Single cell analysis of transposase accessible chromatin

• US 10,400,235 B2
• Filed: 12/14/2017
• Issued: 09/03/2019
• Est. Priority Date: 05/26/2017
• Status: Active Grant

• Current Assignee: 10X Genomics Incorporated
• Original Assignee: 10X Genomics Incorporated
• Inventors: Belhocine, Kamila, McDermott, Geoffrey, Meschi, Francesca, Zheng, Xinying
• Primary Examiner(s): Gross, Christopher M
• Application Number:
  US15/842,687
• Publication Number:
  US 20180340170A1
• Time in Patent Office: 628 Days
• Field of Search: None
• 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: C12N 15/1068 Template (nucleic acid) mediated chemical library synthesis, e.g. chemical and enzymatical DNA-templated organic molecule synthesis, libraries prepared by non ribosomal polypeptide synthesis [NRPS], DNA/RNA-polymerase mediated polypeptide synthesis
• CPC Class Codes: C12N 15/1075 by coupling phenotype to genotype, not provided for in other groups of this subclass
• CPC Class Codes: C12N 15/1096 cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid

Abstract

Methods and systems for sample preparation techniques that allow amplification (e.g., whole genome amplification) and sequencing of chromatin accessible regions of single cells are provided. The methods and systems generally operate by forming or providing partitions (e.g., droplets) including a single biological particle and a single bead comprising a barcoded oligonucleotide. The preparation of barcoded next-generation sequencing libraries prepared from a single cell is facilitated by the transposon-mediated transposition and fragmentation of a target nucleic acid sequence. The methods and systems may be configured to allow the implementation of single-operation or multi-operation chemical and/or biochemical processing within the partitions.

Claims

1. A method of generating barcoded nucleic acid fragments, comprising:

(a) generating a plurality of partitions, wherein at least a subset of said plurality of partitions comprises;

(i) a single biological particle from a plurality of biological particles, wherein said single biological particle comprises template deoxyribonucleic acid (DNA) molecules and template ribonucleic acid (RNA) molecules;

(ii) a plurality of first barcode oligonucleotide molecules comprising a first barcode sequence;

(iii) a plurality of transposon end oligonucleotide molecules comprising a transposon end sequence;

(iv) a plurality of transposase molecules;

(v) a plurality of second barcode oligonucleotide molecules comprising a second barcode sequence and a capture sequence; and

(vi) a plurality of reverse transcriptase molecules;

(b) generating a plurality of template DNA fragments by subjecting said subset of said plurality of partitions to conditions sufficient to cause transposition of said transposon end oligonucleotide molecules into said template DNA molecules with the aid of a transposase-nucleic acid complex comprising a transposase molecule of said plurality of transposase molecules and a transposon end oligonucleotide molecule of said plurality of transposon end oligonucleotide molecules;

(c) generating a barcoded DNA fragment using a first barcode oligonucleotide molecule of said plurality of first barcode oligonucleotide molecules and a template DNA fragment of said plurality of template DNA fragments; and

(d) generating a barcoded complementary DNA molecule from said template RNA molecules by reverse transcription using a second barcode oligonucleotide molecule of said plurality of second barcode oligonucleotide molecules.

2. The method of claim 1, wherein said capture sequence is an oligo(dT) sequence.

3. The method of claim 1, wherein (d) comprises generating a first strand barcoded cDNA molecule and the method further comprises generating a second strand barcoded cDNA molecule.

4. The method of claim 3, wherein said plurality of partitions further comprises a plurality of template switching oligonucleotides and wherein said second strand barcoded cDNA molecule is generated using a template switching oligonucleotide of said plurality of template switching oligonucleotides.

5. The method of claim 4, wherein said template switching oligonucleotide comprises a barcode sequence.

6. The method of claim 5, wherein said barcode sequence of said template switching oligonucleotide is the same as said first barcode sequence of said first barcode oligonucleotide and said second barcode sequence of said second barcode oligonucleotide.

7. The method of claim 1, wherein said subset of said plurality of partitions are subjected to conditions sufficient to generate said transposase-nucleic acid complex using a transposase molecule of said plurality of transposase molecules and a transposon end oligonucleotide molecule of said plurality of transposon end oligonucleotide molecules.

8. The method of claim 1, wherein said transposase-nucleic acid complex is partitioned into said subset of said plurality of partitions.

9. The method of claim 1, wherein, prior to (b), said subset of said plurality of partitions are subjected to conditions sufficient to cause release of said template DNA molecules and said template RNA molecules from said single biological particle.

10. The method of claim 1, wherein, subsequent to (b), said subset of said plurality of partitions are subjected to conditions sufficient to cause release of said template DNA fragments and said template RNA molecules from said single biological particle.

11. The method of claim 1, wherein (i) said plurality of first barcode oligonucleotide molecules is attached to a first bead, said plurality of second barcode oligonucleotide molecules is attached to a second bead, and said subset of said plurality of partitions comprises said first bead and said second bead;

or (ii) said plurality of first barcode oligonucleotide molecules and said plurality of second barcode oligonucleotide molecules are attached to a bead, and said plurality of partitions comprises said bead.

12. The method of claim 11, wherein said first bead, said second bead of (i), or said bead of (ii) is a gel bead.

13. The method of claim 1, wherein said plurality of first barcode oligonucleotide molecules and said plurality of second barcode oligonucleotide molecules are releasably attached to a gel bead.

14. The method of claim 13, further comprising releasing said first barcode oligonucleotide molecule and said second barcode oligonucleotide molecule from said gel bead.

15. The method of claim 14, wherein said gel bead is depolymerized to release said first barcode oligonucleotide molecule and said second barcode oligonucleotide molecule from said gel bead.

16. The method of claim 15, wherein said subset of said plurality of partitions further comprises a reducing agent that depolymerizes said gel bead.

17. The method of claim 1, wherein said single biological particle is a single cell and wherein said plurality of biological particles is a plurality of cells.

18. The method of claim 1, wherein said single biological particle is a single nucleus and wherein said plurality of biological particles is a plurality of nuclei harvested from a plurality of cells.

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

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

21. The method of claim 1, wherein said plurality of first barcode oligonucleotide molecules is attached to a gel bead, wherein said plurality of second barcode oligonucleotide molecules is attached to a plurality of magnetic beads, and wherein said plurality of magnetic beads are embedded within said gel bead, and wherein said subset of said plurality of partitions comprises said gel bead and said plurality of magnetic beads.

22. The method of claim 21, wherein said plurality of first barcode oligonucleotide molecules is releasably attached to said gel bead.

23. The method of claim 22, further comprising releasing said first barcode oligonucleotide molecule from said gel bead and magnetic beads of said plurality of magnetic beads from said gel bead.

24. The method of claim 23, wherein said gel bead is depolymerized to release said first barcode oligonucleotide molecule from said gel bead and said magnetic beads from said gel bead.

25. The method of claim 24, wherein said subset of said plurality of partitions further comprise a reducing agent to depolymerize said gel bead.

26. The method of claim 1, wherein said barcoded DNA fragment and said barcoded cDNA molecule are generated in said subset of said plurality of partitions, and wherein said barcoded DNA fragment and said barcoded cDNA molecule are released from said subset of said plurality of partitions.

27. The method of claim 1, further comprising sequencing said barcoded DNA fragment, or a derivative thereof.

28. The method of claim 1, further comprising sequencing said barcoded cDNA molecule, or a derivative thereof.

29. The method of claim 1, wherein said first barcode sequence and said second barcode sequence are the same.

30. The method of claim 1, wherein said first barcode sequence and said second barcode sequence are different.