Methods and systems for processing polynucleotides

Patent metadata

Field	Value
Publication number	US-12084716-B2
Application number	US-202117473760-A
Country	US
Kind code	B2
Filing date	Sep 13, 2021
Priority date	Dec 22, 2016
Publication date	Sep 10, 2024
Grant date	Sep 10, 2024

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.

First claim

What is claimed is:

1. A method, comprising:
(a) providing:
(i) a first molecule comprising (1) a lipophilic moiety and (2) a first nucleic acid barcode molecule, wherein the first nucleic acid barcode molecule comprises a first nucleic acid barcode sequence, and
(ii) a second molecule comprising a second nucleic acid barcode molecule, wherein the second nucleic acid barcode molecule comprises a second nucleic acid barcode sequence; and

(b) using the first nucleic acid barcode molecule and the second nucleic acid barcode molecule to generate a barcoded nucleic acid molecule comprising (1) the first nucleic acid barcode sequence or a reverse complement thereof and (2) the second nucleic acid barcode sequence or a reverse complement thereof.

2. The method of claim 1 , wherein (a) comprises providing a cell comprising the first molecule.

3. The method of claim 2 , wherein the method comprises, prior to (b), partitioning the cell and the second molecule into a partition.

4. The method of claim 3 , wherein the method comprises, prior to (b) and after the partitioning, lysing the cell.

5. The method of claim 3 , further comprising:
(c) sequencing the barcoded nucleic acid molecule to identify (1) the first nucleic acid barcode sequence, and (2) the second nucleic acid barcode sequence.

6. The method of claim 1 , wherein the lipophilic moiety is an amphiphilic molecule, a cholesterol molecule, a tocopherol or derivative thereof, a steryl lipid, lignoceric acid, or palmitic acid.

7. 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, wherein a first partition nucleic acid barcode molecule of the plurality of partition nucleic acid barcode molecules comprises a partition nucleic acid barcode sequence, and a second partition nucleic acid barcode molecule of the plurality of partition nucleic acid barcode molecules comprises the partition nucleic acid barcode sequence;

(b) using the first cell nucleic acid barcode molecule and the first partition nucleic acid barcode molecule of the plurality of partition nucleic acid barcode molecules to generate a first barcoded nucleic acid molecule comprising (1) the first cell nucleic acid barcode sequence or a reverse complement thereof and (2) the partition nucleic acid barcode sequence or a reverse complement thereof; and
(c) using the second cell nucleic acid barcode molecule and the second partition nucleic acid barcode molecule of the plurality of partition nucleic acid barcode molecules to generate a second barcoded nucleic acid molecule comprising (1) the second cell nucleic acid barcode sequence or a reverse complement thereof and (2) the partition nucleic acid barcode sequence or a reverse complement thereof.

8. The method of claim 7 , 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.

9. The method of claim 7 , wherein (b) and (c) are performed in the partition.

10. The method of claim 9 , wherein the method further comprises:
(d) releasing the first barcoded nucleic acid molecule and the second barcoded nucleic acid molecule from the partition, thereby providing a released first barcoded nucleic acid molecule and a released second barcoded nucleic acid molecule; and
(e) subjecting the released first barcoded nucleic acid molecule and the released second barcoded nucleic acid molecule to one or more reactions selected from the group consisting of: a nucleic acid extension reaction, a polymerase chain reaction, and a ligation reaction.

11. The method of claim 7 , wherein the method further comprises:
(d) sequencing (i) the first barcoded nucleic acid molecule or derivative thereof, and (ii) the second barcoded nucleic acid molecule or derivative thereof.

12. The method of claim 11 , 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.

13. The method of claim 7 , wherein:
(i) 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
(ii) 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.

14. The method of claim 7 , wherein:
(i) the first cell surface labeling molecule comprises a first lipophilic moiety; and
(ii) the second cell surface labeling molecule comprises a second lipophilic moiety.

15. The method of claim 14 , wherein:
(i) 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
(ii) 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.

16. The method of claim 14 , wherein:
(i) the first lipophilic moiety is a cholesterol moiety; and
(ii) the second lipophilic moiety is a cholesterol moiety.

17. The method of claim 7 , wherein:
(i) 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 comprising a molecular weight of no greater than 1,000 Daltons (Da), a pro-body, an aptamer, a monobody, an affimer, a darpin, and a protein scaffold; and
(ii) 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 comprising a molecular weight of no greater than 1,000 Daltons (Da), a pro-body, an aptamer, a monobody, an affimer, a darpin, and a protein scaffold.

18. The method of claim 7 , wherein (b) comprises synthesizing the first barcoded nucleic acid molecule by one or more primer extension reactions and (c) comprises synthesizing the second barcoded nucleic acid molecule by one or more primer extension reactions.

19. The method of claim 7 , wherein (b) comprises synthesizing the first barcoded nucleic acid molecule by one or more ligation reactions and (c) comprises synthesizing the second barcoded nucleic acid molecule by one or more ligation reactions.

Assignees

• 10X Genomics Inc

Inventors

• Belgrader Phillip
• Bent Zachary
• Bharadwaj Rajiv
• Sreenivasa Gopalan Vijay Kumar
• Harada Josephine
• Hindson Christopher
• Rahimi Lenji Mohammad
• Lucero Michael Ybarra
• Mcdermott Geoffrey
• Meer Elliott
• Mikkelsen Tarjei Sigurd
• O'Keeffe Christopher Joachim
• Pfeiffer Katherine
• Price Andrew D
• Ryvkin Paul
• Saxonov Serge
• Stuelpnagel John R
• Terry Jessica Michele
• Wheeler Tobias Daniel
• Wu Indira
• Ziraldo Solongo Batjargal
• Boutet Stephane Claude
• Taylor Sarah
• Srinivas Niranjan

Classifications

• C12Q1/6806 Primary

  Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) · CPC title

• C12Q2525/191

  incorporating an adaptor · CPC title

• C12Q2537/143

  Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis · CPC title

• C12Q2563/179

  the label being a nucleic acid · CPC title

• C12Q1/683

  involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP] · CPC title