Systems and methods for mapping a plurality of sequence reads to a genomic region are provided. A plurality of sequence reads mappable to the genomic region are obtained. An initial Markov model for the genomic region is obtained. The initial Markov model comprises at least (i) a first repeat for a first repeat region, (ii) a second repeat for a second repeat region, and (iii) an intermediate region linking the first repeat to the second repeat. The initial Markov model is refined using the plurality of sequence reads, thereby obtaining a refined Markov model. For each respective sequence read in the plurality of sequences, the respective sequence read is used to find a highest probability path through the Markov model. This highest probability path is then used to map the respective sequence read to the genomic region.
Disclosed herein are blocked asymmetric hairpin adaptors that include a spacer that prevents extension by a strand-displacing polymerase. Such adaptors can be utilized for creating libraries of templates that can be processed into template concatemers and/or interrogated by various sequencing methods.
The present invention provides methods and compositions for carrying out nucleic acid sequencing, particularly paired-end sequencing. The methods use concatemeric sequencing templates that can be produced by rolling circle amplification of asymmetric circular nucleic acids having a central double- stranded region comprising a target nucleic acid sequence that is connected at each end to form a circular construct.
Provided include methods, compositions, kits, and systems for replenishing a rolling circle amplification (RCA) reaction in a vessel. The RCA reaction can be initiated by contacting a nucleic acid template and a primer with a loading buffer comprising a DNA polymerase and polymerase extension agents including a divalent metal cation and a polyelectrolyte, followed by replenishing with an amplification buffer to continue the nucleic acid amplification through primer extension. The amplification buffer is different in composition from the loading buffer and does not comprise any DNA polymerase.
Provided includes methods, compositions and systems for single molecule seeding and amplification on a flow cell. In some embodiments, nucleic acids are isothermally seeded and amplified on a flow cell comprising multiple binding areas (e.g., pads), resulting in an ensemble of substantially the same amplified molecules on each of the binding areas.
Arrays of integrated analytical devices are provided. The arrays are useful in the analysis of highly multiplexed optical reactions in large numbers at high densities, including biochemical reactions, such as nucleic acid sequencing reactions. In particular, the arrays provide increased efficiency of optical collection and decreased background signal as the lateral dimensions of the unit cell of devices within the array are decreased, for example as they are decreased to 2 µm, or even less.
Disclosed herein include methods of specifying sites (e.g., sites for colony formation) on a surface (e.g., a planar surface) and generating a flow cell having the sites specified on a surface. Also disclosed are methods of performing sequencing (e.g., sequencing-by-synthesis and sequencing-by-binding) using the flow cell generated and processing (e.g., aligning, orienting, sorting, and assessing quality) images of the flow cell captured during sequencing.
Provided herein are highly multiplexed optical analytical systems for improved nucleic acid sequencing. The systems include a plurality of highly multiplexed optical chips, at least one optical source, and a plurality of optical delivery devices for illuminating an array of nanoscale rection regions on each of the optical chips. In use, the reaction regions contain fluorescent nucleic acid sequencing reagents and are arranged to report nucleic acid sequence information to optical detectors associated with the multiplexed optical chips in real time. The systems enable a massive increase in the scale of nucleic acid sequencing reactions capable of being performed within a single instrument without a corresponding increase in size, complexity, or cost of the instrument.
G02B 6/12 - Light guides; Structural details of arrangements comprising light guides and other optical elements, e.g. couplings of the optical waveguide type of the integrated circuit kind
C12Q 1/6874 - Methods for sequencing involving nucleic acid arrays, e.g. sequencing by hybridisation [SBH]
G01N 21/00 - Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
G01N 21/17 - Systems in which incident light is modified in accordance with the properties of the material investigated
G01N 21/63 - Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
G01N 21/78 - Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
11.
NUCLEIC ACID SEQUENCING CARTRIDGES, PACKAGED DEVICES, AND SYSTEMS
Provided herein are cartridges, packaged devices, and systems for improved nucleic acid sequencing. The cartridges, devices, and systems include a highly multiplexed optical chip comprising a plurality of nanoscale reaction regions that is configured to perform and report nucleic acid sequencing reactions. The chips are, in some embodiments, packaged for use in analytical nucleic acid sequencing reactions. The chips may be attached to a printed circuit board, may be surrounded by a protective enclosure, may include a flow cell, and may include optical features to minimize or block photobleaching of the sequencing reagents and background fluorescent signals. Also provided are analytical systems for nucleic acid sequencing that comprise the disclosed cartridges and packaged devices. The systems comprise an analytical instrument with electronic, optical, mechanical, fluidic, and/or thermal connectors designed to interact with the corresponding connectors on an associated cartridge or packaged device in a highly precise but reversible manner.
G01N 21/77 - Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
12.
COMPOSITIONS AND METHODS FOR IMPROVED CDNA SYNTHESIS
Modified template switching oligonucleotides (TSOs), compositions containing modified TSOs, and methods for employing modified TSOs to synthesize cDNA from RNA templates, where the cDNA includes an adapter region at the 3' end, are provided. The modified TSOs include at least one 2'-fluoro-ribonucleotide in the 3' annealing region and provide for improved conversion of RNA into full-length cDNA, resulting in increased yield and complexity as compared to non-modified TSOs and thereby finding use in generating cDNA from samples having low RNA input.
inter aliainter alia, methods, compositions, and computer implemented processes for resolving long and highly similar, but non-identical, genomic regions to improve assembly quality, especially for polyploid genomes. Aspects of the disclosure are draw to using exact string matching of homopolymer-collapsed sequence reads to determine whether two sequences overlap and thus represent the same genomic region (e.g., the same haplotype in polyploid genomes) or whether the sequences represent different genomic regions.
G16B 15/00 - ICT specially adapted for analysing two-dimensional or three-dimensional molecular structures, e.g. structural or functional relations or structure alignment
G16B 40/00 - ICT specially adapted for biostatistics; ICT specially adapted for bioinformatics-related machine learning or data mining, e.g. knowledge discovery or pattern finding
14.
SYSTEMS AND METHODS FOR GRAPH BASED MAPPING OF NUCLEIC ACID FRAGMENTS
Technical solutions for mapping long nucleic acid sequence reads to a target sequence are provided. A directed graph, representing all or some of a genome and comprising one or more nonlinear topological components, is obtained for an organism having a heterozygous genome. Each nonlinear topological component has an initiating node and a terminal node connected by at least a first branch and a second branch. One of these branches corresponds to the target sequence. The directed graph uses a plurality of sequence reads from a biological sample of the organism. The sequence reads are overlapped by an unrestricted overhang amount, provided there is a minimum consensus region between each two sequence reads. A query sequence, encompassing at least the initiating node or the terminal node of a first nonlinear topological component, is obtained. The directed graph is used to form a mapping of the query sequence to the directed graph.
The present invention provides methods, compositions, and systems for distributing molecules and complexes into reaction sites. In particular, the methods, compositions, and systems of the present invention result in loading of polymerase enzyme complexes into a predetermined number of reaction sites, including nanoscale wells.
Disclosed are methods and compositions for enriching nucleic acid fragments from a sample that include one or more target region of interest. In certain aspects, a sample of double stranded nucleic acid fragments having a strand-linking adapter at one end and a non- strand-linking adapter at the other end are denatured and contacted with capture probes specific for a target sequence of interest. Capture probe-bound fragments are isolated from the sample, e.g., using a solid substrate specific for the binding moiety on the capture probes, and are renatured for downstream processing, thus maintaining the original double-stranded region. This enrichment process does not require amplification and as such maintains the nucleic acids in their native states. The disclosed enrichment process and compositions are suitable for analyzing nucleic acids that are fragmented and/or damaged, e.g., cell-free DNA such as circulating tumor DNA, as well as nucleic acids that are many kilobases in length.
Compositions comprising covalently modified and mutated biotin-binding proteins, particularly biotin-binding proteins having a negative charge at physiological pH, are provided. Methods of producing such proteins are also provided, as are methods of immobilizing, sequencing, and making nucleic acids employing such proteins.
G01N 33/544 - Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
Provided herein are systems, devices, and methods for improved optical waveguide transmission and alignment in an analytical system. Waveguides in optical analytical systems can exhibit variable and increasing back reflection of single-wavelength illumination over time, thus limiting their effectiveness and reliability. The systems are also subject to optical interference under conditions that have been used to overcome the back reflection. Novel systems and approaches using broadband illumination light with multiple longitudinal modes have been developed to improve optical transmission and analysis in these systems. Novel systems and approaches for the alignment of a target waveguide device and an optical source are also disclosed.
G02B 6/124 - Geodesic lenses or integrated gratings
G01N 21/17 - Systems in which incident light is modified in accordance with the properties of the material investigated
G01N 21/63 - Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
G01N 21/77 - Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
G02B 6/34 - Optical coupling means utilising prism or grating
19.
METHODS AND COMPOSITIONS FOR ISOLATING ASYMMETRIC NUCLEIC ACID COMPLEXES
The present disclosure provides improved methods for isolating asymmetrically- primed and/or asymmetrically-tagged nucleic acid complexes that find use in downstream analytical analyses, including sequence analysis. Compositions comprising such complexes and kits and systems for generating such complexes are also provided.
Aspects of the present disclosure provide methods, systems, and computer program products for generating one or more extended contigs. Aspects of the exemplary embodiment include receiving input contigs for a genome; generating local assembly subgraphs including the ends of each contig; identifying subgraphs that unambiguously connect two contigs; and generating an extended contig in which the orientation and order of at least two contigs is determined. Extended contigs can include any number of linearly ordered and linked contigs.
Labeled nucleotide analogs comprising at least one avidin protein, at least one dye-labeled compound, and at least one nucleotide compound are provided. The analogs are useful in various fluorescence-based analytical methods, including the analysis of highly multiplexed optical reactions in large numbers at high densities, such as single molecule real time nucleic acid sequencing reactions. The analogs are detectable with high sensitivity at desirable wavelengths. They contain structural components that modulate the interactions of the analogs with DNA polymerase, thus decreasing photodamage and improving the kinetic and other properties of the analogs in sequencing reactions. Also provided are nucleotide and dye-labeled compounds of the subject analogs, as well as intermediates useful in the preparation of the compounds and analogs. Compositions comprising the compounds, methods of synthesis of the intermediates, compounds, and analogs, and mutant DNA polymerases are also provided.
Disclosed are packaging methods for the fabrication of analytical device packages and fabricated analytical device packages. The methods include providing a sensor wafer and mounting individual plates or a plate wafer on the sensor wafer. The sensor wafer includes sensor chips with aperture regions and is treated with selective depositions, either prior to or during the fabrication of the analytical device packages, to produce different surface characteristics at different portions of the aperture regions. Before dicing the sensor wafer, openings of the individual plates or the plate wafer are covered by a protective layer to protect surface characteristics at different portions of the aperture regions. A fabricated analytical device package includes a sensor chip with different surface characteristics, a plate, a packaging substrate and an optional cover.
The present disclosure provides methods, compositions, and systems for distributing polymerase compositions into array regions. In particular, the described methods, compositions, and systems utilize density differentials and/or additives to increase efficiency in the distribution of polymerase compositions to a surface as compared to methods utilizing only diffusion control.
Methods, compositions, and systems for distributing nucleic acids into array regions are provided. The methods, compositions, and systems utilize nucleic acid condensing agents to increase efficiency of distribution of the nucleic acids into the array regions. Various methods for facilitating distribution of the nucleic acids to the array regions are provided.
Compositions, devices, systems and methods for increasing the signal to noise ratio (SNR) and/or enhancing photoprotection in an illuminated analytical reaction by addition of one or more signal detection assay (SDA)-enhancing agents to the reaction mixture.
Labeled nucleotide analogs comprising at least one avidin protein, at least one dye-labeled compound, and at least one nucleotide compound are provided. The analogs are useful in various fluorescence-based analytical methods, including the analysis of highly multiplexed optical reactions in large numbers at high densities, such as single molecule real time nucleic acid sequencing reactions. The analogs are detectable with high sensitivity at desirable wavelengths. They contain structural components that modulate the interactions of the analogs with DNA polymerase, thus decreasing photodamage and improving the kinetic and other properties of the analogs in sequencing reactions. Also provided are nucleotide and dye-labeled compounds of the subject analogs, as well as intermediates useful in the preparation of the compounds and analogs. Compositions comprising the compounds, methods of synthesis of the intermediates, compounds, and analogs, and mutant DNA polymerases are also provided.
Labeled nucleotide analogs comprising at least one avidin protein, at least one dye-labeled compound, and at least one nucleotide compound are provided. The analogs are useful in various fluorescence-based analytical methods, including the analysis of highly multiplexed optical reactions in large numbers at high densities, such as single molecule real time nucleic acid sequencing reactions. The analogs are detectable with high sensitivity at desirable wavelengths. They contain structural components that modulate the interactions of the analogs with DNA polymerase, thus decreasing photodamage and improving the kinetic and other properties of the analogs in sequencing reactions. Also provided are nucleotide and dye-labeled compounds of the subject analogs, as well as intermediates useful in the preparation of the compounds and analogs. Compositions comprising the compounds, methods of synthesis of the intermediates, compounds, and analogs, and mutant DNA polymerases are also provided.
Arrays of integrated optical devices and their methods for production are provided. The devices include an integrated bandpass filter layer that comprises at least two multi-cavity filter elements with different central bandpass wavelengths. The device arrays are useful in the analysis of highly multiplexed optical reactions in large numbers at high densities, including biochemical reactions, such as nucleic acid sequencing reactions. The devices provide for the efficient and reliable coupling of optical excitation energy from an optical source to the optical reactions. Optical signals emitted from the reactions can thus be measured with high sensitivity and discrimination. The device arrays are well suited for miniaturization and high throughput.
Real time electronic sequencing methods, devices, and systems are described. Arrays of nanoFET devices are used to provide sequence information about a template nucleic acid in a polymerase-template complex bound to the nanoFET. A sequencing reaction mixture comprising nucleotide analogs having conductivity labels is introduced to the array of nanoscale electronic elements comprising nanoFETs under conditions of polymerase mediated nucleic acid synthesis. The polymerase enzyme template complex is attached to the gate of the nanoFET in an orientation whereby the nucleotide exit region of the polymerase enzyme is directed toward the gate of the nanoFET. Methods for producing nanoFET arrays are provided.
A circuit comprising a substrate with sectors on the substrate is provided, each sector comprising clock and data lines, a controller in electrical communication with the clock and data lines, a counter bias line, an amplifier input line and nano-electronic measurement devices on the substrate. A source of each device is coupled to the counter bias line and a drain of each device is coupled to the amplifier input line to obtain an electrical signal on the drain, the identity of which is determined by electrical interaction between the device and a charge label. Each device drain is gated by a corresponding switch between an on state, in which the drain is connected to the amplifier input line, and an off state, in which the drain is isolated from the amplifier input line. The controller controls switch states responsive to clock signal line pulses and data input line data.
C12Q 1/68 - Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
B01L 3/00 - Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
C12M 1/34 - Measuring or testing with condition measuring or sensing means, e.g. colony counters
G01N 27/22 - Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating impedance by investigating capacitance
H01L 31/109 - Devices sensitive to infrared, visible or ultraviolet radiation characterised by only one potential barrier or surface barrier the potential barrier being of the PN heterojunction type
Exemplary embodiments provide methods and systems for string graph assembly of polyploid genomes. Aspects of the exemplary embodiment include receiving a string graph generated from sequence reads of at least.5 kb in length; identifying unitigs in the string graph and generating a unitig graph; and identifying string bundles in the unitig graph by: determining a primary contig from each of the string bundles; and determining associated contigs that contain structural variations compared to the primary contig.
G06F 19/22 - for sequence comparison involving nucleotides or amino acids, e.g. homology search, motif or Single-Nucleotide Polymorphism [SNP] discovery or sequence alignment
G06F 19/18 - for functional genomics or proteomics, e.g. genotype-phenotype associations, linkage disequilibrium, population genetics, binding site identification, mutagenesis, genotyping or genome annotation, protein-protein interactions or protein-nucleic acid interactions
C12P 19/34 - Polynucleotides, e.g. nucleic acids, oligoribonucleotides
32.
INTEGRATED TARGET WAVEGUIDE DEVICES AND SYSTEMS FOR OPTICAL COUPLING
Integrated target waveguide devices and optical analytical systems comprising such devices are provided. The target devices include an optical coupler that is optically coupled to an integrated waveguide and that is configured to receive optical input from an optical source through free space, particularly through a low numerical aperture interface. The devices and systems are useful in the analysis of highly multiplexed optical reactions in large numbers at high densities, including biochemical reactions, such as nucleic acid sequencing reactions. The devices provide for the efficient and reliable coupling of optical excitation energy from an optical source to the optical reactions. Optical signals emitted from the reactions can thus be measured with high sensitivity and discrimination. The devices and systems are well suited for miniaturization and high throughput.
Exemplary embodiments provide methods and systems for diploid genome assembly and haplotype sequence reconstruction. Aspects of the exemplary embodiment include generating a fused assembly graph from reads of both haplotypes, the fused assembly graph including identified primary contigs and associated contigs; generating haplotype-specific assembly graphs using phased reads and haplotype aware overlapping of the phased reads; merging the fused assembly graph and haplotype- specific assembly graphs to generate a merged assembly haplotype graph; removing cross-phasing edges from the merged assembly haplotype graph to generate a final haplotype-resolved assembly graph; and reconstructing haplotype-specific contigs from the final haplotype-resolved assembly graph resulting in haplotype-specific contigs.
G06F 19/22 - for sequence comparison involving nucleotides or amino acids, e.g. homology search, motif or Single-Nucleotide Polymorphism [SNP] discovery or sequence alignment
G06F 19/18 - for functional genomics or proteomics, e.g. genotype-phenotype associations, linkage disequilibrium, population genetics, binding site identification, mutagenesis, genotyping or genome annotation, protein-protein interactions or protein-nucleic acid interactions
C12P 19/34 - Polynucleotides, e.g. nucleic acids, oligoribonucleotides
A multiprocessor pipeline architecture that converts signals from sequencing sample acquisition into sequence data, comprising: a custom coprocessor card configured to directly receive a stream of serialized sensor data generated by an image sensor, wherein the sensor data represents frame-by-frame intensity values for pixels comprising the image sensor; a first coprocessor that continually receives the stream of serialized sensor data and transposes the frame-by-frame intensity values into reaction cell chunks; a buffer that repeatedly receives the reaction cell chunks and stores in contiguous memory locations the reaction cell chunks for each respective reaction cell over a larger predetermined time window to create larger reaction cell chunks; and a plurality of second coprocessors that retrieve the larger reaction cell chunks from the buffer and convert, in parallel, the pixel intensity values into base-by-base sequence data.
The present invention provides methods, compositions, and systems for distributing single polymerase molecules into array regions. In particular, the methods, compositions, and systems of the present invention result in a distribution of single polymerase molecules into array regions at a percentage that is larger than the percentage expected to be occupied under a Poisson distribution.
Optical delivery devices and integrated analytical systems comprising the optical delivery devices are provided. The optical delivery devices include optical inputs, optical outputs, and integrated optical waveguides that are configured for coupling of optical energy to a target waveguide device through free space. The integrated analytical systems include the optical delivery devices in combination with the target waveguide device. The devices and systems are useful in the analysis of highly multiplexed optical reactions in large numbers at high densities, including biochemical reactions, such as nucleic acid sequencing reactions. The devices provide for the efficient coupling of optical excitation energy from an optical source to the optical reactions. Optical signals emitted from the reactions can thus be measured with high sensitivity and discrimination. The devices and systems are well suited for miniaturization and high throughput.
G02B 6/12 - Light guides; Structural details of arrangements comprising light guides and other optical elements, e.g. couplings of the optical waveguide type of the integrated circuit kind
Multimeric protected fluorescent reagents and their methods of synthesis are provided. The reagents are useful in various fluorescence-based analytical methods, including the analysis of highly multiplexed optical reactions in large numbers at high densities, such as single molecule real time nucleic acid sequencing reactions. The reagents contain fluorescent dye elements, that allow the compounds to be detected with high sensitivity at desirable wavelengths, binding elements, that allow the compounds to be recognized specifically by target biomolecules, and protective shield elements, that decrease undesirable contacts between the fluorescent dye elements and the bound target biomolecules and that therefore decrease photodamage of the bound target biomolecules by the fluorescent dye elements. The reagents also contain coupling elements connect monomeric compounds into multimeric forms, thereby increasing brightness.
Arrays of integrated analytical devices and their methods for production are provided. The arrays are useful in the analysis of highly multiplexed optical reactions in large numbers at high densities, including biochemical reactions, such as nucleic acid sequencing reactions. The devices allow the highly sensitive discrimination of optical signals using features such as spectra, amplitude, and time resolution, or combinations thereof. The devices include an integrated diffractive beam shaping element that provides for the spatial separation of light emitted from the optical reactions.
G01N 21/77 - Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
39.
COMPOSITIONS AND METHODS FOR ENRICHMENT OF NUCLEIC ACIDS
Methods are provided for reducing the complexity of a population of nucleic acids prior to performing an analysis of the nucleic acids, e.g., sequence analysis. The methods result in a subset of the initial population enriched for a target region, which is typically located within one or more target fragments. The methods are particularly useful for analyzing populations having a high degree of complexity, e.g., chromosomal-derived DNA, whole genomic DNA, or mRNA populations.
Protected fluorescent reagent compounds and their methods of synthesis are provided. The compounds are useful in various fluorescence-based analytical methods, including the analysis of highly multiplexed optical reactions in large numbers at high densities, such as single molecule real time nucleic acid sequencing reactions. The compounds contain fluorescent dye elements, that allow the compounds to be detected with high sensitivity at desirable wavelengths, binding elements, that allow the compounds to be recognized specifically by target biomolecules, and protective shield elements, that decrease undesirable contacts between the fluorescent dye elements and the bound target biomolecules and that therefore decrease photodamage of the bound target biomolecules by the fluorescent dye elements.
G01N 23/223 - Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups , or by measuring secondary emission from the material by irradiating the sample with X-rays or gamma-rays and by measuring X-ray fluorescence
Multi-biotinylated reactants are provided which can be used in divalent complexes for various applications such as colocalization, labeling, immobilization, and purification. Methods for constructing, purifying, and using the bis-biotinylated reactants are also provided. In certain embodiments, two bis-biotinylated reactants are bound to a single streptavidin tetramer to provide a complex having a 1 :1 stoichiometry with respect to the bis-biotinylated reactants.
Apparatus, systems and methods for use in analyzing discrete reactions are provided. The analytical devices of the invention use an array of nanoscale regions (a chip) that has discrete patches of nanoscale regions. The chip mates with a collection device comprising an array of compact lens trains (CLTs) where each of the CLTs corresponds to a single patch of nanoscale regions. Each CLT collects the emitted light from a patch on the chip, collimates the light, performs color separation on the collimated emitted light, and focuses the separated light onto a portion of pixels on the detector below the CLT. Such systems are useful for monitoring many analytical reactions at one time including single molecule sequencing reactions.
Real time electronic sequencing methods, devices, and systems are described. Arrays of nanoscale electronic elements comprising capacitive devices with one or two electrodes, or arrays of nanoFET devices are used to provide sequence information about a template nucleic acid in a polymerase-template complex bound proximate to the nanoscale electronic elements or to the substrate proximate to the nanoscale electronic element. A sequencing reaction mixture comprising nucleotide analogs having impedance labels, capacitive labels, or conductivity labels is introduced to the array of nanoscale electronic elements comprising capacitive devices or nanoFETs under conditions of polymerase mediated nucleic acid synthesis. The time sequence of incorporation of nucleotide analogs is determined by identifying the types of labels of the nucleotide analogs that are incorporated into the growing strand using measured impedance, conductivity, or capacitance.
The present invention is directed to methods, devices, compositions and systems for obtaining sequence data from nucleic acid templates by utilizing electronic sensing elements.
Optical analytical devices and their methods of use are provided. The devices are useful in the analysis of highly multiplexed optical reactions in large numbers at high densities, including biochemical reactions, such as nucleic acid sequencing reactions. The devices include integrated illumination elements and optical waveguides for illumination of the optical reactions. The devices further provide for the efficient coupling of optical excitation energy from the waveguides to the optical reactions. Optical signals emitted from the reactions can thus be measured with high sensitivity and discrimination using features such as spectra, amplitude, and time resolution, or combinations thereof. The devices of the invention are well suited for miniaturization and high throughput.
G01N 21/17 - Systems in which incident light is modified in accordance with the properties of the material investigated
G01N 21/63 - Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
Optical analytical devices and their methods of use are provided. The devices are useful in the analysis of highly multiplexed optical reactions in large numbers at high densities, including biochemical reactions, such as nucleic acid sequencing reactions. The devices include optical waveguides for illumination of the optical reactions. The devices further provide for the efficient coupling of optical excitation energy from the waveguides to the optical reactions. Optical signals emitted from the reactions can thus be measured with high sensitivity and discrimination using features such as spectra, amplitude, and time resolution, or combinations thereof. The devices of the invention are well suited for miniaturization and high throughput.
G02B 6/10 - Light guides; Structural details of arrangements comprising light guides and other optical elements, e.g. couplings of the optical waveguide type
47.
COMPOSITIONS AND METHODS FOR SELECTION OF NUCLEIC ACIDS
Methods are provided for reducing the complexity of a population of nucleic acids prior to performing an analysis of the nucleic acids, e.g., sequence analysis. The methods result in a subset of the initial population enriched for a target region, which is typically located within one or more target fragments. The methods are particularly useful for analyzing populations having a high degree of complexity, e.g., chromosomal-derived DNA, whole genomic DNA, or mRNA populations.
An approach to the design of the set of filters which allows for the collection of a larger portion of the optical signal while still distinguishing the presence of the various fluorophores is described. In some embodiments, the filter sets of the invention each block a smaller portion of the spectrum, allowing for a larger portion of the emitted light to be detected. The combined information from the light passing through two or more of the filters is then used to determine the presence of a given fluorophore. The filter sets of the invention can be particularly useful in integrated devices in which the light from a single molecule reaction in a small reaction region is directed to a detector or to a specific portion of a detector.
Methods, compositions, and systems are provided for characterization of modified nucleic acids. Nanopore sequencing is performed using a processive enzyme to control the rate of translation of a single strand of a nucleic acid through a nanopore. The presence and identity of a modified base can be determined by monitoring the kinetics of translation through the pore even though the events that lead to the kinetic changes are separated in time and space from the translation of the modified base or it's compliment through the nanopore. The invention also comprises the use of hemi-genomic DNA in nanopore sequencing.
The present invention provides heteroaryl functionalized cyanine dyes including a reactive functional moiety, or which are conjugated to a carrier molecule.
A method for identifying a 5-MeC in a template nucleic is provided. The method comprises providing a template having 5-MeC, converting the 5-MeC into a further modification selected from 5-caC and 5-FC. The converted template is then sequenced, and a change in sequencing is detected that is indicative of the further modification, allowing for identifying the 5-MeC in the template nucleic acid.
C12Q 1/68 - Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
C12N 15/11 - DNA or RNA fragments; Modified forms thereof
C12Q 1/00 - Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
52.
METHODS AND COMPOSITION FOR SEQUENCING MODIFIED NUCLEIC ACIDS
Methods, compositions, and systems are provided for characterization of modified nucleic acids. In certain preferred embodiments, single molecule sequencing methods are provided for identification of modified nucleotides within nucleic acid sequences. Modifications detectable by the methods provided herein include chemically modified bases, enzymatically modified bases, a basic sites, non-natural bases, secondary structures, and agents bound to a template nucleic acid.
Compositions and methods are provided for nucleotide analogs comprising protein shields for improving enzyme photostability in single molecule real time sequencing. Nucleotide analogs of the invention have a protein shield between the dye moieties and nucleotide moieties of the analog. The protein prevents the direct interaction of the dye moiety with the enzyme carrying out nucleotide synthesis preventing photodamage to the enzyme. The nucleotide analogs of the invention can have multiple dyes and multiple nucleotide moieties.
Real time redox sequencing methods, devices, and systems are described. Arrays of redox devices comprising one or two electrodes are used to provide sequence information about a template nucleic acid in a polymerase-template complex bound proximate to the electrode(s). A sequencing reaction mixture comprising nucleotide analogs comprising redox labels is introduced to the array of redox devices under conditions of polymerase mediated nucleic acid synthesis. The time sequence of incorporation of nucleotide analogs is determined by electrochemically identifying the redox labels of the nucleotide analogs that are incorporated into the growing strand.
Compositions, methods and systems are provided for the isolation of polymerase-nucleic acid complexes. Complexes can be separated from free enzyme by using hook molecules to target single stranded regions on the nucleic acid. Active complexes can be isolated from mixtures having both active and inactive complexes by initiating nucleic acid synthesis so as to open up a portion of a double stranded region rendering that region single stranded. Hook molecules are targeted to bind the sequences that are thus exposed. The hook molecules bound to active polymerase-nucleic acid complex are isolated, and the active polymerase-nucleic acid complexes released. Also disclosed are compositions, devices, and methods for loading molecules-of-interest onto a substrate by contacting beads having molecules-of-interest attached to them with the substrate, for example by providing a field that brings the beads into proximity or contact with the substrate and moves the beads with respect to the substrate. Such molecules-of-interest can be deposited onto substrates for single-molecule analysis.
Methods, compositions, and systems are provided for characterization of modified nucleic acids. In certain preferred embodiments, single molecule sequencing methods are provided for identification of modified nucleotides within nucleic acid sequences. Modifications detectable by the methods provided herein include chemically modified bases, enzymatically modified bases, abasic sites, non-natural bases, secondary structures, and agents bound to a template nucleic acid.
C12Q 1/68 - Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
C12N 15/11 - DNA or RNA fragments; Modified forms thereof
C12Q 1/34 - Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
C12Q 1/48 - Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
C12Q 1/25 - Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving enzymes not classifiable in groups
The invention provides a novel class of cyanine dyes that are functionalized with sulfonic acid groups and a linker moiety that facilitates their conjugation to other species and substituent groups which increase the water-solubility, and optimize the optical properties of the dyes. Also provided are conjugates of the dyes, methods of using the dyes and their conjugates and kits including the dyes and their conjugates.
C07D 403/06 - Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
C07D 209/10 - Indoles; Hydrogenated indoles with substituted hydrocarbon radicals attached to carbon atoms of the hetero ring
C07D 209/18 - Radicals substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
C07D 209/30 - Indoles; Hydrogenated indoles with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to carbon atoms of the hetero ring
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
C09B 23/00 - Methine or polymethine dyes, e.g. cyanine dyes
The invention provides a novel class of scaffold-based labeled polymerase enzyme substrates. The polymerase enzyme substrates have a multivalent core or scaffold to which is attached fluorescent dye moieties and nucleoside phosphate moities. The polymerase enzyme substrates have multiple fluorescent dye moities and/or multiple nucleoside phosphate moieties. Preferred multivalent cores comprise trifunctional six membered aromatic moities. The invention also provides for sequencing methods and kits with scaffold-based labeled polymerase enzyme substrates.
C12N 11/00 - Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
C07D 403/04 - Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group containing two hetero rings directly linked by a ring-member-to-ring- member bond
C07D 251/12 - Heterocyclic compounds containing 1,3,5-triazine rings not condensed with other rings having three double bonds between ring members or between ring members and non-ring members
C12Q 1/68 - Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
C12Q 1/25 - Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving enzymes not classifiable in groups
G01N 33/52 - Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper
The invention provides a novel class of cyanine dyes that are functionalized with a linker moiety that facilitates their conjugation to other species and substituent groups which increase the water-solubility, and optimize the optical properties of the dyes. Also provided are conjugates of the dyes, methods of using the dyes and their conjugates and kits including the dyes and their conjugates.
C07D 403/06 - Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
C07D 209/10 - Indoles; Hydrogenated indoles with substituted hydrocarbon radicals attached to carbon atoms of the hetero ring
C07D 209/18 - Radicals substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
C07D 209/30 - Indoles; Hydrogenated indoles with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to carbon atoms of the hetero ring
C09B 23/00 - Methine or polymethine dyes, e.g. cyanine dyes
Compositions, devices, systems and methods for reducing and/or preventing photo-induced damage of one or more reactants in an illuminated analytical reaction by addition of one or more photoprotective compounds to the reaction mixture and allowing the reaction to proceed for a period that is less than a photo-induced damage threshold period.
G01N 33/52 - Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper
C12Q 1/25 - Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving enzymes not classifiable in groups
C12Q 1/68 - Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
61.
SEQUENCING REACTIONS WITH ALKALI METAL CATIONS FOR PULSE WIDTH CONTROL
Compositions, kits, methods and systems for single molecule nucleotide sequencing comprising producing polymerase reactions having monovalent cations that control the median pulse width for incorporated nucleotides are disclosed. The levels of alkali metals such as lithium, sodium, potassium, rubidium, and cesium in the polymerization are used to control pulse width while allowing other sequencing parameters to remain within a desirable range.
Methods, compositions and arrays for non-random loading of single analyte molecules into array structures are provided. Arrays of confined regions are produced wherein each confined region comprises a single island within the confined region. The island can be selectively functionalized with a coupling agent to couple a single molecule of interest within the confined region.
G01N 33/48 - Biological material, e.g. blood, urine; Haemocytometers
G01N 37/00 - INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES - Details not covered by any other group of this subclass
G01N 33/50 - Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
B82Y 30/00 - Nanotechnology for materials or surface science, e.g. nanocomposites
Methods, arrays, and systems for the optical analysis of multiple chemical, biological, or biochemical reactions are provided. The invention includes methods for producing arrays of micromirrors on transparent substrates, each micromirror comprising a nanostructure or optical confinement on its top. The arrays are produced by a process in which lateral dimensions of both the nanostructures and micromirrors are defined in a single step, allowing for control of the relative placement of the features on the substrate, minimizing the process-related defects, allowing for improved optical performance and consistency. In some aspects, the invention provides methods of selectively etching large features on a substrate while not concurrently etching small features. In some aspects, the invention provides methods of etching large features on a substrate using hard mask materials.
An analytical assembly within a unified device structure for integration into an analytical system. The analytical assembly is scalable and includes a plurality of analytical devices, each of which includes a reaction cell, an optical sensor, and at least one optical element positioned in optical communication with both the reaction cell and the sensor and which delivers optical signals from the cell to the sensor. Additional elements are optionally integrated into the analytical assembly. Methods for forming and operating the analytical system are also disclosed.
Optics collection and detection systems are provided for measuring optical signals from an array of optical sources over time. Methods of using the optics collection and detection systems are also described.
An analytical device including an optically opaque cladding, a sequencing layer including a substrate disposed below the cladding, and a waveguide assembly for receiving optical illumination and introducing illumination into the device. The illumination may be received from a top, a side edge, and a bottom of the device. The waveguide assembly may include a nanoscale aperture disposed in the substrate and extending through the cladding. The aperture defines a reaction cell for receiving a set of reactants. In various aspects, the device includes a sensor element and the illumination pathway is through the sensor element. Waveguides and illumination devices, such as plasmonic illumination devices, are also disclosed. Methods for forming and operating the devices are also disclosed.
G02B 6/10 - Light guides; Structural details of arrangements comprising light guides and other optical elements, e.g. couplings of the optical waveguide type
67.
GENERATION OF MODIFIED POLYMERASES FOR IMPROVED ACCURACY IN SINGLE MOLECULE SEQUENCING
Provided are compositions comprising recombinant DNA polymerases that include amino acid substitutions, insertions, deletions and/or heterologous or exogenous features that confer modified properties upon the polymerase for enhanced single molecule sequencing. Such properties can include reduced reaction rates at one or more steps of the polymerase kinetic cycle, increased closed polymerase/DNA complex stability, enhanced metal ion coordination, reduced exonuclease activity, decreased branching fractions, and the like. Also provided are nucleic acids which encode the polymerases with the aforementioned phenotypes, as well as methods of using such polymerases to make a DNA or to sequence a DNA template.
The application relates to improved optical containment structures, methods of manufacture and use, and systems for employing same. The optical containment structures generally comprise zero-mode waveguide structures having non-reflective walls. The non-reflective walls allow the preparation of optical containment regions in which the optical containment dimensions can be decoupled from the solution containment dimensions. The application also relates to methods for producing islands of functionality within nanoscale apertures.
G02B 6/122 - Basic optical elements, e.g. light-guiding paths
G02B 6/28 - Optical coupling means having data bus means, i.e. plural waveguides interconnected and providing an inherently bidirectional system by mixing and splitting signals
The present invention is generally directed to compositions, methods, and systems for performing single-molecule, real-time analysis of a variety of different biological reactions, and for determining various characteristics of the different biological reactions. The ability to analyze such reactions provides an opportunity to study those reactions as well as to potentially identify factors and/or approaches for impacting such reactions, e.g., to stimulate, enhance, or inhibit such reactions.
The present invention is generally directed to compositions, methods, and systems for performing single-molecule, real-time analysis of analytical reactions in which protein synthesis is occurring. The ability to analyze such reactions provides an opportunity to study those reactions as well as to potentially identify factors and/or approaches for impacting such reactions, e.g., to either enhance, inhibit, or otherwise affect such reactions including, but not limited to, affecting the reaction rate, processivity, fidelity, duration, and the like.
The present invention is generally directed to compositions, methods, and systems for performing single-molecule, real-time analysis of a variety of different biological reactions. The ability to analyze such reactions provides an opportunity to study those reactions as well as to potentially identify factors and/or approaches for impacting such reactions, e.g., to either enhance or inhibit such reactions. In certain preferred embodiments, RNA templates are used in single-molecule real-time sequencing reactions.
FRET-labeled compounds are provided for use in analytical reactions. In certain embodiments, FRET-labeled nucleotide analogs are used in place of naturally occurring nucleoside triphosphates or other analogs in analytical reactions comprising nucleic acids, for example, template-directed nucleic acid synthesis, DNA sequencing, RNA sequencing, single-base identification, hybridization, binding assays, and other analytical reactions.
The invention relates to devices and methods for nanopore sequencing. The invention includes arrays of nanopores having incorporated electronic circuits, for example, in CMOS. In some cases, the arrays of nanopores comprise resistive openings for isolating the electronic signals for improved sequencing. Methods for controlling translocation of through the nanopore are disclosed.
C12Q 1/68 - Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
B82B 3/00 - Manufacture or treatment of nanostructures by manipulation of individual atoms or molecules, or limited collections of atoms or molecules as discrete units
G01N 33/48 - Biological material, e.g. blood, urine; Haemocytometers
Methods, compositions, and systems are provided for characterization of modified nucleic acids. In certain preferred embodiments, single molecule sequencing methods are provided for identification of modified nucleotides within nucleic acid sequences. Modifications detectable by the methods provided herein include chemically modified bases, enzymatically modified bases, abasic sites, non-natural bases, secondary structures, and agents bound to a template nucleic acid.
Nucleic acid compositions, methods of making and using such compositions that comprise modular functional groups that can be configured to provide desired functionality to different nucleotide types through a swappable and preferably non-covalent linkage component. Such compositions are useful in a variety of applications including nucleic acid analyses.
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
C07H 21/04 - Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
The present invention is generally directed to powerful and flexible methods and systems for consensus sequence determination from replicate biomolecule sequence data. It is an object of the present invention to improve the accuracy of consensus biomolecule sequence determination from replicate sequence data by providing methods for assimilating replicate sequence into a final consensus sequence more accurately than any one-pass sequence analysis system.
G06F 19/00 - Digital computing or data processing equipment or methods, specially adapted for specific applications (specially adapted for specific functions G06F 17/00;data processing systems or methods specially adapted for administrative, commercial, financial, managerial, supervisory or forecasting purposes G06Q;healthcare informatics G16H)
77.
PHOTO-INDUCED DAMAGE MITIGATING AGENTS AND PREPARATION AND METHODS OF USE THEREOF
Compositions, devices, systems and methods for reducing and/or preventing photo-induced damage of one or more reactants in an illuminated analytical reaction by addition of one or more photo-induced damage mitigating agents to the reaction mixture and allowing the reaction to proceed for a period that is less than a photo-induced damage threshold period.
The present invention provides labeled phospholink nucleotides that can be used in place of naturally occurring nucleotide triphosphates or other analogs in template directed nucleic acid synthesis reactions and other nucleic acid reactions and various analyses based thereon, including DNA sequencing, single base identification, hybridization assays, and others.
Apparatus, systems and methods for use in analyzing discrete reactions at ultra high multiplex with reduced optical noise, and increased system flexibility. Apparatus include substrates having integrated optical components that increase multiplex capability by one or more of increasing density of reaction regions, improving transmission of light to or collection of light from discrete reactions regions. Integrated optical components include reflective optical elements which re-direct illumination light and light emitted from the discrete regions to more efficiently collect emitted light. Particularly preferred applications include single molecule reaction analysis, such as polymerase mediated template dependent nucleic acid synthesis and sequence determination.
G02B 6/12 - Light guides; Structural details of arrangements comprising light guides and other optical elements, e.g. couplings of the optical waveguide type of the integrated circuit kind
G01N 21/00 - Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
80.
INTERMITTENT DETECTION DURING ANALYTICAL REACTIONS
Methods, devices, and systems for performing intermittent detection during analytical reactions are provided. Such methods facilitate collection of reaction data from disparate reaction times. Further, such methods are useful for reducing photo-induced damage of one or more reactants in an illuminated analytical reaction at a given reaction time. In preferred embodiments, the reaction mixture is subjected to at least one illuminated and non-illuminated period and allowed to proceed such that the time in which the reaction mixture is illuminated is less than a photo-induced damage threshold period.
G01N 33/52 - Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper
C12Q 1/25 - Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving enzymes not classifiable in groups
81.
INTERMITTENT DETECTION DURING ANALYTICAL REACTIONS
Methods, devices, and systems for performing intermittent detection during analytical reactions are provided. Such methods facilitate collection of reaction data from disparate reaction times. Further, such methods are useful for reducing photo-induced damage of one or more reactants in an illuminated analytical reaction at a given reaction time. In preferred embodiments, the reaction mixture is subjected to at least one illuminated and non-illuminated period and allowed to proceed such that the time in which the reaction mixture is illuminated is less than a photo-induced damage threshold period.
This invention provides substrates for use in various applications, including single-molecule analytical reactions. Methods for propagating optical energy within a substrate are provided. Devices comprising waveguide substrates and dielectric omnidirectional reflectors are provided. Waveguide substrates with improved uniformity of optical energy intensity across one or more waveguides and enhanced waveguide illumination efficiency within an analytic detection region of the arrays are provided.
Provided are methods for enhanced sequencing of nucleic acid templates. Also provided are reaction conditions that increase branching fractions during polymerization reactions. Also provided are compositions comprising modified recombinant polymerases that exhibit branching fractions that are higher than the branching fractions of the polymerases from which they were derived. Provided are compositions comprising modified recombinant polymerases that exhibit delayed translocation relative to the polymerases from which they were derived. Also provided are compositions comprising modified recombinant polymerases that exhibit increased nucleotide or nucleotide analog residence time at an active site of the polymerase. Provided are methods for generating polymerases with the aforementioned phenotypes and methods of using such polymerases to sequence a DNA template or make a DNA. Also provided are methods and nucleic acid sequencing systems for determining which labeled nucleotide is incorporated at a site during a template-dependent polymerization reaction.
C12Q 1/68 - Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
C12N 15/11 - DNA or RNA fragments; Modified forms thereof
C12Q 1/25 - Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving enzymes not classifiable in groups
84.
PREPARATIONS, COMPOSITIONS, AND METHODS FOR NUCLEIC ACID SEQUENCING
Preparations, compositions and methods of sequencing nucleic acids are provided. The preparations, methods and compositions employ multiple priming sites on a target nucleic acid and typically utilize single molecule sequencing processes to identify sequence portions of the target which are then assembled to obtain the sequence of the target.
Provided are compositions comprising modified recombinant polymerases that exhibit branching fractions that are less than the branching fractions of the polymerases from which they were derived, or branching fractions that are less than about 25% for a phosphate-labeled nucleotide analog. Also provided are compositions comprising modified recombinant polymerases that exhibit closed polymerase/DNA complexes with increased stability relative to the parental polymerases. Also provided are compositions comprising modified recombinant polymerases that exhibit decreased rate constants relative to the parental polymerases. Provided are methods for generating polymerases with the aforementioned phenotypes. Provided are methods of using such polymerases to make a DNA or to sequence a DNA template.
Compositions, kits, methods and systems for nucleotide sequencing comprising producing polymerase reactions that exhibit two kinetically observable steps within an observable phase of the polymerase reaction. Two slow step systems can be produced, for example, by selecting the appropriate polymerase enzyme, polymerase reaction conditions including cofactors, and polymerase reaction substrates including the primed template and nucleotides.
Compositions and methods for nucleic acid sequencing include template constructs that comprise double stranded portions in a partially or completely contiguous constructs, to provide for redundant sequence determination through one or both of sequencing sense and antisense strands, and iteratively sequencing the entire construct multiple times. Additional sequence components are also optionally included within such template constructs. Methods are also provided for the use and preparation of these constructs as well as sequencing compositions for their application.
Provided are methods and compositions for the production of linear single- stranded nucleic acids, which can be used as templates in high-throughput sequencing systems. Also provided are methods and compositions for the production of closed single- stranded nucleic acid loops, which can be used as templates in high-throughput sequencing systems.
C12N 15/10 - Processes for the isolation, preparation or purification of DNA or RNA
C12Q 1/68 - Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
C12N 15/11 - DNA or RNA fragments; Modified forms thereof
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
Labeled reactant compositions, and particularly labeled nucleic acid reaction compositions, that include structural components that maintain potentially damaging labeling components sufficiently distal from the reactant portion of the molecule such that damaging effects of the label group on other reaction components, such as enzymes, are reduced, minimized and/or eliminated.
Compositions, methods, substrates and systems for use in analysis of single molecule reactions and particularly single molecule nucleic acid sequence analysis. Compositions that include non-reactive, distinguishable or undetectable competitive substrates for the reaction system of interest are provided, as well as their use in systems and substrates for such applications, such compounds typically preferably polyphosphate chains or analogous structures.
The present invention provides methods and compositions for performing illuminated reactions, particularly sequencing reactions, while mitigating and/or preventing photodamage to reactants that can result from prolonged illumination. In particular, the invention provides methods and compositions for incorporating photoprotective agents into conjugates comprising reporter molecules and nucleoside polyphosphates.
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
93.
METHODS AND SYSTEMS FOR ANALYSIS OF FLUORESCENT REACTIONS WITH MODULATED EXCITATION
Methods, systems and their components for monitoring fluorescent signals and particularly transient fluorescent signals from reaction mixtures of interest, which methods and systems employ modulated excitation light sources to reduce impacts of excessive illumination on the reaction components or the data obtained therefrom.
Systems and methods of enhancing fluorescent labeling strategies as well as systems and methods of using non-fluorescent and/or non-optic labeling strategies, e.g., as with single molecule sequencing using ZMWs, are described.
Methods of preparing nucleic acid templates and providing the nucleic acid templates to low copy number reaction volumes are provided. Related compositions of nucleic acid templates are also provided.
C12Q 1/68 - Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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
96.
HIGHLY MULTIPLEXED CONFOCAL DETECTION SYSTEMS AND METHODS OF USING SAME
Systems and methods for analyzing highly multiplexed sample arrays using highly multiplexed, high density optical systems to illuminate high density sample arrays and/or provide detection and preferably confocal detection off signals emanating from such high density arrays. Systems and methods are applied in a variety of different analytical operations, including analysis of biological and biochemical reactions, including nucleic acid synthesis and derivation of sequence information from such synthesis.
Methods, systems and compositions where a target nucleic acid includes a registration sequence disposed therein for identification of the number or relative position of determined sequence from the template sequence. Particularly preferred aspects include a registration sequence in a circular template nucleic acid sequence which is, in turn, used in sequence by incorporation processes that rely upon template dependent, polymerase mediated primer extension in the identification of the sequence of the template.
C12Q 1/68 - Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
C07H 21/02 - 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 ribosyl as saccharide radical
98.
METHODS AND PROCESSES FOR CALLING BASES IN SEQUENCE BY INCORPORATION METHODS
Computer implemented methods, and systems performing such methods for processing signal data from analytical operations and systems, and particularly in processing signal data from sequence-by-incorporation processes to identify nucleotide sequences of template nucleic acids and larger nucleic acid molecules, e.g., genomes or fragments thereof.
Mitigative and remedial approaches to reduction of autofluorescence background noise are applied in analytical systems that rely upon sensitive measurement of fluorescent signals from arrays of fluorescent signal sources. Such systems are for particular use in fluorescence based sequencing by incorporation systems that rely upon small numbers or individual fluorescent molecules in detecting incorporation of nucleotides in primer extension reactions. Systems and methods for analyzing highly multiplexed sample arrays using highly multiplexed, high-density optical systems to illuminate high- density sample arrays and/or provide detection and preferably confocal detection off signals emanating from such high-density arrays. Systems and methods are applied in a variety of different analytical operations, including analysis of biological and biochemical reactions, including nucleic acid synthesis and derivation of sequence information from such synthesis.
Composition, systems, apparatus and methods of enhancing fluorescent signals in biochemical are described. Metal particle proximity to enzymes that produce fluorescent products provide enhanced fluorescence of the product and plasmon resonance of the metal particle. Multi-labeled nucleotides enhance signal production. Reflectance of illumination light and emitted fluorescence increase signal strength for a given illumination light.
C12Q 1/68 - Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
C12P 19/34 - Polynucleotides, e.g. nucleic acids, oligoribonucleotides
G02B 6/10 - Light guides; Structural details of arrangements comprising light guides and other optical elements, e.g. couplings of the optical waveguide type
G02B 6/00 - Light guides; Structural details of arrangements comprising light guides and other optical elements, e.g. couplings
G01N 21/00 - Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
patents
