MOLECULAR INDEXING OF INTERNAL SEQUENCES

• US 20170342405A1
• Filed: 05/16/2017
• Published: 11/30/2017
• Est. Priority Date: 05/31/2016
• Status: Active Grant

• Current Assignee: Becton, Dickinson & Co
• Original Assignee: Cellular Research, Inc. (Becton, Dickinson & Co)
• Inventors: Fu, Glenn, Shum, Eleen
• Granted Patent: US 10,640,763 B2
• Time in Patent Office: 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 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid
• 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 relates to compositions, methods and kits for labeling an internal sequence of a target nucleic acid molecule with molecular barcodes. In some embodiments, the methods comprise intramolecular circulation of a labeled target nucleic acid molecule. Further provided methods for generating sequencing libraries comprising overlapping fragments covering the full length of a target nucleic acid molecule, sequencing the libraries using the methods disclosed herein, and methods of analyzing sequencing results therefrom.

Claims

1. A method of labeling a target nucleic acid in a sample with a molecular barcode, comprising:

hybridizing an oligonucleotide comprising a molecular barcode with a first nucleic acid molecule comprising the target nucleic acid;

extending the oligonucleotide to generate a second nucleic acid molecule comprising the molecular barcode and the target nucleic acid;

circularizing the second nucleic acid molecule or complement thereof to generate a circularized nucleic acid molecule comprising the molecular barcode in close proximity to the target nucleic acid; and

amplifying the circularized nucleic acid molecule to generate a plurality of amplicons comprising the molecular barcode in close proximity to the target nucleic acid.

2. The method of claim 1, further comprising synthesizing a complementary strand of the second nucleic acid molecule to generate a double-stranded nucleic acid molecule.

3. The method of claim 2, wherein the circularizing comprises circularizing the double-stranded nucleic acid molecule.

4. The method of claim 1, further comprising amplifying the second nucleic acid molecule or complement thereof to generate a copy of the second nucleic acid molecule or complement thereof.

5. The method of claim 4, wherein the circularizing comprises circularizing a copy of the second nucleic acid molecule or complement thereof.

6. The method of claim 1, further comprising sequencing the plurality of amplicons.

7. The method of claim 1, wherein the first nucleic acid is an mRNA.

8. The method of claim 1, wherein the oligonucleotide specifically binds to a binding site on the first nucleic acid molecule.

9. The method of claim 8, wherein the binding site is a gene-specific sequence.

10. The method of claim 8, wherein the binding site is a poly-A sequence.

11. The method of claim 1, wherein the target nucleic acid comprises about 20 nt.

12. The method of claim 1, wherein the target nucleic acid comprises about 30 nt.

13. The method of claim 1, wherein the target nucleic acid comprises about 40 nt.

14. The method of claim 8, wherein the binding site is at least 200 nt away from the target nucleic acid on the first nucleic acid molecule.

15. The method of claim 8, wherein the binding site is at least 500 nt away from the target nucleic acid on the first nucleic acid molecule.

16. The method of claim 8, wherein the binding site is at least 1,000 nt away from the target nucleic acid on the first nucleic acid molecule.

17. The method of claim 8, wherein the binding site is at least 2,000 nt away from the target nucleic acid on the first nucleic acid molecule.

18. The method of claim 1, wherein the molecular barcode comprises a sample label, a cellular label, a molecular label, or a combination thereof.

19. The method of claim 1, wherein the molecular barcode comprises a binding site for a primer.

20. The method of claim 19, wherein the primer is a universal primer.