Sequencing of nucleic acids via barcoding in discrete entities

Patent metadata

Field	Value
Publication number	US-11111519-B2
Application number	US-201615015015-A
Country	US
Kind code	B2
Filing date	Feb 3, 2016
Priority date	Feb 4, 2015
Publication date	Sep 7, 2021
Grant date	Sep 7, 2021

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 of introducing multiple copies of a first nucleic acid barcode to nucleic acids of a discrete entity, the method comprising:
encapsulating a plurality of nucleic acid target molecules in a discrete entity;
flowing the discrete entity in a first channel to a junction;
flowing a continuous, unsegmented reagent flow comprising multiple copies of a first nucleic acid barcode and multiple copies of a second nucleic acid barcode in a second channel to the junction;
flowing a carrier fluid in a third channel to the junction, wherein the second channel comprising the continuous, unsegmented reagent flow is positioned between the first channel comprising the discrete entity and the third channel comprising the carrier fluid;
merging the discrete entity into the continuous, unsegmented reagent flow downstream of the junction; and
downstream of the junction, inducing the continuous, unsegmented reagent flow to break off one individual microdroplet comprising the plurality of nucleic acid target molecules of the discrete entity, multiple copies of the first nucleic acid barcode, and no copies of the second nucleic acid barcode, such that substantially all of the plurality of nucleic acid target molecules of the discrete entity are broken off into the one individual microdroplet.

2. The method of claim 1 , wherein encapsulating a plurality of nucleic acid target molecules in a discrete entity comprises lysing a cell in an intermediate droplet, thereby encapsulating nucleic acid target molecules of the cell in the intermediate droplet.

3. The method of claim 1 , wherein the multiple copies of a first nucleic acid barcode are anchored on at least one solid substrate comprising a bead.

4. The method of claim 3 , wherein the merging comprises using an electric field to merge the discrete entity to the continuous, unsegmented reagent flow.

5. The method of claim 1 , further comprising flowing the continuous, unsegmented reagent flow under drip-producing conditions while merging the discrete entity into the reagent flow.

6. The method of claim 1 , further comprising flowing the continuous, unsegmented reagent flow under jetting conditions while merging the discrete entity into the continuous, unsegmented reagent flow.

7. The method of claim 1 , further comprising enzymatically incorporating a copy of the first nucleic acid barcode into a nucleic acid target molecule within the individual microdroplet, and synthesizing a complement of the nucleic acid target molecule tagged with the first nucleic acid barcode.

8. The method of claim 7 , wherein synthesizing the complement comprises amplification.

9. The method of claim 7 , wherein synthesizing the complement comprises reverse transcription.

10. The method of claim 7 , wherein synthesizing comprises synthesizing within a microdroplet.

11. The method of claim 1 , wherein each of the multiple copies of first barcode sequence comprises a corresponding unique molecular identifier.

12. The method of claim 1 , further comprising tagging the plurality of nucleic acid target molecules with a unique molecular identifier prior to the encapsulating.

13. The method of claim 12 , wherein the first nucleic acid barcode comprises a longer sequence length than the unique molecular identifier.

14. The method of claim 12 , further comprising amplifying the plurality of nucleic acid target molecules comprising the unique molecular identifier prior to the encapsulating.

15. The method of claim 1 , further comprising:
at the junction, contacting the discrete entity from the first channel with a first side of the continuous, unsegmented reagent flow from the second channel, and contacting the carrier fluid from the third channel with the continuous, unsegmented reagent flow from the second channel.

16. The method of claim 1 , further comprising:
prior to merging the discrete entity into the continuous, unsegmented reagent flow, co-flowing the discrete entity in contact with the continuous, unsegmented reagent flow downstream of the junction.

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

• C12Q1/6806 Primary

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

• C12Q1/6869

  Methods for sequencing · CPC title

• C12Q1/6855

  Ligating adaptors · CPC title

• G01N2015/1497

  Particle shape · CPC title