Sequencing of nucleic acids via barcoding in discrete entities

Patent metadata

Field	Value
Publication number	US-12448643-B2
Application number	US-202318212868-A
Country	US
Kind code	B2
Filing date	Jun 22, 2023
Priority date	Feb 4, 2015
Publication date	Oct 21, 2025
Grant date	Oct 21, 2025

Abstract

Microfluidic methods for barcoding nucleic acid target molecules to be analyzed, e.g., via nucleic acid sequencing techniques, are provided. Also provided are microfluidic, droplet-based methods of preparing nucleic acid barcodes for use in various barcoding applications. The methods described herein facilitate high-throughput sequencing of nucleic acid target molecules as well as single cell and single virus genomic, transcriptomic, and/or proteomic analysis/profiling. Systems and devices for practicing the subject methods are also provided.

First claim

What is claimed is:

1. A method comprising:
encapsulating, within a discrete entity,
a nucleic acid released from a single cell or a product of the released nucleic acid;
an oligonucleotide including a first nucleic acid barcode serving as an indicator of an affinity reagent that was bound to a molecular target of the single cell;
a plurality of primer sequences;
a plurality of copies of a sequence comprising both a second nucleic acid barcode and a universal sequence,
wherein the second nucleic acid barcode serves as an indicator of the discrete entity in which the oligonucleotide including the first nucleic acid barcode and the released nucleic acid or the product of the released nucleic acid are encapsulated;
incorporating a first copy of the sequence comprising the second nucleic acid barcode and the universal sequence with the oligonucleotide including the first nucleic acid barcode by performing nucleic acid amplification using the universal sequence of the first copy and at least a first primer sequence;
incorporating a second copy of the sequence comprising the second nucleic acid barcode and the universal sequence with the released nucleic acid or the product of the released nucleic acid by performing nucleic acid amplification using the universal sequence of the second copy and at least a second primer sequence; and
sequencing the first nucleic acid barcode, the first copy of the sequence comprising the second nucleic acid barcode, and the second copy of the sequence comprising the second nucleic acid barcode.

2. The method of claim 1 , wherein the affinity reagent comprises a peptide.

3. The method of claim 1 , wherein the affinity reagent comprises an antibody or an antigen binding antibody fragment.

4. The method of claim 1 , wherein the affinity reagent does not comprise an antibody.

5. The method of claim 1 , wherein the affinity reagent comprises a drug.

6. The method of claim 1 , wherein the molecular target is a component of a cell.

7. The method of claim 1 , wherein the product of the released nucleic acid is generated by performing reverse transcription on the released nucleic acid.

8. The method of claim 7 , wherein the reverse transcription occurs without nucleic acid amplification.

9. The method of claim 7 , wherein the product of the released nucleic acid is further generated by performing nucleic acid amplification.

10. The method of claim 9 , further comprising sequencing the amplification product.

11. The method of claim 1 , further comprising amplifying the oligonucleotide before incorporating the second nucleic acid barcode.

12. The method of claim 1 , wherein the oligonucleotide including the first nucleic acid barcode further comprises a unique molecular identifier that uniquely identifies the affinity reagent from a plurality of affinity reagents.

13. The method of claim 12 , further comprising: sequencing the unique molecular identifier; and correcting for amplification bias using the sequenced unique molecular identifier.

14. The method of claim 1 , wherein the universal sequence is complementary to a portion of the first primer sequence and a portion of the second primer sequence,
wherein performing nucleic acid amplification using the universal sequence of the first copy and at least the first primer sequence comprises hybridizing the universal sequence of the first copy to the portion of the first primer sequence, and
wherein performing nucleic acid amplification using the universal sequence of the second copy and at least the second primer sequence comprises hybridizing the universal sequence of the second copy to the portion of the second primer sequence.

15. The method of claim 1 , further comprising:
prior to encapsulating the nucleic acid released from the single cell or the product of the released nucleic acid,
encapsulating the single cell with an affinity reagent bound molecular target comprising the oligonucleotide including the first nucleic acid barcode in the discrete entity;
lysing the single cell within the discrete entity such that the discrete entity comprises a released nucleic acid from within the single cell and the oligonucleotide including the first nucleic acid barcode.

Assignees

• Univ California

Inventors

• Abate Adam R
• Haliburton John R
• Lan Freeman
• Sciambi Adam R

Classifications

• C12Q1/6874

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

• B01F21/00

  Dissolving (separating by dissolving B01D; dissolving to effect cooling F25D5/00) · CPC title

• B01F33/3021

  the components to be mixed being combined in a single independent droplet, e.g. these droplets being divided by a non-miscible fluid or consisting of independent droplets · CPC title

• B01F25/4338

  Mixers with a succession of converging-diverging cross-sections, i.e. undulating cross-section · CPC title

• B01L2200/0647

  Handling flowable solids, e.g. microscopic beads, cells, particles · CPC title