Patent Grant
12209280
The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jul. 6, 2021, is named 47706-0220001SEQ.txt and is 15,891 bytes in size.
A Sequence Listing in ASCII text format, submitted under 37 C.F.R. § 1.821, entitled 47706-0220001_SL_ST26, 16,299 bytes in size, generated on Mar. 7, 2023 and filed via EFS-Web, is provided in lieu of a paper copy.
Cells within a tissue have differences in cell morphology and/or function due to varied analyte levels (e.g., gene and/or protein expression) within the different cells. The specific position of a cell within a tissue (e.g., the cell's position relative to neighboring cells or the cell's position relative to the tissue microenvironment) can affect, e.g., the cell's morphology, differentiation, fate, viability, proliferation, behavior, signaling, and cross-talk with other cells in the tissue.
Spatial heterogeneity has been previously studied using techniques that typically provide data for a handful of analytes in the context of intact tissue or a portion of a tissue (e.g., tissue section), or provide significant analyte data from individual, single cells, but fails to provide information regarding the position of the single cells from the originating biological sample (e.g., tissue).
RNA sequencing libraries generated from tissue samples can pose some challenges. A targeted approach to insert a sequencing adapter directly to the second-strand DNA which is synthesized on the cDNA previously generated directly on the spatial array would increase efficiency.
RNA sequencing libraries generated from formalin-fixed paraffin-embedded tissue samples on spatial arrays are generally short and cDNA could be sequenced directly if it was possible to insert a second sequencing adaptor at the 3′-end of the cDNA. The methods provided herein provide for an efficient, targeted approach for inserting a sequencing adapter directly to the second-strand DNA which is synthesized using the cDNA previously generated directly on the spatial array as a template.
Provided herein are methods of determining abundance and/or location of an RNA molecule in a biological sample. In some instances, the methods include: (a) capturing the RNA molecule from the biological sample on an array comprising a plurality of capture probes, wherein a capture probe of the plurality of capture probes comprises a capture domain and a spatial barcode; (b) extending an end of the capture probe using the RNA molecule as a template, thereby generating an extended capture probe hybridized to the RNA molecule; (c) contacting the extended capture probe with a primer comprising in a 5′ to a 3′ direction: (i) an adapter sequence and (ii) a sequence that specifically binds to the extended capture probe; (d) extending the 3′ end of the primer using the extended capture probe as a template, thereby generating a DNA molecule hybridized to the extended capture probe; and (e) determining (i) all or a part of the sequence of the DNA molecule or a complement thereof, or (ii) all or a part of the sequence of the spatial barcode or a complement thereof, and using the determined sequences of (i) and (ii) to identify the abundance and/or the location of the RNA molecule in the biological sample.
In some instances, the extending in step (b) comprises the use of a reverse transcriptase. In some instances, the methods further include, between steps (b) and (c), digesting the RNA molecule hybridized to the extended capture probe. In some instances, the digesting comprises use of RNAase H or a functional equivalent thereof. In some instances, the extending in step (e) comprises the use of a DNA polymerase.
In some instances, the methods further include releasing the DNA molecule from the extended capture probe, wherein the releasing the DNA molecule comprises heating the DNA molecule to de-hybridize the DNA molecule from the extended capture probe
In some instances, the determining in step (e) comprises sequencing (i) all or a part of the sequence of the RNA molecule or a complement thereof, or (ii) all or a part of the sequence of the spatial barcode or a complement thereof.
In some instances, the adaptor sequence comprises SEQ ID NO:1 (CCTTGGCACACCCGAGAATTCCA). In some instances, the primer sequence comprises a sequence that is complementary to the RNA molecule, or a complement thereof. In some instances, the RNA molecule is an mRNA molecule. In some instances, the capture domain comprises a poly(T) sequence. In some instances, the capture probe further comprises one or more functional domains, a unique molecular identifier, a cleavage domain, and combinations thereof.
In some instances, the capturing in step (a) comprises permeabilizing the biological sample using a permeabilization agent, wherein the permeabilization agent comprises proteinase K or pepsin, thereby releasing the RNA molecule from the biological sample.
In some instances, the biological sample is a tissue section. In some instances, the tissue section is a formalin-fixed paraffin-embedded tissue section. In some instances, the tissue section is a fresh frozen tissue section.
In some instances, the method further comprising imaging the biological sample.
In some instances, the primer is in a primer pool, wherein the primer pool is at a concentration of about 1 μM.
In some instances, the abundance of the RNA molecule is increased by at least about 10% compared to a method that does not utilize the primer.
Also provided herein are methods of identifying a location of an RNA in a biological sample that include: (a) capturing RNA from the biological sample on an array comprising a plurality of capture probes, wherein a capture probe of the plurality of capture probes comprises a capture domain and a spatial barcode; (b) extending an end of the capture probe using the RNA specifically bound by the capture domain as a template, thereby generating an extended capture probe hybridized to the RNA; (c) digesting the RNA hybridized to the extended capture probe; (d) contacting the extended capture probe with a primer comprising in a 5′ to a 3′ direction: (i) an adapter sequence and (ii) a sequence that specifically binds to the extended capture probe; (e) extending the 3′ end of the primer using the extended capture probe as a template, thereby generating a DNA hybridized to the extended capture probe; (f) releasing the generated DNA from the extended capture probe, and (g) determining (i) all or a part of the sequence of the RNA bound by the capture domain or a complement thereof, or (ii) all or a part of the sequence of the spatial barcode or a complement thereof, and using the determined sequences of (i) and (ii) to identify the location of the RNA in the biological sample.
In some embodiments of any of the methods described herein, the extending in step (b) comprises the use of a reverse transcriptase. In some embodiments of any of the methods described herein, the digesting in step (c) comprises the use of RNAase H. In some embodiments of any of the methods described herein, the extending in step (e) comprises the use of a DNA polymerase. In some embodiments of any of the methods described herein, the determining in step (g) comprises sequencing (i) all or a part of the sequence of the RNA or a complement thereof, or (ii) all or a part of the sequence of the spatial barcode or a complement thereof.
In some embodiments of any of the methods described herein, the RNA is an mRNA molecule. In some embodiments of any of the methods described herein, the capture domain comprises a poly(T) sequence. In some embodiments of any of the methods described herein, the capture domain is positioned 3′ relative to the spatial barcode in the capture probe. In some embodiments of any of the methods described herein, the capture probe further comprises a unique molecular identifier. In some embodiments of any of the methods described herein, the capture probe further comprises a cleavage domain. In some embodiments of any of the methods described herein, the capturing in step (a) comprises permeabilizing the biological sample, thereby releasing the RNA from the biological sample.
In some embodiments of any of the methods described herein, the array is a slide. In some embodiments of any of the methods described herein, the slide comprises beads. In some embodiments of any of the methods described herein, the slide comprises wells.
In some embodiments of any of the methods described herein, the biological sample is a tissue sample. In some embodiments of any of the methods described herein, the tissue sample is a tissue section. In some embodiments of any of the methods described herein, the tissue section is a fixed tissue section. In some embodiments of any of the methods described herein, the fixed tissue section is a formalin-fixed paraffin-embedded tissue section. In some embodiments of any of the methods described herein, the tissue section is a fresh, frozen tissue section. Some embodiments of any of the methods described herein further include imaging the biological sample.
Also provided herein are reaction mixtures that include: an array comprising a plurality of capture probes, where a capture probe of the plurality comprises a capture domain that binds specifically to an RNA and a spatial barcode; a reverse transcriptase; RNAse H or a functional equivalent thereof, and a DNA polymerase. In some embodiments of any of the reaction mixtures described herein, the DNA polymerase is DNA polymerase I. Some embodiments of any of the reaction mixtures described herein further include an RNA from a biological sample.
In some embodiments of any of the reaction mixtures described herein, the array is a slide. In some embodiments of any of the reaction mixtures described herein, the slide comprises beads. In some embodiments of any of the reaction mixtures described herein, the slide comprises wells. Some embodiments of any of the reaction mixtures described herein, the reaction mixture further comprises a primer comprising in a 5′ to a 3′ direction: (i) an adapter sequence and (ii) a sequence or a complement thereof present in a 5′ region of the RNA that is specifically bound to the capture domain.
Also provided herein are compositions. In some instances, the compositions include one or more of the following (and any combination thereof): (a) an array comprising a plurality of capture probes, wherein a capture probe of the plurality comprises a capture domain that binds specifically to an RNA and a spatial barcode; (b) a reverse transcriptase enzyme; (c) RNAse H or a functional equivalent thereof, (d) a DNA polymerase; (e) a primer comprising in a 5′ to a 3′ direction: (i) an adapter sequence and (ii) a sequence or a complement thereof present in a 5′ region of the RNA molecule that is specifically bound to the capture domain; and (f) an RNA molecule from a biological sample.
Also provided herein are kits. In some instances, the kits include one or more of the following (and any combination thereof): (a) an array comprising a plurality of capture probes, wherein a capture probe of the plurality comprises a capture domain that binds specifically to an RNA and a spatial barcode; (b) a reverse transcriptase; (c) RNAse H or a functional equivalent thereof; (d) a DNA polymerase; (e) a primer comprising in a 5′ to a 3′ direction: (i) an adapter and (ii) a sequence or a complement thereof present in a 5′ region of the RNA molecule that is specifically bound to the capture domain; and (f) instructions for performing any of the methods described herein.
In some instances, the kits include: an array comprising a plurality of capture probes, where a capture probe of the plurality comprises a capture domain that binds specifically to an RNA and a spatial barcode; a reverse transcriptase; RNAse H or a functional equivalent thereof; and a DNA polymerase. In some embodiments of any of the kits described herein, the DNA polymerase is DNA polymerase I. In some embodiments of any of the kits described herein, the capture domain is positioned 3′ of the spatial barcode in the capture domain. In some embodiments of any of the kits described herein, the capture probe further comprises a unique molecular identifier. In some embodiments of any of the kits described herein, the capture probe further comprises a cleavage domain. Some embodiments of any of the kits described herein further include an RNA from a biological sample.
In some embodiments of any of the kits described herein, the array is a slide. In some embodiments of any of the kits described herein, the slide comprises beads. In some embodiments of any of the kits described herein, the slide comprises wells. Some embodiments of any of the kits described herein further include a primer comprising in a 5′ to a 3′ direction: (i) an adapter and (ii) a sequence or a complement thereof present in a 5′ region of the RNA that is specifically bound to the capture domain.
All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, patent application, or item of information was specifically and individually indicated to be incorporated by reference. To the extent publications, patents, patent applications, and items of information incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and/or take precedence over any such contradictory material.
Where values are described in terms of ranges, it should be understood that the description includes the disclosure of all possible sub-ranges within such ranges, as well as specific numerical values that fall within such ranges irrespective of whether a specific numerical value or specific sub-range is expressly stated.
The term “each,” when used in reference to a collection of items, is intended to identify an individual item in the collection but does not necessarily refer to every item in the collection, unless expressly stated otherwise, or unless the context of the usage clearly indicates otherwise.
The singular form “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a cell” includes one or more cells, comprising mixtures thereof. “A and/or B” is used herein to include all of the following alternatives: “A”, “B”, “A or B”, and “A and B”.
Various embodiments of the features of this disclosure are described herein. However, it should be understood that such embodiments are provided merely by way of example, and numerous variations, changes, and substitutions can occur to those skilled in the art without departing from the scope of this disclosure. It should also be understood that various alternatives to the specific embodiments described herein are also within the scope of this disclosure.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
The following drawings illustrate certain embodiments of the features and advantages of this disclosure. These embodiments are not intended to limit the scope of the appended claims in any manner. Like reference symbols in the drawings indicate like elements.
Spatial analysis methodologies and compositions described herein can provide a vast amount of analyte and/or expression data for a variety of analytes within a biological sample at high spatial resolution, while retaining native spatial context. Spatial analysis methods and compositions can include, e.g., the use of a capture probe including a spatial barcode (e.g., a nucleic acid sequence that provides information as to the location or position of an analyte within a cell or a tissue sample (e.g., mammalian cell or a mammalian tissue sample) and a capture domain that is capable of binding to an analyte (e.g., a protein and/or a nucleic acid) produced by and/or present in a cell. Spatial analysis methods and compositions can also include the use of a capture probe having a capture domain that captures an intermediate agent for indirect detection of an analyte. For example, the intermediate agent can include a nucleic acid sequence (e.g., a barcode) associated with the intermediate agent. Detection of the intermediate agent is therefore indicative of the analyte in the cell or tissue sample.
Non-limiting aspects of spatial analysis methodologies and compositions are described in U.S. Pat. Nos. 10,774,374, 10,724,078, 10,480,022, 10,059,990, 10,041,949, 10,002,316, 9,879,313, 9,783,841, 9,727,810, 9,593,365, 8,951,726, 8,604,182, 7,709,198, U.S. Patent Application Publication Nos. 2020/239946, 2020/080136, 2020/0277663, 2020/024641, 2019/330617, 2019/264268, 2020/256867, 2020/224244, 2019/194709, 2019/161796, 2019/085383, 2019/055594, 2018/216161, 2018/051322, 2018/0245142, 2017/241911, 2017/089811, 2017/067096, 2017/029875, 2017/0016053, 2016/108458, 2015/000854, 2013/171621, WO 2018/091676, WO 2020/176788, Rodriques et al., Science 363(6434):1463-1467, 2019; Lee et al., Nat. Protoc. 10(3):442-458, 2015; Trejo et al., PLOS ONE 14(2):e0212031, 2019; Chen et al., Science 348(6233):aaa6090, 2015; Gao et al., BMC Biol. 15:50, 2017; and Gupta et al., Nature Biotechnol. 36:1197-1202, 2018; the Visium Spatial Gene Expression Reagent Kits User Guide (e.g., Rev D, dated October 2020), and/or the Visium Spatial Tissue Optimization Reagent Kits User Guide (e.g., Rev D, dated October 2020), both of which are available at the 10× Genomics Support Documentation website, and can be used herein in any combination. Further non-limiting aspects of spatial analysis methodologies and compositions are described herein.
Some general terminologies that may be used in this disclosure can be found in Section (I)(b) of WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663. Typically, a “barcode” is a label, or identifier, that conveys or is capable of conveying information (e.g., information about an analyte in a sample, a bead, and/or a capture probe). A barcode can be part of an analyte, or independent of an analyte. A barcode can be attached to an analyte. A particular barcode can be unique relative to other barcodes. For the purpose of this disclosure, an “analyte” can include any biological substance, structure, moiety, or component to be analyzed. The term “target” can similarly refer to an analyte of interest.
Analytes can be broadly classified into one of two groups: nucleic acid analytes, and non-nucleic acid analytes. Examples of non-nucleic acid analytes include, but are not limited to, lipids, carbohydrates, peptides, proteins, glycoproteins (N-linked or O-linked), lipoproteins, phosphoproteins, specific phosphorylated or acetylated variants of proteins, amidation variants of proteins, hydroxylation variants of proteins, methylation variants of proteins, ubiquitylation variants of proteins, sulfation variants of proteins, viral proteins (e.g., viral capsid, viral envelope, viral coat, viral accessory, viral glycoproteins, viral spike, etc.), extracellular and intracellular proteins, antibodies, and antigen binding fragments. In some embodiments, the analyte(s) can be localized to subcellular location(s), including, for example, organelles, e.g., mitochondria, Golgi apparatus, endoplasmic reticulum, chloroplasts, endocytic vesicles, exocytic vesicles, vacuoles, lysosomes, etc. In some embodiments, analyte(s) can be peptides or proteins, including without limitation antibodies and enzymes. Additional examples of analytes can be found in Section (I)(c) of WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663. In some embodiments, an analyte can be detected indirectly, such as through detection of an intermediate agent, for example, a connected probe (e.g., a ligation product) or an analyte capture agent (e.g., an oligonucleotide-conjugated antibody), such as those described herein.
A biological sample is typically obtained from the subject for analysis using any of a variety of techniques including, but not limited to, biopsy, surgery, and laser capture microscopy (LCM), and generally includes cells and/or other biological material from the subject. In some embodiments, a biological sample can be a tissue section. In some embodiments, a biological sample can be a fixed and/or stained biological sample (e.g., a fixed and/or stained tissue section). Non-limiting examples of stains include histological stains (e.g., hematoxylin and/or eosin) and immunological stains (e.g., fluorescent stains). In some embodiments, a biological sample (e.g., a fixed and/or stained biological sample) can be imaged. Biological samples are also described in Section (I)(d) of WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663.
In some embodiments, a biological sample is permeabilized with one or more permeabilization reagents. For example, permeabilization of a biological sample can facilitate analyte capture. Exemplary permeabilization agents and conditions are described in Section (I)(d)(ii)(13) or the Exemplary Embodiments Section of WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663.
Array-based spatial analysis methods involve the transfer of one or more analytes from a biological sample to an array of features on a substrate, where each feature is associated with a unique spatial location on the array. Subsequent analysis of the transferred analytes includes determining the identity of the analytes and the spatial location of the analytes within the biological sample. The spatial location of an analyte within the biological sample is determined based on the feature to which the analyte is bound (e.g., directly or indirectly) on the array, and the feature's relative spatial location within the array.
A “capture probe” refers to any molecule capable of capturing (directly or indirectly) and/or labelling an analyte (e.g., an analyte of interest) in a biological sample. In some embodiments, the capture probe is a nucleic acid or a polypeptide. In some embodiments, the capture probe includes a barcode (e.g., a spatial barcode and/or a unique molecular identifier (UMI)) and a capture domain). In some embodiments, a capture probe can include a cleavage domain and/or a functional domain (e.g., a primer-binding site, such as for next-generation sequencing (NGS)).
The functional sequences can generally be selected for compatibility with any of a variety of different sequencing systems, e.g., Ion Torrent™ Proton or PGM (i.e., ion semiconductor sequencing), Illumina™ sequencing instruments (e.g., sequencing by synthesis), PacBio™ (e.g., HiFi sequencing), OXFORD NANOPORE, etc., and the requirements thereof. In some embodiments, functional sequences can be selected for compatibility with non-commercialized sequencing systems. Examples of such sequencing systems and techniques, for which suitable functional sequences can be used, include (but are not limited to) Ion Torrent™ Proton or PGM sequencing (i.e., ion semiconductor sequencing), Illumina™ sequencing (e.g., sequencing by synthesis), PacBio™ SMRT™ sequencing (e.g., HiFi sequencing), and OXFORD NANOPORE sequencing. Further, in some embodiments, functional sequences can be selected for compatibility with other sequencing systems, including non-commercialized sequencing systems.
In some embodiments, the spatial barcode 105 and functional sequences 104 are common to all of the probes attached to a given feature. In some embodiments, the UMI sequence 106 of a capture probe attached to a given feature is different from the UMI sequence of a different capture probe attached to the given feature.
In some embodiments, more than one analyte type (e.g., nucleic acids and proteins) from a biological sample can be detected (e.g., simultaneously or sequentially) using any appropriate multiplexing technique, such as those described in Section (IV) of WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663.
In some embodiments, detection of one or more analytes (e.g., protein analytes) can be performed using one or more analyte capture agents. As used herein, an “analyte capture agent” refers to an agent that interacts with an analyte (e.g., an analyte in a biological sample) and with a capture probe (e.g., a capture probe attached to a substrate or a feature) to identify the analyte. In some embodiments, the analyte capture agent includes: (i) an analyte binding moiety (e.g., that binds to an analyte), for example, an antibody or antigen-binding fragment thereof; (ii) analyte binding moiety barcode; and (iii) a capture handle sequence. As used herein, the term “analyte binding moiety barcode” refers to a barcode that is associated with or otherwise identifies the analyte binding moiety. As used herein, the term “analyte capture sequence” or “capture handle sequence” refers to a region or moiety configured to hybridize to, bind to, couple to, or otherwise interact with a capture domain of a capture probe. In some embodiments, a capture handle sequence is complementary to a capture domain of a capture probe. In some cases, an analyte binding moiety barcode (or portion thereof) may be able to be removed (e.g., cleaved) from the analyte capture agent.
Additional description of analyte capture agents can be found in Section (II)(b)(ix) of WO 2020/176788 and/or Section (II)(b)(viii) U.S. Patent Application Publication No. 2020/0277663.
There are at least two methods to associate a spatial barcode with one or more neighboring cells, such that the spatial barcode identifies the one or more cells, and/or contents of the one or more cells, as associated with a particular spatial location. One method is to promote analytes or analyte proxies (e.g., intermediate agents) out of a cell and towards a spatially-barcoded array (e.g., including spatially-barcoded capture probes). Another method is to cleave spatially-barcoded capture probes from an array and promote the spatially-barcoded capture probes towards and/or into or onto the biological sample.
In some cases, capture probes may be configured to prime, replicate, and consequently yield optionally barcoded extension products from a template (e.g., a DNA or RNA template, such as an analyte or an intermediate agent (e.g., a connected probe (e.g., a ligation product) or an analyte capture agent), or a portion thereof), or derivatives thereof (see, e.g., Section (II)(b)(vii) of WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663 regarding extended capture probes). In some cases, capture probes may be configured to form a connected probe (e.g., a ligation product) with a template (e.g., a DNA or RNA template, such as an analyte or an intermediate agent, or portion thereof), thereby creating ligations products that serve as proxies for a template.
As used herein, an “extended capture probe” refers to a capture probe having additional nucleotides added to the terminus (e.g., 3′ or 5′ end) of the capture probe thereby extending the overall length of the capture probe. For example, an “extended 3′ end” indicates additional nucleotides were added to the most 3′ nucleotide of the capture probe to extend the length of the capture probe, for example, by polymerization reactions used to extend nucleic acid molecules including templated polymerization catalyzed by a polymerase (e.g., a DNA polymerase or a reverse transcriptase). In some embodiments, extending the capture probe includes adding to a 3′ end of a capture probe a nucleic acid sequence that is complementary to a nucleic acid sequence of an analyte or intermediate agent specifically bound to the capture domain of the capture probe. In some embodiments, the capture probe is extended using reverse transcription. In some embodiments, the capture probe is extended using one or more DNA polymerases. The extended capture probes include the sequence of the capture probe and the sequence of the spatial barcode of the capture probe.
In some embodiments, extended capture probes are amplified (e.g., in bulk solution or on the array) to yield quantities that are sufficient for downstream analysis, e.g., via DNA sequencing. In some embodiments, extended capture probes (e.g., DNA molecules) act as templates for an amplification reaction (e.g., a polymerase chain reaction).
Additional variants of spatial analysis methods, including in some embodiments, an imaging step, are described in Section (II)(a) of WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663. Analysis of captured analytes (and/or intermediate agents or portions thereof), for example, including sample removal, extension of capture probes, sequencing (e.g., of a cleaved extended capture probe and/or a nucleic acid molecule complementary to an extended capture probe), sequencing on the array (e.g., using, for example, in situ hybridization or in situ ligation approaches), temporal analysis, and/or proximity capture, is described in Section (II)(g) of WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663. Some quality control measures are described in Section (II)(h) of WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663.
Spatial information can provide information of biological and/or medical importance. For example, the methods and compositions described herein can allow for: identification of one or more biomarkers (e.g., diagnostic, prognostic, and/or for determination of efficacy of a treatment) of a disease or disorder; identification of a candidate drug target for treatment of a disease or disorder; identification (e.g., diagnosis) of a subject as having a disease or disorder; identification of stage and/or prognosis of a disease or disorder in a subject; identification of a subject as having an increased likelihood of developing a disease or disorder; monitoring of progression of a disease or disorder in a subject; determination of efficacy of a treatment of a disease or disorder in a subject; identification of a patient subpopulation for which a treatment is effective for a disease or disorder; modification of a treatment of a subject with a disease or disorder; selection of a subject for participation in a clinical trial; and/or selection of a treatment for a subject with a disease or disorder.
Spatial information can provide information of biological importance. For example, the methods and compositions described herein can allow for: identification of transcriptome and/or proteome expression profiles (e.g., in healthy and/or diseased tissue); identification of multiple analyte types in close proximity (e.g., nearest neighbor analysis); determination of up- and/or down-regulated genes and/or proteins in diseased tissue; characterization of tumor microenvironments; characterization of tumor immune responses; characterization of cells types and their co-localization in tissue; and identification of genetic variants within tissues (e.g., based on gene and/or protein expression profiles associated with specific disease or disorder biomarkers).
Typically, for spatial array-based methods, a substrate functions as a support for direct or indirect attachment of capture probes to features of the array. A “feature” is an entity that acts as a support or repository for various molecular entities used in spatial analysis. In some embodiments, some or all of the features in an array are functionalized for analyte capture. Exemplary substrates are described in Section (II)(c) of WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663. Exemplary features and geometric attributes of an array can be found in Sections (II)(d)(i), (II)(d)(iii), and (II)(d)(iv) of WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663.
Generally, analytes and/or intermediate agents (or portions thereof) can be captured when contacting a biological sample with a substrate including capture probes (e.g., a substrate with capture probes embedded, spotted, printed, fabricated on the substrate, or a substrate with features (e.g., beads, wells) comprising capture probes). As used herein, “contact,” “contacted,” and/or “contacting,” a biological sample with a substrate refers to any contact (e.g., direct or indirect) such that capture probes can interact (e.g., bind covalently or non-covalently (e.g., hybridize)) with analytes from the biological sample. Capture can be achieved actively (e.g., using electrophoresis) or passively (e.g., using diffusion). Analyte capture is further described in Section (II)(e) of WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663.
In some cases, spatial analysis can be performed by attaching and/or introducing a molecule (e.g., a peptide, a lipid, or a nucleic acid molecule) having a barcode (e.g., a spatial barcode) to a biological sample (e.g., to a cell in a biological sample). In some embodiments, a plurality of molecules (e.g., a plurality of nucleic acid molecules) having a plurality of barcodes (e.g., a plurality of spatial barcodes) are introduced to a biological sample (e.g., to a plurality of cells in a biological sample) for use in spatial analysis. In some embodiments, after attaching and/or introducing a molecule having a barcode to a biological sample, the biological sample can be physically separated (e.g., dissociated) into single cells or cell groups for analysis. Some such methods of spatial analysis are described in Section (III) of WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663.
During analysis of spatial information, sequence information for a spatial barcode associated with an analyte is obtained, and the sequence information can be used to provide information about the spatial distribution of the analyte in the biological sample. Various methods can be used to obtain the spatial information. In some embodiments, specific capture probes and the analytes they capture are associated with specific locations in an array of features on a substrate. For example, specific spatial barcodes can be associated with specific array locations prior to array fabrication, and the sequences of the spatial barcodes can be stored (e.g., in a database) along with specific array location information, so that each spatial barcode uniquely maps to a particular array location.
Alternatively, specific spatial barcodes can be deposited at predetermined locations in an array of features during fabrication such that at each location, only one type of spatial barcode is present so that spatial barcodes are uniquely associated with a single feature of the array. Where necessary, the arrays can be decoded using any of the methods described herein so that spatial barcodes are uniquely associated with array feature locations, and this mapping can be stored as described above.
When sequence information is obtained for capture probes and/or analytes during analysis of spatial information, the locations of the capture probes and/or analytes can be determined by referring to the stored information that uniquely associates each spatial barcode with an array feature location. In this manner, specific capture probes and captured analytes are associated with specific locations in the array of features. Each array feature location represents a position relative to a coordinate reference point (e.g., an array location, a fiducial marker) for the array. Accordingly, each feature location has an “address” or location in the coordinate space of the array.
Some exemplary spatial analysis workflows are described in the Exemplary Embodiments section of WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663. See, for example, the Exemplary embodiment starting with “In some non-limiting examples of the workflows described herein, the sample can be immersed . . . ” of WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663. See also, e.g., the Visium Spatial Gene Expression Reagent Kits User Guide (e.g., Rev D, dated October 2020), and/or the Visium Spatial Tissue Optimization Reagent Kits User Guide (e.g., Rev D, dated October 2020). In some embodiments, spatial analysis can be performed using dedicated hardware and/or software, such as any of the systems described in Sections (II)(e)(ii) and/or (V) of WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663, or any of one or more of the devices or methods described in Sections Control Slide for Imaging, Methods of Using Control Slides and Substrates for, Systems of Using Control Slides and Substrates for Imaging, and/or Sample and Array Alignment Devices and Methods, Informational labels of WO 2020/123320.
Suitable systems for performing spatial analysis can include components such as a chamber (e.g., a flow cell or sealable, fluid-tight chamber) for containing a biological sample. The biological sample can be mounted for example, in a biological sample holder. One or more fluid chambers can be connected to the chamber and/or the sample holder via fluid conduits, and fluids can be delivered into the chamber and/or sample holder via fluidic pumps, vacuum sources, or other devices coupled to the fluid conduits that create a pressure gradient to drive fluid flow. One or more valves can also be connected to fluid conduits to regulate the flow of reagents from reservoirs to the chamber and/or sample holder.
The systems can optionally include a control unit that includes one or more electronic processors, an input interface, an output interface (such as a display), and a storage unit (e.g., a solid state storage medium such as, but not limited to, a magnetic, optical, or other solid state, persistent, writeable and/or re-writeable storage medium). The control unit can optionally be connected to one or more remote devices via a network. The control unit (and components thereof) can generally perform any of the steps and functions described herein. Where the system is connected to a remote device, the remote device (or devices) can perform any of the steps or features described herein. The systems can optionally include one or more detectors (e.g., CCD, CMOS) used to capture images. The systems can also optionally include one or more light sources (e.g., LED-based, diode-based, lasers) for illuminating a sample, a substrate with features, analytes from a biological sample captured on a substrate, and various control and calibration media.
The systems can optionally include software instructions encoded and/or implemented in one or more of tangible storage media and hardware components such as application specific integrated circuits. The software instructions, when executed by a control unit (and in particular, an electronic processor) or an integrated circuit, can cause the control unit, integrated circuit, or other component executing the software instructions to perform any of the method steps or functions described herein.
In some cases, the systems described herein can detect (e.g., register an image) the biological sample on the array. Exemplary methods to detect the biological sample on an array are described in PCT Application No. 2020/061064 and/or U.S. patent application Ser. No. 16/951,854.
Prior to transferring analytes from the biological sample to the array of features on the substrate, the biological sample can be aligned with the array. Alignment of a biological sample and an array of features including capture probes can facilitate spatial analysis, which can be used to detect differences in analyte presence and/or level within different positions in the biological sample, for example, to generate a three-dimensional map of the analyte presence and/or level. Exemplary methods to generate a two- and/or three-dimensional map of the analyte presence and/or level are described in PCT Application No. 2020/053655 and spatial analysis methods are generally described in WO 2020/061108 and/or U.S. patent application Ser. No. 16/951,864.
In some cases, a map of analyte presence and/or level can be aligned to an image of a biological sample using one or more fiducial markers, e.g., objects placed in the field of view of an imaging system which appear in the image produced, as described in the Substrate Attributes Section, Control Slide for Imaging Section of WO 2020/123320, PCT Application No. 2020/061066, and/or U.S. patent application Ser. No. 16/951,843. Fiducial markers can be used as a point of reference or measurement scale for alignment (e.g., to align a sample and an array, to align two substrates, to determine a location of a sample or array on a substrate relative to a fiducial marker) and/or for quantitative measurements of sizes and/or distances.
RNA sequencing libraries generated from formalin-fixed paraffin-embedded (FFPE) tissue samples on spatial arrays are generally short and cDNA could be sequenced directly if it was possible to insert a second sequencing adaptor at the 3′-end of the cDNA. The methods provided herein provide for an efficient, targeted approach for inserting a sequencing adapter directly to the second-strand DNA which is synthesized using the cDNA previously generated directly on the spatial array as a template. However, the methods are not limited to FFPE tissues as the methods are equally amenable with other tissue types, such as fresh frozen samples or alternatively fixed samples (e.g., methanol, acetone, etc.). Thus, in some instances, the biological sample is taken from a sample fixed with formalin (e.g., an FFPE sample). In other instances, the biological sample is not fixed, and can be a freshly-obtained sample or a frozen sample.
In some workflows of spatial analyses, gene-specific primers containing a universal sequence are utilized in a targeted approach for second strand synthesis. An exemplary embodiment of the methods on FFPE tissue described herein is depicted in
Provided herein are methods of identifying a location of an RNA in a biological sample that include: (a) contacting a biological sample (e.g., any of the exemplary biological samples described herein) with an array comprising a plurality of capture probes, wherein a capture probe of the plurality of capture probes comprises a capture domain (e.g., any of the exemplary capture domains described herein), a spatial barcode and a first adaptor sequence (e.g., a first sequencing primer sequence); (b) extending an end of the capture probe using the captured RNA (e.g., any of the exemplary types of RNA described herein, e.g., mRNA) specifically bound by the capture domain as a template, thereby generating an extended capture probe hybridized to the RNA; (c) digesting the RNA hybridized to the extended capture probe; (d) contacting the extended capture probe with a primer comprising in a 5′ to a 3′ direction: (i) an adapter sequence (e.g., a second sequencing adapter sequence, e.g., a universal sequencing adapter sequence) and (ii) a sequence that specifically binds to (e.g., at least a portion of) the extended capture probe; (e) extending the 3′ end of the primer using the extended capture probe as a template, thereby generating a DNA hybridized to the extended capture probe; (f) releasing the generated DNA from the extended capture probe; and (g) determining (i) all or a part of the sequence of the RNA bound by the capture domain or a complement thereof, or (ii) all or a part of the sequence of the spatial barcode or a complement thereof, and using the determined sequences of (i) and (ii) to identify the location of the RNA in the biological sample.
In some instances, after preparing the biological sample for spatial analysis, the analyte (e.g., mRNA) is captured by a capture probe on an array. In some embodiments of any of the methods described herein, the capture domain comprises a poly(T) sequence. In some instances, the analyte hybridizes to the poly(T) sequence. In some embodiments, the capture domain does not comprise a poly(T) sequence. In some embodiments, the capture domain comprises a sequence that is substantially complementary to a contiguous sequence present in the RNA. The capture domain can be about 5 to about 40 nucleotides (e.g., about 5 to about 35 nucleotides, about 5 to about 30 nucleotides, about 5 to about 25 nucleotides, about 5 to about 20 nucleotides, about 5 to about 15 nucleotides, about 5 to about 10 nucleotides, about 10 to about 40 nucleotides, about 10 to about 35 nucleotides, about 10 to about 30 nucleotides, about 10 to about 25 nucleotides, about 10 to about 20 nucleotides, about 10 to about 15 nucleotides, about 15 to about 40 nucleotides, about 15 to about 35 nucleotides, about 15 to about 30 nucleotides, about 15 to about 25 nucleotides, about 15 to about 20 nucleotides, about 20 to about 40 nucleotides, about 20 to about 35 nucleotides, about 20 to about 30 nucleotides, about 20 to about 25 nucleotides, about 25 to about 40 nucleotides, about 25 to about 35 nucleotides, about 25 to about 30 nucleotides, about 30 to about 40 nucleotides, about 30 to about 35 nucleotides, or about 35 to about 40 nucleotides) in length. In some instances, one or more capture probes on the spatial array further include a spatial barcode and/or a unique molecular identifier (UMI).
In some embodiments of any of the methods described herein, the plurality of capture probes are affixed (i.e., attached) to an array. In some embodiments of any of the methods described herein, the array is a slide (e.g., a slide comprising beads or a slide comprising wells (e.g., microwells)). An array can also have one or more of any of the exemplary characteristics of arrays described herein.
In some embodiments, the capture domain is positioned 3′ relative to the spatial barcode in the capture probe. In some embodiments of any of the methods provided herein, the capture probe further includes a unique molecular identifier, a cleavage domain (e.g., any of the exemplary cleavage domains described herein), or both.
In some embodiments, after contacting a biological sample with a substrate that includes capture probes, a removal step can optionally be performed to remove all or a portion of the biological sample from the substrate. In some embodiments, the removal step includes enzymatic and/or chemical degradation of cells of the biological sample. For example, the removal step can include treating the biological sample with an enzyme (e.g., a proteinase, e.g., proteinase K) to remove at least a portion of the biological sample from the substrate. In some embodiments, the removal step can include ablation of the tissue (e.g., laser ablation).
In some embodiments, a biological sample is not removed from the substrate. For example, the biological sample is not removed from the substrate prior to releasing a capture probe (e.g., a capture probe bound to an analyte) from the substrate. In some embodiments, such releasing comprises cleavage of the capture probe from the substrate (e.g., via a cleavage domain). In some embodiments, such releasing does not comprise releasing the capture probe from the substrate (e.g., a copy of the capture probe bound to an analyte can be made and the copy can be released from the substrate, e.g., via denaturation). In some embodiments, the biological sample is not removed from the substrate prior to analysis of an analyte bound to a capture probe after it is released from the substrate. In some embodiments, the biological sample remains on the substrate during removal of a capture probe from the substrate and/or analysis of an analyte bound to the capture probe after it is released from the substrate. In some embodiments, the biological sample remains on the substrate during removal (e.g., via denaturation) of a copy of the capture probe (e.g., complement). In some embodiments, analysis of an analyte bound to capture probe from the substrate can be performed without subjecting the biological sample to enzymatic and/or chemical degradation of the cells (e.g., permeabilized cells) or ablation of the tissue (e.g., laser ablation).
In some embodiments, at least a portion of the biological sample is not removed from the substrate. For example, a portion of the biological sample can remain on the substrate prior to releasing a capture probe (e.g., a capture prove bound to an analyte) from the substrate and/or analyzing an analyte bound to a capture probe released from the substrate. In some embodiments, at least a portion of the biological sample is not subjected to enzymatic and/or chemical degradation of the cells (e.g., permeabilized cells) or ablation of the tissue (e.g., laser ablation) prior to analysis of an analyte bound to a capture probe from the substrate.
In some embodiments, after analyte capture, the capture probe can be extended (an “extended capture probe,” e.g., as described herein). In some embodiments, the capture probe is extended at the 3′ end. For example, extending a capture probe can include generating cDNA from a captured (hybridized) RNA. This process involves synthesis of a complementary strand of the hybridized nucleic acid, e.g., generating cDNA based on the captured RNA template (the RNA hybridized to the capture domain of the capture probe). Thus, in an initial step of extending a capture probe, e.g., the cDNA generation, the captured (hybridized) nucleic acid, e.g., RNA, acts as a template for the extension, e.g., reverse transcription, step.
In some embodiments, the capture probe is extended using reverse transcription. For example, reverse transcription includes synthesizing cDNA (complementary or copy DNA) from RNA, e.g., (messenger RNA), using a reverse transcriptase. In some embodiments, the capture probe is extended using fluorescently labeled nucleotides. In some embodiments, reverse transcription is performed while the tissue is still in place, generating an analyte library, where the analyte library includes the spatial barcodes from the adjacent capture probes. In some embodiments, the capture probe is extended using one or more DNA polymerases.
In some embodiments, digesting the RNA from the RNA:DNA hybrid comprises the use of an RNase that digests RNA from a RNA:DNA hybrid, for example, RNAse H or a functional equivalent thereof.
After extension of the capture probe and degradation of the analyte, target-specific primers are added to the sample. In some instances, a target-specific primer as described herein comprises a sequence that is complementary to the extended capture probe. In some instances, a target-specific primer comprises a sequence that is complementary to the extended capture probe at the sequence complementary to the analyte. Thus, in some instances, the primer includes a sequence that is specific for one or more targets of interest.
The sequence in the primer that specifically binds to (e.g., at least a portion of) the extended capture probe can about 15 to about 50 nucleotides (e.g., about 15 to about 45 nucleotides, about 15 to about 40 nucleotides, about 15 to about 35 nucleotides, about 15 to about 30 nucleotides, about 15 to about 25 nucleotides, about 15 to about 20 nucleotides, about 20 to about 50 nucleotides, about 20 to about 45 nucleotides, about 20 to about 40 nucleotides, about 20 to about 35 nucleotides, about 20 to about 30 nucleotides, about 20 to about 25 nucleotides, about 25 to about 50 nucleotides, about 25 to about 45 nucleotides, about 25 to about 40 nucleotides, about 25 to about 35 nucleotides, about 25 to about 30 nucleotides, about 30 to about 50 nucleotides, about 30 to about 45 nucleotides, about 30 to about 40 nucleotides, about 30 to about 35 nucleotides, about 35 to about 50 nucleotides, about 35 to about 45 nucleotides, about 35 to about 40 nucleotides, about 40 to about 50 nucleotides, about 40 to about 45 nucleotides, or about 45 to about 50 nucleotides) long. In some embodiments, the sequence in the primer that specifically binds to the extended capture probe comprises a sequence corresponding to a contiguous sequence present in the RNA that is specifically bound to the capture domain. For example, the sequence in the primer that specifically binds to the extended capture probe corresponds to a contiguous sequence in the RNA (that is specifically bound to the capture domain) that is about 20 to about 1,000 nucleotides (e.g., about 20 to about 1000 nucleotides, about 20 to about 900 nucleotides, about 20 to about 800 nucleotides, about 20 to about 700 nucleotides, about 20 to about 600 nucleotides, about 20 to about 500 nucleotides, about 20 to about 400 nucleotides, about 20 to about 300 nucleotides, about 20 to about 200 nucleotides, about 20 to about 150 nucleotides, about 20 to about 100 nucleotides, about 20 to about 80 nucleotides, about 20 to about 60 nucleotides, about 20 to about 40 nucleotides,) 5′ to the 3′ end of the RNA that is specifically bound to the capture domain.
Primers (and groups of primers) can be designed to be specific to only a few analytes (e.g., about 2 analytes to about 20 analytes) or more. The specificity of primers depends on the design of the sequence that hybridizes to the extended capture probe. In some instances, primers can be designed to target about 100 analytes, about 500 analytes, about 1000 analytes, and even the entire genome.
In some instances, at the 5′ end, the primer further includes an adaptor sequence. In some instances, the adapter sequence in the primer can include a sequencing adapter sequence (e.g., an adapter sequence that can be used to perform sequencing using any of the exemplary sequencing methods described herein). In some embodiments, the adapter sequence can be an Illumina™ sequencing adapter sequence (e.g., via sequencing by synthesis). In some embodiments, the adapter sequence can be about 15 to about 45 nucleotides (e.g., about 15 to about 45 nucleotides, about 15 to about 40 nucleotides, about 15 to about 35 nucleotides, about 15 to about 30 nucleotides, about 15 to about 25 nucleotides, or about 15 to about 20 nucleotides, about 20 to about 45 nucleotides, about 20 to about 40 nucleotides, about 20 to about 35 nucleotides, about 20 to about 30 nucleotides, about 20 to about 25 nucleotides, about 25 to about 45 nucleotides, about 25 to about 40 nucleotides, about 25 to about 35 nucleotides, about 25 to about 30 nucleotides, about 30 to about 45 nucleotides, about 30 to about 40 nucleotides, about 30 to about 35 nucleotides, about 35 to about 45 nucleotides, about 35 to about 40 nucleotides, or about 40 to about 45 nucleotides) long. In some embodiments, the adapter sequence comprises a sequence of CCTTGGCACACCCGAGAATTCCA (SEQ ID NO:1). In some embodiments, the adapter sequence can be a universal sequence.
In some embodiments, the step of extending the 3′ end of the primer using the extended capture probe as a template, thereby generating a DNA hybridized to the extended capture probe, includes the use of a DNA polymerase, e.g., DNA polymerase I or any of the other exemplary DNA polymerases described herein or known in the art.
In some embodiments, a full-length DNA (e.g., cDNA) molecule is generated. In some embodiments, a “full-length” DNA molecule refers to the whole of the captured nucleic acid molecule. However, if a nucleic acid (e.g., RNA) was partially degraded in the tissue sample, then the captured nucleic acid molecules will not be the same length as the initial RNA in the tissue sample. In some embodiments, the 3′ end of the extended probes, e.g., first strand cDNA molecules, is modified. For example, a linker or adaptor can be ligated to the 3′ end of the extended probes. This can be achieved using single stranded ligation enzymes such as T4 RNA ligase or Circligase™ (available from Lucigen, Middleton, WI). In some embodiments, template switching oligonucleotides are used to extend cDNA in order to generate a full-length cDNA (or as close to a full-length cDNA as possible). In some embodiments, a second strand synthesis helper probe (a partially double stranded DNA molecule capable of hybridizing to the 3′ end of the extended capture probe), can be ligated to the 3′ end of the extended probe, e.g., first strand cDNA, molecule using a double stranded ligation enzyme such as T4 DNA ligase. Other enzymes appropriate for the ligation step are known in the art and include, e.g., Tth DNA ligase, Taq DNA ligase, Thermococcus sp. (strain 9° N) DNA ligase (9° N™ DNA ligase, New England Biolabs), Ampligase™ (available from Lucigen, Middleton, WI), and SplintR (available from New England Biolabs, Ipswich, MA). In some embodiments, a polynucleotide tail, e.g., a poly(A) tail, is incorporated at the 3′ end of the extended probe molecules. In some embodiments, the polynucleotide tail is incorporated using a terminal transferase active enzyme.
In some embodiments of any of the methods described herein, the releasing of the generated DNA from the extended capture probe can be performed using heat and/or a solution (e.g., a solution having an increased salt concentration).
After release of the generated DNA molecule, the resulting generated DNA molecule—as shown in
In some instances, the methods of generating a DNA molecule from the extended capture probe comprises one or more steps of heating the samples. In some instances, the heating step is performed prior to second strand synthesis. In some instances, the heating step performed prior to second strand synthesis is performed at about 98° C. In some instances, the heating step performed prior to second strand synthesis is performed from about 80° C. to about 100° C. (e.g., about 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100° C.). In some instances, the heating step is performed during second strand synthesis. In some instances, the temperature of the heating step during second strand synthesis is about 65° C. and can range from 50° C. to 80° C. (e.g., 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80° C.). In some instance, any of the heating steps can be performed from 1 to 30 minutes (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 minutes). In some instances, the buffer for second strand synthesis a Hot Start Master Mix (e.g., a Hot Start Taq 2X Master Mix; e.g., New England Biolabs®, Inc.
In some embodiments, the methods further include a determining (e.g., sequencing) step. In some instances, the determining step comprises determining the sequence of (i) all or a part of the sequence of generated DNA or a complement thereof, or (ii) all or a part of the sequence of the spatial barcode or a complement thereof. In some embodiments, the sequencing can be performed using any of the exemplary sequencing methods described herein (e.g., high throughput sequencing). In some instance, the generated DNA (e.g., the second strand molecule) can be amplified via PCR prior to library construction. The generated DNA can then be enzymatically fragmented and size-selected in order to optimize for amplicon size. P5 and P7 sequences directed to capturing the amplicons on a sequencing flowcell (Illumina™ sequencing instruments (e.g., sequencing by synthesis)) can be appended to the amplicons, i7, and i5 can be used as sample indexes, and TruSeq™ Read 2 (e.g., an RNA-seq library preparation primer) can be added via End Repair, A-tailing, Adaptor Ligation, and PCR. The cDNA fragments can then be sequenced using paired-end sequencing using TruSeq™ Read 1 and TruSeq™ Read 2 (e.g., an RNA-seq library preparation primers) as sequencing primer sites. The additional sequences are directed toward Illumina™ sequencing instruments (e.g., sequencing by synthesis) or sequencing instruments that utilize those sequences; however a skilled artisan will understand that additional or alternative sequences used by other sequencing instruments or technologies are also equally applicable for use in the aforementioned methods.
A wide variety of different sequencing methods can be used to analyze barcoded analyte. In general, sequenced polynucleotides can be, for example, nucleic acid molecules such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), including variants or derivatives thereof (e.g., single stranded DNA or DNA/RNA hybrids, and nucleic acid molecules with a nucleotide analog).
Sequencing of polynucleotides can be performed by various systems. More generally, sequencing can be performed using nucleic acid amplification, polymerase chain reaction (PCR) (e.g., digital PCR and droplet digital PCR (ddPCR), quantitative PCR, real time PCR, multiplex PCR, PCR-based single plex methods, emulsion PCR), and/or isothermal amplification. Non-limiting examples of methods for sequencing genetic material include, but are not limited to, DNA hybridization methods (e.g., Southern blotting), restriction enzyme digestion methods, Sanger sequencing methods, next-generation sequencing methods (e.g., single-molecule real-time sequencing, nanopore sequencing, and Polony sequencing), ligation methods, and microarray methods.
Methods disclosed herein can be performed on any type of sample (also interchangeably called “biological sample”). In some embodiments, the sample is a fresh tissue. In some embodiments, the sample is a frozen sample. In some embodiments, the sample was previously frozen. In some embodiments, the sample is a formalin-fixed, paraffin embedded (FFPE) sample.
Subjects from which biological samples can be obtained can be healthy or asymptomatic individuals, individuals that have or are suspected of having a disease (e.g., cancer) or a pre-disposition to a disease, and/or individuals that are in need of therapy or suspected of needing therapy. In some instances, the biological sample can include one or more diseased cells. A diseased cell can have altered metabolic properties, gene expression, protein expression, and/or morphologic features. Examples of diseases include inflammatory disorders, metabolic disorders, nervous system disorders, and cancer. In some instances, the biological sample includes cancer or tumor cells. Cancer cells can be derived from solid tumors, hematological malignancies, cell lines, or obtained as circulating tumor cells. In some instances, the biological sample is a heterogenous sample. In some instances, the biological sample is a heterogenous sample that includes tumor or cancer cells and/or stromal cells,
In certain embodiments, the cancer is squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, gastrointestinal cancer, Hodgkin's or non-Hodgkin's lymphoma, pancreatic cancer, glioblastoma, glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, colorectal cancer, endometrial carcinoma, myeloma, salivary gland carcinoma, kidney cancer, basal cell carcinoma, melanoma, prostate cancer, vulval cancer, thyroid cancer, testicular cancer, esophageal cancer, or a type of head or neck cancer. In certain embodiments, the cancer treated is desmoplastic melanoma, inflammatory breast cancer, thymoma, rectal cancer, anal cancer, or surgically treatable or non-surgically treatable brain stem glioma. In some embodiments, the subject is a human.
FFPE samples generally are heavily cross-linked and fragmented, and therefore this type of sample allows for limited RNA recovery using conventional detection techniques. In certain embodiments, methods of targeted RNA capture provided herein are less affected by RNA degradation associated with FFPE fixation than other methods (e.g., methods that take advantage of oligo-dT capture and reverse transcription of mRNA). In certain embodiments, methods provided herein enable sensitive measurement of specific genes of interest that otherwise might be missed with a whole transcriptomic approach.
In some instances, FFPE samples are stained (e.g., using H&E). The methods disclosed herein are compatible with H&E will allow for morphological context overlaid with transcriptomic analysis. However, depending on the need some samples may be stained with only a nuclear stain, such as staining a sample with only hematoxylin and not eosin, when location of a cell nucleus is needed.
In some embodiments, a biological sample (e.g. tissue section) can be fixed with methanol, stained with hematoxylin and eosin, and imaged. In some embodiments, fixing, staining, and imaging occurs before one or more probes are hybridized to the sample. Some embodiments of any of the workflows described herein can further include a destaining step (e.g., a hematoxylin and eosin destaining step), after imaging of the sample and prior to permeabilizing the sample. For example, destaining can be performed by performing one or more (e.g., one, two, three, four, or five) washing steps (e.g., one or more (e.g., one, two, three, four, or five) washing steps performed using a buffer including HCl). The images can be used to map spatial gene expression patterns back to the biological sample. A permeabilization enzyme can be used to permeabilize the biological sample directly on the slide.
In some embodiments, the FFPE sample is deparaffinized, permeabilized, equilibrated, and blocked before target probe oligonucleotides are added. In some embodiments, deparaffinization using xylenes. In some embodiments, deparaffinization includes multiple washes with xylenes. In some embodiments, deparaffinization includes multiple washes with xylenes followed by removal of xylenes using multiple rounds of graded alcohol followed by washing the sample with water. In some aspects, the water is deionized water. In some embodiments, equilibrating and blocking includes incubating the sample in a pre-Hyb buffer. In some embodiments, the pre-Hyb buffer includes yeast tRNA. In some embodiments, permeabilizing a sample includes washing the sample with a phosphate buffer. In some embodiments, the buffer is PBS. In some embodiments, the buffer is PBST.
The biological samples included herein comprise one or more analytes. Analytes can be broadly classified into one of two groups: nucleic acid analytes, and non-nucleic acid analytes.
Examples of non-nucleic acid analytes include, but are not limited to, lipids, carbohydrates, peptides, proteins, glycoproteins (N-linked or O-linked), lipoproteins, phosphoproteins, specific phosphorylated or acetylated variants of proteins, amidation variants of proteins, hydroxylation variants of proteins, methylation variants of proteins, ubiquitylation variants of proteins, sulfation variants of proteins, viral coat proteins, extracellular and intracellular proteins, antibodies, and antigen binding fragments. In some embodiments, the analyte can be an organelle (e.g., nuclei or mitochondria).
Examples of nucleic acid analytes also include RNA analytes such as various types of coding and non-coding RNA. Examples of the different types of RNA analytes include messenger RNA (mRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), microRNA (miRNA), and viral RNA. The RNA can be a transcript (e.g., present in a tissue section). The RNA can be small (e.g., less than 200 nucleic acid bases in length) or large (e.g., RNA greater than 200 nucleic acid bases in length). Small RNAs mainly include 5.8S ribosomal RNA (rRNA), 5S rRNA, transfer RNA (tRNA), microRNA (miRNA), small interfering RNA (siRNA), small nucleolar RNA (snoRNAs), Piwi-interacting RNA (piRNA), tRNA-derived small RNA (tsRNA), and small rDNA-derived RNA (srRNA). The RNA can be double-stranded RNA or single-stranded RNA. The RNA can be circular RNA. The RNA can be a bacterial rRNA (e.g., 16s rRNA or 23s rRNA).
Additional examples of analytes are disclosed in WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663, each of which is incorporated by reference in its entirety.
(i) Imaging and Staining
Prior to analyte migration and capture, in some instances, biological samples can be stained using a wide variety of stains and staining techniques. In some instances, the biological sample is a section on a slide (e.g., a 10 μm section). In some instances, the biological sample is dried after placement onto a glass slide. In some instances, the biological sample is dried at 42° C. In some instances, drying occurs for about 1 hour, about 2, hours, about 3 hours, or until the sections become transparent. In some instances, the biological sample can be dried overnight (e.g., in a desiccator at room temperature).
In some embodiments, a sample can be stained using any number of biological stains, including but not limited to, acridine orange, Bismarck brown, carmine, coomassie blue, cresyl violet, DAPI, eosin, ethidium bromide, acid fuchsine, hematoxylin, Hoechst stains, iodine, methyl green, methylene blue, neutral red, Nile blue, Nile red, osmium tetroxide, propidium iodide, rhodamine, or safranin. In some instances, the methods disclosed herein include imaging the biological sample. In some instances, imaging the sample occurs prior to deaminating the biological sample. In some instances, the sample can be stained using known staining techniques, including Can-Grunwald, Giemsa, hematoxylin and eosin (H&E), Jenner's, Leishman, Masson's trichrome, Papanicolaou, Romanowsky, silver, Sudan, Wright's, and/or Periodic Acid Schiff (PAS) staining techniques. PAS staining is typically performed after formalin or acetone fixation. In some instances, the stain is an H&E stain.
In some embodiments, the biological sample can be stained using a detectable label (e.g., radioisotopes, fluorophores, chemiluminescent compounds, bioluminescent compounds, and dyes) as described elsewhere herein. In some embodiments, a biological sample is stained using only one type of stain or one technique. In some embodiments, staining includes biological staining techniques such as H&E staining. In some embodiments, staining includes identifying analytes using fluorescently-conjugated antibodies. In some embodiments, a biological sample is stained using two or more different types of stains, or two or more different staining techniques. For example, a biological sample can be prepared by staining and imaging using one technique (e.g., H&E staining and brightfield imaging), followed by staining and imaging using another technique (e.g., IHC/IF staining and fluorescence microscopy) for the same biological sample.
In some embodiments, biological samples can be destained. Methods of destaining or discoloring a biological sample are known in the art, and generally depend on the nature of the stain(s) applied to the sample. For example, H&E staining can be destained by washing the sample in HCl, or any other acid (e.g., selenic acid, sulfuric acid, hydroiodic acid, benzoic acid, carbonic acid, malic acid, phosphoric acid, oxalic acid, succinic acid, salicylic acid, tartaric acid, sulfurous acid, trichloroacetic acid, hydrobromic acid, hydrochloric acid, nitric acid, orthophosphoric acid, arsenic acid, selenous acid, chromic acid, citric acid, hydrofluoric acid, nitrous acid, isocyanic acid, formic acid, hydrogen selenide, molybdic acid, lactic acid, acetic acid, carbonic acid, hydrogen sulfide, or combinations thereof). In some embodiments, destaining can include 1, 2, 3, 4, 5, or more washes in an acid (e.g., HCl). In some embodiments, destaining can include adding HCl to a downstream solution (e.g., permeabilization solution). In some embodiments, destaining can include dissolving an enzyme used in the disclosed methods (e.g., pepsin) in an acid (e.g., HCl) solution. In some embodiments, after destaining hematoxylin with an acid, other reagents can be added to the destaining solution to raise the pH for use in other applications. For example, SDS can be added to an acid destaining solution in order to raise the pH as compared to the acid destaining solution alone. As another example, in some embodiments, one or more immunofluorescence stains are applied to the sample via antibody coupling. Such stains can be removed using techniques such as cleavage of disulfide linkages via treatment with a reducing agent and detergent washing, chaotropic salt treatment, treatment with antigen retrieval solution, and treatment with an acidic glycine buffer. Methods for multiplexed staining and destaining are described, for example, in Bolognesi et al., J. Histochem. Cytochem. 2017; 65(8): 431-444, Lin et al., Nat Commun. 2015; 6:8390, Pirici et al., J. Histochem. Cytochem. 2009; 57:567-75, and Glass et al., J. Histochem. Cytochem. 2009; 57:899-905, the entire contents of each of which are incorporated herein by reference.
In some embodiments, immunofluorescence or immunohistochemistry protocols (direct and indirect staining techniques) can be performed as a part of, or in addition to, the exemplary spatial workflows presented herein. For example, tissue sections can be fixed according to methods described herein. The biological sample can be transferred to an array (e.g., capture probe array), wherein analytes (e.g., proteins) are probed using immunofluorescence protocols. For example, the sample can be rehydrated, blocked, and permeabilized (3×SSC, 2% BSA, 0.1% Triton X, 1 U/μl RNAse inhibitor for 10 minutes at 4° C.) before being stained with fluorescent primary antibodies (1:100 in 3×SSC, 2% BSA, 0.1% Triton X, 1 U/μl RNAse inhibitor for 30 minutes at 4° C.). The biological sample can be washed, coverslipped (in glycerol+1 U/μl RNAse inhibitor), imaged (e.g., using a confocal microscope or other apparatus capable of fluorescent detection), washed, and processed according to analyte capture or spatial workflows described herein.
In some instances, a glycerol solution and a cover slip can be added to the sample. In some instances, the glycerol solution can include a counterstain (e.g., DAPI).
As used herein, an antigen retrieval buffer can improve antibody capture in IF/IHC protocols. An exemplary protocol for antigen retrieval can be preheating the antigen retrieval buffer (e.g., to 95° C.), immersing the biological sample in the heated antigen retrieval buffer for a predetermined time, and then removing the biological sample from the antigen retrieval buffer and washing the biological sample.
In some embodiments, optimizing permeabilization can be useful for identifying intracellular analytes. Permeabilization optimization can include selection of permeabilization agents, concentration of permeabilization agents, and permeabilization duration. Tissue permeabilization is discussed elsewhere herein.
In some embodiments, blocking an array and/or a biological sample in preparation of labeling the biological sample decreases nonspecific binding of the antibodies to the array and/or biological sample (decreases background). Some embodiments provide for blocking buffers/blocking solutions that can be applied before and/or during application of the label, wherein the blocking buffer can include a blocking agent, and optionally a surfactant and/or a salt solution. In some embodiments, a blocking agent can be bovine serum albumin (BSA), serum, gelatin (e.g., fish gelatin), milk (e.g., non-fat dry milk), casein, polyethylene glycol (PEG), polyvinyl alcohol (PVA), or polyvinylpyrrolidone (PVP), biotin blocking reagent, a peroxidase blocking reagent, levamisole, Carnoy's solution, glycine, lysine, sodium borohydride, pontamine sky blue, Sudan Black, trypan blue, FITC blocking agent, and/or acetic acid. The blocking buffer/blocking solution can be applied to the array and/or biological sample prior to and/or during labeling (e.g., application of fluorophore-conjugated antibodies) to the biological sample.
(ii) Preparation of Sample for Analyte Migration and Capture
In some instances, the biological sample is deparaffinized. Deparaffinization can be achieved using any method known in the art. For example, in some instances, the biological samples is treated with a series of washes that include xylene and various concentrations of ethanol. In some instances, methods of deparaffinization include treatment of xylene (e.g., three washes at 5 minutes each). In some instances, the methods further include treatment with ethanol (e.g., 100% ethanol, two washes 10 minutes each; 95% ethanol, two washes 10 minutes each; 70% ethanol, two washes 10 minutes each; 50% ethanol, two washes 10 minutes each). In some instances, after ethanol washes, the biological sample can be washed with deionized water (e.g., two washes for 5 minutes each). It is appreciated that one skilled in the art can adjust these methods to optimize deparaffinization.
In some instances, the biological sample is decrosslinked. In some instances, the biological sample is decrosslinked in a solution containing TE buffer (comprising Tris and EDTA). In some instances, the TE buffer is basic (e.g., at a pH of about 9). In some instances, decrosslinking occurs at about 50° C. to about 80° C. In some instances, decrosslinking occurs at about 70° C. In some instances, decrosslinking occurs for about 1 hour at 70° C. Just prior to decrosslinking, the biological sample can be treated with an acid (e.g., 0.1M HCl for about 1 minute). After the decrosslinking step, the biological sample can be washed (e.g., with 1×PBST).
In some instances, the methods of preparing a biological sample for analyte capture include permeabilizing the sample. In some instances, the biological sample is permeabilized using a phosphate buffer. In some instances, the phosphate buffer is PBS (e.g., 1×PBS). In some instances, the phosphate buffer is PBST (e.g., 1×PBST). In some instances, the permeabilization step is performed multiple times (e.g., 3 times at 5 minutes each).
In some instances, the methods of preparing a biological sample for analyte capture include steps of equilibrating and blocking the biological sample. In some instances, equilibrating is performed using a pre-hybridization (pre-Hyb) buffer. In some instances, the pre-Hyb buffer is RNase-free. In some instances, the pre-Hyb buffer contains no bovine serum albumin (BSA), solutions like Denhardt's, or other potentially nuclease-contaminated biological materials.
In some instances, the equilibrating step is performed multiple times (e.g., 2 times at 5 minutes each; 3 times at 5 minutes each). In some instances, the biological sample is blocked with a blocking buffer. In some instances, the blocking buffer includes a carrier such as tRNA, for example yeast tRNA such as from brewer's yeast (e.g., at a final concentration of 10-20 μg/mL). In some instances, blocking can be performed for 5, 10, 15, 20, 25, or 30 minutes.
Any of the foregoing steps can be optimized for performance. For example, one can vary the temperature. In some instances, the pre-hybridization methods are performed at room temperature. In some instances, the pre-hybridization methods are performed at 4° C. (in some instances, varying the timeframes provided herein).
Also provided herein are reaction mixtures that include: an array comprising a plurality of capture probes, where a capture probe of the plurality comprises a capture domain (e.g., any of the exemplary capture domains described herein or known in the art) that binds specifically to an RNA (e.g., any of the exemplary types of RNA described herein or known in the art) and a spatial barcode; a reverse transcriptase (e.g., any of the exemplary reverse transcriptases described herein or known in the art); RNAse H or a functional equivalent thereof, and a DNA polymerase (e.g., any of the exemplary DNA polymerases described herein or known in the art).
Also provided herein are kits that include: an array comprising a plurality of capture probes, where a capture probe of the plurality comprises a capture domain (e.g., any of the exemplary capture domains described herein or known in the art) that binds specifically to an RNA (e.g., any of the exemplary types of RNA described herein or known in the art) and a spatial barcode; a reverse transcriptase (e.g., any of the exemplary reverse transcriptases described herein or known in the art); RNAse H or a functional equivalent thereof, and a DNA polymerase (e.g., any of the exemplary DNA polymerases described herein or known in the art).
In some embodiments of any of the reaction mixtures or kits described herein, the capture domain can be any of the capture domains described herein. In some embodiments, the capture domain can comprise a poly(T) sequence. In some embodiments, the capture domain does not comprise a poly(T) sequence. In some embodiments, the capture domain comprises a sequence that is substantially complementary to a contiguous sequence present in the RNA. The capture domain can be about 5 to about 40 nucleotides (e.g., or any of the subranges of this range described herein) in length.
In some embodiments, the capture domain is positioned 3′ relative to the spatial barcode in the capture probe. In some embodiments of any of the reaction mixtures or kits provided herein, the capture probe further includes a unique molecular identifier, a cleavage domain (e.g., any of the exemplary cleavage domains described herein), or both.
In some embodiments of any of the reaction mixtures or kits described herein, the plurality of capture probes are affixed (i.e., attached) to an array. In some embodiments of any of the reaction mixtures or kits described herein, the array is a slide (e.g., a slide comprising beads or a slide comprising wells (e.g., microwells)). An array can also have one or more of any of the exemplary characteristics of arrays described herein.
Some embodiments of any of the reaction mixtures or kits described herein can further include a primer comprising in a 5′ to a 3′ direction: (i) an adapter sequence (e.g., any of the exemplary adapter sequences described herein) and (ii) a sequence or a complement thereof present in a 5′ region of the RNA that is specifically bound to the capture domain.
The sequence or complement thereof present in a 5′ region of the RNA that is specifically bound to the capture domain can be about 15 to about 50 nucleotides (e.g., or any of the subranges of this range described herein) long. In some embodiments, the sequence present in the 5′ region of the RNA (that is specifically bound to the capture domain) is about 20 to about 1,000 nucleotides (e.g., or any of the subranges of this range described herein) 5′ to the 3′ end of the RNA that is specifically bound to the capture domain.
Some embodiments of the kits described herein further include a solution that can be used to dissociate two strands of DNA (e.g., an extended capture probe and a DNA that is hybridized to the extended capture probe). In some embodiments, the solution that can be used to dissociate two strands of DNA can have an increased salt concentration.
In some embodiments of any of the reaction mixtures described herein, the reaction mixture can include an RNA from a biological sample (e.g., an mRNA or any of the other types of RNA described herein or known in the art).
In some embodiments of any of the kits or reaction mixtures described herein, the kit or reaction mixture can further include one or more permeabilization reagents (e.g., one or more of any of the permeabilization reagents described herein).
Some embodiments of any of the kits described herein can further include a staining agent. In some embodiments, a staining agent can include an optical label, e.g., a fluorescent, a radioactive, a chemiluminescent, a calorimetric, or a colorimetric detectable label. In some embodiments, a staining agent can be a fluorescent antibody directed to a target analyte (e.g., cell surface or intracellular proteins). In some embodiments, a staining agent can be a chemical stain, such as hematoxylin and eosin (H&E) or periodic acid-schiff (PAS).
Some embodiments of any of the kits described herein can further include instructions for performing any of the methods described herein.
It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.
An experiment was performed to demonstrate the performance of the methods described herein in determining the location of 20 exemplary RNA molecules in FFPE mouse brain tissue.
Briefly, a FFPE mouse brain tissue section was placed on a spatial array comprising a plurality of capture probes. The tissue section was permeabilized to release mRNA from the sample. After permeabilization, mRNA molecules were captured by capture probes via hybridization of the poly(A) tail of the mRNA to the poly(T) sequence of the capture probe. The capture probe was extended using a polymerase to generate a first strand cDNA molecule, using the mRNA bound to capture domain as a template. After cDNA synthesis, the RNA that was used as a template for first strand cDNA synthesis was digested by RNase H, leaving a single-stranded extended capture probe. The extended capture probes were contacted with primers comprising an adapter sequence (e.g., a second sequencing adapter sequence; e.g., SEQ ID NO:1 (CCTTGGCACACCCGAGAATTCCA)) and a sequence that specifically binds to the extended capture probe (e.g., binding to the target sequences shown in Table 2). The primers were extended using the extended capture probe as a template, thereby generating a DNA molecule that is hybridized to the extended capture probe and that is termed the second strand. The second strands were recovered and were used to prepare libraries for subsequent processing and analysis (e.g., sequencing using any of the methods described herein, e.g., high throughput sequencing, e.g., Illumina™ sequencing (e.g., sequencing by synthesis)).
Tables 1 and 2 show the list of the 20 exemplary RNA sequences and the sequences that specifically binds to the extended capture probe (“target sequence;” SEQ ID NOs: 2-21) using a primer sequence (e.g., one of SEQ ID NOs: 22-41) in Table 2. The captured sequences for each analyte are shown in Table 3 (SEQ ID NOs: 42-61).
All primers include sequences that correspond to sequences in the 3′ UTR of the target RNAs, except two primers which include a sequence that spans an exon and the 3′ UTR of the target RNA. The four groups of genes shown in Table 1 are based on varying levels of analyte expression and UMI detection, with Group 1 having the highest expression and abundance of detection. The primer sequences were blasted and checked for self-dimer and cross-primer dimers. The data in Table 1 demonstrate the ability of these methods to add a sequencing adapter to a 5′ end of a DNA that is complementary to the extended capture probe (“the second strand”), and the subsequent successful sequencing of the second strand. The 20 exemplary RNAs listed in Table 1 include mRNAs for the housekeeping genes of GAPDH, ACTB, B2M, and FGB.
Given the ability to detect a cohort of genes from Example 1, optimal conditions for second strand synthesis were determined. After analyte capture, capture probe extension, and analyte digestion as described in Example 1, second strand cDNA synthesis was performed for 30 minutes on three-day old tissue sections. The amplification reaction was carried out for 22 cycles. Table 4 shows four experimental condition groups (Groups A-D) that were tested while varying the primer concentration and whether the tissue was removed.
As shown in
These data demonstrate a proof of concept that one can optimize the conditions (e.g., with or without tissue removal; primer concentration) to increase analyte detection. Because of the results in Group A, additional analysis was performed on this Group. In particular, a sequencing comparison was performed looking at detection of the original cDNA compared to detection of the targeted second strand (TSS). As shown in Tables 6 and 7, sequencing results for the targeted second strand indicated that Group A (compared to the original cDNA) had an increase in sequencing saturation, an increase in reads mapped confidently to intergenic regions, an increase in cDNA PCR Duplication, and a decrease in the fraction of UMI counts that were mapped to ribosomal protein. Taken together, these data provide proof of concept that the methods using second strand amplification using an adaptor and a primer as disclosed herein readily target and detect sequences of interest.
Using a fresh mouse brain tissue sample, parameters were adjusted to test whether using Hot Start Taq DNA Polymerase would affect target-specific detection using a primer comprising an adaptor as described herein. Briefly, a fresh mouse brain tissue sample was sectioned and placed on an array comprising a plurality of capture probes. After permeabilization, analyte capture, capture probe extension, and second strand synthesis was performed for 15 minutes followed by 22 cycles of PCR.
Second strand synthesis was performed using either second strand mix (i.e., the same buffer from Examples 1 and 2), or using Hot Start AMP Mix Buffer. See Table 7. As shown in
The experimental settings were modified to adjust the temperature either before extension (to 98° C.) or during extension (to 65° C.). See Table 9.
As shown in
Taken together, these data show that using a Hot-Start Amp Mix Buffer while increasing the temperature before and during second strand synthesis can increase specific detection of target analytes while decreasing off-target capture.
It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.
This application claims priority to U.S. Provisional Patent Application No. 63/048,584, filed on Jul. 6, 2020, the contents of which are incorporated herein by reference in its entirety.
| Number | Name | Date | Kind |
|---|---|---|---|
| 4683195 | Mullis | Jul 1987 | A |
| 4683202 | Mullis | Jul 1987 | A |
| 4800159 | Mullis | Jan 1989 | A |
| 4883867 | Lee | Nov 1989 | A |
| 4965188 | Mullis | Oct 1990 | A |
| 4988617 | Landegren et al. | Jan 1991 | A |
| 5002882 | Lunnen | Mar 1991 | A |
| 5130238 | Malek | Jul 1992 | A |
| 5308751 | Ohkawa | May 1994 | A |
| 5321130 | Yue | Jun 1994 | A |
| 5410030 | Yue | Apr 1995 | A |
| 5436134 | Haugland | Jul 1995 | A |
| 5455166 | Walker | Oct 1995 | A |
| 5494810 | Barany et al. | Feb 1996 | A |
| 5503980 | Cantor | Apr 1996 | A |
| 5512439 | Hornes | Apr 1996 | A |
| 5512462 | Cheng | Apr 1996 | A |
| 5582977 | Yue | Dec 1996 | A |
| 5599675 | Brenner | Feb 1997 | A |
| 5641658 | Adams | Jun 1997 | A |
| 5648245 | Fire et al. | Jul 1997 | A |
| 5658751 | Yue | Aug 1997 | A |
| 5695940 | Drmanac et al. | Dec 1997 | A |
| 5750341 | Macevicz | May 1998 | A |
| 5763175 | Brenner | Jun 1998 | A |
| 5830711 | Barany et al. | Nov 1998 | A |
| 5837832 | Chee et al. | Nov 1998 | A |
| 5854033 | Lizardi | Dec 1998 | A |
| 5863753 | Haugland | Jan 1999 | A |
| 5871921 | Landegren et al. | Feb 1999 | A |
| 5912148 | Eggerding | Jun 1999 | A |
| 5925545 | Reznikoff et al. | Jul 1999 | A |
| 5928906 | Koester et al. | Jul 1999 | A |
| 5958775 | Wickstrrom | Sep 1999 | A |
| 5962272 | Chenchik et al. | Oct 1999 | A |
| 5965443 | Reznikoff et al. | Oct 1999 | A |
| 6013440 | Lipshutz | Jan 2000 | A |
| 6027889 | Barany et al. | Feb 2000 | A |
| 6054274 | Sampson et al. | Apr 2000 | A |
| 6060240 | Kamb et al. | May 2000 | A |
| 6130073 | Eggerding | Oct 2000 | A |
| 6143496 | Brown | Nov 2000 | A |
| 6153389 | Haarer | Nov 2000 | A |
| 6159736 | Reznikoff et al. | Dec 2000 | A |
| 6165714 | Lane et al. | Dec 2000 | A |
| 6210891 | Nyren | Apr 2001 | B1 |
| 6210894 | Brennan | Apr 2001 | B1 |
| 6214587 | Dattagupta | Apr 2001 | B1 |
| 6251639 | Kurn | Jun 2001 | B1 |
| 6258568 | Nyren | Jul 2001 | B1 |
| 6266459 | Walt | Jul 2001 | B1 |
| 6268148 | Barany et al. | Jul 2001 | B1 |
| 6274320 | Rothberg | Aug 2001 | B1 |
| 6291180 | Chu | Sep 2001 | B1 |
| 6291187 | Kingsmore et al. | Sep 2001 | B1 |
| 6300063 | Lipshutz et al. | Oct 2001 | B1 |
| 6309824 | Drmanac | Oct 2001 | B1 |
| 6323009 | Lasken et al. | Nov 2001 | B1 |
| 6344316 | Lockhart | Feb 2002 | B1 |
| 6344329 | Lizardi et al. | Feb 2002 | B1 |
| 6355431 | Chee | Mar 2002 | B1 |
| 6368801 | Faruqi | Apr 2002 | B1 |
| 6401267 | Drmanac | Jun 2002 | B1 |
| 6404907 | Gilchrist | Jun 2002 | B1 |
| 6432360 | Church et al. | Aug 2002 | B1 |
| 6503713 | Rana | Jan 2003 | B1 |
| 6506561 | Cheval et al. | Jan 2003 | B1 |
| 6534266 | Singer | Mar 2003 | B1 |
| 6544732 | Chee | Apr 2003 | B1 |
| 6573043 | Cohen et al. | Jun 2003 | B1 |
| 6579695 | Lambalot | Jun 2003 | B1 |
| 6620584 | Chee | Sep 2003 | B1 |
| 6632641 | Brennan | Oct 2003 | B1 |
| 6737236 | Pieken et al. | May 2004 | B1 |
| 6770441 | Dickinson | Aug 2004 | B2 |
| 6773886 | Kaufman | Aug 2004 | B2 |
| 6787308 | Balasubramanian | Sep 2004 | B2 |
| 6797470 | Barany et al. | Sep 2004 | B2 |
| 6800453 | Labaer | Oct 2004 | B2 |
| 6812005 | Fan et al. | Nov 2004 | B2 |
| 6828100 | Ronaghi | Dec 2004 | B1 |
| 6833246 | Balasubramanian | Dec 2004 | B2 |
| 6852487 | Barany et al. | Feb 2005 | B1 |
| 6859570 | Walt | Feb 2005 | B2 |
| 6864052 | Drmanac | Mar 2005 | B1 |
| 6867028 | Janulaitis | Mar 2005 | B2 |
| 6872816 | Hall et al. | Mar 2005 | B1 |
| 6875572 | Prudent et al. | Apr 2005 | B2 |
| 6890741 | Fan et al. | May 2005 | B2 |
| 6897023 | Fu | May 2005 | B2 |
| 6913881 | Aizenstein et al. | Jul 2005 | B1 |
| 6942968 | Dickinson et al. | Sep 2005 | B1 |
| 7011944 | Prudent et al. | Mar 2006 | B2 |
| 7057026 | Barnes | Jun 2006 | B2 |
| 7083980 | Reznikoff et al. | Aug 2006 | B2 |
| 7115400 | Adessi | Oct 2006 | B1 |
| 7118883 | Inoue | Oct 2006 | B2 |
| 7166431 | Chee et al. | Jan 2007 | B2 |
| 7192735 | Lambalot | Mar 2007 | B2 |
| 7211414 | Hardin | May 2007 | B2 |
| 7255994 | Lao | Aug 2007 | B2 |
| 7258976 | Mitsuhashi | Aug 2007 | B2 |
| 7282328 | Kong et al. | Oct 2007 | B2 |
| 7297518 | Quake | Nov 2007 | B2 |
| 7329492 | Hardin | Feb 2008 | B2 |
| 7358047 | Hafner et al. | Apr 2008 | B2 |
| 7361488 | Fan et al. | Apr 2008 | B2 |
| 7378242 | Hurt | May 2008 | B2 |
| 7393665 | Brenner | Jul 2008 | B2 |
| 7405281 | Xu | Jul 2008 | B2 |
| 7407757 | Brenner | Aug 2008 | B2 |
| 7473767 | Dimitrov | Jan 2009 | B2 |
| 7499806 | Kermani et al. | Mar 2009 | B2 |
| 7537897 | Brenner | May 2009 | B2 |
| 7563576 | Chee | Jul 2009 | B2 |
| 7579153 | Brenner | Aug 2009 | B2 |
| 7582420 | Oliphant et al. | Sep 2009 | B2 |
| 7601498 | Mao | Oct 2009 | B2 |
| 7608434 | Reznikoff et al. | Oct 2009 | B2 |
| 7611869 | Fan | Nov 2009 | B2 |
| 7635566 | Brenner | Dec 2009 | B2 |
| 7666612 | Johnsson | Feb 2010 | B2 |
| 7674752 | He | Mar 2010 | B2 |
| 7709198 | Luo et al. | May 2010 | B2 |
| 7776547 | Roth | Aug 2010 | B2 |
| 7776567 | Mao | Aug 2010 | B2 |
| 7803943 | Mao | Sep 2010 | B2 |
| 7888009 | Barany et al. | Feb 2011 | B2 |
| 7892747 | Barany et al. | Feb 2011 | B2 |
| 7910304 | Drmanac | Mar 2011 | B2 |
| 7914981 | Barany et al. | Mar 2011 | B2 |
| 7955794 | Shen et al. | Jun 2011 | B2 |
| 7960119 | Chee | Jun 2011 | B2 |
| 7985565 | Mayer et al. | Jul 2011 | B2 |
| 8003354 | Shen et al. | Aug 2011 | B2 |
| 8076063 | Fan | Dec 2011 | B2 |
| 8092784 | Mao | Jan 2012 | B2 |
| 8148068 | Brenner | Apr 2012 | B2 |
| 8206917 | Chee | Jun 2012 | B2 |
| 8268554 | Schallmeiner | Sep 2012 | B2 |
| 8288103 | Oliphant | Oct 2012 | B2 |
| 8288122 | O'Leary et al. | Oct 2012 | B2 |
| 8383338 | Kitzman | Feb 2013 | B2 |
| 8431691 | McKernan et al. | Apr 2013 | B2 |
| 8460865 | Chee | Jun 2013 | B2 |
| 8481257 | Van Eijk | Jul 2013 | B2 |
| 8481258 | Church et al. | Jul 2013 | B2 |
| 8481292 | Casbon | Jul 2013 | B2 |
| 8481698 | Lieberman et al. | Jul 2013 | B2 |
| 8507204 | Pierce et al. | Aug 2013 | B2 |
| 8519115 | Webster et al. | Aug 2013 | B2 |
| 8551710 | Bernitz et al. | Oct 2013 | B2 |
| 8568979 | Stuelpnagel et al. | Oct 2013 | B2 |
| 8586310 | Mitra | Nov 2013 | B2 |
| 8597891 | Barany et al. | Dec 2013 | B2 |
| 8603743 | Liu et al. | Dec 2013 | B2 |
| 8604182 | Luo et al. | Dec 2013 | B2 |
| 8614073 | Van Eijk | Dec 2013 | B2 |
| 8624016 | Barany et al. | Jan 2014 | B2 |
| 8685889 | Van Eijk | Apr 2014 | B2 |
| 8741564 | Seligmann | Jun 2014 | B2 |
| 8741606 | Casbon | Jun 2014 | B2 |
| 8771950 | Church et al. | Jul 2014 | B2 |
| 8785353 | Van Eijk | Jul 2014 | B2 |
| 8790873 | Namsaraev et al. | Jul 2014 | B2 |
| 8809238 | Livak et al. | Aug 2014 | B2 |
| 8815512 | Van Eijk | Aug 2014 | B2 |
| 8835358 | Fodor | Sep 2014 | B2 |
| 8865410 | Shendure | Oct 2014 | B2 |
| 8906626 | Oliphant et al. | Dec 2014 | B2 |
| 8911945 | Van Eijk | Dec 2014 | B2 |
| 8936912 | Mitra | Jan 2015 | B2 |
| 8951726 | Luo et al. | Feb 2015 | B2 |
| 8951728 | Rasmussen | Feb 2015 | B2 |
| 8986926 | Ferree et al. | Mar 2015 | B2 |
| 9005891 | Sinicropi et al. | Apr 2015 | B2 |
| 9005935 | Belyaev | Apr 2015 | B2 |
| 9023768 | Van Eijk | May 2015 | B2 |
| 9062348 | Van Eijk | Jun 2015 | B1 |
| 9080210 | Van Eijk | Jul 2015 | B2 |
| 9194001 | Brenner | Nov 2015 | B2 |
| 9201063 | Sood et al. | Dec 2015 | B2 |
| 9273349 | Nguyen et al. | Mar 2016 | B2 |
| 9290808 | Fodor | Mar 2016 | B2 |
| 9290809 | Fodor | Mar 2016 | B2 |
| 9328383 | Van Eijk | May 2016 | B2 |
| 9334536 | Van Eijk | May 2016 | B2 |
| 9371563 | Geiss et al. | Jun 2016 | B2 |
| 9371598 | Chee | Jun 2016 | B2 |
| 9376716 | Van Eijk | Jun 2016 | B2 |
| 9376717 | Gao et al. | Jun 2016 | B2 |
| 9376719 | Eijk | Jun 2016 | B2 |
| 9416409 | Hayden | Aug 2016 | B2 |
| 9447459 | Van Eijk | Sep 2016 | B2 |
| 9453256 | Van Eijk | Sep 2016 | B2 |
| 9493820 | Van Eijk | Nov 2016 | B2 |
| 9506061 | Brown et al. | Nov 2016 | B2 |
| 9512422 | Barnard et al. | Dec 2016 | B2 |
| 9574230 | Van Eijk | Feb 2017 | B2 |
| 9593365 | Frisen et al. | Mar 2017 | B2 |
| 9598728 | Barany et al. | Mar 2017 | B2 |
| 9624538 | Church et al. | Apr 2017 | B2 |
| 9644204 | Hindson et al. | May 2017 | B2 |
| 9657335 | Van Eijk | May 2017 | B2 |
| 9670542 | Van Eijk | Jun 2017 | B2 |
| 9694361 | Bharadwaj | Jul 2017 | B2 |
| 9702004 | Van Eijk | Jul 2017 | B2 |
| 9714446 | Webster et al. | Jul 2017 | B2 |
| 9714937 | Dunaway | Jul 2017 | B2 |
| 9727810 | Fodor et al. | Aug 2017 | B2 |
| 9745627 | Van Eijk | Aug 2017 | B2 |
| 9777324 | Van Eijk | Oct 2017 | B2 |
| 9783841 | Nolan et al. | Oct 2017 | B2 |
| 9790476 | Gloeckner et al. | Oct 2017 | B2 |
| 9816134 | Namsaraev | Nov 2017 | B2 |
| 9834814 | Peter et al. | Dec 2017 | B2 |
| 9850536 | Oliphant et al. | Dec 2017 | B2 |
| 9856521 | Stevens et al. | Jan 2018 | B2 |
| 9868979 | Chee et al. | Jan 2018 | B2 |
| 9879313 | Chee et al. | Jan 2018 | B2 |
| 9896721 | Van Eijk | Feb 2018 | B2 |
| 9898576 | Van Eijk | Feb 2018 | B2 |
| 9898577 | Van Eijk | Feb 2018 | B2 |
| 9902991 | Sinicropi et al. | Feb 2018 | B2 |
| 9909167 | Samusik et al. | Mar 2018 | B2 |
| 9938566 | Shepard et al. | Apr 2018 | B2 |
| 9957550 | Yeakley et al. | May 2018 | B2 |
| 10002316 | Fodor et al. | Jun 2018 | B2 |
| 10023907 | Van Eijk | Jul 2018 | B2 |
| 10030261 | Frisen et al. | Jul 2018 | B2 |
| 10035992 | Gloeckner et al. | Jul 2018 | B2 |
| 10041949 | Bendall et al. | Aug 2018 | B2 |
| 10059989 | Giresi et al. | Aug 2018 | B2 |
| 10059990 | Boyden et al. | Aug 2018 | B2 |
| 10095832 | Van Eijk | Oct 2018 | B2 |
| 10144966 | Cantor | Dec 2018 | B2 |
| 10208982 | Bannish et al. | Feb 2019 | B2 |
| 10227639 | Levner et al. | Mar 2019 | B2 |
| 10266876 | Cai et al. | Apr 2019 | B2 |
| 10273541 | Hindson et al. | Apr 2019 | B2 |
| 10308982 | Chee | Jun 2019 | B2 |
| 10357771 | Bharadwaj | Jul 2019 | B2 |
| 10370698 | Nolan et al. | Aug 2019 | B2 |
| 10415080 | Dunaway et al. | Sep 2019 | B2 |
| 10465235 | Gullberg et al. | Nov 2019 | B2 |
| 10472669 | Chee | Nov 2019 | B2 |
| 10480022 | Chee | Nov 2019 | B2 |
| 10480029 | Bent et al. | Nov 2019 | B2 |
| 10494667 | Chee | Dec 2019 | B2 |
| 10495554 | Deisseroth et al. | Dec 2019 | B2 |
| 10501777 | Beechem et al. | Dec 2019 | B2 |
| 10501791 | Church et al. | Dec 2019 | B2 |
| 10510435 | Cai et al. | Dec 2019 | B2 |
| 10544403 | Gloeckner et al. | Jan 2020 | B2 |
| 10550429 | Harada et al. | Feb 2020 | B2 |
| 10590244 | Delaney et al. | Mar 2020 | B2 |
| 10633648 | Seelig et al. | Apr 2020 | B2 |
| 10640816 | Beechem et al. | May 2020 | B2 |
| 10640826 | Church et al. | May 2020 | B2 |
| 10669569 | Gullberg et al. | Jun 2020 | B2 |
| 10724078 | Van Driel et al. | Jul 2020 | B2 |
| 10725027 | Bell | Jul 2020 | B2 |
| 10774372 | Chee et al. | Sep 2020 | B2 |
| 10774374 | Frisen et al. | Sep 2020 | B2 |
| 10787701 | Chee | Sep 2020 | B2 |
| 10815519 | Husain et al. | Oct 2020 | B2 |
| 10829803 | Terbrueggen et al. | Nov 2020 | B2 |
| 10844426 | Daugharthy et al. | Nov 2020 | B2 |
| 10858698 | Church et al. | Dec 2020 | B2 |
| 10858702 | Lucero et al. | Dec 2020 | B2 |
| 10913975 | So et al. | Feb 2021 | B2 |
| 10914730 | Chee et al. | Feb 2021 | B2 |
| 10927403 | Chee et al. | Feb 2021 | B2 |
| 10961566 | Chee | Mar 2021 | B2 |
| 11001879 | Chee | May 2021 | B1 |
| 11008607 | Chee | May 2021 | B2 |
| 11046996 | Chee et al. | Jun 2021 | B1 |
| 11067567 | Chee | Jul 2021 | B2 |
| 11104936 | Zhang et al. | Aug 2021 | B2 |
| 11118216 | Koshinsky et al. | Sep 2021 | B2 |
| 11156603 | Chee | Oct 2021 | B2 |
| 11162132 | Frisen et al. | Nov 2021 | B2 |
| 11208684 | Chee | Dec 2021 | B2 |
| 11286515 | Chee et al. | Mar 2022 | B2 |
| 11293917 | Chee | Apr 2022 | B2 |
| 11299774 | Frisen et al. | Apr 2022 | B2 |
| 11313856 | Chee | Apr 2022 | B2 |
| 11332790 | Chell et al. | May 2022 | B2 |
| 11352659 | Frisen et al. | Jun 2022 | B2 |
| 11352667 | Hauling et al. | Jun 2022 | B2 |
| 11359228 | Chee et al. | Jun 2022 | B2 |
| 11365442 | Chee | Jun 2022 | B2 |
| 11371086 | Chee | Jun 2022 | B2 |
| 11384386 | Chee | Jul 2022 | B2 |
| 11390912 | Frisen et al. | Jul 2022 | B2 |
| 11401545 | Chee | Aug 2022 | B2 |
| 11407992 | Dadhwal | Aug 2022 | B2 |
| 11408029 | Katiraee et al. | Aug 2022 | B2 |
| 11434524 | Ramachandran Iyer et al. | Sep 2022 | B2 |
| 11459607 | Terry et al. | Oct 2022 | B1 |
| 11479809 | Frisen et al. | Oct 2022 | B2 |
| 11479810 | Chee | Oct 2022 | B1 |
| 11492612 | Dadhwal | Nov 2022 | B1 |
| 11501440 | Weisenfeld et al. | Nov 2022 | B2 |
| 11505828 | Chell et al. | Nov 2022 | B2 |
| 11512308 | Gallant et al. | Nov 2022 | B2 |
| 11519022 | Chee | Dec 2022 | B2 |
| 11519033 | Schnall-Levin et al. | Dec 2022 | B2 |
| 11519138 | Meier | Dec 2022 | B2 |
| 11530438 | Persson et al. | Dec 2022 | B2 |
| 11535887 | Gallant et al. | Dec 2022 | B2 |
| 11542543 | Chee | Jan 2023 | B2 |
| 11549138 | Chee | Jan 2023 | B2 |
| 11560587 | Chee | Jan 2023 | B2 |
| 11560592 | Chew et al. | Jan 2023 | B2 |
| 11560593 | Chell et al. | Jan 2023 | B2 |
| 11592447 | Uytingco et al. | Feb 2023 | B2 |
| 11608498 | Gallant et al. | Mar 2023 | B2 |
| 11608520 | Galonska et al. | Mar 2023 | B2 |
| 11613773 | Frisen et al. | Mar 2023 | B2 |
| 11618897 | Kim et al. | Apr 2023 | B2 |
| 11618918 | Chee et al. | Apr 2023 | B2 |
| 11624063 | Dadhwal | Apr 2023 | B2 |
| 11624086 | Uytingco et al. | Apr 2023 | B2 |
| 11634756 | Chee | Apr 2023 | B2 |
| 11649485 | Yin et al. | May 2023 | B2 |
| 11661626 | Katiraee et al. | May 2023 | B2 |
| 11680260 | Kim et al. | Jun 2023 | B2 |
| 11692218 | Engblom et al. | Jul 2023 | B2 |
| 11702693 | Bharadwaj | Jul 2023 | B2 |
| 11702698 | Stoeckius | Jul 2023 | B2 |
| 11732292 | Chee | Aug 2023 | B2 |
| 11732299 | Ramachandran Iyer | Aug 2023 | B2 |
| 11732300 | Bava | Aug 2023 | B2 |
| 11733238 | Chee | Aug 2023 | B2 |
| 11739372 | Frisen et al. | Aug 2023 | B2 |
| 11739381 | Chew et al. | Aug 2023 | B2 |
| 11753673 | Chew et al. | Sep 2023 | B2 |
| 11753674 | Chee et al. | Sep 2023 | B2 |
| 11753675 | Ramachandran Iyer | Sep 2023 | B2 |
| 11761030 | Chee | Sep 2023 | B2 |
| 11761038 | Stoeckius | Sep 2023 | B1 |
| 11767550 | Chee | Sep 2023 | B2 |
| 11768175 | Kim et al. | Sep 2023 | B1 |
| 11773433 | Gallant et al. | Oct 2023 | B2 |
| 11781130 | Dadhwal | Oct 2023 | B2 |
| 11788122 | Frisen et al. | Oct 2023 | B2 |
| 11795498 | Frisen et al. | Oct 2023 | B2 |
| 11795507 | Chell et al. | Oct 2023 | B2 |
| 11808769 | Uytingco et al. | Nov 2023 | B2 |
| 11821024 | Chee et al. | Nov 2023 | B2 |
| 11821035 | Bent et al. | Nov 2023 | B1 |
| 11827935 | Ramachandran Iyer et al. | Nov 2023 | B1 |
| 11835462 | Bava | Dec 2023 | B2 |
| 11840687 | Gallant et al. | Dec 2023 | B2 |
| 11840724 | Chew et al. | Dec 2023 | B2 |
| 11845979 | Engblom et al. | Dec 2023 | B2 |
| 11859178 | Gallant et al. | Jan 2024 | B2 |
| 11866767 | Uytingco et al. | Jan 2024 | B2 |
| 11866770 | Chee | Jan 2024 | B2 |
| 11873482 | Kim et al. | Jan 2024 | B2 |
| 11891654 | Alvarado Martinez et al. | Feb 2024 | B2 |
| 11898205 | Bava | Feb 2024 | B2 |
| 11926822 | Gohil et al. | Mar 2024 | B1 |
| 11926863 | Boutet | Mar 2024 | B1 |
| 11926867 | Yin et al. | Mar 2024 | B2 |
| 11933957 | Tentori et al. | Mar 2024 | B1 |
| 11952627 | Stoeckius | Apr 2024 | B2 |
| 11959076 | Kim et al. | Apr 2024 | B2 |
| 11959130 | Galonska et al. | Apr 2024 | B2 |
| 11965213 | Williams | Apr 2024 | B2 |
| 11970739 | Chew et al. | Apr 2024 | B2 |
| 11981958 | Galonska | May 2024 | B1 |
| 11981960 | Lin et al. | May 2024 | B1 |
| 11981965 | Chell et al. | May 2024 | B2 |
| 20010055764 | Empendocles et al. | Dec 2001 | A1 |
| 20020040275 | Cravatt | Apr 2002 | A1 |
| 20020051986 | Baez et al. | May 2002 | A1 |
| 20020055100 | Kawashima | May 2002 | A1 |
| 20020058250 | Firth | May 2002 | A1 |
| 20020086441 | Baranov et al. | Jul 2002 | A1 |
| 20020164611 | Bamdad | Nov 2002 | A1 |
| 20030017451 | Wang et al. | Jan 2003 | A1 |
| 20030022207 | Balasubramanian | Jan 2003 | A1 |
| 20030064398 | Barnes | Apr 2003 | A1 |
| 20030092624 | Wang et al. | May 2003 | A1 |
| 20030138879 | Lambalot | Jul 2003 | A1 |
| 20030148335 | Shen et al. | Aug 2003 | A1 |
| 20030162216 | Gold | Aug 2003 | A1 |
| 20030165948 | Alsmadi et al. | Sep 2003 | A1 |
| 20030211489 | Shen et al. | Nov 2003 | A1 |
| 20030224419 | Corcoran | Dec 2003 | A1 |
| 20030232348 | Jones et al. | Dec 2003 | A1 |
| 20030232382 | Brennan | Dec 2003 | A1 |
| 20030235854 | Chan et al. | Dec 2003 | A1 |
| 20040033499 | Ilsley et al. | Feb 2004 | A1 |
| 20040067492 | Peng et al. | Apr 2004 | A1 |
| 20040082059 | Webb et al. | Apr 2004 | A1 |
| 20040096853 | Mayer | May 2004 | A1 |
| 20040106110 | Balasubramanian | Jun 2004 | A1 |
| 20040235103 | Reznikoff et al. | Nov 2004 | A1 |
| 20040248325 | Bukusoglu et al. | Dec 2004 | A1 |
| 20040259105 | Fan et al. | Dec 2004 | A1 |
| 20050003431 | Wucherpfennig | Jan 2005 | A1 |
| 20050014203 | Darfler et al. | Jan 2005 | A1 |
| 20050037393 | Gunderson et al. | Feb 2005 | A1 |
| 20050048580 | Labaer | Mar 2005 | A1 |
| 20050064460 | Holliger et al. | Mar 2005 | A1 |
| 20050095627 | Kolman et al. | May 2005 | A1 |
| 20050100900 | Kawashima et al. | May 2005 | A1 |
| 20050130173 | Leamon et al. | Jun 2005 | A1 |
| 20050136414 | Gunderson et al. | Jun 2005 | A1 |
| 20050164292 | Farooqui | Jul 2005 | A1 |
| 20050170373 | Monforte | Aug 2005 | A1 |
| 20050191656 | Drmanac et al. | Sep 2005 | A1 |
| 20050191698 | Chee et al. | Sep 2005 | A1 |
| 20050202433 | Van Beuningen | Sep 2005 | A1 |
| 20050227271 | Kwon | Oct 2005 | A1 |
| 20050239119 | Tsukada et al. | Oct 2005 | A1 |
| 20050260653 | LaBaer | Nov 2005 | A1 |
| 20050266417 | Barany et al. | Dec 2005 | A1 |
| 20060041385 | Bauer et al. | Feb 2006 | A1 |
| 20060046313 | Roth | Mar 2006 | A1 |
| 20060084078 | Zhao | Apr 2006 | A1 |
| 20060105352 | Qiao et al. | May 2006 | A1 |
| 20060154286 | Kong et al. | Jul 2006 | A1 |
| 20060188901 | Barnes et al. | Aug 2006 | A1 |
| 20060199183 | Valat et al. | Sep 2006 | A1 |
| 20060211001 | Yu et al. | Sep 2006 | A1 |
| 20060216775 | Burkart et al. | Sep 2006 | A1 |
| 20060240439 | Smith et al. | Oct 2006 | A1 |
| 20060263789 | Kincaid | Nov 2006 | A1 |
| 20060275782 | Gunderson et al. | Dec 2006 | A1 |
| 20060281109 | Barr Ost et al. | Dec 2006 | A1 |
| 20070020640 | McCloskey et al. | Jan 2007 | A1 |
| 20070020669 | Ericsson | Jan 2007 | A1 |
| 20070026430 | Andersen et al. | Feb 2007 | A1 |
| 20070054288 | Su et al. | Mar 2007 | A1 |
| 20070087360 | Boyd | Apr 2007 | A1 |
| 20070099208 | Drmanac et al. | May 2007 | A1 |
| 20070128624 | Gormley et al. | Jun 2007 | A1 |
| 20070128656 | Agrawal | Jun 2007 | A1 |
| 20070134723 | Kozlov et al. | Jun 2007 | A1 |
| 20070161020 | Luo et al. | Jul 2007 | A1 |
| 20070166705 | Milton et al. | Jul 2007 | A1 |
| 20070172873 | Brenner et al. | Jul 2007 | A1 |
| 20070207482 | Church et al. | Sep 2007 | A1 |
| 20070254305 | Paik et al. | Nov 2007 | A1 |
| 20070269805 | Hogers | Nov 2007 | A1 |
| 20080003586 | Hyde et al. | Jan 2008 | A1 |
| 20080009420 | Schroth et al. | Jan 2008 | A1 |
| 20080108082 | Rank et al. | May 2008 | A1 |
| 20080108804 | Hayashizaki et al. | May 2008 | A1 |
| 20080132429 | Perov et al. | Jun 2008 | A1 |
| 20080160580 | Adessi et al. | Jul 2008 | A1 |
| 20080220434 | Thomas | Sep 2008 | A1 |
| 20080261204 | Lexow | Oct 2008 | A1 |
| 20080286795 | Kawashima et al. | Nov 2008 | A1 |
| 20080293046 | Allawi et al. | Nov 2008 | A1 |
| 20090005252 | Drmanac et al. | Jan 2009 | A1 |
| 20090006002 | Honisch et al. | Jan 2009 | A1 |
| 20090018024 | Church et al. | Jan 2009 | A1 |
| 20090026082 | Rothberg et al. | Jan 2009 | A1 |
| 20090036323 | van Eijk et al. | Feb 2009 | A1 |
| 20090082212 | Williams | Mar 2009 | A1 |
| 20090099041 | Church et al. | Apr 2009 | A1 |
| 20090105959 | Braverman et al. | Apr 2009 | A1 |
| 20090117573 | Fu et al. | May 2009 | A1 |
| 20090127589 | Rothberg et al. | May 2009 | A1 |
| 20090155781 | Drmanac et al. | Jun 2009 | A1 |
| 20090170713 | van Eijk et al. | Jul 2009 | A1 |
| 20090202998 | Schlumpberger et al. | Aug 2009 | A1 |
| 20090233802 | Bignell et al. | Sep 2009 | A1 |
| 20090253581 | van Eijk et al. | Oct 2009 | A1 |
| 20090270273 | Burns et al. | Oct 2009 | A1 |
| 20090283407 | Van Eijk | Nov 2009 | A1 |
| 20090291854 | Weisinger-Mayr et al. | Nov 2009 | A1 |
| 20090312193 | Kim et al. | Dec 2009 | A1 |
| 20100035249 | Hayashizaki et al. | Feb 2010 | A1 |
| 20100069263 | Shendure et al. | Mar 2010 | A1 |
| 20100105052 | Drmanac et al. | Apr 2010 | A1 |
| 20100120097 | Matz et al. | May 2010 | A1 |
| 20100120098 | Grunenwald et al. | May 2010 | A1 |
| 20100129874 | Mitra et al. | May 2010 | A1 |
| 20100145037 | Brive et al. | Jun 2010 | A1 |
| 20100173384 | Johnsson et al. | Jul 2010 | A1 |
| 20100184618 | Namsaraev et al. | Jul 2010 | A1 |
| 20100210475 | Lee et al. | Aug 2010 | A1 |
| 20100227329 | Cuppens | Sep 2010 | A1 |
| 20100273219 | May et al. | Oct 2010 | A1 |
| 20110028685 | Purkayastha et al. | Feb 2011 | A1 |
| 20110033854 | Drmanac et al. | Feb 2011 | A1 |
| 20110045462 | Fu et al. | Feb 2011 | A1 |
| 20110059436 | Hardin et al. | Mar 2011 | A1 |
| 20110111409 | Sinicropi et al. | May 2011 | A1 |
| 20110152111 | Fan et al. | Jun 2011 | A1 |
| 20110245101 | Chee et al. | Oct 2011 | A1 |
| 20110245111 | Chee | Oct 2011 | A1 |
| 20110287435 | Grunenwald et al. | Nov 2011 | A1 |
| 20120021930 | Schoen et al. | Jan 2012 | A1 |
| 20120046175 | Rodesch et al. | Feb 2012 | A1 |
| 20120046178 | Van Den Boom et al. | Feb 2012 | A1 |
| 20120065081 | Chee | Mar 2012 | A1 |
| 20120135871 | van Eijk et al. | May 2012 | A1 |
| 20120202698 | van Eijk et al. | Aug 2012 | A1 |
| 20120202704 | Fan et al. | Aug 2012 | A1 |
| 20120220479 | Ericsson et al. | Aug 2012 | A1 |
| 20120245053 | Shirai et al. | Sep 2012 | A1 |
| 20120252702 | Muratani et al. | Oct 2012 | A1 |
| 20120258871 | Kozlov et al. | Oct 2012 | A1 |
| 20120289414 | Mitra et al. | Nov 2012 | A1 |
| 20120301925 | Belyaev | Nov 2012 | A1 |
| 20130005594 | Terbrueggen et al. | Jan 2013 | A1 |
| 20130005600 | Olek | Jan 2013 | A1 |
| 20130023433 | Luo et al. | Jan 2013 | A1 |
| 20130035239 | Kong et al. | Feb 2013 | A1 |
| 20130040842 | Lim et al. | Feb 2013 | A1 |
| 20130065768 | Zheng et al. | Mar 2013 | A1 |
| 20130079232 | Kain et al. | Mar 2013 | A1 |
| 20130171621 | Luo et al. | Jul 2013 | A1 |
| 20130244884 | Jacobson et al. | Sep 2013 | A1 |
| 20130261019 | Lin et al. | Oct 2013 | A1 |
| 20130302801 | Asbury et al. | Nov 2013 | A1 |
| 20130338042 | Shen et al. | Dec 2013 | A1 |
| 20140066318 | Frisen et al. | Mar 2014 | A1 |
| 20140121118 | Warner | May 2014 | A1 |
| 20140270435 | Dunn | Sep 2014 | A1 |
| 20140274731 | Raymond et al. | Sep 2014 | A1 |
| 20140323330 | Glezer et al. | Oct 2014 | A1 |
| 20140342921 | Weiner | Nov 2014 | A1 |
| 20140378350 | Hindson et al. | Dec 2014 | A1 |
| 20150000854 | Gann-Fetter et al. | Jan 2015 | A1 |
| 20150292988 | Bharadwaj et al. | Oct 2015 | A1 |
| 20150344942 | Frisen et al. | Dec 2015 | A1 |
| 20160019337 | Roberts et al. | Jan 2016 | A1 |
| 20160024576 | Chee | Jan 2016 | A1 |
| 20160041159 | Labaer et al. | Feb 2016 | A1 |
| 20160060687 | Zhu et al. | Mar 2016 | A1 |
| 20160108458 | Frei et al. | Apr 2016 | A1 |
| 20160122817 | Jarosz et al. | May 2016 | A1 |
| 20160138091 | Chee et al. | May 2016 | A1 |
| 20160145677 | Chee et al. | May 2016 | A1 |
| 20160194692 | Gore et al. | Jul 2016 | A1 |
| 20160201125 | Samuels et al. | Jul 2016 | A1 |
| 20160253584 | Fodor et al. | Sep 2016 | A1 |
| 20160289740 | Fu et al. | Oct 2016 | A1 |
| 20160298180 | Chee | Oct 2016 | A1 |
| 20160305856 | Boyden et al. | Oct 2016 | A1 |
| 20160333403 | Chee | Nov 2016 | A1 |
| 20160376642 | Landegren et al. | Dec 2016 | A1 |
| 20170009278 | Söderberg et al. | Jan 2017 | A1 |
| 20170016053 | Beechem et al. | Jan 2017 | A1 |
| 20170029875 | Zhang et al. | Feb 2017 | A1 |
| 20170058339 | Chee | Mar 2017 | A1 |
| 20170058340 | Chee | Mar 2017 | A1 |
| 20170058345 | Chee | Mar 2017 | A1 |
| 20170067096 | Wassie et al. | Mar 2017 | A1 |
| 20170088881 | Chee | Mar 2017 | A1 |
| 20170089811 | Tillberg et al. | Mar 2017 | A1 |
| 20170159109 | Zheng et al. | Jun 2017 | A1 |
| 20170166962 | van Eijk et al. | Jun 2017 | A1 |
| 20170220733 | Zhuang et al. | Aug 2017 | A1 |
| 20170233722 | Seelig et al. | Aug 2017 | A1 |
| 20170241911 | Rockel et al. | Aug 2017 | A1 |
| 20170283860 | Kool et al. | Oct 2017 | A1 |
| 20170335297 | Ha et al. | Nov 2017 | A1 |
| 20170335410 | Driscoll et al. | Nov 2017 | A1 |
| 20170342405 | Fu et al. | Nov 2017 | A1 |
| 20170349940 | Morin et al. | Dec 2017 | A1 |
| 20180051322 | Church et al. | Feb 2018 | A1 |
| 20180057873 | Zhou et al. | Mar 2018 | A1 |
| 20180080019 | Blainey et al. | Mar 2018 | A1 |
| 20180094316 | Oliphant et al. | Apr 2018 | A1 |
| 20180112261 | Van Driel et al. | Apr 2018 | A1 |
| 20180127817 | Borchert et al. | May 2018 | A1 |
| 20180163265 | Zhang et al. | Jun 2018 | A1 |
| 20180179591 | van Eijk | Jun 2018 | A1 |
| 20180201925 | Steemers et al. | Jul 2018 | A1 |
| 20180201980 | Chee et al. | Jul 2018 | A1 |
| 20180208967 | Larman et al. | Jul 2018 | A1 |
| 20180216161 | Chen et al. | Aug 2018 | A1 |
| 20180216162 | Belhocine et al. | Aug 2018 | A1 |
| 20180237864 | Imler et al. | Aug 2018 | A1 |
| 20180245142 | So et al. | Aug 2018 | A1 |
| 20180247017 | van Eijk et al. | Aug 2018 | A1 |
| 20180291427 | Edelman | Oct 2018 | A1 |
| 20180291439 | van Eijk et al. | Oct 2018 | A1 |
| 20180305681 | Jovanovich et al. | Oct 2018 | A1 |
| 20180312822 | Lee et al. | Nov 2018 | A1 |
| 20180320226 | Church et al. | Nov 2018 | A1 |
| 20180334670 | Bharadwaj et al. | Nov 2018 | A1 |
| 20190055594 | Samusik et al. | Feb 2019 | A1 |
| 20190064173 | Bharadwaj et al. | Feb 2019 | A1 |
| 20190071656 | Chang et al. | Mar 2019 | A1 |
| 20190085383 | Church et al. | Mar 2019 | A1 |
| 20190119735 | Deisseroth et al. | Apr 2019 | A1 |
| 20190135774 | Orbai | May 2019 | A1 |
| 20190145982 | Chee et al. | May 2019 | A1 |
| 20190161796 | Hauling et al. | May 2019 | A1 |
| 20190177777 | Chee | Jun 2019 | A1 |
| 20190177778 | Chee | Jun 2019 | A1 |
| 20190177789 | Hindson et al. | Jun 2019 | A1 |
| 20190177800 | Boutet et al. | Jun 2019 | A1 |
| 20190194709 | Church et al. | Jun 2019 | A1 |
| 20190203275 | Frisen et al. | Jul 2019 | A1 |
| 20190218276 | Regev et al. | Jul 2019 | A1 |
| 20190218608 | Daugharthy et al. | Jul 2019 | A1 |
| 20190233878 | Delaney et al. | Aug 2019 | A1 |
| 20190233880 | Mir | Aug 2019 | A1 |
| 20190249226 | Bent et al. | Aug 2019 | A1 |
| 20190262831 | West et al. | Aug 2019 | A1 |
| 20190264268 | Frisen et al. | Aug 2019 | A1 |
| 20190271028 | Khafizov et al. | Sep 2019 | A1 |
| 20190271030 | Chee | Sep 2019 | A1 |
| 20190271031 | Chee | Sep 2019 | A1 |
| 20190300943 | Chee et al. | Oct 2019 | A1 |
| 20190300944 | Chee et al. | Oct 2019 | A1 |
| 20190300945 | Chee et al. | Oct 2019 | A1 |
| 20190309353 | Chee | Oct 2019 | A1 |
| 20190309354 | Chee | Oct 2019 | A1 |
| 20190309355 | Chee | Oct 2019 | A1 |
| 20190323071 | Chee | Oct 2019 | A1 |
| 20190323088 | Boutet et al. | Oct 2019 | A1 |
| 20190330617 | Church et al. | Oct 2019 | A1 |
| 20190338353 | Belgrader et al. | Nov 2019 | A1 |
| 20190360034 | Zhou et al. | Nov 2019 | A1 |
| 20190360043 | Pham et al. | Nov 2019 | A1 |
| 20190367969 | Belhocine et al. | Dec 2019 | A1 |
| 20190367982 | Belhocine et al. | Dec 2019 | A1 |
| 20190367997 | Bent et al. | Dec 2019 | A1 |
| 20200002763 | Belgrader et al. | Jan 2020 | A1 |
| 20200010891 | Beechem et al. | Jan 2020 | A1 |
| 20200024641 | Nolan et al. | Jan 2020 | A1 |
| 20200047010 | Lee et al. | Feb 2020 | A1 |
| 20200048690 | Chee | Feb 2020 | A1 |
| 20200063191 | Kennedy-Darling et al. | Feb 2020 | A1 |
| 20200063195 | Chee | Feb 2020 | A1 |
| 20200063196 | Chee | Feb 2020 | A1 |
| 20200071751 | Daugharthy et al. | Mar 2020 | A1 |
| 20200080136 | Zhang et al. | Mar 2020 | A1 |
| 20200109443 | Chee | Apr 2020 | A1 |
| 20200123597 | Daniel | Apr 2020 | A1 |
| 20200140920 | Pierce et al. | May 2020 | A1 |
| 20200173985 | Dong et al. | Jun 2020 | A1 |
| 20200199565 | Chen et al. | Jun 2020 | A1 |
| 20200199572 | Kuersten et al. | Jun 2020 | A1 |
| 20200224244 | Nilsson et al. | Jul 2020 | A1 |
| 20200239874 | Mikkelsen | Jul 2020 | A1 |
| 20200239946 | Dewal | Jul 2020 | A1 |
| 20200256867 | Hennek et al. | Aug 2020 | A1 |
| 20200277663 | Iyer | Sep 2020 | A1 |
| 20200277664 | Frenz | Sep 2020 | A1 |
| 20200283852 | Oliphant et al. | Sep 2020 | A1 |
| 20200299757 | Chee et al. | Sep 2020 | A1 |
| 20200325531 | Chee | Oct 2020 | A1 |
| 20200362398 | Kishi et al. | Nov 2020 | A1 |
| 20200370095 | Farmer et al. | Nov 2020 | A1 |
| 20200399687 | Frisen et al. | Dec 2020 | A1 |
| 20200407781 | Schnall-Levin | Dec 2020 | A1 |
| 20210010068 | Chee et al. | Jan 2021 | A1 |
| 20210010070 | Schnall-Levin et al. | Jan 2021 | A1 |
| 20210017587 | Cai et al. | Jan 2021 | A1 |
| 20210095331 | Fan et al. | Apr 2021 | A1 |
| 20210115504 | Cai et al. | Apr 2021 | A1 |
| 20210123040 | Macosko et al. | Apr 2021 | A1 |
| 20210130881 | Cox | May 2021 | A1 |
| 20210140982 | Uytingco et al. | May 2021 | A1 |
| 20210150707 | Weisenfeld et al. | May 2021 | A1 |
| 20210155982 | Yin et al. | May 2021 | A1 |
| 20210158522 | Weisenfeld et al. | May 2021 | A1 |
| 20210172007 | Chee et al. | Jun 2021 | A1 |
| 20210189475 | Tentori et al. | Jun 2021 | A1 |
| 20210190770 | Delaney et al. | Jun 2021 | A1 |
| 20210198741 | Williams | Jul 2021 | A1 |
| 20210199660 | Williams et al. | Jul 2021 | A1 |
| 20210207202 | Chee | Jul 2021 | A1 |
| 20210214785 | Stoeckius | Jul 2021 | A1 |
| 20210222235 | Chee | Jul 2021 | A1 |
| 20210222241 | Bharadwaj | Jul 2021 | A1 |
| 20210222242 | Ramachandran Iyer | Jul 2021 | A1 |
| 20210222253 | Uytingco | Jul 2021 | A1 |
| 20210223227 | Stoeckius | Jul 2021 | A1 |
| 20210230584 | Mikkelsen et al. | Jul 2021 | A1 |
| 20210230681 | Patterson et al. | Jul 2021 | A1 |
| 20210230692 | Daugharthy et al. | Jul 2021 | A1 |
| 20210237022 | Bava | Aug 2021 | A1 |
| 20210238581 | Mikkelsen et al. | Aug 2021 | A1 |
| 20210238664 | Bava et al. | Aug 2021 | A1 |
| 20210238675 | Bava | Aug 2021 | A1 |
| 20210238680 | Bava | Aug 2021 | A1 |
| 20210247316 | Bava | Aug 2021 | A1 |
| 20210255175 | Chee et al. | Aug 2021 | A1 |
| 20210262018 | Bava et al. | Aug 2021 | A1 |
| 20210262019 | Alvarado Martinez et al. | Aug 2021 | A1 |
| 20210269864 | Chee | Sep 2021 | A1 |
| 20210270822 | Chee | Sep 2021 | A1 |
| 20210285036 | Yin et al. | Sep 2021 | A1 |
| 20210285046 | Chell et al. | Sep 2021 | A1 |
| 20210292748 | Frisen et al. | Sep 2021 | A1 |
| 20210292822 | Frisen et al. | Sep 2021 | A1 |
| 20210317510 | Chee et al. | Oct 2021 | A1 |
| 20210317524 | Lucero et al. | Oct 2021 | A1 |
| 20210324457 | Ramachandran Iyer et al. | Oct 2021 | A1 |
| 20210332424 | Schnall-Levin | Oct 2021 | A1 |
| 20210332425 | Pfeiffer et al. | Oct 2021 | A1 |
| 20210348221 | Chell et al. | Nov 2021 | A1 |
| 20220002791 | Frisen et al. | Jan 2022 | A1 |
| 20220003755 | Chee | Jan 2022 | A1 |
| 20220010367 | Ramachandran Iyer et al. | Jan 2022 | A1 |
| 20220017951 | Ramachandran Iyer et al. | Jan 2022 | A1 |
| 20220025446 | Shah | Jan 2022 | A1 |
| 20220025447 | Tentori et al. | Jan 2022 | A1 |
| 20220033888 | Schnall-Levin et al. | Feb 2022 | A1 |
| 20220049293 | Frenz et al. | Feb 2022 | A1 |
| 20220064630 | Bent et al. | Mar 2022 | A1 |
| 20220081728 | Williams | Mar 2022 | A1 |
| 20220090058 | Frisen et al. | Mar 2022 | A1 |
| 20220090175 | Uytingco et al. | Mar 2022 | A1 |
| 20220090181 | Gallant et al. | Mar 2022 | A1 |
| 20220098576 | Dadhwal | Mar 2022 | A1 |
| 20220098661 | Chew et al. | Mar 2022 | A1 |
| 20220106632 | Galonska et al. | Apr 2022 | A1 |
| 20220106633 | Engblom et al. | Apr 2022 | A1 |
| 20220112486 | Ramachandran Iyer et al. | Apr 2022 | A1 |
| 20220112545 | Chee | Apr 2022 | A1 |
| 20220119869 | Ramachandran Iyer et al. | Apr 2022 | A1 |
| 20220127659 | Frisen et al. | Apr 2022 | A1 |
| 20220127666 | Katiraee et al. | Apr 2022 | A1 |
| 20220127672 | Stoeckius | Apr 2022 | A1 |
| 20220145361 | Frenz et al. | May 2022 | A1 |
| 20220154255 | Chee et al. | May 2022 | A1 |
| 20220170083 | Khaled et al. | Jun 2022 | A1 |
| 20220195422 | Gallant et al. | Jun 2022 | A1 |
| 20220195505 | Frisen et al. | Jun 2022 | A1 |
| 20220196644 | Chee | Jun 2022 | A1 |
| 20220213526 | Frisen et al. | Jul 2022 | A1 |
| 20220220544 | Ach et al. | Jul 2022 | A1 |
| 20220241780 | Tentori et al. | Aug 2022 | A1 |
| 20220267844 | Ramachandran Iyer et al. | Aug 2022 | A1 |
| 20220282329 | Chell et al. | Sep 2022 | A1 |
| 20220290217 | Frenz et al. | Sep 2022 | A1 |
| 20220290219 | Chee | Sep 2022 | A1 |
| 20220298560 | Frisen et al. | Sep 2022 | A1 |
| 20220325325 | Chee et al. | Oct 2022 | A1 |
| 20220326251 | Uytingco et al. | Oct 2022 | A1 |
| 20220333171 | Chee | Oct 2022 | A1 |
| 20220333191 | Mikkelsen et al. | Oct 2022 | A1 |
| 20220333192 | Uytingco | Oct 2022 | A1 |
| 20220333195 | Schnall-Levin et al. | Oct 2022 | A1 |
| 20220334031 | Delaney et al. | Oct 2022 | A1 |
| 20220348905 | Dadhwal | Nov 2022 | A1 |
| 20220348992 | Stoeckius et al. | Nov 2022 | A1 |
| 20220356464 | Kim et al. | Nov 2022 | A1 |
| 20220364163 | Stahl et al. | Nov 2022 | A1 |
| 20220389491 | Chee | Dec 2022 | A1 |
| 20220389503 | Mikkelsen et al. | Dec 2022 | A1 |
| 20220389504 | Chew et al. | Dec 2022 | A1 |
| 20220403455 | Ramachandran Iyer et al. | Dec 2022 | A1 |
| 20220404245 | Chell et al. | Dec 2022 | A1 |
| 20230002812 | Stoeckius et al. | Jan 2023 | A1 |
| 20230014008 | Shastry | Jan 2023 | A1 |
| 20230017773 | Kim et al. | Jan 2023 | A1 |
| 20230416807 | Chee | Jan 2023 | A1 |
| 20230416808 | Sukovich et al. | Jan 2023 | A1 |
| 20230033960 | Gallant et al. | Feb 2023 | A1 |
| 20230034039 | Shahjamali | Feb 2023 | A1 |
| 20230034216 | Bava | Feb 2023 | A1 |
| 20230040363 | Chee | Feb 2023 | A1 |
| 20230042088 | Chee | Feb 2023 | A1 |
| 20230042817 | Mignardi | Feb 2023 | A1 |
| 20230047782 | Tentori et al. | Feb 2023 | A1 |
| 20230056549 | Dadhwal | Feb 2023 | A1 |
| 20230064372 | Chell et al. | Mar 2023 | A1 |
| 20230069046 | Chew et al. | Mar 2023 | A1 |
| 20230077364 | Patterson et al. | Mar 2023 | A1 |
| 20230080543 | Katiraee et al. | Mar 2023 | A1 |
| 20230081381 | Chew et al. | Mar 2023 | A1 |
| 20230100497 | Frisen et al. | Mar 2023 | A1 |
| 20230107023 | Chee | Apr 2023 | A1 |
| 20230111225 | Chew et al. | Apr 2023 | A1 |
| 20230113230 | Kim et al. | Apr 2023 | A1 |
| 20230126825 | Nagendran et al. | Apr 2023 | A1 |
| 20230129552 | Ramachandran Iyer | Apr 2023 | A1 |
| 20230135010 | Tentori et al. | May 2023 | A1 |
| 20230143569 | Iyer et al. | May 2023 | A1 |
| 20230145575 | Gallant et al. | May 2023 | A1 |
| 20230147726 | Hadrup et al. | May 2023 | A1 |
| 20230151412 | Chee | May 2023 | A1 |
| 20230159994 | Chee | May 2023 | A1 |
| 20230159995 | Iyer et al. | May 2023 | A1 |
| 20230160008 | Chell et al. | May 2023 | A1 |
| 20230175045 | Katsori et al. | Jun 2023 | A1 |
| 20230183785 | Frisen et al. | Jun 2023 | A1 |
| 20230194469 | Tentori et al. | Jun 2023 | A1 |
| 20230194470 | Kim et al. | Jun 2023 | A1 |
| 20230203478 | Kim et al. | Jun 2023 | A1 |
| 20230183684 | Gallant et al. | Jul 2023 | A1 |
| 20230212650 | Chew et al. | Jul 2023 | A1 |
| 20230212655 | Chee | Jul 2023 | A1 |
| 20230220368 | Kim | Jul 2023 | A1 |
| 20230220454 | Bent et al. | Jul 2023 | A1 |
| 20230220455 | Galonska et al. | Jul 2023 | A1 |
| 20230227811 | Dadhwal | Jul 2023 | A1 |
| 20230228762 | Uytingco et al. | Jul 2023 | A1 |
| 20230242973 | Frisen et al. | Aug 2023 | A1 |
| 20230242976 | Tentori et al. | Aug 2023 | A1 |
| 20230265488 | Gohil et al. | Aug 2023 | A1 |
| 20230265489 | Uytingco et al. | Aug 2023 | A1 |
| 20230265491 | Tentori et al. | Aug 2023 | A1 |
| 20230267625 | Tentori et al. | Aug 2023 | A1 |
| 20230279474 | Katiraee | Sep 2023 | A1 |
| 20230279477 | Kvastad et al. | Sep 2023 | A1 |
| 20230279481 | Marrache et al. | Sep 2023 | A1 |
| 20230287399 | Gallant et al. | Sep 2023 | A1 |
| 20230287475 | Chell et al. | Sep 2023 | A1 |
| 20230287481 | Katsori et al. | Sep 2023 | A1 |
| 20230295699 | Sukovich et al. | Sep 2023 | A1 |
| 20230295722 | Bharadwaj | Sep 2023 | A1 |
| 20230304072 | Gohil et al. | Sep 2023 | A1 |
| 20230304074 | Chee et al. | Sep 2023 | A1 |
| 20230304078 | Frisen et al. | Sep 2023 | A1 |
| 20230313279 | Giacomello et al. | Oct 2023 | A1 |
| 20230323340 | Dadhwal | Oct 2023 | A1 |
| 20230323434 | Yin et al. | Oct 2023 | A1 |
| 20230323436 | Chee | Oct 2023 | A1 |
| 20230323447 | Schnall-Levin et al. | Oct 2023 | A1 |
| 20230323453 | Stoeckius | Oct 2023 | A1 |
| 20230332138 | Kim et al. | Oct 2023 | A1 |
| 20230332211 | Chee | Oct 2023 | A1 |
| 20230332212 | Chew et al. | Oct 2023 | A1 |
| 20230332227 | Ramachandran Iyer | Oct 2023 | A1 |
| 20230332247 | Singh et al. | Oct 2023 | A1 |
| 20230351619 | Tentori et al. | Nov 2023 | A1 |
| 20230358733 | Chee | Nov 2023 | A1 |
| 20230366008 | Chew et al. | Nov 2023 | A1 |
| 20230383285 | Kim et al. | Nov 2023 | A1 |
| 20230383344 | Stoeckius | Nov 2023 | A1 |
| 20230392204 | Chell et al. | Dec 2023 | A1 |
| 20230393071 | Bava | Dec 2023 | A1 |
| 20230407404 | Baumgartner et al. | Dec 2023 | A1 |
| 20230416850 | Singh et al. | Dec 2023 | A1 |
| 20240002931 | Bava | Jan 2024 | A1 |
| 20240011081 | Chee | Jan 2024 | A1 |
| 20240011090 | Chew et al. | Jan 2024 | A1 |
| 20240018572 | Mignardi | Jan 2024 | A1 |
| 20240018575 | Gallant et al. | Jan 2024 | A1 |
| 20240018589 | Schnall-Levin et al. | Jan 2024 | A1 |
| 20240026445 | Ramachandran Iyer et al. | Jan 2024 | A1 |
| 20240033743 | Tentori et al. | Feb 2024 | A1 |
| 20240035937 | Cox et al. | Feb 2024 | A1 |
| 20240043908 | Chew et al. | Feb 2024 | A1 |
| 20240043925 | Bent et al. | Feb 2024 | A1 |
| 20240052343 | Gallant et al. | Feb 2024 | A1 |
| 20240053351 | Uytingco et al. | Feb 2024 | A1 |
| 20240060115 | Chee et al. | Feb 2024 | A1 |
| 20240067953 | Mikkelsen et al. | Feb 2024 | A1 |
| 20240068016 | Frisen et al. | Feb 2024 | A1 |
| 20240068017 | Lundeberg et al. | Feb 2024 | A1 |
| 20240076723 | Mignardi | Mar 2024 | A1 |
| 20240080346 | Engblom et al. | Mar 2024 | A1 |
| 20240084365 | Frisen et al. | Mar 2024 | A1 |
| 20240084366 | Chee | Mar 2024 | A1 |
| 20240084383 | Ramachandran Iyer et al. | Mar 2024 | A1 |
| 20240093274 | Frisen et al. | Mar 2024 | A1 |
| 20240093290 | Stahl et al. | Mar 2024 | A1 |
| 20240110228 | Uytingco et al. | Apr 2024 | A1 |
| 20240124933 | Chell et al. | Apr 2024 | A1 |
| 20240125772 | Delaney et al. | Apr 2024 | A1 |
| 20240141327 | Kim et al. | May 2024 | A1 |
| 20240158838 | Alvarado Martinez et al. | May 2024 | A1 |
| 20240175080 | Galonska et al. | May 2024 | A1 |
| 20240182968 | Bava | Jun 2024 | A1 |
| 20240191286 | Boutet et al. | Jun 2024 | A1 |
| Number | Date | Country |
|---|---|---|
| 2003200718 | Oct 2006 | AU |
| 1273609 | Nov 2000 | CN |
| 1537953 | Oct 2004 | CN |
| 1680604 | Oct 2005 | CN |
| 1749752 | Mar 2006 | CN |
| 1898398 | Jan 2007 | CN |
| 101142325 | Mar 2008 | CN |
| 101221182 | Jul 2008 | CN |
| 101522915 | Sep 2009 | CN |
| 107849606 | Mar 2018 | CN |
| 108949924 | Dec 2018 | CN |
| 1782737 | May 2007 | EP |
| 1910562 | Apr 2008 | EP |
| 1923471 | May 2008 | EP |
| 1929039 | Jun 2008 | EP |
| 2002017 | Dec 2008 | EP |
| 2292788 | Mar 2011 | EP |
| 2302070 | Mar 2011 | EP |
| 2580351 | Apr 2013 | EP |
| 2881465 | Jun 2015 | EP |
| 3013984 | May 2016 | EP |
| 3511423 | Jul 2019 | EP |
| 3541956 | Sep 2019 | EP |
| 2520765 | Jun 2015 | GB |
| 2007-014297 | Jan 2007 | JP |
| 2007-074967 | Mar 2007 | JP |
| 2009-036694 | Feb 2009 | JP |
| WO 1989010977 | Nov 1989 | WO |
| WO 1991006678 | May 1991 | WO |
| WO 1993004199 | Mar 1993 | WO |
| WO 1995023875 | Sep 1995 | WO |
| WO 1995025116 | Sep 1995 | WO |
| WO 1995035505 | Dec 1995 | WO |
| WO 1997031256 | Aug 1997 | WO |
| WO 1998044151 | Oct 1998 | WO |
| WO 2000017390 | Mar 2000 | WO |
| WO 2000063437 | Oct 2000 | WO |
| WO 2001006012 | Jan 2001 | WO |
| WO 2001009363 | Feb 2001 | WO |
| WO 2001012862 | Feb 2001 | WO |
| WO 2001042796 | Jun 2001 | WO |
| WO 2001046402 | Jun 2001 | WO |
| WO 2001059161 | Aug 2001 | WO |
| WO 2001090415 | Nov 2001 | WO |
| WO 2001096608 | Dec 2001 | WO |
| WO 2002040874 | May 2002 | WO |
| WO 2002059355 | Aug 2002 | WO |
| WO 2002059364 | Aug 2002 | WO |
| WO 2002077283 | Oct 2002 | WO |
| WO 2003002979 | Jan 2003 | WO |
| WO 2003008538 | Jan 2003 | WO |
| WO 2003010176 | Feb 2003 | WO |
| WO 2003102233 | Dec 2003 | WO |
| WO 2004015080 | Feb 2004 | WO |
| WO 2004067759 | Aug 2004 | WO |
| WO 2004081225 | Sep 2004 | WO |
| WO 2005007814 | Jan 2005 | WO |
| WO 2005010145 | Feb 2005 | WO |
| WO 2005026387 | Mar 2005 | WO |
| WO 2005042759 | May 2005 | WO |
| WO 2005113804 | Dec 2005 | WO |
| WO 2006020515 | Feb 2006 | WO |
| WO 2006124771 | Nov 2006 | WO |
| WO 2006137733 | Dec 2006 | WO |
| WO 2007037678 | Apr 2007 | WO |
| WO 2007041689 | Apr 2007 | WO |
| WO 2007060599 | May 2007 | WO |
| WO 2007073171 | Jun 2007 | WO |
| WO 2007076726 | Jul 2007 | WO |
| WO 2007139766 | Dec 2007 | WO |
| WO 2007145612 | Dec 2007 | WO |
| WO 2008069906 | Jun 2008 | WO |
| WO 2008093098 | Aug 2008 | WO |
| WO 2009032167 | Mar 2009 | WO |
| WO 2009036525 | Mar 2009 | WO |
| WO 2009137521 | Nov 2009 | WO |
| WO 2009152928 | Dec 2009 | WO |
| WO 2010019826 | Feb 2010 | WO |
| WO 2010027870 | Mar 2010 | WO |
| WO 2010126614 | Nov 2010 | WO |
| WO 2010127186 | Nov 2010 | WO |
| WO 2011008502 | Jan 2011 | WO |
| WO 2011062933 | May 2011 | WO |
| WO 2011068088 | Jun 2011 | WO |
| WO 2011127006 | Oct 2011 | WO |
| WO 2011155833 | Dec 2011 | WO |
| WO 2012049316 | Apr 2012 | WO |
| WO 2012061832 | May 2012 | WO |
| WO 2012071428 | May 2012 | WO |
| WO 2012129242 | Sep 2012 | WO |
| WO 2012159089 | Nov 2012 | WO |
| WO 2013123442 | Aug 2013 | WO |
| WO 2013131962 | Sep 2013 | WO |
| WO 2013138510 | Sep 2013 | WO |
| WO 2013142389 | Sep 2013 | WO |
| WO 2013150082 | Oct 2013 | WO |
| WO 2013150083 | Oct 2013 | WO |
| WO 2014044724 | Mar 2014 | WO |
| WO 2014060483 | Apr 2014 | WO |
| WO 2014071361 | May 2014 | WO |
| WO 2014130576 | Aug 2014 | WO |
| WO 2014144713 | Sep 2014 | WO |
| WO 2014152397 | Sep 2014 | WO |
| WO 2014210223 | Dec 2014 | WO |
| WO 2014210353 | Dec 2014 | WO |
| WO 2015031691 | Mar 2015 | WO |
| WO 2015069374 | May 2015 | WO |
| WO 2015161173 | Oct 2015 | WO |
| WO 2016077763 | May 2016 | WO |
| WO 2016138496 | Sep 2016 | WO |
| WO 2016138500 | Sep 2016 | WO |
| WO 2016162309 | Oct 2016 | WO |
| WO 2016166128 | Oct 2016 | WO |
| WO 2016168825 | Oct 2016 | WO |
| WO 2016172362 | Oct 2016 | WO |
| WO 2017019456 | Feb 2017 | WO |
| WO 2017019481 | Feb 2017 | WO |
| WO 2017075293 | May 2017 | WO |
| WO 2017096158 | Jul 2017 | WO |
| WO 2017143155 | Aug 2017 | WO |
| WO 2017156336 | Sep 2017 | WO |
| WO 2017184984 | Oct 2017 | WO |
| WO 2017192633 | Nov 2017 | WO |
| WO 2018023068 | Feb 2018 | WO |
| WO 2018026873 | Feb 2018 | WO |
| WO 2018045181 | Mar 2018 | WO |
| WO 2018064640 | Apr 2018 | WO |
| WO 2018085599 | May 2018 | WO |
| WO 2018089550 | May 2018 | WO |
| WO 2018091676 | May 2018 | WO |
| WO 2018136397 | Jul 2018 | WO |
| WO 2018136856 | Jul 2018 | WO |
| WO 2018144582 | Aug 2018 | WO |
| WO 2018175779 | Sep 2018 | WO |
| WO 2018209398 | Nov 2018 | WO |
| WO 2019023214 | Jan 2019 | WO |
| WO 2019032760 | Feb 2019 | WO |
| WO 2019068880 | Apr 2019 | WO |
| WO 2019113457 | Jun 2019 | WO |
| WO 2019126313 | Jun 2019 | WO |
| WO 2019140201 | Jul 2019 | WO |
| WO 2019165318 | Aug 2019 | WO |
| WO 2019213254 | Nov 2019 | WO |
| WO 2019213294 | Nov 2019 | WO |
| WO 2019241290 | Dec 2019 | WO |
| WO 2020028194 | Feb 2020 | WO |
| WO 2020047002 | Mar 2020 | WO |
| WO 2020047010 | Mar 2020 | WO |
| WO 2020053655 | Mar 2020 | WO |
| WO 2020056381 | Mar 2020 | WO |
| WO 2020061064 | Mar 2020 | WO |
| WO 2020061066 | Mar 2020 | WO |
| WO 2020061108 | Mar 2020 | WO |
| WO 2020076979 | Apr 2020 | WO |
| WO 2020099640 | May 2020 | WO |
| WO 2020112604 | Jun 2020 | WO |
| WO 2020117914 | Jun 2020 | WO |
| WO 2020123301 | Jun 2020 | WO |
| WO 2020123305 | Jun 2020 | WO |
| WO 2020123311 | Jun 2020 | WO |
| WO 2020123316 | Jun 2020 | WO |
| WO 2020123317 | Jun 2020 | WO |
| WO 2020123318 | Jun 2020 | WO |
| WO 2020123319 | Jun 2020 | WO |
| WO 2020123320 | Jul 2020 | WO |
| WO 2020160044 | Aug 2020 | WO |
| WO 2020167862 | Aug 2020 | WO |
| WO 2020176788 | Sep 2020 | WO |
| WO 2020176882 | Sep 2020 | WO |
| WO 2020190509 | Sep 2020 | WO |
| WO 2020198071 | Oct 2020 | WO |
| WO 2020206285 | Oct 2020 | WO |
| WO 2020240025 | Dec 2020 | WO |
| WO 2020243579 | Dec 2020 | WO |
| WO 2020254519 | Dec 2020 | WO |
| WO 2021041974 | Mar 2021 | WO |
| WO 2021067246 | Apr 2021 | WO |
| WO 2021067514 | Apr 2021 | WO |
| WO 2021091611 | May 2021 | WO |
| WO 2021092433 | May 2021 | WO |
| WO 2021097255 | May 2021 | WO |
| WO 2021102003 | May 2021 | WO |
| WO 2021102005 | May 2021 | WO |
| WO 2021102039 | May 2021 | WO |
| WO 2021116715 | Jun 2021 | WO |
| WO 2021119320 | Jun 2021 | WO |
| WO 2021133842 | Jul 2021 | WO |
| WO 2021133845 | Jul 2021 | WO |
| WO 2021133849 | Jul 2021 | WO |
| WO 2021142233 | Jul 2021 | WO |
| WO 2021168261 | Aug 2021 | WO |
| WO 2021168278 | Aug 2021 | WO |
| WO 2021207610 | Oct 2021 | WO |
| WO 2021216708 | Oct 2021 | WO |
| WO 2021225900 | Nov 2021 | WO |
| WO 2021236625 | Nov 2021 | WO |
| WO 2021236929 | Nov 2021 | WO |
| WO 2021237056 | Nov 2021 | WO |
| WO 2021237087 | Nov 2021 | WO |
| WO 2021242834 | Dec 2021 | WO |
| WO 2021247543 | Dec 2021 | WO |
| WO 2021247568 | Dec 2021 | WO |
| WO 2021252499 | Dec 2021 | WO |
| WO 2021252576 | Dec 2021 | WO |
| WO 2021252591 | Dec 2021 | WO |
| WO 2021252747 | Dec 2021 | WO |
| WO 2021263111 | Dec 2021 | WO |
| WO 2022025965 | Feb 2022 | WO |
| WO 2022060798 | Mar 2022 | WO |
| WO 2022060953 | Mar 2022 | WO |
| WO 2022061150 | Mar 2022 | WO |
| WO 2022061152 | Mar 2022 | WO |
| WO 2022087273 | Apr 2022 | WO |
| WO 2022099037 | May 2022 | WO |
| WO 2022103712 | May 2022 | WO |
| WO 2022109181 | May 2022 | WO |
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| Entry |
|---|
| Sharma et. al. Melanopsichium pennsylvanicum 4 genomic scaffold, scaffold SCAFFOLD32. GenBank:HG529673.1. Ecology and Evolution, Biodiversity and Climate Research Center. 2016. [retrieved on Jun. 15, 2023]. Retrieved from the Internet <URL:https://www.ncbi.nlm.nih.gov/nuccore/hg529673> (Year: 2016). |
| Jiang et. al. Skewer: a fast and accurate adapter trimmer for next-generation sequencing paired-end reads. BMC Bioinformatics. 15:182, 2014, 1-12. [online], [retrieved on Jun. 15, 2023]. Retrieved from the Internet <URL:https://bmcbioinformatics.biomedcentral.com/articles/10.1186/1471-2105-15-182> (Year: 2014). |
| “Trimming adapter sequences—is it necessary?” ecSEQ Bioinformatics. 2016. [online] [retrieved on Jul. 15, 2023]. Retrieved from the Internet <URL:https://www.ecseq.com/support/ngs/trimming-adapter-sequences-is-it-necessary> (Year: 2016). |
| Appella, “Non-natural nucleic acids for synthetic biology,” Current Opinion in Chemical Biology, Dec. 2009, 13(5-6): 687-696. |
| Bunt et al., “FRET from single to multiplexed signaling events,” Biophys Rev. Apr. 2017, 9(2): 119-129. |
| Grünweller et al., “Locked Nucleic Acid Oligonucleotides,” BioDrugs, Jul. 2007, 21(4): 235-243. |
| Gu et al., “Multiplex single-molecule interaction profiling of DNA-barcoded proteins,” Nature, Sep. 21, 2014, 515:554-557. |
| Ma et al., “Isothermal amplification method for next-generation sequencing,” PNAS, Aug. 12, 2013, 110(35):14320-14323. |
| Orenstein et al., “γPNA FRET Pair Miniprobes for Quantitative Fluorescent In Situ Hybridization to Telomeric DNA in Cells and Tissue,” Molecules, Dec. 2, 2017, 22(12):2117, 15 pages. |
| Marx, “Method of the Year: spatially resolved transcriptomics,” Nature Methods, 2021, 18(1):9-14. |
| Altschul et al., “Basic local alignment search tool,” J. Mol. Biol., Oct. 5, 1990, 215(3):403-410. |
| Arslan et al., “Engineering of a superhelicase through conformational control (Supplementary Materials),” Science, Apr. 17, 2015, 348(6232):344-347, 18 pages. |
| Arslan et al., “Engineering of a superhelicase through conformational control,” Science, Apr. 17, 2015, 348(6232):344-347. |
| Baner et al., “Signal amplification of padlock probes by rolling circle replication,” Nucleic Acids Res., 1998, 26(22):5073-5078. |
| Borm et al., “High throughput Human embryo spatial transcriptome mapping by surface transfer of tissue RNA,” Abstracts Selected Talks, Single Cell Genomics mtg, (SCG2019), 2019, 1 pages (Abstract Only). |
| Bychkov et al., “Deep learning based tissue analysis predicts outcome in colorectal cancer,” Scientific Reports, Feb. 21, 2018, 8:3395, 12 pages. |
| Chen et al., “Efficient in situ barcode sequencing using padlock probe-based BaristaSeq,” Nucleic Acids Res., 2018, 46(4): e22, 11 pages. |
| Codeluppi et al., “Spatial organization of the somatosensory cortex revealed by osmFISH,” Nature Methods, Nov. 2018, 15:932-935. |
| Dean et al., “Rapid Amplification Of Plasmid And Phage DNA Using Phi29 DNA Polymerase And Multiply-Primed Rolling Circle Amplification,” Genome Research, Jun. 2001, 11:1095-1099. |
| Eng et al., “Transcriptome-scale super-resolved imaging in tissues by RNA seqFISH+,” Nature, Apr. 2019, 568(7751):235-239, 37 pages. |
| Faruqi et al., “High-throughput genotyping of single nucleotide polymorphisms with rolling circle amplification,” BMC Genomics, Aug. 2001, 2:4, 10 pages. |
| Gao et al., “A highly homogeneous expansion microscopy polymer composed of tetrahedron-like monomers,” bioRxiv, Oct. 22, 2019, 23 pages (Preprint). |
| Georgieva et al., “Optimization of DMD-based independent amplitude and phase modulation: a spatial resolution and quantization,” arXiv:2010.00955v1, Oct. 2, 2020, 16 pages. |
| Gilar et al., “Study of phosphorothioate-modified oligonucleotide resistance to 3′-exonuclease using capillary electrophoresis,” J Chromatogr B Biomed Sci Appl., Aug. 28, 1998, 714(1):13-20. |
| Goh et al., “Highly Specific Multiplexed RNA Imaging In Tissues With Split-FISH,” Nat Methods, Jun. 15, 2020, 17(7):689-693, 21 pages. |
| Goransson et al., “A single molecule array for digital targeted molecular analyses,” Nucleic Acids Res., Nov. 25, 2009, 37(1):e7, 9 pages. |
| Li et al., “A new GSH-responsive prodrug of 5-aminolevulinic acid for photodiagnosis and photodynamic therapy of tumors,” European Journal of Medicinal Chemistry, Nov. 2019, 181:111583, 9 pages. |
| Liu et al., “High-Spatial-Resolution Multi-Omnics Sequencing via Deterministic Barcoding in Tissue,” Cell, Nov. 13, 2020, 183(6):1665-1681, 36 pages. |
| Liu et al., “Spatial transcriptome sequencing of FFPE tissues at cellular level,” bioRxiv 788992, Oct. 14, 2020, 39 pages. |
| Mignardi et al., “Oligonucleotide gap-fill ligation for mutation detection and sequencing in situ,” Nucleic Acids Research, Aug. 3, 2015, 43(22):e151, 12 pages. |
| Mohsen et al., “The Discovery of Rolling Circle Amplification and Rolling Circle Transcription,” Acc Chem Res., Nov. 15, 2016, 49(11):2540-2550, 25 pages. |
| Schweitzer et al., “Immunoassays with rolling circle DNA amplification: A versatile platform for ultrasensitive antigen detection,” Proc. Natl Acad. Sci. USA, May 22, 2000, 97:10113-119. |
| Takei et al., “Integrated Spatial Genomics Reveals Global Architecture Of Single Nuclei,” Nature, Jan. 27, 2021, 590(7845):344-350, 53 pages. |
| Chen et al., “Large field of view-spatially resolved transcriptomics at nanoscale resolution,” bioRxiv, Jan. 19, 2021, retrieved from URL <https://www.biorxiv.org/node/1751045.abstract>, 37 pages. |
| Cho et al., “Seq-Scope: Submicrometer-resolution spatial transcriptomics for single cell and subcellular studies,” bioRxiv, Jan. 27, 2021, retrieved from URL <https://www.biorxiv.org/node/1754517.abstract>, 50 pages. |
| Ergin et al., “Proteomic Analysis of PAXgene-Fixed Tissues,” J Proteome Res., 2010, 9(10):5188-96. |
| Mathieson et al., “A Critical Evaluation of the PAXgene Tissue Fixation System: Morphology, Immunohistochemistry, Molecular Biology, and Proteomics,” Am J Clin Pathol., Jul. 8, 2016, 146(1):25-40. |
| Nallur et al., “Signal amplification by rolling circle amplification on DNA microarrays,” Nucleic Acids Res., Dec. 1, 2001, 29(23):e118, 9 pages. |
| Raj et al., “Imaging individual mRNA molecules using multiple singly labeled probes,” Nature Methods, Oct. 2008, 5(10):877-879, 9 pages. |
| Xia et al., “Spatial transcriptome profiling by MERFISH reveals subcellular RNA compartmentalization and cell cycle-dependent gene expression”, Proceedings of the National Academy of Sciences, Sep. 2019, 116(39):19490-19499. |
| [No Author Listed], “Chromium Next GEM Single Cell 3′ Reagent Kits v3.1—User Guide,” 10x Genomics, Document No. CG000204, Nov. 2019, 58 pages. |
| [No Author Listed], “Chromium Next GEM Single Cell 3′ Reagent Kits v3.1 (Dual Index)—User Guide,” 10x Genomics, Mar. 2021, Document No. CG000315, 61 pages. |
| [No Author Listed], “HuSNP Mapping Assay User's Manual,” Affymetrix Part No. 90094 (Affymetrix, Santa Clara, Calif.), GeneChip, 2000, 104 pages. |
| [No Author Listed], “Microarray technologies have excellent possibilities in genomics-related researches,” Science Tools From Amersham Pharmacia Biotech, 1998, 3(4): 8 pages (with English Translation). |
| [No Author Listed], “Proseek® Multiplex 96×96 User Manual,” Olink Proteomics, Olink Bioscience, Uppsala, Sweden, 2017, 20 pages. |
| 10xGenomics.com, [online], “Visium Spatial Gene Expression Reagent Kits—Tissue Optimization—User Guide,” Jul. 2020, retrieved on May 25, 2021, retrieved from URL<https://assets.ctfassets.net/an68im79xiti/5UJrN0CH17rEk0UXwd19It/e54d99fb08a8f1500aba503005a04a56/CG000238_VisiumSpatialTissueOptimizationUserGuide_RevD.pdf>, 42 pages. |
| 10xGenomics.com, [online], “Visium Spatial Gene Expression Reagent Kits—Tissue Optimization,” Nov. 2019, retrieved on Jan. 25, 2022, retrieved from URL<https://assets.ctfassets.net/an68im79xiti/4q03w6959AJFxffSw5lee9/6a2ac61cf6388a72564eeb96bc294967/CG000238_VisiumSpatialTissueOptimizationUserGuide_Rev_A.pdf>, 46 pages. |
| 10xGenomics.com, [online], “Visium Spatial Gene Expression Reagent Kits—Tissue Optimization,” Oct. 2020, retrieved on Dec. 28, 2021, retrieved from URL<https://assets.ctfassets.net/an68im79xiti/5UJrN0CH17rEk0UXwd19It/e54d99fb08a8f1500aba503005a04a56/CG000238_VisiumSpatialTissueOptimizationUserGuide_RevD.pdf>, 43 pages. |
| 10xGenomics.com, [online], “Visium Spatial Gene Expression Reagent Kits—User Guide,” Jun. 2020, retrieved on May 25, 2021, retrieved from URL<https://assets.ctfassets.net/an68im79xiti/3GGIfH3RWpd1bFVha1pexR/8baa08d9007157592b65b2cdc7130990/CG000239_VisiumSpatialGeneExpression_UserGuide_RevD.pdf>, 69 pages. |
| 10xGenomics.com, [online], “Visium Spatial Gene Expression Reagent Kits—User Guide,” Oct. 2020, retrieved on Dec. 28, 2021, retrieved from URL<https://assets.ctfassets.net/an68im79xiti/3GGIfH3RWpd1bFVha1pexR/8baa08d9007157592b65b2cdc7130990/CG000239_VisiumSpatialGeneExpression_UserGuide_RevD.pdf>, 70 pages. |
| Adessi et al., “Solid phase DNA amplification: characterisation of primer attachment and amplification mechanisms,” Nucl. Acids Res., 2000, 28(20):E87, 8 pages. |
| Adiconis et al., “Comparative analysis of RNA sequencing methods for degraded or low-input samples, ” Nat Methods, Jul. 2013, 10(7):623-9. |
| Affymetrix, “GeneChip Human Genome U133 Set,” retrieved from the Internet: on the World Wide Web at affymetrix.com/support/technical/datasheets/hgu133_datasheet.pdf, retrieved on Feb. 26, 2003, 2 pages. |
| Affymetrix, “Human Genome U95Av2,” Internet Citation, retrieved from the internet: on the World Wide Web affymetrix.com, retrieved on Oct. 2, 2002, 1 page. |
| Alam, “Proximity Ligation Assay (PLA),” Curr Protoc Immunol., Nov. 2018, 123(1):e58, 8 pages. |
| Albretsen et al., “Applications of magnetic beads with covalently attached oligonucleotides in hybridization: Isolation and detection of specific measles virus mRNA from a crude cell lysate,” Anal. Biochem., 1990, 189(1):40-50. |
| Allawi et al., “Thermodynamics and NMR of Internal GâT Mismatches in DNA,” Biochemistry, 1996, 36(34):10581-10594. |
| Amidzadeh et al., “Assessment of different permeabilization methods of minimizing damage to the adherent cells for detection of intracellular RNA by flow cytometry,” Avicenna J Med Biotechnol., Jan. 2014, 6(1):38-46. |
| Andresen et al., “Helicase-dependent amplification: use in OnChip amplification and potential for point-of-care diagnostics,” Expert Rev Mol Diagn., Oct. 2009, 9(7):645-650. |
| Aran et al., “xCell: digitally portraying the tissue cellular heterogeneity landscape,” Genome Biol., Nov. 2017, 18(1):220, 14 pages. |
| Archer et al., “Selective and flexible depletion of problematic sequences from RNA-seq libraries at the cDNA stage,” BMC Genomics, May 2014, 15(1):401, 9 pages. |
| Armani et al., “2D-PCR: a method of mapping DNA in tissue sections,” Lab Chip, 2009, 9(24):3526-34. |
| Asp et al., “Spatially Resolved Transcriptomes-Next Generation Tools for Tissue Exploration,” Bioessays, Oct. 2020, 42(10):e1900221, 16 pages. |
| Atkinson et al., “An Updated Protocol for High Throughput Plant Tissue Sectioning,” Front Plant Sci, 2017, 8:1721, 8 pages. |
| Atkinson, “Overview of Translation: Lecture Manuscript,” U of Texas, 2000, DD, pp. 6.1-6.8. |
| Bains et al., “A novel method for nucleic acid sequence determination,” Journal of Theoretical Biology, 1988, 135(3), 303-7. |
| Balakrishnan et al., “Flap endonuclease 1,” Annu Rev Biochem., Jun. 2013, 82:119-138. |
| Barnes, “PCR amplification of up to 35-kb DNA with high fidelity and high yield from lambda bacteriophage templates,” Proc. Natl. Acad. Sci USA, 1994, 91(6):2216-2220. |
| Barnett et al., “ATAC-Me Captures Prolonged DNA Methylation of Dynamic Chromatin Accessibility Loci during Cell Fate Transitions,” Mol Cell., Mar. 2020, 77(6):1350-1364.e6. |
| Bartosovic et al., “Single-cell CUT&Tag profiles histone modifications and transcription factors in complex tissues,” Nat Biotechnol., Jul. 2021, 39(7):825-835, Abstract. |
| Baugh et al., “Quantitative analysis of mRNA amplification by in vitro transcription,” Nucleic Acids Res., 2001, 29(5):e29, 9 pages. |
| Beattie et al., “Advances in genosensor research,” Clin Chem., May 1995, 41(5):700-6. |
| Beechem et al., “High-Plex Spatially Resolved RNA and Protein Detection Using Digital Spatial Profiling: A Technology Designed for Immuno-oncology Biomarker Discovery and Translational Research,” Methods Mol Biol, 2020, Chapter 25, 2055:563-583. |
| Bell, “A simple way to treat PCR products prior to sequencing using ExoSAP-IT,” Biotechniques, 2008, 44(6):834, 1 page. |
| Bentley et al., “Accurate whole human genome sequencing using reversible terminator chemistry,” Nature, 2008, 456(7218):53-59. |
| Bergenstråhle et al., “Seamless integration of image and molecular analysis for spatial transcriptomics workflows,” BMC Genomics, Jul. 2020, 21(1):482, 7 pages. |
| Berger et al., “Universal bases for hybridization, replication and chain termination,” Nucleic Acid Res., Aug. 2000, 28(15):2911-2914. |
| Birney et al., “Identification and analysis of functional elements in 1% of the human genome by the ENCODE pilot project,” Nature, 2007, 447(7146):799-816. |
| Blair et al., “Microarray temperature optimization using hybridization kinetics,” Methods Mol Biol., 2009, 529:171-96. |
| Blanchard et al., “High-density oligonucleotide arrays,” Biosensors & Bioelectronics, 1996, 11(6-7):687-690. |
| Blanco et al., “A practical approach to FRET-based PNA fluorescence in situ hybridization,” Methods, Dec. 2010, 52(4):343-51. |
| Blokzijl et al., “Profiling protein expression and interactions: proximity ligation as a tool for personalized medicine,” J Intern. Med., 2010, 268(3):232-245. |
| Blow, “Tissue Issues,” Nature, 2007, 448(7156):959-962. |
| Bolognesi et al., “Multiplex Staining by Sequential Immunostaining and Antibody Removal on Routine Tissue Sections,” J. Histochem. Cytochem., Aug. 2017, 65(8):431-444. |
| Bolotin et al., “MiXCR: software for comprehensive adaptive immunity profiling,” Nat Methods., May 2015, 12(5):380-1. |
| Boulé et al., “Terminal deoxynucleotidyl transferase indiscriminately incorporates ribonucleotides and deoxyribonucleotides,” J Biol Chem., Aug. 2001, 276(33):31388-93. |
| Brandon et al., “Mitochondrial mutations in cancer,” Oncogene, 2006, 25(34):4647-4662. |
| Brenner et al., “Gene expression analysis by massively parallel signature sequencing (MPSS) on microbead arrays,” Nat. Biotech., 2000, 18(6):630-634. |
| Brenner et al., “In vitro cloning of complex mixtures of DNA on microbeads: physical separation of differentially expressed cDNAs,” Proc. Natl. Acad. Sci. USA, 2000, 97(4):1665-1670. |
| Brow, “35—The Cleavase I enzyme for mutation and polymorphism scanning,” PCR Applications Protocols for Functional Genomics, 1999, pp. 537-550. |
| Brown et al., “Retroviral integration: structure of the initial covalent product and its precursor, and a role for the viral IN protein,” Proc Natl Acad Sci USA, Apr. 1989, 86(8):2525-9. |
| Buenrostro et al., “Transposition of native chromatin for multimodal regulatory analysis and personal epigenomics,” Nat Methods, Dec. 2013, 10(12):1213-1218. |
| Bullard et al., “Direct comparison of nick-joining activity of the nucleic acid ligases from bacteriophage T4,” Biochem. J. 2006, 398(1):135-144. |
| Burgess, “A space for transcriptomics,” Nature Reviews Genetics, 2016, 17(8):436-7. |
| Burgess, “Finding structure in gene expression,” Nature Reviews Genetics, 2018, 19(5):249, 1 page. |
| Burgess, “Spatial transcriptomics coming of age,” Nat Rev Genet., Jun. 2019, 20(6):317, 1 page. |
| Ertsey et al., “Coverslip Mounted-Immersion Cycled in Situ RT-PCR for the Localization of mRNA in Tissue Sections,” Biotechniques, 1998, 24(1):92-100. |
| Caliari et al., “A practical guide to hydrogels for cell culture,” Nat Methods., Apr. 2016, 13(5):405-14. |
| Cha et al., “Specificity, efficiency, and fidelity of PCR,” Genome Res., 1993, 3(3):S18-29. |
| Chandra et al., “Cell-free synthesis-based protein microarrays and their applications,” Proteomics, 2009, 5(6):717-30. |
| Chatterjee et al., “Mitochondrial DNA mutations in human cancer. Oncogene,” 2006, 25(34):4663-4674. |
| Chen et al., “DNA hybridization detection in a microfluidic Channel using two fluorescently labelled nucleic acid probes,” Biosensors and Bioelectronics, 2008, 23(12):1878-1882. |
| Chen et al., “Expansion microscopy,” Science, 2015, 347(6221):543-548. |
| Chen et al., “Nanoscale imaging of RNA with expansion microscopy,” Nat Methods, Aug. 2016, 13(8):679-84. |
| Chen et al., “Parallel single nucleotide polymorphism genotyping by surface invasive cleavage with universal detection,” Anal Chem., Apr. 2005, 77(8):2400-5. |
| Chen et al., “RNA imaging. Spatially resolved, highly multiplexed RNA profiling in single cells,” Science, Apr. 2015, 348(6233):aaa6090, 21 pages. |
| Chen et al., “Spatial Transcriptomics and In Situ Sequencing to Study Alzheimer's Disease,” Cell, Aug. 2020, 182(4):976-991. |
| Chen et al., “uCB-seq: microfluidic cell barcoding and sequencing for high-resolution imaging and sequencing of single cells,” Lab Chip, Nov. 2020, 20(21):3899-3913. |
| Chester et al., “Dimethyl sulfoxide-mediated primer Tm reduction: a method for analyzing the role of renaturation temperature in the polymerase chain reaction,” Anal Biochem, Mar. 1993, 209(2):284-90. |
| Chrisey et al., “Covalent attachment of synthetic DNA to self-assembled monolayer films,” Nucleic Acids Res., Aug. 1996, 24(15):3031-9. |
| Ciaccio et al., “Systems analysis of EGF receptor signaling dynamics with microwestern arrays,” Nat Methods, Feb. 2010, 7(2):148-55. |
| Constantine et al., “Use of genechip high-density oligonucleotide arrays for gene expression monitoring,” Life Science News, Amersham Life Science, 1998, pp. 11-14. |
| Corces et al., “An improved ATAC-seq protocol reduces background and enables interrogation of frozen tissues,” Nat. Methods, 2017, 14(10):959-962. |
| Credle et al., “Multiplexed analysis of fixed tissue RNA using Ligation in situ Hybridization,” Nucleic Acids Research, 2017, 45(14):e128, 9 pages. |
| Crosetto et al., “Spatially resolved transcriptomics and beyond,” Nature Review Genetics, 2015, 16(1):57-66. |
| Cruz et al., “Methylation in cell-free DNA for early cancer detection,” Ann Oncol., Jun. 2018, 29(6):1351-1353. |
| Cujec et al., “Selection of v-Abl tyrosine kinase substrate sequences from randomized peptide and cellular proteomic libraries using mRNA display,” Chemistry and Biology, 2002, 9(2):253-264. |
| Czarnik, “Encoding methods for combinatorial chemistry,” Curr Opin Chem Biol., Jun. 1997, 1(1):60-6. |
| Dahl et al., “Circle-to-circle amplification for precise and sensitive DNA analysis,” Proc. Natl. Acad. Sci., 2004, 101(13):4548-4553. |
| Dalma-Weiszhausz et al., “The affymetrix GeneChip platform: an overview,” Methods Enzymol., 2006, 410:3-28. |
| Darmanis et al., “ProteinSeq: High-Performance Proteomic Analyses by Proximity, Ligation and Next Generation Sequencing,” PLos One, 2011, 6(9):e25583, 10 pages. |
| Daubendiek et al., “Rolling-Circle RNA Synthesis: Circular Oligonucleotides as Efficient Substrates for T7 RNA Polymerase,” J. Am. Chem. Soc., 1995, 117(29):7818-7819. |
| Davies et al., “How best to identify chromosomal interactions: a comparison of approaches,” Nat. Methods, 2017, 14(2):125-134. |
| Deamer et al., “Characterization of nucleic acids by nanopore analysis,” Acc Chem Res., Oct. 2002, 35(10):817-25. |
| Dean et al., “Comprehensive human genome amplification using multiple displacement amplification,” Proc Natl. Acad. Sci. USA, 2002, 99(8):5261-66. |
| Deng et al., “Spatial Epigenome Sequencing at Tissue Scale and Cellular Level,” BioRxiv, Mar. 2021, 40 pages. |
| Dressman et al., “Transforming single DNA molecules into fluorescent magnetic particles for detection and enumeration of genetic variations,” Proc. Natl. Acad. Sci. USA, 2003, 100(15):8817-8822. |
| Drmanac et al., “CoolMPS™: Advanced massively parallel sequencing using antibodies specific to each natural nucleobase,” BioRxiv, 2020, 19 pages. |
| Druley et al., “Quantification of rare allelic variants from pooled genomic DNA,” Nat. Methods, 2009, 6(4):263-65. |
| Duncan et al., “Affinity chromatography of a sequence-specific DNA binding protein using Teflon-linked oligonucleotides,” Anal. Biochem., 1988, 169(1):104-108. |
| Eberwine, “Amplification of mRNA populations using aRNA generated from immobilized oligo(dT)-T7 primed cDNA,” BioTechniques, 1996, 20(4):584-91. |
| Eguiluz et al., “Multitissue array review: a chronological description of tissue array techniques, applications and procedures,” Pathology Research and Practice, 2006, 202(8):561-568. |
| Eldridge et al., “An in vitro selection strategy for conferring protease resistance to ligand binding peptides,” Protein Eng Des Sel., 2009, 22(11):691-698. |
| Ellington et al., “Antibody-based protein multiplex platforms: technical and operational challenges,” Clin Chem, 2010, 56(2):186-193. |
| Eng et al., “Profiling the transcriptome with RNA SPOTs,” Nat Methods., 2017, 14(12):1153-1155. |
| Evers et al., “The effect of formaldehyde fixation on RNA: optimization of formaldehyde adduct removal,” J Mol Diagn., May 2011, 13(3):282-8. |
| Fire et al., “Rolling replication of short DNA circles,” Proc. Natl. Acad. Sci., 1995, 92(10):4641-4645. |
| Flanigon et al., “Multiplex protein detection with DNA readout via mass spectrometry,” N. Biotechnol., 2013, 30(2):153-158. |
| Fluidigm, “Equivalence of Imaging Mass Cytometry and Immunofluorescence on FFPE Tissue Sections,” White Paper, 2017, 12 pages. |
| Fodor et al., “Light-directed, spatially addressable parallel chemical synthesis,” Science, 1995, 251(4995):767-773. |
| Forster et al., “A human gut bacterial genome and culture collection for improved metagenomic analyses,” Nature Biotechnology, 2019, 37(2):186-192. |
| Frese et al., “Formylglycine aldehyde Tag—protein engineering through a novel post-translational modification,” ChemBioChem., 2009, 10(3):425-27. |
| Fu et al., “Counting individual DNA molecules by the stochastic attachment of diverse labels,” PNAS, 2011, 108(22):9026-9031. |
| Fu et al., “Repeat subtraction-mediated sequence capture from a complex genome,” Plant J., Jun. 2010, 62(5):898-909. |
| Fu et al., “Continuous Polony Gels for Tissue Mapping with High Resolution and RNA Capture Efficiency,” bioRxiv, 2021, 20 pages. |
| Fullwood et al., “Next-generation DNA sequencing of paired-end tags (PET) for transcriptome and genome analyses,” Genome Res., 2009, 19(4):521-532. |
| Ganguli et al., “Pixelated spatial gene expression analysis from tissue,” Nat Commun., Jan. 2018, 9(1):202, 9 pages. |
| Gansauge et al., “Single-stranded DNA library preparation from highly degraded DNA using T4 DNA ligase,” Nucleic Acids Res., Jun. 2017, 45(10):e79, 10 pages. |
| Gao et al., “Q&A: Expansion microscopy,” BMC Biology, 15:50, 9 pages, 2017. |
| Gene@arrays[online], BeadArray Technology, available on or before Feb. 14, 2015, via Internet Archive: Wayback Machine URL <https://web.archive.org/web/20150214084616/http://genearrays.com/services/microarrays/illumina/beadarray-technology/>, [retrieved on Jan. 30, 2020], 3 pages. |
| Gerard et al., “Excess dNTPs minimize RNA hydrolysis during reverse transcription,” Biotechniques, Nov. 2002, 33(5):984, 986, 988, 990. |
| Gill et al., “Nucleic acid isothermal amplification technologies: a review,” Nucleosides Nucleotides Nucleic Acids, Mar. 2008, 27(3):224-43. |
| Glass et al., “SIMPLE: a sequential immunoperoxidase labeling and erasing method,” J. Histochem. Cytochem., Oct. 2009, 57(10):899-905. |
| Gloor, “Gene targeting in Drosophila,” Methods Mol Biol., 2004, 260:97-114. |
| Gnanapragasam, “Unlocking the molecular archive: the emerging use of formalin-fixed paraffin-embedded tissue for biomarker research in urological cancer,” BJU International, 2009, 105(2):274-278. |
| Goldkorn et al., “A simple and efficient enzymatic method for covalent attachment of DNA to cellulose. Application for hybridization-restriction analysis and for in vitro synthesis of DNA probes,” Nucleic Acids Res., 1986, 14(22):9171-9191. |
| Goryshin et al., “Tn5 in vitro transposition,” J Biol Chem., Mar. 1998, 273(13):7367-74. |
| Gracia Villacampa et al., “Genome-wide Spatial Expression Profiling in FFPE Tissues,” bioRxiv, 2020, pp. 38 pages. |
| Grokhovsky, “Specificity of DNA cleavage by ultrasound,” Molecular Biology, 2006, 40(2):276-283. |
| Gu et al., “Protein tag-mediated conjugation of oligonucleotides to recombinant affinity binders for proximity ligation,” N Biotechnol., 2013, 30(2):144-152. |
| Gunderson et al., “Decoding randomly ordered DNA arrays,” Genome Research, 2004, 14(5):870-877. |
| Guo et al., “Direct fluorescence analysis of genetic polymorphisms by hybridization with oligonucleotide arrays on glass supports,” Nucleic Acids Res., Dec. 1994, 22(24):5456-65. |
| Gupta et al., “Single-cell isoform RNA sequencing characterizes isoforms in thousands of cerebellar cells,” Nature Biotechnol., Oct. 2018, 36:1197-1202. |
| Hafner et al., “Identification of microRNAs and other small regulatory RNAs using cDNA library sequencing,” Methods, Jan. 2008, 44(1):3-12. |
| Hahnke et al., “Striptease on glass: validation of an improved stripping procedure for in situ microarrays,” J Biotechnol., Jan. 2007, 128(1):1-13. |
| Hamaguchi et al., “Direct reverse transcription-PCR on oligo(dT)-immobilized polypropylene microplates after capturing total mRNA from crude cell lysates,” Clin Chem., Nov. 1998, 44(11):2256-63. |
| Hanauer et al., “Separation of nanoparticles by gel electrophoresis according to size and shape,” Nano Lett., Sep. 2007, 7(9):2881-5. |
| Hardenbol et al., “Highly multiplexed molecular inversion probe genotyping: over 10,000 targeted SNPs genotyped in a single tube assay,” Genome Res., Feb. 2005, 15(2):269-75. |
| Hardenbol et al., “Multiplexed genotyping with sequence-tagged molecular inversion probes,” Nature Biotechnol., Jun. 2003, 21(6):673-678. |
| Hayes et al., “Electrophoresis of proteins and nucleic acids: I-Theory,” BMJ, Sep. 1989, 299(6703):843-6. |
| He et al., “In situ synthesis of protein arrays,” Current Opinion in Biotechnology, 2008, 19(1):4-9. |
| He et al., “Printing protein arrays from DNA arrays,” Nature Methods, 2008, 5(2):175-77. |
| He, “Cell-free protein synthesis: applications in proteomics and biotechnology,” New Biotechnology, 2008, 25(2-3):126-132. |
| Healy, “Nanopore-based single-molecule DNA analysis,” Nanomedicine (Lond), Aug. 2007, 2(4):459-81. |
| Hejatko et al., “In situ hybridization technique for mRNA detection in whole mount Arabidopsis samples,” Nature Protocols, 2006, 1(4):1939-1946. |
| Hessner et al., “Genotyping of factor V G1691A (Leiden) without the use of PCR by invasive cleavage of oligonucleotide probes,” Clin Chem., Aug. 2000, 46(8 Pt 1):1051-6. |
| Hiatt et al., “Parallel, tag-directed assembly of locally derived short sequence reads,” Nature Methods, 2010, 7(2):119-25. |
| Ho et al., “Bacteriophage T4 RNA ligase 2 (gp24.1) exemplifies a family of RNA ligases found in all phylogenetic domains,” PNAS, Oct. 2002, 99(20):12709-14. |
| Ho et al., “Characterization of an ATP-Dependent DNA Ligase Encoded by Chlorella Virus PBCV-1,” Journal of Virology, Mar. 1997, 71(3):1931-1937. |
| Hoffman et al., “Formaldehyde crosslinking: a tool for the study of chromatin complexes,” J Biol Chem., Oct. 2015, 290(44):26404-11. |
| Hsuih et al., “Novel, Ligation-Dependent PCR Assay for Detection of Hepatitis C Virus in Serum,” Journal of Clinical Microbiology, Mar. 1996, 34(3):501-507. |
| Hu et al., “High reproducibility using sodium hydroxide-stripped long oligonucleotide DNA microarrays,” Biotechniques, Jan. 2005, 38(1):121-4. |
| Hughes et al., “Microfluidic Western blotting,” PNAS, Dec. 2012, 109(52):21450-21455. |
| Hycultbiotech.com, [online], “Immunohistochemistry, Paraffin” Apr. 2010, retrieved on Apr. 16, 2020, retrieved from URL<https://www.hycultbiotech.com/media/wysiwyg/Protocol_Immunohistochemistry_Paraffin_2.pdf>, 3 pages. |
| Ichikawa et al., “In vitro transposition of transposon Tn3,” J Biol. Chem., Nov. 1990, 265(31):18829-32, Abstract. |
| Illumina.com [online], “Ribo-Zero® rRNA Removal Kit Reference Guide,” Aug. 2016, retrieved on Apr. 26, 2022, retrieved from URL<https://jp.support.illumina.com/content/dam/illumina-support/documents/documentation/chemistry_documentation/ribosomal-depletion/ribo-zero/ribo-zero-reference-guide-15066012-02.pdf>, 36 pages. |
| Jamur et al., “Permeabilization of cell membranes.,” Method Mol. Biol., 2010, 588:63-66. |
| Jemt et al., “An automated approach to prepare tissue-derived spatially barcoded RNA-sequencing libraries,” Scientific Reports, 2016, 6:37137, 10 pages. |
| Jensen et al., “Zinc fixation preserves flow cytometry scatter and fluorescence parameters and allows simultaneous analysis of DNA content and synthesis, and intracellular and surface epitopes,” Cytometry A., Aug. 2010, 77(8):798-804. |
| Jucá et al., “Effect of dimethyl sulfoxide on reverse transcriptase activity,” Braz. J. Med. Biol. Res., Mar. 1995, 28(3):285-90. |
| Kalantari et al., “Deparaffinization of formalin-fixed paraffin-embedded tissue blocks using hot water instead of xylene,” Anal Biochem., Aug. 2016, 507:71-3. |
| Kap et al., “Histological assessment of PAXgene tissue fixation and stabilization reagents,” PLoS One, 2011, 6:e27704, 10 pages. |
| Kapteyn et al., “Incorporation of non-natural nucleotides into template-switching oligonucleotides reduces background and improves cDNA synthesis from very small RNA samples,” BMC Genomics, Jul. 2010, 11:413, 9 pages. |
| Karmakar et al., “Organocatalytic removal of formaldehyde adducts from RNA and DNA bases,” Nature Chemistry, Aug. 3, 2015, 7(9):752-758. |
| Kaya-Okur et al., “CUT&Tag for efficient epigenomic profiling of small samples and single cells,” Apr. 2019, 10(1):1930, 10 pages. |
| Ke et al., “In situ sequencing for RNA analysis in preserved tissue and cells,” Nat Methods., Sep. 2013, Supplementary Materials, 29 pages. |
| Kennedy-Darling et al., “Measuring the Formaldehyde Protein-DNA Cross-Link Reversal Rate,” Analytical Chemistry, 2014, 86(12):5678-5681. |
| Kent et al., “Polymerase θ is a robust terminal transferase that oscillates between three different mechanisms during end-joining” Elife, Jun. 2016, 5:e13740, 25 pages. |
| Kirby et al., “Cryptic plasmids of Mycobacterium avium: Tn552 to the rescue,” Mol Microbiol., Jan. 2002, 43(1):173-86. |
| Kleckner et al., “Tn10 and IS10 transposition and chromosome rearrangements: mechanism and regulation in vivo and in vitro,” Curr Top Microbiol Immunol., 1996, 204:49-82. |
| Korbel et al., “Paired-end mapping reveals extensive structural variation in the human genome,” Science, 2007, 318(5849):420-426. |
| Kozlov et al., “A highly scalable peptide-based assay system for proteomics,” PLoS One, 2012, 7(6):e37441, 10 pages. |
| Kozlov et al., “A method for rapid protease substrate evaluation and optimization,” Comb Chem High Throughput Screen, 2006, 9(6):481-87. |
| Kristensen et al., “High-Throughput Methods for Detection of Genetic Variation,” BioTechniques, Feb. 2001, 30(2):318-332. |
| Krzywkowski et al., “Chimeric padlock and iLock probes for increased efficiency of targeted RNA detection,” RNA, Jan. 2019, 25(1):82-89. |
| Krzywkowski et al., “Fidelity of RNA templated end-joining by chlorella virus DNA ligase and a novel iLock assay with improved direct RNA detection accuracy,” Nucleic Acids Research, Oct. 2017, 45(18):e161, 9 pages. |
| Kumar et al., “Template-directed oligonucleotide strand ligation, covalent intramolecular DNA circularization and catenation using click chemistry,” J Am Chem Soc., May 2007, 129(21):6859-64. |
| Kurz et al., “cDNA—protein fusions: covalent protein—gene conjugates for the in vitro selection of peptides and proteins,” ChemBioChem., 2001, 2(9):666-72. |
| Kwok, “High-throughput genotyping assay approaches,” Pharmocogenomics, Feb. 2000, 1(1):95-100. |
| Lage et al., “Whole genome analysis of genetic alterations in small DNA samples using hyperbranched strand displacement amplification and array-CGH,” Genome Research, 2003, 13(2):294-307. |
| Lahiani et al., “Enabling Histopathological Annotations on Immunofluorescent Images through Virtualization of Hematoxylin and Eosin,” J Pathol Inform., Feb. 2018, 9:1, 8 pages. |
| Lampe et al., “A purified mariner transposase is sufficient to mediate transposition in vitro,” EMBO J., Oct. 1996, 15(19):5470-9. |
| Landegren et al., “Reading bits of genetic information: methods for single-nucleotide polymorphism analysis,” Genome Res., Aug. 1998, 8(8):769-76. |
| Langdale et al., “A rapid method of gene detection using DNA bound to Sephacryl,” Gene, 1985, 36(3):201-210. |
| Larman et al., “Sensitive, multiplex and direct quantification of RNA sequences using a modified RASL assay,” Nucleic Acids Research, 2014, 42(14):9146-9157. |
| Lee et al., “Fluorescent in situ sequencing (FISSEQ) of RNA for gene expression profiling in intact cells and tissues,” Nature Protocols, 2015, 10(3):442-458. |
| Lee et al., “Improving the efficiency of genomic loci capture using oligonucleotide arrays for high throughput resequencing,” BMC Genomics, Dec. 2009, 10:646, 12 pages. |
| Leriche et al., “Cleavable linkers in chemical biology,” Bioorganic & Medicinal Chemistry, 2012, 20:571-582. |
| Li et al., “A photocleavable fluorescent nucleotide for DNA sequencing and analysis,” Proc. Natl. Acad. Sci., 2003, 100(2):414-419. |
| Li et al., “An activity-dependent proximity ligation platform for spatially resolved quantification of active enzymes in single cells,” Nat Commun, Nov. 2017, 8(1):1775, 12 pages. |
| Li et al., “RASL-seq for Massively Parallel and Quantitative Analysis of Gene Expression,” Curr Protoc Mol Biol., Apr. 2012, 4(13):1-10. |
| Li et al., “Review: a comprehensive summary of a decade development of the recombinase polymerase amplification,” Analyst, Dec. 2018, 144(1):31-67. |
| Lin et al., “Highly multiplexed imaging of single cells using a high-throughput cyclic immunofluorescence method,” Nat Commun., Sep. 2015, 6:8390, 7 pages. |
| Linnarsson, “Recent advances in DNA sequencing methods—general principles of sample preparation,” Experimental Cell Research, 2010, 316(8):1339-1343. |
| Liu et al., “High-Spatial-Resolution Multi-Omics Atlas Sequencing of Mouse Embryos via Deterministic Barcoding in Tissue,” BioRxiv, 2019, 55 pages. |
| Lizardi et al., “Mutation detection and single-molecule counting using isothermal rolling-circle amplification,” Nat. Genet., 1998, 19(3):225-232. |
| Lou et al., “A review of room temperature storage of biospecimen tissue and nucleic acids for anatomic pathology laboratories and biorepositories,” Clin Biochem., Mar. 2014, 47(4-5):267-73. |
| Lovatt et al., “Transcriptome in vivo analysis (TIVA) of spatially defined single cells in live tissue,” Nature Methods, 2013, 11(2):190-196. |
| Lund et al., “Assessment of methods for covalent binding of nucleic acids to magnetic beads, Dynabeads, and the characteristics of the bound nucleic acids in hybridization reactions,” Nucleic Acids Res., 1988, 16(22):10861-80. |
| Lundberg et al., “High-fidelity amplification using a thermostable DNA polymerase isolated from Pyrococcus furiosus,” Gene, 1991, 108(1):1-6. |
| Lundberg et al., “Homogeneous antibody-based proximity extension assays provide sensitive and specific detection of low-abundant proteins in human blood,” Nucleic Acids Res., 2011, 39(15):e102, 8 pages. |
| Lundberg et al., “Multiplexed homogeneous proximity ligation assays for high-throughput protein biomarker research in serological material,” Mol Cell Proteomics, 2011, 10(4):M110.004978, 11 pages. |
| Lundin et al., “Increased throughput by parallelization of library preparation for massive sequencing,” PLoS One, Apr. 2010, 5(4):e10029, 7 pages. |
| Lyamichev et al., “Invader assay for SNP genotyping,” Methods Mol Biol., 2003, 212:229-40. |
| Lyamichev et al., “Polymorphism identification and quantitative detection of genomic DNA by invasive cleavage of oligonucleotide probes,” Nat Biotechnol., Mar. 1999, 17(3):292-6. |
| Lyck et al., “Immunohistochemical markers for quantitative studies of neurons and glia in human neocortex,” J Histochem Cytochem, 2008, 56(3):201-21. |
| Lykidis et al., “Novel zinc-based fixative for high quality DNA, RNA and protein analysis,” Nucleic Acids Res., Jun. 2007, 35(12):e85, 10 pages. |
| MacBeath et al., “Printing proteins as microarrays for high-throughput function determination,” Science, Sep. 2000, 289(5485):1760-1763. |
| MacIntyre, “Unmasking antigens for immunohistochemistry.,” Br J Biomed Sci., 2001, 58(3):190-6. |
| McCloskey et al., “Encoding PCR products with batch-stamps and barcodes,” Biochem. Genet., 2007, 45(11-12):761-767. |
| Meers et al., “Improved CUT&RUN chromatin profiling tools,” Elife, Jun. 2019, 8:e46314, 16 pages. |
| Merritt et al., “Multiplex digital spatial profiling of proteins and RNA in fixed tissue,” Nat Biotechnol, May 2020, 38(5):586-599. |
| Metzker, “Sequencing technologies—the next generation,” Nature Reviews Genetics, 2010, 11(1):31-46. |
| Miele et al., “Mapping cis- and trans-chromatin interaction networks using chromosome conformation capture (3C),” Methods Mol Biol., 2009, 464:105-21. |
| Miller et al., “Basic concepts of microarrays and potential applications in clinical microbiology,” Clinical Microbiology Reviews, 2009, 22(4):611-633. |
| Miller et al., “Chapter 11—Solid and Suspension Microarrays for Microbial Diagnostics,” Methods in Microbiology, 2015, 42:395-431. |
| Miner et al., “Molecular barcodes detect redundancy and contamination in hairpin-bisulfite PCR,” Nucleic Acids Res., Sep. 2004, 32(17):e135, 4 pages. |
| Mishra et al., “Three-dimensional genome architecture and emerging technologies: looping in disease,” Genome Medicine, 2017, 9(1):87, 14 pages. |
| Mitra et al., “Digital genotyping and haplotyping with polymerase colonies,” Proc. Natl. Acad. Sci. USA, May 2003, 100(10):5926-5931. |
| Miura et al., “Highly efficient single-stranded DNA ligation technique improves low-input whole-genome bisulfite sequencing by post-bisulfite adaptor tagging,” Nucleic Acids Res., Sep. 2019, 47(15):e85, 10 pages. |
| Mizusawa et al., “A bacteriophage lambda vector for cloning with BamHI and Sau3A,” Gene, 1982, 20(3):317-322. |
| Morlan et al., “Selective depletion of rRNA enables whole transcriptome profiling of archival fixed tissue,” PLoS One, Aug. 2012, 7(8):e42882, 8 pages. |
| Motea et al., “Terminal deoxynucleotidyl transferase: the story of a misguided DNA polymerase,” Biochim Biophys Acta., May 2010, 1804(5):1151-66. |
| Mulder et al., “CapTCR-seq: hybrid capture for T-cell receptor repertoire profiling,” Blood Advances, Dec. 2018, 2(23):3506-3514. |
| Nadji et al., “Immunohistochemistry of tissue prepared by a molecular-friendly fixation and processing system,” Appl Immunohistochem Mol Morphol., Sep. 2005, 13(3):277-82. |
| Nandakumar et al., “How an RNA Ligase Discriminates RNA versus DNA Damage,” Molecular Cell, 2004, 16:211-221. |
| Nandakumar et al., “RNA Substrate Specificity and Structure-guided Mutational Analysis of Bacteriophage T4 RNA Ligase 2,” Journal of Biological Chemistry, Jul. 2004, 279(30):31337-31347. |
| Ncbi.nlm.nih.gov, [online], “Molecular Inversion Probe Assay,” available on or before Oct. 14, 2014, via Internet Archive: Wayback Machine URL<https://web.archive.org/web/20141014124037/https://www.ncbi.nlm.nih.gov/probe/docs/techmip/>, retrieved on Jun. 16, 2021, retrieved from URL<https://www.ncbi.nlm.nih.gov/probe/docs/techmip/>, 2 pages. |
| Ng et al., “Gene identification signature (GIS) analysis for transcriptome characterization and genome annotation,” Nature Methods, 2005, 2(2):105-111. |
| Nichols et al., “RNA Ligases,” Curr Protoc Mol Biol., Oct. 2008, 84(1):3.15.1-3.15.4. |
| Niedringhaus et al., “Landscape of next-generation sequencing technologies,” Anal Chem., Jun. 2011, 83(12):4327-41. |
| Nikiforov et al., “The use of 96-well polystyrene plates for DNA hybridization-based assays: an evaluation of different approaches to oligonucleotide immobilization,” Anal Biochem, May 1995, 227(1):201-9. |
| Niklas et al., “Selective permeabilization for the high-throughput measurement of compartmented enzyme activities in mammalian cells,” Anal Biochem, Sep. 2011, 416(2):218-27. |
| Nilsson et al., “RNA-templated DNA ligation for transcript analysis,” Nucleic Acids Res., Jan. 2001, 29(2):578-81. |
| Nowak et al., “Entering the Postgenome Era,” Science, 1995, 270(5235):368-71. |
| Ohtsubo et al., “Bacterial insertion sequences,” Curr Top Microbiol Immunol., 1996, 204:1-26. |
| Olivier, “The Invader assay for SNP genotyping,” Mutat. Res., Jun. 2005, 573(1-2):103-110. |
| Ozsolak et al., “Digital transcriptome profiling from attomole-level RNA samples,” Genome Res., Apr. 2010, 20(4):519-25. |
| Pandey et al., “Inhibition of terminal deoxynucleotidyl transferase by adenine dinucleotides. Unique inhibitory action of Ap5A,” FEBS Lett., Mar. 1987, 213(1):204-8. |
| Park et al., “Single cell trapping in larger microwells capable of supporting cell spreading and proliferation,” Microfluid Nanofluid, 2010, 8:263-268. |
| Passow et al., “RNAlater and flash freezing storage methods nonrandomly influence observed gene expression in RNAseq experiments,” bioRxiv, Jul. 2018, 28 pages. |
| PCT International Preliminary Report on Patentability in International Appln. No. PCT/EP2016/057355, dated Oct. 10, 2017, 7 pages. |
| PCT International Preliminary Report on Patentability in International Appln. No. PCT/US2019/048434, dated Mar. 2, 2021, 15 pages. |
| PCT International Preliminary Report on Patentability in International Appln. No. PCT/US2021/018795, dated Sep. 1, 2022, 10 pages. |
| PCT International Preliminary Report on Patentability in International Appln. No. PCT/US2021/018816, dated Sep. 1, 2022, 9 pages. |
| PCT International Search Report and Written Opinion in International Appln. No. PCT/US2020/066681, dated Apr. 14, 2021, 17 pages. |
| PCT International Search Report and Written Opinion in International Appln. No. PCT/US2021/012659, dated Apr. 16, 2021, 15 pages. |
| PCT International Search Report and Written Opinion in International Appln. No. PCT/US2022/028071, dated Aug. 25, 2022, 13 pages. |
| Pellestor et al., “The peptide nucleic acids (PNAs), powerful tools for molecular genetics and cytogenetics,” Eur J Hum Genet., Sep. 2004, 12(9):694-700. |
| Pemov et al., “DNA analysis with multiplex microarray-enhanced PCR,” Nucl. Acids Res., Jan. 2005, 33(2):e11, 9 pages. |
| Penno et al., “Stimulation of reverse transcriptase generated cDNAs with specific indels by template RNA structure: retrotransposon, dNTP balance, RT-reagent usage,” Nucleic Acids Res., Sep. 2017, 45(17):10143-10155. |
| Perler et al., “Intervening sequences in an Archaea DNA polymerase gen,” Proc Natl Acad Sci USA, Jun. 1992, 89(12):5577-5581. |
| Perocchi et al., “Antisense artifacts in transcriptome microarray experiments are resolved by actinomycin D,” Nucleic Acids Res., 2007, 35(19):e128, 7 pages. |
| Petterson et al., “Generations of sequencing technologies,” Genomics, 2009, 93(2):105-111. |
| Picelli et al., “Full-length RNA-seq from single cells using Smart-seq2,” Nat Protoc., Jan. 2014, 9(1):171-81. |
| Picelli et al., “Tn5 transposase and tagmentation procedures for massively scaled sequencing projects,” Genome Res., Dec. 2014, 24(12):2033-40. |
| Pipenburg et al., “DNA detection using recombination proteins,” PLoS Biol., Jul. 2006, 4(7):e204, 7 pages. |
| Pirici et al., “Antibody elution method for multiple immunohistochemistry on primary antibodies raised in the same species and of the same subtypem,” J. Histochem. Cytochem., Jun. 2009, 57(6):567-75. |
| Plasterk, “The Tcl/mariner transposon family,” Curr Top Microbiol Immunol., 1996, 204:125-43. |
| Plongthongkum et al., “Advances in the profiling of DNA modifications: cytosine methylation and beyond,” Nature Reviews Genetics, Aug. 2014, 15(10):647-661. |
| Polsky-Cynkin et al., “Use of DNA immobilized on plastic and agarose supports to detect DNA by sandwich hybridization,” Clin. Chem., 1985, 31(9):1438-1443. |
| Porreca et al., “Polony DNA sequencing,” Curr Protoc Mol Biol., Nov. 2006, Chapter 7, Unit 7.8, pp. 7.8.1-7.8.22. |
| U.S. Appl. No. 61/267,363, filed Dec. 7, 2009, 33 pages. |
| Qiu et al., “Combination probes with intercalating anchors and proximal fluorophores for DNA and RNA detection,” Nucleic Acids Research, Sep. 2016, 44(17):e138, 12 pages. |
| Raab et al., “Human tRNA genes function as chromatin insulators,” EMBO J., Jan. 2012, 31(2):330-50. |
| Ranki et al., “Sandwich hybridization as a convenient method for the detection of nucleic acids in crude samples,” Gene, 1983, 21(1-2):77-85. |
| Reinartz et al., “Massively parallel signature sequencing (MPSS) as a tool for in-depth quantitative gene expression profiling in all organisms,” Brief Funct Genomic Proteomic, Feb. 2002, 1(1):95-104. |
| Reznikoff, “Tn5 as a model for understanding DNA transposition,” Mol Microbiol., Mar. 2003, 47(5):1199-206. |
| Ristic et al., “Detection of Protein-Protein Interactions and Posttranslational Modifications Using the Proximity Ligation Assay: Application to the Study of the SUMO Pathway,” Methods Mol. Biol., 2016, 1449:279-90. |
| Rodriques et al., “Slide-seq: A scalable technology for measuring genome-wide expression at high spatial resolution,” Science, 2019, 363(6434):1463-1467. |
| Ronaghi et al., “A sequencing method based on real-time pyrophosphate,” Science, Jul. 1998, 281(5375):363-365. |
| Ronaghi et al., “Real-time DNA sequencing using detection of pyrophosphate release,” Analytical Biochemistry, Nov. 1996, 242(1):84-89. |
| Ronaghi, “Pyrosequencing sheds light on DNA sequencing,” Genome Res, Jan. 2001, 11(1):3-11. |
| Roy et al., “Assessing long-distance RNA sequence connectivity via RNA-templated DNA-DNA ligation,” eLife, 2015, 4:e03700, 21 pages. |
| Salmén et al., “Barcoded solid-phase RNA capture for Spatial Transcriptomics profiling in mammalian tissue sections,” Nature Protocols, Oct. 2018, 13(11):2501-2534. |
| Saxonov et al., “10x Genomics, Mastering Biology to Advance Human Health,” PowerPoint, 10x, 2020, 41 pages. |
| Schena et al., “Quantitative monitoring of gene expression patterns with a complementary DNA microarray,” Science, Oct. 1995, 270(5235):467-470. |
| Schouten et al., “Relative quantification of 40 nucleic acid sequences by multiplex ligation-dependent probe amplification,” Nucleic Acids Res., Jun. 2002, 30(12):e57, 13 pages. |
| Schweitzer et al., “Multiplexed protein profiling on microarrays by rolling-circle amplification,” Nature Biotechnology, Apr. 2002, 20(4):359-365. |
| Schwers et al., “A high-sensitivity, medium-density, and target amplification-free planar waveguide microarray system for gene expression analysis of formalin-fixed and paraffin-embedded tissue,” Clin. Chem., Nov. 2009, 55(11):1995-2003. |
| Shalon et al., “A DNA microarray system for analyzing complex DNA samples using two-color fluorescent probe hybridization,” Genome Res., Jul. 1996, 6(7):639-45. |
| Shelbourne et al., “Fast copper-free click DNA ligation by the ring-strain promoted alkyne-azide cycloaddition reaction,” Chem. Commun., 2011, 47(22):6257-6259. |
| Shendure et al., “Accurate multiplex polony sequencing of an evolved bacterial genome,” Science, 2005, 309(5741):1728-1732. |
| Simonis et al., “Nuclear organization of active and inactive chromatin domains uncovered by chromosome conformation capture-on-chip (4C),” Nat Genet., Nov. 2006, 38(11):1348-54. |
| Singh et al., “High-throughput targeted long-read single cell sequencing reveals the clonal and transcriptional landscape of lymphocytes,” Nat Commun., Jul. 2019, 10(1):3120, 13 pages. |
| Skene et al., “An efficient targeted nuclease strategy for high-resolution mapping of DNA binding sites,” Elife, Jan. 2017, 6:e21856, 35 pages. |
| Slomovic et al., “Addition of poly(A) and poly(A)-rich tails during RNA degradation in the cytoplasm of human cells,” Proc Natl Acad Sci USA, Apr. 2010, 107(16):7407-12. |
| Sountoulidis et al., “SCRINSHOT, a spatial method for single-cell resolution mapping of cell states in tissue sections,” PLoS Biol., Nov. 2020, 18(11):e3000675, 32 pages. |
| Spiess et al., “A highly efficient method for long-chain cDNA synthesis using trehalose and betaine,” Anal. Biochem., Feb. 2002, 301(2):168-74. |
| Spitale et al., “Structural imprints in vivo decode RNA regulatory mechanisms,” Nature, 2015, 519(7544):486-90. |
| Stahl et al., “Visualization and analysis of gene expression in tissue sections by spatial transcriptomics,” Science, Jul. 2016, 353(6294):78-82. |
| Stahl et al., “Visualization and analysis of gene expression in tissue sections by spatial transcriptomics,” Supplementary Materials, Science, Jul. 2016, 353(6294):78-82, 41 pages. |
| Stimpson et al., “Real-time detection of DNA hybridization and melting on oligonucleotide arrays by using optical wave guides,” Proc Natl Acad Sci USA, Jul. 1995, 92(14):6379-83. |
| Stoddart et al., “Single-nucleotide discrimination in immobilized DNA oligonucleotides with a biological nanopore,” PNAS USA., May 2009, 106(19):7702-7707. |
| Strell et al., “Placing RNA in context and space—methods for spatially resolved transcriptomics,” The FEBS Journal, 2019, 286(8):1468-1481. |
| Stroh et al., “Quantum dots spectrally distinguish multiple species within the tumor milieu in vivo,” Nat Med., Jun. 2005, 11(6):678-82. |
| Sutherland et al., “Utility of formaldehyde cross-linking and mass spectrometry in the study of protein-protein interactions,” J. Mass Spectrom., Jun. 2008, 43(6):699-715. |
| Taylor et al., “Mitochondrial DNA mutations in human disease,” Nature Reviews Genetics, May 2005, 6(5):389-402. |
| Tentori et al., “Detection of Isoforms Differing by a Single Charge Unit in Individual Cells,” Chem. Int. Ed., 2016, 55(40):12431-5. |
| Tian et al., “Antigen peptide-based immunosensors for rapid detection of antibodies and antigens,” Anal Chem, 2009, 81(13):5218-5225. |
| Tijssen et al., “Overview of principles of hybridization and the strategy of nucleic acid assays” in Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Acid Probes, 1993, 24(Chapter 2), 65 pages. |
| Tolbert et al., “New methods for proteomic research: preparation of proteins with N-terminal cysteines for labeling and conjugation,” Angewandte Chemie International Edition, Jun. 2002, 41(12):2171-4. |
| Toubanaki et al., “Dry-reagent disposable biosensor for visual genotyping of single nucleotide polymorphisms by oligonucleotide ligation reaction: application to pharmacogenetic analysis,” Hum Mutat., Aug. 2008, 29(8):1071-8. |
| Trejo et al., “Extraction-free whole transcriptome gene expression analysis of FFPE sections and histology-directed subareas of tissue,” PLoS One, Feb. 2019, 14(2):e0212031, 22 pages. |
| Tu et al., “TCR sequencing paired with massively parallel 3′ RNA-seq reveals clonotypic T cell signatures,” Nature Immunology, Dec. 2019, 20(12):1692-1699. |
| Twyman et al., “Techniques Patents for SNP Genotyping,” Pharmacogenomics, Jan. 2003, 4(1):67-79. |
| Ulery et al., “Biomedical Applications of Biodegradable Polymers,” J Polym Sci B Polym Phys., Jun. 2011, 49(12):832-864. |
| U.S. Appl. No. 60/416,118 Fan et al., Multiplex Nucleic Acid Analysis Using Archived or Fixed Samples, filed Oct. 3, 2002, 22 pages. |
| Van Gelder et al., “Amplified RNA synthesized from limited quantities of heterogeneous cDNA,” Proc. Natl. Acad. Sci. USA, 1990, 87(5):1663-1667. |
| Vandenbroucke et al., “Quantification of splice variants using real-time PCR,” Nucleic Acids Research, 2001, 29(13):e68, 7 pages. |
| Vandernoot et al., “cDNA normalization by hydroxyapatite chromatography to enrich transcriptome diversity in RNA-seq applications,” Biotechniques, Dec. 2012, 53(6):373-80. |
| Vasiliskov et al., “Fabrication of microarray of gel-immobilized compounds on a chip by copolymerization,” Biotechniques, Sep. 1999, 27(3):592-606. |
| Vázquez Bernat et al., “High-Quality Library Preparation for NGS-Based Immunoglobulin Germline Gene Inference and Repertoire Expression Analysis,” Front Immunol., Apr. 2019, 10:660, 12 pages. |
| Velculescu et al., “Serial analysis of gene expression,” Science, Oct. 1995, 270(5235):484-7. |
| Vickovic et al., “High-definition spatial transcriptomics for in situ tissue profiling,” Nat Methods, Oct. 2019, 16(10):987-990. |
| Vickovic et al., “SM-Omics: An automated Platform for High-Throughput Spatial Multi-Omics,” bioRxiv, Oct. 2020, 40 pages. |
| Vincent et al., “Helicase-dependent isothermal DNA amplification,” EMBO Rep., Aug. 2004, 5(8):795-800. |
| Viollet et al., “T4 RNA ligase 2 truncated active site mutants: improved tools for RNA analysis,” BMC Biotechnol., Jul. 2011, 11:72, 14 pages. |
| Vogelstein et al., “Digital PCR,” Proceedings of the National Academy of Sciences, Aug. 1999, 96(16):9236-9241. |
| Waichman et al., “Functional immobilization and patterning of proteins by an enzymatic transfer reaction,” Analytical chemistry, 2010, 82(4):1478-85. |
| Walker et al., “Strand displacement amplification—an isothermal, in vitro DNA amplification technique,” Nucleic Acids Research, 1992, 20(7):1691-1696. |
| Wang et al., “Concentration gradient generation methods based on microfluidic systems,” RSC Adv., 2017, 7:29966-29984. |
| Wang et al., “Imaging-based pooled CRISPR screening reveals regulators of lncRNA localization,” Proc Natl Acad Sci USA, May 2019, 116(22):10842-10851. |
| Wang et al., “Optimization of Process Conditions for Infected Animal Tissues by Alkaline Hydrolysis Technology,” Procedia Environmental Sciences, 2016, 31:366-374. |
| Wang et al., “Tagmentation-based whole-genome bisulfite sequencing,” Nature Protocols, Oct. 2013, 8(10):2022-2032. |
| Wang et al., “High-fidelity mRNA amplification for gene profiling,” Nature Biotechnology, Apr. 2000, 18(4):457-459. |
| Wang, “RNA amplification for successful gene profiling analysis,” J Transl Med., Jul. 2005, 3:28, 11 pages. |
| Weinreich et al., “Evidence that the cis Preference of the Tn5 Transposase is Caused by Nonproductive Multimerization,” Genes and Development, Oct. 1994, 8(19):2363-2374. |
| Wiedmann et al., “Ligase chain reaction (LCR)—overview and applications,” PCR Methods Appl., Feb. 1994, 3(4):S51-64. |
| Wilson et al., “New transposon delivery plasmids for insertional mutagenesis in Bacillus anthracis,” J Microbiol Methods, Dec. 2007, 71(3):332-5. |
| Wohnhaas et al., “DMSO cryopreservation is the method of choice to preserve cells for droplet-based single-cell RNA sequencing,” Scientific Reports, Jul. 2019, 9(1):10699, 14 pages. |
| Wolf et al., “Rapid hybridization kinetics of DNA attached to submicron latex particles,” Nucleic Acids Res, 1987, 15(7):2911-2926. |
| Wong et al., “Direct Site-Selective Covalent Protein Immobilization Catalyzed by a Phosphopantetheinyl Transferase,” J. Am. Chem Soc., 2008, 130(37):12456-64. |
| Worthington et al., “Cloning of random oligonucleotides to create single-insert plasmid libraries,” Anal Biochem, 2001, 294(2):169-175. |
| Wu et al., “Detection DNA Point Mutation with Rolling-Circle Amplification Chip,” IEEE, 2010 4th International Conference on Bioinformatics and Biomedical Engineering, Jun. 2010, 4 pages. |
| Wu et al., “RollFISH achieves robust quantification of single-molecule RNA biomarkers in paraffin-embedded tumor tissue samples,” Commun Biol., Nov. 2018, 1:209, 8 pages. |
| Yasukawa et al., “Effects of organic solvents on the reverse transcription reaction catalyzed by reverse transcriptases from avian myeloblastosis virus and Moloney murine leukemia virus,” Biosci Biotechnol Biochem., 2010, 74(9):1925-30. |
| Yeakley et al., “Profiling alternative splicing on fiber-optic arrays,” Nature biotechnology, 2002, 20:353-358. |
| Yeakley et al., “A trichostatin A expression signature identified by TempO-Seq targeted whole transcriptome profiling,” PLoS One, May 2017, 12(5):e0178302, 22 pages. |
| Yershov et al., “DNA analysis and diagnostics on oligonucleotide microchips,” Proc. Natl. Acad. Sci. USA, May 1996, 93(10):4913-4918. |
| Yin et al., “Genetically encoded short peptide tag for versatile protein labeling by Sfp phosphopantetheinyl transferase,” PNAS, 2005, 102(44):15815-20. |
| Zhang et al., “Archaeal RNA ligase from Thermoccocus kodakarensis for template dependent ligation,” RNA Biol., Jan. 2017, 14(1):36-44. |
| Zhang et al., “Assembling DNA through Affinity Binding to Achieve Ultrasensitive Protein Detection,” Angew Chem Int Ed Engl., 2013, 52(41):10698-705. |
| Zhang et al., “Binding-induced DNA assembly and its application to yoctomole detection of proteins,” Anal Chem, 2012, 84(2):877-884. |
| Zhang et al., “Genome-wide open chromatin regions and their effects on the regulation of silk protein genes in Bombyx mori,” Sci Rep., Oct. 2017, 7(1):12919, 9 pages. |
| Zhang et al., “Multiplex ligation-dependent probe amplification (MLPA) for ultrasensitive multiplexed microRNA detection using ribonucleotide-modified DNA probest,” Chem. Commun., 2013, 49:10013-10015. |
| Zhao et al., “Isothermal Amplification of Nucleic Acids,” Chemical Reviews, Nov. 2015, 115(22):12491-12545. |
| Zheng et al., “Origins of human mitochondrial point mutations as DNA polymerase gamma-mediated errors,” Mutat. Res., 2006, 599(1-2):11-20. |
| Zhou et al., “Genetically encoded short peptide tags for orthogonal protein labeling by Sfp and AcpS phosphopantetheinyl transferases,” ACS Chemical Biol., 2007, 2(5):337-346. |
| Zhu et al., “Reverse transcriptase template switching: a SMART approach for full-length cDNA library construction,” Biotechniques, Apr. 2001, 30(4):892-897. |
| Goldmeyer et al., “Development of a novel one-tube isothermal reverse transcription thermophilic helicase-dependent amplification platform for rapid RNA detection,” Journal of Molecular Diagnostics, American Society for Investigative Pathology and the Association for Molecular Pathology, Nov. 1, 2007, 9(5):639-644. |
| Zahra et al., “Assessment of Different Permeabilization Methods of Minimizing Damage to the Adherent Cells for Detection of Intracellular RNA by Flow Cytometry,” Avicenna Journal of Medical Biotechnology, Jan. 1, 2014, 6(1):38-46. |
| Belaghzal et al., “Hi—C 2.0: An Optimized Hi-C Procedure for High-Resolution Genome-Wide Mapping of Chromosome Conformation,” Methods, Jul. 1, 2017, 123:56-65, 20 pages. |
| Belton et al., “Hi—C: A comprehensive technique to capture the conformation of genomes,” Methods, Nov. 2012, 58(3):268-276, 16 pages. |
| Bentzen et al., “Large-scale detection of antigen-specific T cells using peptide-MHC-I multimers labeled with DNA barcodes,” Nat Biotechnol., Oct. 2016, 34(10):1037-1045, 12 pages. |
| Bibikova et al., “Quantitative gene expression profiling in formalin-fixed paraffin-embedded tissues using universal bead arrays,” The American Journal of Pathology, Nov. 1, 2004, 165(5):1799-1807. |
| Chen et al. “Arrayed profiling of multiple glycans on whole living cell surfaces.” Analytical chemistry, Oct. 15, 2013, 85(22):11153-11158. |
| Choi et al., “Multiplexed detection of mRNA using porosity-tuned hydrogel microparticles,” Analytical chemistry, Sep. 28, 2012, 84(21):9370-9378. |
| Fan et al., “A versatile assay for high-throughput gene expression profiling on universal array matrices,” Genome Research, May 1, 2004, 14(5):878-885. |
| Fan et al., “Illumina Universal Bead Arrays,” Methods in Enzymology, 2006, 410:57-73. |
| Hadrup et al., “Parallel detection of antigen-specific T-cell responses by multidimensional encoding of MHC multimers,” Nat. Methods., Jul. 2009, 6(7), 520-526. |
| Hobro et al., “An evaluation of fixation methods: Spatial and compositional cellular changes observed by Raman imaging,” Vibrational Spectroscopy, Jul. 2017, 91:31-45. |
| Landegren et al., “A Ligase-Mediated Gene Detection Technique,” Science, 1988, 241(4869):1077-1080. |
| Mamedov et al., “Preparing unbiased T-cell receptor and antibody cDNA libraries for the deep next generation sequencing profiling,” Frontiers in Immunol., Dec. 23, 2013, 4(456):1-10. |
| Oksuz et al., “Systematic evaluation of chromosome conformation capture assays,” Nature Methods, Sep. 2021, 18:1046-1055. |
| Rohland et al., “Partial uracil-DNA-glycosylase treatment for screening of ancient DNA,” Phil. Trans. R. Soc. B, Jan. 19, 2015, 370(1660): Jun. 24, 2013, 11 pages. |
| Schmidl et al., “ChIPmentation: fast, robust, low-input ChIP-seq for histones and transcription factors,” Nature Methods, Oct. 2015, 12:963-965. |
| Su et al., “Restriction enzyme selection dictates detection range sensitivity in chromatin conformation capture-based variant-to-gene mapping approaches,” bioRxiv, Dec. 15, 2020, 22 pages. |
| Sun et al., “Statistical Analysis of Spatial Expression Pattern for Spatially Resolved Transcriptomic Studies,” Nature Methods, Jan. 27, 2020, 17(2): 193-200. |
| Svensson et al., “SpatialDE: identification of spatially variable genes,” Nature Methods, May 2018, 15:343-346, 15 pages. |
| Howell et al., “iFRET: An Improved Fluorescence System for DNA-Melting Analysis,” Genome Research, 2002, 12:1401-1407. |
| Nam et al., “Nanoparticle-Based Bio-Bar Codes for the Ultrasensitive Detection of Proteins,” Science, Sep. 26, 2003, 301(5641):1884-1886. |
| Redmond et al., “Single-cell TCRseq: paired recovery of entire T-cell alpha and beta chain transcripts in T-cell receptors from single-cell RNAseq,” Genome Med, 2016, 8:80, 12 pages. |
| Asp et al., “A spatiotemporal organ-wide gene expression and cell atlas of the developing human heart,” Cell, Dec. 12, 2019, 179(7):1647-1660. |
| Lubeck et al., “Single-cell in situ RNA profiling by sequential hybridization,” Nature Methods, Apr. 2014, 11(4):360-361, 2 pages (Supplemental Materials). |
| Kuhn et al., “A novel, high-performance random array platform for quantitative gene expression profiling,” Genome Res, 2004, 14:2347-2356. |
| Number | Date | Country | |
|---|---|---|---|
| 63048584 | Jul 2020 | US |
