Molecular indexing of internal sequences

Patent metadata

Field	Value
Publication number	US-10640763-B2
Application number	US-201715596364-A
Country	US
Kind code	B2
Filing date	May 16, 2017
Priority date	May 31, 2016
Publication date	May 5, 2020
Grant date	May 5, 2020

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.

First claim

What is claimed is:

1. A method of labeling a target nucleic acid sequence 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 sequence;
extending the oligonucleotide to generate a second nucleic acid molecule comprising the molecular barcode and the target nucleic acid sequence;
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 sequence; 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 sequence, wherein one or more amplification reactions use at least one primer comprising a binding site for a sequencing primer, wherein the sequencing primer does not comprise a sequence homologous to the sequence of the first nucleic acid molecule.

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 sequence comprises 20 nt to 30 nt.

12. The method of claim 1 , wherein the target nucleic acid sequence comprises 30 nt to 40 nt.

13. The method of claim 1 , wherein the target nucleic acid sequence comprises 40 nt to 50 nt.

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

21. The method of claim 1 , wherein the sequencing primer is a sequence associated with a high-throughput sequencing platform.

Assignees

• Cellular Res Inc

Inventors

• Fu Glenn
• Shum Eleen

Classifications

• C12Q1/6874

  involving nucleic acid arrays, e.g. sequencing by hybridisation · CPC title

• C12N15/1065 Primary

  Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags · CPC title

• C12Q1/6806

  Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) · CPC title

• C12Q1/6844

  Nucleic acid amplification reactions · CPC title

• C12Q2565/514

  characterised by the use of the arrayed oligonucleotides as identifier tags, e.g. universal addressable array, anti-tag or tag complement array · CPC title