Molecular indexing of internal sequences

Patent metadata

Field	Value
Publication number	US-11220685-B2
Application number	US-202016836750-A
Country	US
Kind code	B2
Filing date	Mar 31, 2020
Priority date	May 31, 2016
Publication date	Jan 11, 2022
Grant date	Jan 11, 2022

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, wherein the oligonucleotide specifically binds to a binding site on the first nucleic acid molecule, wherein the binding site is at least 200 nt away from the target nucleic acid sequence on the first nucleic acid molecule, wherein the oligonucleotide comprises a first universal primer binding site and a second universal primer binding site, wherein the first universal primer binding site is 5′ of the molecular barcode, and wherein the second universal primer binding site is 3′ of the molecular barcode;
extending the oligonucleotide to generate a second nucleic acid molecule comprising the molecular barcode and the target nucleic acid sequence;
amplifying the second nucleic acid molecule or complement thereof using a first target-specific primer and the first universal primer to generate a copy of the second nucleic acid molecule or complement thereof;
circularizing the amplified copy of 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;
amplifying the circularized nucleic acid molecule using the second universal primer and a second target-specific primer to generate a plurality of amplicons comprising the molecular barcode in close proximity to the target nucleic acid sequence; and
sequencing the plurality of amplicons to generate a plurality of sequencing reads, wherein the sequencing reads comprise at most 200 nucleotides, wherein the sequencing reads comprise at least a portion of the target nucleic acid sequence and at least a portion of the molecular barcode.

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 amplified copy of the double-stranded nucleic acid molecule.

4. The method of claim 1 , wherein the target nucleic acid sequence comprises an unknown sequence.

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

6. The method of claim 1 , further comprising aligning the plurality of sequencing reads to determine the full length target nucleic acid sequence.

7. The method of claim 1 , wherein the binding site is a gene-specific sequence.

8. The method of claim 1 , wherein the binding site is a poly-A sequence.

9. The method of claim 1 , wherein the target nucleic acid sequence is 20 nt to 500 nt in length.

10. The method of claim 1 , wherein the first nucleic acid molecule comprises a T cell receptor gene or an immunoglobulin gene.

11. The method of claim 1 , wherein the target nucleic acid sequence comprises a complementarity determining region (CDR) coding region of a T cell receptor gene or an immunoglobulin gene.

12. The method of claim 1 , wherein the target nucleic acid sequence comprises a mutation site, a splicing junction, a coding region, an untranslated region, or any combination thereof.

13. The method of claim 1 , wherein the binding site is at least 500 nt away from the target nucleic acid sequence on the first nucleic acid molecule.

14. The method of claim 1 , wherein the sample comprises a single cell.

15. The method of claim 14 , wherein the single cell is an immune cell.

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

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

18. The method of claim 1 , wherein the binding site is at least 2,000 nt away from the target nucleic acid sequence on the first nucleic acid molecule.

19. The method of claim 1 , wherein the second universal primer binding site is oriented in the opposite direction of the oligonucleotide.

Assignees

• Cellular Res Inc
• Becton Dickinson Co

Inventors

• Fu Glenn
• Shum Eleen

Classifications

• C12Q1/6806

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

• C12Q1/6874

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

• C12Q1/6844

  Nucleic acid amplification reactions · CPC title

• C12N15/1065 Primary

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