Methods and systems for processing polynucleotides

Patent metadata

Field	Value
Publication number	US-2018094313-A1
Application number	US-201715831847-A
Country	US
Kind code	A1
Filing date	Dec 5, 2017
Priority date	Jun 26, 2014
Publication date	Apr 5, 2018
Grant date	—

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.

First claim

What is claimed is:

1 . A device for generating a plurality of droplets comprising a plurality of cells and a plurality of beads, comprising:
a first channel in fluid communication with a first source comprising a suspension of said plurality of cells; a second channel in fluid communication with a second source comprising a suspension of said plurality of beads; a third channel in fluid communication with a third source comprising an oil; and a fourth channel in fluid communication with said first channel, said second channel and said third channel, wherein said fourth channel directs a plurality of droplets generated upon an aqueous fluid comprising said plurality of cells and said plurality of beads coming in contact with a continuous phase comprising said oil that is immiscible with said aqueous fluid, wherein (i) said plurality of cells have sizes that are less than a cross-section of said first channel, or (ii) said plurality of beads have sizes that are less than a cross-section of said second channel, 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 oligonucleotide molecules comprising a common barcode sequence.

2 . The device of claim 1 , wherein said oligonucleotide molecules comprises at least 1,000 oligonucleotide molecules.

3 . The device of claim 1 , wherein said oligonucleotide molecules comprises at least 100,000 oligonucleotide molecules.

4 . The device of claim 1 , wherein said plurality of beads comprises oligonucleotide molecules having different barcode sequences.

5 . The device of claim 1 , wherein said first channel, said second channel, said third channel and said fourth channel are part of a channel structure.

6 . The device of claim 1 , 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.

7 . The device of claim 6 , wherein said plurality of beads flows through said second channel at a flow rate of at least about 50 Hz.

8 . The device of claim 7 , wherein said plurality of beads flows through said second channel at a flow rate of at least about 1,000 Hz

9 . The device of claim 8 , wherein said plurality of beads flows through said second channel at a flow rate of at least about 5,000 Hz.

10 . The device of claim 1 , wherein said plurality of beads flows through 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 device of claim 10 , wherein said coefficient of variation is less than 20%.

12 . The device of claim 1 , wherein (i) said plurality of cells have sizes that are less than a cross-section of said first channel, and (ii) said plurality of beads have sizes that are less than a cross-section of said second channel.

13 . The device of claim 1 , wherein said oligonucleotide molecules are releasable from said single bead.

14 . The device of claim 13 , wherein said given droplet comprises a chemical stimulus that subjects said oligonucleotide molecules to release from said single bead.

15 . The device of claim 1 , wherein said single bead is a gel bead.

16 . The device of claim 1 , wherein said single cell and said single bead are partitioned in said given droplet in the absence of droplet coalescence.

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

18 . The device of claim 1 , wherein said aqueous fluid comprises said plurality of beads.

19 . The device of claim 1 , wherein said given droplet comprises a lysis agent that subjects said single cell in said given droplet to lysis.

20 . The device of claim 1 , wherein said plurality of droplets is generated at a rate of at least about 50 Hz.

21 . The device of claim 20 , wherein said plurality of droplets is generated at a rate of at least about 1,000 Hz.

22 . The device of claim 21 , wherein said plurality of droplets is generated at a rate of at least about 5,000 Hz.

23 . The device of claim 1 , wherein each of said oligonucleotide molecules comprises said common barcode sequence and a unique molecular sequence, wherein said common barcode sequence is constant across said oligonucleotide molecules, and wherein said unique molecular sequence varies across said oligonucleotide molecules.

24 . A system for nucleic acid analysis, comprising a device comprising (1) a first channel in fluid communication with a first source comprising a suspension of a plurality of cells; (2) a second channel in fluid communication with a second source comprising a suspension of a plurality of beads; (3) a third channel in fluid communication with a third source comprising an oil; and (4) a fourth channel in fluid communication with said first channel, said second channel and said third channel, wherein said fourth channel directs a plurality of droplets generated upon an aqueous fluid comprising said plurality of cells and said plurality of beads coming in contact with a continuous phase comprising said oil that is immiscible with said aqueous fluid, wherein (i) said plurality of cells have sizes that are less than a cross-section of said first channel, or (ii) said plurality of beads have sizes that are less than a cross-section of said second channel,
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 oligonucleotide molecules comprising a common barcode sequence.

25 . The system of claim 24 , further comprising a sequencer for subjecting complementary deoxyribonucleic acid (cDNA) molecules, generated from ribonucleic acid (RNA) molecules from said single cell and said oligonucleotide molecules, to sequencing to generate a plurality of sequences comprising sequences corresponding to said RNA molecules and said common barcode sequence.

26 . The system of claim 24 , further comprising a flow controller that directs said plurality of droplets along said fourth channel.

27 . The system of claim 24 , wherein (i) said plurality of cells have sizes that are less than a cross-section of said first channel, and (ii) said plurality of beads have sizes that are less than a cross-section of said second channel.

Assignees

• 10X Genomics Inc

Inventors

• Hindson Benjamin
• Hindson Christopher
• Schnall-Levin Micheal
• Ness Kevin
• Jarosz Mirna
• Saxonov Serge
• Hardenbol Paul
• Bharadwaj Rajiv
• Zheng Xinying
• Belgrader Phillip

Classifications

• C12Q1/6804

  Nucleic acid analysis using immunogens (immunoassay G01N33/53) · CPC title

• C12Q2537/143

  Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis · CPC title

• C12Q2565/629

  being a microfluidic device · CPC title

• C12Q1/683 Primary

  involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP] · CPC title

• C12Q2525/191

  incorporating an adaptor · CPC title