# US 12,110,541 B2 · Methods for preparing high-resolution spatial arrays

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- Number: US 12,110,541 B2
- Document ID: US12110541B2
- Status: Granted
- Kind: B2
- Application number: 17/165,453
- Filed: 2021-02-02
- Published: 2021-08-05
- Granted: 2024-10-08
- Earliest claimed: 2020-02-03
- Assignees: 10x Genomics, Inc.
- Applicants: 10x Genomics, Inc.
- Inventors: Felice Alessio Bava
- Web page: https://patentgrep.com/patent/US12110541B2

## CPC

- C12Q 1/6837: using probe arrays or probe chips (C12Q1/6874 takes precedence)
- C12Q 1/6816: characterised by the detection means (C12Q1/6804 takes precedence)
- C12Q 1/6874: involving nucleic acid arrays, e.g. sequencing by hybridisation
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES

## Abstract

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

## Claims

### Claim 1

A method for preparing a spatial array, the method comprising:
(a) providing a substrate comprising a plurality of primers attached to a surface of the substrate, wherein a primer of the plurality of primers comprises a first hybridization domain;
(b) contacting the substrate with a plurality of first features, wherein a first feature of the plurality of first features comprises an oligonucleotide, a first capture probe, and a first bridging probe, wherein:
(i) the oligonucleotide comprises a second hybridization domain, wherein the second hybridization domain is capable of hybridizing to the first hybridization domain;
(ii) the first capture probe comprises a first spatial barcode and a first capture domain, wherein the first capture domain is capable of hybridizing to a first analyte from a biological sample; and
(iii) the first bridging probe comprises a first bridging domain, wherein the first bridging domain is capable of hybridizing to a second bridging domain; and
(c) attaching the plurality of first features to the plurality of primers by coupling the second hybridization domain to the first hybridization domain.

### Claim 2 (depends on 1)

The method of claim 1, further comprising:
(d) contacting the substrate with a plurality of second features, wherein a second feature of the plurality of second features comprises a second capture probe and a second bridging probe, wherein:
(i) the second capture probe comprises a second spatial barcode and a second capture domain, wherein the second capture domain is capable of hybridizing to a second analyte from the biological sample; and
(ii) the second bridging probe comprises a second bridging domain, wherein the second bridging domain is capable of hybridizing to the first bridging domain; and
(e) attaching the plurality of second features to the plurality of first features by coupling the second bridging probe to the first bridging probe.

### Claim 3 (depends on 2)

The method of claim 2, wherein step (d) further comprises increasing temperature of the spatial array compared to temperature of the spatial array in steps (a)-(c), wherein the increasing the temperature is associated with increased hybridization of the first bridging domain and the second bridging domain.

### Claim 4 (depends on 2)

The method of claim 2, wherein the second feature comprises a known combination of the second capture probe and the second bridging probe, wherein determining location of the second feature is based on the known combination.

### Claim 5 (depends on 2)

The method of claim 2, wherein the first feature comprises a first bead, and the second feature comprises a second bead.

### Claim 6 (depends on 5)

The method of claim 5, wherein the first bead and/or the second bead has a diameter of about 0.1 μm to about 5 μm, 0.5 μm to about 4 μm, about 1 μm to about 10 μm, about 1 μm to about 20 μm, about 1 μm to about 30 μm, about 1 μm to about 40 μm, about 1 m to about 50 μm, about 1 μm to about 60 μm, about 1 μm to about 70 μm, about 1 μm to about 80 m, about 1 μm to about 90 μm, about 90 μm to about 100 μm, about 80 μm to about 100 μm, about 70 μm to about 100 μm, about 60 μm to about 100 μm, about 50 μm to about 100 μm, about 40 μm to about 100 μm, about 30 μm to about 100 μm, about 20 μm to about 100 μm, or about 10 μm to about 100 μm.

### Claim 7 (depends on 2)

The method of claim 2, further comprising determining abundance and location of the first analyte and the second analyte by:
(f) contacting the spatial array with the biological sample;
(g) hybridizing the first analyte to the first capture probe and the second analyte to the second capture probe; and
(h) determining (i) all or a part of the sequence of the first analyte and the second analyte, or a complement thereof, and (ii) the sequence of the first spatial barcode and/or the second spatial barcode, or a complement thereof, and using the determined sequence of (i) and (ii) to determine the abundance and the location of the first analyte and/or the second analyte in the biological sample.

### Claim 8 (depends on 7)

The method of claim 7, wherein the determining step (h) comprises amplifying all or part of the first analyte specifically bound to the first capture domain and/or all or part of the second analyte specifically bound to the second capture domain, wherein the amplifying creates an amplification product comprising (i) all or part of the first analyte specifically bound to the first capture domain and/or all or part of the second analyte specifically bound to the second capture domain, or a complement thereof, and (ii) the sequence of the first spatial barcode and/or the second spatial barcode, or a complement thereof.

### Claim 9 (depends on 7)

The method of claim 7, wherein the determining step comprises sequencing.

### Claim 10 (depends on 7)

The method of claim 7, further comprising imaging the biological sample.

### Claim 11 (depends on 7)

The method of claim 7, wherein the first capture probe and/or the second capture probe further comprises one or more functional domains, a unique molecular identifier, a cleavage domain, and combinations thereof.

### Claim 12 (depends on 7)

The method of claim 7, wherein the method further comprises extending the one or more capture probes via a nucleic acid extension reaction using the first analyte and/or the second analyte as a template to generate an extended one or more capture probes comprising the one or more capture probes and a reverse complement of the first analyte and/or the second analyte.

### Claim 13 (depends on 7)

The method of claim 7, further comprising removing (i) all or a part of the sequence of the first analyte and/or the second analyte, or a complement thereof from the spatial array, and removing (ii) the sequence of the first spatial barcode and/or the second spatial barcode, or a complement thereof, from the spatial array and, determining the sequence of (i) and (ii) by sequencing.

### Claim 14 (depends on 1)

The method of claim 1, wherein the primer is affixed to the substrate at a 5′ end of the primer.

### Claim 15 (depends on 1)

The method of claim 1, wherein the primer is deposited onto the substrate in a manner where the primer has a known location on the substrate using a method selected from the group consisting of printing, photolithography, synthesis, and ligation.

### Claim 16 (depends on 1)

The method of claim 1, wherein the method further comprises amplifying all or part of the primer.

### Claim 17 (depends on 16)

The method of claim 16, wherein the amplifying comprises rolling circle amplification.

### Claim 18 (depends on 17)

The method of claim 17, wherein the amplifying is performed prior to step (b).

### Claim 19 (depends on 1)

The method of claim 1, wherein the first bridging domain is about 10 nucleotides to about 90 nucleotides in length, and the second bridging domain is about 10 nucleotides to about 90 nucleotides in length.

### Claim 20 (depends on 1)

The method of claim 1, wherein the method further comprises washing the substrate after step (c), thereby removing unattached first features and/or washing the substrate after step (e), thereby removing unattached second features.

### Claim 21 (depends on 1)

The method of claim 1, wherein the first feature comprises a known combination of the first capture probe, the oligonucleotide, and the first bridging probe, wherein determining location of the first feature is based on the known combination.