Barcoding systems and methods for gene sequencing and other applications

Patent metadata

Field	Value
Publication number	US-11746367-B2
Application number	US-201615566904-A
Country	US
Kind code	B2
Filing date	Apr 15, 2016
Priority date	Apr 17, 2015
Publication date	Sep 5, 2023
Grant date	Sep 5, 2023

Abstract

The present invention generally relates to microfluidics and labeled nucleic acids. In one aspect, the present invention is generally directed to a method, wherein the method includes providing a plurality of droplets comprising particles, the particles comprising oligonucleotides, and attaching a nucleic acid sequence to the oligonucleotides. Certain embodiments are generally directed to systems and methods for splitting a droplet into two or more droplets. Certain embodiments are generally directed to systems and methods for sorting fluidic droplets in a liquid.

First claim

What is claimed is:

1. A method, comprising:
providing a plurality of droplets comprising particles such that at least about 90% of the droplets contains one particle or no particle, the particles comprising oligonucleotides, the oligonucleotides comprising an adapter sequence that is identical for all of the plurality of droplets and particles, and a barcode sequence comprising first barcode selected from a pre-defined pool of first barcodes and a second barcode selected from a pre-defined pool of second barcodes, such that substantially each of the particles comprises distinguishable barcode sequences, wherein the barcode sequences of the pre-defined pool of first barcodes and the barcode sequences of the pre-defined pool of second barcodes are separated by a specific distance; and
attaching nucleic acid sequences to the oligonucleotides, wherein at least some of the nucleic acid sequences comprise a recognition sequence that is at least 80% complementary to a nucleic acid present within the droplet containing the respective nucleic acid sequence, wherein attaching a nucleic acid sequence to the oligonucleotides comprises exposing the adapter sequence to a sequence comprising (1) a sequence complementary to the adapter sequence, and (2) two or more primers, wherein the two or more primers comprise a gene-specific inner forward primer comprising the sequence complementary to the adapter sequence and a sequence complementary to the nucleic acid sequence, and a gene-specific reverse primer, and wherein the concentration of the gene-specific inner forward primer is lower than the concentration of the gene-specific reverse primer;
exposing the two or more primers to a nucleic acid sequence comprising a target of the two or more primers; and
applying amplification using the two or more primers to produce an oligonucleotide comprising the first barcode, the second barcode, and the nucleic acid sequence, wherein a gene-specific amplicon is generated using the gene-specific inner forward primer and the gene-specific reverse primer, and wherein the amplicon is further amplified using the oligonucleotide and the gene-specific reverse primer.

2. The method of claim 1 , wherein the pre-defined pool of first barcodes comprises at least about 300 distinguishable barcodes.

3. The method of claim 1 , wherein the pre-defined pool of second barcodes comprises at least about 300 distinguishable barcodes.

4. The method of claim 1 , wherein the nucleic acid sequence is configured to bind to genomic DNA.

5. The method of claim 1 , wherein at least some of the oligonucleotides are attached to the surface of the particles.

6. The method of claim 1 , wherein at least some of the oligonucleotides comprise a cleavable linker.

7. The method of claim 6 , wherein the cleavable linker is a photocleavable linker.

8. The method of claim 6 , wherein the cleavable linker is a chemically cleavable linker.

9. The method of claim 6 , wherein the cleavable linker is an enzymatically cleavable linker.

10. The method of claim 1 , further comprising releasing at least some of the oligonucleotides from the particles.

11. The method of claim 1 , further comprising exposing the nucleic acid sequence attached to the oligonucleotides to nucleic acids arising from a plurality of cells which are present in at least some of the plurality of droplets.

12. The method of claim 11 , wherein the plurality of cells is present in the plurality of droplets at no more than 1 cell/droplet.

13. The method of claim 11 , further comprising lysing at least some of the cells within the droplets.

14. The method of claim 1 , wherein the adapter sequence comprises no more than 10 nucleotides.

15. The method of claim 1 , wherein the adapter sequence comprises at least 5 nucleotides.

16. A method, comprising:
providing a plurality of at least 10,000 microfluidic droplets containing cells, at least about 90% of the plurality of droplets containing one cell or no cell;
lysing the cells within the plurality of microfluidic droplets to release nucleic acid from the cells;
attaching oligonucleotides to the nucleic acids within the droplets, wherein an oligonucleotide comprises an adapter sequence that is identical for all of the plurality of droplets and particles, and a barcode sequence comprising a first barcode selected from a pre-defined pool of first barcodes, a second barcode selected from a pre-defined pool of second barcodes, by exposing the adapter sequence to a sequence comprising (1) a sequence complementary to the adapter sequence, and (2) two or more primers, wherein the two or more primers comprise a gene-specific inner forward primer comprising the sequence complementary to the adapter sequence and a sequence complementary to the nucleic acid, and a gene-specific reverse primer, and wherein the concentration of the gene-specific inner forward primer is lower than the concentration of the gene-specific reverse primer;
exposing the two or more primers to a nucleic acid sequence comprising a target of the two or more primers, and
applying amplification to produce amplified oligonucleotides comprising the first barcode, the second barcode, and the nucleic acid sequence, wherein a gene-specific amplicon is generated using the gene-specific inner forward primer and the gene-specific reverse primer, and wherein the amplicon is further amplified using the oligonucleotide and the gene-specific reverse primer, thereby producing selectively amplified nucleic acids within the droplets bound to oligonucleotides, wherein for at least about 90% of the droplets, the oligonucleotide within the droplet is distinguishable from oligonucleotides within other droplets of the plurality of droplets.

Assignees

• Harvard College

Inventors

• Weitz David A
• Zhang Huidan
• Heyman John
• Klein Allon Moshe

Classifications

• C12P19/34 Primary

  Polynucleotides, e.g. nucleic acids, oligoribonucleotides · CPC title

• C12N15/1006

  by means of a solid support carrier, e.g. particles, polymers · CPC title

• C12N15/1065

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

• C12N15/1093

  General methods of preparing gene libraries, not provided for in other subgroups · CPC title

• C12Q1/6869

  Methods for sequencing · CPC title