Methods and systems for processing polynucleotides

Patent metadata

Field	Value
Publication number	US-10273541-B2
Application number	US-201816052431-A
Country	US
Kind code	B2
Filing date	Aug 1, 2018
Priority date	Aug 14, 2012
Publication date	Apr 30, 2019
Grant date	Apr 30, 2019

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 method for processing messenger ribonucleic acid (mRNA) molecules from a single cell, comprising:
(a) partitioning a plurality of cells and a plurality of beads in a microwell array comprising a plurality of wells, wherein a well of said plurality of wells comprises said single cell from said plurality of cells and a single bead from said plurality of beads, and wherein said single bead comprises nucleic acid barcode molecules each comprising a common barcode sequence;
(b) in said well comprising said single cell and said single bead, releasing messenger ribonucleic acid (mRNA) molecules from said single cell, wherein upon release from said single cell, said released mRNA molecules attach to said nucleic acid barcode molecules;
(c) subjecting said released mRNA molecules attached to said nucleic acid barcode molecules to reverse transcription to yield complementary deoxyribonucleic acid (cDNA) molecules each comprising said common barcode sequence or a complement thereof; and
(d) subjecting said cDNA molecules to one or more reactions to generate a set of nucleic acid molecules for nucleic acid sequencing.

2. The method of claim 1 , wherein (c) is performed in said well comprising said single cell and said single bead, and wherein subsequent to (c), said cDNA molecules, or derivatives thereof, are removed from said well.

3. The method of claim 1 , wherein, prior to (c), said released mRNA molecules attached to said nucleic acid barcode molecules are removed from said well comprising said single cell and said single bead.

4. The method of claim 1 , wherein each of said nucleic acid barcode molecules comprises a universal primer sequence.

5. The method of claim 1 , wherein said single bead is a single magnetic bead.

6. The method of claim 5 , wherein said nucleic acid barcode molecules are attached to said single magnetic bead.

7. The method of claim 6 , wherein prior to (c), said single magnetic bead comprising said released mRNA molecules attached to said nucleic acid barcode molecules are removed from said well comprising said single cell and said single bead.

8. The method of claim 7 , wherein said single magnetic bead is removed using a magnetic field.

9. The method of claim 1 , wherein each of said nucleic acid barcode molecules comprises a sequence for priming the synthesis of cDNA.

10. The method of claim 1 , wherein said plurality of cells comprises at least 100 cells.

11. The method of claim 1 , wherein said plurality of cells comprises at least 1,000 cells.

12. The method of claim 1 , wherein said plurality of cells comprises at least 10,000 cells.

13. The method of claim 1 , wherein said released mRNA molecules attach to said nucleic acid barcode molecules by hybridization.

14. The method of claim 1 , wherein said plurality of beads comprises a plurality of nucleic acid barcode molecules comprising barcode sequences that are different across said plurality of beads.

15. The method of claim 1 , further comprising, prior to (c), (i) pooling said released mRNA molecules attached to said nucleic acid barcode molecules and (ii) performing said one or more reactions in bulk.

16. The method of claim 1 , wherein said one or more reactions comprise nucleic acid amplification that generates amplified products from said plurality of cDNA molecules.

17. The method of claim 16 , wherein said nucleic acid amplification adds functional sequences to said amplified products, wherein said functional sequences permit attachment of said amplified products to a flow cell of a sequencer for said nucleic acid sequencing.

18. The method of claim 16 , further comprising ligating functional sequences to said amplified products, wherein said functional sequences permit attachment of said amplified products to a flow cell of a sequencer for said nucleic acid sequencing.

19. The method of claim 16 , wherein said nucleic acid amplification is polymerase chain reaction.

20. The method of claim 1 , wherein said one or more reactions comprise addition of functional sequences to said plurality of cDNA molecules, wherein said functional sequences permit attachment to a flow cell of a sequencer for said nucleic acid sequencing.

21. The method of claim 1 , further comprising performing said nucleic acid sequencing on said set of nucleic acid molecules, or derivatives thereof, to generate a plurality of sequences comprising sequences corresponding to said released mRNA molecules and said common barcode sequence.

22. The method of claim 1 , wherein said plurality of beads have substantially monodisperse cross-sectional dimensions.

23. The method of claim 1 , wherein said nucleic acid barcode molecules further comprise functional sequences that facilitate sequencing of said set of nucleic acid molecules.

24. The method of claim 4 , wherein said universal primer sequence is a random N-mer.

25. The method of claim 1 , wherein said single bead is a single gel bead.

26. The method of claim 1 , wherein said microwell array comprises 100,000 wells.

27. The method of claim 1 , wherein said microwell array comprises 200,000 wells.

28. The method of claim 1 , wherein said microwell array comprises 1,000,000 wells.

29. The method of claim 1 , wherein a subset of said plurality of wells in said microwell array does not include a cell.

30. The method of claim 1 , wherein a subset of said plurality of wells in said microwell array does not include a bead.

31. The method of claim 25 , wherein said nucleic acid barcode molecules are attached to said single gel bead.

32. The method of claim 25 , wherein prior to (c), said single gel bead comprising said released mRNA molecules attached to said nucleic acid barcode molecules is removed from said well.

33. The method of claim 1 , wherein releasing said mRNA molecules from said single cell comprises lysing said single cell.

34. The method of claim 33 , wherein lysing said single cell comprises treating said single cell with a detergent.

35. The method of claim 33 , wherein lysing said single cell comprises heating said single cell.

36. The method of claim 1 , wherein said nucleic acid barcode molecules comprise a sequence complementary to said released mRNA molecules.

37. The method of claim 1 , wherein said plurality of cells is a plurality of cancer cells.

38. The method of claim 1 , wherein said nucleic acid barcode molecules comprise uracil.

39. The method of claim 1 , wherein said nucleic acid barcode molecules comprise 1,000 nucleic acid barcode molecules.

40. The method of claim 1 , wherein said nucleic acid barcode molecules comprise 100,000 nucleic acid barcode molecules.

41. The method of claim 1 , wherein said plurality of beads comprises 1,000 beads.

42. The method of claim 1 , wherein said plurality of beads comprises 100,000 beads.

43. The method of claim 1 , wherein said plurality of wells comprises 1,000 wells.

44. The method of claim 1 , wherein said plurality of wells comprises 100,000 wells.

45. The method of claim 1 , wherein said plurality of wells comprises 1,000,000 well

Assignees

• 10X Genomics Inc

Inventors

• Hindson Benjamin
• Hindson Christopher
• Schnall-Levin Michael
• Ness Kevin
• Jarosz Mirna
• Saxonov Serge
• Hardenbol Paul

Classifications

• C12Q1/6855

  Ligating adaptors · CPC title

• C12Q2521/107

  RNA dependent DNA polymerase,(i.e. reverse transcriptase) · CPC title

• C12Q1/6874 Primary

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

• C12Q2563/159

  Microreactors, e.g. emulsion PCR or sequencing, droplet PCR, microcapsules, i.e. non-liquid containers with a range of different permeability's for different reaction components · CPC title

• C12Q2563/143

  Magnetism, e.g. magnetic label · CPC title