METHODS AND COMPOSITIONS FOR LABELING CELLS

• US 20190177800A1
• Filed: 08/21/2018
• Published: 06/13/2019
• Est. Priority Date: 12/08/2017
• Status: Abandoned Application

• Current Assignee: 10X Genomics Incorporated
• Original Assignee: 10X Genomics Incorporated
• Inventors: Boutet, Stephane Claude, Lucero, Michael Ybarra, Mikkelsen, Tarjei Sigurd, Pfeiffer, Katherine
• Application Number:
  US16/107,685
• Publication Number:
  US 20190177800A1
• Time in Patent Office: Days
• CPC Class Codes: C12N 15/1013 by using magnetic beads
• 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: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• 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 1/6881 for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
• CPC Class Codes: C12Q 1/6886 for cancer immunoassay for cancer G01N33/574
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• 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 2563/185 Nucleic acid dedicated to use as a hidden marker/bar code, e.g. inclusion of nucleic acids to mark art objects or animals
• CPC Class Codes: C12Q 2600/158 Expression markers
• CPC Class Codes: G16B 30/00 ICT specially adapted for sequence analysis involving nucleotides or amino acids
• CPC Class Codes: G16B 50/00 ICT programming tools or database systems specially adapted for bioinformatics

Abstract

The present disclosure provides methods, systems, and compositions for parallel processing of nucleic acid samples. Methods and systems of the present disclosure comprise the use of sample-specific barcode sequences, which facilitate the multiplexing of samples, detection of discrete cell populations within a pooled population, and detection of partitions comprising more than one cell.

Claims

1. A method for analyzing cellular occupancy of partitions, comprising:

(a) contacting a plurality of cells with a plurality of labelling molecules comprising a plurality of cell nucleic acid barcode sequences to generate a plurality of labelled cells, wherein each of said plurality of labelled cells comprises a different cell nucleic acid barcode sequence;

(b) generating a plurality of partitions comprising said plurality of labelled cells and a plurality of beads, wherein said plurality of beads comprise a plurality of partition nucleic acid barcode sequences coupled thereto, wherein each of said plurality of partitions comprises a different partition nucleic barcode sequence, and wherein at least a fraction of said plurality of partitions comprises more than one labelled cell of said plurality of labelled cells;

(c) within said plurality of partitions, (i) using partition nucleic acid barcode sequences of said plurality of partition nucleic acid barcode sequences and cell nucleic acid barcode sequences of said plurality of cell nucleic acid barcode sequences to synthesize a plurality of barcoded nucleic acid products, and (ii) releasing or recovering said plurality of barcoded nucleic acid products or derivatives thereof from said plurality of partitions; and

(d) identifying-sequencing said plurality of barcoded nucleic acid products or derivatives thereof to determine that at least two labelled cells of said plurality of labelled cells originate from a same partition using (i) said cell nucleic acid barcode sequences of said plurality of cell nucleic acid barcode sequences or complements thereof and (ii) said partition nucleic acid barcode sequences of said plurality of partition nucleic acid barcode sequences or complements thereof.

2. The method of claim 1, wherein a given cell nucleic acid barcode sequence of said plurality of cell nucleic acid barcode sequences identifies a sample from which an associated cell of said plurality of labelled cells originates.

3. The method of claim 1, wherein a given barcoded nucleic acid product of said plurality of barcoded nucleic acid products comprises (iii) a cell identification sequence comprising a given cell nucleic acid barcode sequence of said plurality of cell nucleic acid barcode sequences or a complement of said given cell nucleic acid barcode sequence;

and (iv) a partition identification sequence comprising a given partition nucleic acid barcode sequence of said plurality of partition nucleic acid barcode sequences or a complement of said given partition nucleic acid barcode sequence.

4. The method of claim 3, wherein (v) a plurality of partition nucleic acid barcode molecules comprises said plurality of partition nucleic acid barcode sequences, such that a given partition nucleic acid barcode molecule of said plurality of partition nucleic acid barcode molecules comprises a single partition nucleic acid barcode sequence of said plurality of partition nucleic acid barcode sequences;

and (vi) a plurality of cell nucleic acid barcode molecules comprises said plurality of cell nucleic acid barcode sequences, such that a given cell nucleic acid barcode molecule of said plurality of cell nucleic acid barcode molecules comprises a single cell nucleic acid barcode sequence of said plurality of cell nucleic acid barcode sequences.

5. The method of claim 4, wherein a given partition nucleic acid barcode molecule of said plurality of partition nucleic acid barcode molecules comprises a priming sequence that is capable of hybridizing to a sequence of a given cell nucleic acid barcode molecule of said plurality of cell nucleic acid barcode molecules.

7. The method of claim 1, wherein said plurality of barcoded nucleic acid products is synthesized via one or more primer extension reactions.

8. The method of claim 1, wherein said plurality of barcoded nucleic acid products is synthesized via one or more ligation reactions.

9. The method of claim 1, wherein said plurality of barcoded nucleic acid products is synthesized via one or more nucleic acid amplification reactions.

10. The method of claim 3, further comprising sequencing said plurality of barcoded nucleic acid products or derivatives thereof to yield a plurality of sequencing reads.

11. The method of claim 10, further comprising associating each sequencing read of said plurality of sequencing reads with a labelled cell of said plurality of labelled cells via its respective cell identification sequence, and associating each sequencing read of said plurality of sequencing reads with a partition of said plurality of partitions via its respective partition identification sequence.

12. The method of claim 1, wherein each and of said plurality of beads comprises a different partition nucleic acid barcode sequence of said plurality of partition nucleic acid barcode sequences.

13. The method of claim 1, wherein each partition of said plurality of partitions comprises a single bead of said plurality of beads.

14. The method of claim 1, wherein each bead of said plurality of beads comprises a plurality of partition nucleic acid barcode molecules, wherein each partition nucleic acid barcode molecule of said plurality of partition nucleic acid barcode molecules coupled to a given bead of said plurality of beads comprises a single partition nucleic acid barcode sequence of said plurality of partition nucleic acid barcode sequences.

15. The method of claim 12, wherein each partition nucleic acid barcode sequence of said plurality of partition nucleic acid barcode sequences is releasably coupled to its respective bead of said plurality of beads.

16. The method of claim 15, further comprising, after (b), releasing partition nucleic acid barcode sequences of said plurality of nucleic acid barcode sequences from said plurality of beads.

17. The method of claim 16, further comprising degrading each bead of said plurality of beads to release said partition nucleic acid barcode sequences of said plurality of partition nucleic acid barcode sequences from each bead of said plurality of beads.

18. The method of claim 17, wherein each partition of said plurality of partitions comprises an agent that is capable of degrading each bead of said plurality of beads.

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

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

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

22. The method of claim 1, wherein, in (a), said plurality of cells is labelled with said plurality of cell nucleic acid barcode sequences by binding a plurality of cell binding moieties to each cell of said plurality of cells, wherein a cell binding moiety of said plurality of cell binding moieties is coupled to a cell nucleic acid barcode sequence of said plurality of cell nucleic acid barcode sequences.

23. The method of claim 22, wherein said plurality of cell binding moieties comprises a plurality of antibodies, cell surface receptor binding molecules, receptor ligands, small molecules, pro-bodies, aptamers, monobodies, affimers, darpins, or protein scaffolds.

24. The method of claim 23, wherein said plurality of cell binding moieties comprises a plurality of antibodies.

25. The method of claim 22, wherein said plurality of cell binding moieties bind to proteins or cell surface species of cells of said plurality of cells.

27. The method of claim 22, wherein each cell binding moiety of said plurality of cell binding moieties binds to a species common to each cell of said plurality of cells.

29. The method of claim 1, wherein, in (a), said plurality of cells is labelled with said plurality of labelling molecules with the aid of liposomes, nanoparticles, electroporation, or mechanical force.

31. The method of claim 1, wherein said plurality of labelling molecules comprises a plurality of lipophilic moieties.

32. The method of claim 1, wherein, prior to (d), said plurality of barcoded nucleic acid products or derivatives thereof are not coupled to said plurality of beads.

6. (canceled)

26. (canceled)

28. (canceled)

30. A method for analyzing cellular occupancy of a partition, comprising:

(a) labelling a first cell with a first cell nucleic acid barcode sequence and a second cell with a second cell nucleic acid barcode sequence to generate a first labelled cell and a second labelled cell, wherein said first cell nucleic acid barcode sequence has a different sequence than said second cell nucleic acid barcode sequence;

(b) generating a partition comprising said first labelled cell and said second labelled cell, wherein said partition further comprises a plurality of partition nucleic acid barcode molecules coupled to a bead, wherein each of said plurality of partition nucleic acid barcode molecules comprises a partition nucleic acid barcode sequence;

(c) within said partition, using said first cell nucleic acid barcode sequence, said second cell nucleic acid barcode sequence, and said plurality of partition nucleic acid barcode molecules to generate (i) a first barcoded nucleic acid molecule comprising said first cell nucleic acid barcode sequence or a complement thereof and said partition nucleic acid barcode sequence or a complement thereof, and (ii) a second barcoded nucleic acid molecule comprising said second cell nucleic acid barcode sequence or a complement thereof and said partition nucleic acid barcode sequence or a complement thereof, and

(d) sequencing said first barcoded nucleic acid molecule and said second barcoded nucleic acid molecule, or a derivative of said first barcoded nucleic acid molecule or said second barcoded nucleic acid molecule, to (i) identify said first cell nucleic acid barcode sequence and said second cell nucleic acid barcode sequence, or a complement of said first cell nucleic acid barcode sequence or said second cell nucleic acid barcode sequence, thereby identifying said first labelled cell and said second labelled cell, and (ii) identify said partition nucleic acid barcode sequence or complement thereof, thereby identifying said first labelled cell and said second labelled cell as originating from said partition based on said first barcoded nucleic acid molecule and said second barcoded nucleic acid molecule having said partition nucleic acid barcode sequence or a complement thereof.

33. The method of claim 30, wherein, prior to (d), said first barcoded nucleic acid molecule and said second barcoded nucleic acid molecule or derivatives thereof are not coupled to said bead.