Granted Patent B2

US 12,742,166 · App. 17/692,758 · Granted Sep 22, 2026

Inventors: Tarjei Sigurd Mikkelsen (Dublin, CA); Phillip Belgrader (Livermore, CA); Xinying Zheng (San Jose, CA)

Assignee: 10x Genomics, Inc.

C12N15/1003C12N5/0602C12N15/1062C12N15/1065C12N15/1075C12N15/1096C12Q1/6804C12Q1/6806C12Q1/6816C12Q1/6834C12Q1/6853C12Q1/686C12Q1/6869C12Q1/6874C12Q1/6855

Abstract

Claims (35)

1 . A method for nucleic acid sequencing, comprising:

(a) providing a plurality of partitions, wherein a partition of the plurality of partitions comprises:

(i) a plurality of ribonucleic acid (RNA) molecules from a single cell; and

(ii) a particle comprising nucleic acid barcode molecules coupled thereto,

wherein the nucleic acid barcode molecules coupled to the particle comprise a first subset and a second subset,

wherein the first subset comprises a partition-specific barcode sequence and a first primer region comprising a poly-T sequence, and

wherein the second subset comprises the partition-specific barcode sequence and a second primer region comprising a sequence that is complementary to a sequence encoding a T cell receptor (TCR) or B cell receptor (BCR) constant region;

(b) using the plurality of RNA molecules and the first subset of nucleic acid barcode molecules to generate a first plurality of barcoded nucleic acid molecules, each comprising: 1) the partition-specific barcode sequence or a complement thereof, and 2) a sequence of a messenger RNA (mRNA) molecule of the plurality of RNA molecules or a complement thereof; and using the plurality of RNA molecules and the second subset of nucleic acid barcode molecules to generate a second plurality of barcoded nucleic acid molecules, each comprising: 1) the partition-specific barcode sequence or a complement thereof, and 2) a TCR or BCR variable region-encoding sequence or a complement thereof; wherein the generating the first plurality of barcoded nucleic acid molecules and the second plurality of barcoded nucleic acid molecules comprises:

(i) hybridizing the first subset of nucleic acid barcode molecules to poly-A sequences in mRNA molecules of the plurality of RNA molecules in the partition, and hybridizing the second subset of nucleic acid barcode molecules to sequences encoding TCR or BCR constant regions in mRNA molecules of the plurality of RNA molecules in the partition;

(ii) extending the first and second subsets of the nucleic acid barcode molecules using the hybridized mRNA molecules as template with a reverse transcriptase having terminal transferase activity to generate first and second barcoded cDNA molecules comprising 3′ terminal sequences added by the reverse transcriptase;

(iii) hybridizing the 3′ terminal sequences of the first and second barcoded cDNA molecules to hybridization regions of template switching oligonucleotides (TSOs), and

(iv) extending the first and second barcoded cDNA molecules using the TSOs as templates, thereby generating the first plurality of barcoded nucleic acid molecules and the second plurality of barcoded nucleic acid molecules;

(c) amplifying barcoded nucleic acid molecules of the first plurality of barcoded nucleic acid molecules or derivatives thereof to generate an unenriched sequencing library; and

(d) amplifying barcoded nucleic acid molecules of the second plurality of barcoded nucleic acid molecules or derivatives thereof to generate an immune receptor-enriched sequencing library.

2 . The method of claim 1 , wherein the particle is a bead.

3 . The method of claim 1 , wherein the particle is a gel bead and/or a magnetic bead.

4 . The method of claim 3 , wherein the partition initially comprises the single cell, and the method comprises lysing the single cell to release the RNA molecules.

5 . The method of claim 1 , wherein the nucleic acid barcode molecules are released from the particle upon application of a stimulus.

6 . The method of claim 5 , wherein the stimulus comprises a biological stimulus, a chemical stimulus, a thermal stimulus, an electrical stimulus, a magnetic stimulus, or a photo stimulus.

7 . The method of claim 1 , wherein the nucleic acid barcode molecules are released from the particle prior to step (b), and/or upon formation of the partition.

8 . The method of claim 1, 2, or 3 , wherein the nucleic acid barcode molecules further comprise unique molecule identifier (UMI) sequences.

9 . The method of claim 8 , wherein the method further comprises sequencing the unenriched sequencing library, sequencing the immune receptor-enriched sequencing library, and associating sequenced molecules from the unenriched sequencing library and sequenced molecules from the immune receptor-enriched sequencing library with individual partitions of the plurality of partitions and/or single cells.

10 . The method of claim 9 , wherein the immune receptor-enriched sequencing library comprises sequences corresponding to an immune receptor variable region of a TCR or BCR.

11 . The method of claim 1 , wherein the nucleic acid barcode molecules further comprise a sequencing primer sequence.

12 . The method of claim 1 , wherein the partition initially comprises the single cell, and the method comprises lysing the single cell to release the RNA molecules.

13 . The method of claim 1 , wherein the partition is a droplet, a microwell, or a well.

14 . The method of claim 1, 2, or 3 , wherein amplifying the barcoded nucleic acid molecules of the second plurality of barcoded nucleic acid molecules or derivatives thereof comprises using a target-specific primer to amplify TCR or BCR variable region-encoding sequences.

15 . The method of claim 1 or 2 , wherein the hybridization regions of the TSOs comprise a polyG sequence, and the 3′ terminal sequence added by the reverse transcriptase comprises a polyC sequence.

16 . The method of claim 1 , wherein (c) comprises adding a functional sequence to the barcoded nucleic acid molecules of the first plurality of barcoded nucleic acid molecules, and (d) comprises adding a functional sequence to the barcoded nucleic acid molecules of the second plurality of barcoded nucleic acid molecules or derivatives thereof.

17 . The method of claim 16 , wherein the functional sequence in (c) or (d) comprises a flow cell attachment sequence and/or a sequencing primer binding sequence.

18 . The method of claim 1 , wherein the first and second primer regions are at the 3′ ends of the first and second subsets of nucleic acid barcode molecules, respectively.

19 . The method of claim 1, 2, or 3 , wherein the method further comprises sequencing the unenriched sequencing library, sequencing the immune receptor-enriched sequencing library, and associating sequenced molecules from the unenriched sequencing library and sequenced molecules from the immune receptor-enriched sequencing library with individual partitions of the plurality of partitions and/or single cells.

20 . The method of claim 19 , wherein the immune receptor-enriched sequencing library comprises sequences corresponding to an immune receptor variable region of a TCR or BCR.

21 . The method of claim 1 , wherein step (b) is carried out in the partition after lysis of the single cell.

22 . The method of claim 1 , wherein the method comprises analyzing both immune receptor expression and non-immune receptor gene expression in the same single cell for one or more single cells.

Assignments (1)

Continuity (6)

References Cited (400)