Methods and systems for processing polynucleotides

Patent metadata

Field	Value
Publication number	US-12110549-B2
Application number	US-202217822117-A
Country	US
Kind code	B2
Filing date	Aug 24, 2022
Priority date	Dec 22, 2016
Publication date	Oct 8, 2024
Grant date	Oct 8, 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 a partition comprising:
(i) a labeled cell or a labeled organelle, wherein the labeled cell or the labeled organelle comprises a surface labeling agent, wherein the surface labeling agent is a small molecule attached to a reporter nucleic acid barcode molecule comprising a reporter barcode sequence, wherein the small molecule comprises a molecular weight of no greater than 1,000 Daltons (Da); and
(ii) a partition nucleic acid barcode molecule comprising a partition barcode sequence; and

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

2. The method of claim 1 , wherein the surface labeling agent is directly linked to the reporter nucleic acid barcode molecule.

3. The method of claim 1 , wherein the surface labeling agent is indirectly linked to the reporter nucleic acid barcode molecule.

4. The method of claim 1 , further comprising: (c) sequencing the barcoded nucleic acid molecule or a derivative or an amplification product thereof to identify ( 1 ) the reporter barcode sequence, and ( 2 ) the partition barcode sequence.

5. The method of claim 1 , wherein the labeled cell or the labeled organelle further comprises a messenger ribonucleic nucleic acid (mRNA) molecule comprising a mRNA nucleic acid sequence, and wherein:
the partition of (a) further comprises a second partition nucleic acid barcode molecule comprising the partition barcode sequence; and
(b) further comprises using the mRNA molecule and the second partition nucleic acid barcode molecule to generate a second barcoded nucleic acid molecule comprising ( 1 ) the mRNA nucleic acid sequence or a reverse complement thereof and ( 2 ) the partition barcode sequence or a reverse complement thereof.

6. The method of claim 5 , further comprising:
(c) sequencing the second barcoded nucleic acid molecule or a derivative or an amplification product thereof.

7. The method of claim 1 , wherein the labeled cell or the labeled organelle further comprises a second labeling agent comprising ( 1 ) an antibody or binding fragment thereof and ( 2 ) a second reporter nucleic acid barcode molecule comprising a second reporter barcode sequence, and wherein:
the partition of (a) further comprises a second partition nucleic acid barcode molecule comprising the partition barcode sequence; and
(b) further comprises using the second reporter nucleic acid barcode molecule and the second partition nucleic acid barcode molecule to generate a second barcoded nucleic acid molecule comprising ( 1 ) the second reporter barcode sequence or a reverse complement thereof and ( 2 ) the partition barcode sequence or a reverse complement thereof.

8. The method of claim 7 , further comprising:
(c) sequencing the second barcoded nucleic acid molecule or a derivative or an amplification product thereof.

9. The method of claim 7 , wherein the antibody or binding fragment thereof is directly attached to the second reporter nucleic acid barcode molecule.

10. The method of claim 7 , wherein the antibody or binding fragment thereof is indirectly attached to the second reporter nucleic acid barcode molecule.

11. The method of claim 1 , wherein the barcoded nucleic acid molecule is generated via one or more extension reactions.

12. The method of claim 1 , wherein the partition nucleic acid barcode molecule is coupled to a support.

13. The method of claim 12 , wherein the partition nucleic acid barcode molecule is releasably coupled to the support.

14. The method of claim 12 , wherein the support is a bead.

15. The method of claim 14 , wherein the bead is a solid bead.

16. The method of claim 15 , wherein the solid bead is a magnetic bead.

17. The method of claim 1 , wherein the partition is a well.

18. The method of claim 1 , wherein the partition is a droplet in an emulsion.

19. The method of claim 1 , wherein the surface labeling agent is attached to a surface feature of the labeled cell or the labeled organelle.

20. The method of claim 1 , wherein the method further comprises, before (b), lysing the labeled cell or the labeled organelle.

21. The method of claim 1 , wherein the small molecule is selected from the group consisting of cholesterol, tocopherol, lignoceric acid, palmitic acid, and derivatives thereof.

22. The method of claim 1 , wherein the small molecule is an organic compound.

23. The method of claim 1 , wherein the surface labelling agent is linked to the reporter nucleic acid barcode molecule via a click chemistry linker.

24. The method of claim 1 , wherein the partition nucleic acid barcode molecule comprises a unique molecular identification (UMI) sequence.

25. The method of claim 19 , wherein the surface feature is a surface protein.

26. A method of analyzing a cell or an organelle, comprising:
(a) partitioning a labeled cell or a labeled organelle and a bead comprising a plurality of partition nucleic acid barcode molecules into a partition, wherein:
(i) the labeled cell or the labeled organelle comprises ( 1 ) a surface labeling agent that is a small molecule linked to a reporter nucleic acid barcode molecule comprising a reporter nucleic acid barcode sequence, and ( 2 ) a messenger ribonucleic acid (mRNA) analyte comprising an mRNA sequence, wherein the small molecule comprises a molecular weight of no greater than 1,000 Daltons (Da), and
(ii) the plurality of partition nucleic acid barcode molecules comprises ( 1 ) a first partition nucleic acid barcode molecule comprising a partition barcode sequence, a first unique molecular identifier (UMI) sequence, and a poly-T sequence and ( 2 ) a second partition nucleic acid barcode molecule comprising the partition barcode sequence, a second unique molecular identifier (UMI) sequence, and the poly-T sequence;

(b) hybridizing the poly-T sequence of the first partition nucleic acid barcode molecule to the reporter nucleic acid barcode molecule and extending the first partition nucleic acid barcode molecule using the reporter nucleic acid barcode molecule as a template to generate a first barcoded nucleic acid molecule comprising ( 1 ) the partition barcode sequence, ( 2 ) the first UMI sequence, and ( 3 ) a reverse complement of the reporter nucleic acid barcode sequence;
(c) hybridizing the poly-T sequence of the second partition nucleic acid barcode molecule to the mRNA analyte and extending the second partition nucleic acid barcode molecule using the mRNA analyte as a template to generate a second barcoded nucleic acid molecule comprising ( 1 ) the partition barcode sequence, ( 2 ) the second UMI sequence, and ( 3 ) a reverse complement of the mRNA sequence; and
(d) sequencing (i) the first barcoded nucleic acid molecule or a derivative or an amplification product thereof, and (ii) the second barcoded nucleic acid molecule or a derivative or an amplification product thereof.

27. The method of claim 26 , wherein the partition is a droplet in an emulsion or a well.

28. The method of claim 26 , wherein the hybridizing of (b) and (c) occurs in the partition.

29. The method of claim 26 , wherein the hybridizing and the extending of (b) and (c)

Assignees

• 10X Genomics Inc

Inventors

• Belgrader Phillip
• Bent Zachary
• Bharadwaj Rajiv
• Gopalan Vijay Kumar Sreenivasa
• Harada Josephine
• Hindson Christopher
• Lenji Mohammad Rahimi
• 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