Droplet-Based Method And Apparatus For Composite Single-Cell Nucleic Acid Analysis

• US 20180030515A1
• Filed: 03/08/2017
• Published: 02/01/2018
• Est. Priority Date: 09/09/2014
• Status: Active Grant

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation), Massachusetts Institute of Technology, The Broad Institute, Inc.
• Original Assignee: President and Fellows of Harvard College (Harvard Corporation), Massachusetts Institute of Technology, The Broad Institute, Inc.
• Inventors: Regev, Aviv, MACOSKO, Evan Zane, MCCARROLL, Steven Andrew, SHALEK, Alexander K., BASU, Anindita, FORD, Christopher B., PARK, Hongkun, WEITZ, David A.
• Granted Patent: US 11,597,964 B2
• Time in Patent Office: Days
• CPC Class Codes: C12N 15/1096 cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2521/107 RNA dependent DNA polymerase,(i.e. reverse transcriptase)
• CPC Class Codes: C12Q 2525/15 incorporating a consensus or conserved sequence
• CPC Class Codes: C12Q 2525/155 incorporating/generating a new priming site
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2525/173 incorporating a polynucleotide run, e.g. polyAs, polyTs
• 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
• CPC Class Codes: G06K 19/06 characterised by the kind of the digital marking, e.g. shape, nature, code

Abstract

The present invention generally relates to a combination of molecular barcoding and emulsion-based microfluidics to isolate, lyse, barcode, and prepare nucleic acids from individual cells in a high-throughput manner.

Claims

Please. Please add the following claims:

1-97. -97. (canceled)

12. A method for simultaneously preparing a thousand or more nucleotide- or oligonucleotide-adorned beads wherein a uniform or near-uniform nucleotide or oligonucleotide sequence is synthesized upon any individual bead while a plurality of different nucleotide or oligonucleotide sequences are simultaneously synthesized on different beads, comprising:

(a) forming a mixture comprising a plurality of beads;

(b) separating the beads into subsets;

(c) extending the nucleotide or oligonucleotide sequence on the surface of the beads by adding an individual nucleotide via chemical synthesis;

(d) pooling the subsets of beads in (c) into a single common pool;

(e) repeating steps (b), (c) and (d) multiple times to produce a combinatorially large number of nucleotide or oligonucleotide sequences; and

(f) collecting the nucleotide- or oligonucleotide-adorned beads;

(g) performing polynucleotide synthesis on the surface of the plurality of beads in a pool-and-split synthesis, such that in each cycle of synthesis the beads are split into a plurality of subsets wherein each subset is subjected to different chemical reactions;

(h) repeating the pool-and-split synthesis multiple times.

98. A nucleotide- or oligonucleotide-adorned bead wherein said bead comprises:

(a) a linker;

(b) an identical sequence for use as a sequencing priming site;

(c) a uniform or near-uniform nucleotide or oligonucleotide sequence;

(d) a Unique Molecular Identifier which differs for each priming site;

(e) optionally an oligonucleotide redudant sequence for capturing polyadenylated mRNAs and priming reverse transcription; and

(f) optionally at least one other oligonucleotide barcode which provides an additional substrate for identification.

99. The nucleotide- or oligonucleotide-adorned bead of claim 98, wherein:

the nucleotide or oligonucleotide sequence on the surface of the bead is a molecular barcode;

or

the nucleotide or oligonucleotide sequence on the surface of the bead is a molecular barcode from 4 to 1000 nucleotides in length;

or

the oligonucleotide sequence for capturing polyadenylated mRNAs and priming reverse transcription is an oligo dT sequence;

or

the linker is a non-cleavable, straight-chain polymer;

or

the linker is a chemically-cleavable, straight-chain polymer;

or

the linker is a non-cleavable optionally substituted hydrocarbon polymer;

or

the linker is a photolabile optionally substituted hydrocarbon polymer;

or

the linker is a polyethylene glycol;

or

the linker is a PEG-C_3-24.

100. A mixture comprising a plurality of nucleotide- or oligonucleotide- adorned beads, wherein said beads comprises;

(a) a linker;

(b) an identical sequence for use as a sequencing priming site;

(c) a uniform or near-uniform nucleotide or oligonucleotide sequence;

(d) a Unique Molecular Identifier which differs for each priming site;

(e) an oligonucleotide redudant sequence for capturing polyadenylated mRNAs and priming reverse transcription; and

(f) optionally at least one additional oligonucleotide sequences which provide substrates for downstream molecular-biological reactions;

wherein the uniform or near-uniform nucleotide or oligonucleotide sequence is the same across all the priming sites on any one bead, but varies among the oligonucleotides on an individual bead.

101. The mixture of claim 100,

wherein the nucleotide or oligonucleotide sequence on the surface of the bead is a molecular barcode;

of

wherein the nucleotide or oligonucleotide sequence on the surface of the bead is a molecular barcode from 4 to 1000 nucleotides in length;

or

wherein the oligonucleotide sequence for capturing polyadenylated mRNAs and priming reverse transcription is an oligo dT sequence;

or

which comprises at least one oligonucleotide sequences, which provide for substrates for downstream molecular-biological reactions;

or

wherein the downstream molecular biological reactions are for reverse transcription of mature mRNAs;

capturing specific portions of the transcriptome, priming for DNA polymerases and/or similar enzymes;

or priming throughout the transcriptome or genome;

or

wherein the additional oligonucleotide sequence comprises a oligio-dT sequence;

or

wherein the additional oligonucleotide sequence comprises a primer sequence;

or

wherein the additional oligonucleotide sequence comprises a oligio-dT sequence and a primer sequence.

102. An error-correcting barcode bead wherein said bead comprises:

(a) a linker;

(b) an identical sequence for use as a sequencing priming site;

(c) a uniform or near-uniform nucleotide or oligonucleotide sequence which comprises at least a nucleotide base duplicate;

(d) a Unique Molecular Identifier which differs for each priming site; and

(e) an oligonucleotide redudant for capturing polyadenylated mRNAs and priming reverse transcription;

103. A method wherein the barcode beads of claim 102, fail to hybridize to the mRNA thereby failing to undergo reverse transcription.

105. A method for creating a composite single-cell sequencing library comprising:

(a) merging one uniquely barcoded RNA capture microbead with a single-cell in an emulsion droplet having a diameter from 50 μ

m to 210 μ

m;

(b) lysing the cell thereby capturing the RNA on the RNA capture microbead;

(c) performing a reverse transcription reaction to convert the cells'"'"' RNA to first strand cDNA that is covalently linked to the RNA capture microbead;

or conversely reverse transcribing within droplets and thereafter breaking droplets and collecting cDNA-attached beads;

(d) preparing and sequencing a single composite RNA-Seq library, containing cell barcodes that record the cell-of-origin of each RNA, and molecular barcodes that distinguish among RNAs from the same cell.

106. A method for creating a composite single-cell sequencing library comprising:

(a) merging one uniquely barcoded RNA capture microbead with a single-cell in an emulsion droplet having a diameter from 50 μ

m to 210 μ

m;

(b) lysing the cell thereby capturing the RNA on the RNA capture microbead;

(c) breaking droplets and pooling beads in solution;

(d) performing a reverse transcription reaction to convert the cells'"'"' RNA to first strand cDNA that is covalently linked to the RNA capture microbead;

or conversely reverse transcribing within droplets and thereafter breaking droplets and collecting cDNA-attached beads;

(e) preparing and sequencing a single composite RNA-Seq library, containing cell barcodes that record the cell-of-origin of each RNA, and molecular barcodes that distinguish among RNAs from the same cell.

107. The method of claim 106, wherein

the method of amplifying the cDNA-attached beads is template switch amplification;

or

the method of amplifying the cDNA-attached beads is T7 linear application;

or

the method of amplifying the cDNA-attached beads is exponential isothermal amplification;

or

the emulsion droplet is formed via co-encapsulation comprising RNA capture microbead and composite single-cell;

or

the emulsion droplet is at least 1.25 μ

m;

or

the diameter of the RNA capture microbeads is from 10 μ

m to 95 μ

m.

108. A method for preparing a plurality of beads with unique nucleic acid sequence comprising:

(a) performing polynucleotide synthesis on the surface of the plurality of beads in a pool-and-split process, such that in each cycle of synthesis the beads are split into a plurality of subsets wherein each subset is subjected to different chemical reactions;

(b) repeating the pool-and-split process from anywhere from 2 cycles to 200 cycles.

109. The method of claim 108, wherein

the polynucleotide synthesis is phosphoramidite synthesis;

or

the polynucleotide synthesis is reverse direction phosphoramidite chemistry;

or

each subset is subjected to a different nucleotide;

or

each subset is subjected to a different canonical nucleotide;

or

the method is repeated three times;

or

the method is repeated four times;

or

the method is repeated twelve times;

or

the linker covalently connecting the microbead to the oligonucleotide is polyethylene glycol.

110. The method of claim 109, wherein

the diameter of the RNA capture microbeads is from 10 μ

m to 95 μ

m;

or multiple steps is twelve steps.

111. A method for simultaneously preparing a plurality of nucleotide- or oligonucleotide-adorned beads wherein a uniform, near-uniform, or patterned nucleotide or oligonucleotide sequence is synthesized upon any individual bead while vast numbers of different nucleotide or oligonucleotide sequences are simultaneously synthesized on different beads, comprising:

(a) forming a mixture comprising a plurality of beads;

(b) separating the beads into subsets;

(c) extending the nucleotide or oligonucleotide sequence on the surface of the beads by adding an individual nucleotide via chemical synthesis;

(d) pooling the subsets of beads in (c) into a single common pool;

(e) repeating steps (b), (c) and (d) multiple times to produce combinatorially a thousand or more nucleotide or oligonucleotide sequences; and

(f) collecting the nucleotide- or oligonucleotide-adorned beads.

113. The method of claim 112, wherein

the nucleotide or oligonucleotide sequence on the surface of the bead is a molecular barcode;

or

the pool-and-split synthesis steps occur every 2-10 cycles, rather than every cycle;

or

the barcode contains built-in error correction;

or -p1 the barcode ranges from 4 to 1000 nucleotides in length;

or

the polynucleotide synthesis is phosphoramidite synthesis;

or

the polynucleotide synthesis is reverse direction phosphoramidite chemistry;

or

each subset is subjected to a different nucleotide;

or

the method further comprises one or more subsets receive a cocktail of two nucleotides;

or

each subset is subjected to a different canonical nucleotide;

or

the bead is a microbead;

or

the bead is a nanoparticle;

or

the bead is a macrobead;

or

the oligonucleotide barcoded bead is a dinucleotide;

or

the oligonucleotide barcoded bead is a trinucleotide;

or

the pool-and-split synthesis is repeated twelve times;

or

the diameter of the complexed bead is from 10 μ

m to 95 μ

m.

114. An apparatus for creating a composite single-cell sequencing library via a microfluidic system, comprising:

an oil-surfactant inlet comprising a filter and a carrier fluid channel, wherein said carrier fluid channel further comprises a resistor;

an inlet for an analyte comprising a filter and a carrier fluid channel, wherein said carrier fluid channel further comprises a resistor;

an inlet for mRNA capture microbeads and lysis reagent comprising a filter and a carrier fluid channel, wherein said carrier fluid channel further comprises a resistor;

said carrier fluid channels have a carrier fluid flowing therein at an adjustable or predetermined flow rate;

wherein each said carrier fluid channels merge at a junction; and

said junction being connected to a mixer, which contains an outlet for drops.

115. The apparatus of claim 114, wherein

the analyte comprises a chemical reagent, a protein, a drug, an antibody, an enzyme, a nucleic acid, an organelle;

a cell or any combination thereof;

or

said junction is connected to said mixer by a fluid carrier channel with a constriction for droplet pinch-off;

or

the analyte is a cell;

or

the analyte is a mammalian cell;

or

the cell is a brain cell;

or

the cell is a retina cell;

or

the cell is a human bone marrow cell;

or

the cell is a host-pathogen cell

the analyte is complex tissue;

or

the lysis reagent comprises an anionic surfactant, such as sodium lauroyl sarcosine, or a chaotropic salt, such as guanidiunium thiocyanate;

or

the filter comprises square PDMS;

or

the resistor is serpentine having a length from 7000 9000, width of 50-75 μ

m and depth of 100-150 mm;

or

the resistor has a diameter of 50 μ

m;

or

the channels having a length of length of 8000-12,000 μ

m and width of 12.5-250 mm, and depth of 100-150 mm;

or

wherein the channel has a diameter of 125 μ

m;

or

wherein the mixer has a length of 7000-9000 μ

m and a width of 110-140 μ

m;

or

wherein the mixer width is 125 μ

m;

or

the oil-surfactant is a PEG block polymer;

or

the carrier fluid is water-glycerol mixture.