Methods and systems for processing polynucleotides

Abstract

The present disclosure provides compositions, methods, systems, and devices for polynucleotide processing and analyte characterization. Such polynucleotide processing may be useful for a variety of applications, including analyte characterization by polynucleotide sequencing. The compositions, methods, systems, and devices disclosed herein generally describe barcoded oligonucleotides, which can be bound to a bead, such as a gel bead, useful for characterizing one or more analytes including, for example, protein (e.g., cell surface or intracellular proteins), genomic DNA, and RNA (e.g., mRNA or CRISPR guide RNAs). Also described herein, are barcoded labelling agents and oligonucleotide molecules useful for “tagging” analytes for characterization

What is claimed: 
     
         1 . A method of analyzing nucleic acids, comprising:
 (a) providing a partition comprising:
 (1) a labeled cell comprising:
 (i) a messenger ribonucleic acid (mRNA) molecule comprising a sequence encoding for a B-cell receptor or a portion thereof, 
 (ii) a labeling agent, wherein said labeling agent comprises an MHC-peptide complex comprising a peptide, an MHC molecule, and a reporter barcode molecule, wherein said reporter barcode molecule comprises a reporter barcode sequence that identifies said peptide, 
 
 (2) a plurality of partition nucleic acid barcode molecules, wherein a partition nucleic acid barcode molecule of said plurality of partition nucleic acid barcode molecules comprises a partition barcode sequence; 
   (b) using a first partition nucleic acid barcode molecule of said plurality of partition nucleic acid barcode molecules and said reporter barcode molecule to generate a first barcoded nucleic acid molecule comprising (1) said partition barcode sequence or reverse complement thereof and (2) said reporter barcode sequence or reverse complement thereof; and   (c) using a second partition nucleic acid barcode molecule of said plurality of partition nucleic acid barcode molecules and said mRNA molecule to generate a second barcoded nucleic acid molecule comprising (1) said partition barcode sequence or reverse complement thereof and (2) said sequence encoding for said B-cell receptor or the portion thereof, or a reverse complement of the said sequence encoding for said B-cell receptor or the portion thereof.   
     
     
         2 . The method of  claim 1 , wherein said MHC-peptide complex is bound to said labeled cell. 
     
     
         3 . The method of  claim 1 , wherein said method further comprises:
 (d) determining a sequence of said first barcoded nucleic acid molecule to identify (1) said partition barcode sequence or reverse complement thereof and (2) said reporter barcode sequence or reverse complement thereof; and   (e) determining a sequence of said second barcoded nucleic acid molecule to identify (1) said partition barcode sequence or reverse complement thereof and (2) said sequence encoding for said B-cell receptor or the portion thereof.   
     
     
         4 . The method of  claim 1 , wherein said sequence encoding said B-cell receptor or the portion thereof comprises a CDR1 sequence, a CDR2 sequence, and a CDR3 sequence. 
     
     
         5 . The method of  claim 1 , wherein:
 said sequence encoding said B-cell receptor or the portion thereof comprises a heavy chain variable region sequence; and   said labeled cell further comprises a second messenger ribonucleic acid (mRNA) molecule comprising a second sequence encoding for said B-cell receptor or a portion thereof comprising light chain variable region sequence, wherein (c) further comprises using a third partition nucleic acid barcode molecule of said plurality of partition nucleic acid barcode molecules and said second mRNA molecule to generate a third barcoded nucleic acid molecule comprising (1) said partition barcode sequence or reverse complement thereof and (2) said second sequence encoding for said B-cell receptor or the portion thereof, or a reverse complement of the said sequence encoding for said B-cell receptor or the portion thereof.   
     
     
         6 . The method of  claim 5 , wherein said heavy chain variable region comprises a heavy chain CDR1 sequence, a heavy chain CDR2 sequence, and a heavy chain CDR3 sequence; and wherein the said light chain variable region comprises a light chain CDR1 sequence, a light chain CDR2 sequence, and a light chain CDR3 sequence. 
     
     
         7 . The method of  claim 6 , wherein the method further comprises:
 (d) determining a sequence of said first barcoded nucleic acid molecule to identify (1) said partition barcode sequence or reverse complement thereof and (2) said reporter barcode sequence or reverse complement thereof;   (e) determining a sequence of said second barcoded nucleic acid molecule to identify (1) said partition barcode sequence or reverse complement thereof and (2) said heavy chain CDR1 sequence, said heavy chain CDR2 sequence, or said heavy chain CDR3 sequence; and   (f) determining a sequence of said third barcoded nucleic acid molecule to identify (1) said partition barcode sequence or reverse complement thereof and (2) said light chain CDR1 sequence, said light chain CDR2 sequence, or said light chain CDR3 sequence.   
     
     
         8 . The method of  claim 1 , wherein said partition is an aqueous droplet in an emulsion. 
     
     
         9 . The method of  claim 1 , wherein said partition is a well. 
     
     
         10 . The method of  claim 1 , wherein said partition comprises a support. 
     
     
         11 . The method of  claim 10 , wherein said plurality of partition nucleic acid barcode molecules are attached to said support. 
     
     
         12 . The method of  claim 11 , wherein said support is a bead. 
     
     
         13 . The method of  claim 12 , wherein said method further comprises releasing said plurality of partition nucleic acid barcode molecules from said support. 
     
     
         14 . The method of  claim 12 , wherein said bead is a gel bead. 
     
     
         15 . The method of  claim 1 , wherein the labeled cell is a B cell. 
     
     
         16 . A method of analyzing nucleic acids, comprising:
 (a) providing a partition comprising:
 (1) a labeled cell comprising:
 (i) a messenger ribonucleic acid (mRNA) molecule comprising a sequence encoding for a B-cell receptor or a portion thereof, 
 (ii) a labeling agent, wherein said labeling agent comprises a T-cell receptor molecule and a reporter barcode molecule, wherein said reporter barcode molecule comprises a reporter barcode sequence that identifies said T-cell receptor molecule, 
 
 (2) a plurality of partition nucleic acid barcode molecules, wherein a partition nucleic acid barcode molecule of said plurality of partition nucleic acid barcode molecules comprises a partition barcode sequence; 
   (b) using a first partition nucleic acid barcode molecule of said plurality of partition nucleic acid barcode molecules and said reporter barcode molecule to generate a first barcoded nucleic acid molecule comprising (1) said partition barcode sequence or reverse complement thereof and (2) said reporter barcode sequence or reverse complement thereof; and   (c) using a second partition nucleic acid barcode molecule of said plurality of partition nucleic acid barcode molecules and said mRNA molecule to generate a second barcoded nucleic acid molecule comprising (1) said partition barcode sequence or reverse complement thereof and (2) said sequence encoding for said B-cell receptor or the portion thereof, or a reverse complement of the said sequence encoding for said B-cell receptor or the portion thereof.   
     
     
         17 . The method of  claim 16 , wherein said T-cell receptor is bound to said labeled cell. 
     
     
         18 . The method of  claim 16 , wherein said method further comprises:
 (d) determining a sequence of said first barcoded nucleic acid molecule to identify (1) said partition barcode sequence or reverse complement thereof and (2) said reporter barcode sequence or reverse complement thereof; and   (e) determining a sequence of said second barcoded nucleic acid molecule to identify (1) said partition barcode sequence or reverse complement thereof and (2) said sequence encoding for said B-cell receptor or the portion thereof.   
     
     
         19 . The method of  claim 16 , wherein said sequence encoding said B-cell receptor or the portion thereof comprises a CDR1 sequence, a CDR2 sequence, and a CDR3 sequence. 
     
     
         20 . The method of  claim 16 , wherein:
 said sequence encoding said B-cell receptor or the portion thereof comprises a heavy chain variable region sequence; and   said labeled cell further comprises a second messenger ribonucleic acid (mRNA) molecule comprising a second sequence encoding for said B-cell receptor or a portion thereof comprising light chain variable region sequence, wherein (c) further comprises using a third partition nucleic acid barcode molecule of said plurality of partition nucleic acid barcode molecules and said second mRNA molecule to generate a third barcoded nucleic acid molecule comprising (1) said partition barcode sequence or reverse complement thereof and (2) said second sequence encoding for said B-cell receptor or the portion thereof, or a reverse complement of the said sequence encoding for said B-cell receptor or the portion thereof.   
     
     
         21 . The method of  claim 20 , wherein said heavy chain variable region comprises a heavy chain CDR1 sequence, a heavy chain CDR2 sequence, and a heavy chain CDR3 sequence; and wherein the said light chain variable region comprises a light chain CDR1 sequence, a light chain CDR2 sequence, and a light chain CDR3 sequence. 
     
     
         22 . The method of  claim 21 , wherein the method further comprises:
 (d) determining a sequence of said first barcoded nucleic acid molecule to identify (1) said partition barcode sequence or reverse complement thereof and (2) said reporter barcode sequence or reverse complement thereof;   (e) determining a sequence of said second barcoded nucleic acid molecule to identify (1) said partition barcode sequence or reverse complement thereof and (2) said heavy chain CDR1 sequence, said heavy chain CDR2 sequence, or said heavy chain CDR3 sequence; and   (f) determining a sequence of said third barcoded nucleic acid molecule to identify (1) said partition barcode sequence or reverse complement thereof and (2) said light chain CDR1 sequence, said light chain CDR2 sequence, or said light chain CDR3 sequence.   
     
     
         23 . The method of  claim 16 , wherein said partition is an aqueous droplet in an emulsion. 
     
     
         24 . The method of  claim 16 , wherein said partition is a well. 
     
     
         25 . The method of  claim 16 , wherein said partition comprises a support. 
     
     
         26 . The method of  claim 16 , wherein said plurality of partition nucleic acid barcode molecules are attached to said support. 
     
     
         27 . The method of  claim 26 , wherein said support is a bead. 
     
     
         28 . The method of  claim 27 , wherein said method further comprises releasing said plurality of partition nucleic acid barcode molecules from said support. 
     
     
         29 . The method of  claim 27 , wherein said bead is a gel bead. 
     
     
         30 . The method of  claim 16 , wherein the labeled cell is a B cell.