METHODS AND APPLICATIONS FOR STITCHED DNA BARCODES

• US 20090098555A1
• Filed: 09/24/2008
• Published: 04/16/2009
• Est. Priority Date: 09/26/2007
• Status: Active Grant

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Original Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Inventors: Suzuki, Yo, Mellor, Joseph, Roth, Frederick P.
• Granted Patent: US 8,268,564 B2
• Time in Patent Office: Days
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 2531/113 PCR
• CPC Class Codes: C12Q 2535/101 Sanger sequencing method, i.e. oligonucleotide sequencing using primer elongation and dideoxynucleotides as chain terminators
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid

Abstract

Methods of identifying the genotypes of a plurality of single cells, wherein each cell includes a plurality of DNA barcodes, each associated with a genetic mutation or marker, are provided. In particular, methods including linking a plurality of DNA barcodes together to create a stitched barcode, amplifying the stitched barcode and sequencing the stitched barcode are provided. Also provided are methods of determining the presence of at least one genetic mutation in a population of cells.

Claims

1. A method of determining the presence of at least one genetic mutation in a population of organisms, comprising the steps of:

(a) creating one or more aqueous emulsion droplets, wherein at least one emulsion droplet includes an organism having at least one barcode-associated mutation;

(b) performing a polymerase chain reaction (PCR) in the emulsion droplet to amplify at least one fused DNA product of one or more barcodes;

(c) recovering the aqueous phase from the emulsion droplet, wherein the aqueous phase includes the amplified, barcode-associated mutation; and

(d) detecting the presence of the amplified, barcode-associated mutation.

2. The method of claim 1, wherein the population of organisms is S. cerevisiae.

3. The method of claim 1, wherein the barcode-associated mutation includes a detectable label.

4. The method of claim 1, wherein the population of organisms includes a detectable label.

5. The method of claim 1, wherein the step of detecting is performed by sequencing the amplified, barcode-associated mutation.

6. The method of claim 1, wherein the step of detecting is performed by identifying the presence of a detectable label.

7. The method of claim 1, wherein the amplified, barcode-associated mutation is a genetic deletion or a genetic insertion.

8. The method of claim 1, wherein the barcode-associated mutation is present on a plasmid.

9. The method of claim 1, wherein the population of organisms includes at least 10 different barcode-associated mutations, and wherein for at least 95% of the organisms, each organism contains at least one barcode-associated mutation.

10. The method of claim 9, wherein for at least 95% of the organisms, each organism contains at least two barcode-associated mutations.

11. The method of claim 9, wherein for at least 95% of the organisms, each organism contains at least three barcode-associated mutations.

12. The method of claim 1, wherein the population of organisms includes at least 10 different barcode-associated mutations, and wherein for at least 95% of the organisms, each organism contains at least one barcode-associated mutation.

13. The method of claim 1, wherein the population of organisms includes at least 100 different barcode-associated mutations and wherein for at least 95% of the organisms, each organism contains at least one barcode-associated mutation.

14. The method of claim 1, wherein the population of organisms includes at least 1000 different barcode-associated mutations, and wherein for at least 95% of the organisms, each organism contains at least one barcode-associated mutation.

15. A method of identifying the genotype of an organism, wherein the organism comprises a plurality of barcode-associated mutations, comprising the steps of:

(a) covalently linking a plurality of barcode-associated mutations together to create a stitched barcode;

(b) amplifying the stitched barcode; and

(c) determining the identity of the stitched barcode.

16. The method of claim 15, wherein the step of determining the identity is performed by detecting the presence of at least one detectable label in the stitched barcode.

17. The method of claim 15, wherein each barcode-associated mutation includes a detectable label.

18. The method of claim 15, wherein the organism is S. cerevisiae.

19. The method of claim 15, wherein the plurality of barcode-associated mutations includes at least one vector that expresses a gene encoding at an RNA that is capable of inhibiting expression an endogenous gene.

20. The method of claim 19, wherein the organism is H. sapiens and the step of determining is performed on a cell derived from said H. sapiens.

21. The method of claim 19, wherein the organism is a mammal and the step of determining is performed on a cell derived from said mammal.

22. A method of making a library of organisms each organism carrying a combination of barcode-associated mutations, comprising the steps of:

(a) providing a first population of organisms each of which contains at least one of a first plurality of barcode-associated mutations;

(b) providing a second population of organisms each of which contains at least one of a second plurality of barcode-associated mutations; and

(c) combining the first population of organisms and the second population of organisms such that at least one resulting organism comprises a combination of at least one of the first plurality of barcode-associated mutations and at least one of the second plurality of barcode-associated mutations.

23. The method of claim 22, further comprising the step of:

(d) selecting the at least one resulting organism comprising a combination of at least one of the first plurality of barcode-associated mutations and the at least one of the second plurality of barcode-associated mutations.

24. The method of claim 22, wherein the first population of organisms and the second population of organisms are combined by mating.

25. The method of claim 22, further comprising the step of:

(d) subjecting the at least one resulting organism comprising a combination of at least one of the first plurality of barcode-associated mutations and at least one of the second plurality of barcode-associated mutations to a selection that alters the relative abundance of organisms within the library in a manner that is dependent upon at least one phenotype of at least one combination of barcode-associated mutations.

26. The method of claim 22, further comprising the step of:

(d) determining relative abundance of organisms of each population of steps (a)-(c) by sequencing linked barcodes from a cell derived from each of said populations.

27. The method of claim 25, further comprising the step of:

(e) determining relative abundance of organisms of each population of steps (a)-(d) by sequencing linked barcodes from a cell derived from each of said populations.