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US 2021/0277460 A1Published application
Three-dimensional spatial transcriptomics with sequencing readout
Assignee10x Genomics, Inc.
InventorFelice Alessio BAVA
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Abstract
In some embodiments described herein are methods for three-dimensional analysis of a biological sample, comprising migrating a population of spatial probes into the biological sample in each of three dimensions, wherein each spatial probe comprises a targeting domain and a migration domain. Also provided are kits and compositions for use according to any of the methods described herein.
Claims33
A method for three-dimensional analysis of a biological sample, comprising:
- (a) in a first dimension, migrating a first population of spatial probes into the biological sample, wherein a first-dimension spatial probe of the first population is targeted to a target molecule immobilized in the biological sample;
- (b) in a second dimension, migrating a second population of spatial probes into the biological sample, wherein a second-dimension spatial probe of the second population is targeted to the target molecule and/or the first-dimension spatial probe targeted thereto;
- (c) in a third dimension, migrating a third population of spatial probes into the biological sample, wherein a third-dimension spatial probe of the third population is targeted to the target molecule, the first-dimension spatial probe targeted thereto, and/or the second-dimension spatial probe targeted thereto,
- wherein the first-dimension spatial probe comprises
- a targeting domain that binds the target molecule and
- wherein the first-dimension spatial probe, the second-dimension spatial probe, and the third-dimension spatial probe each comprises:
- (i) a migration domain corresponding to a location in the biological sample to which the spatial probe is migrated in the respective dimension, and
- (ii) a migration barcode sequence that identifies the migration domain;
- (d) generating a product in the biological sample, the product comprising:
- (1) a sequence or complement thereof of the migration domains and/or the migration barcode sequences of the first-dimension spatial probe, the second-dimension spatial probe, and the third-dimension spatial probe;
- and
- (2) a sequence corresponding to the target molecule;
- and
- (e) determining a sequence of the product, thereby identifying the location of the target molecule in a three-dimensional space in the biological sample.
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A method for three-dimensional analysis of a biological sample, comprising:
- (a) generating a target nucleic acid molecule in the biological sample, wherein the target nucleic acid molecule is (i) a product of an endogenous nucleic acid molecule, (ii) a product of one or more nucleic acid probes that directly or indirectly hybridize to the endogenous nucleic acid molecule or to the product of (i), or (iii) a product of the product of (i) or (ii);
- (b) embedding the biological sample in a matrix, wherein the target nucleic acid molecule is crosslinked to the matrix, thereby immobilizing the target nucleic acid molecule in the matrix-embedded biological sample;
- (c) electrophoresing a first population of spatial probes for distribution by size in a first dimension, wherein a first-dimension spatial probe of the first population is targeted to the target nucleic acid molecule immobilized in the matrix-embedded biological sample;
- (d) electrophoresing a second population of spatial probes for distribution by size in a second dimension, wherein a second-dimension spatial probe of the second population is targeted to the target nucleic acid molecule and/or the first-dimension spatial probe targeted thereto;
- (e) electrophoresing a third population of spatial probes for distribution by size in a third dimension, wherein a third-dimension spatial probe of the third population is targeted to the target nucleic acid molecule, the first-dimension spatial probe targeted thereto, and/or the second-dimension spatial probe targeted thereto,
- wherein the first-dimension spatial probe, the second-dimension spatial probe, and the third-dimension spatial probe each comprises:
- (i) a targeting sequence for targeting,
- (ii) a variable-length sequence corresponding to a location in the matrix-embedded biological sample to which the spatial probe is distributed in the respective dimension, and
- (iii) a length-barcode sequence corresponding to the variable-length sequence;
- (f) generating a product in the matrix-embedded biological sample, the product comprising:
- (1) a sequence or complement thereof of the length-barcode sequences of the first-dimension spatial probe, the second-dimension spatial probe, and the third-dimension spatial probe;
- and
- (2) a sequence or complement thereof of the target nucleic acid molecule;
- (g) releasing, removing, or isolating the product from the matrix-embedded biological sample;
- and
- (h) subjecting the released, removed, or isolated product to nucleic acid sequencing, thereby identifying the location of the target nucleic acid molecule in the three dimensions of the matrix-embedded biological sample.
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Dependencies are inferred from the claim text.