Methods and systems for processing polynucleotides

• US 10,400,280 B2
• Filed: 12/21/2018
• Issued: 09/03/2019
• Est. Priority Date: 08/14/2012
• Status: Active Grant

• Current Assignee: 10X Genomics Incorporated
• Original Assignee: 10X Genomics Incorporated
• Inventors: Hindson, Benjamin, Hindson, Christopher, Schnall-Levin, Michael, Ness, Kevin, Jarosz, Mirna, Saxonov, Serge, Hardenbol, Paul
• Primary Examiner(s): Thomas, David C
• Application Number:
  US16/231,185
• Publication Number:
  US 20190177788A1
• Time in Patent Office: 256 Days
• CPC Class Codes: C12Q 1/6804 Nucleic acid analysis using immunogens immunoassay G01N33/53
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6855 Ligating adaptors
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: C12Q 2525/185 incorporating bases where the precise position of the bases in the nucleic acid string is important
• CPC Class Codes: C12Q 2527/125 Specific component of sample, medium or buffer
• CPC Class Codes: C12Q 2531/10 the purpose being amplify/increase the copy number of target nucleic acid
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2563/149 Particles, e.g. beads
• CPC Class Codes: C12Q 2563/159 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 Class Codes: C12Q 2563/179 the label being a nucleic acid
• CPC Class Codes: C12Q 2565/519 characterised by the capture moiety being a single stranded oligonucleotide
• CPC Class Codes: C12Q 2565/629 being a microfluidic device

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.

Claims

1. A method of single cell gene expression analysis, comprising:

(a) providing a plurality of beads, wherein a bead of said plurality of beads comprises a plurality of nucleic acid barcode molecules each comprising;

(i) a common barcode sequence configured to identify a cellular origin of mRNA molecules; and

(ii) an identification barcode sequence configured to quantify mRNA molecules;

(b) partitioning a plurality of cells and said plurality of beads in a system comprising a plurality of partitions, wherein said system comprises 1,000 occupied partitions each comprising a single cell of said plurality of cells and a single bead of said plurality of beads, wherein different occupied partitions have different common barcode sequences;

(c) releasing mRNA molecules from single cells of said 1,000 occupied partitions;

(d) using said plurality of nucleic acid barcode molecules to generate a plurality of barcoded nucleic acid molecules from the mRNA molecules released in (c), wherein a barcoded nucleic acid molecule of said plurality of barcoded nucleic acid molecules comprises;

(i) a sequence of an mRNA molecule of said released mRNA molecules, or a reverse complement of said sequence of said mRNA molecule; and

(ii) a sequence of a nucleic acid barcode molecule of said plurality of nucleic acid barcode molecules, including said common barcode sequence and said identification barcode sequence, or a reverse complement of said sequence of said nucleic acid barcode molecule;

(e) determining sequences of said plurality of barcoded nucleic acid molecules;

(f) using common barcode sequences from said sequences determined in (e) to identify cellular origins of said plurality of barcoded nucleic acid molecules, thereby identifying cellular origins of said mRNA molecules from which said plurality of barcoded nucleic acid molecules were generated; and

(g) using identification barcode sequences from said sequences determined in (e) to identify molecular origins of said plurality of barcoded nucleic acid molecules, thereby quantifying said mRNA molecules from which said plurality of barcoded nucleic acid molecules were generated.

2. The method of claim 1, wherein said system comprises 5,000 occupied partitions.

3. The method of claim 1, wherein said system comprises 10,000 occupied partitions.

4. The method of claim 1, wherein no more than 5% of said 1,000 occupied partitions have more than one cell.

5. The method of claim 1, wherein no more than 1% of said 1,000 occupied partitions have more than one cell.

6. The method of claim 1, wherein no more than 5% of said 1,000 occupied partitions have more than one bead.

7. The method of claim 1, wherein no more than 1% of said 1,000 occupied partitions have more than one bead.

8. The method of claim 1, wherein at least 10% of said plurality of partitions are occupied partitions.

9. The method of claim 1, wherein at least 50% of said plurality of partitions are occupied partitions.

10. The method of claim 1, wherein said releasing in (c) comprises lysing said single cells of said 1,000 occupied partitions.

11. The method of claim 1, wherein said nucleic acid barcode molecule of said plurality of nucleic acid barcode molecules comprises a poly-T sequence for capturing mRNA molecules, and (d) comprises hybridizing said poly-T sequence to a poly-A tail of said mRNA molecule of said released mRNA molecules.

12. The method of claim 11, wherein (d) comprises performing a reverse transcription reaction using said nucleic acid barcode molecule as a primer, thereby generating a complementary deoxyribonucleic acid (cDNA) molecule that incorporates at least a portion of said nucleic acid barcode molecule.

13. The method of claim 1, wherein (d) comprises ligating said nucleic acid barcode molecule to said mRNA molecule.

14. The method of claim 1, further comprising pooling barcoded nucleic acid molecules derived from different cells prior to (e).

15. The method of claim 14, wherein (d) further comprises performing two or more polymerase chain reactions (PCR) subsequent to said pooling.

16. The method of claim 1, wherein (e) comprises determining said sequences of said plurality of barcoded nucleic acid molecules using a sequencer.

17. The method of claim 1, wherein (e) comprises determining said sequences of said plurality of barcoded nucleic acid molecules using an array.

18. The method of claim 1, wherein said plurality of nucleic acid barcode molecules comprises 1,000,000 different common barcode sequences.

19. The method of claim 1, wherein said plurality of nucleic acid barcode molecules comprises 10,000,000 different common barcode sequences.

20. The method of claim 1, wherein said plurality of nucleic acid barcode molecules comprises common barcode sequences that are modular sequences.

21. The method of claim 1, wherein said plurality of nucleic acid barcode molecules comprises 100,000 different identification barcode sequences.

22. The method of claim 1, wherein said plurality of nucleic acid barcode molecules comprises 1,000,000 different identification barcode sequences.

23. The method of claim 1, wherein said plurality of nucleic acid barcode molecules comprises identification barcode sequences that are random sequences.

24. The method of claim 1, wherein said system is a microwell array and said plurality of partitions is a plurality of wells.

25. The method of claim 24, wherein said microwell array comprises 100,000 wells.

26. The method of claim 24, wherein a well of said microwell array has a volume of less than about 5 nanoliters (nL).

27. The method of claim 1, wherein said system is a droplet microfluidic system and said plurality of partitions is a plurality of droplets.

28. The method of claim 1, wherein said plurality of beads is a plurality of gel beads.

29. The method of claim 1, wherein said plurality of beads is a plurality of magnetic beads.

30. The method of claim 1, wherein an occupied partition of said 1,000 occupied partitions comprises an antibody.

31. The method of claim 24, wherein said microwell array further comprises an inlet port in fluid communication with said plurality of wells.

32. The method of claim 31, wherein said microwell array further comprises an outlet port in fluid communication with said plurality of wells.

33. The method of claim 32, wherein said partitioning comprises flowing a solution from said plurality of wells via said outlet port.

34. The method of claim 31, wherein said partitioning comprises flowing said plurality of cells to said plurality of wells via said inlet port.

35. The method of claim 24, wherein said plurality of wells are hexagonal in cross- sectional dimension.

36. The method of claim 1, wherein said plurality of nucleic acid barcode molecules are releasably attached to said plurality of beads.

37. The method of claim 1, wherein said plurality of nucleic acid barcode molecules are attached to said plurality of beads via one or more linkers.

38. The method of claim 37, wherein said one or more linkers comprise a disulfide moiety.

39. The method of claim 1, wherein said plurality of nucleic acid barcode molecules each comprises a primer binding sequence.

40. The method of claim 1, wherein (d) is performed in said plurality of partitions.

41. The method of claim 1, wherein (d) is performed outside said plurality of partitions.

42. The method of claim 41, wherein, prior to (d), contents of said plurality of partitions are pooled.

43. The method of claim 1, wherein said mRNA molecule of said released mRNA molecules is hybridized to said nucleic acid barcode molecule of said plurality of nucleic acid barcode molecules, thereby attaching said mRNA molecule to said bead, and said bead is removed from said partition prior to (d).

44. The method of claim 43, wherein said nucleic acid barcode molecule comprises a poly-T sequence, and wherein a poly-A sequence of said mRNA molecule is hybridized to said poly-T sequence, thereby attaching said mRNA molecule to said bead.

45. The method of claim 41, wherein a mRNA molecule of said mRNA molecules is released from a single cell of said single cells in a partition of said 1,000 occupied partitions during said partitioning in (b).

46. The method of claim 41, wherein a mRNA molecule of said mRNA molecules is released from a single cell of said single cells in a partition of said 1,000 occupied partitions subsequent to said partitioning in (b).