Molecular indexing of internal sequences

Patent metadata

Field	Value
Publication number	US-2020332284-A1
Application number	US-202016836750-A
Country	US
Kind code	A1
Filing date	Mar 31, 2020
Priority date	May 31, 2016
Publication date	Oct 22, 2020
Grant date	—

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

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; 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; 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; and sequencing the plurality of amplicons to generate a plurality of sequencing reads, wherein the sequencing reads comprise at most about 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 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 , wherein the target nucleic acid sequence comprises an unknown sequence.

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

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

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

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

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

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

13 . 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.

14 . 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.

15 . 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.

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

17 . The method of claim 16 , wherein the single cell is an immune cell.

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.

Assignees

• Cellular Res Inc

Inventors

• Fu Glenn
• Shum Eleen

Classifications

• 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

• C12Q1/6806

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