Polynucleotide barcode generation

Abstract

The present disclosure provides compositions, methods, systems, and devices for polynucleotide processing. Such polynucleotide processing may be useful for a variety of applications, including polynucleotide sequencing. In some cases, this disclosure provides methods for the generation of polynucleotide barcode libraries, and for the attachment of such polynucleotides to target polynucleotides.

What is claimed is: 
     
         1 . A method of synthesizing a library of polynucleotides comprising barcode sequences, said method comprising:
 a. partitioning a plurality of polynucleotides comprising at least 1,000 different nucleic acid barcode sequences into a plurality of droplets in an emulsion comprising at least 1,000 droplets, which at least 1,000 droplets comprise target polynucleotides, wherein each of said at least 1,000 different nucleic acid barcode sequences is disposed within a separate droplet of said at least 1,000 droplets in said emulsion, and wherein said at least 1,000 droplets (i) have a single nucleic acid barcode sequence of said 1,000 different nucleic barcode sequences and (ii) comprise at least 10% of said plurality of droplets in said emulsion; and   b. subjecting said target polynucleotides to amplification under conditions that are sufficient to generate amplified polynucleotides.   
     
     
         2 . The method of  claim 1 , wherein at least one droplet of said plurality of droplets does not include a nucleic acid barcode sequence. 
     
     
         3 . The method of  claim 1 , wherein said at least 1,000 droplets lack components necessary to perform any polymerase chain reaction (PCR). 
     
     
         4 . The method of  claim 1 , wherein said amplification is performed by polymerase chain reaction, asymmetric polymerase chain reaction, emulsion PCR (ePCR), ePCR including the use of a bead, ePCR including the use of a hydrogel, multiple annealing and looping-based amplification cycles (MALBAC), single primer isothermal amplification, or a combination thereof. 
     
     
         5 . The method of  claim 1 , wherein said amplification is performed using a ribonucleic acid primer. 
     
     
         6 . The method of  claim 5 , further comprising exposing said amplified polynucleotides to RNase H. 
     
     
         7 . The method of  claim 1 , wherein a hairpin structure is formed from a polynucleotide selected from the group consisting of said plurality of polynucleotides and said amplified polynucleotides. 
     
     
         8 . The method of  claim 1 , wherein said plurality of polynucleotides comprising at least 1,000 different barcode sequences or said amplified polynucleotides is attached to a bead. 
     
     
         9 . The method of  claim 8 , wherein said bead comprises a covalent bond that is cleavable upon application of a stimulus. 
     
     
         10 . The method of  claim 9 , wherein said covalent bond is a disulfide bond. 
     
     
         11 . The method of  claim 1 , wherein upon subjecting said target polynucleotides to said amplification, a given nucleic acid barcode sequence of said at least 1,000 different nucleic acid barcode sequences couples to a given target polynucleotide of said target polynucleotides. 
     
     
         12 . The method of  claim 1 , wherein said target polynucleotides are fragments of larger polynucleotides. 
     
     
         13 . The method of  claim 12 , wherein said larger polynucleotides are fragmented by mechanical shear or treatment with an enzyme. 
     
     
         14 . The method of  claim 13 , wherein said mechanical shear is induced by ultrasound. 
     
     
         15 . The method of  claim 13 , wherein said enzyme is selected from the group consisting of a restriction enzyme, a fragmentase, and a transposase. 
     
     
         16 . The method of  claim 1 , further comprising attaching additional nucleic acid sequences to said amplified polynucleotides. 
     
     
         17 . The method of  claim 16 , wherein said additional nucleic acid sequences comprise nucleic acid barcode sequences. 
     
     
         18 . The method of  claim 16 , wherein said attaching comprises ligation or additional amplification. 
     
     
         19 . The method of  claim 1 , wherein a given droplet of said at least 1,000 droplets comprises multiple copies of a given nucleic acid barcode sequence of said at least 1,000 different nucleic acid barcode sequences. 
     
     
         20 . The method of  claim 1 , wherein a given polynucleotide of said plurality of polynucleotides comprises a nucleic acid sequence selected from the group consisting of an immobilization sequence, an annealing sequence for a sequencing primer, and a primer sequence. 
     
     
         21 . The method of  claim 1 , wherein said at least 1,000 droplets comprise at least 50% of said plurality of droplets in said emulsion. 
     
     
         22 . The method of  claim 1 , wherein said plurality of polynucleotides comprises at least about 10,000 different barcode sequences. 
     
     
         23 . The method of  claim 1 , wherein said plurality of polynucleotides comprises at least about 100,000 different barcode sequences. 
     
     
         24 . The method of  claim 1 , wherein said plurality of polynucleotides comprises at least about 500,000 different barcode sequences. 
     
     
         25 . The method of  claim 1 , wherein said plurality of polynucleotides comprises at least about 1,000,000 different barcode sequences. 
     
     
         26 . The method of  claim 1 , wherein said plurality of polynucleotides comprises at least about 10,000,000 different barcode sequences. 
     
     
         27 . The method of  claim 1 , wherein said plurality of polynucleotides comprises at least about 50,000,000 different barcode sequences. 
     
     
         28 . The method of  claim 1 , wherein said plurality of polynucleotides comprises at least about 100,000,000 different barcode sequences. 
     
     
         29 . The method of  claim 1 , further comprising, prior to (a), synthesizing said plurality of polynucleotides. 
     
     
         30 . The method of  claim 1 , further comprising collecting droplets comprising said amplified polynucleotides.