Methods and systems for droplet-based single cell barcoding

• US 10,428,326 B2
• Filed: 02/02/2018
• Issued: 10/01/2019
• Est. Priority Date: 01/30/2017
• Status: Active Grant

• Current Assignee: 10X Genomics Incorporated
• Original Assignee: 10X Genomics Incorporated
• Inventors: Belhocine, Kamila, Bharadwaj, Rajiv, Hindson, Christopher, Schnall-Levin, Michael, Lin, Bill, Makarewicz, Anthony, Patel, Pranav, Pfeiffer, Katherine, Price, Andrew D., Rahimi Lenji, Mohammad, Wheeler, Tobias Daniel, Yin, Yifeng
• Primary Examiner(s): Dunston, Jennifer
• Application Number:
  US15/887,947
• Publication Number:
  US 20180216162A1
• Time in Patent Office: 606 Days
• CPC Class Codes: B01L 2200/0652 Sorting or classification of particles or molecules
• CPC Class Codes: B01L 2200/0668 Trapping microscopic beads
• CPC Class Codes: B01L 2200/0673 Handling of plugs of fluid surrounded by immiscible fluid
• CPC Class Codes: B01L 2300/021 Identification, e.g. bar codes
• CPC Class Codes: B01L 3/502761 specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules investigating characteristics of particles G01N15/00
• CPC Class Codes: B01L 3/502784 specially adapted for droplet or plug flow, e.g. digital microfluidics automatic analysis using a stream of discrete samples in a tube system G01N35/08
• CPC Class Codes: C12N 15/09 Recombinant DNA-technology
• CPC Class Codes: C12N 15/1006 by means of a solid support carrier, e.g. particles, polymers
• CPC Class Codes: C12N 15/1065 Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags
• 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 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2525/179 incorporating arbitrary or random nucleotide sequences
• CPC Class Codes: C12Q 2527/156 Permeability
• CPC Class Codes: C12Q 2531/113 PCR
• 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 2565/514 characterised by the use of the arrayed oligonucleotides as identifier tags, e.g. universal addressable array, anti-tag or tag complement array
• CPC Class Codes: C12Q 2565/625 : being a nucleic acid test strip device, e.g. dipsticks, strips, tapes, CD plates
• CPC Class Codes: C12Q 2565/629 : being a microfluidic device

Abstract

Methods and systems are provided for sample preparation techniques and sequencing of macromolecular constituents of cells and other biological materials.

Claims

1. A method for processing or analyzing one or more components from a cellular sample, comprising:

(a) providing a plurality of cell beads and a plurality of barcode beads, wherein (i) a cell bead of said plurality of cell beads comprises a cell, wherein said cell comprises a nucleic acid molecule in an interior region of said cell, and (ii) a barcode bead of said plurality of barcode beads comprises a plurality of nucleic acid barcode molecules, wherein nucleic acid barcode molecules of said plurality of nucleic acid barcode molecules comprise a common barcode sequence;

(b) subjecting said plurality of cell beads to bulk processing, which bulk processing comprises;

(i) rendering said nucleic acid molecule accessible to a species external to said interior region and (ii) denaturing said nucleic acid molecule to generate a denatured nucleic acid molecule in said cell bead; and

(c) partitioning said plurality of cell beads and said plurality of barcode beads into a plurality of partitions, wherein upon partitioning, a partition of said plurality of partitions comprises said cell bead and said barcode bead.

2. The method of claim 1, further comprising performing one or more reactions on said denatured nucleic acid molecule.

3. The method of claim 2, wherein said one or more reactions comprise a reaction selected from the group consisting of nucleic acid extension, nucleic acid amplification, nucleic acid ligation, reverse transcription, and a combination thereof.

4. The method of claim 2, wherein said one or more reactions are performed in said partition.

5. The method of claim 1, further comprising using a nucleic acid barcode molecule from said plurality of nucleic acid barcode molecules and said denatured nucleic acid molecule or a derivative thereof to generate a barcoded nucleic acid molecule comprising a first sequence corresponding to said denatured nucleic acid molecule and a second sequence corresponding to said common barcode sequence.

6. The method of claim 5, further comprising subjecting said barcoded nucleic acid molecule or derivative thereof to sequencing.

7. The method of claim 1, wherein said cell bead is subjected to conditions sufficient to lyse said cell to render said nucleic acid molecule accessible to a species external to said interior region.

8. The method of claim 7, wherein said conditions sufficient to lyse said cell comprise contacting said cell beads with a lysis agent.

9. The method of claim 1, wherein said barcode bead is a gel bead.

10. The method of claim 1, wherein said plurality of partitions is a plurality of droplets.

11. The method of claim 1, wherein said plurality of partitions is a plurality of wells.

12. The method of claim 1, wherein said nucleic acid barcode molecules of said plurality of nucleic acid barcode molecules are attached to said barcode bead.

13. The method of claim 1, wherein said nucleic acid molecule is a genomic deoxyribonucleic acid molecule.

14. The method of claim 5, further comprising recovering said barcoded nucleic acid molecule or said derivative thereof from said partition.

15. The method of claim 9, wherein said gel bead is degradable upon application of a stimulus.

16. The method of claim 15, further comprising degrading said gel bead upon application of said stimulus.

17. The method of claim 15, wherein said stimulus is a chemical or biological stimulus, and wherein said partition comprises said stimulus.

18. The method of claim 15, wherein said stimulus is a reducing agent.

19. The method of claim 1, wherein said cell bead is degradable upon application of a stimulus.

20. The method of claim 19, wherein said stimulus is a chemical or biological stimulus, and wherein said partition comprises said stimulus.

21. The method of claim 19, wherein said stimulus is a reducing agent.

22. The method of claim 6, further comprising subjecting said barcoded nucleic acid molecule or derivative thereof to one or more reactions prior to said sequencing.

23. The method of claim 22, further comprising recovering said barcoded nucleic acid molecule or derivative thereof from said partition and subsequently performing said one or more reactions.

24. The method of claim 22, wherein said one or more reactions comprises adding a functional sequence to said barcoded nucleic acid molecule or derivative thereof, which functional sequence permits attachment of said barcoded nucleic acid molecule or derivative thereof to a flow cell of a sequencer during said sequencing.

25. The method of claim 7, wherein said conditions sufficient to lyse said cell comprise exposing said cell beads to light.

26. The method of claim 1, further comprising:

prior to (a), partitioning a plurality of cells comprising said cell and polymer or gel precursors into a plurality of first partitions; and

subjecting said plurality of first partitions to conditions sufficient to polymerize or crosslink said polymer or gel precursors in said plurality of first partitions to generate said plurality of cell beads.

27. The method of claim 26, further comprising recovering said plurality of cell beads from said plurality of first partitions.

28. The method of claim 1, wherein said plurality of nucleic acid barcode molecules is a plurality of double-stranded nucleic acid barcode molecules.

29. The method of claim 28, further comprising performing one or more nucleic acid extension reactions on said denatured nucleic acid molecule to generate a double-stranded nucleic acid molecule comprising a sequence corresponding to said denatured nucleic acid molecule and ligating said nucleic acid barcode molecule to said double-stranded nucleic acid molecule to generate a barcoded nucleic acid molecule.

30. The method of claim 29, wherein said one or more nucleic acid extension reactions comprise:

(i) annealing a first primer to said denatured nucleic acid molecule and performing a first nucleic acid extension reaction in the presence of uracil to generate a first nucleic acid extension product comprising a uracil-containing moiety;

(ii) excising said uracil-containing moiety to generate a nick in said first nucleic acid extension product;

(iii) performing a second nucleic acid extension reaction on said first nucleic acid extension product comprising said nick to generate a plurality of single-stranded nucleic acid fragments;

(iv) annealing a second primer to a single-stranded nucleic acid fragment of said plurality of single-stranded nucleic acid fragments; and

(v) performing a third nucleic acid extension reaction to generate a second nucleic acid extension product.

31. The method of claim 30, wherein said first primer or said second primer comprises a random primer sequence.

32. The method of claim 30, wherein said second nucleic acid extension reaction is completed with use of a polymerase having strand displacement activity.

33. The method of claim 30, wherein said second nucleic acid extension reaction is a primer-independent extension process.

34. The method of claim 1, wherein a nucleic acid barcode molecule of said plurality of nucleic acid barcode molecules comprises one or more functional sequences.

35. The method of claim 34, wherein said one or more functional sequences are selected from the group consisting of:

a unique molecular index (UMI), a target-specific primer sequence, a random primer sequence, a sequencing primer sequence, and a sequence configured to attach to the flow cell of a sequencer.

36. The method of claim 1, wherein said bulk processing comprises contacting said plurality of cell beads with a chemical agent.

37. The method of claim 36, wherein said chemical agent is sodium hydroxide.