The present disclosure provides systems and methods for preparing a DNA library from a sample comprising shelf-stable lyophilized microspheres comprising DNA library preparation reagents providing a streamlined workflow for DNA library preparation. The present disclosure also provides a smart consumable container for collecting and transporting the sample.
Systems for sequencing biopolymers and methods of manufacturing the systems are disclosed. In one example, such a system may include an application specific integrated circuit (ASIC) layer, a post array layer beneath the AISC layer, and a nanopore layer above the ASIC layer. The ASIC layer is formed by building active circuitry on a front side of a semiconductor wafer and polishing a back side of the semiconductor wafer. The post array layer is formed by etching a front side of a support substrate and the post array layer provides mechanical support to the ASIC layer. The nanopore layer contains membrane and nanopores. The membrane inhibits passage of water-soluble molecules and the nanopores permit passage of water-soluble molecules. In some embodiments, the system may have short through-substrate vias extending through the ASIC layer. In some embodiments, wafer bonding processes may be used when fabricating the system.
In some examples, a nucleoside triphosphate analogue may include a sugar, a nucleobase coupled to the sugar, a triphosphate group coupled to the sugar, a heteroatom coupled to an alpha phosphate of the triphosphate group, and a first substituent coupled to the heteroatom. The heteroatom may be selected from the group consisting of oxygen, nitrogen, and carbon. The first substituent may include at least one of an alkyl chain or a polymer.
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
4.
A CIRCUIT DESIGN TO APPLY DIFFERENT VOLTAGES IN A NANOPORE ARRAY
In one aspect, the disclosed technology relates to systems and methods for sequencing polynucleotides. In one embodiment, the disclosed system for sequencing polynucleotides includes: a plurality of sequencing cells, each of the plurality of sequencing cells comprising a nanopore for sensing a polynucleotide; a plurality of electronic circuits, each of the plurality of electronic circuits associated with one of the plurality of sequencing cells; and at least one voltage source operably connected to at least one shift register, the output terminals of the at least one shift register operably connected to the plurality of electronic circuits.
An apparatus includes a fluidic circuit, a bypass fluidic circuit, a first set of fluid wells, a second set of fluid wells, a first valve, and a second valve. The first valve operatively associated with the first set of fluid wells such that the first selectively fluidly connects any one of the first set of fluid wells to a first valve outlet. The second valve operatively associated with the fluidic circuit, the bypass fluidic circuit, the first valve outlet, and the second set of fluid wells such that the second valve selectively fluidly connects any one of the second set of fluid wells and the first valve outlet to the fluidic circuit or the first valve outlet to the bypass fluidic circuit.
Automated methods conducted in a sequencing flowcell, and kits for reusing a flowcell, are provided herein. In some examples, an automated method conducted in a sequencing flowcell may include, at a surface of the sequencing flowcell coupled to a first moiety, using a reagent to decouple a first complex from the first moiety. In some examples, the first complex may include a second moiety which couples to the first moiety and a polynucleotide coupled to the second moiety. In some examples, the method may further include using a nuclease to polynucleotides in the sequencing flowcell. The method may include, after using the reagent and after using the nuclease, coupling a second complex to the first moiety. The second complex may include a third moiety which couples with the first moiety and an oligonucleotide coupled to the third moiety.
An example of a kit includes a flow cell assembly. The flow cell assembly includes a reaction chamber, a temperature controlled flow channel in selective fluid communication with an inlet of the reaction chamber, and a filter positioned in the temperature controlled flow channel. The reaction chamber includes depressions separated by interstitial regions and capture primers attached within each of the depressions. The filter is i) to block concentrated biological sample-polymer complexes generated in the temperature controlled flow channel at a first temperature, and ii) to allow passage of concentrated biological sample and polymer released from the complexes in the temperature controlled flow channel at a second temperature.
Well assemblies enabling optical access therein and related systems and methods are disclosed. In accordance with an implementation, an apparatus includes a body, dry reagent, and a cover. The body defines a well and has an opening, an aperture, and a field of view (FOV) enabling optical access from the aperture to the well. The dry reagent is contained within the well. The cover is coupled to the body and covering the opening.
In an example method, a grafting solution is applied to a patterned substrate using a liquid-phase thin-film deposition technique. The patterned substrate includes a lane surrounded by, or a plurality of depressions separated by interstitial regions; and a polymer in the lane or in each of the plurality of depressions. The polymer is functionalized with a first click reaction moiety. The grafting solution includes a solvent; a polymer matrix material dissolved in the solvent; and primers of a primer set dissolved in the solvent, each of the primers being terminated with a second click reaction moiety. The applied grafting solution is dried. During drying, a solid polymer matrix is formed and at least some of the primers attach to the polymer i) via the first and second click reaction moieties and ii) in at least a portion of the lane or in at least some of the plurality of depressions.
An example of a flow cell includes a substrate; a first primer set attached to a first region on the substrate, the first primer set including an un-cleavable first primer and a cleavable second primer; and a second primer set attached to a second region on the substrate, the second primer set including a cleavable first primer and an un-cleavable second primer.
G03F 7/00 - Photomechanical, e.g. photolithographic, production of textured or patterned surfaces, e.g. printed surfaces; Materials therefor, e.g. comprising photoresists; Apparatus specially adapted therefor
In accordance with embodiments herein a method for capturing cells of interest in a digital microfluidic system is provided, comprising utilizing a droplet actuator to transport a sample droplet to a microwell device. The microwell device includes a substrate having a plurality of microwells that open onto a droplet operations surface of the microwell device. The sample droplet includes cells of interest that enter the microwells. The method introduces capture beads to the microwells, and the capture elements are immobilized on the capture beads. The method utilizes the droplet actuator to transport a cell lysis reagent droplet to the microwell device. Portions of the cell lysis reagent droplet enter the microwells and, during an incubation period, cause the cells of interest to release analyte that is captured by the capture elements on the capture beads.
Presented herein are techniques for identifying and/or validating sequence variants in genomic sequence data. The techniques include generating an error rate reflective of sequence errors present in the genomic sequence data. The error rate may be used to validate potential sequence variants. The error rate may be based on errors identified during consensus sequence confirmation for sequence reads associated with individual unique molecular identifiers.
G16B 20/20 - Allele or variant detection, e.g. single nucleotide polymorphism [SNP] detection
G16B 30/00 - ICT specially adapted for sequence analysis involving nucleotides or amino acids
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
Two-phase flushing systems and methods. An example method includes moving a valve to a first position to fluidly connect a first reagent reservoir containing a first reagent to a flow cell and flowing the first reagent from the first reagent reservoir to the flow cell to perform a biochemical reaction. The method includes moving the valve to a second position to fluidly connect a gas to the flow cell and flowing gas into the flow cell to expel at least a portion of the first reagent from the biochemical reaction from the flow cell. The method includes moving the valve to a third position to fluidly connect a buffer reagent reservoir containing a buffer reagent to the flow cell and flowing the buffer reagent into the flow cell.
Devices for sequencing biopolymers, methods of manufacturing the devices, and methods of using the devices are disclosed. In one example, such a device has a nanopore and a horizontal nanochannel. In some embodiments, the horizontal nanochannel may take a tortuous path. In some embodiments, such a device includes gas or air bubble generators or pressure pulse generators to block or unblock the horizontal nanochannel.
Embodiments provided herein relate to methods and compositions for next generation sequencing. Some embodiments include the preparation of a template library from a target nucleic acid using one-sided transposition, sequencing the template library, and capturing the contiguity information.
Embodiments of the present disclosure relates to various bisulfite-free chemical methods for detecting methylation of cytosine in the DNA sample. These methods convert methylated and hydroxymethylated cytosine in the nucleic acid sequence to a modified or pseudo thymine or a uracil moiety which then can be detected in sequencing.
In an example of a method for making a flow cell, a sacrificial layer is deposited over a substrate including depressions separated by interstitial regions. The sacrificial layer is dry etched from the depressions, and the sacrificial layer remains on the interstitial regions. A functionalized layer is deposited over the depressions and over the sacrificial layer. The sacrificial layer is removed from the interstitial regions, which also removes the functionalized layer that overlies the interstitial regions.
G03F 7/00 - Photomechanical, e.g. photolithographic, production of textured or patterned surfaces, e.g. printed surfaces; Materials therefor, e.g. comprising photoresists; Apparatus specially adapted therefor
C23C 14/18 - Metallic material, boron or silicon on other inorganic substrates
Genomic library preparation using Cas-gRNA RNPs, and targeted epigenetic assays, are provided herein. Some compositions include, from a first species, substantially only single-stranded polynucleotides; from a second species, substantially only double-stranded polynucleotides; and amplification primers ligated to ends of the second double-stranded polynucleotides and substantially not ligated to any ends of the first double-stranded polynucleotides. Some compositions include first and second molecules of a target polynucleotide having a sequence, the first molecule having a first end at a first subsequence, the second molecule having a first end at a second subsequence, wherein the first subsequence only partially overlaps with the second subsequence. Some examples provide a composition that includes a target polynucleotide and a first fusion protein including a Cas-gRNA RNP coupled to a transposase having an amplification adapter coupled thereto. The Cas-gRNA RNP may be hybridized to a subsequence in the target polynucleotide.
This disclosure describes methods, non-transitory-computer readable media, and systems that can identify and apply a temperature weight to a pathogenicity prediction for an amino-acid variant at a particular protein position to calibrate and improve an accuracy of such a prediction. For example, in some cases, a variant pathogenicity machine-learning model generates an initial pathogenicity score for a protein or a target amino acid at a particular protein position based on an amino-acid sequence of the protein. The disclosed system further identifies a temperature weight that estimates a degree of certainty for pathogenicity scores output by the variant pathogenicity machine-learning model. To generate such a weight, the disclosed system can use a new triangle attention neural network as a temperature prediction machine-learning model. Based on the temperature weight and the initial pathogenicity score, the disclosed system generates a calibrated pathogenicity score for the target amino acid at the particular protein position.
G16H 50/30 - ICT specially adapted for medical diagnosis, medical simulation or medical data mining; ICT specially adapted for detecting, monitoring or modelling epidemics or pandemics for individual health risk assessment
24.
CONCURRENT SEQUENCING OF FORWARD AND REVERSE COMPLEMENT STRANDS ON CONCATENATED POLYNUCLEOTIDES FOR METHYLATION DETECTION
Some embodiments relate to methods and compositions for preparing combinatorially indexed beads. Some embodiments include sequential addition of different indexes to polynucleotides attached to beads. In some embodiments, indexes are added by chemical ligation, polymerase extension, ligation of partially double-stranded adaptors, or short splint ligation.
Aptamer detection techniques with dynamic range compression are described that permit removal of a portion of more abundant aptamers in an aptamer-based assay. In an embodiment, a mixture of tagged probes and dummy probes can be used such that the dummy probes bind abundant aptamers and in turn are not captured or amplified for detection in downstream steps. Other techniques are also contemplated, including targeted removal of or cleavage of probes that bind to excess aptamers.
Reagent cartridges and related systems and methods are disclosed. In accordance with an implementation, an apparatus includes a first flexible container, a second flexible container, and a coupling. The first flexible container has an end and defines a first interior containing reagent. The second flexible container has an end and defines a second interior. The first flexible container is positioned within the second interior. The coupling has a first portion coupled to the end of the first flexible container and a second portion coupled to the end of the second flexible container. The coupling has a reagent port fluidly coupled to the first interior of the first flexible container and a pressure port fluidly coupled to the second interior of the second flexible container.
The determination of pharmacogenomics gene star alleles using high-throughput targeted genotyping includes obtaining input genetic sequence variation data from a high-throughput genotyping platform based on a pharmacogenomic genotyping of a sample, applying a Bayesian graphical model to determine a plurality of different star allele calls corresponding to the sample, and providing a respective quality score for each star allele call of the plurality of different star allele calls. For instance, the application of the Bayesian graphical model uses multi-solution integer programming to explore a model space of the Bayesian graphical model in a first phase that includes structural variant candidate identification and a second phase that includes star allele candidate identification based on the structural variant candidate identification, to determine the plurality of different star allele calls.
The assembly includes a docking console and a manifold. The docking console includes a cartridge support surface having a first end and a second end. The manifold has one or more wells defined therein. The docking console further includes a manifold retention bracket to releasably hold the manifold against a fluid cartridge supported on the cartridge support surface at an interface position such that the one or more wells are in fluid communication with the fluid cartridge and a biased seal bar to press the fluid cartridge against the manifold held by the manifold retention bracket. A hydrophilic porous frit disposed within at least one of the wells and is to permit liquid to flow through the outlet aperture but prevent gas from passing through the outlet aperture.
The invention provides systems and methods for making sequencing libraries that are useful for quantitatively analyzing nucleic acids in a sample. Sample nucleic acids are randomly cleaved at, and PCR handled are attached to, a random cut site. The nucleic acid is amplified into a sequencing library in which a sequencing primer generates a sequence read from adjacent the random cut site. The sequence reads can be mapped to a reference, but they will also include a unique identifier sequence that comes from within the nucleic acid molecule being analyzed, i.e., an intrinsic molecular identifier (IMI). The IMI is unique for each molecule and can thus be used to deduplicate sequence reads originating from the same molecule.
Array-based targeted copy number detection, for instance detection on contaminated and/or variable concentration samples, includes obtaining a collection of intensity signals from assays of a set of input samples, performing a cross-sample calibration on the intensity signals based on reference sample(s), which calibration includes constructing a reference signal distribution based on intensity signals of the reference sample(s) and for one or more input samples calibrating a set of intensity signals corresponding to the input sample based on the reference signal distribution, determining, for the one or more input samples, and from a respective one or more calibrated sets of intensity signals corresponding to the one or more input samples, a respective at least one aggregated calibrated signal from targeted genomic region(s) to produce a collection of aggregated calibrated signals, and detecting variant(s) in the targeted genomic region(s) based on the collection of aggregated calibrated signals.
Polypeptide nanopores synthetically functionalized with positively charged species, and methods of making and using the same, are provided herein. In some examples, a polypeptide nanopore includes a first side, a second side, a channel extending through the first and second sides, and a mutated amino acid residue. The mutated amino acid residue may be synthetically functionalized with a positively charged species that inhibits translocation of cations through the channel.
G01N 33/487 - Physical analysis of biological material of liquid biological material
B82B 1/00 - Nanostructures formed by manipulation of individual atoms or molecules, or limited collections of atoms or molecules as discrete units
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
The present disclosure is directed to decoupling library capture (template seeding) from cluster generation to optimise both processes. This is achieved by introducing orthogonality between the seeding and clustering primer.
Nucleic acid techniques are disclosed. Embodiments include modified nucleotides with oligonucleotide adapters that are coupled via cleavable linkers. Incorporation of the modified nucleotide at a 3′ end of a nucleic acid permits end-adapterization via ligation of a free 5′ end of the oligonucleotide adapter to a 3′ reactive group of the modified nucleotide and cleavage at the cleavable linker to liberate a free 3′ end.
Disclosed herein include methods, compositions, reaction mixtures, kits and systems for identification of methylated cytosines in nucleic acids using a bisulfite-free, one-step chemoenzymatic modification of methylated cytosines.
An array includes a support including a plurality of discrete wells, a gel material positioned in each of the plurality of discrete wells, and a quality control tracer grafted to the gel material in each of the plurality of discrete wells. The quality control tracer comprises (a) a cleavable nucleotide sequence comprising a cleavage site and (b) a detectable label; and in some aspects, is a cleavable nucleotide sequence with a detectable label and a non-reactive nucleotide sequence or a primer nucleotide sequence.
Embodiments of the present disclosure relate to nucleotides with 3′ allyl blocking groups. Also provided herein are methods of sequencing using nucleotides with 3′ allyl blocking groups described herein, and sequencing kits.
The disclosed embodiments concern methods, systems and computer program products for determining sequences of interest using unique molecular indexes (UMIs) that are uniquely associable with individual polynucleotide fragments, including sequences with low allele frequencies or long sequence length. In some implementations, the UMIs include variable-length nonrandom UMIs (vNRUMIs). Methods and systems for making and using sequencing adapters comprising vNRUMIs are also provided.
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
39.
DETERMINING AND REMOVING INTER-CLUSTER LIGHT INTERFERENCE
This disclosure describes embodiments of methods, systems, and non-transitory computer readable media that accurately estimates the crosstalk from an adjacent cluster of oligonucleotides onto a target cluster of oligonucleotides and removes or reduces the crosstalk emitted by the adjacent cluster of oligonucleotides from the target cluster of oligonucleotides. For instance, the disclosed systems can detect the intensity values for a target cluster and the adjacent cluster. Based on the intensity values of the adjacent cluster, the disclosed systems can determine an inter-cluster-interference metric that estimates the crosstalk emitted from the adjacent cluster. The disclosed systems can remove the inter-cluster-interference metric from the intensity value of the target cluster and generate modified intensity values for the target cluster.
Systems and methods for identifying conditions in a sample obtain a set of sample sequence reads from the sample. For each respective read, or respective sample contig derived from a respective subset of the set, a corresponding sequence comparison between the respective read or contig and each reference sequence in a set of reference sequences is performed. There is calculated, from these sequence comparisons, a respective probability that the respective read or contig corresponds to a particular reference sequence in the set of reference sequences thereby computing a plurality of probabilities. The presence or an absence of each of the conditions in the sample is identified based at least in part on these probabilities. One condition is identification of a species present in the sample, and the percentage of the genome of this species identified in the reads is provided.
The present disclosure is related to methods and materials for depleting unwanted RNA species from a nucleic acid sample. In particular, the present disclosure describes how to remove unwanted rRNA, tRNA, mRNA or other RNA species that could interfere with the analysis, manipulation and study of target RNA molecules in a sample.
C12N 15/10 - Processes for the isolation, preparation or purification of DNA or RNA
C12Q 1/6806 - Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
C12Q 1/6848 - 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
Systems, methods, and apparatuses are described herein. For instance, a detection apparatus may comprise memory and at least one processor. The detection apparatus may be configured to obtain an image comprising at least one feature and a plurality of fiducials. The plurality of fiducials may be arranged in a pattern. The detection apparatus may be configured to determine a plurality of sub-regions of the image. Each sub-region comprises a subset of the fiducials comprised in the image. The detection apparatus may be configured to perform a geometric transform on each sub-region to generate a respective local transform associated with each sub-region. The detection apparatus may be configured to register respective locations of the fiducials comprised in the image based on the respective local transform associated with each sub-region. A size of each sub-region may be selected such that each sub-region is substantially invariant to stage jitter.
Improved copy number variant (CNV) calling in a genomic sequence, and potential recovery, includes (i) obtaining genetic sequence variant data that includes records indicating structural variant(s) (SVs) and records indicating CNV(s) in the genomic sequence, (ii) determining, based on an initial CNV indicated in the genetic sequence variant data and on initial SV(s) indicated in the genetic sequence variant data, an SV-informed CNV call as an updated version of the initial CNV, where the determining uses information from the initial SV(s) to determine a start breakpoint position and an end breakpoint position for the SV-informed CNV call, at least one of the start breakpoint position and end breakpoint position being updated, informed by the initial SV(s), in comparison to a corresponding start breakpoint position and/or end breakpoint position of the initial CNV, and (ii) writing the determined SV-informed CNV call as record(s) in a genetic sequence variant data file.
Techniques are described for processing a text document or passage to derive a suitable set of phrases from the document or passage. These phrases may in turn be related to codes or other labels useful to a reviewer, such as insurance, diagnostic, or clinical codes, genes related to identified phenotypes, and so forth. In certain embodiments, one or more tries generated based on respective ontologies may be used to process and parse the input text passage or document to derive candidate phrases. To improve performance, a limited number of skips may be allowed. The candidate phrases and corresponding intervals may, in one implementation, be used to populate a graph having nodes and edges and from which a set of phrases may be determined that provides maximal coverage of the text passage or document and having limited (or no) overlaps.
Systems and methods for identifying a host of an AMR marker from one or more samples are provided, which include obtaining short-read sequence data derived from one or more samples; identifying one or more AMR markers from the short-read sequence data to obtain short-read metrics, the short-read metrics comprising quantitative metrics such as RPKM, median depth, read count, or others of any one or more of the AMR markers identified in the short-reads; obtaining one or more reference sequence data; identifying one or more AMR markers from the reference sequence data to obtain reference metrics, the reference metrics comprising quantitative metrics such as RPKM, median depth, read count, or others of any one or more of the AMR markers identified in the reference sequence; identifying a host of the one or more AMR markers in the sample when average ratios between the short-read metrics and the reference metrics are below a threshold ratio.
C12Q 1/70 - Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
C12Q 1/6874 - Methods for sequencing involving nucleic acid arrays, e.g. sequencing by hybridisation [SBH]
C12Q 1/6888 - Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
47.
PALLADIUM CATALYST COMPOSITIONS AND METHODS FOR SEQUENCING BY SYNTHESIS
The present application relates to palladium catalyst composition and uses in sequencing by synthesis. In particular, the Pd catalyst composition comprises one or more macrocycles (e.g., cyclodextrin or analogs thereof) as additives for improving thermal or oxidative stability of the active Pd(0) species.
Embodiments of the present invention relate to analyzing components of a cell. In some embodiments, the present invention relate to analyzing components of a single cell. In some embodiments, the methods and compositions relate to sequencing nucleic acids. In some embodiments, the methods and compositions relate to identifying and/or quantitating nucleic acid, proteins, organelles, and/or cellular metabolites.
A composition for amplifying a polynucleotide is provided that includes a substrate comprising a first region and a second region. A first plurality of capture primers is coupled to the first region of the substrate. A second plurality of capture primers is coupled to the second region of the substrate. The capture primers of the second plurality of capture primers are longer than the capture primers of the first plurality of capture primers. A first plurality of orthogonal capture primers are coupled to the first region of the substrate. A second plurality of orthogonal capture primers are coupled to the second region of the substrate. The orthogonal capture primers of the second plurality of orthogonal capture primers are shorter than the orthogonal capture primers of the first plurality of orthogonal capture primers.
We propose a neural network-implemented method for base calling analytes. The method includes accessing a sequence of per-cycle image patches for a series of sequencing cycles, where pixels in the image patches contain intensity data for associated analytes, and applying three-dimensional (3D) convolutions on the image patches on a sliding convolution window basis such that, in a convolution window, a 3D convolution filter convolves over a plurality of the image patches and produces at least one output feature. The method further includes beginning with output features produced by the 3D convolutions as starting input, applying further convolutions and producing final output features and processing the final output features through an output layer and producing base calls for one or more of the associated analytes to be base called at each of the sequencing cycles.
G06V 10/75 - Image or video pattern matching; Proximity measures in feature spaces using context analysis; Selection of dictionaries
G06V 10/764 - Arrangements for image or video recognition or understanding using pattern recognition or machine learning using classification, e.g. of video objects
G06V 10/82 - Arrangements for image or video recognition or understanding using pattern recognition or machine learning using neural networks
51.
Deep Learning-Based Pathogenicity Classifier for Promoter Single Nucleotide Variants (pSNVs)
We disclose computational models that alleviate the effects of human ascertainment biases in curated pathogenic non-coding variant databases by generating pathogenicity scores for variants occurring in the promoter regions (referred to herein as promoter single nucleotide variants (pSNVs)). We train deep learning networks (referred to herein as pathogenicity classifiers) using a semi-supervised approach to discriminate between a set of labeled benign variants and an unlabeled set of variants that were matched to remove biases.
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
G06N 3/044 - Recurrent networks, e.g. Hopfield networks
Embodiments of the present disclosure relate to nucleotide and nucleoside molecules with 3′ vinyl or isonitrile containing blocking groups and/or tetrazine or strained unsaturated ring containing cleavable linkers. Additionally, the present disclosure provides methods of using the nucleoside/nucleotide in oligonucleotide synthesis, and methods of sequencing using the nucleotide described herein.
The present application relates to compositions and methods for sequencing by synthesis. A blocking group of a nucleotide may be removed by a transition metal catalyst, the transition metal catalyst activated by a non-reducing ligand and a reducing agent.
B01J 31/14 - Catalysts comprising hydrides, coordination complexes or organic compounds containing organic compounds or metal hydrides containing organo-metallic compounds or metal hydrides of aluminium or boron
This application relates to methods of denaturing double-stranded DNA (dsDNA). In some examples, the methods utilize dried sodium hydroxide. In some examples, the method includes loading dsDNA into a first portion of a cartridge, wherein the second portion of the cartridge contains sodium hydroxide in a dry form; and mixing the dsDNA with the sodium hydroxide, thereby denaturing the dsDNA.
The technology disclosed corrects inter-cluster intensity profile variation for improved base calling on a cluster-by-cluster basis. The technology disclosed accesses current intensity data and historic intensity data of a target cluster, where the current intensity data is for a current sequencing cycle and the historic intensity data is for one or more preceding sequencing cycles. A first accumulated intensity correction parameter is determined by accumulating distribution intensities measured for the target cluster at the current and preceding sequencing cycles. A second accumulated intensity correction parameter is determined by accumulating intensity errors measured for the target cluster at the current and preceding sequencing cycles. Based on the first and second accumulated intensity correction parameters, next intensity data for a next sequencing cycle is corrected to generate corrected next intensity data, which is used to base call the target cluster at the next sequencing cycle.
G06F 18/2411 - Classification techniques relating to the classification model, e.g. parametric or non-parametric approaches based on the proximity to a decision surface, e.g. support vector machines
G06F 18/2135 - Feature extraction, e.g. by transforming the feature space; Summarisation; Mappings, e.g. subspace methods based on approximation criteria, e.g. principal component analysis
The present application relates to palladium compositions, methods for sequencing by synthesis using nucleotides with 3′ blocking groups, and sequencing kits, where one or more palladium scavengers were used to improve sequencing metrics such phasing and prephasing values.
Examples of flow cells include substrates. Embodiments of the present disclosure also relate to methods of fabricating flow cell substrates. Some example workflows exploit light blocking properties of an imprint layer such that the process does not include etch steps. Such processes may be used to create substrates compatible with simultaneous paired-end sequencing methods.
G03F 7/11 - Photosensitive materials - characterised by structural details, e.g. supports, auxiliary layers having cover layers or intermediate layers, e.g. subbing layers
B01J 19/00 - Chemical, physical or physico-chemical processes in general; Their relevant apparatus
B01L 3/00 - Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
G03F 7/038 - Macromolecular compounds which are rendered insoluble or differentially wettable
58.
AMPLIFICATION TECHNIQUES FOR NUCLEIC ACID CHARACTERIZATION
Nucleic acid amplification techniques are disclosed. Embodiments include generating concatenated nucleic acids using rolling circle amplification of templates, e.g., starting from a cDNA of a full-length mRNA or from synthetic templates, and sequencing and/or detecting the concatenated nucleic acids. In some embodiments, the technology disclosed includes amplification reactions that include CRISPR-Cas interactions that generate primers as a result of the CRISPR-Cas interactions, whereby primers are in turn used as part of detectable amplification reactions. The disclosed amplification techniques may use synthetic oligonucleotides or primers.
Biological samples on multiple surfaces of a support structure may be imaged using a machine comprising a lens, a flow cell and a controller. Such a machine may capture light emitted from nucleic acids disposed on first and second surfaces of the flow cell when the lens is, respectively, at first and second distances from the flow cell. In such a machine, the lens may be immersed in a first fluid, and the first and second surfaces of the flow cell may be separated by a second fluid. Additionally, in such a machine, differences between marginal and axial light rays in the field of view of the lens may be substantially equal when the lens is at the first and second distances from the flow cell.
The technology disclosed is directed to context-dependent base calling. The technology disclosed describes a system including memory storing k-mer-specific centroids for k-mers. The k-mer-specific centroids are learned by training a base calling pipeline to represent base calls of an already base called sequence in k-mer-specific time series, transform the k-mer-specific time series into predicted k-mer-specific centroids, merge the predicted k-mer-specific centroids on a sequencing cycle-by-sequencing cycle basis to generate predicted per-sequencing cycle intensity values, determine a training loss (e.g., a transformation loss) based on comparing the predicted per-sequencing cycle intensity values against known intensity values of the base calls, update the predicted k-mer-specific centroids based on the determined training loss, and store the updated centroids as the k-mer-specific centroids. The system also includes runtime logic that uses the k-mer-specific centroids to base call bases in a yet-to-be base called sequence in dependence upon k-mer context.
G16B 30/00 - ICT specially adapted for sequence analysis involving nucleotides or amino acids
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
61.
METHODS OF SEQUENCING USING 3' BLOCKED NUCLEOTIDES
Embodiments of the present disclosure relate to nucleotide and nucleoside molecules with acetal or allyl 3′ blocking groups. Also provided herein are methods to prepare such nucleotide and nucleoside molecules, and the uses of fully functionalized nucleotides containing the 3′ blocking groups for sequencing applications.
In an example method, a positive photoresist is deposited over a substrate that includes depressions separated by interstitial regions. The positive photoresist is exposed to ultraviolet light at an angle that is non-perpendicular, non-parallel, and offset from a surface plane of the depressions such that a first portion of the positive photoresist in each depression remains soluble and a second portion of the positive photoresist in each depression is rendered insoluble. The soluble portions of the positive photoresist are removed, which exposes a first substrate portion in each depression. A first functionalized layer is deposited over the first substrate portion in each depression. The insoluble portions of the positive photoresist are removed, which exposes a second substrate portion in each depression. The second functionalized layer is selectively deposited over the second substrate portion in each depression.
G03F 7/039 - Macromolecular compounds which are photodegradable, e.g. positive electron resists
G03F 7/038 - Macromolecular compounds which are rendered insoluble or differentially wettable
G03F 7/11 - Photosensitive materials - characterised by structural details, e.g. supports, auxiliary layers having cover layers or intermediate layers, e.g. subbing layers
Methods for on-flow cell selective capture and enrichment of clustered beads, general capture strategies on bead mobility on flow cell surfaces, sorting clustered and unclustered beads, and flow cell reusability for bead immobilization onto flow cells.
An example of a flow cell includes a substrate; a plurality of reactive regions spatially separated from one another across the substrate; and a plurality of independently removable coatings respectively positioned over each of the plurality of reactive regions. Each of the plurality of reactive regions includes a polymeric hydrogel layer; and a reactive entity attached to the polymeric hydrogel layer.
An example of a flow cell includes a substrate; a plurality of reactive regions spatially separated from one another across the substrate; and a plurality of independently removable coatings respectively positioned over each of the plurality of reactive regions. Each of the plurality of reactive regions includes a polymeric hydrogel layer; and a reactive entity attached to the polymeric hydrogel layer. At least one of the independently removable coatings is a composite that includes a thermo-responsive polymer and a photo-thermal additive.
An example of a flow cell includes a substrate; a plurality of reactive regions spatially separated from one another across the substrate; and a plurality of independently removable coatings respectively positioned over each of the plurality of reactive regions. Each of the plurality of reactive regions includes a polymeric hydrogel layer; and a reactive entity attached to the polymeric hydrogel layer. At least one of the independently removable coatings is a gas-dissolvable coating.
A method for modifying an interstitial surface separating recesses from one another can include flowing a first fluid over the interstitial surface and into the recesses, such that the interstitial surface is substantially coated with the first fluid and the recesses are substantially filled with the first fluid; and while the first fluid remains within the recesses, replacing the first fluid coating the interstitial surface with a second fluid comprising a reagent.
B05D 1/38 - Successively applying liquids or other fluent materials, e.g. without intermediate treatment with intermediate treatment
B05D 7/24 - Processes, other than flocking, specially adapted for applying liquids or other fluent materials to particular surfaces or for applying particular liquids or other fluent materials for applying particular liquids or other fluent materials
69.
ARRAYS INCLUDING A RESIN FILM AND A PATTERNED POLYMER LAYER
An example of an array includes a support, a cross-linked epoxy polyhedral oligomeric silsesquioxane (POSS) resin film on a surface of the support, and a patterned hydrophobic polymer layer on the cross-linked epoxy POSS resin film. The patterned hydrophobic polymer layer defines exposed discrete areas of the cross-linked epoxy POSS resin film, and a polymer coating is attached to the exposed discrete areas. Another example of an array includes a support, a modified epoxy POSS resin film on a surface of the support, and a patterned hydrophobic polymer layer on the modified epoxy POSS resin film. The modified epoxy POSS resin film includes a polymer growth initiation site, and the patterned hydrophobic polymer layer defines exposed discrete areas of the modified epoxy POSS resin film. A polymer brush is attached to the polymer growth initiation site in the exposed discrete areas.
The current document discusses electromechanical sequence detectors that transduce changes in the shape of a shape-change sensor component into an electrical signal from which one or more derived values are generated. In a disclosed implementation, the sequence-detection system comprises a mechanical-change sensor that changes shape when specifically interacting with entities within a target, a shape-to-signal-transduction component that transduces changes in the shape of the mechanical-change sensor into an electrical signal, an analysis subsystem that determines the types of entities within the target using the electrical signal, and a control subsystem that continuously monitors operational characteristics of the sequence-detection system and adjusts sequence-detection system operational parameters.
Systems, methods, and apparatus are described herein for aggregating genome data into bins with summary data at various levels. As described herein, a computing device may be configured to receive genome data associated with a genome. The computing device may be configured to generate an aggregate file using the received genome data. The aggregate file may include a plurality of bins at a plurality of depths. The computing device may be configured to determine summary data for respective reads associated with one or more respective portions of the genome covered by respective bins of the plurality of bins. The computing device may be configured to store the summary data for the respective reads in respective bins of the plurality of bins. The computing device may be configured to display a portion of the summary data in response to a selection of a genomic region by a user.
Reusable flow cells for sequencing which exhibit signal intensity retention over numerous use cycles, the active surface of which contains reactive sulfur moieties for reversible primer binding, methods of using such flow cells, reagents therefor, and kits containing the same.
The invention provides methods for annotating cell types using non-parametric statistical scoring of gene expression levels from RNA sequencing (RNA-seq). Expression levels for cells are measured by RNA-seq and a non-parametric statistic such as a Mann-Whitney U score or Wilcoxon score is generated for the expression levels and correlated to such scores from reference data from known cell types. When test cells in the RNA-seq data have a score that correlate highly with such a score from cells of a known type in the reference, those test cells are annotated as being of the known type from the reference.
Library preparation systems and methods are disclosed. In an implementation, a modular bay for preparing a library of samples for sequencing, the modular bay comprising a contact dispenser and a working area comprising a working plate receptacle adapted to receive a working plate, a thermocycler, a magnet, and a drawer. The drawer comprising a consumables area adapted to receive a sample plate adapted to contain a sample, and a plurality of consumables for interacting with the sample in the working plate. The contact dispenser is linearly movable in a longitudinal direction between the consumables area and the working area, such that the contact dispenser is configured to (i) move the sample from the sample plate in the consumables area to the working plate in the working area, and (ii) move the plurality of consumables between the consumables area and the working plate in the working area.
An example flow cell includes a substrate including depressions separated by interstitial regions; a polymeric hydrogel positioned within each of the depressions; a primer set attached to the polymeric hydrogel; and one of: a passivation component attached to the interstitial regions, or a passivation component attached to the polymeric hydrogel, or respective passivation components attached to each of the interstitial regions and the polymeric hydrogel.
A fluidic device holder configured to orient a fluidic device. The device holder includes a support structure configured to receive a fluidic device. The support structure includes a base surface that faces in a direction along the Z-axis and is configured to have the fluidic device positioned thereon. The device holder also includes a plurality of reference surfaces facing in respective directions along an XY-plane. The device holder also includes an alignment assembly having an actuator and a movable locator arm that is operatively coupled to the actuator. The locator arm has an engagement end. The actuator moves the locator arm between retracted and biased positions to move the engagement end away from and toward the reference surfaces. The locator arm is configured to hold the fluidic device against the reference surfaces when the locator arm is in the biased position.
The disclosure relates to methods, compositions, and kits for the selective depletion of non-desirable fragments from amplified libraries using blocking oligonucleotides.
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 1/48 - Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
Embodiments of the present disclosure relate to nucleotide and nucleoside molecules with 3′ acetal, thiocarbamate or allyl blocking groups. Also provided herein are methods to prepare such nucleotide and nucleoside molecules, and the uses of fully functionalized nucleotides containing the 3′-OH blocking group for sequencing applications.
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
In one example, a flow cell includes a substrate, an electrode positioned on the substrate, and a patterned material positioned on the electrode. In this example, the patterned material includes depressions separated by interstitial regions, and a functionalized surface of the electrode is exposed at each of the depressions. In this example, the flow cell further includes a primer grafted to the functionalized surface in each of the depressions. In another example, a flow cell includes a substrate and a patterned electrode positioned on the substrate. In this other example, the patterned electrode includes depressions separated by interstitial regions, and a functionalized surface of the substrate exposed at each of the depressions. In this other example, a primer is grafted to the functionalized surface in each of the depressions.
Some examples herein provide a hydrogel on a substrate. The hydrogel includes a three-dimensional network of polymer chains; first functional groups coupled to the polymer chains; amplification primers coupled to the polymer chains via the first functional groups; and second functional groups coupled to the polymer chains and reversibly cross-linking the polymer chains to one another. Some examples herein provide a method of using a hydrogel. The method includes hybridizing a target polynucleotide to an amplification primer coupled to a hydrogel; cleaving cross-linkages within the hydrogel within which the target polynucleotide is hybridized to the amplification primer; and amplifying the target polynucleotide using additional amplification primers within the hydrogel within which the cross-linkages have been cleaved.
The present disclosure is concerned with proteins, methods, compositions, and kits for mapping of methylation status of nucleic acids, including 5-methylcytosine and 5-hydroxymethyl cytosine (5 hmC). In one embodiment, proteins are provided that selectively act on certain modified cytosines of target nucleic acids and converts them to thymidine or modified thymidine analogues. In another embodiment, proteins are provided that selectively act on certain modified cytosines of target nucleic acids and converts them to uracil or thymidine and selectively do not act on other certain modified cytosines of target nucleic acids. Also provided are compositions and kits that include one or more of the proteins and methods for using one or more of the proteins.
The technology disclosed is directed to cluster segmentation and base calling. The technology disclosed describes a computer-implemented method including segmenting a population of clusters into a plurality of subpopulations of clusters based on one or more prior bases called at one or more prior sequencing cycles of a sequencing run. At a current sequencing cycle of the sequencing run, the method includes applying a mixture of four distributions to current sequenced data of each subpopulation of clusters in the plurality of subpopulations of clusters, the four distributions corresponding to four bases adenine (A), cytosine (C), guanine (G), and thymine (T), and the current sequenced data being generated at the current sequencing cycle. The method further includes base calling clusters in a particular subpopulation of clusters using a corresponding mixture of four distributions.
G16B 40/00 - ICT specially adapted for biostatistics; ICT specially adapted for bioinformatics-related machine learning or data mining, e.g. knowledge discovery or pattern finding
G16B 30/00 - ICT specially adapted for sequence analysis involving nucleotides or amino acids
Disclosed herein is a modified transposon end sequence comprising a mosaic end sequence, wherein the mosaic end sequence comprises one or more mutation as compared to a wild-type mosaic end sequence, wherein the mutation comprises a substitution with a uracil, an inosine, a ribose, an 8-oxoguanine, a thymine glycol, a modified purine, or a modified pyrimidine. Also disclosed are transposome complexes comprising these modified transposon end sequences and methods of library preparation using these modified transposon end sequences.
This disclosure describes methods, non-transitory computer readable media, and systems that can utilize methylation sequencing assay data to generate genotype calls efficiently and accurately for a target genomic sample. In some implementations, the disclosed system identifies the target genomic sample's nucleotide reads comprising nucleobases converted by a methylation sequencing assay. The disclosed system can determine variant calls based on aligning the nucleotide reads with a reference genome. To account for errors introduced by the methylation sequencing assay, in some cases, the disclosed system corrects or modifies genotype-likelihood metrics for a subset of candidate variant calls. The disclosed system further imputes genotype calls based on such modified genotype-likelihood metrics and a comparison of a subset of variant calls with marker variants from a reference panel.
A system includes a reagent selector valve controllable to select a reagent flow path from a plurality of reagent flow paths, and a pump coupled to the reagent flow path to draw a liquid through the reagent flow path in accordance with a prescribed test protocol. The system includes a discharge flow path to expel the drawn liquid, and a flow meter to measure liquid displaced by the pump and that outputs data representative of the measured flow. The system also includes a processor to access the data and to determine a volume of the liquid displaced by the pump.
A system, method and apparatus for executing an HMM analysis on genetic sequence data includes an integrated circuit formed of a set of hardwired digital logic circuits that are interconnected by physical electrical interconnects. One of the physical electrical interconnects forms an input to the integrated circuit that may be connected with an electronic data source for receiving reads of genomic data. The hardwired digital logic circuits may be arranged as a set of processing engines, each processing engine being formed of a subset of the hardwired digital logic circuits to perform one or more steps in the HMM analysis on the reads of genomic data. Each subset of the hardwired digital logic circuits may be formed in a wired configuration to perform the one or more steps in the HMM analysis.
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
Methods and compositions for characterizing a target polynucleotide, including, characterizing the sequence of the target polynucleotide, using the fractional translocation steps of the target polynucleotide's translocation through a pore.
C07K 14/35 - Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Mycobacteriaceae (F)
Non-contact dispenser assemblies and related systems and methods are disclosed. An implementation of an apparatus is disclosed that includes a reagent cartridge including a body defining a plurality of reservoirs. Each reservoir has an outlet and a distal end defining an opening, a manifold assembly including an outlet, a common fluidic line fluidly coupled to the outlet, a plurality of reagent fluidic lines coupled to the corresponding outlet of the reservoirs, and a plurality of membrane valves selectively fluidly coupling the common fluidic line and a corresponding one of the plurality of reagent fluidic lines.
Analyzing expression of protein-coding variants in cells is provided herein. A method may include replacing a protein coding-region of the DNA in a cell with a donor vector including a variant of the protein-coding region and a first barcode identifying that variant. The cell may generate mRNA including an expression of the variant and an expression of the first barcode. A second barcode corresponding to the cell may be coupled to the mRNA. The mRNA. having the second barcode coupled thereto, may be reverse transcribed into complementary cDNA. The cDNA may be sequenced. The donor vector or cDNA may be sequenced using amplicon sequencing. The donor vector sequence and the cDNA sequence may be correlated to identify the variant and the cell's expression of the variant.
Provided herein are compositions and methods for sequencing using metal-coated polymers. In some examples, a bridge spans a space between first and second electrodes and includes a polymer chain having a first metal-coated region contacting the first electrode, a second metal-coated region contacting the second electrode, and an exposed region located between the first and second regions. The composition includes first and second polynucleotides; a plurality of nucleotides, each nucleotide coupled to a corresponding label; and a polymerase to add nucleotides of the plurality of nucleotides to the first polynucleotide using at least a sequence of the second polynucleotide. The composition includes detection circuitry to detect a sequence in which the polymerase adds the nucleotides to the first polynucleotide using at least changes in an electrical signal through the bridge, the changes being responsive to contact between the labels corresponding to those nucleotides and the exposed region.
In some examples, novel photochemically-reversible hydrogels and nanogel particles are described comprising copolymer chains including at least one reactive alkene or reactive 1,4-diene end group capable of [2+2] or [2+2+2+2] photodimerization, respectively, at wavelengths >270 nm. In various examples, the photochemically-reversible hydrogels comprise copolymer chains including at least one —N3, —C≡CH or —CO2H end group for dual functionality and/or pH responsiveness. For nucleic acid sequencing, amplification primers are grafted to photochemically-reversible hydrogels or nanogel particles reversibly bound to surfaces within a flow cell. After sequencing is complete, the photochemically-reversible hydrogel or nanogel particles is/are removable from the flow cell surfaces by irradiation, enabling the flow cell to be reusable.
Disclosed herein include systems and methods for designing probes for depleting abundant transcripts from a sample. Abundant sequence reads can be determined in a species-agnostic manner, and probes for depleting abundant transcripts can be designed based on the sequences of the top abundant sequences. Also disclosed herein include compositions and kits for depleting abundant transcripts and methods for depleting abundant transcripts.
C12Q 1/6876 - Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
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
95.
METHODS OF ISOTHERMAL COMPLEMENTARY DNA AND LIBRARY PREPARATION
Described herein are compositions and methods for preparing double-stranded complementary DNA (cDNA) from RNA. In some embodiments, these methods allow isothermal preparation of cDNA. In some embodiments, these methods allow mesophilic or thermostable preparation of cDNA. Also described herein are compositions and methods for preparing cDNA and a library of double-stranded cDNA fragments in a single reaction vessel.
Disclosed in one example is an apparatus including a substrate, a sensor over the substrate including an active surface and a sensor bond pad, a molding layer over the substrate and covering sides of the sensor, the molding layer having a molding height relative to a top surface of the substrate that is greater than a height of the active surface of the sensor relative to the top surface of the substrate, and a lidding layer over the molding layer and over the active surface. The lidding layer and the molding layer form a space over the active surface of the sensor that defines a flow channel.
Embodiments of the present disclosure relate to cyclooctatetraene containing dyes and their uses as fluorescent labels. Also provided are composition containing cyclooctatetraene. The dyes and compositions may be used in various biological applications, such as nucleic acid sequencing.
C07D 413/04 - Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings directly linked by a ring-member-to-ring- member bond
Systems, methods and compositions provided herein relate to the preparation of nucleic acid libraries. Some embodiments include the preparation of nucleic acid libraries by ligation of single-stranded nucleic acids.
Presented herein are altered polymerase enzymes for improved incorporation of nucleotides and nucleotide analogues, in particular altered polymerases that maintain low error rate, low phasing rate, or increased incorporation rate for a second generation ffN under reduced incorporation times, as well as methods and kits using the same.
The present disclosure provides methods and systems for detecting multiple different nucleotides in a sample. In particular, the disclosure provides for detection of multiple different nucleotides in a sample utilizing fewer detection moieties than the number of nucleotides being detected and/or fewer imaging events than the number of nucleotides being detected.
patents
