The present disclosure in some aspects relates to methods and compositions for accurately detecting and quantifying multiple analytes present in a biological sample. In some aspects, the methods and compositions provided herein address issues associated with the heterogeneity of analyte abundance (e.g., gene expression levels) and variations among reactions at different locations of a sample (e.g., amplification reaction starting earlier at one location than another location). In some aspects, a method disclosed herein provides a tighter distribution of signal spot size and intensity in a sample, as compared to methods that result in a wide and heterogeneous size and intensity distribution of signal spots.
Provided herein is an integrated assay of a biological sample comprising an in situ assay module and a spatial assay module. The in situ assay comprises analyzing binding between nucleic acid probes and a first analyte at a spatial location of the biological sample. The method further comprises providing conditions to allow spatially barcoded capture agents to capture a second analyte for analysis in the spatial assay module.
Systems and methods for spatial analysis of analytes include placing a sample on a substrate having fiducial markers and capture spots. Then, an image of the sample is acquired and sequence reads are obtained from the capture spots. Each capture probe plurality in a set of capture probe pluralities is (i) at a different capture spot, (ii) directly or indirectly associates with analytes from the sample and (iii) has a unique spatial barcode. The sequencing reads serve to detect the analytes. Sequencing reads include a spatial barcode of the corresponding capture probe plurality. Spatial barcodes localize reads to corresponding capture spots, thereby dividing them into subsets, each subset for a respective capture spot. Fiducial markers facilitate a composite representation comprising (i) the image aligned to the capture spots and (ii) a representation of each subset of sequence reads at respective positions within the image mapping to the corresponding capture spots.
Systems and methods for spatial analysis of analytes are provided. A data structure is obtained comprising an image, as an array of pixel values, of a sample on a substrate having an identifier, fiducial markers and a set of capture spots. The pixel values are used to identify derived fiducial spots. The substrate identifier identifies a template having reference positions for reference fiducial spots and a corresponding coordinate system. The derived fiducial spots are aligned with the reference fiducial spots using an alignment algorithm to obtain a transformation between the derived and reference fiducial spots. The transformation and the template corresponding coordinate system are used to register the image to the set of capture spots. The registered image is then analyzed in conjunction with spatial analyte data associated with each capture spot, thereby performing spatial analysis of analytes.
Systems and methods for tissue classification are provided. An image of tissue on a substrate is obtained as a plurality of pixels. Fiducial markers are on the substrate boundary. Pixels are assigned to a first class, indicating tissue sample, or a second class, indicating background. The assigning uses the fiducial markers to define a bounding box within the image and disregards pixels outside the box. Then, heuristic classifiers are applied to the pixels: for each respective pixel in the plurality of pixels, each heuristic classifier votes for the respective pixel between the first and second class, thereby forming an aggregated score for each pixel that in one of first class, likely first class, likely second class, and obvious second class. The aggregated score and intensity of each pixel is applied to a segmentation algorithm to assign a probability to each pixel of being tissue sample or background.
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 10/26 - Segmentation of patterns in the image field; Cutting or merging of image elements to establish the pattern region, e.g. clustering-based techniques; Detection of occlusion
G06V 20/69 - Microscopic objects, e.g. biological cells or cellular parts
The present application provides methods for detecting a nucleic acid molecule involving the use of a signal code sequence which corresponds to said nucleic acid molecule and a plurality of labelled detection probes which yield signals which make up the signal code sequence. In particular, the invention provides a sequential barcoding and decoding scheme which utilises a sequencing-by-hybridisation (SBH) strategy to sequence and decode a nucleotide barcode sequence, and to differentiate the nucleotide barcode sequence from other nucleotide barcode sequences. In an extension of the method, the application also provides a new coding scheme for providing a target nucleic acid with a detectable "colour" (or similar signal)-based code.
The present application provides methods for detecting a target nucleic acid molecule in a sample comprising contacting said sample with a ligatable probe comprising one or more parts and allowing said probe to hybridise to the target nucleic acid molecule, ligating any probe which has hybridised to the target nucleic acid molecule, amplifying the ligated probe, and detecting the amplification product, thereby to detect the target nucleic acid molecule, wherein said probes comprise at least one ribonucleotide at or near to a ligation site and/or wherein the probe or a probe part comprises an additional sequence 5' to a target-specific binding site which is not hybridised to the target nucleic acid molecule upon hybridisation of the probe to the target nucleic acid molecule and forms a 5' flap containing one or more nucleotides at its 3' end that is cleaved prior to ligation, and methods of synthesising a DNA molecule with Phi29 DNA polymerase using a template nucleic acid molecule comprising at least one ribonucleotide. Probes for use in the detection methods are provided.
This disclosure provides methods for preparing a sequencing library including the steps of providing a template nucleic acid sequence, dNTPs, dUTP, a primer, a polymerase, a dUTP excising enzyme, and a plurality of beads including oligonucleotide adapter sequence segments; amplifying the template nucleic acid with the polymerase, dNTPs, dUTP and random hexamer to provide a complementary nucleic acid sequence including occasional dUTPs; and excising the incorporated dUTPs with the dUTP excising enzyme to provide nicks in the complementary nucleic acid sequence to provide a sequencing library.
Methods, compositions and systems for analyzing individual cells or cell populations through the partitioned analysis of contents of individual cells or cell populations. Individual cells or cell populations are co-partitioned with processing reagents for accessing cellular contents, and for uniquely identifying the contents of a given cell or cell population, and subsequently analyzing the cell's contents and characterizing it as having derived from an individual cell or cell population, including analysis and characterization of the cell's nucleic acids through sequencing.
The disclosure provides devices, systems and methods for the generation of encapsulated reagents and the partitioning of encapsulated reagents for use in subsequent analyses and/or processing, such as in the field of biological analyses and characterization.
G01N 35/08 - Automatic analysis not limited to methods or materials provided for in any single one of groups ; Handling materials therefor using a stream of discrete samples flowing along a tube system, e.g. flow injection analysis
This disclosure provides methods and compositions for sample processing, particularly for sequencing applications. Included within this disclosure are bead compositions, such as diverse libraries of beads attached to large numbers of oligonucleotides containing barcodes. Often, the beads provides herein are degradable. For example, they may contain disulfide bonds that are susceptible to reducing agents. The methods provided herein include methods of making libraries of barcoded beads as well as methods of combining the beads with a sample, such as by using a microfluidic device.
C40B 40/06 - Libraries containing nucleotides or polynucleotides, or derivatives thereof
C12N 15/10 - Processes for the isolation, preparation or purification of DNA or RNA
C12P 19/34 - Polynucleotides, e.g. nucleic acids, oligoribonucleotides
C12Q 1/68 - Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
C40B 20/00 - Methods specially adapted for identifying library members
C40B 30/04 - Methods of screening libraries by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
C40B 50/14 - Solid phase synthesis, i.e. wherein one or more library building blocks are bound to a solid support during library creation; Particular methods of cleavage from the solid support
C40B 70/00 - Tags or labels specially adapted for combinatorial chemistry or libraries, e.g. fluorescent tags or barcodes
12.
PARTITIONING AND PROCESSING OF ANALYTES AND OTHER SPECIES
Provided is a composition comprising a plurality of capsules, the plurality of capsules situated within droplets in an emulsion, wherein capsules of the plurality of capsules are configured to release their contents into the droplets upon application of a stimulus. Also provided are devices comprising the composition and methods to cause the plurality of capsules to release their contents into the droplets.
C12M 1/40 - Apparatus specially designed for the use of free, immobilised, or carrier-bound enzymes, e.g. apparatus containing a fluidised bed of immobilised enzymes
C12Q 1/25 - Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving enzymes not classifiable in groups
The present disclosure provides compositions, methods, systems, and devices for polynucleotide processing. Such polynucleotide processing may be useful for a variety of applications, including polynucleotide sequencing. In some cases, this disclosure provides methods for the generation of polynucleotide barcode libraries, and for the attachment of such polynucleotides to target polynucleotides.
C40B 40/06 - Libraries containing nucleotides or polynucleotides, or derivatives thereof
C07H 21/00 - Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
C40B 20/04 - Identifying library members by means of a tag, label, or other readable or detectable entity associated with the library members, e.g. decoding processes
C40B 30/04 - Methods of screening libraries by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
C40B 50/06 - Biochemical methods, e.g. using enzymes or whole viable microorganisms
C40B 50/14 - Solid phase synthesis, i.e. wherein one or more library building blocks are bound to a solid support during library creation; Particular methods of cleavage from the solid support
C40B 70/00 - Tags or labels specially adapted for combinatorial chemistry or libraries, e.g. fluorescent tags or barcodes
14.
METHODS AND SYSTEMS FOR PROCESSING POLYNUCLEOTIDES
The present disclosure provides compositions, methods, systems, and devices for polynucleotide processing. Such polynucleotide processing may be useful for a variety of applications, including polynucleotide sequencing.
This disclosure provides microwell capsule array devices. The microwell capsule array devices are generally capable of performing one or more sample preparation operations. Such sample preparation operations may be used as a prelude to one more or more analysis operations. For example, a device of this disclosure can achieve physical partitioning and discrete mixing of samples with unique molecular identifiers within a single unit in preparation for various analysis operations. The device may be useful in a variety of applications and most notably nucleic-acid- based sequencing, detection and quantification of gene expression and single- cell analysis.
This disclosure provides microwell capsule array devices. The microwell capsule array devices are generally capable of performing one or more sample preparation operations. Such sample preparation operations may be used as a prelude to one more or more analysis operations. For example, a device of this disclosure can achieve physical partitioning and discrete mixing of samples with unique molecular identifiers within a single unit in preparation for various analysis operations. The device may be useful in a variety of applications and most notably nucleic-acid-based sequencing, detection and quantification of gene expression and single-cell analysis.
C12Q 1/68 - Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
C12Q 1/6806 - Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
B01J 13/02 - Making microcapsules or microballoons
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
C08K 5/529 - Esters containing heterocyclic rings not representing cyclic esters of phosphoric or phosphorous acids
C08L 33/26 - Homopolymers or copolymers of acrylamide or methacrylamide
C12M 1/34 - Measuring or testing with condition measuring or sensing means, e.g. colony counters
C12N 15/10 - Processes for the isolation, preparation or purification of DNA or RNA
C12P 19/34 - Polynucleotides, e.g. nucleic acids, oligoribonucleotides
C40B 20/04 - Identifying library members by means of a tag, label, or other readable or detectable entity associated with the library members, e.g. decoding processes
C40B 30/04 - Methods of screening libraries by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
C40B 70/00 - Tags or labels specially adapted for combinatorial chemistry or libraries, e.g. fluorescent tags or barcodes
G01N 33/48 - Biological material, e.g. blood, urine; Haemocytometers
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