Granted Patent B2

US 12,559,791 · App. 18/313,256 · Granted Feb 24, 2026

Inventor: Malte Kühnemund (Stockholm, SE)

Assignee: 10X GENOMICS, INC.

C12Q1/6841C12Q1/682C12Q1/6876

Abstract

Claims (35)

1 . A method for analyzing a biological sample, comprising:

(a) generating a plurality of nucleic acid molecules using a plurality of primers, wherein the generation comprises reverse transcribing an antigen receptor transcript at a location in the biological sample using a polymerase having strand displacement activity, wherein the plurality of primers specifically target different sequences of the antigen receptor transcript;

(b) immobilizing at least a nucleic acid molecule of the plurality of nucleic acid molecules at the location,

wherein the nucleic acid molecule comprises a V (D) J join comprising a V (variable) segment, a J (joint) segment, and optionally a D (diversity) segment between the V and J segments;

(c) circularizing the nucleic acid molecule or a circularizable probe or probe set hybridized to the nucleic acid molecule to generate a circularized molecule;

(d) generating a rolling circle amplification (RCA) product of the circularized molecule; and

(e) detecting a signal associated with the RCA product, thereby detecting the antigen receptor transcript or a sequence thereof at the location in the biological sample.

2 . The method of claim 1 , wherein the nucleic acid molecule of the plurality of nucleic acid molecules comprises a complementary deoxyribonucleic acid (cDNA) sequence complementary to a sequence of the antigen receptor transcript.

3 . The method of claim 1 , wherein the antigen receptor transcript is reverse transcribed in the presence of a helicase having strand displacement activity.

4 . The method of claim 1 , wherein one or more primers of the plurality of primers comprise a 5′ overhang upon hybridization to the antigen receptor transcript.

5 . The method of claim 1 , wherein the antigen receptor transcript is reverse transcribed using at least two primers of the plurality of primers that hybridize to adjacent sequences in a C (constant) region in the antigen receptor transcript.

6 . The method of claim 5 , wherein the adjacent sequences are non-overlapping with one another, and extension of a particular 5′ primer displaces an extension product of one or more 3′ primers hybridized to the antigen receptor transcript.

7 . The method of claim 6 , wherein the extension product is linked to one or more molecules at the location in the biological sample.

8 . The method of claim 1 , wherein the biological sample is contacted with the circularizable probe or probe set, wherein the circularizable probe or probe set comprises a barcode region comprising one or more barcode sequences, and the circularizable probe or probe set is ligated using the nucleic acid molecule as a template to generate the circularized molecule.

9 . The method of claim 8 , wherein the circularizable probe or probe set comprises: a 3′ region that hybridizes to a sequence in, or 3′ to, the V segment of the nucleic acid molecule; and a 5′ region that hybridizes to a sequence in, or 5′ to, the J segment of the nucleic acid molecule.

10 . The method of claim 9 , comprising using a polymerase to extend the 3′ region using the nucleic acid molecule as a template to generate an extended 3′ region, and using template-dependent ligation to ligate the extended 3′ region to the 5′ region, thereby filling a gap between the 3′ region and the 5′ region hybridized to the nucleic acid molecule.

11 . The method of claim 9 , comprising hybridizing an oligonucleotide comprising a 5′ end and a 3′ end to the nucleic acid molecule between the 3′ region and the 5′ region of the circularizable probe or probe set, and using a ligase to ligate the 3′ region and the 5′ region to the 5′ end and the 3′ end, respectively, of the oligonucleotide, thereby filling a gap between the 3′ region and the 5′ region hybridized to the nucleic acid molecule.

12 . The method of claim 11 , wherein the oligonucleotide comprises a sequence complementary to the V (D) J join or a portion thereof.

13 . The method of claim 1 , wherein the nucleic acid molecule is ligated to generate the circularized molecule.

14 . The method of claim 1 , wherein the circularized molecule comprises the D segment or a complement thereof.

15 . The method of claim 1 , wherein the RCA product is generated in situ in the biological sample or a matrix embedding the biological sample.

16 . The method of claim 1 , wherein the method comprises imaging the biological sample to detect the RCA product in situ in the biological sample or a matrix embedding the biological sample.

17 . The method of claim 1 , wherein the method comprises detecting the RCA product using sequential hybridization of detectable probes, sequencing by hybridization, sequencing by ligation, sequencing by synthesis, sequencing by binding, or a combination thereof.

18 . The method of claim 1 , wherein the RCA product comprises multiple copies of a unit sequence comprising a sequence of the V(D)J join, wherein a sequence of the V(D)J join in the unit sequence is assigned a signal code sequence, and detecting the sequence of the V(D)J join comprises:

(i) contacting the biological sample with a first detectable probe and a first detectably labeled oligonucleotide to generate a first complex comprising the first detectable probe hybridized to the unit sequence of the RCA product and the first detectably labeled oligonucleotide hybridized to the first detectable probe,

wherein the first detectable probe comprises: a recognition sequence complementary to the unit sequence, and a first overhang sequence, and

wherein the first detectably labeled oligonucleotide comprises: a sequence complementary to the first overhang sequence, and a first optically detectable moiety;

(ii) imaging the biological sample to detect a first signal from the first optically detectable moiety, wherein the first signal corresponds to a first signal code in the signal code sequence;

(iii) contacting the biological sample with a second detectable probe and a second detectably labeled oligonucleotide to generate a second complex comprising the second detectable probe hybridized to the unit sequence of the RCA product and the second detectably labeled oligonucleotide hybridized to the second detectable probe,

wherein the second detectable probe comprises: a recognition sequence complementary to the unit sequence, and a second overhang sequence, and

wherein the second detectably labeled oligonucleotide comprises: a sequence complementary to the second overhang sequence, and a second optically detectable moiety; and

(iv) imaging the biological sample to detect a second signal from the second optically detectable moiety, wherein the second signal corresponds to a second signal code in the signal code sequence,

wherein the signal code sequence comprising at least the first signal code and the second signal code is determined at a location in the biological sample, thereby detecting the sequence of the V(D)J join in the unit sequence and detecting the corresponding antigen receptor transcript at the location in the biological sample.

19 . The method of claim 1 , wherein the biological sample is a tissue section.

20 . The method of claim 1 , wherein the method comprises detecting the RCA product using sequencing by synthesis.

Assignments (2)

Continuity (2)

References Cited (360)