Granted Patent B2

US 12,692,536 · App. 18/428,058 · Granted Jul 28, 2026

Inventors: Rajiv Bharadwaj (Pleasanton, CA); Zachary Bent (Pleasanton, CA)

Assignee: 10x Genomics, Inc.

C12Q1/6806C12N15/1006C12Q1/686C12Q2531/113C12Q2563/149C12Q2565/629

Abstract

Claims (23)

1 . A method, comprising:

(a) hybridizing a bead and a probe to generate an optically-tagged bead, wherein the optically-tagged bead comprises a binding sequence,

wherein the probe hybridizes to the binding sequence,

wherein the probe comprises: (i) a fluorescent tag; and (ii) at a 3′ end of the probe, a moiety selected from the group consisting of a fluorophore, a carbon spacer, a biotin molecule, an inverted terminal base, a sequence that destabilizes hybridization between the bead and the probe, and an unlocked nucleic acid, and

wherein the moiety renders the probe hybridized to the bead at least 60% less reactive in a base incorporation reaction as compared to an equivalent nucleic acid probe lacking the moiety; and

(b) detecting one or more signals indicative of the fluorescent tag from the optically-tagged bead,

wherein the optically-tagged bead comprises: i) a molecule comprising the binding sequence hybridized to the probe, and ii) a nucleic acid barcode molecule, wherein the molecule is different from the nucleic acid barcode molecule.

2 . The method of claim 1 , wherein the probe comprises the fluorescent tag at a 5′ end of the probe.

3 . The method of claim 1 , further comprising, prior to (a), subjecting a nucleic acid probe comprising the fluorescent tag to conditions sufficient to modify the nucleic acid probe to yield the probe.

4 . The method of claim 3 , further comprising adding the moiety to the nucleic acid probe.

5 . The method of claim 1 , further comprising conducting the base incorporation reaction with or in a presence of the optically-tagged bead.

6 . The method of claim 5 , wherein the base incorporation reaction is an extension reaction.

7 . The method of claim 5 , wherein (b) is performed prior to conducting the base incorporation reaction with or in the presence of the optically-tagged bead.

8 . The method of claim 5 , wherein (b) is performed subsequent to conducting the base incorporation reaction with or in the presence of the optically-tagged bead.

9 . The method of claim 1 , wherein the optically-tagged bead comprises a gel bead.

10 . The method of claim 1 , the method further comprises determining a parameter of the optically-tagged bead based on the one or more signals.

11 . The method of claim 10 , wherein the optically-tagged bead comprises a plurality of primers and wherein the parameter is a concentration of the plurality of primers on the optically-tagged bead, and wherein the probe comprises a probe sequence configured to hybridize to a sequence common to the plurality of primers.

12 . The method of claim 10 , wherein the parameter is a size of the optically-tagged bead.

13 . The method of claim 1 , further comprising, subsequent to (a), partitioning the optically-tagged bead into a partition among a plurality of partitions.

14 . The method of claim 13 , further comprising using the one or more signals to assess a bead occupancy of the partition.

15 . The method of claim 13 , wherein the partition is a droplet or a well.

16 . The method of claim 1 , further comprising, subsequent to (a), co-partitioning the optically-tagged bead with an analyte in a partition among a plurality of partitions.

17 . The method of claim 1 , wherein the carbon spacer comprises a 3 carbon atom spacer, a 6 carbon spacer, a 9 carbon spacer, or an 18 carbon spacer.

Assignments (1)

Continuity (3)

References Cited (400)