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

• US 10,106,789 B2
• Filed: 02/13/2017
• Issued: 10/23/2018
• 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:
  US15/431,660
• Publication Number:
  US 20170166889A1
• Time in Patent Office: 617 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 creating a library of polynucleotides, comprising the steps of:

introducing multiple sets of initial probes into a plurality of compartments under conditions selected such that more than a half of the plurality of compartments contain one or less than one set of the initial probes, wherein each set of the initial probes comprises (1) an initial forward probe, wherein the initial forward probe (i) is affixed to a bead or a solid surface, and (ii) comprises a sequence complementary to a first subsequence of a first target sequence and one of at least 1,000 unique barcode sequences, and (2) an initial reverse probe, wherein the initial reverse probe comprises (i) a sequence complementary to a second subsequence of the first target sequence and (ii) a sequence complementary to a non-human, exogenous sequence;

introducing multiple sets of second probes into the plurality of compartments, wherein each set of the second probes comprises (1) a second forward probe, wherein the second forward probe comprises (i) the non-human, exogenous sequence and (ii) a sequence that is complementary to a first subsequence of a second target sequence, and (2) a second reverse probe comprising a sequence that is complementary to a second subsequence to the second target sequence;

amplifying the first target sequence using the multiple sets of the initial probes;

amplifying the second target sequence using the multiple sets of the second probes;

hybridizing the non-human exogenous sequence to its complement; and

amplifying a fused sequence comprising one of the at least 1,000 barcode sequences, the first target sequence and the second target sequence, thereby generating a library of fused polynucleotides, wherein each of the fused polynucleotides comprises one of the at least 1,000 unique barcode sequences.

2. The method of claim 1, further comprising the step of:

bulk sequencing the library of fused polynucleotides.

3. The method of claim 2, further comprising the step of:

separating a plurality of single cells from a population of at least 10,000 cells into the plurality of compartments.

4. The method of claim 3, further comprising the step of:

identifying at least one of the plurality of compartments based on the unique barcode sequences.

5. The method of claim 3, further comprises the step of:

identifying a sequence that comprises one of the unique barcode sequences; and

tracing a cell that the sequence originates from.

6. The method of claim 3, wherein the plurality of compartments comprises emulsion microdroplets.

7. The method of claim 1, wherein the first target sequence and the second target sequence are RNA molecules.

8. The method of claim 7, wherein the steps of amplifying the first target sequence and the second target sequence comprise:

performing RT-PCR.

9. The method of claim 1, wherein the first target sequence and the second target sequence are DNA molecules.

10. The method of claim 9, wherein the steps of amplifying the first target sequence and the second target sequence comprise:

performing a polymerase chain reaction.

11. The method of claim 9, wherein the steps of amplifying the first target sequence and the second target sequence comprise:

a ligase chain reaction.

12. The method of claim 3, wherein the single cell is contained in a population of at least 25,000 cells.

13. The method of claim 12, wherein the single cell is contained in a population of at least 50,000 cells.

14. The method of claim 13, wherein the single cell is contained in a population of at least 75,000 cells.

15. The method of claim 14, wherein the single cell is contained in a population of at least 100,000 cells.

16. The method of claim 1, wherein the first target sequence and the second target sequence are human sequences.