METHODS AND SYSTEMS FOR PROCESSING POLYNUCLEOTIDES

• US 20180258482A1
• Filed: 05/15/2018
• Published: 09/13/2018
• Est. Priority Date: 12/14/2012
• Status: Active Grant

• Current Assignee: 10X Genomics Incorporated
• Original Assignee: 10X Genomics Incorporated
• Inventors: Hindson, Benjamin, Hindson, Christopher, Schnall-Levin, Michael, Ness, Kevin, Jarosz, Mima, Saxonov, Serge, Hardenbol, Paul, Bharadwaj, Rajiv, Zheng, Xinying, Belgrader, Phillip
• Granted Patent: US 10,253,364 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 generating a sequencing set for nucleic acid sequencing, comprising:

(a) bringing an aqueous fluid comprising a plurality of cells and a plurality of beads in contact with a continuous phase comprising an oil that is immiscible with said aqueous fluid, to generate a plurality droplets, wherein a given droplet of said plurality of droplets comprises a single cell from said plurality of cells and a single bead from said plurality of beads, and wherein said single bead comprises nucleic acid molecules each comprising a common barcode sequence;

(b) in said given droplet, subjecting messenger ribonucleic acid (mRNA) molecules from said single cell to reverse transcription in the presence of said nucleic acid molecules to yield complementary deoxyribonucleic acid (cDNA) molecules comprising said common barcode sequence;

(c) breaking said plurality of droplets, thereby releasing a plurality of cDNA molecules from said plurality of droplets, wherein said plurality of cDNA molecules comprises said cDNA molecules or derivatives thereof; and

(d) subjecting said plurality of cDNA molecules to one or more reactions to generate said sequencing set.

2. The method of claim 1, wherein said nucleic acid molecules comprise at least 1,000 nucleic acid molecules.

3. The method of claim 1, wherein said nucleic acid molecules comprise at least 100,000 nucleic acid molecules.

4. The method of claim 1, wherein said plurality of droplets is generated at a flow rate of at least about 1,000 Hz.

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

6. The method of claim 1, further comprising digesting nucleic acid molecules from said given droplet subsequent to breaking said plurality of droplets.

7. The method of claim 1, further comprising, subsequent to (d), pooling said plurality of cDNA molecules and performing said one or more reactions in bulk.

8. The method of claim 1, wherein said single cell and said single bead are partitioned in said given droplet using a channel structure comprising a first channel in fluid communication with a first source comprising a suspension of said plurality of cells and a second channel in fluid communication with a second source comprising a suspension of said plurality of beads.

9. The method of claim 8, wherein said second source is in fluid communication with a flow regulator that provides a regular flow of said plurality of beads in said second channel.

10. The method of claim 9, wherein said plurality of beads flow in said second channel at a flow rate having a coefficient of variation of less than 30% as determined by number of beads that flow past a given point in said second channel within a one second period of time.

11. The method of claim 10, wherein said coefficient of variation is less than 20%.

12. The method of claim 8, wherein individual cells of said plurality of cells have sizes that are less than a cross-section of said first channel.

13. The method of claim 8, wherein individual beads of said plurality of beads have sizes that are less than a cross-section of said second channel.

14. The method of claim 1, further comprising, prior to (b), releasing said nucleic acid molecules from said single bead.

15. The method of claim 14, wherein said nucleic acid molecules are released from said single bead upon exposure to a chemical stimulus in said droplet.

16. The method of claim 1, wherein said single bead is a gel bead.

17. The method of claim 1, wherein said single cell is in a polymer matrix.

18. The method of claim 1, wherein said continuous phase comprises a fluorinated oil and a fluorosurfactant that inhibits coalescence of said plurality of droplets.

19. 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.

20. The method of claim 19, further comprising purifying said amplified products.

21. The method of claim 19, further comprising ligating functional sequences to said amplified products, wherein said functional sequences permit attachment of said sequencing set to a flow cell of a sequencer for said nucleic acid sequencing.

22. The method of claim 19, wherein said nucleic acid amplification is polymerase chain reaction.

23. 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 of said sequencing set to a flow cell of a sequencer for nucleic acid sequencing.

24. The method of claim 1, wherein said aqueous fluid comprises said plurality of beads.

25. The method of claim 1, further comprising using a sequencer to subject said sequencing set to said nucleic acid sequencing to generate a plurality of sequences comprising sequences corresponding to said mRNA molecules and said common barcode sequence.

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

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

28. The method of claim 27, wherein each of said nucleic acid molecules comprises an RNA specific priming sequence.

29. The method of claim 27, wherein said nucleic acid molecules comprise functional sequences that facilitate sequencing of said sequencing set.

30. The method of claim 1, wherein said plurality of droplets comprises transposases.