Granted Patent B2

US 12,735,743 · App. 17/272,033 · Granted Sep 15, 2026

Inventors: Michael Ybarra Lucero (South San Francisco, CA); Michael Schnall-Levin (San Francisco, CA); Tarjei Sigurd Mikkelsen (Pleasanton, CA); Eswar Prasad Ramachandran Iyer (Pleasanton, CA); Paul Giresi (Palo Alto, CA); Benjamin Hindson (Pleasanton, CA)

Assignee: 10x Genomics, Inc.

C12Q1/6874

Abstract

Claims (20)

1 . A method of determining a location of a feature in a spatial array, the method comprising:

(a) providing the spatial array comprising a plurality of features on a substrate, wherein the substrate of the spatial array comprises both:

(i) a plurality of oligonucleotides on the substrate, wherein an oligonucleotide of the plurality of oligonucleotides is attached to a first site of the substrate and comprises: a first spatial barcode and a first capture domain; and

(ii) the plurality of features randomly affixed on the substrate, wherein the feature of the plurality of features is affixed to the first site and comprises a plurality of capture probes, wherein a capture probe of the plurality of capture probes comprises: a second spatial barcode and a second capture domain;

(b) determining a nucleotide sequence of the first spatial barcode, thereby determining the location of the oligonucleotide on the substrate;

(c) hybridizing the first capture domain of the oligonucleotide attached to the first site to the second capture domain of the capture probe of the feature affixed to the first site, thereby producing a second capture probe comprising the first spatial barcode of the oligonucleotide and the second spatial barcode of the capture probe, wherein the first spatial barcode and second spatial barcode comprise different sequences;

(d) determining: (i) the sequence of the first spatial barcode of the second capture probe, or a complement thereof; and (ii) the sequence of the second spatial barcode of the second capture probe, or a complement thereof; and

(e) associating the second spatial barcode of the second capture probe with the first spatial barcode of the oligonucleotide having a known location on the substrate, thereby determining the location of the feature in the spatial array of the plurality of features randomly affixed to the substrate.

2 . The method of claim 1 , wherein the plurality of features comprises a plurality of beads.

3 . The method of claim 1 , wherein the first capture domain comprises a poly (A) sequence.

4 . The method of claim 1 , wherein the second capture domain comprises a poly (T) sequence.

5 . The method of claim 1 , wherein the determining in (b) comprises sequencing.

6 . The method of claim 1 , wherein the oligonucleotide further comprises a constant sequence for initiating a sequencing reaction.

7 . The method of claim 6 , further comprising hybridizing a sequencing anchor to the constant sequence prior to hybridizing a sequencing probe to the oligonucleotide downstream of the sequencing anchor.

8 . The method of claim 1 , wherein the determining in (d) comprises sequencing.

9 . The method of claim 1 , wherein the capture probe further comprises a constant sequence for initiating a sequencing reaction.

10 . The method of claim 9 , further comprising hybridizing a sequencing anchor to the constant sequence prior to hybridizing a sequencing probe to the capture probe downstream of the sequencing anchor.

11 . The method of claim 1 , wherein the capture probe further comprises one or more functional domains, a unique molecular identifier, a cleavage domain, or combinations thereof.

12 . The method of claim 1 , wherein, further comprising extending the oligonucleotide using the capture probe as a template, and optionally, extending the capture probe using the oligonucleotide as a template.

13 . The method of claim 1 , wherein the second spatial barcode of the capture probe on the feature is unique relative to the second spatial barcode of capture probes on other features in the spatial array.

Assignments (1)

Continuity (44)

References Cited (400)