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US 2023/0416808 A1Published application
Methods, compositions, and systems for improved in situ detection of analytes and spatial analysis
Assignee10x Genomics, Inc.
InventorsDavid Sukovich, Felice Alessio Bava, Augusto Manuel Tentori, Hanyoup Kim, Amanda Janesick
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Abstract
Provided herein are methods, compositions, and systems for improved in situ detection of analytes and spatial analysis using, e.g., a sequencing readout.
Claims30
A method for analyzing multiple analytes in a biological sample placed on a first substrate, the method comprising:
- (a) contacting the biological sample with one or more nucleic acid probes that directly or indirectly hybridize to a set of first analytes or complements or amplification products thereof in the biological sample;
- (b) detecting in the biological sample the one or more nucleic acid probes at a spatial location of the biological sample;
- (c) hybridizing a first probe and a second probe to a second analyte, wherein the first probe comprises a sequence that is substantially complementary to a first sequence of the second analyte, the second probe comprises a sequence that is substantially complementary to a second sequence of the second analyte, and wherein the second probe comprises a capture probe binding domain;
- (d) coupling the first probe and the second probe, thereby generating a connected probe;
- (e) mounting the first substrate on a first member of a support device, the first member configured to retain the first substrate;
- (f) mounting a second substrate comprising an array on a second member of the support device, the second member configured to retain the second substrate, the array comprising a plurality of capture probes, wherein a capture probe of the plurality of capture probes comprises (i) a spatial barcode and (ii) a capture domain;
- (g) applying a reagent medium to the first substrate and/or the second substrate;
- (h) operating an alignment mechanism of the support device to move the first member and/or the second member such that at least a portion of the biological sample is aligned with at least a portion of the array and the portion of the biological sample and the portion of the array contact the reagent medium, and such that the first substrate and the second substrate are separated by a distance of less than 50 micrometers;
- (i) when the biological sample is aligned with at least a portion of the array, (i) releasing the connected probe from the second analyte and (ii) migrating the connected probe from the biological sample to the array;
- and
- (j) hybridizing the connected probe to the capture domain.
8 more elements
A method for analyzing multiple analytes in a biological sample placed on a first substrate, the method comprising:
- (a) contacting the biological sample with an analyte capture agent that binds to a first analyte in the biological sample, wherein the analyte capture agent comprises an analyte binding moiety and a capture agent barcode domain, and wherein the capture agent barcode domain comprises a nucleic acid sequence;
- (b) detecting in the biological sample all or a portion of the nucleic acid sequence of the capture agent barcode domain at a spatial location of the biological sample;
- (c) hybridizing a first probe and a second probe to a second analyte, wherein the first probe comprises a sequence that is substantially complementary to a first sequence of the second analyte, the second probe comprises a sequence that is substantially complementary to a second sequence of the second analyte, and wherein the second probe comprises a capture probe binding domain;
- (d) coupling the first probe and the second probe, thereby generating a connected probe;
- (e) mounting the first substrate on a first member of a support device, the first member configured to retain the first substrate;
- (f) mounting a second substrate comprising an array on a second member of the support device, the second member configured to retain the second substrate, the array comprising a plurality of capture probes, wherein a capture probe of the plurality of capture probes comprises (i) a spatial barcode and (ii) a capture domain;
- (g) applying a reagent medium to the first substrate and/or the second substrate, the reagent medium comprising a permeabilization agent;
- (h) operating an alignment mechanism of the support device to move the first member and/or the second member such that at least a portion of the biological sample is aligned with at least a portion of the array and the portion of the biological sample and the portion of the array contact the reagent medium, and such that the first substrate and the second substrate are separated by a distance of less than 50 micrometers;
- (i) when the biological sample is aligned with at least a portion of the array, (i) releasing the connected probe from the second analyte and (ii) migrating the connected probe from the biological sample to the array;
- and
- (j) hybridizing the connected probe to the capture domain.
8 more elements
Dependencies are inferred from the claim text.