METHODS AND SYSTEMS FOR PROCESSING POLYNUCLEOTIDES

• US 20180112266A1
• Filed: 12/19/2017
• Published: 04/26/2018
• Est. Priority Date: 06/26/2014
• 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, Bharadwaj, Rajiv, Zheng, Grace, Belgrader, Phillip
• Granted Patent: US 10,480,028 B2
• Time in Patent Office: 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 1/683 involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2537/149 Sequential reactions
• 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 2565/629 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 for processing messenger ribonucleic acid (mRNA) molecules from a single cell, comprising:

(a) partitioning a plurality of cells and a plurality of beads in a plurality of wells, wherein a well of said plurality of wells comprises said single cell from said plurality of cells and a single bead from said plurality of beads, and wherein said single bead comprises nucleic acid barcode molecules each comprising a common barcode sequence;

(b) in said well comprising said single cell and said single bead, releasing messenger ribonucleic acid (mRNA) molecules from said single cell, wherein upon release from said single cell, said mRNA molecules attach to said nucleic acid barcode molecules;

(c) subjecting said mRNA molecules attached to said nucleic acid barcode molecules to reverse transcription to yield complementary deoxyribonucleic acid (cDNA) molecules each comprising said common barcode sequence; and

(d) subjecting said cDNA molecules to one or more reactions to generate a set of nucleic acid molecules for nucleic acid sequencing.

2. The method of claim 1, wherein (c) is performed in said well comprising said single cell and said single bead, and wherein subsequent to (c), said cDNA molecules, or derivatives thereof, are removed from said given well.

3. The method of claim 1, wherein, prior to (c), said mRNA molecules attached to said nucleic acid barcode molecules are removed from said well comprising said single cell and said single bead.

4. The method of claim 1, wherein each of said nucleic acid barcode molecules comprises a universal primer sequence.

5. The method of claim 1, wherein said single bead is a single magnetic bead.

6. The method of claim 5, wherein said nucleic acid barcode molecules are attached to said single magnetic bead.

7. The method of claim 6, wherein prior to (c), said single magnetic bead comprising said mRNA molecules attached to said nucleic acid barcode molecules are removed from said well comprising said single cell and said single bead.

8. The method of claim 7, wherein said single magnetic bead is removed using a magnetic field.

9. The method of claim 1, wherein each of said nucleic acid barcode molecules comprises a sequence for priming the synthesis of cDNA.

10. The method of claim 9, wherein said RNA priming sequence is an oligo(dT) sequence.

11. The method of claim 1, wherein said plurality of cells comprises at least 100 cells.

12. The method of claim 1, wherein said plurality of cells comprises at least 1,000 cells

13. The method of claim 1, wherein said plurality of cells comprises at least 10,000 cells.

14. The method of claim 1, wherein said mRNA molecules attach to said nucleic acid barcode molecules by hybridization.

15. The method of claim 1, wherein said plurality of beads comprises a plurality of nucleic acid barcode molecules comprising barcode sequences that are different across said plurality of beads.

16. The method of claim 1, further comprising, prior to (c), (i) pooling said mRNA molecules attached to said nucleic acid barcode molecules and (ii) performing said one or more reactions in bulk.

17. The method of claim 1, wherein said one or more reactions comprise nucleic acid amplification that generates amplified products from said plurality of cDNA molecules.

18. The method of claim 17, further comprising purifying said amplified products.

19. The method of claim 17, wherein said nucleic acid amplification adds functional sequences to said amplified products, wherein said functional sequences permit attachment of said amplified products to a flow cell of a sequencer for said nucleic acid sequencing.

20. The method of claim 17, further comprising ligating functional sequences to said amplified products, wherein said functional sequences permit attachment of said amplified products to a flow cell of a sequencer for said nucleic acid sequencing.

21. The method of claim 17, wherein said nucleic acid amplification is polymerase chain reaction.

22. The method of claim 1, wherein said one or more reactions comprise addition of functional sequences to said plurality of cDNA molecules, wherein said functional sequences permit attachment to a flow cell of a sequencer for said nucleic acid sequencing.

23. The method of claim 1, further comprising performing said nucleic acid sequencing on said set of nucleic acid molecules, or derivatives thereof, to generate a plurality of sequences comprising sequences corresponding to said mRNA molecules and said common barcode sequence.

24. The method of claim 1, wherein said plurality of beads have substantially monodisperse cross-sectional dimensions.

25. The method of claim 1, wherein each of said nucleic acid barcode molecules comprise said common barcode sequence and a unique molecular sequence, wherein said common barcode sequence is constant across said nucleic acid barcode molecules, and wherein said unique molecular sequence varies across said nucleic acid barcode molecules.

26. The method of claim 25, wherein each of said nucleic acid barcode molecules further comprise a sequence for priming the synthesis of cDNA.

27. The method of claim 26, wherein said RNA priming sequence is an oligo(dT) sequence.

28. The method of claim 1, wherein said nucleic acid barcode molecules further comprise functional sequences that facilitate sequencing of said set of nucleic acid molecules.