Whole transcriptome analysis of single cells using random priming

Patent metadata

Field	Value
Publication number	US-11932849-B2
Application number	US-201916677012-A
Country	US
Kind code	B2
Filing date	Nov 7, 2019
Priority date	Nov 8, 2018
Publication date	Mar 19, 2024
Grant date	Mar 19, 2024

Abstract

Disclosed herein include systems, methods, compositions, and kits for whole transcriptome analysis (WTA) with random priming and extension (RPE). The RPE-based WTA method can comprise hybridizing random primers with a plurality of first strand barcoded polynucleotides associated with a solid support and extending the random primers to generate a plurality of extension products. The method can comprise amplifying the plurality of extension products to generate a sequencing library.

First claim

What is claimed is:

1. A method of labeling nucleic acid targets in a sample, the method comprising:
contacting copies of a nucleic acid target with a plurality of oligonucleotide barcodes, wherein each oligonucleotide barcode comprises a first universal sequence, a molecular label, and a target-binding region capable of hybridizing to the copies of the nucleic acid target, wherein the nucleic acid target is an mRNA of a gene;
extending the plurality of oligonucleotide barcodes hybridized to the copies of the nucleic acid target to generate a plurality of first strand barcoded polynucleotides;
contacting random primers with the plurality of first strand barcoded polynucleotides, wherein each of the random primers comprises a second universal sequence, or a complement thereof,
extending the random primers hybridized to the plurality of first strand barcoded polynucleotides to generate a plurality of extension products;
amplifying the plurality of extension products using primers capable of hybridizing to the first universal sequence or complements thereof, and primers capable of hybridizing to the second universal sequence or complements thereof, thereby generating a first plurality of barcoded amplicons; and
synthesizing a plurality of gene-specific barcoded amplicons using the plurality of first strand barcoded polynucleotides as templates, wherein synthesizing a plurality of gene-specific barcoded amplicons comprises PCR amplification using primers capable of hybridizing to the first universal sequence, or a complement thereof, and a gene-specific primer.

2. The method of claim 1 , wherein amplifying the plurality of extension products comprises adding sequences of binding sites of sequencing primers and/or sequencing adaptors, complementary sequences thereof, and/or portions thereof, to the plurality of extension products.

3. The method of claim 1 , comprising obtaining sequence information of the plurality of first plurality of barcoded amplicons, or products thereof, and wherein the number of distinct sequences associated with the first plurality of barcoded amplicons, or products thereof, indicates the copy number of the nucleic acid target in the sample.

4. The method of claim 3 ,
wherein contacting copies of a nucleic acid target comprises contacting copies of a plurality of nucleic acid targets with a plurality of oligonucleotide barcodes,
wherein extending the plurality of oligonucleotide barcodes comprises extending the plurality of oligonucleotide barcodes hybridized to the copies of the plurality nucleic acid targets to generate a plurality of first strand barcoded polynucleotides, and
wherein the number of the molecular labels with distinct sequences associated with barcoded amplicons of the first plurality of barcoded amplicons comprising a sequence of the each of the plurality of nucleic acid targets indicates the number of each of the plurality of nucleic acid targets in the sample.

5. The method of claim 1 , comprising amplifying the first plurality of barcoded amplicons using primers capable of hybridizing to the first universal sequence or complements thereof, and primers capable of hybridizing to the second universal sequence or complements thereof, thereby generating a second plurality of barcoded amplicons, wherein amplifying the first plurality of barcoded amplicons comprises adding sequences of binding sites of sequencing primers and/or sequencing adaptors, complementary sequences thereof, and/or portions thereof, to the first plurality of barcoded amplicons.

6. The method of claim 5 , comprising obtaining sequence information of the plurality of second plurality of barcoded amplicons, or products thereof, and wherein the number of distinct sequences associated with the second plurality of barcoded amplicons, or products thereof, indicates the copy number of the nucleic acid target in the sample.

7. The method of claim 5 ,
wherein contacting copies of a nucleic acid target comprises contacting copies of a plurality of nucleic acid targets with a plurality of oligonucleotide barcodes,
wherein extending the plurality of oligonucleotide barcodes comprises extending the plurality of oligonucleotide barcodes hybridized to the copies of the plurality nucleic acid targets to generate a plurality of first strand barcoded polynucleotides, and
wherein the number of the molecular labels with distinct sequences associated with barcoded amplicons of the second plurality of barcoded amplicons comprising a sequence of the each of the plurality of nucleic acid targets indicates the number of each of the plurality of nucleic acid targets in the sample.

8. The method of claim 1 , wherein the first universal sequence and the second universal sequence are different.

9. The method of claim 1 , wherein extending the plurality of oligonucleotide barcodes hybridized to the copies of the nucleic acid target comprises extending the plurality of oligonucleotide barcodes hybridized to the copies of the nucleic acid target using a reverse transcriptase.

10. The method of claim 1 , wherein extending the plurality of oligonucleotide barcodes hybridized to the copies of the nucleic acid target comprises extending the plurality of oligonucleotide barcodes hybridized to the copies of the nucleic acid target using a DNA polymerase lacking at least one of 5′ to 3′ exonuclease activity and 3′ to 5′ exonuclease activity.

11. The method of claim 1 , wherein extending the random primers hybridized to the plurality of first strand barcoded polynucleotides comprises extending the random primers hybridized to the plurality of first strand barcoded polynucleotides using a DNA polymerase lacking at least one of 5′ to 3′ exonuclease activity and 3′ to 5′ exonuclease activity.

12. The method of claim 1 , wherein said random primers comprise a random sequence of nucleotides, and wherein the random sequence of nucleotides is 4 to 30 nucleotides in length.

13. The method of claim 1 , comprising:
providing additional random primers;
contacting the additional random primers with the plurality of first strand barcoded polynucleotides, wherein each of the additional random primers comprises a second universal sequence, or a complement thereof,
extending the additional random primers hybridized to the plurality of first strand barcoded polynucleotides to generate an additional plurality of extension products; and
amplifying the additional plurality of extension products using primers capable of hybridizing to the first universal sequence or complements thereof, and primers capable of hybridizing to the second universal sequence or complements thereof.

14. The method of claim 1 , wherein the sample comprises peripheral blood mononuclear cells or immune cells, and optionally the immune cells comprise B cells, T cells or a combination thereof.

15. The method of claim 1 , wherein the nucleic acid target encodes an immune receptor.

16. The method of claim 15 , wherein the immune receptor is a T cell receptor (TCR) or a B cell receptor (BCR).

17. The method of claim 1 , wherein the sample comprises a single cell, the method comprising physically associating a synthetic particle comprising the plurality of the oligonucleotide barcodes with the single cell in the sample.

18. The method of claim 17 , comprising lysing the single cell after physically associating the synthetic particle with the single cell.

19. The method of claim 17 , wherein the synthetic particle and the single cell are in the same partition.

20. The method of claim 1 , compris

Assignees

• Cellular Res Inc
• Becton Dickinson Co

Inventors

• Shum Eleen

Classifications

• C12N15/1096 Primary

  cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR · CPC title

• C12N15/1006

  by means of a solid support carrier, e.g. particles, polymers · CPC title

• C40B40/06

  Libraries containing nucleotides or polynucleotides, or derivatives thereof · CPC title

• C40B50/04

  using dynamic combinatorial chemistry techniques · CPC title

• C40B50/06

  Biochemical methods, e.g. using enzymes or whole viable microorganisms · CPC title