System and methods for massively parallel analysis of nucleic acids in single cells

• US 9,695,474 B2
• Filed: 05/19/2016
• Issued: 07/04/2017
• Est. Priority Date: 12/16/2010
• Status: Active Grant

• Current Assignee: GigaGen Inc.
• Original Assignee: GigaGen Inc.
• Inventors: Johnson, David Scott, Meyer, Everett Hurteau
• Primary Examiner(s): Zhang, Kaijiang
• Application Number:
  US15/159,674
• Publication Number:
  US 20160326583A1
• Time in Patent Office: 411 Days
• Field of Search: None
• CPC Class Codes: C07K 16/00 Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies antibodies with enzymatic activity, e.g. abzymes C12N9/0002
• CPC Class Codes: C07K 2317/622 Single chain antibody (scFv)
• CPC Class Codes: C12N 15/1006 by means of a solid support carrier, e.g. particles, polymers
• 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: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6846 Common amplification features
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 1/6881 for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
• CPC Class Codes: C12Q 1/6883 for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 1/6886 for cancer immunoassay for cancer G01N33/574
• CPC Class Codes: C12Q 1/6888 for detection or identification of organisms
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2525/307 Circular oligonucleotides
• 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 2600/156 Polymorphic or mutational markers
• CPC Class Codes: C12Q 2600/158 Expression markers
• CPC Class Codes: C40B 50/06 Biochemical methods, e.g. using enzymes or whole viable microorganisms

Abstract

Methods and systems are provided for massively parallel genetic analysis of single cells in emulsion droplets or reaction containers. Genetic loci of interest are targeted in a single cell using a set of probes, and a fusion complex is formed by molecular linkage and amplification techniques. Methods are provided for high-throughput, massively parallel analysis of the fusion complex in a single cell in a population of at least 10,000 cells. Also provided are methods for tracing genetic information back to a cell using barcode sequences.

Claims

1. A method for analyzing at least two nucleic acid sequences in a single cell contained within a population of at least 10,000 cells, comprising:

isolating each of a plurality of single cells from the population of at least 10,000 cells in an emulsion microdroplet or a reaction container;

introducing a unique barcode sequence affixed to a bead or a solid surface into the emulsion microdroplet or the reaction container, wherein the unique barcode sequence comprises at least six nucleotides and wherein the unique barcode sequence is selected from a pool of barcode sequences with greater than 1000-fold diversity in sequence;

for each of the plurality of single cells,

providing at least one set of nucleic acid probes, the set comprising (i) a first probe comprising a sequence that is complementary to a nucleic acid sequence that is located at the 5′

end of the unique barcode sequence, (ii) a second probe comprising a sequence that is complementary to a nucleic acid sequence that is located at the 3′

end of the barcode sequence and a second region of sequence that is complementary to a non-human, exogenous sequence, (iii) a third probe comprising a sequence that comprises the non-human, exogenous sequence and a sequence that is complementary to a first subsequence of a target nucleic acid sequence, and (iv) a fourth probe comprising a sequence that is complementary to a second subsequence of the target nucleic acid sequence;

amplifying the unique barcode sequence and the target nucleic acid sequence independently, wherein the unique barcode sequence is amplified using the first probe and the second probe, and wherein the target nucleic acid sequence is amplified using the third probe and the fourth probe;

hybridizing the non-human exogenous sequence to its complement;

amplifying the unique barcode sequence, the target nucleic acid sequence, and the non-human exogenous sequence using the first probe and the fourth probe, thereby generating fused complexes;

performing bulk sequencing of the fused complexes; and

identifying a single cell for each of the fused complexes based on the unique barcode sequence.

2. The method of claim 1, further comprising isolating the unique barcode sequence affixed to the bead or the solid surface in a different emulsion microdroplet or a different reaction container.

3. The method of claim 2, wherein the step of introducing the unique barcode sequence comprises fusing the emulsion microdroplet or the reaction container comprising the single cell with the different emulsion microdroplet or the different reaction container comprising the barcode sequence affixed to the bead or the solid surface.

4. The method of claim 1, wherein the target nucleic acid sequence is complementary to an RNA sequence.

5. The method of claim 1, wherein the target nucleic acid sequence is complementary to a DNA sequence.

6. The method of claim 1, wherein the step of amplifying the unique barcode sequence and the target nucleic acid sequence comprises performing a polymerase chain reaction.

7. The method of claim 1, wherein the step of amplifying the unique barcode sequence and the target nucleic acid sequence comprises performing a ligase chain reaction.

8. The method of claim 1, wherein amplifying comprises performing a ligase chain reaction followed by a polymerase chain reaction.

9. The method of claim 1, wherein the single cell is contained within a population of at least 25,000 cells.

10. The method of claim 9, wherein the single cell is contained within a population of at least 50,000 cells.

11. The method of claim 10, wherein the single cell is contained within a population of at least 75,000 cells.

12. The method of claim 11, wherein the single cell is contained within a population of at least 100,000 cells.

13. The method of claim 1, further comprising quantifying the fused complexes.

14. The method of claim 1, wherein the fused complexes are circular.

15. The method of claim 1, further comprising:

providing N sets of nucleic acid probes, wherein each of the N sets of nucleic acid probes comprise (i) an I₁ probe comprising a sequence that is complementary a first subsequence of a barcode sequence, (ii) an I₂ probe comprising a sequence that is complementary to a second subsequence of the barcode sequence and a second sequence that is complementary to an I exogenous sequence, (iii) an I₃ probe comprising the I exogenous sequence and a sequence that is complementary to an I_b target nucleic acid first subsequence, and (iv) an I₄ probe comprising a sequence that is complementary to an I_b target nucleic acid second subsequence, wherein I is a natural number from 1 to N;

identifying the emulsion microdroplet or the reaction container comprising one of the plurality of single cells and the N sets of nucleic acid probes;

amplifying for all values of I, the barcode sequence and the I_b target nucleic acid sequences, wherein the barcode sequence is amplified using the I₁ probe and the I₂ probe and the I_b target nucleic acid sequence is amplified using the I₃ probe and the I₄ probe;

hybridizing the I exogenous sequence to its complement;

amplifying for each I, the barcode sequence, the I_b target sequence and the I exogenous sequence using the I₁ and I₄ probes, thereby generating N fused complexes; and

performing a bulk sequencing reaction to generate sequence information for at least 100,000 fused complexes from at least 10,000 cells within the population of cells, wherein the sequence information is sufficient to co-localize the barcode sequence and the I_b target nucleic acid sequence to a single cell from the population of at least 10,000 cells.

16. The method of claim 15, wherein N is less than or equal to 10.

17. The method of claim 15, wherein N is less than or equal to 100.

18. The method of claim 15, wherein N is less than or equal to 1000.

19. The method of claim 15, wherein N is less than or equal to 10,000.

20. The method of claim 15, wherein N is less than or equal to 100,000.

21. The method of claim 15, wherein N represents all of the number of different polyadenylated transcripts in a cell.

22. The method of claim 15, wherein the barcode sequence is the same sequence for all N.