# US 12,110,548 B2 · Bi-directional in situ analysis > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 12,110,548 B2 - Document ID: US12110548B2 - Status: Granted - Kind: B2 - Application number: 17/165,802 - 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/US12110548B2 ## CPC - C12Q 1/6841: In situ hybridisation - C12Q 1/6874: involving nucleic acid arrays, e.g. sequencing by hybridisation - C12Q 1/6844: Nucleic acid amplification reactions - C12Q 1/6876: Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes - 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 In some aspects, provided herein are methods for analyzing a nucleic acid comprising first and second regions of interest flanking an adaptor region, comprising hybridizing an anchor to the adaptor region, analyzing the first region of interest from one end of the anchor using probe ligation (e.g., sequencing-by-ligation), and binding a polymerase to the other end of the anchor and optionally incorporating a nucleotide and/or analog thereof into the anchor by the polymerase using the second region of interest or a probe bound thereto as a template. In some embodiments, the second region of interest is used as a template for sequencing-by-synthesis. In some embodiments, spatially resolved detections of analytes are performed at a cellular or sub-cellular resolution which involve correlating signals associated with analytes with specific spatial locations in a biological sample. ## Claims ### Claim 1 A method for analyzing a sample, comprising: (a) contacting the sample with a plurality of detection probes, wherein: the sample comprises a nucleic acid comprising non-overlapping first and second regions of interest flanking an adaptor region that hybridizes to an anchor, each detection probe comprises an interrogatory region and a detectable label, and wherein the plurality of detection probes comprise detection probes labeled with different detectable labels, wherein the detectable label of each detection probe in the plurality of detection probes corresponds to a nucleotide or sequence in the interrogatory region of the respective detection probe; ligating a detection probe of the plurality of detection probes to the anchor to generate a ligation product, wherein the detection probe of the plurality of detection probes is complementary to the first region of interest; and detecting a signal associated with the detectable label of the ligation product; and (b) binding a polymerase to the other end of the anchor and incorporating a nucleotide and/or analog thereof into the anchor by the polymerase to generate an extended anchor (i) using the second region of interest as a template, or (ii) using a primary probe bound to the second region of interest as a template, wherein the primary probe comprises a region that hybridizes to the anchor. ### Claim 2 (depends on 1) The method of claim 1, further comprising detecting a signal associated with the polymerase and/or the nucleotide or analog thereof incorporated into the other end of the anchor. ### Claim 3 (depends on 2) The method of claim 2, wherein the incorporation of nucleotide and/or analog thereof and signal detection are repeated to perform sequencing by synthesis, thereby sequencing all or a portion of the second region of interest or primary probe bound thereto. ### Claim 4 (depends on 1) The method of claim 1, wherein step (b) comprises contacting the sample with a pool of nucleotides and/or analogs thereof. ### Claim 5 (depends on 4) The method of claim 4, wherein the pool of nucleotides and/or analogs comprises a terminator nucleotide or analog thereof. ### Claim 6 (depends on 1) The method of claim 1, further comprising incorporating one or more nucleotides or analogs thereof into the extended anchor. ### Claim 7 (depends on 1) The method of claim 1, wherein the first region of interest, the adaptor region, and the second region of interest are in the same molecule. ### Claim 8 (depends on 1) The method of claim 1, wherein the first region of interest and the second region of interest are in a first and a second molecule, respectively. ### Claim 9 (depends on 1) The method of claim 1, wherein the contacting, ligating, and detecting steps in (a) are repeated to perform sequencing by ligation, thereby sequencing all or a portion of the first region of interest. ### Claim 10 (depends on 1) The method of claim 1, wherein the method comprises repeating the contacting, ligating, and detecting steps in (a) and repeating the binding in (b). ### Claim 11 (depends on 10) The method of claim 10, wherein the anchor remains hybridized to the nucleic acid during one or more cycles of the repeated steps in (a) and/or (b). ### Claim 12 (depends on 1) The method of claim 1, wherein the nucleic acid is endogenous in the sample, and the contacting, ligating, and/or detecting steps in (a) and/or the binding step in (b) are performed in situ. ### Claim 13 (depends on 1) The method of claim 1, wherein the nucleic acid in the sample is a product of an endogenous molecule in the sample. ### Claim 14 (depends on 1) The method of claim 1, wherein the sample comprises a support. ### Claim 15 (depends on 14) The method of claim 14, wherein the support comprises a bead. ### Claim 16 A method of analyzing a sample, comprising: (a) contacting the sample with a plurality of detection probes, wherein the sample comprises a nucleic acid comprising a first region of interest and a second region of interest flanking an adaptor region that hybridizes to an anchor, wherein each detection probe comprises an interrogatory region and a detectable label, wherein the plurality of detection probes comprise detection probes labeled with different detectable labels, wherein the detectable label of each detection probe in the plurality of detection probes corresponds to a nucleotide or sequence in the interrogatory region of the respective detection probe; ligating a detection probe of the plurality of detection probes to the anchor to generate a ligation product, wherein the detection probe of the plurality of detection probes is complementary to the first region of interest; and detecting a signal from the detectable label of the ligation product; and (b) contacting the sample with a primary probe comprising a sequence complementary to the second region of interest, wherein the primary probe comprises a region that hybridizes to the anchor; and extending the anchor with a polymerase using the primary probe as a template.