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US-20260125747-A1 - PROBES COMPRISING A SPLIT BARCODE REGION AND METHODS OF USE

US20260125747A1US 20260125747 A1US20260125747 A1US 20260125747A1US-20260125747-A1

Abstract

In some aspects, the present disclosure relates to methods for reducing the detection of false positive ligation events. In some aspects, the method comprises use of a double split (or “split split”) probe. The methods herein have particular applicability in reducing the detection of false positive ligation events when using ligases that have high ligation efficiency but low specificity (e.g., SplintR® ligase). Also provided are kits comprising probes for use in such methods.

Inventors

  • Malte KÜHNEMUND

Assignees

  • 10X GENOMICS, INC.

Dates

Publication Date
20260507
Application Date
20250912

Claims (20)

  1. 1 - 87 . (canceled)
  2. 88 . A method for analyzing a biological sample, comprising: (a) contacting the biological sample with: (i) a first probe comprising a first hybridization region and a first portion of a barcode region, and (ii) a second probe comprising a second hybridization region and a second portion of the barcode region, wherein the first and second hybridization regions are complementary to target sequences in a target nucleic acid molecule in the biological sample, wherein the first probe and the second probe hybridize to the target nucleic acid molecule in the biological sample, and wherein the barcode region comprises one or more barcode sequences corresponding to the target nucleic acid molecule; (b) ligating the first and second probes hybridized to the target nucleic acid molecule to form a composite probe by connecting the first and second portions of the barcode region and by connecting the ends of the first hybridization region and the second hybridization region; and (c) contacting the biological sample with a detectable probe that hybridizes to the barcode region at sequences corresponding to both the first portion of the barcode region and the second portion of the barcode region, or a complement thereof, wherein a signal associated with the detectable probe is detected in the biological sample.
  3. 89 . The method of claim 88 , wherein the target nucleic acid molecule comprises a reporter oligonucleotide of a labelling agent and the labelling agent comprises a binder that directly or indirectly binds a non-nucleic acid analyte or a portion thereof in the biological sample.
  4. 90 . The method of claim 89 , wherein the non-nucleic acid analyte comprises a protein or a polypeptide.
  5. 91 . The method of claim 89 , wherein the binder comprises an antibody or antigen binding fragment thereof.
  6. 92 . The method of claim 88 , wherein the ends of the first hybridization region and the second hybridization region are directly ligated using enzymatic ligation.
  7. 93 . The method of claim 88 , wherein the ends of the first and second portions of the barcode region are directly ligated using enzymatic ligation.
  8. 94 . The method of claim 88 , wherein the ends of the first and second portions of the barcode region are ligated using a splint that hybridizes to (i) the first portion or a subportion thereof and (ii) the second portion or a subportion thereof.
  9. 95 . The method of claim 88 , comprising removing a detectable probe comprising a mismatch with (i) the first portion or a subportion thereof or (ii) the second portion or a subportion thereof, whereas under the same conditions, the detectable probe complementary to both (i) the first portion or a subportion thereof and (ii) the second portion or a subportion thereof remain hybridized to the barcode region or complement thereof.
  10. 96 . The method of claim 88 , comprising circularizing the composite probe and generating a rolling circle amplification (RCA) product of the circularized composite probe, wherein the RCA product comprises multiple copies of the complement of the barcode region.
  11. 97 . The method of claim 88 , wherein the detectable probe comprises a detectable label or a sequence complementary to a sequence of a detectably labelled probe.
  12. 98 . The method of claim 97 , wherein the detectably labelled probe that hybridizes to a sequence in a 3′ overhang or a 5′ overhang of the detectable probe.
  13. 99 . The method of claim 88 , wherein the composite probe or a product thereof is generated in situ in the biological sample or in a matrix embedding the biological sample or molecules thereof.
  14. 100 . The method of claim 88 , wherein the method comprises imaging the biological sample to detect the signal associated with the detectable probe.
  15. 101 . The method of claim 88 , wherein the biological sample is a cell or tissue sample.
  16. 102 . A system, comprising: (a) a labelling agent comprising a binder configured to bind a non-nucleic acid analyte, wherein the labelling agent comprises a reporter oligonucleotide; (b) a first probe comprising a first hybridization region and a first portion of a barcode sequence; (c) a second probe comprising a second hybridization region and a second portion of the barcode sequence, wherein the first and second hybridization regions are complementary to adjacent target sequences in the reporter oligonucleotide, and the barcode sequence corresponds to the labelling agent; (d) a splint comprising complimentary sequences to both (i) the first portion of the barcode sequence or a subportion thereof and (ii) the second portion of the barcode sequence or a subportion thereof; and (e) a detectable probe configured to hybridize to sequences corresponding to both the first portion of the barcode region and the second portion of the barcode sequence or a complement thereof.
  17. 103 . The system of claim 102 , comprising a ligase configured to connect the first and second probes to form a composite probe and a polymerase configured to perform rolling circle amplification (RCA) using the composite probe.
  18. 104 . The system of claim 102 , further comprising a cell or tissue sample attached to a substrate, wherein the cell or tissue sample comprises the non-nucleic acid analyte.
  19. 105 . The system of claim 102 , further comprising an optical detection system for imaging the cell or tissue sample.
  20. 106 . The system of claim 102 , wherein the non-nucleic acid analyte comprises a protein or a polypeptide.

Description

CROSS-REFERENCE TO RELATED APPLICATION This application is a continuation of U.S. patent application Ser. No. 17/888,361, filed on Aug. 15, 2022, issued on Oct. 7, 2025, as U.S. Pat. No. 12,435,364, which claims priority to U.S. Provisional Patent Application No. 63/233,599, filed on Aug. 16, 2021, entitled “PROBES COMPRISING A SPLIT BARCODE REGION AND METHODS OF USE,” each of which is hereby incorporated herein by reference in its entirety for all purposes. REFERENCE TO AN ELECTRONIC SEQUENCE LISTING The contents of the electronic sequence listing (202412006701SEQLIST.xml; Size: 4,718 bytes; and Date of Creation: Nov. 24, 2025) is herein incorporated by reference in its entirety. FIELD The present disclosure generally relates to methods and compositions for in situ analysis for detection of analytes in a sample. BACKGROUND Methods are available for analyzing nucleic acids in a biological sample in situ, such as a cell or a tissue. For instance, advances in single molecule fluorescent hybridization (smFISH) have enabled nanoscale-resolution imaging of RNA in cells and tissues. However, oligonucleotide probe-based assay methods for in situ analysis may suffer from low sensitivity, specificity, and/or detection efficiency and may require careful and laborious optimization. Improved methods for in situ analysis are needed. The present disclosure addresses these and other needs. BRIEF SUMMARY In some embodiments, disclosed herein is a method for analyzing a biological sample, comprising: (a) contacting the biological sample with: (i) a first probe comprising a first hybridization region and a first portion of a barcode region, and (ii) a second probe comprising a second hybridization region and a second portion of the barcode region, wherein the first and second hybridization regions are complementary to target sequences in a target nucleic acid molecule in the biological sample. In some embodiments, the barcode region comprises one or more barcode sequences corresponding to the target nucleic acid molecule. For instance, the first and second probes comprise a split barcode sequence, a portion of which is provided in the first portion of the barcode region, whereas another portion of the barcode sequence is provided in the second portion of the barcode region. In any of the embodiments herein, the method may comprise connecting the first and second probes hybridized to the target nucleic acid molecule to form a composite probe. In some embodiments, formation of the composite probe comprises connecting the first and second portions of the barcode region. In any of the embodiments herein, the method may comprise contacting the biological sample with a detectable probe that hybridizes to a sequence of the barcode region or a complement thereof. In any of the embodiments herein, the method may comprise contacting the biological sample with one or more detectable probes that hybridize to the barcode region at sequences corresponding to both the first portion of the barcode region and the second portion of the barcode region, or a complement thereof. In some embodiments, a signal associated with the one or more detectable probes is detected in the biological sample, thereby detecting the target nucleic acid molecule in the biological sample. In any of the embodiments herein, the method may comprise detecting a signal associated with the one or more detectable probes in situ. In any of the embodiments herein, the first and second hybridization regions can hybridize to adjacent target sequences in the target nucleic acid molecule. In any of the embodiments herein, the first and second hybridization regions can hybridize to target sequences that are separated by 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotides in the target nucleic acid molecule. In some embodiments, the first and second hybridization regions in the target nucleic acid molecule are directly linked by a phosphodiester bond. In some embodiments, the first and second hybridization regions in the target nucleic acid molecule are linked by a nucleic sequence of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotides in length. In any of the embodiments herein, the first and second hybridization regions can be equal in length. In any of the embodiments herein, the first hybridization region can be longer or shorter than the second hybridization region. In any of the embodiments herein, the melting temperature of the first hybridization region hybridized to the target nucleic acid molecule can the same as the melting temperature of the second hybridization region hybridized to the target nucleic acid molecule. In any of the embodiments herein, the melting temperature of the first hybridization region hybridized to the target nucleic acid molecule can be higher or lower than the melting temperature of the second hybridization region hybridized to the target nucleic acid molecule. In any of the embodiments herein, the melting temperatures may differ by no more t