Abstract: The invention described herein solves challenges in providing a proficient, rapid and meaningful analysis of sequencing data. Methods and computer program products of the invention allow for a system to receive, analyze, and display sequencing data in real-time. The invention provides solutions to several difficulties encountered in assembling short sequencing-reads, and by doing so the invention improves the worth and significance of sequencing data.

Abstract: Quantitative methods for optimizing the permeabilization of cellular tissues for spatial transcriptomics are provided. Also provided is an instrument for quantitatively optimizing the permeabilization of cellular tissues used for spatial transcriptomics.

Abstract: A discrete attribute value dataset is obtained that is associated with a plurality of probe spots each assigned a different probe spot barcode. The dataset comprises spatial projections, each comprising images of a biological sample. Each image includes a corresponding plurality of discrete attribute values for the probe spots. Each such value is associated with a probe spot in the plurality of probes spots based on the probe spot barcodes. The dataset is clustered using the discrete attribute values, or dimension reduction components thereof, for a plurality of loci at each respective probe spot across the images of the projections thereby assigning each probe spot to a cluster in a plurality of clusters. Morphological patterns are identified from the spatial arrangement of the probe spots in the various clusters.

Abstract: The present disclosure relates in some aspects to recombinant Bacillus phage AP50 polymerases, modified AP50 polymerases, and compositions thereof. Also provided are methods of using the recombinant and/or modified AP50 polymerases for nucleic acid amplification (e.g., rolling circle amplification). In some aspects, the compositions and methods disclosed herein provide more robust amplification (e.g., RCA) reactions for improved in vitro and in situ analysis.

Abstract: Methods and systems for cell and bead processing or analysis are provided. A cell or bead can be subjected to conditions sufficient to change a first characteristic or set of characteristics (e.g., cell or bead size). The cell or bead can further be subjected to conditions sufficient to change a second characteristic or set of characteristics. In some cases, crosslinks are formed within the cell or bead.

Abstract: The present disclosure provides compositions and methods for using fixed biological samples in partition-based assays. In at least one embodiment, the disclosure provides a composition comprising a fixed biological sample and an un-fixing agent contained in a partition, such as a discrete droplet. In some embodiments, the disclosure provides un-fixing agent compounds capable of catalytically cleaving crosslinks in fixed biological samples, particularly crosslinked nucleic acids, such as RNA.

Abstract: The present disclosure provides compositions, methods, systems, and devices for polynucleotide processing and analyte characterization. Such polynucleotide processing may be useful for a variety of applications, including analyte characterization by polynucleotide sequencing. The compositions, methods, systems, and devices disclosed herein generally describe barcoded oligonucleotides, which can be bound to a bead, such as a gel bead, useful for characterizing one or more analytes including, for example, protein (e.g., cell surface or intracellular proteins), genomic DNA, and RNA (e.g., mRNA or CRISPR guide RNAs). Also described herein, are barcoded labelling agents and oligonucleotide molecules useful for “tagging” analytes for characterization.

Abstract: In some aspects, provided herein are methods for analyzing a nucleic acid comprising first and second regions of interest flanking an adaptor region, comprising hybridizing an anchor to the adaptor region, analyzing the first region of interest from one end of the anchor using probe ligation (e.g., sequencing-by-ligation), and binding a polymerase to the other end of the anchor and optionally incorporating a nucleotide and/or analog thereof into the anchor by the polymerase using the second region of interest or a probe bound thereto as a template. In some embodiments, the second region of interest is used as a template for sequencing-by-synthesis. In some embodiments, spatially resolved detections of analytes are performed at a cellular or sub-cellular resolution which involve correlating signals associated with analytes with specific spatial locations in a biological sample.

Abstract: Methods for index hopping sequence read filtering are provided. Each read in a plurality of reads from a multiplexed reaction comprises an insert portion, and first (molecular identifier) and second (sample index) non-insert portions. For each of a plurality of hashes, a hash data structure is formed with a representation of each read. Each representation comprises a hash of the first non-insert portion of the corresponding read. Read pairs are identified in the hash data structures. Each pair includes a first and second read sharing a common hash value but differing index values. An entry is added into a heterogeneous data structure, for each such pair, that includes the first and second non-insert portions of the first and second reads of the pair. Reads with first non-insert portion values appearing more than a threshold number of times in the heterogeneous data structure are removed from the plurality of reads.

Abstract: Provided herein are methods of preparing a spatial array and methods for associating specific sample analytes with spatial locations in the spatial array. Provided herein are methods for preparing a spatial array using a plurality of primers attached to a substrate to guide a plurality of features to specific locations on the spatial array. In a non-limiting example, a plurality of primers on a substrate can be used to guide a plurality of first features that include capture probes onto the substrate. A plurality of second features that are configured to hybridize to the first features and also include capture probes are added to the substrate.

Abstract: The present disclosure provides methods and systems for processing nucleic acid molecules from one or more cells. A cell may be comprised in a cell bead. Processing may comprise uniquely identifying nucleic acid molecules. Nucleic acid molecules may be identified by barcoding. Barcoding may be combinatorial barcoding. Combinatorial barcoding may allow for generation of a large number of barcodes, thereby uniquely identifying nucleic acids from a large number of single cells. Combinatorial barcoding may be performed in successive operations. Successive operations may be performed in partitions.

Abstract: A substrate holder includes a base configured to receive a substrate; a cover configured to mateably engage with the base, the cover defining an opening formed by inner sidewalls; and a removable insert defining a surface, the removable insert being configured to be received within the opening of the cover. The removable insert includes a gasket; a projection coupled to the gasket; and at least two insert tabs extending from opposite sides of the removable insert, each insert tab being configured to engage with at least one of the inner sidewalls forming the opening of the cover.

Abstract: 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.

Abstract: Provided herein are methods, compositions, and kits for the detection of immune cell clonotypes and immune cell analytes within a biological sample.

Abstract: The present disclosure provides methods of processing or analyzing a sample. A method for processing a sample may comprise hybridizing a probe molecule to a target region of a nucleic acid molecule (e.g., a ribonucleic acid (RNA) molecule), barcoding the probe-nucleic acid molecule complex, and performing extension, denaturation, and amplification processes. A method for processing a sample may comprise hybridizing first and second probes to adjacent or non-adjacent target regions of a nucleic acid molecule (e.g., an RNA molecule), linking the first and second probes to provide a probe-linked nucleic acid molecule, and barcoding the probe-linked nucleic acid molecule. One or more processes of the methods described herein may be performed within a partition, such as a droplet or well. One or more processes of the methods described herein may be performed on a cell, such as a permeabilized cell.

Abstract: Provided herein are methods of identifying a location of an RNA in a sample that include: (a) contacting the sample with an array comprising capture probes, where a capture probe comprises a capture domain and a spatial barcode; (b) releasing the RNA from the sample; (c) extending a 3? end of the capture probe using the capture domain-bound RNA as a template; (d) generating nick(s) in the extended capture probe-hybridized RNA and performing random-primed DNA synthesis; (e) performing end repair on the second strand DNA molecule; (f) adding a single adenosine nucleotide to the 3? end of the extended capture probe; (g) ligating a double-stranded sequencing adaptor to the double-stranded DNA product; and (h) determining all or a part of the sequence of the RNA, and the sequence of the spatial barcode, or complements thereof, and using the determined sequences to identify the location of the RNA in the sample.

Abstract: The present disclosure provides compositions, methods, systems, and devices for polynucleotide processing and analyte characterization. Such polynucleotide processing may be useful for a variety of applications, including analyte characterization by polynucleotide sequencing. The compositions, methods, systems, and devices disclosed herein generally describe barcoded oligonucleotides, which can be bound to a bead, such as a gel bead, useful for characterizing one or more analytes including, for example, protein (e.g., cell surface or intracellular proteins), genomic DNA, and RNA (e.g., mRNA or CRISPR guide RNAs). Also described herein, are barcoded labelling agents and oligonucleotide molecules useful for “tagging” analytes for characterization.

Abstract: Disclosed herein is a method of preventing the amplification of artifactual antisense products when sequencing a target polynucleotide. Incorporation of modified nucleotides into handle polynucleotide sequences can allow for the selective degradation of artifact antisense products generated by mis-priming, prior to amplification, thus preventing them from appearing in the amplification library. Selective degradation can be accomplished using one or more agents configured to specifically cleave the handle polynucleotide at the modified nucleotide. Amplification can be performed using a primer that is configured to hybridize to the handle sequence and a second primer that is configured to hybridize to a complement of a target nucleotide.