Systems and methods for barcoding nucleic acids

Abstract

The present invention generally relates to microfluidics and labeled nucleic acids. For example, certain aspects are generally directed to systems and methods for labeling nucleic acids within microfluidic droplets. In one set of embodiments, the nucleic acids may include “barcodes” or unique sequences that can be used to distinguish nucleic acids in a droplet from those in another droplet, for instance, even after the nucleic acids are pooled together. In some cases, the unique sequences may be incorporated into individual droplets using particles and attached to nucleic acids contained within the droplets (for example, released from lysed cells). In some cases, the barcodes may be used to distinguish tens, hundreds, or even thousands of nucleic acids, e.g., arising from different cells or other sources.

What is claimed is: 
     
         1 . A method of barcoding nucleic acids, the method comprising:
 encapsulating a cell and a particle within a microfluidic droplet, the particle having attached thereto a barcoded nucleic acid;   cleaving the barcoded nucleic acid from the particle to release the barcoded nucleic acid into the microfluidic droplet;   lysing the cell within the microfluidic droplet to release cellular nucleic acid from the cell into the microfluidic droplet; and   attaching the released cellular nucleic acid to the released barcoded nucleic acid within the microfluidic droplet.   
     
     
         2 . The method of  claim 1 , wherein the particle is a hydrogel particle. 
     
     
         3 . The method of  claim 1 , comprising photocleaving the barcoded nucleic acid from the particle. 
     
     
         4 . The method of  claim 1 , comprising encapsulating only a single cell and only a single particle within the microfluidic droplet. 
     
     
         5 . The method of  claim 1 , wherein the microfluidic droplet is one of a plurality of microfluidic droplets, wherein, after encapsulation, the microfluidic droplets contain cells and particles such that at least about 90% of the plurality of microfluidic droplets contains only one particle and at least about 90% of the plurality of microfluidic droplets contains only one cell. 
     
     
         6 . The method of  claim 5 , wherein, after cleavage, at least 90% of the microfluidic droplets contains only a single type of barcoded nucleic acid. 
     
     
         7 . The method of  claim 5 , further comprising breaking at least some of the microfluidic droplets to release and combine the attached cellular and barcoded nucleic acids. 
     
     
         8 . The method of  claim 7 , further comprising amplifying and/or sequencing the attached cellular and barcoded nucleic acids. 
     
     
         9 . The method of  claim 1 , wherein after cleavage, the droplet has a concentration of the barcoded nucleic acids of at least 1 micromolar. 
     
     
         10 . The method of  claim 1 , wherein at least some of the barcoded nucleic acids, prior to cleaving, are covalently bonded to the particles via an acrylic phosphoramidite linkage, an amino linkage, or a biotin-steptavidin linkage. 
     
     
         11 . The method of  claim 1 , comprising enzymatically attaching the released cellular nucleic acid to the released barcoded nucleic acid. 
     
     
         12 . A method of containing barcoded nucleic acids within microfluidic droplets, the method comprising:
 attaching first oligonucleotides to a plurality of particles such that at least about 90% of the particles has covalently bonded thereto only one first oligonucleotide, wherein the first oligonucleotides are taken from a first pool of at least 100 unique oligonucleotides;   attaching second oligonucleotides to the first oligonucleotides such that at least about 90% of the first oligonucleotides has covalently bonded thereto only one second oligonucleotide, wherein the second oligonucleotides are taken from a second pool of at least 100 unique oligonucleotides;   encapsulating the plurality of particles within a plurality of microfluidic droplets such that at least about 90% of the plurality of microfluidic droplets contains only one particle; and   cleaving the first and second oligonucleotides from the particles into the microfluidic droplets.   
     
     
         13 . The method of  claim 12 , wherein the particle is a hydrogel particle. 
     
     
         14 . The method of  claim 12 , wherein at least some of the barcoded nucleic acids, prior to cleaving, are covalently bonded to the particles via an acrylic phosphoramidite linkage, an amino linkage, or a biotin-steptavidin linkage. 
     
     
         15 . The method of  claim 12 , wherein the first pool comprises at least 1,000 unique oligonucleotides and the second pool comprises at least 1,000 unique oligonucleotides. 
     
     
         16 . The method of  claim 12 , further comprising encapsulating a plurality of cells in the plurality of microfluidic droplets such that at least about 90% of the plurality of microfluidic droplets contains only one cell. 
     
     
         17 . The method of  claim 12 , wherein after cleavage, the droplets containing particles have an average concentration of the first and second oligonucleotides of at least 1 micromolar. 
     
     
         18 . An article for barcoding nucleic acids, comprising:
 a plurality of at least 10,000 microfluidic droplets, at least some of the droplets containing cell lysate comprising nucleic acid fragments, a plurality of the nucleic acid fragments within a droplet being bound to an oligonucleotide tag, wherein the oligonucleotide tag within the droplet is distinguishable from oligonucleotide tags within the other droplets of the plurality of 10,000 microfluidic droplets.   
     
     
         19 . The article of  claim 18 , wherein the microfluidic droplets comprise a plurality of at least distinguishable 1,000,000 oligonucleotide tags within the microfluidic droplets. 
     
     
         20 . The article of  claim 18 , wherein at least some of the distinguishable oligonucleotide tags each comprise a first oligonucleotide and a second oligonucleotide, wherein the first oligonucleotides are taken from a first pool of at least 100 unique first oligonucleotides, and wherein the second oligonucleotides are taken from a second pool of at least 100 unique first oligonucleotides. 
     
     
         21 . The article of  claim 18 , wherein in at least about 90% of the plurality of microfluidic droplets, the cell lysate arises from only one cell.