Methods and systems for processing polynucleotides

• US 10,550,429 B2
• Filed: 05/30/2019
• Issued: 02/04/2020
• Est. Priority Date: 12/22/2016
• Status: Active Grant

• Current Assignee: 10X Genomics Incorporated
• Original Assignee: 10X Genomics Incorporated
• Inventors: Harada, Josephine, Mikkelsen, Tarjei Sigurd, Pfeiffer, Katherine, Saxonov, Serge, Stuelpnagel, John R.
• Primary Examiner(s): Thomas, David C
• Application Number:
  US16/426,762
• Publication Number:
  US 20190338353A1
• Time in Patent Office: 250 Days
• CPC Class Codes: C12N 15/1075 by coupling phenotype to genotype, not provided for in other groups of this subclass
• CPC Class Codes: C12Q 1/6804 Nucleic acid analysis using immunogens immunoassay G01N33/53
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/683 involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2521/107 RNA dependent DNA polymerase,(i.e. reverse transcriptase)
• CPC Class Codes: C12Q 2525/121 incorporating both deoxyribonucleotides and ribonucleotides
• CPC Class Codes: C12Q 2525/155 incorporating/generating a new priming site
• CPC Class Codes: C12Q 2525/173 incorporating a polynucleotide run, e.g. polyAs, polyTs
• CPC Class Codes: C12Q 2525/179 incorporating arbitrary or random nucleotide sequences
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2533/101 Primer extension
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2563/149 Particles, e.g. beads
• CPC Class Codes: C12Q 2563/159 Microreactors, e.g. emulsion PCR or sequencing, droplet PCR, microcapsules, i.e. non-liquid containers with a range of different permeability's for different reaction components
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid

Abstract

The present disclosure provides compositions, methods, systems, and devices for polynucleotide processing and analyte characterization. Such polynucleotide processing may be useful for a variety of applications, including analyte characterization by polynucleotide sequencing. The compositions, methods, systems, and devices disclosed herein generally describe barcoded oligonucleotides, which can be bound to a bead, such as a gel bead, useful for characterizing one or more analytes including, for example, protein (e.g., cell surface or intracellular proteins), genomic DNA, and RNA (e.g., mRNA or CRISPR guide RNAs). Also described herein, are barcoded labelling agents and oligonucleotide molecules useful for “tagging” analytes for characterization.

Claims

1. A method of immune receptor analysis, comprising:

(a) contacting a plurality of cells with a plurality of peptide-major histocompatibility complex (MHC) molecules, wherein each peptide-MHC molecule of said plurality of peptide-MHC molecules comprises a nucleic acid reporter molecule comprising a first barcode sequence that identifies a peptide bound to an MHC molecule of said plurality of peptide-MHC molecules;

(b) partitioning cells of said plurality of cells and a plurality of nucleic acid barcode molecules comprising barcode sequences into a plurality of partitions such that a partition of said plurality of partitions comprises (i) a cell bound to a peptide-MHC molecule; and

(ii) nucleic acid barcode molecules comprising a second barcode sequence, wherein said cell comprises a messenger ribonucleic acid (mRNA) molecule encoding for an immune receptor;

(c) generating (i) a first barcoded nucleic acid molecule comprising said first barcode sequence or a complementary sequence thereof and said second barcode sequence or a complementary sequence thereof; and

(ii) a second barcoded nucleic acid molecule comprising a sequence of said mRNA molecule or a complementary sequence thereof and said second barcode sequence or a complementary sequence thereof.

2. The method of claim 1, wherein said reporter molecule is covalently bound to said peptide-MHC molecule.

3. The method of claim 1, wherein said reporter molecule is indirectly coupled to said peptide-MHC molecule.

4. The method of claim 1, wherein said sequence of said mRNA molecule comprises a VDJ region or VJ region.

5. The method of claim 1, wherein an MHC molecule of said plurality of peptide-MHC molecules comprises a biotin moiety and wherein said MHC molecule is coupled to a streptavidin complex though a biotin-streptavidin interaction.

6. The method of claim 5, wherein said reporter molecule comprises biotin and is coupled to said streptavidin complex though a biotin-streptavidin interaction.

7. The method of claim 5, wherein said reporter molecule is covalently coupled to said streptavidin complex.

8. The method of claim 1, wherein said plurality of peptide-MHC molecules is a plurality of MHC multimers.

9. The method of claim 8, wherein said plurality of MHC multimers is a plurality of MHC tetramers, a plurality of MHC pentamers, or a plurality of MHC dextramers.

10. The method of claim 1, further comprising (d) sequencing (i) said first barcoded nucleic acid molecule or a derivative generated therefrom and (ii) said second barcoded nucleic acid molecule or a derivative generated therefrom, thereby generating sequencing information corresponding to said first barcoded nucleic acid molecule and said second barcoded nucleic acid molecule.

11. The method of claim 10, further comprising using said sequencing information to associate a peptide of said plurality of peptide-MHC molecules and said immune receptor with said cell.

12. The method of claim 1, wherein said cell is a T cell.

13. The method of claim 1, wherein said cell comprises a first mRNA molecule encoding for a T cell receptor alpha chain (TRA) and a second mRNA molecule encoding for a T cell receptor beta chain (TRB).

14. The method of claim 13, wherein said sequence of said mRNA molecule or a complementary sequence thereof comprises a sequence of said first mRNA molecule or a complementary sequence thereof;

and wherein (c) further comprises generating a third barcoded nucleic acid molecule comprising a sequence of said second mRNA molecule or a complementary sequence thereof and said second barcode sequence or a complementary sequence thereof.

15. The method of claim 14, further comprising (d) sequencing (i) said first barcoded nucleic acid molecule or a derivative generated therefrom, (ii) said second barcoded nucleic acid molecule or a derivative generated therefrom, and (iii) said third barcoded nucleic acid molecule or a derivative generated therefrom, thereby generating sequencing information corresponding to said first barcoded nucleic acid molecule, said second barcoded nucleic acid molecule, and said third barcoded nucleic acid molecule.

16. The method of claim 15, further comprising using said sequencing information to associate said peptide, said TRA, and said TRB with said cell.

17. The method of claim 1, wherein said plurality of nucleic acid barcode molecules are attached to a plurality of beads and wherein said partition comprises a bead of said plurality of beads.

18. The method of claim 17, wherein said plurality of beads is a plurality of gel beads.

19. The method of claim 18, wherein said plurality of gel beads is a plurality of degradable gel beads.

20. The method of claim 17, wherein said plurality of nucleic acid barcode molecules is releasably attached to said plurality of beads.

21. The method of claim 17, wherein each bead of said plurality of beads comprises a common barcode sequence that is distinct from barcode sequences of other barcode molecules attached to other beads of said plurality of beads.

22. The method of claim 17, wherein said partition comprises at most a single bead.

23. The method of claim 1, wherein said partition comprises at most a single cell.

24. The method of claim 1, wherein said reporter molecule further comprises an adapter sequence;

wherein, in (b), said partition comprises a bead comprising (1) a first nucleic acid barcode molecule comprising said second barcode sequence and a capture sequence complementary to said adapter sequence, and (2) a second nucleic acid barcode molecule comprising said second barcode sequence, and a template switch oligonucleotide (TSO) sequence; and

wherein (c) comprises using said reporter molecule and said first nucleic acid barcode molecule to generate said first barcoded nucleic acid molecule; and

using said mRNA molecule and said second nucleic acid barcode molecule to generate said second barcoded nucleic acid molecule.

25. The method of claim 24, wherein (c) comprises hybridizing said adapter sequence of said reporter molecule to said capture sequence of said first nucleic acid molecule and performing a nucleic acid extension reaction to generate said first barcoded nucleic acid barcode molecule;

and generating a complementary deoxyribonucleic acid (cDNA) molecule from said mRNA molecule and using said TSO sequence of said second nucleic acid barcode molecule to perform a template switching reaction, thereby generating said second barcoded nucleic acid molecule.

26. The method of claim 25, wherein said TSO sequence comprises three terminal guanine nucleotides and wherein said cDNA is generated using a reverse transcriptase with terminal transferase activity.

27. The method of claim 26, wherein said three terminal guanine nucleotides are ribonucleotides.

28. The method of claim 1, wherein said plurality of partitions is a plurality of droplets in an emulsion.

29. The method of claim 1, wherein said plurality of partitions is a plurality of microwells in a microwell array, wherein said microwell array comprises at least 1,000 microwells.

30. A method of immune receptor analysis, comprising:

(a) contacting a plurality of cells with a plurality of peptide-major histocompatibility complex (MHC) multimers, wherein each MHC multimer of said plurality of peptide-MHC multimers comprises;

(i) a plurality of peptide-MHC molecules comprising a common peptide, and

(ii) a nucleic acid reporter molecule comprising (1) a first adapter sequence, (2) a reporter sequence that identifies said common peptide, and (3) a second adapter sequence;

(b) partitioning cells of said plurality of cells and a plurality of beads into a plurality of partitions, wherein said plurality of beads comprises a plurality of barcode sequences, and wherein a partition of said plurality of partitions comprises;

(i) a cell bound to an MHC multimer, wherein said cell comprises a messenger ribonucleic acid (mRNA) molecule encoding for an immune receptor;

(ii) a bead comprising a plurality of nucleic acid barcode molecules attached thereto, wherein said plurality of nucleic acid barcode molecules comprises (1) a common barcode sequence and (2) a capture sequence configured to perform a template switching reaction; and

(iii) a primer comprising a poly-T sequence;

(c) in said partition, hybridizing said first adapter sequence of said reporter molecule to said capture sequence and performing a nucleic acid extension reaction to generate a first barcoded nucleic acid molecule comprising (i) said common barcode sequence or a complementary sequence thereof, (ii) said reporter sequence or a complementary sequence thereof, and (iii) said second adapter sequence or a complementary sequence thereof;

(d) in said partition, (i) hybridizing a poly-A sequence of said mRNA molecule to said poly-T sequence of said primer, (ii) performing a reverse transcription reaction to generate a complementary deoxyribonucleic acid (cDNA) molecule, and (iii) performing a template switching reaction onto a nucleic acid barcode molecule of said plurality of nucleic acid barcode molecules to generate a second barcoded nucleic acid molecule comprising (1) said common barcode sequence or a complementary sequence thereof, and (2) a sequence of said mRNA molecule or a complementary sequence thereof.

31. The method of claim 30, wherein said cDNA molecule is generated using a reverse transcriptase with terminal transferase activity.

32. The method of claim 30, wherein said mRNA molecule comprises an mRNA molecule encoding for a T cell receptor alpha chain (TRA) or an mRNA molecule encoding for a T cell receptor beta chain (TRB).

33. The method of claim 32, further comprising (e) sequencing (i) said first barcoded nucleic acid molecule or a derivative generated therefrom and (ii) said second barcoded nucleic acid molecule or a derivative generated therefrom, thereby generating sequencing information corresponding to said first barcoded nucleic acid molecule and said second barcoded nucleic acid molecule.

34. The method of claim 33, further comprising using said sequencing information to associate said common peptide, said TRA, and said TRB with said cell.