Barcoding nucleic acids

• US 10,155,942 B2
• Filed: 11/05/2013
• Issued: 12/18/2018
• Est. Priority Date: 11/05/2012
• Status: Active Grant

• Current Assignee: Takara Bio Usa, Inc. (Ningbo Junsheng Investment Group Co., Ltd.)
• Original Assignee: Takara Bio Usa, Inc. (Ningbo Junsheng Investment Group Co., Ltd.)
• Inventors: Kurihara, Takao, Kamberov, Emmanuel, Tesmer, Tim, Langmore, John
• Primary Examiner(s): Priest, Aaron A
• Application Number:
  US14/438,280
• Publication Number:
  US 20150284712A1
• Time in Patent Office: 1,869 Days
• 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: C12Q 1/6855 Ligating adaptors
• CPC Class Codes: C12Q 2525/155 incorporating/generating a new priming site
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid

Abstract

Methods of barcoding nucleic acids, such as genomic DNA, are provided herein. In some embodiments, a fragment of genomic DNA may comprise a first and a second barcode.

Claims

1. A method of making a dual barcoded nucleic acid molecule comprising:

(a) ligating one strand of a stem-loop oligonucleotide adaptor of a population of stem-loop oligonucleotide adaptors, wherein each stem-loop oligonucleotide adaptor of the population comprises a loop and a double-stranded stem region that forms an intramolecular inverted repeat, the double-stranded stem region comprising a first stem strand and a second stem strand that comprises a unique barcode comprising a random or partially random sequence, to each end of a nucleic acid molecule to form a first barcode-coupled nucleic acid molecule;

(b) displacing the first stem strand of the stem-loop oligonucleotide adaptor from the first barcode-coupled nucleic acid molecule by strand displacement or by nick translation polymerization to form a first barcoded nucleic acid molecule that lacks the intramolecular inverted repeat and comprises the unique barcode;

(c) annealing a primer to the first barcoded nucleic acid molecule, wherein the primer comprises a first portion complementary to the first barcoded nucleic acid molecule and a second portion comprising a second barcode; and

(d) extending the annealed primer to form a dual barcoded nucleic acid molecule, the dual barcoded nucleic acid molecule comprising the second barcode, the unique barcode, and at least a portion of the nucleic acid molecule.

2. The method of claim 1, wherein the first portion of the primer does not anneal within the unique barcode in the first barcoded nucleic acid molecule.

3. The method of claim 1, wherein the nucleic acid molecule is genomic DNA, cDNA, amplified DNA, a nucleic acid library, or a fragment thereof.

4. The method of claim 1, wherein ligating one strand of a stem-loop oligonucleotide adaptor to at least one end of a nucleic acid molecule is further defined as ligating the 3′

end of the stem-loop oligonucleotide adaptor to the 5′

end of the nucleic acid molecule.

5. The method of claim 1, wherein the primer is a member of a population of primers.

6. The method of claim 1, wherein extending comprises primer extension or polymerase chain reaction.

7. The method of claim 1, further comprising amplification of at least part of the dual barcoded nucleic acid molecule.

8. The method of claim 4, wherein ligation is further defined as blunt-end ligation.

9. The method of claim 1, wherein the unique barcode and the second barcode are not immediately adjacent.

10. The method of claim 1, wherein the unique barcode comprises a known or partially known sequence.

11. The method of claim 1, wherein the second barcode comprises a known or partially known sequence.

12. The method of claim 1, wherein the 5′

end of the stem-loop oligonucleotide adaptor lacks a phosphate.

13. The method of claim 1, wherein the nick is removed from the first barcoded nucleic acid molecule following nick translation polymerization and prior to step (c).

14. The method of claim 1, wherein the loop of the stem-loop oligonucleotide adaptor or a region of the double-stranded stem region adjacent to the loop between the first and second stem strands of the stem-loop oligonucleotide adaptor comprises a replication stop.

15. The method of claim 14, wherein the replication stop is a cleavable replication stop.

16. The method of claim 15, wherein the nick is removed from the first barcoded nucleic acid molecule and the cleavable replication stop is cleaved following nick translation polymerization and prior to step (c).

17. The method of claim 1, wherein the loop of the stem-loop oligonucleotide comprises a cleavable base.

18. The method of claim 17, wherein the cleavable base is deoxyuridine.

19. The method of claim 17, wherein the cleavable base is cleaved prior to step (b) to produce an abasic site.

20. The method of claim 1, wherein the ligating comprises blunt-end ligation.

21. The method of claim 11, wherein the known or partially known sequence comprises an indexing sequence.

22. The method of claim 21, wherein the indexing sequence is a sequencing platform index sequence.