Provided herein are methods for library preparation that may be applied to duplex Sequencing by Expansion. In particular, the present invention relates to methods for generating duplex nucleic acid constructs for use as templates for Xpandomer synthesis and nanopore sequence determination thereof that provide sequence information from both strands of a DNA target fragment in a single run. The present invention also provides methods for epigenetic analysis using duplex template constructs that include a parental strand derived from a library fragment that may include modified nucleobases and a newly synthesized complementary daughter copy strand that includes native nucleobases. The present invention also relates to improved reaction conditions for synthesizing Xpandomer copies of the duplex nucleic acid templates. Compositions and kits for use in the methods are also provided.
A system for quality control of a medical device or software product including a plurality of quality control (QC) modules, each QC module configured for one or more QC task(s) of a QC process and configured for access by a plurality of users assigned roles and responsibilities for the respective QC task(s), the quality control library configured to store a plurality of QC records including information associated with the QC task(s) and governmental rule(s) or regulation(s) applicable to the respective QC module and medical device product.
G16H 40/20 - ICT specially adapted for the management or administration of healthcare resources or facilitiesICT specially adapted for the management or operation of medical equipment or devices for the management or administration of healthcare resources or facilities, e.g. managing hospital staff or surgery rooms
G06Q 10/0639 - Performance analysis of employeesPerformance analysis of enterprise or organisation operations
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.
in vitroin 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.
C07D 249/06 - 1,2,3-TriazolesHydrogenated 1,2,3-triazoles with aryl radicals directly attached to ring atoms
C07D 401/14 - Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing three or more hetero rings
C07D 403/14 - Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group containing three or more hetero rings
C07D 401/04 - Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings directly linked by a ring-member-to-ring- member bond
C07D 405/14 - Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing three or more hetero rings
C07D 409/14 - Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing three or more hetero rings
C07D 421/14 - Heterocyclic compounds containing two or more hetero rings, at least one ring having selenium, tellurium, or halogen atoms as ring hetero atoms containing three or more hetero rings
C07D 473/00 - Heterocyclic compounds containing purine ring systems
C07D 519/00 - Heterocyclic compounds containing more than one system of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring system not provided for in groups or
The present invention relates to methods and compositions for generating duplex nucleic acid template constructs that find use in duplex Sequencing by Expansion and improved reaction conditions for synthesizing Xpandomer copies of the duplex nucleic acid template constructs for nanopore sequencing. Also provided are novel adapter compositions for generating the duplex nucleic acid templates. In particular, provided are extendable Y adapter, cleavable hairpin adapters, and Y-hairpin hybrid adapters. The methods and compositions of the present invention may be used for genetic and epigenetic analysis in a single experiment.
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.
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.
A method of analyzing a molecule is disclosed. A voltage source is selectively connected to or disconnected from a capacitor using a switch controlled by a reset signal. A charge is stored in a capacitor when the voltage source is connected to the capacitor. The capacitor is discharged through a nanopore in a membrane when the voltage source is disconnected from the capacitor. A duty cycle of the reset signal is determined such that the voltage source and the capacitor is connected for at least a one tenth portion of a reset signal period and disconnected for a remaining portion of the reset signal period, such that a voltage across the nanopore is maintained at a higher level during the portion of the reset signal period in which the connection is maintained than during the remaining portion of the reset signal period in which the connection is not maintained.
G01N 27/22 - Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating impedance by investigating capacitance
The present disclosure relates to performing endpoint genotyping based on a Gaussian mixture model (GMM). As one example, a method includes: obtaining, using a processor, cross-talk corrected fluorescent PCR data for an assay; determining, using the processor, a plurality of values based on the cross-talk corrected fluorescent PCR data; generating, using the processor, an assay based on the plurality of values; determining, using the processor, a genotype of a sample based on one or more gray-zones and at least one value associated with the sample; and displaying the genotype determination on a user display.
The present disclosure relates to classifying genotypes on an assay plate according to predefined standards. As one example, a method includes: obtaining all raw melt curves for an assay plate from a memory; determining melting peak curves for all standards and unknown samples; calculating a median or mean peak curve for each of the standards based on a plurality of replicate melting peak curves of each standard; calculating at least one correlation coefficient between each of the unknown samples and the median standards; comparing the correlation coefficient to a threshold level for each standard; and assigning the unknown sample a genotype of the standard when the unknown sample has a correlation coefficient greater than the threshold level.
The present disclosure relates to determining crosstalk coefficients for an analyzer based on customer data. In an aspect, a method includes obtaining a manufacturer defined temperature matrix for an analyzer. The method also includes obtaining a customer defined temperature matrix. The method also includes generating a custom temperature dependent crosstalk matrix for a customer. The method further includes performing, based on the custom temperature dependent crosstalk matrix, a matrix inversion and dot matrix multiplication to generate a crosstalk corrected customer fluorescence vector. The method further includes modifying manufacturer defined temperature dependent crosstalk coefficients on the analyzer based on the crosstalk corrected customer fluorescence vector.
G01N 21/27 - ColourSpectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands using photo-electric detection
A method for efficiently processing data using a parallel processor includes storing in memory data in an array in an interleaved format. The first two bits of data stored in the memory for each read is accessed using a plurality of threads, with each thread processing data from one sequence read. Each thread in parallel performs the same operation on the accessed first two bits of data. These steps are repeated until all the data in the array is processed.
The present disclosure is directed to hybridization buffers, reaction mixtures, and master mixes suitable for enrichment of DNA oligonucleotides. In some embodiments, the hybridization buffers, reaction mixtures, and master mixes are free of formamide.
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.
Techniques for replacing nanopores within a nanopore based sequencing chip are provided. A first electrolyte solution is added to the external reservoir of the sequencing chip, introducing an osmotic imbalance between the reservoir and the well chamber located on the opposite side of a lipid bilayer membrane. The osmotic imbalance causes the membrane to change shape, and a nanopore within the membrane to be ejected. A second electrolyte solution is then added to the external reservoir to provide replacement nanopores and to restore the membrane shape. The replacement nanopores can be inserted into the membrane, effectively replacing the initial pore without causing the destruction of the membrane.
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.
A collaboration platform is provided to analyze one or more biomedical data sets. The collaboration platform may include a project management engine, a data ingestion engine, and a data analysis engine. The project management engine may generate a plurality of project environments for analyzing biomedical data sets of interest, and designate a set of authorized users with access to given project environments. The data ingestion engine may process data sets and store them in a central data storage system for access within the project environments. The data analysis engine may perform analyses and special computing on the ingested data and generate updated output data that may be re-ingested for further analyses in the project environments.
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 50/70 - ICT specially adapted for medical diagnosis, medical simulation or medical data miningICT specially adapted for detecting, monitoring or modelling epidemics or pandemics for mining of medical data, e.g. analysing previous cases of other patients
G06F 21/62 - Protecting access to data via a platform, e.g. using keys or access control rules
A method of analyzing molecules using a nanopore array including a plurality of cells included on a chip is disclosed. Nanopores are caused to be formed in at least a portion of the plurality of the cells. A first physical measurement of the nanopores is evaluated. It is determined whether to cause the molecules to interact with the nanopores. At least a portion of the nanopores is caused to interact with the molecules. A second physical measurement of the nanopores that indicates a property of the molecules is evaluated. It is determined whether to cause the nanopores to be reformed so that the cells may be reused to interact with additional molecules.
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 15/12 - Investigating individual particles by measuring electrical or magnetic effects by observing changes in resistance or impedance across apertures when traversed by individual particles, e.g. by using the Coulter principle
The present disclosure provides methods of sequencing one or more target nucleic acid molecules, where each of the one or more target nucleic acid molecules includes one or more modified nucleotides, and wherein the method does not require conversion of any of the one or more modified nucleotides prior to sequencing, and/or does not require any amplification (PCR) cycles prior to sequencing In some embodiments, sequencing is performed with a sequencing-by-tag sequencing device. In other embodiments, sequencing is performed with a Single Molecule Real Time sequencing device.
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.
C12N 15/10 - Processes for the isolation, preparation or purification of DNA or RNA
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
Nucleic acid sequencing methods and related products are disclosed. Methods for sequencing a target nucleic acid comprise providing a daughter strand produced by a template-directed synthesis, the daughter strand comprising a plurality of subunits coupled in a sequence corresponding to a contiguous nucleotide sequence of all or a portion of the target nucleic acid, wherein the individual subunits comprise a tether, at least one probe or nucleobase residue, and at least one selectively cleavable bond. The selectively cleavable bond(s) is/are cleaved to yield an Xpandomer of a length longer than the plurality of the subunits of the daughter strand, the Xpandomer comprising the tethers and reporter elements for parsing genetic information in a sequence corresponding to the contiguous nucleotide sequence of all or a portion of the target nucleic acid. Reporter elements of the Xpandomer are then detected. Corresponding products, including Xpandomers are also disclosed.
C12Q 1/6897 - Measuring or testing processes involving enzymes, nucleic acids or microorganismsCompositions thereforProcesses of preparing such compositions involving nucleic acids involving reporter genes operably linked to promoters
C07H 21/00 - Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
C12Q 1/68 - Measuring or testing processes involving enzymes, nucleic acids or microorganismsCompositions thereforProcesses of preparing such compositions involving nucleic acids
A system includes a circuit configured to detect a voltage corresponding to an electrical measurement of a nanopore. The system also includes a component configured to compare the voltage to another voltage. Based at least in part on the comparison, a one bit indicator is determined. The one bit indicator indicates whether the voltage indicates a change in a state of the nanopore. In the event it is determined that the voltage indicates the change in the state of the nanopore, a multiple bit signal is provided for output.
Described herein are alpha-hemolysin nanopores having relatively narrow channels and D127G and D128K substitutions relative to SEQ ID NO: 1. The narrow channel reduces the extent to which the nucleic acid template threads through the nanopore, while the D127G and D128K substitutions improve the lifetime and arrival rate of the narrow channel pores. Also disclosed herein are polypeptides for forming such nanopores, systems comprising such nanopores, and methods of making and using such nanopores.
C07K 14/31 - Peptides having more than 20 amino acidsGastrinsSomatostatinsMelanotropinsDerivatives thereof from bacteria from Micrococcaceae (F) from Staphylococcus (G)
C12N 15/10 - Processes for the isolation, preparation or purification of DNA or RNA
Disclosed herein are ruthenium-containing materials, such as ruthenium containing materials having a double layer capacitance ranging from between about 180 pF/um2 to about 320 pF/um2. In some embodiments, the ruthenium-containing materials are suitable for use in electrodes. In some embodiments, the ruthenium-containing materials are suitable for use in nanopore sequencing devices.
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.
G01N 27/26 - Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variablesInvestigating or analysing materials by the use of electric, electrochemical, or magnetic means by using electrolysis or electrophoresis
B82Y 35/00 - Methods or apparatus for measurement or analysis of nanostructures
G01N 33/487 - Physical analysis of biological material of liquid biological material
27.
ENHANCEMENT OF NUCLEIC ACID POLYMERIZATION BY AROMATIC COMPOUNDS
The invention relates to compounds, 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 - Nucleic acid amplification reactions characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
C07D 249/06 - 1,2,3-TriazolesHydrogenated 1,2,3-triazoles with aryl radicals directly attached to ring atoms
C07D 401/14 - Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing three or more hetero rings
C07D 403/14 - Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group containing three or more hetero rings
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 of undefined number of amino acidsDerivatives thereof
C07K 14/00 - Peptides having more than 20 amino acidsGastrinsSomatostatinsMelanotropinsDerivatives thereof
C07K 14/31 - Peptides having more than 20 amino acidsGastrinsSomatostatinsMelanotropinsDerivatives thereof from bacteria from Micrococcaceae (F) from Staphylococcus (G)
C12N 15/01 - Preparation of mutants without inserting foreign genetic material thereinScreening processes therefor
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.
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 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.
G01N 33/487 - Physical analysis of biological material of liquid biological material
G01R 27/26 - Measuring inductance or capacitanceMeasuring quality factor, e.g. by using the resonance methodMeasuring loss factorMeasuring dielectric constants
34.
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 - Processes for the preparation of sugar derivatives
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
C12Q 1/6806 - Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
36.
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 - Nucleic acid amplification reactions characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
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.
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 acidsGastrinsSomatostatinsMelanotropinsDerivatives thereof from bacteria from Micrococcaceae (F) from Staphylococcus (G)
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 microorganismsCompositions thereforProcesses 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
40.
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 (South Africa)
Inventor
Klass, Daniel
Chang, Shwu Shin
Graf Grachet, Nathalia
Garcia-Montoya, Gladys
Saelee, Seng Lor
Ristow, Peter
Abstract
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.
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.
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 - Investigating individual particles by measuring electrical or magnetic effects by observing changes in resistance or impedance across apertures when traversed by individual particles, e.g. by using the Coulter principle
45.
MODIFIED TRIBLOCK COPOLYMER COMPOUNDS AND METHODS OF USE THEREOF
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.
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.
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 1/6876 - Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
C40B 40/06 - Libraries containing nucleotides or polynucleotides, or derivatives thereof
C40B 50/08 - Liquid phase synthesis, i.e. wherein all library building blocks are in liquid phase or in solution during library creationParticular methods of cleavage from the liquid support
C40B 80/00 - Linkers or spacers specially adapted for combinatorial chemistry or libraries, e.g. traceless linkers or safety-catch linkers
49.
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.
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.
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.
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 - 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
G06N 3/0895 - Weakly supervised learning, e.g. semi-supervised or self-supervised learning
G06V 10/50 - Extraction of image or video features by performing operations within image blocksExtraction of image or video features by using histograms, e.g. histogram of oriented gradients [HoG]Extraction of image or video features by summing image-intensity valuesProjection analysis
58.
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 - 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
59.
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 - Nucleic acid amplification reactions characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
C12Q 1/6876 - Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
60.
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 - 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
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.
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 - 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
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.
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.
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.
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 having more than 20 amino acidsGastrinsSomatostatinsMelanotropinsDerivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci
A61K 47/64 - Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
C07K 1/107 - General processes for the preparation of peptides by chemical modification of precursor peptides
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 - 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
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 - 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 microorganismsCompositions thereforProcesses 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
81.
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 - 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 microorganismsCompositions thereforProcesses 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
84.
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 - 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
85.
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.
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 - Biochemical methods, e.g. using enzymes or whole viable microorganisms
C12Q 1/68 - Measuring or testing processes involving enzymes, nucleic acids or microorganismsCompositions thereforProcesses 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
91.
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.
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 - Physical analysis of biological material of liquid biological material
B81B 1/00 - Devices without movable or flexible elements, e.g. microcapillary devices
G01N 15/12 - Investigating individual particles by measuring electrical or magnetic effects by observing changes in resistance or impedance across apertures when traversed by individual particles, e.g. by using the Coulter principle
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.
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.
patents
