-length DNA template constructs.
Aspects provide a method of isolating RNA from a biological sample. The method may include adding the biological sample to a first electrolyte to form a first mixture. The method may include applying a voltage difference between a first electrode and a second electrode. A gel may include a portion of a second electrolyte. The method may include flowing, using the voltage difference, the first subset of RNA molecules into one or more focused zones within the second electrolyte to the second electrode. The method may include separating the second subset of RNA molecules from the first subset. The method may include collecting the first subset of RNA molecules by collecting a second mixture comprising the one or more focused zones. The concentration of the first subset in the second mixture is higher than the concentration of the first subset in the biological sample. Related systems are also described.
Provided are lipid binding molecules and/or combinations of the lipid binding protein with a lipid component (i.e., a mispid) that are used to modify the interaction of a target molecule with a lipid membrane. This includes use of the lipid binding molecules and/or mispids, for example, to improve sequencing efficiency and throughput of nanopore-based sequencing systems. To sequence a target molecule, such as a nucleic acid sequence or a surrogate nucleic acid polymer derived therefrom, lipid binding molecules and/or mispids thereof are combined with the target molecule. The mixture is then applied to a nanopore-based sequencing chip. The target molecule is then sequenced in the presence of the lipid binding molecules and/or nanodiscs, thereby improving the capture, arrival time, and effective concentration of the target molecule across the membrane of the chip. Such improved efficiency is particularly beneficial, for example, when concentrations of a target molecule are low.
Epitachophoresis (ETP) methods and systems described herein allow for efficient and improved extraction of DNA and RNA molecules from a biological sample. The extraction may involve fragmenting nucleic acid molecules to smaller sizes and then running the fragmented sample through an ETP device. The fragmentation improves the extraction of nucleic acid molecules when using a gel with ETP. Fragmentation may also reduce extraction of undesired ribosomal RNA with gel ETP. Nucleic acid molecules are fragmented for preparing a library, and therefore the fragmentation of nucleic acid molecules before extraction rather than after extraction does not negatively impact library prep. In order to facilitate fragmentation, nucleic acid molecules may be treated so that the nucleic acid molecules are not protected from fragmentation.
Provided herein is a composition comprising a mixture of barcoded nucleic acid molecules made from a plurality of cells or cell organelles, wherein the mixture comprises: (a) a population of first nucleic acid molecules each comprising: a sequence of a nucleic acid from a cell or cell organelle, a complement of the sequence, or a barcode identifying the sequence, and a cell-origination barcode; and (b) a population of second nucleic acid molecules each comprising: an epitope specific barcode and a cell-origination barcode. In this composition, the first and second nucleic acid molecules from the same cell or cell organelle have the same cell-origination barcode and first and second nucleic acid molecules from different cells or cell organelles have different cell-origination barcodes.
C12Q 1/68 - Procédés de mesure ou de test faisant intervenir des enzymes, des acides nucléiques ou des micro-organismesCompositions à cet effetProcédés pour préparer ces compositions faisant intervenir des acides nucléiques
C12Q 1/6806 - Préparation d’acides nucléiques pour analyse, p. ex. pour test de réaction en chaîne par polymérase [PCR]
C12Q 1/6816 - Tests d’hybridation caractérisés par les moyens de détection
C12Q 1/686 - Réaction en chaine par polymérase [PCR]
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.
C12N 9/12 - Transférases (2.) transférant des groupes contenant du phosphore, p. ex. kinases (2.7)
C12Q 1/68 - Procédés de mesure ou de test faisant intervenir des enzymes, des acides nucléiques ou des micro-organismesCompositions à cet effetProcédés pour préparer ces compositions faisant intervenir des acides nucléiques
C12Q 1/6874 - Méthodes de séquençage faisant intervenir des réseaux d’acides nucléiques, p. ex. séquençage par hybridation [SBH]
G01N 33/68 - Analyse chimique de matériau biologique, p. ex. de sang ou d'urineTest par des méthodes faisant intervenir la formation de liaisons biospécifiques par ligandsTest immunologique faisant intervenir des protéines, peptides ou amino-acides
28.
EFFICIENT EXECUTION OF MACHINE LEARNING MODELS ON SPECIALIZED HARDWARE
Systems and methods of executing a machine learning model on a specialized computing device can comprise obtaining raw input data by a first computing device; obtaining the machine learning model including a function that applies a set of M model parameters to at least one channel of the raw input data; determining a configuration parameter K for the specialized computing device; configuring the raw input data based on the configuration parameter to obtain configured input data; configuring the machine learning model based on the configuration parameter to obtain a configured machine learning model with a configured model dimension corresponding to the data size of the acceleration path; executing the configured machine learning model with the configured model parameter using the configured input data to obtain output data; and providing the output data.
G06N 3/063 - Réalisation physique, c.-à-d. mise en œuvre matérielle de réseaux neuronaux, de neurones ou de parties de neurone utilisant des moyens électroniques
29.
DEVICES AND METHODS FOR ELECTROPHORETIC EXTRACTION OF NUCLEIC ACIDS FROM BIOLOGICAL SAMPLES
The invention relates to a device methods and an assembly for isolating biological polymers from a sample, the device comprising a top reservoir, a bottom reservoir, a collection chamber located between the top and the bottom reservoirs and operably connected to the top and bottom reservoirs, a sieving matrix capable of passing the biological polymers to be extracted, a semipermeable membrane not capable of passing the biological polymers to be extracted, and at least one set of a working electrode and a counter electrode.
A method of detecting a biomarker by a detection system based on machine learning includes identifying, by the detection system, a plurality of tiles corresponding to whole-slide image data of a tissue sample; generating, by the detection system, tile-level embeddings data based on the plurality of tiles; generating, by the detection system, cell-level embeddings data based on the plurality of tiles; and generating, by the detection system, a slide-level prediction based on the tile-level embeddings data and the cell-level embeddings data, the slide-level prediction indicating presence or absence of the biomarker in the tissue sample.
G16H 10/40 - TIC spécialement adaptées au maniement ou au traitement des données médicales ou de soins de santé relatives aux patients pour des données relatives aux analyses de laboratoire, p. ex. pour des analyses d’échantillon de patient
G06N 3/0895 - Apprentissage faiblement supervisé, p. ex. apprentissage semi-supervisé ou auto-supervisé
G06V 10/50 - Extraction de caractéristiques d’images ou de vidéos en effectuant des opérations dans des blocs d’imagesExtraction de caractéristiques d’images ou de vidéos en utilisant des histogrammes, p. ex. l’histogramme de gradient orienté [HoG]Extraction de caractéristiques d’images ou de vidéos en utilisant l’addition des valeurs d’intensité d’imageAnalyse de projection
31.
IMMUME CELL COUNTING BASED ON IMMUNE REPERTOIRE SEQUENCING
The disclosure includes methods and compositions for accurately detecting subject's immune cell repertoire based on sequencing genomic DNA of immune cells.
C12Q 1/6881 - Produits d’acides nucléiques utilisés dans l’analyse d’acides nucléiques, p. ex. amorces ou sondes pour le typage de tissu ou de cellule, p. ex. sondes d’antigène leucocytaire humain [HLA]
32.
TARGETED DEPLETION OF NON-TARGET LIBRARY MOLECULES USING POISON PRIMERS DURING TARGET CAPTURE OF NEXT-GENERATION SEQUENCING LIBRARIES
The present disclosure is directed to compositions, kits, and methods of target enrichment by unidirectional primer extension, whereby the compositions, kits, and methods utilize both poison primers and target capture primers.
C12Q 1/6848 - Réactions d’amplification d’acides nucléiques caracterisées par les moyens d’empêcher la contamination ou d’augmenter la spécificité ou la sensibilité d’une réaction d’amplification
C12Q 1/6876 - Produits d’acides nucléiques utilisés dans l’analyse d’acides nucléiques, p. ex. amorces ou sondes
A method of barcoding is provided. The method comprises: (a) obtaining a population of cells or cell organelles in a first volume, wherein the cells or cell organelles comprise target molecules that are associated with a first assayable oligonucleotide subunit; (b) separating the cells or cell organelles into a plurality of second volumes, wherein at least some of second volumes receive a single cell or cell organelle from the population of cells or cell organelles; and (c) associating a plurality of second assayable oligonucleotide subunits with the first assayable oligonucleotide subunit in the second volumes, wherein at least some of the second volumes each receive a different second assayable oligonucleotide subunit, to produce at least some nucleic acid molecules that comprise a first assayable oligonucleotide subunit and a second assayable oligonucleotide subunit.
C12Q 1/68 - Procédés de mesure ou de test faisant intervenir des enzymes, des acides nucléiques ou des micro-organismesCompositions à cet effetProcédés pour préparer ces compositions faisant intervenir des acides nucléiques
C12Q 1/6806 - Préparation d’acides nucléiques pour analyse, p. ex. pour test de réaction en chaîne par polymérase [PCR]
C12Q 1/6816 - Tests d’hybridation caractérisés par les moyens de détection
C12Q 1/686 - Réaction en chaine par polymérase [PCR]
34.
TARGETED NEXT-GENERATION SEQUENCING VIA ANCHORED PRIMER EXTENSION
The present disclosure is directed to compositions, kits, and methods of and methods which facilitate the amplification of a unidirectional primer extension product. In particular, the compositions, kits, and methods described herein facilitate the amplification of a unidirectional primer extension product without the need to incorporate a second polymerase chain reaction primer binding target on a distal end of an initial single-stranded nucleic acid molecule primer extension product.
C12Q 1/6886 - Produits d’acides nucléiques utilisés dans l’analyse d’acides nucléiques, p. ex. amorces ou sondes pour les maladies provoquées par des altérations du matériel génétique pour le cancer
Provided herein are methods and compositions for detecting and/or quantitating target analytes, including nucleic acids and polypeptides, using nanopore detectable barcodes.
For high sequencing throughput, circuitry can compress read data generated in real-time by a sequencing device. Various compression techniques can be used. A stream of raw data can be processed to generate raw read data stream. The raw read data stream may include sub-streams of data comprising a header data sub-stream, a basecall sub-stream, and a quality score sub-stream. The sub-streams can be extracted and compressed using separate threads, and the compressed data can be recombined. Sequence reads corresponding to different copies of the same nucleic acid molecule may be clustered and used to generate a consensus read. The number of sequence reads that are used to generate the consensus read can be limited to a threshold when a consensus read is substantially accurate. After the limit is reached, data from any new raw read data corresponding to the same nucleic acid molecule may be discarded.
The invention includes improved methods and compositions for nucleic acid hybridization wherein the improvement comprises the use of enhancer oligonucleotides. Target enrichment is performed using probe oligonucleotides, wherein each probe oligonucleotide comprising a target-binding region, and a first and a second primer-binding region, and one or more enhancer oligonucleotides capable of hybridizing to at least one of the primer binding regions. The forward and reverse primer binding sites can be universal primer binding sites.
C12Q 1/6886 - Produits d’acides nucléiques utilisés dans l’analyse d’acides nucléiques, p. ex. amorces ou sondes pour les maladies provoquées par des altérations du matériel génétique pour le cancer
Described are methods of detecting modified nucleotide bases in a DNA sample using specific DNA glycosylases to excise a modified nucleobase of interest. Prior to glycosylase treatment, DNA target fragments are copied by a DNA polymerase to produce a complementary copy strand that preserves the genetic information of the DNA target strand. Following glycosylase treatment, the DNA target fragments are repaired by either ligating across the gaps to produce a deletion at each position of the modified nucleobase of interest or filling in the gaps with a single non-native nucleotide to produce a base substitution at each position of the modified nucleobase of interest. Comparison of the DNA sequences of the two strands of the target fragments enables identification of the positions of the modified nucleotide base in the DNA target fragment.
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.
A nanopore-based sequencing system includes a plurality of nanopore-based sequencing chips. Each of the nanopore-based sequencing chips comprises a plurality of nanopore sensors. The system comprises at least one flow cell coupled to at least one of the plurality of nanopore-based sequencing chips, wherein the flow cell coupled to the at least one of the plurality of nanopore-based sequencing chips comprises one or more fluidic flow channels that allow a fluid external to the system to flow on top of the nanopore-based sequencing chip and out of the system. The system further comprises a printed circuit board electrically connected to the plurality of nanopore-based sequencing chips.
The present disclosure relates to compositions and methods based on polypeptide-tagged nucleotide, and the use of such polypeptide-tagged nucleotides in nanopore devices and methods.
Disclosed is a novel structure of a nucleic acid template and the method of making and using the structure. The structure consists of a double-stranded circle with a single-stranded gap. The circular gapped structure includes an extendable end from which copying or sequencing can be initiated.
Epitachophoresis (ETP) methods and devices that improve concentrating samples and/or separating components of samples. ETP methods and devices allow for electromigration in two dimensions. Electromigration of a sample may first occur in a first dimension along a single plane. Electromigration may then continue in a second dimension, which may be different from the first dimension. The volume where the electromigration occurs may significantly reduce from the first dimension to the second dimension. This smaller dimension may allow for increased concentration of samples or improved separation of components of a sample.
Methods and systems for constructing cfDNA sequence libraries, including methods and systems for sequencing 5' and/or 3' cfDNA overhangs to identify overhang length and sequence topology data are described herein. The method can comprise, for example, the use of the cfDNA topology data to generate cfDNA overhang sequence libraries.
Translocation control for sensing by a nanopore, as well as methods and products related to the same, are provided. Such methods optimize duplex stability to provide high fill rate (of the hybridization sites) but do not prevent rapid dissociation required for high read rates, as well as controlling the translocation of a target molecule for sensing by a nanopore by use of a selective pulsed voltage. Products related to the same include a reporter construct comprising two or more phosphoramidites.
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 relates methods and associated compositions that provide fast, efficient site-selective conjugation of a protein, such as the pore-forming protein α-hemolysin, to a biomolecule, such as a DNA polymerase, and the use of such site-selective protein-biomolecule conjugates in nanopore devices and methods.
C07K 14/315 - Peptides ayant plus de 20 amino-acidesGastrinesSomatostatinesMélanotropinesLeurs dérivés provenant de bactéries provenant de Streptococcus (G), p. ex. Enterocoques
A61K 47/64 - Conjugués médicament-peptide, médicament-protéine ou médicament-acide polyaminé, c.-à-d. l’agent de modification étant un peptide, une protéine ou un acide polyaminé lié par covalence ou complexé à un agent thérapeutiquement actif
C07K 1/107 - Procédés généraux de préparation de peptides par modification chimique de peptides précurseurs
C12N 15/62 - Séquences d'ADN codant pour des protéines de fusion
C12Q 1/68 - Procédés de mesure ou de test faisant intervenir des enzymes, des acides nucléiques ou des micro-organismesCompositions à cet effetProcédés pour préparer ces compositions faisant intervenir des acides nucléiques
48.
NUCLEOSIDE-5'-OLIGOPHOSPHATES HAVING A CATIONICALLY-MODIFIED NUCELOBASE
Disclosed herein are base-modified nucleoside-5′-oligophosphates (bm-N5OP) that include a positively charged moiety at least at one position of the base, compositions comprising the same, compositions made from the same, methods of making the same, and methods of using the same. The bm-N5OP disclosed herein are useful, for example, as tagged nucleotides for use in nanoSBS methods and for generating primers and/or templates for use in nanoSBS methods. When incorporated into a polynucleotide, the disclosed bm-N5OPs can neutralize at least a portion of the negative charge of the overall polynucleotide molecule.
The present disclosure provides a method for enrichment of at least one target nucleic acid in a library of nucleic acids. A first oligonucleotide is hybridized to a target nucleic acid in library of nucleic acids having first and second adapters. The hybridized first oligonucleotide is extended with a first polymerase, thereby producing a first primer extension complex including the target nucleic acid and the extended first oligonucleotide. The first primer extension complex is captured, enriched relative to the library of nucleic acids, and a second oligonucleotide is hybridized to the target nucleic acid. The hybridized second oligonucleotide is extended with a second polymerase, thereby producing a second primer extension complex including the target nucleic acid and the extended second oligonucleotide, and further liberating the extended first oligonucleotide from the first primer extension complex.
C12Q 1/6881 - Produits d’acides nucléiques utilisés dans l’analyse d’acides nucléiques, p. ex. amorces ou sondes pour le typage de tissu ou de cellule, p. ex. sondes d’antigène leucocytaire humain [HLA]
C12N 15/10 - Procédés pour l'isolement, la préparation ou la purification d'ADN ou d'ARN
NUCLEOSIDE-5'-OLIGOPHOSPHATES TAGGED WITH POSTIVIELY-CHARGED POLYMERS, NANOPORES INCORPORATING NEGATIVE CHARGES, AND METHODS AND SYSTEMS USING THE SAME
The present disclosure relates to tagged nucleoside-5′-oligophosphates having a positively charged polymer tag structure and components thereof. Such nucleoside-5′-oligophosphates are useful, for example, in nanopore-based sequencing-by-synthesis applications. Also disclosed herein are nanopore constructs engineered to have additional negatively-charged moieties in the channel of the nanopore. Such nanopores can be useful, for example, for providing a repellant force against template and/or primer nucleic acids inserting into the pore during a nucleic sequence-by-synthesis process. The tagged nucleoside-5′-oligophosphates and nanopores disclosed herein can be used together to provide nanopore-based nucleic acid sequencing-by-synthesis systems and processes having reduced background tag levels and improved throughput.
Described are methods of detecting modified nucleotide bases in a nucleic acid sample using specific DNA glycosylases to excise a modified nucleobase of interest. Prior to glycosylase treatment, DNA target fragment templates are copied by a DNA polymerase to produce a first complementary copy strand that preserves the genetic information of the DNA target fragment. Following glycosylase treatment, the DNA target fragment templates are copied by an abasic bypass polymerase to produce a second complementary copy strand that preserves the epigenetic information of the DNA target fragment. Comparison of the DNA sequences of the two complementary copy strands enables identification of the positions of the modified nucleobases in the DNA target fragment.
C40B 50/06 - Procédés biochimiques, p. ex. utilisant des enzymes ou des micro-organismes viables entiers
C12N 15/10 - Procédés pour l'isolement, la préparation ou la purification d'ADN ou d'ARN
C12Q 1/68 - Procédés de mesure ou de test faisant intervenir des enzymes, des acides nucléiques ou des micro-organismesCompositions à cet effetProcédés pour préparer ces compositions faisant intervenir des acides nucléiques
C12Q 1/6881 - Produits d’acides nucléiques utilisés dans l’analyse d’acides nucléiques, p. ex. amorces ou sondes pour le typage de tissu ou de cellule, p. ex. sondes d’antigène leucocytaire humain [HLA]
C12Q 1/6886 - Produits d’acides nucléiques utilisés dans l’analyse d’acides nucléiques, p. ex. amorces ou sondes pour les maladies provoquées par des altérations du matériel génétique pour le cancer
54.
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.
Methods of sequencing by expansion and related improvements to the sequencing of surrogate polymers in a nanopore are described. The surrogate polymer is formed from a template nucleic acid molecule. A surrogate polymer includes multiple units. Each unit includes a reporter code portion. The reporter codes correspond to the different nucleotides. surrogate polymers may get stuck in the nanopore. Embodiments described herein address these stuck surrogate polymers. In order to allow for multiple reads on the surrogate polymer, a processive consensus technique can be applied. The surrogate polymer may be moved a few units forward and then fewer units backward so that some of the same reporter codes are identified again. This method allows for multiple reads of the same reporter codes. The surrogate polymer eventually passes through the nanopore in the forward direction. Periodically, higher clearing voltages may be applied to clear any stuck surrogate polymer in the nanopore.
C40B 50/06 - Procédés biochimiques, p. ex. utilisant des enzymes ou des micro-organismes viables entiers
C12N 15/10 - Procédés pour l'isolement, la préparation ou la purification d'ADN ou d'ARN
C12Q 1/68 - Procédés de mesure ou de test faisant intervenir des enzymes, des acides nucléiques ou des micro-organismesCompositions à cet effetProcédés pour préparer ces compositions faisant intervenir des acides nucléiques
C12Q 1/6881 - Produits d’acides nucléiques utilisés dans l’analyse d’acides nucléiques, p. ex. amorces ou sondes pour le typage de tissu ou de cellule, p. ex. sondes d’antigène leucocytaire humain [HLA]
C12Q 1/6886 - Produits d’acides nucléiques utilisés dans l’analyse d’acides nucléiques, p. ex. amorces ou sondes pour les maladies provoquées par des altérations du matériel génétique pour le cancer
57.
IMPROVEMENTS TO NEXT-GENERATION TARGET ENRICHMENT PERFORMANCE
The present disclosure is directed to compositions and kits for PCR amplification. The present disclosure is also directed to methods of amplifying nucleic acid molecules to improve upon uniformity of coverage and/or to reduce GC bias during downstream sequencing operations.
C12Q 1/6806 - Préparation d’acides nucléiques pour analyse, p. ex. pour test de réaction en chaîne par polymérase [PCR]
C12Q 1/6848 - Réactions d’amplification d’acides nucléiques caracterisées par les moyens d’empêcher la contamination ou d’augmenter la spécificité ou la sensibilité d’une réaction d’amplification
58.
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.
C12Q 1/68 - Procédés de mesure ou de test faisant intervenir des enzymes, des acides nucléiques ou des micro-organismesCompositions à cet effetProcédés pour préparer ces compositions faisant intervenir des acides nucléiques
C12Q 1/6806 - Préparation d’acides nucléiques pour analyse, p. ex. pour test de réaction en chaîne par polymérase [PCR]
C12Q 1/6816 - Tests d’hybridation caractérisés par les moyens de détection
C12Q 1/686 - Réaction en chaine par polymérase [PCR]
59.
Method for labeling ligation products with cell-specific barcodes II
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.
C12Q 1/68 - Procédés de mesure ou de test faisant intervenir des enzymes, des acides nucléiques ou des micro-organismesCompositions à cet effetProcédés pour préparer ces compositions faisant intervenir des acides nucléiques
C12Q 1/6806 - Préparation d’acides nucléiques pour analyse, p. ex. pour test de réaction en chaîne par polymérase [PCR]
C12Q 1/6816 - Tests d’hybridation caractérisés par les moyens de détection
C12Q 1/686 - Réaction en chaine par polymérase [PCR]
60.
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 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.
A method of forming a plurality of lipid bilayers over an array of cells in a nanopore based sequencing chip is disclosed. Each of the cells comprises a well. A salt buffer solution is flowed over the array of cells in the nanopore based sequencing chip to substantially fill the wells in the cells with the salt buffer solution. A lipid and solvent mixture is flowed over the array of cells to deposit the lipid and solvent mixture over at least some of the wells in the cells. A first portion of the cells, each having a lipid bilayer over its well, is detected. A second portion of the cells, each having a lipid membrane but not a lipid bilayer over its well, is detected. An electrical lipid-thinning stimulus is selectively applied to the second portion of the cells but not to the first portion of the cells.
C40B 50/06 - Procédés biochimiques, p. ex. utilisant des enzymes ou des micro-organismes viables entiers
C12Q 1/68 - Procédés de mesure ou de test faisant intervenir des enzymes, des acides nucléiques ou des micro-organismesCompositions à cet effetProcédés pour préparer ces compositions faisant intervenir des acides nucléiques
C12Q 1/6881 - Produits d’acides nucléiques utilisés dans l’analyse d’acides nucléiques, p. ex. amorces ou sondes pour le typage de tissu ou de cellule, p. ex. sondes d’antigène leucocytaire humain [HLA]
C12N 15/10 - Procédés pour l'isolement, la préparation ou la purification d'ADN ou d'ARN
C12Q 1/6886 - Produits d’acides nucléiques utilisés dans l’analyse d’acides nucléiques, p. ex. amorces ou sondes pour les maladies provoquées par des altérations du matériel génétique pour le cancer
64.
VARIANT ALLELE ENRICHMENT BY UNIDIRECTIONAL DUAL PROBE PRIMER EXTENSION
The present disclosure provides a method for enrichment of at least one target nucleic acid in a library of nucleic acids. This present disclosure is also directed to a faster and easier method of target capture using primer extension reactions that can improve ease of use, turnaround time, and variant allele specificity by designing target enrichment primers to specifically enrich library fragments based on the relative location of the variant base(s) in the primer, the utilization of polymerases with better priming specificity, designing the variant bases in the capture primer, designing the variant bases in the release primer, and/or designing variant specific primers to the both the plus and minus strands of the target library fragment.
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.
C12Q 1/68 - Procédés de mesure ou de test faisant intervenir des enzymes, des acides nucléiques ou des micro-organismesCompositions à cet effetProcédés pour préparer ces compositions faisant intervenir des acides nucléiques
C12N 9/12 - Transférases (2.) transférant des groupes contenant du phosphore, p. ex. kinases (2.7)
C12Q 1/6874 - Méthodes de séquençage faisant intervenir des réseaux d’acides nucléiques, p. ex. séquençage par hybridation [SBH]
G01N 33/68 - Analyse chimique de matériau biologique, p. ex. de sang ou d'urineTest par des méthodes faisant intervenir la formation de liaisons biospécifiques par ligandsTest immunologique faisant intervenir des protéines, peptides ou amino-acides
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.
G01N 33/487 - Analyse physique de matériau biologique de matériau biologique liquide
B81B 1/00 - Dispositifs sans éléments mobiles ou flexibles, p. ex. dispositifs capillaires microscopiques
G01N 15/12 - Recherche de particules individuelles en mesurant des effets électriques ou magnétiques en observant des changements de résistance ou d’impédance à travers des fentes traversées par des particules individuelles, p. ex. en utilisant le principe de Coulter
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.
The invention includes improved methods and compositions for reduction of a C5-C6 double bond of a cytosine. In particular, the improved methods and compositions for reduction of a C5-C6 double bond of a cytosine is via enzymatic means, not via chemical means. In particular, the disclosure is directed to methods of converting 5,6-dihydro-fC (fC) and/or 5,6-dihydro-caC to 5,6-dihydro-U (DHU). In particular, the disclosure is directed to methods of converting 5fC and/or 5caC to DHU. In addition, the disclosure is directed to methods for detection of epigenetic cytosine modification, particularly cytosine methylation, using ene reductases to reduce the C5-C6 double bond of cytosine.
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.
C12Q 1/68 - Procédés de mesure ou de test faisant intervenir des enzymes, des acides nucléiques ou des micro-organismesCompositions à cet effetProcédés pour préparer ces compositions faisant intervenir des acides nucléiques
C12Q 1/6806 - Préparation d’acides nucléiques pour analyse, p. ex. pour test de réaction en chaîne par polymérase [PCR]
C12Q 1/6816 - Tests d’hybridation caractérisés par les moyens de détection
C12Q 1/686 - Réaction en chaine par polymérase [PCR]
72.
Kit for split-pool barcoding target molecules that are in or on cells or cell organelles
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.
C12Q 1/68 - Procédés de mesure ou de test faisant intervenir des enzymes, des acides nucléiques ou des micro-organismesCompositions à cet effetProcédés pour préparer ces compositions faisant intervenir des acides nucléiques
C12Q 1/6806 - Préparation d’acides nucléiques pour analyse, p. ex. pour test de réaction en chaîne par polymérase [PCR]
C12Q 1/6816 - Tests d’hybridation caractérisés par les moyens de détection
C12Q 1/686 - Réaction en chaine par polymérase [PCR]
Ecole Polytechnique Federale De Lausanne (EPFL) (Suisse)
Roche Sequencing Solutions, Inc. (USA)
Inventeur(s)
Feng, Jiandong
Liu, Ke
Radenovic, Aleksandra
Astier, Yann
Abrégé
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 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.
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.
This application discloses electrochemical cells, nanopore devices, and associated buffer compositions useful for nanopore-based nucleic acid sequencing. Also disclosed are methods for using the electrochemical cells, devices, and compositions in nanopore-based nucleic acid sequencing methods, such as nanopore Sequencing-by-Expansion (Nano-SBX) and nanopore Sequencing-by-Synthesis (Nano-SBS) methods.
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 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.
A method of detecting a state of a lipid membrane in a cell of a nanopore based sequencing chip is disclosed. A lipid membrane is coupled with an integrating capacitor, 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 an intermediate change in the AC voltage is determined. A state of the lipid membrane is determined based on the determined change in the sampled voltage across the integrating capacitor in response to the intermediate change in the AC voltage.
C12Q 1/68 - Procédés de mesure ou de test faisant intervenir des enzymes, des acides nucléiques ou des micro-organismesCompositions à cet effetProcédés pour préparer ces compositions faisant intervenir des acides nucléiques
G01N 33/487 - Analyse physique de matériau biologique de matériau biologique liquide
G01R 27/26 - Mesure de l'inductance ou de la capacitanceMesure du facteur de qualité, p. ex. en utilisant la méthode par résonanceMesure de facteur de pertesMesure des constantes diélectriques
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 14/00 - Peptides ayant plus de 20 amino-acidesGastrinesSomatostatinesMélanotropinesLeurs dérivés
A61K 38/02 - Peptides à nombre indéterminé d'amino-acidesLeurs dérivés
C07K 14/31 - Peptides ayant plus de 20 amino-acidesGastrinesSomatostatinesMélanotropinesLeurs dérivés provenant de bactéries provenant de Micrococcaceae (F) provenant de Staphylococcus (G)
C12N 15/01 - Préparation de mutants sans introduction de matériel génétique étrangerProcédés de criblage à cet effet
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 present disclosure relates to a process for the regiochemically and enantiomerically controlled synthesis of phosphoramidite-containing monomers, and to intermediate products of this process. In some embodiments, the phosphoramidite-containing monomers or their precursors are regioisomerically and/or enantiomerically pure and may be polymerized into polymers or copolymers.
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.
C12Q 1/68 - Procédés de mesure ou de test faisant intervenir des enzymes, des acides nucléiques ou des micro-organismesCompositions à cet effetProcédés pour préparer ces compositions faisant intervenir des acides nucléiques
C12Q 1/6806 - Préparation d’acides nucléiques pour analyse, p. ex. pour test de réaction en chaîne par polymérase [PCR]
C12Q 1/6816 - Tests d’hybridation caractérisés par les moyens de détection
C12Q 1/686 - Réaction en chaine par polymérase [PCR]
87.
A METHOD FOR DETECTING REACTION VOLUME DEVIATIONS IN A DIGITAL POLYMERASE CHAIN REACTION
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.
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 ayant plus de 20 amino-acidesGastrinesSomatostatinesMélanotropinesLeurs dérivés provenant de bactéries provenant de Micrococcaceae (F) provenant de Staphylococcus (G)
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 - Réseaux récurrents, p. ex. réseaux de Hopfield caractérisés par la présence de mémoire ou de portes, p. ex. mémoire longue à court terme [LSTM] ou unités récurrentes à porte [GRU]
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 - Produits d’acides nucléiques utilisés dans l’analyse d’acides nucléiques, p. ex. amorces ou sondes pour les maladies provoquées par des altérations du matériel génétique pour le cancer
C12N 15/10 - Procédés pour l'isolement, la préparation ou la purification d'ADN ou d'ARN
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.
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.
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 - Méthodes de séquençage faisant intervenir des réseaux d’acides nucléiques, p. ex. séquençage par hybridation [SBH]
C12Q 1/6883 - Produits d’acides nucléiques utilisés dans l’analyse d’acides nucléiques, p. ex. amorces ou sondes pour les maladies provoquées par des altérations du matériel génétique
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