METHODS AND SYSTEMS FOR BARCODING NUCLEIC ACID MOLECULES FROM INDIVIDUAL CELLS OR CELL POPULATIONS

• US 20180105808A1
• Filed: 11/29/2017
• Published: 04/19/2018
• Est. Priority Date: 10/19/2016
• Status: Abandoned Application

• Current Assignee: 10X Genomics Incorporated
• Original Assignee: 10X Genomics Incorporated
• Inventors: Mikkelsen, Tarjei Sigurd, Belgrader, Phillip, Zheng, Xinying
• Application Number:
  US15/825,740
• Publication Number:
  US 20180105808A1
• Time in Patent Office: Days
• CPC Class Codes: C12N 15/1003 Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
• CPC Class Codes: C12N 15/1062 mRNA-Display, e.g. polypeptide and encoding template are connected covalently
• CPC Class Codes: C12N 15/1065 Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags
• CPC Class Codes: C12N 15/1075 by coupling phenotype to genotype, not provided for in other groups of this subclass
• CPC Class Codes: C12N 15/1096 cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR
• CPC Class Codes: C12N 5/0602 Vertebrate cells
• 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 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 1/6855 Ligating adaptors
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• 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 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
• CPC Class Codes: C12Q 2565/514 characterised by the use of the arrayed oligonucleotides as identifier tags, e.g. universal addressable array, anti-tag or tag complement array

Abstract

The present disclosure provides methods, compositions and systems for analyzing individual cells or cell populations through a partitioned analysis of contents of individual cells or cell populations, such as cancer cells and cells of the immune system. Individual cells or cell populations may be co-partitioned with processing reagents for accessing cellular contents, and for uniquely identifying the content of a given cell or cell population, and subsequently analyzing the content of the cell and characterizing it as having derived from an individual cell or cell population, including analysis and characterization of nucleic acid(s) from the cell through sequencing.

Claims

1. A method for nucleic acid sequencing, comprising:

(a) providing a plurality of droplets, wherein a droplet of said plurality of droplets comprises (i) a ribonucleic acid (RNA) molecule comprising a nucleic acid sequence, and (ii) a bead comprising a nucleic acid barcode molecule coupled thereto, wherein said nucleic acid barcode molecule comprises a barcode sequence;

(b) using said RNA molecule and said nucleic acid barcode molecule to generate a barcoded nucleic acid molecule comprising, from a 5′

end to a 3′

end, a sequence corresponding to said nucleic acid sequence of said RNA molecule and a complement of said barcode sequence; and

(c) sequencing said barcoded nucleic acid molecule or a derivative thereof.

2. The method of claim 1, wherein said RNA molecule is from a cell.

3. The method of claim 2, wherein said droplet comprises said cell.

4. The method of claim 3, further comprising releasing said RNA molecule from said cell prior to (b).

5. The method of claim 1, wherein said bead comprises a plurality of nucleic acid molecules coupled thereto, wherein said plurality of nucleic acid molecules comprises said nucleic acid barcode molecule.

6. The method of claim 5, wherein each of said plurality of nucleic acid molecules comprises said barcode sequence.

7. The method of claim 6, wherein each of said plurality of nucleic acid molecules comprises an additional barcode sequence that varies across said plurality of nucleic acid molecules.

8. The method of claim 1, wherein said nucleic acid barcode molecule comprises a template switching sequence.

9. The method of claim 1, further comprising, prior to (c), subjecting said barcoded nucleic acid molecule or derivative thereof to nucleic acid amplification.

10. The method of claim 9, wherein said nucleic acid amplification is performed subsequent to releasing said barcoded nucleic acid molecule or derivative thereof from said droplet.

11. The method of claim 9, wherein said nucleic acid amplification is polymerase chain reaction.

12. The method of claim 1, wherein said RNA molecule is a messenger ribonucleic acid (mRNA) molecule.

13. The method of claim 1, wherein in (a) said droplet comprises (i) an additional nucleic acid molecule comprising an additional nucleic acid sequence, and (ii) an additional nucleic acid barcode molecule comprising an additional barcode sequence, and wherein in (b) said additional nucleic acid molecule and said additional nucleic acid barcode molecule are used to generate an additional barcoded nucleic acid molecule comprising, from a 5′

end to a 3′

end, said additional barcode sequence and an additional sequence corresponding to said additional nucleic acid sequence.

14. The method of claim 13, wherein said additional nucleic acid barcode molecule is coupled to said bead.

15. The method of claim 1, wherein (b) comprises extending a primer hybridized to a region at a 3′

end of the RNA molecule in a primer extension reaction, said nucleic acid barcode molecule acting as a template switching oligonucleotide in said primer extension reaction, thereby generating said barcoded nucleic acid molecule comprising, from the 5′

end to the 3′

end, the sequence corresponding to the nucleic acid sequence of the RNA molecule and the complement of the barcode sequence.

16. The method of claim 1, wherein (b) is performed in said droplet.

17. The method of claim 15, further comprising releasing said barcoded nucleic acid molecule or a derivative thereof from said droplet.

18. The method of claim 1, wherein said barcoded nucleic acid molecule further comprises, towards a 5′

end, a functional sequence for permitting said barcoded nucleic acid molecule or a derivative thereof to couple to a flow cell of a sequencer.

19. The method of claim 1, wherein said sequence is a reverse complement of said nucleic acid sequence.

20. The method of claim 1, further comprising, prior to (c), using said barcoded nucleic acid molecule or a derivative thereof and a pair of primers to generate nucleic acid molecules having a target nucleic acid sequence.

21. The method of claim 20, wherein said target nucleic acid sequence comprises a T cell receptor variable region sequence, a B cell receptor variable region sequence, or an immunoglobulin variable region sequence.

22. The method of claim 20, wherein at least one of said pair of primers hybridizes to a constant region of a T cell receptor nucleic acid sequence, a constant region of a B cell receptor nucleic acid sequence, or a constant region of an immunoglobulin nucleic acid sequence.

23. The method of claim 20, wherein said nucleic acid molecules having said target nucleic acid sequence or derivatives thereof are sequenced in (c).

24. The method of claim 1, further comprising releasing said nucleic acid barcode molecule from said bead.

25. The method of claim 24, wherein said nucleic acid barcode molecule is released from said bead before said barcoded nucleic acid molecule is generated.

26. The method of claim 24, wherein said nucleic acid barcode molecule is released from said bead while said barcoded nucleic acid molecule is generated.

27. The method of claim 24, wherein said nucleic acid barcode molecule is released from said bead after said barcoded nucleic acid molecule is generated.

28. The method of claim 1, wherein said bead is a gel bead.

29. The method of claim 1, wherein said barcode sequence is a combinatorial assembly of a plurality of barcode segments.

30. The method of claim 29, wherein plurality of barcode segments comprises at least three segments.