Functionalized gel beads

Patent metadata

Field	Value
Publication number	US-10876147-B2
Application number	US-201916680343-A
Country	US
Kind code	B2
Filing date	Nov 11, 2019
Priority date	Nov 15, 2017
Publication date	Dec 29, 2020
Grant date	Dec 29, 2020

Abstract

The present disclosure provides methods of generating supports (e.g., beads) comprising barcode molecules coupled thereto. A barcode molecule coupled to a support may comprise a barcode sequence and a functional sequence. A barcode molecule may be generated using two or more ligation reactions in a combinatorial fashion. A support comprising two or more different barcode molecules may be useful for analyzing or processing one or more analytes such as nucleic acid molecules, proteins, and/or perturbation agents.

First claim

What is claimed is:

1. A composition comprising a support coupled to a nucleic acid molecule comprising a sequence selected from the group consisting of SEQ ID NO: 1-163.

2. The composition of claim 1 , wherein said nucleic acid molecule further comprises a nucleic acid barcode sequence.

3. The composition of claim 1 , wherein said sequence is SEQ ID NO: 155.

4. The composition of claim 1 , wherein said sequence is SEQ ID NO: 156.

5. The composition of claim 1 , wherein said support is a bead.

6. The composition of claim 5 , wherein said bead is a gel bead.

7. The composition of claim 1 , wherein said nucleic acid molecule is coupled to said support via a labile moiety.

8. The composition of claim 7 , wherein said labile moiety is a disulfide bond.

9. The composition of claim 1 , wherein said support comprises a labile moiety.

10. The composition of claim 9 , wherein said labile moiety is a disulfide bond.

11. The composition of claim 1 , wherein said support is coupled to an additional nucleic acid molecule.

12. The composition of claim 11 , wherein said additional nucleic acid molecule comprises an additional sequence selected from the group consisting of SEQ ID NO: 1-163.

13. The composition of claim 12 , wherein said sequence is different than said additional sequence.

14. The composition of claim 13 , wherein said sequence is SEQ ID NO: 155 and said additional sequence is SEQ ID NO: 156.

15. The composition of claim 11 , wherein said nucleic acid molecule comprises a first nucleic acid barcode sequence and said additional nucleic acid molecule comprises a second nucleic acid barcode sequence.

16. The composition of claim 15 , wherein said first nucleic acid barcode sequence and said second nucleic acid barcode sequence are identical.

17. The composition of claim 15 , wherein said first nucleic acid barcode sequence and said second nucleic acid barcode sequence are different.

18. The composition of claim 15 , wherein said first nucleic acid barcode sequence and said second nucleic acid barcode sequence comprise identical barcode sequence segments.

19. The composition of claim 11 , wherein said nucleic acid molecule is free of an exonuclease-resistant bond.

20. The composition of claim 11 , wherein said additional nucleic acid molecule comprises an exonuclease-resistant bond.

21. The composition of claim 20 , wherein said exonuclease-resistant bond is a phosphorothioate bond.

22. The composition of claim 20 , wherein said exonuclease-resistant bond is disposed at an end of said additional nucleic acid molecule.

23. The composition of claim 1 , wherein said nucleic acid molecule comprises a unique molecular identifier sequence.

24. The composition of claim 1 , further comprising an additional nucleic acid molecule that is different from said nucleic acid molecule.

25. The composition of claim 24 , wherein said additional nucleic acid molecule comprises an additional sequence selected from the group consisting of SEQ ID NO: 1-163.

26. The composition of claim 24 , wherein said nucleic acid molecule comprises SEQ ID NO: 155 and said additional nucleic acid molecule comprises SEQ ID NO: 156.

27. The composition of claim 1 , wherein said nucleic acid molecule is releasably coupled to said support.

28. The composition of claim 7 , wherein said labile moiety comprises a restriction recognition site.

29. The composition of claim 7 , wherein said labile moiety is chemically-labile, thermally-labile, enzymatically-labile, labile via a change in pH, or photo-labile.

30. The composition of claim 7 , wherein said labile moiety is selected from the group consisting of an ester linkage, a vicinal diol linkage, a Diels-Alder linkage, a sulfone linkage, a silyl ether linkage, a glycosidic linkage, a peptide linkage, and a phosphodiester linkage.

31. The composition of claim 1 , wherein said support is a degradable support.

32. The composition of claim 31 , wherein said degradable support is degradable chemically, thermally, or via a change in pH.

33. The composition of claim 1 , wherein said nucleic acid molecule further comprises a functional sequence.

34. The composition of claim 33 , wherein said functional sequence is partial read sequence or a sequence for attachment to a sequencing flow cell.

35. The composition of claim 24 , wherein said additional nucleic acid molecule comprises a poly-thymine (poly-T) sequence.

36. The composition of claim 1 , further comprising a plurality of partitions, wherein a partition of said plurality of partitions comprises said support.

37. The composition of claim 35 , wherein said partition is a well.

38. The composition of claim 35 , wherein said partition is a droplet.

39. The composition of claim 2 , wherein said nucleic acid barcode sequence comprises between 6 and 20 nucleotides.

40. The composition of claim 1 , wherein said nucleic acid molecule comprises a uracil base.

41. The composition of claim 1 , wherein said support further comprises 1000 or more nucleic acid molecules.

Assignees

• 10X Genomics Inc

Inventors

• Bent Zachary
• Meer Elliott
• Riordan Daniel
• Ryvkin Paul
• Srinivas Niranjan
• Terry Jessica

Classifications

• C12Q1/6811

  Selection methods for production or design of target specific oligonucleotides or binding molecules · CPC title

• C12Q1/6834

  Enzymatic or biochemical coupling of nucleic acids to a solid phase · CPC title

• C40B20/04

  Identifying library members by means of a tag, label, or other readable or detectable entity associated with the library members, e.g. decoding processes · CPC title

• C12Q1/6806 Primary

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

• G06K19/06093

  the marking being constructed out of a plurality of similar markings, e.g. a plurality of barcodes randomly oriented on an object · CPC title