Methods of sequencing by expansion and related improvements to the sequencing of surrogate polymers in a nanopore are described. The surrogate polymer is formed from a template nucleic acid molecule. A surrogate polymer includes multiple units. Each unit includes a reporter code portion. The reporter codes correspond to the different nucleotides. surrogate polymers may get stuck in the nanopore. Embodiments described herein address these stuck surrogate polymers. In order to allow for multiple reads on the surrogate polymer, a processive consensus technique can be applied. The surrogate polymer may be moved a few units forward and then fewer units backward so that some of the same reporter codes are identified again. This method allows for multiple reads of the same reporter codes. The surrogate polymer eventually passes through the nanopore in the forward direction. Periodically, higher clearing voltages may be applied to clear any stuck surrogate polymer in the nanopore.
The present disclosure is directed to compositions and kits for PCR amplification. The present disclosure is also directed to methods of amplifying nucleic acid molecules to improve upon uniformity of coverage and/or to reduce GC bias during downstream sequencing operations.
C12Q 1/6806 - Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
C12Q 1/6848 - Nucleic acid amplification reactions characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
3.
VARIANT ALLELE ENRICHMENT BY UNIDIRECTIONAL DUAL PROBE PRIMER EXTENSION
The present disclosure provides a method for enrichment of at least one target nucleic acid in a library of nucleic acids. This present disclosure is also directed to a faster and easier method of target capture using primer extension reactions that can improve ease of use, turnaround time, and variant allele specificity by designing target enrichment primers to specifically enrich library fragments based on the relative location of the variant base(s) in the primer, the utilization of polymerases with better priming specificity, designing the variant bases in the capture primer, designing the variant bases in the release primer, and/or designing variant specific primers to the both the plus and minus strands of the target library fragment.
The invention includes improved methods and compositions for reduction of a C5-C6 double bond of a cytosine. In particular, the improved methods and compositions for reduction of a C5-C6 double bond of a cytosine is via enzymatic means, not via chemical means. In particular, the disclosure is directed to methods of converting 5,6-dihydro-fC (fC) and/or 5,6-dihydro-caC to 5,6-dihydro-U (DHU). In particular, the disclosure is directed to methods of converting 5fC and/or 5caC to DHU. In addition, the disclosure is directed to methods for detection of epigenetic cytosine modification, particularly cytosine methylation, using ene reductases to reduce the C5-C6 double bond of cytosine.
This application discloses electrochemical cells, nanopore devices, and associated buffer compositions useful for nanopore-based nucleic acid sequencing. Also disclosed are methods for using the electrochemical cells, devices, and compositions in nanopore-based nucleic acid sequencing methods, such as nanopore Sequencing-by-Expansion (Nano-SBX) and nanopore Sequencing-by-Synthesis (Nano-SBS) methods.
The present disclosure relates to a process for the regiochemically and enantiomerically controlled synthesis of phosphoramidite-containing monomers, and to intermediate products of this process. In some embodiments, the phosphoramidite-containing monomers or their precursors are regioisomerically and/or enantiomerically pure and may be polymerized into polymers or copolymers.
Described herein is a variant pol6 polymerase having improved thermostability. The disclosed polymerases each contain one or more substitutions relative to SEQ ID NO: 1 selected from the group consisting of G12W/Y/F, K114W/F/I, L117L/F/P, N194F/W/V, M232W/G/R, G313E/Q/L, A451F/W/Y, K490W/F/Y, Q565Y/I/V, Q590P/V/Y, and D681G/N/H. Additionally, the present specification discloses substitutions that can reduce stuttering in such polymerases, the substitutions selected from the group consisting of N298L, L538R, P542A, I570H/T/W/R/N/G, N574L, E633W/F, S636F, E639K, and K655G.
For high sequencing throughput, circuitry can compress read data generated in real-time by a sequencing device. Various compression techniques can be used. A stream of raw data can be processed to generate raw read data stream. The raw read data stream may include sub-streams of data comprising a header data sub-stream, a basecall sub-stream, and a quality score sub-stream. The sub-streams can be extracted and compressed using separate threads, and the compressed data can be recombined. Sequence reads corresponding to different copies of the same nucleic acid molecule may be clustered and used to generate a consensus read. The number of sequence reads that are used to generate the consensus read can be limited to a threshold when a consensus read is substantially accurate. After the limit is reached, data from any new raw read data corresponding to the same nucleic acid molecule may be discarded.
Described herein are alpha-hemolysin nanopores having relatively narrow channels and D127G and D128K substitutions relative to SEQ ID NO: 1. The narrow channel reduces the extent to which the nucleic acid template threads through the nanopore, while the D127G and D128K substitutions improve the lifetime and arrival rate of the narrow channel pores. Also disclosed herein are polypeptides for forming such nanopores, systems comprising such nanopores, and methods of making and using such nanopores.
C07K 14/31 - Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F) from Staphylococcus (G)
Epitachophoresis (ETP) methods and systems described herein allow for efficient and improved extraction of DNA and RNA molecules from a biological sample. The extraction may involve fragmenting nucleic acid molecules to smaller sizes and then running the fragmented sample through an ETP device. The fragmentation improves the extraction of nucleic acid molecules when using a gel with ETP. Fragmentation may also reduce extraction of undesired ribosomal RNA with gel ETP. Nucleic acid molecules are fragmented for preparing a library, and therefore the fragmentation of nucleic acid molecules before extraction rather than after extraction does not negatively impact library prep. In order to facilitate fragmentation, nucleic acid molecules may be treated so that the nucleic acid molecules are not protected from fragmentation.
Aspects provide a method of isolating RNA from a biological sample. The method may include adding the biological sample to a first electrolyte to form a first mixture. The method may include applying a voltage difference between a first electrode and a second electrode. A gel may include a portion of a second electrolyte. The method may include flowing, using the voltage difference, the first subset of RNA molecules into one or more focused zones within the second electrolyte to the second electrode. The method may include separating the second subset of RNA molecules from the first subset. The method may include collecting the first subset of RNA molecules by collecting a second mixture comprising the one or more focused zones. The concentration of the first subset in the second mixture is higher than the concentration of the first subset in the biological sample. Related systems are also described.
Disclosed herein are base-modified nucleoside-5 '-oligophosphates (bm-N5OP) that include a positively charged moiety at least at one position of the base, compositions comprising the same, compositions made from the same, methods of making the same, and methods of using the same. The bm-N50P disclosed herein are useful, for example, as tagged nucleotides for use in nanoSBS methods and for generating primers and/or templates for use in nanoSBS methods, When incorporated into a polynucleotide, the disclosed bm-N50Ps can neutralize at least a portion of the negative charge of the overall polynucleotide molecule.
C07H 19/10 - Pyrimidine radicals with the saccharide radical being esterified by phosphoric or polyphosphoric 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
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
B82Y 5/00 - Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
Nanopores having charged polymers linked thereto are provided. At least one end of the charged polymer is fixed to or near one end of the channel of the nanopore in a position that enables the charged polymer to enter into the channel. The charged polymer may optionally be fixed in the threaded configuration. When the charged polymer is in the threaded configuration, it enhances the conductivity of the nanopore while still permitting other polymers (such as nucleic acids or polymer tags of tagged nucleotides) to flow through the channel of the nanopore.
The invention includes improved methods and compositions for nucleic acid hybridization wherein the improvement comprises the use of enhancer oligonucleotides. Target enrichment is performed using probe oligonucleotides, wherein each probe oligonucleotide comprising a target-binding region, and a first and a second primer-binding region, and one or more enhancer oligonucleotides capable of hybridizing to at least one of the primer binding regions. The forward and reverse primer binding sites can be universal primer binding sites.
Epitachophoresis (ETP) methods and devices that improve concentrating samples and/or separating components of samples. ETP methods and devices allow for electromigration in two dimensions. Electromigration of a sample may first occur in a first dimension along a single plane. Electromigration may then continue in a second dimension, which may be different from the first dimension. The volume where the electromigration occurs may significantly reduce from the first dimension to the second dimension. This smaller dimension may allow for increased concentration of samples or improved separation of components of a sample.
The disclosure includes methods and compositions for accurately detecting subject's immune cell repertoire based on sequencing genomic DNA of immune cells.
C12Q 1/6853 - Nucleic acid amplification reactions using modified primers or templates
C12Q 1/6881 - Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
The present disclosure is directed to hybridization buffers, reaction mixtures, and master mixes suitable for enrichment of DNA oligonucleotides. In some embodiments, the hybridization buffers, reaction mixtures, and master mixes are free of formamide.
The present disclosure is directed to compositions, kits, and methods of and methods which facilitate the amplification of a unidirectional primer extension product. In particular, the compositions, kits, and methods described herein facilitate the amplification of a unidirectional primer extension product without the need to incorporate a second polymerase chain reaction primer binding target on a distal end of an initial single-stranded nucleic acid molecule primer extension product.
The invention relates to a device methods and an assembly for isolating biological polymers from a sample, the devic comprising a top reservoir, a bottom reservoir, a collection chamber located between the top and the bottom reservoirs and operably connected to the top and bottom reservoirs, a sieving matrix capable of passing the biological polymers to be extracted, a semipermeable membrane not capable of passing the biological polymers to be extracted, and at least one set of a working electrode and a counter electrode.
The invention related to forming nucleic acid templates including control templates for sequencing using a nanopore-based method, wherein the templates of the novel structure disclosed herein are limited or prevented from threading into the nanopore during sequencing.
The invention includes improved methods and compositions for detecting methylation in nucleic acids. In particular, the disclosure is directed to methods of converting 5-hydroxymethyl cytosine (5hmC) and/or 5-methylcytosine (5mC) to Thymine (T). In addition, the disclosure is also directed to methods of detecting 5hmC and/or 5mC in a sample.
The invention is a method of single cell transcriptome analysis. The method comprises detecting multiple transcripts in each individual cell of the plurality of cells by barcoding the transcripts with a cell-specific compound barcode formed using a DNA polymerase and a terminal transferase, optionally in a single enzyme such as a reverse transcriptase.
The present disclosure is directed to automated systems including an electrophoretic device including one or more separation conduits. In some embodiments, the automated systems are suitable for use in sample cleanup and/or target enrichment processes, such as sample cleanup and/or target enrichment processes conducted prior to sequencing, e.g., next generation sequencing.
Some embodiments relate to methods, systems, uses, or software for generating a consensus sequence of a particular molecule. A set of sequences of the particular molecule can be accessed, each having been generated independently from other sequences in the set of sequences and each including an ordered set of bases. An alignment process may be performed using the set of sequences to generate an alignment result associating, for each base of the ordered sets of bases of the sets of sequences. The base may have a reference position. For each reference position of a set of reference positions, a feature vector for the reference position may be generated that represents each base from the ordered sets of bases aligned to the reference position. The feature vectors for the set of references positions may be processed using a machine learning model to generate the consensus sequence for the particular molecule.
NUCLEOSIDE-5'-OLIGOPHOSPHATES TAGGED WITH POSITIVELY-CHARGED POLYMERS, NANOPORES INCORPORATING NEGATIVE CHARGES, AND METHODS AND SYSTEMS USING THE SAME
The present disclosure relates to tagged nucleoside-5 '-oligophosphates having a positively charged polymer tag structure and components thereof. Such nucleoside- 5 '-oligophosphates are useful, for example, in nanopore-based sequencing-by-synthesis applications. Also disclosed herein are nanopore constructs engineered to have additional negatively-charged moieties in the channel of the nanopore. Such nanopores can be useful, for example, for providing a repellant force against template and/or primer nucleic acids inserting into the pore during a nucleic sequence-by- synthesis process. The tagged nucleoside-5 '-oligophosphates and nanopores disclosed herein can be used together to provide nanopore-based nucleic acid sequencing-by-synthesis systems and processes having reduced background tag levels and improved throughput.
The present disclosure is directed to compositions, kits, and methods of target enrichment by unidirectional primer extension, whereby the compositions, kits, and methods utilize both poison primers and target capture primers.
C12Q 1/6848 - Nucleic acid amplification reactions characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
27.
SPATIAL ANALYSIS OF MULTIPLE TARGETS IN TISSUE SAMPLES
The invention provides methods and compositions for analysis of single cell in tissue sample allowing for simultaneous detection and localization of multiple targets in the cells.
Described herein are systems and methods for dividing a population of particles into two or more subpopulations, reacting each formed subpopulation of particles with a different reagent, pooling the reacted subpopulations of particles back together.
Disclosed herein are devices, systems, and methods for monitoring single-molecule biological processes using magnetic sensors and magnetic particles (MNP). A MNP is attached to a biopolymer (e.g., a nucleic acid, protein, etc.), and motion of the MNP is detected and/or monitored using a magnetic sensor. Because the MNP is small (e.g., its size is comparable to the size of the molecule being monitored) and is tethered to a biopolymer, changes in the volume of Brownian motion of the MNP in a solution can be monitored to monitor the movement of the MNP and, by inference, the tethered biopolymer. The magnetic sensor is small (e.g., nanoscale or having a size on the order of the sizes of the MNP and the biopolymer) and can be used to detect even small changes in the position of the MNP within the sensing region of the magnetic sensor.
G01N 27/74 - Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating magnetic variables of fluids
The invention provides a method and compositions for single cell analysis of cells present in multiple samples assisted by sample pooling and sample barcoding.
Systems and methods for inserting a single pore (316) into a membrane (314) under faradaic conditions are described herein. A stepped or ramped voltage waveform can be applied across the membranes of the cells of an array, where the voltage waveform starts at first voltage and increases in magnitude over a period of time to a second voltage. The voltage waveform has a polarity that maintains a first species of a redox couple (308) in its current oxidation state. The first voltage is selected to be low enough to reduce the risk of damaging the membrane, while the rate of voltage increase is selected to provide sufficient time for the pores to insert into the membranes. Once a pore is inserted into the membrane, the voltage across the membrane rapidly drops, thereby reducing the risk of damaging the membrane even if the applied voltage between the electrodes is further increased.
Disclosed are compositions, kits, and methods for detecting gene fusions involving an unknown fusion partner using locked nucleic acid primers. In some embodiments, the compositions include a compound including at least two nucleotide sequences which are joined, directly or indirectly, through a 5' to 5' linkage. In some embodiments, the compound further includes a spacer moiety and/or a cleavage moiety.
C12Q 1/6883 - Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
33.
NANOPORE CELL WITH SEAMLESS WORKING ELECTRODE AND METHODS OF FORMING THE SAME
A nanopore cell may include a well having a seamless porous electrode and hydrophobic sidewalls. The seamless porous electrode may be formed by depositing porous electrode material on a planar electrode support layer formed by a conductive layer island and a dielectric layer. The porous electrode material may form uniform seamless columns and may be protected during manufacturing by depositing a selectably removable protective layer thereon. The well may be formed by forming and then patterning hydrophobic cladding over the protective layer. The protective layer may be removed to expose the seamless porous electrode at the bottom of the well.
Described herein is a novel alignment method which leverages multi-stage secondary analysis, with each stage progressively reducing the amount of data to be analyzed in the next stage(s), but increasing exhaustiveness of the search on the remaining data received from previous stage(s). This way, less noisy alignments can be quickly identified from the initially large data-pools in early stage(s), while very noisy alignments can be identified equally fast from smaller data-pools in latter stage(s) of computation, thus maintaining target sensitivity while reducing overall compute times.
The invention provides methods and compositions for removal of undesired or excess oligonucleotides from reaction mixtures using a double hairpin nucleic acid comprising a single nucleic acid strand having: i. a first hairpin at the 5'-end; ii. a second hairpin at the 3'- end; and iii. a single-stranded region between the 5'-end and the 3'-end, wherein the single-stranded region comprises a sequence capable of hybridizing to the oligonucleotide to be removed, e.g, excess primers, subcodes or adaptor molecules.
The present disclosure relates to a method for detection reaction volume deviations in a digital polymerase chain reaction (dPCR) and to a method for determining the amount or concentration of a nucleic acid of interest in a sample with dPCR. The method uses a convolution with kernel function to analyse the optical image, wherein each partition is assigned a convolution value which is compared to a threshold convolution value.
A nanopore-based sequencing chip can have a surface with an array of wells, with each well having a working electrode. Charge can be established within the wells by applying a voltage between the working electrodes and a counter electrode. The charge can then be trapped within the wells by sealing the wells with a membrane. The trapped charge can be used to facilitate pore insertion into the membranes.
Disclosed herein are embodiments of single-molecule array sequencing (SMAS) devices and systems. Each sensor of an array of sensors of the SMAS device is capable of detecting labels attached to nucleotides incorporated into a single nucleic acid strand bound to a respective binding site. Each sensor can detect a single label (e.g., fluorescent, magnetic, organometallic, charged molecule, etc.) attached to the incorporated nucleotide. Also disclosed are methods of using SMAS devices and systems for highly- scalable nucleic acid (e.g., DNA) sequencing based on sequencing by synthesis (SBS) of multiple instances of clonally amplified DNA immobilized on such SMAS devices. Also disclosed are error correction methods that mitigate errors (e.g., errant label detections or non-detections) made in sequencing individual nucleic acid strands.
The present disclosure generally relates to devices and methods for effecting epitachophoresis in order to isolate/purify analytes from urine samples or other samples comprising high salt concentrations, e.g., sodium or potassium salts. Epitachophoresis may be used to effect sample analysis, such as by selective separation, detection, extraction, and/or pre-concentration of target analytes such as, for example, DNA, RNA, and/or other biological molecules. Said target analytes may be collected following epitachophoresis and used for desired downstream applications and further analysis.
Disclosed are methods and compositions for detecting structural rearrangements in a genome using rearrangement-specific enrichment probes or rearrangement- specific amplification primers.
C12Q 1/6883 - Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
The present disclosure generally relates to devices and methods for effecting epitachophoresis. Epitachophoresis may be used to effect sample analysis, such as by selective separation, detection, extraction, and/or pre-concentration of target analytes such as, for example, DNA, RNA, and/or other biological molecules. Said target analytes may be collected following epitachophoresis and used for desired downstream applications and further analysis.
Disclosed herein are apparatuses for nucleic acid sequencing using magnetic labels (e.g., magnetic particles) and magnetic sensors. Also disclosed are methods of making and using such apparatuses. An apparatus for nucleic acid sequencing comprises a plurality of magnetic sensors, a plurality of binding areas disposed above the plurality of magnetic sensors, each of the binding areas for holding fluid, and at least one line for detecting a characteristic of at least a first magnetic sensor of the plurality of magnetic sensors, the characteristic indicating presence or absence of one or more magnetic nanoparticles coupled to a first binding area associated with the first magnetic sensor.
Disclosed is a novel structure of a nucleic acid template and the method of making and using the structure. The structure consists of a double-stranded circle with a single-stranded gap. The circular gapped structure includes an extendable end from which copying or sequencing can be initiated.
This application discloses compositions comprising primer compounds that reduce or block deleterious threading into a nanopore of nucleic acid strands displaced by a nanopore-linked polymerase, for example during the use of a nanopore device for nucleic acid sequencing. Also disclosed are methods for using the compositions to reduce deleterious threading events during nanopore-based nucleic acid detection techniques, such as nanopore sequencing.
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
The present disclosure is directed to automated systems including a microfluidic chip having one or more independently operable processing conduits. In some embodiments, the automated systems are suitable for use in sample cleanup and/or target enrichment processes, such as sample cleanup and/or target enrichment processes conducted prior to sequencing.
Described herein are systems and methods for randomly dividing a population of particles into two or more sub-populations, reacting each formed sub-population of particles with a different reagent, pooling the reacted sub-populations of particles back together.
The present disclosure provides a method for sequencing target polynucleotide molecules. In some embodiments, the present disclosure provides a method of sequencing by synthesis where different subsets of nucleotide-conjugate complexes are sequentially formed and detected during each iterative extension of a plurality of nascent nucleic acid copy strands, where each nascent nucleic acid copy strand is complementary to one of a plurality of target polynucleotide molecules. In some embodiments, the plurality of target polynucleotide molecules are arrayed on a solid support.
C07H 19/00 - Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro derivatives thereof
C12Q 1/6837 - Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips
C12Q 1/6874 - Methods for sequencing involving nucleic acid arrays, e.g. sequencing by hybridisation [SBH]
50.
SYSTEMS AND METHODS FOR CONTAMINATION DETECTION IN NEXT GENERATION SEQUENCING SAMPLES
Here we describe a statistical approach based on beta mixture modelling to detect contamination and report contamination levels as both point estimates and confidence intervals in liquid biopsy samples. We validate our method with both in silico simulation and in vitro contamination spiked samples. Although we focus on liquid biopsy samples, the same strategy is applicable to any generic NGS application with minor modifications. For example, tissue samples from a biopsy can be used according to the systems and methods described herein.
A consumable device used in a nanopore based sequencing system can include a nanopore chip, a flow cell with one or more flow channels, and a flow cell cover. A fluidic interface can be used to deliver fluid to the flow cell. The fluid interface can include a flow cell boss and the flow cell cover can include a receptacle for receiving the flow cell boss. A dispense tip can be used to introduce fluid into the flow cell through the flow cell boss.
The invention is a method of assessing a mammalian immune repertoire by primer extension target enrichment (PETE). Methods and compositions for assessing an immune repertoire and the status of additional genetic markers are disclosed.
The present invention is a method and compositions for forming a library for nucleic acids sequencing simultaneously from DNA and RNA present in a sample.
The present invention relates generally to classification of biological samples, and more specifically to cell of original classification. In particular, some embodiments of the invention relate to diffuse large B cell lymphoma cell of origin classification using machine learning models. The machine learning models can be based on decision trees such as a random forest algorithm or a gradient boosted decision tree. Features for the models can be determined through analysis of variant data from plasma or blood samples from a plurality of subjects with the disease.
C12Q 1/6886 - Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
G16B 20/20 - Allele or variant detection, e.g. single nucleotide polymorphism [SNP] detection
The present technology provides an approach to designing libraries of peptide sequences for discovery and testing of significantly more motifs than would be otherwise available in a given fixed library format. The technology includes a plurality of x-mers embedded in N-mer peptides sequences, where N and x are integers and where N is greater than x. This approach provides for the representation of multiple unique x-mer peptides in a single N-mer peptide feature.
The present disclosure provides, in some embodiments, a computing device comprising an improved user interface. In some embodiments, the improved user interface enables the visualization of clinically relevant information pertaining to interacting gene variants, including therapeutic recommendations and longitudinal data visualization. In some embodiments, the improved user interface facilitates the contemporaneous visualization of clinically relevant information pertaining to individual gene variants and the visualization of clinically relevant information pertaining to an interaction between gene variants, including therapeutic recommendations, over time. In some embodiments, the visualization(s), through the improved user interface, facilitates the rapid interpretation of clinically relevant information by a medical professional such that decisions regarding patient care may be made accurately and efficiently.
The present disclosure generally relates to devices and methods for effecting epitachophoresis. Epitachophoresis may be used to effect sample analysis, such as by selective separation, detection, extraction, and/or pre-concentration of target analytes such as, for example, DNA, RNA, and/or other biological molecules. Said target analytes may be collected following epitachophoresis and used for desired downstream applications and further analysis.
C12N 15/10 - Processes for the isolation, preparation or purification of DNA or RNA
C12Q 1/6827 - Hybridisation assays for detection of mutation or polymorphism
C12Q 1/6883 - Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
58.
SYSTEMS AND METHODS FOR INSERTING A NANOPORE IN A MEMBRANE USING OSMOTIC IMBALANCE
Systems and methods for inserting a nanopore into a membrane covering a well are described herein. The membrane can be bowed outwards by establishing an osmotic gradient across the membrane in order to drive fluid into the well, which will increase the amount of fluid in the well and cause the membrane to bow outwards. Nanopore insertion can then be initiated on the bowed membrane.
Disclosed herein are apparatuses for nucleic acid sequencing, and methods of making and using such apparatuses. In some embodiments, the apparatus comprises a magnetic sensor array comprising a plurality of magnetic sensors, each of the plurality of magnetic sensors coupled to at least one address line, and a fluid chamber adjacent to the magnetic sensor array, the fluid chamber having a proximal wall adjacent to the magnetic sensor array. In some embodiments, a method of sequencing nucleic acid using the apparatus comprises (a) coupling a plurality of molecules of a nucleic acid polymerase to the proximal wall of the fluid chamber; (b) in one or more rounds of addition, adding, to the fluid chamber, (i) a nucleic acid template comprising a primer binding site and an extendable primer, and (ii) a first magnetically-labeled nucleotide precursor comprising a first cleavable magnetic label, a second magnetically-labeled nucleotide comprising a second cleavable magnetic label, a third magnetically-labeled nucleotide comprising a third cleavable magnetic label, and a fourth magnetically-labeled nucleotide comprising a fourth cleavable magnetic label; and (c) sequencing the nucleic acid template, wherein sequencing the nucleic acid template comprises, using the at least one address line, detecting a characteristic of at least a portion of the magnetic sensors in the magnetic sensor array, wherein the characteristic indicates which of the first, second, third, or fourth magnetically-labeled nucleotide precursors has been incorporated into the extendable primer. In some embodiments, a method of sequencing nucleic acid using the apparatus comprises (a) binding a nucleic acid strand to the proximal wall; (b) in one or more rounds of addition, adding, to the fluid chamber, (i) an extendable primer, and (ii) a plurality of molecules of a nucleic acid polymerase; (c) adding, to the fluid chamber, a first magnetically-labeled nucleotide precursor comprising a first cleavable magnetic label; and (d) sequencing the nucleic acid template, wherein sequencing the nucleic acid template comprises, using the at least one address line, detecting a characteristic of at least a first portion of the magnetic sensors in the magnetic sensor array, wherein the characteristic indicates that the first magnetically-labeled nucleotide precursor has bound to at least one molecule of the plurality of molecules of the nucleic acid polymerase or has been incorporated into the extendable primer. In some embodiments, a method of manufacturing a nucleic acid sequencing device having at least one fluid chamber configured to contain fluid comprises fabricating a first addressing line on a substrate, fabricating a plurality of magnetic sensors, each magnetic sensor having a bottom portion and a top portion, wherein each bottom portion is coupled to the first addressing line, depositing a dielectric material between the magnetic sensors, fabricating a plurality of additional addressing lines, each of the plurality of additional addressing lines coupled to the top portion of a respective magnetic sensor of the plurality of magnetic sensors, and removing a portion of the dielectric material adjacent to the plurality of magnetic sensors to create the at least one fluid chamber.
The invention is an improved method of target enrichment and target depletion where probe binding is facilitated by the FANCA protein. Improved workflows for next regeneration sequencing and related methods involving nucleic acid probe hybridization are improved by the use of FANCA protein.
C12Q 1/6832 - Enhancement of hybridisation reaction
C12Q 1/6848 - Nucleic acid amplification reactions characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
61.
TUMOR CLASSIFICATION BASED ON PREDICTED TUMOR MUTATIONAL BURDEN
The present disclosure provides systems and methods of classifying and/or identifying a cancer subtype. The present disclosure also provides methods of enhancing the prediction of a tumor mutational burden by using both synonymous and non-synonymous somatic mutations in the computation method. It is believed that by increasing the number of mutations in the computation of the tumor mutational burden, a comparatively more consistent tumor mutational burden may be derived, especially for targeted-panel sequencing. It is believed that the consistent computation of the tumor mutational burden from targeted panels allows for computationally quicker and less costly analysis of sequencing data as compared with a tumor mutational burden computed from whole exome sequencing data.
Techniques for identification of global cancer-specific sequence features in whole genome sequence data obtained from cell-free DNA (cfDNA) samples. An exemplary technique includes obtaining a plurality of whole genome sequencing reads from a cfDNA sample and determining two or more metrics from at least a majority of the plurality of genome sequencing reads, where a first metric of the two or more metrics is: (i) a fragment size of the cell free DNA, (ii) relative read depth of the plurality of whole genome sequencing reads, or (iii) germline allelic imbalance. The technique further includes inputting the two or more metrics into a classifier to obtain a first prediction for a first class and a second prediction for a second class, and classifying the sample of cell free DNA as the first class or the second class based on the first prediction and the second prediction.
C12Q 1/6886 - Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
G16B 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
The present disclosure provides 3' protected nucleotides, including those 3' protected nucleotides having a detectable tag. Systems and methods of sequencing nucleic acids using the 3' protected nucleotides are also disclosed, such as the sequencing of a nucleic acid using a nanopore or the sequencing of a nucleic acid via sequencing-by-synthesis.
C07H 19/02 - Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro derivatives thereof sharing nitrogen
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
Systems and methods for inserting a single pore into a membrane are described herein. A stepped or ramped voltage waveform can be applied across the membranes of the cells of an array, where the voltage waveform starts at first voltage and increases in magnitude over a period of time to a second voltage. The first voltage is selected to be low enough to reduce the risk of damaging the membrane, while the rate of voltage increase is selected to provide sufficient time for the pores to insert into the membranes. Once a pore is inserted into the membrane, the voltage across the membrane rapidly drops, thereby reducing the risk of damaging the membrane even if the applied voltage between the electrodes is further increased.
Techniques for cancer patient management, and more particularly, to techniques for ultrasensitive detection of circulating nucleic acid with prior knowledge of variants to be monitored in the blood. An exemplary technique includes detecting one or more variants in a sample of cell free DNA from a subject. The one or more variants are selected from a plurality of variants known to be specific to a tumor or disease area of the subject. The technique further includes counting the detected one or more variants, determining a tumor burden based on the count of the one or more variants, and determining a statistical significance of the tumor burden based on whether the detection of the one or more variants is associate with true signals or background noise.
G16B 20/20 - Allele or variant detection, e.g. single nucleotide polymorphism [SNP] detection
C12Q 1/6886 - Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
66.
SPATIALLY ORIENTED QUANTUM BARCODING OF CELLULAR TARGETS
The invention is a method of simultaneously detecting the presence and spatial location of a target in a tissue sample by attaching an anchor to the target and assembling unique positional barcodes on the anchor. The method enables analyzing cellular targets in 3D.
The present disclosure provides for compositions and methods for identifying peptide classifiers. In one embodiment, a peptide classifier for diagnosing rheumatoid arthritis includes a composition comprising a plurality of molecules. Each molecule comprises a peptide having a sequence selected from SEQ ID NOS: 1-8861, wherein the plurality of molecules defines a classifier for rheumatoid arthritis.
Molecules may be analyzed (e.g., sequencing of nucleic acid molecules) by tunneling recognition at a tunneling junction. Embodiments of the present invention may allow detecting individual nucleotides and the sequencing of a nucleic acid molecule using a tunneling junction. By labeling a specific 5 nucleotide with a moiety, tunneling junctions may generate a signal with a suitable signal-to-noise ratio. An electric field may be applied to move the nucleic acid molecule and the moiety close to the tunneling junction so that a current may travel through the moiety. Because a single nucleotide can be detected with a signal with a suitable signal-to-noise ratio resulting from the tunneling current passing through 10 the moiety, embodiments of the present invention may allow for fast detection of nucleotides using a tunneling current.
The present disclosure generally relates to systems comprising devices for effecting epitachophoresis and sample detection and methods of using such systems. Epitachophoresis may be used to effect sample analysis, such as by selective separation, detection, extraction, and/or pre-concentration of target analytes such as, for example, DNA, RNA, and/or other biological molecules. Sample detection may be used to trigger automated target analyte collection. Said target analytes may be used for desired downstream applications and further analysis.
The present disclosure provides a method of preparing a library of nucleic acids having modular end sequences. The method includes combining a pool of different modular nucleic acid tags with a nucleic acid sample, the nucleic acid sample including a plurality of double-stranded target nucleic acids. The method further includes joining the ends of each of the double-stranded target nucleic acids to tags selected from the pool of different modular nucleic acid tags to form a plurality of doubly-tagged target nucleic acids, amplifying each of the doubly-tagged target nucleic acids, thereby preparing a library of nucleic acids having modular end sequences, and detecting the library of amplified nucleic acids having modular end sequences.
The present disclosure provides systems and methods that utilize neural networks such as convolutional neural networks to analyze genomic sequence data generated by a sequencer and generate accurate prediction data identifying and describing germline and/or somatic variants within the sequence data.
The present invention provides a new building block for peptide synthesis, which introduces a cleavage site that can be used to generate cleavable fragments subsequent to a peptide sequence.
C07C 271/22 - Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms to carbon atoms of hydrocarbon radicals substituted by carboxyl groups
C07K 7/06 - Linear peptides containing only normal peptide links having 5 to 11 amino acids
C07K 7/08 - Linear peptides containing only normal peptide links having 12 to 20 amino acids
C07K 14/00 - Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
C07K 1/04 - General processes for the preparation of peptides on carriers
73.
FORMAMIDE FREE TARGET ENRICHMENT COMPOSITIONS FOR NEXT-GENERATION SEQUENCING APPLICATIONS
The invention is a method of determining a likelihood that a cancer patient will respond to immunotherapy based on a mutation metric obtained by sequencing a small panel of nucleic acid targets in patient's cell-free DNA.
C12Q 1/6886 - Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
75.
MULTIPLEXING ANALOG COMPONENT IN BIOCHEMICAL SENSOR ARRAYS
Techniques for increasing the density and the number of cells on a nanopore sensor chip are disclosed. Two or more cells of the nanopore sensor chip share some analog components (e.g., an integration capacitor and/or a read-out transistor) through one or more digital relays. Under the control of various control signals during a sampling period of the sensor chip, the two or more cells are connected one at time to the shared analog components and are measured one at a time using the shared analog components. In this way, the average size of the cells on the sensor chip is reduced to increase the cell density without affecting the analog measurement performance of the cells.
The present disclosure provides, in some embodiments, a computing device comprising an improved user interface. In some embodiments, the improved user interface enables the visualization of clinically relevant information pertaining to interacting gene variants, including therapeutic recommendations. In some embodiments, the improved user interface facilitates the contemporaneous visualization of clinically relevant information pertaining to individual gene variants and the visualization of clinically relevant information pertaining to an interaction between gene variants, including therapeutic recommendations. In some embodiments, the visualization(s), through the improved user interface, facilitates the rapid interpretation of clinically relevant information by a medical professional such that decisions regarding patient care may be made accurately and efficiently.
Methods and systems for detecting MSI are provided. Also provided are methods for enriching human genomic DNA for microsatellite loci. Additional, an oligonucleotide array for detecting MSI is provided.
Molecules may be analyzed (e.g., sequencing of nucleic acid molecules) by tunneling recognition at a tunneling junction. Embodiments of the present invention may allow detecting individual nucleotides and the sequencing of a nucleic acid molecule using a tunneling junction. By labeling a specific nucleotide with a moiety, tunneling junctions may generate a signal with a suitable signal-to-noise ratio. The tunneling recognition uses a tunneling current that is mostly through the moiety rather than mostly through the nucleotide or a portion of the molecule of interest. Because a single nucleotide can be detected with a signal with a suitable signal-to-noise ratio resulting from the tunneling current passing through the moiety, embodiments of the present invention may allow for fast detection of nucleotides using a tunneling current.
The invention is a method of identifying a cognate antigen for a T-cell receptor using neoantigens from a patient's tumor cells combined with the patient's T-cells and using cell sorting, genome sequencing, expressing TCR genes, presenting tumor neoantigens on MHC complex and uniquely barcoding the T-cells where TCR recognition occurs to tag all components of the TCR recognition complex.
G01N 33/50 - Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
G01N 33/569 - Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
C12Q 1/6806 - Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
C12Q 1/6881 - Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
C12Q 1/6883 - Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
C12N 15/10 - Processes for the isolation, preparation or purification of DNA or RNA
80.
ENZYMATIC ENRICHMENT OF DNA-PORE-POLYMERASE COMPLEXES
The present invention provides a method for isolating Sequencing complexes, said method comprising forming a complex between a nanopore covalently linked to a polymerase and an oligonucleotide that is associated with a purification moiety, separating any unbound/uncomplexed nanopores and oligonucleotides from the complexes by use of a solid support capable of binding the purification moiety, and cleaving bound complexes from the solid support with an enzyme composition.
The present invention provides method essentially comprising the steps of (i) providing a DNA sample, (ii) providing at least a first pair of amplification primers which is capable of generating a first amplicon from a LINE sequence that has a size of less than 80 bp, (iii) providing at least a second pair of amplification primers which is capable of generating a second amplicon from a LINE sequence that has a size of more than 160 bp, (iv) performing a qPCR and determining cq values for each of the generated amplicons, and (v) determining the relative concentrations of said amplicons.
The invention is a method of predicting response to therapy in a colorectal cancer patient, the method comprising measuring tumor genetic heterogeneity via analysis of circulating tumor DNA from a patient's sample.
C12Q 1/6886 - Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
83.
SURROGATE MARKER AND METHOD FOR TUMOR MUTATION BURDEN MEASUREMENT
The invention is a method of predicting recurrence of lung cancer in a patient following surgery, the method comprising analysis of circulating tumor DNA from a patient's sample.
C12Q 1/6886 - Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
84.
FABRICATION OF TUNNELING JUNCTIONS WITH NANOPORES FOR MOLECULAR RECOGNITION
Embodiments of the present technology may allow for improved and more reliable tunneling junctions and methods of fabricating the tunneling junctions. Electrical shorting issues may be reduced by depositing electrodes without a sharp sidewall and comer but instead with a sloping or curved sidewall. Layers deposited on top of the electrode layer may then be able to adequately cover the underlying electrode layer and therefore reduce or prevent shorting. Additionally, two insulating materials may be used as the dielectric layer may reduce the possibility of incomplete coverage and the possibility of flaking. Furthermore, the electrodes may be tapered from the contact area to the junction area to provide a thin electrode where the hole is to be patterned, while the thicker contact area reduces sheet resistance. The electrode may also be patterned to be wider at the contact area and narrower at the junction area.
The disclosure relates to tagged nucleoside compounds comprising a nucleotide polyphosphate covalently attached to a tag, wherein the compound is a polymerase substrate and the polymer moiety is capable of entering a nanopore linked to the polymerase and thereby altering the flow of ions through the nanopore. The disclosure also provides methods for preparing the tagged nucleoside compounds and for their use as tags in nanopore-based nucleic acid detection and sequencing.
C07H 19/00 - Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro derivatives thereof
Described herein are variants of alpha-hemolysin having at least one mutation, such as a mutation to a positive charge. In certain examples, the mutation is selected from OK, A1 K, A1 R, D2N, S3K, D4K, D4N, K8R, N47K, E70K, S106K, E1 1 1 N, 127-131 G, D128K, K147N, V149K, E287R, M298A, or combinations thereof in the mature, wild-type alpha-hemolysin amino acid sequence. Also provided are compositions including the variants of alpha-hemolysin, nanopore assemblies including the alpha-hemolysin variants, and methods of sequencing nucleic acids incorporating the same.
C07K 14/31 - Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F) from Staphylococcus (G)
The invention is a novel method of separately sequencing each strand of a nucleic acid involving the use of an adaptor comprising a strand cleavage site or a strand synthesis termination site. The adaptor may also be self-priming at the strand cleavage site.
The invention is a novel method of sequencing nucleic acids involving making and sequencing a library of double stranded target nucleic acids containing a linear partially single-stranded adaptor.
The invention is a method of predicting response to therapy in a cancer patient by serial sampling the patient's cell-free tumor nucleic acids to determine a change in the number of mutations per amount of plasma.
C12Q 1/6886 - Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
90.
METHOD OF PREDICTING RESPONSE TO THERAPY BY ASSESSING TUMOR GENETIC HETEROGENEITY
The invention is a method of predicting response to therapy in a colorectal cancer patient, the method comprising measuring tumor genetic heterogeneity via analysis of circulating tumor DNA from a patient's sample.
C12Q 1/6886 - Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
91.
GENERATION OF SINGLE-STRANDED CIRCULAR DNA TEMPLATES FOR SINGLE MOLECULE
The invention is a novel method of sequencing nucleic acids involving making and sequencing a library of single stranded circular target nucleic acids.
A method of using a sequencing cell (200, 300) includes applying (1310) an alternating signal (228, 328) across a nanopore (216, 316) of the sequencing cell. The method further includes acquiring (1320) a first set of voltage data during a first portion (520) of a plurality of cycles of the alternating signal. The method further includes determining (1330) a time shifted set of voltage data from the first set of voltage data, computing (1340) difference data values by computing differences (730) between data points of the first set of voltage data (710) and corresponding data points of the time shifted set of voltage data (720), identifying (1350) a plurality of noise data points (1401, 1403, 1405) as data points having difference data values that are larger than a first threshold value, and removing (1360) the plurality of noise data points from the first set of voltage data.
The present disclosure provides a method for enrichment of at least one target nucleic acid in a library of nucleic acids. A first oligonucleotide is hybridized to a target nucleic acid in library of nucleic acids having first and second adapters. The 5 hybridized first oligonucleotide is extended with a first polymerase, thereby producing a first primer extension complex including the target nucleic acid and the extended first oligonucleotide. The first primer extension complex is captured, enriched relative to the library of nucleic acids, and a second oligonucleotide is hybridized to the target nucleic acid. The hybridized second oligonucleotide is 10 extended with a second polymerase, thereby producing a second primer extension complex including the target nucleic acid and the extended second oligonucleotide, and further liberating the extended first oligonucleotide from the first primer extension complex.
This disclosure provides chips, systems and methods for sequencing a nucleic acid sample. Tagged nucleotides are provided into a reaction chamber comprising a nanopore in a membrane. An individual tagged nucleotide of the tagged nucleotides can contain a tag coupled to a nucleotide, which tag is detectable with the aid of the nanopore. Next, an individual tagged nucleotide of the tagged nucleotides can be incorporated into a growing strand complementary to a single stranded nucleic acid molecule derived from the nucleic acid sample. With the aid of the nanopore, a tag associated with the individual tagged nucleotide can be detected upon incorporation of the individual tagged nucleotide. The tag can be detected with the aid of the nanopore when the tag is released from the nucleotide.
A method of using a sequencing cell (300) includes applying an alternating voltage (328) across the sequencing cell while sequencing a nucleic acid (332), acquiring a plurality of signal values (P(ti)) from the sequencing cell while a tag molecule (338) is threaded in a nanopore (316) of the sequencing cell, and acquiring a plurality of correlated signal values (X(ti)) that are correlated with respective values of the plurality of acquired signal values ( P(ti )) thereby forming a plurality of two-dimensional data points (1201, 1203, 1205, 1207, 1209, 1303, 1305, 1307, 1309, 1311). The two-dimensional data points comprise a first dimension for the acquired signal values and a second dimension for the correlated signal values. The method further includes computing a plurality of transformed signal values by applying a two-dimensional transformation to the two-dimensional data points.
The present disclosure generally relates to devices for effecting epitachophoresis. Epitachophoresis may be used to effect sample analysis, such as by selective separation, detection, extraction, and/ or pre-concentration of target analytes such as, for example, DNA, RNA, and/or other biological molecules. Said target analytes may be collected following epitachophoresis and used for desired downstream applications and further analysis.
The invention is a novel method of sequencing nucleic acids involving the generation of a library of linear concatenated target nucleic acids, comprising the steps of: (i) ligating at least one end of a double stranded target nucleic acid to a first adaptor to form an adapted target nucleic acid; (ii) separating the strands of the adapted target nucleic acid to form a single stranded adapted target nucleic acid; (iii) circularizing the single stranded adapted target nucleic acid to form a single stranded circle comprising at least one first adaptor sequence; (iv) annealing a primer to the single stranded circle; (v) extending the primer with a DNA polymerase to generate a nucleic acid strand comprising multiple copies of the target nucleic acid; (vi) generating a copy strand of the nucleic acid strand from step e) forming a concatemer comprising multiple copies of the target nucleic acid; and (vii) ligating second adaptor to the concatemer wherein one strand of the second adaptor comprises a primer binding site thereby forming a concatenated nucleic acid template for analysis.
Techniques for replacing nanopores within a nanopore based sequencing chip are provided. A first electrolyte solution is added to the external reservoir of the sequencing chip, introducing an osmotic imbalance between the reservoir and the well chamber located on the opposite side of a lipid bilayer membrane. The osmotic imbalance causes the membrane to change shape, and a nanopore within the membrane to be ejected. A second electrolyte solution is then added to the external reservoir to provide replacement nanopores and to restore the membrane shape. The replacement nanopores can be inserted into the membrane, effectively replacing the initial pore without causing the destruction of the membrane.
The invention is a novel method of making and using a library such as a sequencing library of single stranded circular nucleic acid templates via splint ligation.
A method of detecting a state of a lipid membrane in a cell of a nanopore based sequencing chip is disclosed. A lipid membrane is coupled with an integrating capacitor, wherein the lipid membrane is between a working electrode and a counter electrode. An alternating current (AC) voltage is applied to the counter electrode. A voltage across the integrating capacitor is periodically sampled by an analog-to-digital converter (ADC). An intermediate change in the AC voltage is inserted between two magnitudes of the AC voltage. A change in the sampled voltage across the integrating capacitor in response to the intermediate change in the AC voltage is determined. A state of the lipid membrane is determined based on the determined change in the sampled voltage across the integrating capacitor in response to the intermediate change in the AC voltage.
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