Massively parallel single cell analysis
The disclosure provides for methods, compositions, and kits for multiplex nucleic acid analysis of single cells. The methods, compositions and systems may be used for massively parallel single cell sequencing. The methods, compositions and systems may be used to analyze thousands of cells concurrently. The thousands of cells may comprise a mixed population of cells (e.g., cells of different types or subtypes, different sizes).
1. An instrument system for performing multiplexed single cell assay, comprising:
a microtiter plate or a microfluidic chip; and
a plurality of particles each associated with a plurality of oligonucleotides, wherein each of the plurality of oligonucleotides comprises a cellular label sequence, a molecular label sequence, and a target-binding region, wherein the cellular label sequence of each of the plurality of oligonucleotides is the same on the same particle but different on different particles, and wherein at least 100 of the plurality of oligonucleotides associated with each of the plurality of particles comprise different molecular label sequences.
2. The instrument system of claim 1 , comprising one or more amplification agents.
3. The instrument system of claim 2 , wherein the one or more amplification agents comprise a polymerase, a deoxyribonucleotide triphosphate (dNTP), a primer, a buffer, or any combination thereof.
4. The instrument system of claim 1 , wherein the target-binding region comprises an oligo-dT sequence, a gene-specific sequence, a random multimer sequence, or a combination thereof.
5. The instrument system of claim 1 , wherein each of the plurality of oligonucleotides further comprises at least one of (a) a sample label sequence and (b) a universal label sequence.
6. The instrument system of claim 1 , wherein the molecular label sequences comprise random sequences.
7. The instrument system of claim 1 , wherein at least 10,000 of the plurality of oligonucleotides associated with each of the plurality of particles comprise different molecular label sequences.
8. The instrument system of claim 1 , wherein the plurality of oligonucleotides comprises at least 100,000 oligonucleotides.
9. The instrument system of claim 1 , wherein the microtiter plate or the microfluidic chip comprises a plurality of compartments, and wherein each of at least 10% of the plurality of compartments contains one of the plurality of particles.
10. The instrument system of claim 9 , wherein each of at least 10% of the plurality of compartments contains a single cell or a lysate of a single cell.
11. The instrument system of claim 9 , wherein each of at least 10% of the plurality of compartments contains (1) one of the plurality of particles and (2) a single cell or a lysate of a single cell.
12. The instrument system of claim 11 , wherein the single cell is a rare cell, a cancer cell, an immune cell, a cell from a tissue, a human cell, a cell comprising viral polynucleotides, or a combination thereof.
13. The instrument system of claim 9 , wherein the plurality of compartments comprises microwells, droplets, or a combination thereof.
14. The instrument system of claim 1 , wherein one or more of the plurality of particles are associated with a plurality of peptides.
15. The instrument system of claim 14 , wherein the plurality of peptides are a plurality of antibodies.
16. The instrument system of claim 15 , wherein the plurality of antibodies is attached to the plurality of oligonucleotides.
17. The instrument system of claim 15 , wherein each of at least 10% of the plurality of compartments contains a single cell, and wherein at least one of the plurality of antibodies bind to the surface of the single cell.
18. The instrument system of claim 1 , wherein the plurality of oligonucleotides of each of the plurality of particles is capable of labeling individual occurrences of target molecules of the single cell or the lysate of the single cell.
19. The instrument system of claim 18 , wherein said target molecules comprise a DNA molecule, an RNA molecule, or a combination thereof.
20. The instrument system of claim 1 , wherein the plurality of oligonucleotides of each of the plurality of particles is capable of labeling individual occurrences of mRNA molecules of the single cell or the lysate of the single cell via reverse transcription.
21. The instrument system of claim 1 , wherein the plurality of particles comprises at least 384 particles.
22. The instrument system of claim 1 , wherein each of the plurality of particles comprises polydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose, gelatin, hydrogel, paramagnetic, ceramic, plastic, glass, methylstyrene, acrylic polymer, titanium, latex, sepharose, cellulose, nylon, silicone, silica gel, controlled pore glass, Dynabead, Wang resin, Merrifield resin, Sephadex/Sepharose, cellulose, or a combination thereof.
23. The instrument system of claim 1 , wherein each of the plurality of particles is a microparticle or a nanoparticle.
24. The instrument system of claim 23 , wherein each of the plurality of particles is a bead, and wherein the bead is a streptavidin bead, an agarose bead, a magnetic beads, a hydrogel bead, an antibody conjugated bead, a protein A conjugated bead, a protein G conjugated bead, a protein NG conjugated bead, a protein L conjugated bead, an oligodT conjugated bead, a silica bead, a silica-like bead, an anti-biotin microbead, or an anti-fluorochrome microbead.
25. The instrument system of claim 23 , wherein each of the plurality of particles is a semi-soft bead.
26. The instrument system of claim 1 , further comprising an imaging system, a cell or particle distribution system, a fluidics controller, a cell lysis system, a temperature control controller, a magnetic field controller, a computer or processor programmed to provide a user interface and control of system functions, program code for providing real-time image analysis, an integrated PCR thermal cycler, an integrated sequencer, or a combination thereof.