Methods and systems for processing polynucleotides

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.

1 .- 27 . (canceled) 
     
     
         28 . A method for analyzing cells comprising:
 (a) contacting a cell with an antibody to provide the cell bound to the antibody, wherein the cell comprises a nucleic acid analyte, and wherein the antibody is attached to a reporter nucleic acid molecule comprising a reporter sequence;   (b) co-partitioning into a partition: (i) the cell bound to the antibody attached to the reporter nucleic acid molecule and (ii) a population of oligonucleotides, wherein oligonucleotides of the population of oligonucleotides comprise a partition-specific barcode sequence;   (c) generating a first nucleic acid construct from the reporter nucleic acid molecule and a first oligonucleotide of the population of oligonucleotides, the first nucleic acid construct comprising the reporter sequence or complement thereof, and the partition-specific barcode sequence or complement thereof; and   (d) generating a second nucleic acid construct from the nucleic acid analyte or complement thereof and a second oligonucleotide of the population of oligonucleotides, the second nucleic acid construct comprising a sequence from the nucleic acid analyte or complement thereof and the partition-specific barcode sequence or complement thereof.   
     
     
         29 . The method of  claim 28 , further comprising sequencing the first nucleic acid construct or derivative thereof to obtain sequencing reads, and using the sequencing reads to identify the reporter sequence and the partition-specific barcode sequence. 
     
     
         30 . The method of  claim 28 , further comprising sequencing the second nucleic acid construct or derivative thereof to obtain sequencing reads, and using the sequencing reads to identify the sequence from the nucleic acid analyte and the partition-specific barcode sequence. 
     
     
         31 . The method of  claim 28 , wherein (c) comprises hybridizing the reporter oligonucleotide to the first oligonucleotide of the population of oligonucleotides and performing a nucleic acid extension reaction to generate the first nucleic acid construct. 
     
     
         32 . The method of  claim 28 , wherein (d) comprises hybridizing the nucleic acid analyte or complement thereof to the second oligonucleotide of the population of oligonucleotides and performing a nucleic acid extension reaction to generate the second nucleic acid construct. 
     
     
         33 . The method of  claim 28 , wherein (c) and (d) are performed in the partition. 
     
     
         34 . The method of  claim 28 , wherein, in (b), the population of oligonucleotides is attached to a bead. 
     
     
         35 . The method of  claim 28 , wherein the first oligonucleotide of the population of oligonucleotides is complementary to at least a portion of the reporter oligonucleotide. 
     
     
         36 . The method of  claim 28 , wherein the second oligonucleotide of the population of oligonucleotides is complementary to at least a portion of the nucleic acid analyte or complement thereof. 
     
     
         37 . The method of  claim 28 , wherein the first oligonucleotide and the second oligonucleotide of the population of oligonucleotides comprise differing unique molecular sequences. 
     
     
         38 . The method of  claim 28 , wherein the first oligonucleotide comprises a first primer sequence and the second oligonucleotide comprises a second primer sequence. 
     
     
         39 . The method of  claim 28 , wherein the reporter oligonucleotide comprises a primer sequence. 
     
     
         40 . The method of  claim 28 , further comprising subjecting the first nucleic acid construct or derivative thereof and the second nucleic acid construct or derivative thereof to a nucleic acid amplification reaction. 
     
     
         41 . The method of  claim 28 , further comprising adding one or more first functional sequences to the first nucleic acid construct or derivative thereof and one or more second functional sequences to the second nucleic acid construct or derivative thereof, wherein the one or more first functional sequences and the one or more second functional sequences are configured to permit attachment to a flow cell of a sequencer. 
     
     
         42 . The method of  claim 28 , wherein the partition is a well or a droplet. 
     
     
         43 . The method of  claim 28 , wherein the partition is among a plurality of partitions comprising at least about 1,000 partitions. 
     
     
         44 . The method of  claim 28 , wherein the nucleic acid analyte comprises a messenger ribonucleic acid (mRNA) molecule. 
     
     
         45 . A method for analyzing cells comprising:
 (a) providing a cell comprising a nucleic acid analyte, wherein the cell is bound to an antibody attached to a reporter oligonucleotide comprising a reporter sequence;   (b) providing a population of oligonucleotides, wherein oligonucleotides of the population of oligonucleotides share a common barcode sequence;   (c) co-partitioning: (i) the cell comprising the nucleic acid analyte and bound to the antibody attached to the reporter oligonucleotide and (ii) the population of oligonucleotides into a partition; and   (d) using the reporter oligonucleotide, the nucleic acid analyte, and the population of oligonucleotides to generate a sequencing library.   
     
     
         46 . The method of  claim 45 , wherein the reporter oligonucleotide comprises a poly-A sequence, wherein the population of oligonucleotides is attached to a bead, wherein the oligonucleotides of the population of oligonucleotides further comprise a poly-T sequence, and wherein (e) comprises (i) hybridizing the poly-A sequence of the reporter oligonucleotide to the poly-T sequence of an individual oligonucleotide of the oligonucleotides attached to the bead, to provide the bead comprising the reporter oligonucleotide attached thereto, and (ii) performing a nucleic acid extension reaction to generate a barcoded nucleic acid molecule. 
     
     
         47 . The method of  claim 45 , wherein the oligonucleotides of the population of oligonucleotides further comprise a poly-T sequence, wherein the nucleic acid analyte comprises a messenger ribonucleic acid RNA (mRNA) comprising a poly-A sequence, and wherein (e) comprises hybridizing the poly-A sequence of the mRNA to the poly-T sequence of an individual oligonucleotide of the oligonucleotides to provide the bead comprising the mRNA attached thereto; and (f) generating a nucleic acid construct comprising (i) a sequence corresponding to the mRNA and (ii) the partition-specific barcode sequence. 
     
     
         48 . The method of  claim 47 , further comprising sequencing the nucleic acid construct or a derivative thereof to identify the sequence corresponding to the mRNA and the barcode sequence. 
     
     
         49 . The method of  claim 45 , wherein the antibody is bound to a cell surface feature of the cell. 
     
     
         50 . The method of  claim 45 , wherein the antibody binds to a protein of the cell. 
     
     
         51 . The method of  claim 50 , wherein the protein comprises an intracellular protein. 
     
     
         52 . The method of  claim 50 , wherein the protein comprises a cell surface protein. 
     
     
         53 . The method of  claim 52 , wherein the cell surface protein is a cluster of differentiation (CD) protein. 
     
     
         54 . The method of  claim 45 , wherein the partition is a well. 
     
     
         55 . A method for cell analysis comprising:
 (a) contacting a cell organelle with an antibody to provide the cell organelle bound to the antibody, wherein the cell organelle comprises a nucleic acid analyte, and wherein the antibody is attached to a reporter nucleic acid molecule comprising a reporter sequence;   (b) co-partitioning into a partition: (i) the cell organelle bound to the antibody attached to the reporter nucleic acid molecule and (ii) a population of oligonucleotides, wherein oligonucleotides of the population of oligonucleotides comprise a partition-specific barcode sequence;   (c) generating a first nucleic acid construct from the reporter nucleic acid molecule and a first oligonucleotide of the population of oligonucleotides, the first nucleic acid construct comprising (i) the reporter sequence or complement thereof, and (ii) the partition-specific barcode sequence or complement thereof; and   (d) generating a second nucleic acid construct from the nucleic acid analyte or complement thereof and a second oligonucleotide of the population of oligonucleotides, the second nucleic acid construct comprising a sequence of the nucleic acid analyte or complement thereof and the partition-specific barcode sequence or complement thereof.   
     
     
         56 . The method of  claim 55 , wherein the nucleic acid analyte is a messenger ribonucleic acid RNA (mRNA). 
     
     
         57 . The method of  claim 55 , wherein the antibody binds to a protein of the cell organelle.