METHODS AND SYSTEMS FOR PROCESSING POLYNUCLEOTIDES
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.
1 - 30 . (canceled)
31 . A method of cell analysis, comprising:
(a) providing a partition comprising:
(i) a first labeled cell and a first cell surface labeling molecule, wherein the first cell surface labeling molecule comprises a first cell nucleic acid barcode molecule comprising a first cell nucleic acid barcode sequence,
(ii) a second labeled cell and a second cell surface labeling molecule, wherein the second cell surface labeling molecule comprises a second cell nucleic acid barcode molecule comprising a second cell nucleic acid barcode sequence, and
(iii) a plurality of partition nucleic acid barcode molecules attached thereto, wherein a partition nucleic acid barcode molecule of the plurality of partition nucleic acid barcode molecules comprises a partition nucleic acid barcode sequence;
(b) using the first cell nucleic acid barcode molecule and a first partition nucleic acid barcode molecule of the plurality of partition nucleic acid barcode molecules to generate a first barcoded nucleic acid molecule comprising (i) the first cell nucleic acid barcode sequence or reverse complement thereof and (ii) the partition nucleic acid barcode sequence or reverse complement thereof; and
(c) using the second cell nucleic acid barcode molecule and a second partition nucleic acid barcode molecule of the plurality of partition nucleic acid barcode molecules to generate a second barcoded nucleic acid molecule comprising (i) the second cell nucleic acid barcode sequence or reverse complement thereof and (ii) the partition nucleic acid barcode sequence or reverse complement thereof.
32 . The method of claim 31 , further comprising, prior to (a):
contacting a first cell with the first cell surface labeling molecule, thereby generating the first labeled cell; and
contacting a second cell with the second cell surface labeling molecule, thereby generating the second labeled cell.
33 . The method of claim 31 , wherein the (b) and (c) are performed in the partition.
34 . The method of claim 31 , wherein the method further comprises:
(d) releasing the first barcoded nucleic acid molecule and the second barcoded nucleic acid molecule from the partition; and
(e) subjecting the first barcoded nucleic acid molecule and the second barcoded nucleic acid molecule to one or more reactions elected from the group consisting of: a nucleic acid extension reaction, a polymerase chain reaction, and a ligation reaction.
35 . The method of claim 31 , wherein the method further comprises:
(d) sequencing (i) the first barcoded nucleic acid molecule or derivative thereof, (ii) the second barcoded nucleic acid molecule or derivative thereof.
36 . The method of claim 35 , wherein the method further comprises:
(e) identifying the first labeled cell and the second labeled cell as originating from the partition using (i) the partition nucleic acid barcode sequence of the first barcoded nucleic acid molecule or derivative thereof and (ii) the partition nucleic acid barcode sequence of the second barcoded nucleic acid molecule or derivative thereof.
37 . The method of claim 31 , wherein:
the first cell surface labeling molecule comprises a first cell membrane labeling moiety, and wherein the first cell membrane labeling moiety is selected from the group consisting of a lipid, a fluorophore, a dye, a peptide, and a nanoparticle; and
the second cell surface labeling molecule comprises a second cell membrane labeling moiety, and wherein the second cell membrane labeling moiety is selected from the group consisting of a lipid, a fluorophore, a dye, a peptide, and a nanoparticle.
38 . The method of claim 31 , wherein:
the first cell surface labeling molecule comprises a first lipophilic moiety; and
the second cell surface labeling molecule comprises a second lipophilic moiety.
39 . The method of claim 38 , wherein:
the first lipophilic moiety is selected from the group consisting of an amphiphilic molecule, a tocopherol or derivative thereof, a steryl lipid, lignoceric acid, and palmitic acid; and
the second lipophilic moiety is selected from the group consisting of an amphiphilic molecule, a tocopherol or derivative thereof, a steryl lipid, lignoceric acid, and palmitic acid.
40 . The method of claim 38 , wherein:
the first lipophilic moiety is a cholesterol moiety; and
the second lipophilic moiety is a cholesterol moiety.
41 . The method of claim 31 , wherein:
the first cell surface labeling molecule comprises a first cell binding moiety, wherein the first cell binding moiety is selected from the group consisting of an antibody, a cell surface receptor binding molecule; a receptor ligand; a small molecule; a pro-body, an aptamer, a monobody, an affimer, a darpin, and a protein scaffold; and
the second cell surface labeling molecule comprises a second cell binding moiety, wherein the second cell binding moiety is selected from the group consisting of an antibody, a cell surface receptor binding molecule; a receptor ligand; a small molecule; a pro-body, an aptamer, a monobody, an affimer, a darpin, and a protein scaffold.
42 . The method of claim 31 , wherein the first barcoded nucleic acid molecule and the second barcoded nucleic acid molecule are synthesized by one or more primer extension reactions.
43 . The method of claim 31 , wherein the first barcoded nucleic acid molecule and the second barcoded nucleic acid molecule are synthesized by one or more ligation reactions.
44 . The method of claim 31 , wherein the first barcoded nucleic acid molecule and the second barcoded nucleic acid molecule are synthesized by one or more nucleic acid amplification reactions.
45 . The method of claim 31 , wherein the plurality of partition nucleic acid barcode molecules is coupled to a support.
46 . The method of claim 45 , wherein the support is a bead.
47 . The method of claim 31 , wherein the partition is a droplet.
48 . The method of claim 31 , wherein the partition is a well.
49 . The method of claim 31 , wherein the first labeled cell comprises a first nucleic acid analyte comprising a first analyte nucleic acid sequence and the second labeled cell comprises a second nucleic acid analyte comprising a second analyte nucleic acid sequence, and wherein the method further comprises:
(d) using the first nucleic acid analyte and a third partition nucleic acid barcode molecules of the plurality of partition nucleic acid barcode molecules to generate a third barcoded nucleic acid molecule comprising (i) the first analyte nucleic acid sequence or reverse complement thereof and (ii) the partition nucleic acid barcode sequence or reverse complement thereof; and
(c) using the second nucleic acid analyte and a fourth partition nucleic acid barcode molecules of the plurality of partition nucleic acid barcode molecules to generate a fourth barcoded nucleic acid molecule comprising (i) the second analyte nucleic acid sequence or reverse complement thereof and (ii) the partition nucleic acid barcode sequence or reverse complement thereof.
50 . The method of claim 49 , wherein the first nucleic acid analyte and the second nucleic acid analyte are selected from the group consisting of a messenger ribonucleic acid molecule and a genomic deoxyribonucleic acid molecule.