Methods and systems for processing polynucleotides

Patent metadata

Field	Value
Publication number	US-10760124-B2
Application number	US-201715832547-A
Country	US
Kind code	B2
Filing date	Dec 5, 2017
Priority date	Jun 26, 2014
Publication date	Sep 1, 2020
Grant date	Sep 1, 2020

Abstract

The present disclosure provides compositions, methods, systems, and devices for polynucleotide processing. Such polynucleotide processing may be useful for a variety of applications, including polynucleotide sequencing.

First claim

What is claimed is:

1. A composition comprising: a support comprising a nucleic acid barcode molecule coupled thereto, wherein said nucleic acid barcode molecule comprises (i) a barcode sequence, and (ii) a template switching sequence, wherein said template switching sequence of said nucleic acid barcode molecule is configured to capture a complementary deoxyribonucleic acid (cDNA) molecule from solution and facilitate a template switching reaction of said cDNA molecule onto said nucleic acid barcode molecule as a template, which cDNA molecule is derived from a ribonucleic acid (RNA) molecule.

2. The composition of claim 1 , wherein said template switching sequence is a 3′ terminal template switching sequence.

3. The composition of claim 1 , wherein said nucleic acid barcode molecule further comprises a primer or primer binding sequence.

4. The composition of claim 3 , wherein said primer or primer binding sequence is a sequencing primer or sequencing primer binding sequence.

5. The composition of claim 1 , wherein said template switching sequence comprises a poly-nucleotide sequence at a 3′ terminus.

6. The composition of claim 1 , wherein said template switching sequence comprises a poly-guanine (polyG) sequence at said 3′ terminus of said nucleic acid barcode molecule.

7. The composition of claim 6 , wherein said polyG sequence comprises a ribonucleic acid base.

8. The composition of claim 1 , wherein said template switching sequence is between about 6 nucleotides and about 20 nucleotides in length.

9. A composition comprising: a support comprising a plurality of nucleic acid barcode molecules coupled thereto, wherein a nucleic acid barcode molecule of said plurality of nucleic acid barcode molecules comprises (i) a barcode sequence, and (ii) a template switching sequence, wherein said template switching sequence of said nucleic acid barcode molecule is configured to capture a complementary deoxyribonucleic acid (cDNA) molecule from solution and facilitate a template switching reaction of said cDNA molecule onto said nucleic acid barcode molecule as a template, which cDNA molecule is derived from a ribonucleic acid (RNA) molecule.

10. The composition of claim 9 , wherein said nucleic acid barcode molecule further comprises a unique molecular sequence that varies across individual molecules of said plurality of nucleic acid barcode molecules.

11. The composition of claim 10 , wherein said unique molecular sequence is a random N-mer.

12. The composition of claim 9 , wherein said plurality of nucleic acid barcode molecules comprises 100,000 nucleic acid barcode molecules.

13. The composition of claim 9 , wherein said plurality of nucleic acid barcode molecules comprises 1,000,000 nucleic acid barcode molecules.

14. The composition of claim 1 , wherein said nucleic acid barcode molecule is covalently linked to said support.

15. The composition of claim 14 , wherein said nucleic acid barcode molecule is coupled to said support through a disulfide linker.

16. The composition of claim 1 , wherein said support comprises a cystamine or a modified cystamine.

17. The composition of claim 1 , wherein said support is covalently linked to said plurality of nucleic acid barcode molecules through a cleavable linkage.

18. The composition of claim 17 , wherein said cleavable linkage is one or more members selected from the group consisting of a chemically cleavable linkage, a photocleavable linkage, and a thermally cleavable linkage.

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

20. The composition of claim 19 , wherein said bead is a gel bead.

21. The composition of claim 20 , wherein said gel bead is degradable upon application of a stimulus.

22. The composition of claim 21 , wherein said stimulus is a chemical stimulus or a photo stimulus.

23. The composition of claim 21 , wherein said gel bead comprises cross-links and wherein said stimulus is a chemical stimulus that cleaves said cross-links.

24. The composition of claim 20 , wherein said gel bead comprises disulfide cross-links.

25. The composition of claim 20 , wherein said nucleic acid barcode molecule is coupled to said gel bead through a disulfide linker.

26. A composition comprising: a gel bead comprising 100,000 nucleic acid barcode molecules releasably attached thereto, wherein a nucleic acid barcode molecule of said 100,000 nucleic acid barcode molecules comprises (i) a primer or primer binding sequence, (ii) a barcode sequence, (iii) a random N-mer sequence, and (iv) a template switching sequence configured to facilitate a template switching reaction of a complementary deoxyribonucleic acid (cDNA) molecule derived from a ribonucleic acid (RNA) molecule.

27. The composition of claim 26 , wherein said template switching sequence comprises a 3′ terminal polyG sequence, wherein said polyG sequence comprises a ribonucleic acid base.

28. The composition of claim 26 , wherein said gel bead comprises disulfide cross-links.

29. The composition of claim 26 , wherein said nucleic acid barcode molecule is coupled to said gel bead through a disulfide linker.

30. The composition of claim 26 , further comprising a plurality of gel beads, including said gel bead, wherein said plurality of gel beads comprise a plurality of barcode sequences, including said barcode sequence, wherein said barcode sequence is different than other barcode sequences of other gel beads of said plurality of gel beads.

31. The composition of claim 9 , wherein said template switching sequence is a 3 ′ terminal template switching sequence.

32. The composition of claim 9 , wherein said nucleic acid barcode molecule further comprises a primer or primer binding sequence.

33. The composition of claim 32 , wherein said primer or primer binding sequence is a sequencing primer or sequencing primer binding sequence.

34. The composition of claim 9 , wherein said template switching sequence comprises a poly-nucleotide sequence at a 3′ terminus.

35. The composition of claim 9 , wherein said template switching sequence comprises a poly-guanine (polyG) sequence at said 3′ terminus of said nucleic acid barcode molecule.

36. The composition of claim 35 , wherein said polyG sequence comprises a ribonucleic acid base.

37. The composition of claim 9 , wherein said template switching sequence is between about 6 nucleotides and about 20 nucleotides in length.

38. The composition of claim 9 , wherein said nucleic acid barcode molecule is covalently linked to said support.

39. The composition of claim 38 , wherein said nucleic acid barcode molecule is coupled to said support through a disulfide linker.

40. The composition of claim 9 , wherein said support comprises a cystamine or a modified cystamine.

41. The composition of claim 9 , wherein said support is covalently linked to said plurality of nucleic acid barcode molecules through a cleavable linkage.

42. The composition of claim 41 , wherein said cleavable linkage is one or more members selected from the group consisting of a chemically cleavable linkage, a photocleavable linkage, and a thermally cleavable linkage.

Assignees

• 10X Genomics Inc

Inventors

• Hindson Benjamin
• Hindson Christopher
• Schnall-Levin Michael
• Ness Kevin
• Jarosz Mirna
• Saxonov Serge
• Hardenbol Paul
• Bharadwaj Rajiv
• Zheng Xinying
• Belgrader Phillip

Classifications

• C12Q1/683 Primary

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

• C12Q2537/149

  Sequential reactions · CPC title

• C12Q1/6804

  Nucleic acid analysis using immunogens (immunoassay G01N33/53) · CPC title

• C12Q1/6874 Primary

  involving nucleic acid arrays, e.g. sequencing by hybridisation · CPC title