METHODS AND COMPOSITIONS FOR COMBINATORIAL BARCODING

• US 20160289740A1
• Filed: 03/29/2016
• Published: 10/06/2016
• Est. Priority Date: 03/30/2015
• Status: Active Grant

• Current Assignee: Becton, Dickinson & Co
• Original Assignee: Cellular Research, Inc. (Becton, Dickinson & Co)
• Inventors: Fu, Glenn, Fodor, Stephen P.A.
• Granted Patent: US 11,535,882 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• 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 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2525/173 incorporating a polynucleotide run, e.g. polyAs, polyTs
• CPC Class Codes: C12Q 2525/185 incorporating bases where the precise position of the bases in the nucleic acid string is important
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2525/204 specific length of the oligonucleotides
• CPC Class Codes: C12Q 2531/119 Strand displacement amplification [SDA]
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2563/185 Nucleic acid dedicated to use as a hidden marker/bar code, e.g. inclusion of nucleic acids to mark art objects or animals
• 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 provide compositions, methods and kits for generating a set of combinatorial barcodes, and uses thereof for barcoding samples such as single cells or genomic DNA fragments. Some embodiments disclosed herein provide compositions comprising a set of component barcodes for producing a set of combinatorial barcodes. The set of component barcodes can comprise, for example, n×m unique component barcodes, wherein n and m are integers, each of the component barcodes comprises: one of n unique barcode subunit sequences; and one or two linker sequences or the complements thereof, wherein the component barcodes are configured to connect to each other through the one or two linker sequences or the complements thereof to produce a set of combinatorial barcodes.

Claims

1. A composition comprising a set of component barcodes for producing a set of combinatorial barcodes, comprising:

n×

m unique component barcodes, wherein n and m are positive integers, each of the component barcodes comprises;

one of n unique barcode subunit sequences; and

one or two linker sequences or the complements thereof,

wherein the component barcodes are configured to connect to each other through the one or two linker sequences or the complements thereof to produce a set of combinatorial barcodes.

2. The composition of claim 1, wherein each one of the set of n unique barcode subunit sequences comprises a molecular label, a cellular label, a dimensional label, a universal label, or any combination thereof.

3. The composition of claim 1, wherein the total number of different linker sequences is m−

1 or m−

2.

4. The composition of claim 1, wherein each of the component barcodes comprises:

a) barcode subunit sequence-linker;

b) complement of linker-barcode subunit sequence-linker;

or

c) complement of linker-barcode subunit sequence.

5. The composition of claim 1, wherein each one of the set of combinatorial barcodes comprises an oligonucleotide comprising the formula:

barcode subunit sequence_a-linker₁-barcode subunit sequence_b-linker₂- . . . barcode subunit sequence_c-linker_m-1-barcode subunit sequence_d.

6. The composition of claim 5, wherein the oligonucleotide comprises a target-specific region.

7. The composition of claim 6, wherein the target-specific region is oligo-dT.

8. The composition of claim 1, wherein each one of the set of combinatorial barcodes comprises:

a first oligonucleotide comprising the formula barcode subunit sequence_a-linker₁-barcode subunit sequence_b-linker₂- . . . -barcode subunit sequence_c; and

a second oligonucleotide comprising barcode subunit sequence_d- . . . -linker₃-barcode subunit sequence_e-linker_m-2-barcode subunit sequence_f.

9. The composition of claim 8, wherein each of the first oligonucleotide and the second oligonucleotide comprises a target-specific region.

10. The composition of claim 9, wherein one of the target-specific regions is oligo-dT.

11. The composition of claim 1, wherein each one of the one or two linker sequences is different.

12. The composition of claim 1, wherein the set of combinatorial barcodes has at least 100,000 unique combinatorial barcodes.

13. The composition of claim 1, wherein the set of combinatorial barcodes has at least 1,000,000 unique combinatorial barcodes.

14. A method of barcoding a plurality of partitions comprising:

introducing m component barcodes in each of the plurality of partitions, wherein the m component barcodes are selected from a set of n×

m different component barcodes, wherein n and m are positive integers, each of the component barcodes comprises;

one of n unique barcode subunit sequences; and

one or two linker sequences or the complements thereof; and

connecting the m component barcodes to generate a combinatorial barcode,

whereby each of the plurality of partitions is associated with a unique combinatorial barcode comprising the m component barcodes.

15. The method of claim 14, wherein each of the plurality of the partitions comprises a sample.

16. The method of claim 15, wherein the sample is a DNA sample.

17. The method of claim 15, wherein the sample is a single cell.

18. The method of claim 15, comprising hybridizing one or two of the m component barcodes to a target of the sample.

19. The method of claim 18, comprising extending the one or two of the m component barcodes hybridized to the DNA target to produce an extension product comprising the combinatorial barcode.

20. The method of claim 19, comprising amplifying the extension product.

21. The method of claim 20, comprising pooling the amplification products from the plurality of partitions.

22. The method of claim 21, comprising sequencing the pooled amplification products.

23. The method of claim 22, comprising assembling the sequences using the combinatorial barcodes.

24. The method of claim 23, comprising generating haplotypes using the assembled sequences.

25. A method of spatially barcoding a target in a sample, comprising providing a plurality of oligonucleotides immobilized on a substrate;

contacting a sample with the plurality of oligonucleotides immobilized on the substrate;

hybridizing the target with a probe that specifically binds to the target;

capturing an image showing the locations of the target;

capturing an image of the sample; and

correlating the image showing the locations of the target and the image of the sample to spatially barcode the target in the sample.

26. The method of claim 25, wherein the oligonucleotides immobilized on the substrate are oligo-dT primers.

27. The method of claim 25, wherein the sample comprises a tissue slice, a cell monolayer, fixed cells, a tissue section, or any combination thereof.

28. The method of claim 25, comprising amplifying the target to generate a plurality of amplicons immobilized on the substrate.

29. A method of single cell sequencing comprising:

depositing a first oligonucleotide comprising a first barcode selected from a first set of 40 unique barcodes and a first primer into a plurality of partitions comprising a single cell;

depositing a second oligonucleotide comprising a second barcode selected from a second set of 40 unique barcodes and a second primer into the plurality of partitions comprising the single cell;

contacting the first oligonucleotide with a target from the single cell;

extending the first oligonucleotide to generate a first strand comprising the first barcode;

contacting the second oligonucleotide with the first strand; and

extending the second oligonucleotide to generate a second strand comprising the first barcode and the second barcode,

whereby the target from the single cell is labeled with the first barcode and the second barcode.

30. The method of claim 29, comprising amplifying the second strand to produce amplification products.

31. A kit comprising a set of component barcodes for producing a set of combinatorial barcodes, the set of component barcodes comprises:

n×

m unique component barcodes, wherein n and m are positive integers, each of the component barcodes comprises;

one of n unique barcode subunit sequences; and

one or two linker sequences or the complements thereof,

wherein the component barcodes are configured to connect to each other through the one or two linker sequences or the complements thereof to produce a set of combinatorial barcodes.

32. The kit of claim 31, further comprising a microwell array.