WO2018022581A1

WO2018022581A1 - Barcoded systems with multiple information

Info

Publication number: WO2018022581A1
Application number: PCT/US2017/043660
Authority: WO
Inventors: David A. Weitz; John Heyman; Sarah FORTUNE
Original assignee: Harvard University
Current assignee: Harvard University
Priority date: 2016-07-26
Filing date: 2017-07-25
Publication date: 2018-02-01
Anticipated expiration: 2019-01-26

Abstract

The present invention generally relates to microfluidics and, in particular, to barcoded systems with multiple information. For example, one set of embodiments is generally directed to particles, which may be contained within droplets, containing a first barcode (e.g., which can be associated with a nucleic acid) and a second barcode (e.g., which can be associated with a protein). For instance, the first barcode may be associated with a first oligonucleotide containing a gene-specific reverse transcription primer (e.g., for associating with mRNA), and/or a second barcode may be associated with a second oligonucleotide containing an antibody (e.g., for associating with a protein). In some cases, the droplet may also contain a cell or other sample to be analyzed, e.g., for DNA and/or proteins. Binding of the nucleic acids and/or proteins may then be determined, e.g., using the first and second barcodes. In some cases, a plurality of droplets containing cells, particles, etc., may be burst or otherwise combined; however, specific nucleic acids, proteins, etc., may be uniquely identified, even after combination, using the barcodes.

Description

BARCODED SYSTEMS WITH MULTIPLE INFORMATION

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Patent Application Serial No. 62/366,991, filed July 26, 2016, entitled "Barcoded Systems with Multiple Information," by Weitz, et al., incorporated herein by reference in its entirety.

GOVERNMENT FUNDING

This invention was made with government support under Grant No. HR0011-11-C- 0093 awarded by DARPA. The government has certain rights in the invention.

FIELD

The present invention generally relates to microfluidics and, in particular, to barcoded systems with multiple information.

SUMMARY

The present invention generally relates to microfluidics and, in particular, to barcoded systems with multiple information. The subject matter of the present invention involves, in some cases, interrelated products, alternative solutions to a particular problem, and/or a plurality of different uses of one or more systems and/or articles.

In one aspect, the present invention is generally directed to a composition. In one set of embodiments, the composition comprises a particle contained within a microfluidic droplet. IN some cases, the particle may contain a first oligonucleotide sequence encoding a primer and a second oligonucleotide sequence attached to an antibody.

In another set of embodiments, the composition comprises a particle comprising a first oligonucleotide sequence comprising a first barcode encoding a gene-specific reverse transcription primer and a second oligonucleotide sequence comprising a second barcode encoding an antibody.

The present invention, in another aspect, is generally directed to an article. In one set of embodiments, the article includes a plurality of microfluidic droplets containing particles. In one embodiment, at least some of the particles comprise a first oligonucleotide sequence comprising a first barcode encoding a gene- specific reverse transcription primer and a second oligonucleotide sequence comprising a second barcode encoding an antibody. In another embodiment, at least some of the particles comprise a first oligonucleotide sequence comprising a first barcode encoding a transposase-bound primer and a second oligonucleotide sequence comprising a second barcode encoding an antibody. According to still another embodiment, at least some of the particles comprise a first oligonucleotide sequence comprising a first barcode encoding a oligo-dT primer and a second oligonucleotide sequence comprising a second barcode encoding an antibody.

According to another set of embodiments, the article includes a plurality of microfluidic droplets containing antibodies. In some instances, at least about 90% of the droplets contain one antibody type or no antibody. In certain embodiments, at least some of the antibodies comprise an oligonucleotide sequence. In some cases, the oligonucleotide sequences contained within a droplet are distinguishable from the oligonucleotide sequences contained within other droplets of the plurality of droplets.

According to yet another aspect, the present invention is generally directed to a method. The method, in one set of embodiments, includes acts of encapsulating a plurality of cells and a plurality of particles within a plurality of droplets, at least some of the particles comprising a first oligonucleotide sequence comprising a gene- specific reverse transcription primer and a first barcode encoding the gene- specific reverse transcription primer and a second oligonucleotide sequence attached to an antibody and comprising a second barcode encoding the antibody, such that the first barcode and/or the second barcode contained within a droplet is distinguishable from the first barcodes and/or the second barcodes contained in other droplets of the plurality of droplets; releasing the antibody from the particle internally of the droplet; and lysing at least some of the cells within the droplets to release nucleic acid from the cell internally of the droplet.

In another set of embodiments, the method comprises encapsulating a plurality of cells in a plurality of droplets; adding a plurality of particles to the plurality of droplets, at least some of the particles comprising a first oligonucleotide sequence comprising a gene- specific reverse transcription primer and a first barcode encoding the gene- specific reverse transcription primer and a second oligonucleotide sequence attached to an antibody and comprising a second barcode encoding the antibody, such that the first barcode and/or the second barcode contained within a droplet is distinguishable from the first barcodes and/or the second barcodes contained in other droplets of the plurality of droplets; releasing the antibody from the particle internally of the droplet; and causing the antibody to bind to at least some of the plurality of cells.

The method, in still another set of embodiments, includes acts of encapsulating a plurality of cells in a plurality of droplets; adding a plurality of particles to the plurality of droplets, at least some of the particles comprising a first oligonucleotide sequence comprising a gene-specific reverse transcription primer and a first barcode encoding the gene-specific reverse transcription primer and a second oligonucleotide sequence attached to an antibody and comprising a second barcode encoding the antibody, such that the first barcode and/or the second barcode contained within a droplet is distinguishable from the first barcodes and/or the second barcodes contained in other droplets of the plurality of droplets; releasing the antibody from the particle internally of the droplet; releasing nucleic acid from the cell internally of the droplet; and causing the antibody to bind to at least some of the released nucleic acid.

Another set of embodiments is generally directed to a method comprising acts of providing a plurality of nucleic-acid-coded antibodies; exposing the plurality of antibodies to a plurality of cells, encapsulating the plurality of cells and a plurality of particles containing barcode sequences in a plurality of droplets at no more than about 1 particle/droplet, lysing cells within the droplets to release cellular nucleic acid, and ligating the barcode sequences to the cellular nucleic acid and/or the antibody- attached nucleic acid sequences. In certain instances, some of the antibodies are able to bind to at least some of the cells. In some aspects, the present invention encompasses methods of making one or more of the embodiments described herein. In some aspects, the present invention encompasses methods of using one or more of the embodiments described herein.

Other advantages and novel features of the present invention will become apparent from the following detailed description of various non-limiting embodiments of the invention when considered in conjunction with the accompanying figures. In cases where the present specification and a document incorporated by reference include conflicting and/or inconsistent disclosure, the present specification shall control. If two or more documents incorporated by reference include conflicting and/or inconsistent disclosure with respect to each other, then the document having the later effective date shall control.

BRIEF DESCRIPTION OF THE DRAWINGS

Non-limiting embodiments of the present invention will be described by way of example with reference to the accompanying figures, which are schematic and are not intended to be drawn to scale. In the figures, each identical or nearly identical component illustrated is typically represented by a single numeral. For purposes of clarity, not every component is labeled in every figure, nor is every component of each embodiment of the invention shown where illustration is not necessary to allow those of ordinary skill in the art to understand the invention. In the figures:

Figs. 1A-1B illustrate a microfluidic device for creating barcoding droplets, in accordance with one embodiment of the invention; Fig. 2 illustrates an assay for determining protein and mRNA, in another embodiment of the invention;

Fig. 3 illustrates a method to produce particles having barcodes, in yet another embodiment of the invention;

Fig. 4 illustrates identity-coded antibodies, in still another embodiment of the invention;

Fig. 5 illustrates ligation of identity-coded antibodies to a particle, in another embodiment of the invention;

Fig. 6 illustrates ligation of primers to particles, in yet another embodiment of the invention;

Fig. 7 illustrates certain particles containing particles, in accordance with still another embodiment of the invention;

Fig. 8 illustrates a barcoded antibody, in another embodiment of the invention;

Fig. 9 illustrates a barcoded antibody, in yet another embodiment of the invention;

Fig. 10 illustrates a barcoded antibody, in still another embodiment of the invention;

Fig. 11 illustrates a particle in accordance with one embodiment of the invention;

Fig. 12 illustrates barcoding, in yet another embodiment of the invention; and

Fig. 13 illustrates a photocleavable linker in still another embodiment of the invention.

DETAILED DESCRIPTION

The present invention generally relates to microfluidics and, in particular, to barcoded systems with multiple information. For example, one set of embodiments is generally directed to particles, which may be contained within droplets, containing a first barcode (e.g., which can be associated with a nucleic acid) and a second barcode (e.g., which can be associated with a protein). For instance, the first barcode may be associated with a first oligonucleotide containing a gene-specific reverse transcription primer (e.g., for associating with mRNA), and/or a second barcode may be associated with a second oligonucleotide containing an antibody (e.g., for associating with a protein). In some cases, the droplet may also contain a cell or other sample to be analyzed, e.g., for DNA and/or proteins. Binding of the nucleic acids and/or proteins may then be determined, e.g., using the first and second barcodes. In some cases, a plurality of droplets containing cells, particles, etc., may be burst or otherwise combined; however, specific nucleic acids, proteins, etc., may be uniquely identified, even after combination, using the barcodes. For example, in one embodiment, as is shown in Fig. 11, a particle 5 containing a first oligonucleotide sequence 10 and a second oligonucleotide sequence 20 is shown. (Only two oligonucleotides are shown here for clarity, although in reality, multiple copies of each may be attached to the particle). The oligonucleotides may be attached, for example, covalently. In some cases, the particle is a hydrogel; other materials, dimensions, etc. are discussed in more detail below.

Oligonucleotide sequences 10 and 20 may each be selected to be able to bind to a different analyte. For example, oligonucleotide sequence 10 may contain an entity that can recognize a nucleic acid, e.g., mRNA, rRNA, DNA, etc., a protein, or the like. For instance, oligonucleotide sequence 10 may contain a first recognition entity 15 that is able to bind to a first analyte and oligonucleotide sequence 20 may contain a second recognition entity 25 that is able to bind to a second analyte. The recognition entity may be, for example, amino acids, proteins, sugars, nucleic acid such as DNA, antibodies, antigens, enzymes, or the like. The recognition entities may be the same or different, and may be independently selected. For instance, recognition entity 15 may be a nucleic acid sequence (e.g., a single-stranded nucleic acid sequence) and recognition entity 25 may be an antibody.

In some embodiments, e.g., as is shown in this figure, one or more of oligonucleotide sequences 10 and 20 may contain a barcode or a unique sequence (12 and 22, respectively). The sequence may be used for identification or other purposes, e.g., as discussed herein. For instance, a sequence may be selected such that some or most of the oligonucleotide tags have a unique sequence (or combination of sequences that is unique), but other oligonucleotide tags do not have that unique sequence or combination of sequences. In some cases, barcodes 12 and 22 are identical. However, in other embodiments, barcodes 12 and 22 may not necessarily be identical, and may be related or unrelated. In some cases, more than one barcode may be present on each of oligonucleotide sequences 10 and 20. For instance, each oligonucleotide sequence may contain a first barcode that is identical to the other

oligonucleotide sequence and a second barcode that is not identical.

Other sequences may also be present as well in one or more of oligonucleotide sequences 10 and 20. It should also be noted that oligonucleotide sequences 10 and 20 may have the same structure or geometries, or different structures or geometries. For instance, the barcode sequences may be in the same or different locations within the oligonucleotide sequences. As non-limiting examples, one or more of the oligonucleotide sequences may contain spacers, cleavable linkers, e.g., photocleavable linkers, primers, nonsense or random sequences, or the like, e.g., as discussed in more detail below. It should also be noted that although only two oligonucleotide sequences 10 and 20 are shown in the figure, this is by way of example only and in some cases, more than two unique oligonucleotide sequences may be present, e.g., that are able to bind to a different analyte. Thus, for instance, a particle may contain sequences that are structured to bind to 3, 4, 5, or more different analytes.

In some cases, binding of an analyte to a recognition entity of an oligonucleotide sequence may occur while the oligonucleotide sequence is attached to or otherwise associated with a particle. However, in some cases, the oligonucleotide sequence (or portion thereof) may be released (e.g., cleaved) from the particle prior to binding, for example, using a suitable cleavable linker such as those discussed herein. For instance, in one set of embodiments, the particles may be contained within droplets, and the oligonucleotide sequence (or portion thereof) may be released into droplet after containing the particle within droplets. Thus, for example, as is shown in Fig.

Turning now to Fig. 12, an example of another aspect of the invention is now provided. However, it should be understood that this is by way of example only; other examples and embodiments of the invention are discussed in further detail below. In the non- limiting example of Fig. 12, a population of cells 10 is desired to be analyzed, e.g., by sequencing their DNA, by identifying certain proteins or genes that may be suspected of being present in at least some of the cells, by determining their mRNA or transcriptome, or the like. Although cells are used in this example as a source of nucleic acid material, this is by way of example, and in other embodiments, the nucleic acid may be introduced into the droplets from other sources, or using other techniques.

The cells may first be encapsulated in a series of microfluidic droplets 40. Those of ordinary skill in the art will be aware of techniques for encapsulating cells within

microfluidic droplets; see, for example, U.S. Pat. Nos. 7,708,949, 8,337,778, 8,765,485, or

Int. Pat. Apl. Pub. Nos. WO 2004/091763 and WO 2006/096571, each incorporated herein by reference. In some cases, the cells may be encapsulated at a density of less than 1 cell/droplet (and in some cases, much less than 1 cell/droplet) to ensure that most or all of the droplets have only zero or one cell present in them. Thus, as is shown in Fig. 12, each of droplets 41, 42, 43... have either zero or one cell present in them.

Also encapsulated in the droplets are oligonucleotide tags 20, present on particles 30. Particles 30 may be, for example, microparticles, and may be a hydrogel or a polymeric particle, or other types of particles such as those described herein. The particles and the cells may be encapsulated within the droplets simultaneously or sequentially, in any suitable order. In one set of embodiments, each particle contains a unique oligonucleotide tag, although there may be multiple copies of the tag present on a particle. For instance, each of the oligonucleotide tags may have one or more unique sequences or "barcodes" that are present. Thus, for example, particle 31 contains only copies of oligonucleotide tag 21, particle 32 contains only copies of oligonucleotide tag 22, particle 33 contains only copies of

oligonucleotide tag 33, etc. In some cases, the particles may be present in the droplets at a density of less than 1 particle/droplet (and in some cases, much less than 1 particle/droplet) to ensure that most or all of the droplets have only zero or one particle present in them. In addition, in certain embodiments, the oligonucleotide tags may be cleavable or otherwise releasable from the particles.

It should be noted that according to certain embodiments of the invention, the oligonucleotide tags are initially attached to particles to facilitate the introduction of only one unique oligonucleotide tag to each droplet, as is shown in Fig. 12. (In other embodiments, however, a plurality of oligonucleotide tags may be present, e.g., containing the same unique barcode.) For example, if the particles are present in the droplets at a density of less than 1 particle/droplet, then most or all of the droplets will each have only a single particle, and thus only a single type of oligonucleotide tag, that is present. Accordingly, as is shown in Fig. 12, the oligonucleotide tags may be cleaved or otherwise released from the particles, e.g., such that each droplet 41, 42, 43, ... contains a unique oligonucleotide tag 21, 22, 23, ... that is different than the other oligonucleotide tags that may be present in the other droplets. Thus, each oligonucleotide tag present within a droplet will be distinguishable from the

oligonucleotide tags that are present in the other droplets. Although light (hv) is used in Fig. 12 to cleave the oligonucleotides from the particles, it should be understood that this is by way of example only, and that other methods of cleavage or release can also be used, e.g., as discussed herein. For example, in one set of embodiments, agarose particles containing oligonucleotides (e.g., physically) may be used, and the oligonucleotides may be released by heating the agarose, e.g., until the agarose at least partially liquefies or softens.

In some cases, the cells are lysed to release nucleic acid or other materials 51, 52, 53, ... from the cells. For example, the cells may be lysed using chemicals or ultrasound. The cells may release, for instance, DNA, RNA, mRNA, proteins, enzymes or the like. In some cases, the nucleic acids that are released may optionally undergo amplification, for example, by including suitable reagents specific to the amplification method. Examples of

amplification methods known to those of ordinary skill in the art include, but are not limited to, polymerase chain reaction (PCR), reverse transcriptase (RT) PCR amplification, in vitro transcription amplification (IVT), multiple displacement amplification (MDA), or quantitative real-time PCR (qPCR).

Some or all of the nucleic acid or other material 51, 52, 53, ... may be associated with the oligonucleotide tags present in the droplets, e.g., by covalently bonding. For example, the nucleic acid or other material 51, 52, 53 may be ligated or enzymatically attached to the oligonucleotide tags present in the droplets. Thus, as is shown in Fig. 12, droplet 41 exhibits nucleic acids 51 attached to oligonucleotide tags 21, droplet 42 exhibits nucleic acids 52 attached to oligonucleotide tags 22, droplet 43 exhibits nucleic acids 53 attached to oligonucleotide tags 23, etc. Thus, the nucleic acids within each droplet are distinguishable from the nucleic acids within the other droplets of the plurality of droplets 50 by way of the oligonucleotide tags, which are unique to each droplet in this example.

It should also be understood that although Fig. 12 depicts cleavage of the

oligonucleotide tags from the particles followed by lysis of the cells, in other embodiments, these need not necessarily occur in this order. For example, cell lysis may occur after cleavage, or both may occur simultaneously.

Droplet 41, 42, 43, ... may then be "burst" or "broken" to release their contents, and in some cases, the nucleic acids present in each droplet may be combined or pooled together, as is shown in Fig. 12. However, since the nucleic acids are labeled by the different

oligonucleotide tags, the nucleic acids from one droplet (i.e., from one cell) can still be distinguished from those from other droplets (or other cells) using the oligonucleotide tags. Accordingly, subsequent analysis (e.g., sequencing) of the combined pool of nucleic acids may be performed, and the source of each nucleic acid (e.g., individual cells) may be determined be determining the different oligonucleotide tags.

Thus, for example, a population of normal cells and cancer cells (e.g., arising from a tissue sample or biopsy) may be analyzed in such a fashion, and the cancer cells may be identified as having abnormal DNA, even if present in a large pool of normal cells. For example, due to the ability to track DNA on a cellular level using the oligonucleotide tags, the abnormal DNA can still be identified even if outnumbered by a large volume of normal DNA. As other non-limiting examples, stem cells may be isolated from normal cells, or the isolation of rare cell types in a population of interest may be performed.

In another aspect, the present invention provides systems and methods for the parallel capture and barcoding of DNA or RNA from large numbers of cells, e.g., for the purpose of profiling cell populations, or other purposes such as those described herein. In some embodiments, this relies on the encapsulation of barcoded nucleic acids or other suitable oligonucleotide tags, e.g., attached to particles or microspheres (for example, hydrogel or polymer microspheres) together with cells and/or other reagents that may be used for RNA and/or DNA capture and/or amplification.

In one set of embodiments, the contents arising from substantially each individual cell may be labeled, e.g., with a unique barcode (which may be randomly determined, or determined as discussed herein), which may allow in some cases for hundreds, thousands, tens of thousands, or even hundreds of thousands or more of different cells to be barcoded or otherwise labeled in a single experiment, e.g., to determine or define the heterogeneity between cells in a population or for screening cell populations, etc. Other purposes have been described herein.

In one set of embodiments, a microfluidic system is used to capture single cells into individual droplets (e.g., 50 pL to 10 nL volume), e.g., in a single reaction vessel. Each cell may be lysed and its RNA and/or DNA uniquely barcoded or labeled with a droplet- specific barcode, e.g., through an enzymatic reaction, through ligation, etc. Examples of microfluidic systems, including those with dimensions other than these, are also provided herein. Some embodiments might also be used, in some embodiments, to quantify protein abundance in single cells in parallel to RNA or DNA, e.g., by first treating cells with DNA-tagged antibodies, in which case the DNA tags can be similarly barcoded with a droplet- specific barcode. Once the cell components in droplets have been barcoded, the droplets may be broken or burst and the sample can be processed, e.g., in bulk, for high-throughput sequencing or other applications. After sequencing, the data can be split or otherwise analyzed according to the DNA barcodes.

To perform parallel barcoding of DNA, RNA and/or DNA-antibody tags in single cells, a single hydrogel or polymer particle or microsphere may be encapsulated into each droplet together with biological or chemical reagents and a cell, in accordance with one set of embodiments. Particles or microspheres carrying a high concentration (e.g. 1 to 100 micromolar) of DNA fragments (hereafter "primers") may encode (a) a barcode sequence selected at random from a pool of, e.g., at least 10,000 barcodes (or at least 30,000 barcodes, at least 100,000 barcodes, at least 300,000 barcodes, or at least 1,000,000 barcodes, etc.), with the same barcode found on all nucleic acid fragments on the particles or microspheres; and/or encode (b) one or more a primer sequences used for hybridization and capture of DNA or RNA. The number of distinct barcodes may be at least 10-fold, and in some cases at least 100-fold, larger than the number of cells to be captured, in order to reduce the possibility of two or more cells occupying different droplets with particles or microspheres that carry the same barcode. For example, with 150,000 barcodes and 1,000 cells, on average just 3 cells will acquire a duplicate barcode (resulting in 997 detected barcodes).

In some embodiments, the encapsulation conditions are chosen such droplets contain one particle (or microsphere) and one cell. The presence of empty droplets and/or droplets with single particles but without cells, and/or droplets with cells but without particles, may not substantially affect performance. However, the presence of two or more particles or two or more cells in one droplet may lead to errors that can be difficult to control for, so the incidence of such events is kept to minimum in some instances, for example, less than about 10% or less than about 5%. Excepting the cells and particles, other biological and chemical reagents may be distributed equally among the droplets. The co-encapsulated cells and particles may be collected and processed according to the aim of the particular application. For example, in one particular embodiment, the DNA or RNA of single cells is captured by the primers introduced with particle, and may then be converted into barcoded

complimentary DNA upon reverse transcription or other DNA polymerization reaction.

After purification and optional DNA amplification, the base composition and barcode identity of cellular nucleic acids may be determined, for instance, by sequencing or other techniques. Alternatively, in some embodiments, primers introduced with particles or microspheres can be used for amplification of specific nucleic acid sequences from a genome.

In some embodiments, the barcoded primers introduced using particles or

microspheres can be cleaved therefrom by, e.g., light, chemical, enzymatic or other techniques, e.g., to improve the efficiency of priming enzymatic reactions in droplets.

However, the cleavage of the primers can be performed at any step or point, and can be defined by the user in some cases. Such cleavage may be particularly important in certain circumstances and/or conditions; for example, some fraction of RNA and DNA molecules in single cells might be very large, or might be associated in complexes and therefore will not diffuse efficiently to the surface or interior of the particle or microsphere. However, in other embodiments, cleavage is not essential.

Techniques such as these can be used to analyze, for example, genomes, single nucleotide polymorphisms, specific gene expression levels, non-coding RNA, the whole transcriptome (or a portion thereof), entire genes or their sections, etc. However, the invention should not be limited to only these applications.

In one non-limiting embodiment, the 3' end of a barcoded primer is terminated with a poly-T sequences that may be used to capture cellular mRNA for whole-transcriptome profiling. The resulting library combining all cells can optionally be enriched using PCR- based methods or using hybridization capture-based methods (such as Agilent SureSelect), e.g., to allow sequencing of only a sub-set of genes of interest. In another embodiment, the 3' end of the barcoded primers may terminate with a random DNA sequence that can be used to capture the RNA in the cell. In another embodiment, the 3' end of the barcoded primers may terminate with a specific DNA sequence, e.g., that can be used to capture DNA or RNA species ("genes") of interest, or to hybridize to a DNA probe that is delivered into the droplets in addition to the particles or microspheres, for example, together with the enzyme reagents. In another embodiment, a particle or microsphere may carry a number of different primers to target several genes of interest. Yet another embodiment is directed to

optimization of the size of droplets and the concentration of reaction components required for droplet barcoding.

Still another aspect of the present invention is generally directed to creating barcoded nucleic acids attached to the particles or microspheres. The nucleic acids may be attached to the surface of the particles or microspheres, or in some cases, attached or incorporated within the particle. For instance, the nucleic acids may be incorporated into the particle during formation of the particle, e.g., physically and/or chemically.

For example, one set of embodiments is generally directed to creating particles or microspheres carrying nucleic acid fragments (each encoding a barcode, a primer, and/or other sequences possibly used for capture, amplification and/or sequencing of nucleic acids). Microspheres may refer to a hydrogel particle (polyacrylamide, agarose, etc.), or a colloidal particle (polystyrene, magnetic or polymer particle, etc.) of 1 to 500 micrometer in size, or other dimensions such as those described herein. The microspheres may be porous in some embodiments. Other suitable particles or microspheres that can be used are discussed in more detail herein. In some cases, the barcodes may be synthesized on a particle (e.g., a polymeric particle, a gel particle, a plastic bead, etc.), and in some cases, encapsulated within a droplet, such as is discussed herein.

The preparation of DNA-carrying particles or microspheres, in some cases, may rely on the covalent attachment or other techniques of incorporation of an initial DNA

oligonucleotide to the particles or microspheres, followed by enzymatic extension of each oligonucleotide by one or more barcodes selected, e.g., at random, from a pre-defined pool. The final number of possible unique barcodes may depend in some cases on the size of the pre-defined barcode pool and/or on the number of extension steps. For example, using a pool of 384 pre-defined barcodes and 2 extension steps, each particle or microsphere carries one of 384 =147,456 possible barcodes; using 3 extension steps, each particle or microsphere carries one of 384 =56,623,104 possible barcodes; and so on. Other numbers of steps may also be used in some cases; in addition, each pool may have various numbers of pre-defined barcodes (not just 384), and the pools may have the same or different numbers of pre-defined barcodes. The pools may include the same and/or different sequences.

Accordingly, in some embodiments, the possible barcodes that are used are formed from one or more separate "pools" of barcode elements that are then joined together to produce the final barcode, e.g., using a split- and-pool approach. A pool may contain, for example, at least about 300, at least about 500, at least about 1,000, at least about 3,000, at least about 5,000, or at least about 10,000 distinguishable barcodes. For example, a first pool may contain x₁ elements and a second pool may contain x₂ elements; forming a barcode containing an element from the first pool and an element from the second pool may yield, e.g., xix₂ possible barcodes that could be used. It should be noted that xi and x₂ may or may not be equal. This process can be repeated any number of times; for example, the barcode may include elements from a first pool, a second pool, and a third pool (e.g., producing xix₂x₃ possible barcodes), or from a first pool, a second pool, a third pool, and a fourth pool (e.g., producing xix₂x₃x₄ possible barcodes), etc. There may also be 5, 6, 7, 8, or any other suitable number of pools. Accordingly, due to the potential number of combinations, even a relatively small number of barcode elements can be used to produce a much larger number of distinguishable barcodes.

In some cases, such use of multiple pools, in combination, may be used to create substantially large numbers of useable barcodes, without having to separately prepare and synthesize large numbers of barcodes individually. For example, in many prior art systems, requiring 100 or 1,000 barcodes would require the individual synthesis of 100 or 1,000 barcodes. However, if larger numbers of barcodes are needed, e.g., for larger numbers of cells to be studied, then correspondingly larger numbers of barcodes would need to be synthesized. Such systems become impractical and unworkable at larger numbers, such as 10,000, 100,000, or 1,000,000 barcodes. However, by using separate "pools" of barcodes, larger numbers of barcodes can be achieved without necessarily requiring each barcode to be individually synthesized. As a non-limiting example, a first pool of 1,000 distinguishable barcodes (or any other suitable number) and a second pool of 1,000 distinguishable barcodes can be synthesized, requiring the synthesis of 2,000 barcodes (or only 1,000 if the barcodes are re-used in each pool), yet they may be combined to produce 1,000 x 1,000 = 1,000,000 distinguishable barcodes, e.g., where each distinguishable barcode comprises a first barcode taken from the first pool and a second barcode taken from the second pool. Using 3, 4, or more pools to assemble the barcode may result in even larger numbers of barcodes that may be prepared, without substantially increasing the total number of distinguishable barcodes that would need to be synthesized.

In some aspects, the DNA fragments or oligonucleotides can be released from the particles or microspheres using a variety of techniques including light, temperature, chemical, and/or enzymatic treatment. For example, with light, nucleic acid fragments may be released at a selected time and/or under desirable conditions, thus providing flexibility for their use.

In some embodiments, the particles or microspheres can be stored for long periods of time and used as a reagent for subsequent applications.

In yet another aspect, the present invention provides systems and methods for the parallel capture, barcoding and quantification of a panel of tens to hundreds, or more, of specific DNA and/or RNA sequences from large numbers of single cells, e.g., for the purpose of profiling cell populations or other purposes. Certain embodiments rely on encapsulation of barcoded nucleic acids, e.g., attached to particles such as hydrogel or polymer microspheres, together with cells and/or other reagents for, for example, RNA and/or DNA capture and amplification.

In some cases, systems and methods for labeling specific sets of genes (e.g., tens, or hundreds of genes, or more in some cases) arising from individual cells with a unique, random barcode, allowing hundreds, thousands, or even hundreds of thousands or more of different cells to be labeled or barcoded, e.g., in a single experiment, for the purpose of defining the heterogeneity between cells in a population or for screening cell populations, or for other purposes.

For example, in situations where a large number of cells are to be analyzed through multiplexed high-throughput sequencing, it may be desirable in some embodiments to focus on a sub- set of genes of interest, for example between tens to hundreds of genes, rather than whole-transcriptome or whole-genome capture and sequencing.

Some embodiments are directed to the parallel barcoding of the contents of cells focusing on specific sequences of cellular DNA or RNA. These may include, for example, the synthesis of DNA-barcoded microspheres (or other particles), and/or the use of such microspheres for the capture and barcoding of single cells in individual droplets (for example, 50 pL to 10 nL in volume, or other volumes described herein), e.g., in a single reaction vessel. In some cases, substantially each cell may be lysed and its RNA and/or DNA uniquely barcoded (tagged) with a droplet- specific nucleic acid barcode, e.g., through an enzymatic reaction. In some embodiments, modifying the DNA-barcoded microspheres may be performed in such a way that they target only a specific panel of DNA sequences, rather than either using one sequence of interest or using random sequences. This may allow a high concentration of sequence-specific barcoded primers to be delivered into each droplet, which may, in some instances, allow that the enzymatic barcoding and synthesis of complementary DNA occurs primarily for the sequences of interest. This may be used, for example, with any enzymatic approach in which a panel of sequence- specific primers can be used to capture genes of interest.

Some embodiments of the invention may be used to quantify protein abundance in single cells in parallel to RNA or DNA, for example, by first treating cells with DNA-tagged antibodies, in which case one or more of the sequences or oligonucleotides on the particle or microsphere can be made complementary to the DNA tags delivered by the antibodies. In some cases, once the cell components in droplets have been barcoded, the droplets can be broken or burst and the sample can be processed, e.g., in bulk, for applications such as high- throughput sequencing. After sequencing, the data may be split, in certain embodiments, according to the DNA barcodes thus providing information about the type, sequence, molecule count, origin of nucleic acids and/or proteins of interest, or the like.

The above discussions are non-limiting examples of various embodiments of the present invention. However, other embodiments are also possible. Accordingly, more generally, various aspects of the invention are directed to various systems and methods for systems and methods for labeling nucleic acids within microfluidic droplets, as discussed below.

In one aspect, the present invention is generally directed to systems and methods for labeling nucleic acids within a population of droplets, e.g., microfluidic droplets. In some cases, the microfluidic droplets may have an average diameter of the droplets of less than about 1 mm and/or the microfluidic droplets may be substantially monodisperse, e.g., as discussed herein. However, in other embodiments, the droplets may not be substantially monodisperse.

In some cases, an oligonucleotide tag comprising DNA and/or other nucleic acids may be attached to particles and delivered to the droplets. In some cases, the oligonucleotide tags are attached to particles to control their delivery into droplets, e.g., such that a droplet will typically have at most one particle in it. In some cases, upon delivery into a droplet, the oligonucleotide tags may be removed from the particle, e.g., by cleavage, by degrading the particle, etc. However, it should be understood that in other embodiments, a droplet may contain 2, 3, or any other number of particles, which may have oligonucleotide tags that are the same or different.

The oligonucleotide tags may be of any suitable length or comprise any suitable number of nucelotides. The oligonucleotide tags may comprise DNA, RNA, and/or other nucleic acids such as PNA, and/or combinations of these and/or other nucleic acids. In some cases, the oligonucleotide tag is single stranded, although it may be double stranded in other cases. For example, the oligonucleotide tag may have a length of at least about 10 nt, at least about 30 nt, at least about 50 nt, at least about 100 nt, at least about 300 nt, at least about 500 nt, at least about 1000 nt, at least about 3000 nt, at least about 5000 nt, at least about 10,000 nt, etc. In some cases, the oligonucleotide tag may have a length of no more than about

10,000 nt, no more than about 5000 nt, no more than about 3000 nt, no more than about 1000 nt, no more than about 500 nt, no more than about 300 nt, no more than about 100 nt, no more than about 50 nt, etc. Combinations of any of these are also possible, e.g., the oligonucleotide tag may be between about 10 nt and about 100 nt. The length of the oligonucleotide tag is not critical, and a variety of lengths may be used in various

embodiments.

The oligonucleotide tag may contain a variety of sequences. For example, the oligonucleotide tag may contain one or more primer sequences, one or more unique or "barcode" sequences, one or more promoter sequences, one or more spacer sequences, or the like. The oligonucleotide tag may also contain, in some embodiments one or more cleavable spacers, e.g., photocleavable linker. The oligonucleotide tag may be attached to a particle chemically (e.g., via a linker) or physically (e.g., without necessarily requiring a linker), e.g., such that the oligonucleotide tags can be removed from the particle via cleavage. Other examples include portions that may be used to increase the bulk of the oligonucleotide tag (e.g., using specific sequences or nonsense sequences), to facilitate handling (for example, a tag may include a poly-A tail), to increase selectivity of binding (e.g., as discussed below), to facilitate recognition by an enzyme (e.g., a suitable ligase), to facilitate identification, or the like. Examples of these and/or other sequences are described in further detail herein.

As an example, in some embodiments, the oligonucleotide tags may comprise a "barcode" or a unique sequence. The sequence may be selected such that some or most of the oligonucleotide tags (e.g., present on a particle and/or in a droplet) have the unique sequence (or combination of sequences that is unique), but other oligonucleotide tags (e.g., on other particles or droplets) do not have the unique sequence or combination of sequences. Thus, for example, the sequences may be used to uniquely identify or distinguish a droplet, or nucleic acid contained arising from the droplet (e.g., from a lysed cell) from other droplets, or other nucleic acids (e.g., released from other cells) arising from other droplets.

The sequences may be of any suitable length. The length of the barcode sequence is not critical, and may be of any length sufficient to distinguish the barcode sequence from other barcode sequences. One, two, or more "barcode" sequence may be present in an oligonucleotide tag. A barcode sequence may have a length of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nt. More than 25 nucleotides may also be present in some cases.

In some cases, the unique or barcode sequences may be taken from a "pool" of potential barcode sequences. If more than one barcode sequence is present in an

oligonucleotide tag, the barcode sequences may be taken from the same, or different pools of potential barcode sequences. The pool of sequences may be selected using any suitable technique, e.g., randomly, or such that the sequences allow for error detection and/or correction, for example, by being separated by a certain distance (e.g., Hamming distance) such that errors in reading of the barcode sequence can be detected, and in some cases, corrected. The pool may have any number of potential barcode sequences, e.g., at least 100, at least 300, at least 500, at least 1,000, at least 3,000, at least 5,000, at least 10,000, at least 30,000, at least 50,000, at least 100,000, at least 300,000, at least 500,000, or at least

1,000,000 barcode sequences.

In some cases, the oligonucleotide tag may contain one or more promoter sequences, e.g., to allow for production of the tags, to allow for enzymatic amplification, or the like. Those of ordinary skill in the art will be aware of primer sequences, e.g., P5 or P7. Many such primer sequences are available commercially. Examples of promoters include, but are not limited to, T7 promoters, T3 promoters, or SP6 promoters.

In some cases, the oligonucleotide tag may contain one or more primer sequences.

Typically, a primer is a single- stranded or partially double-stranded nucleic acid (e.g., DNA) that serves as a starting point for nucleic acid synthesis, allowing polymerase enzymes such as nucleic acid polymerase to extend the primer and replicate the complementary strand. A primer may be complementary to and to hybridize to a target nucleic acid. In some embodiments, a primer is a synthetic primer. In some embodiments, a primer is a non- naturally-occurring primer. A primer typically has a length of 10 to 50 nucleotides. For example, a primer may have a length of 10 to 40, 10 to 30, 10 to 20, 25 to 50, 15 to 40, 15 to 30, 20 to 50, 20 to 40, or 20 to 30 nucleotides. In some embodiments, a primer has a length of 18 to 24 nucleotides. Examples of primers include, but are not limited to, P5 primer, P7 primer, PE1 primer, PE2 primer, A19 primer, or other primers discussed herein.

In some cases, the oligonucleotide tag may contain nonsense or random sequences, e.g., to increase the mass or size of the oligonucleotide tag. The random sequence can be of any suitable length, and there may be one or more than one present. As non-limiting examples, the random sequence may have a length of 10 to 40, 10 to 30, 10 to 20, 25 to 50, 15 to 40, 15 to 30, 20 to 50, 20 to 40, or 20 to 30 nucleotides.

In some cases, the oligonucleotide tag may comprise one or more sequences able to specifically bind a gene or other entity. For example, in one set of embodiments, the oligonucleotide tag may comprise a sequence able to recognize mRNA, e.g., one containing a poly-T sequence (e.g., having several T's in a row, e.g., 4, 5, 6, 7, 8, or more T's).

In one set of embodiments, the oligonucleotide tag may contain one or more cleavable linkers, e.g., that can be cleaved upon application of a suitable stimulus. For example, the cleavable sequence may be a photocleavable linker that can be cleaved by applying light or a suitable chemical or enzyme. A non-limiting example of a photocleavable linker can be seen in Fig. 13. In some cases, for example, a plurality of particles (for instance, containing oligonucleotide tags on their surfaces) may be prepared and added to droplets, e.g., such that, on average, each droplet contains one particle, or less (or more) in some cases. After being added to the droplet, the oligonucleotide tags may be cleaved from the particles, e.g., using light or other suitable cleavage techniques, to allow the oligonucleotide tags to become present in solution, i.e., within the interior of the droplet. In such fashion, oligonucleotide tags can be easily loaded into droplets by loading of the particles into the droplets in some embodiments, then cleaved off to allow the oligonucleotide tags to be in solution, e.g., to interact with nucleotides or other species, such as is discussed herein.

In addition, in one set of embodiments, the oligonucleotide tag may comprise an antibody, e.g., that can specifically bind to a protein suspected of being present in the cell (or droplet). For example, the droplet may contain one or more antibodies tagged with an oligonucleotide tag as described herein.

The oligonucleotide tag may be attached to a particle, e.g., as discussed herein. In some embodiments, a particle may comprise only one oligonucleotide tag, although multiple copies of the oligonucleotide tag may be present on the particle; other particles may comprise different oligonucleotide tags that are distinguishable, e.g., using the barcode sequences described herein. Any suitable method may be used to attach the oligonucleotide tag to the particle. The exact method of attachment is not critical, and may be, for instance, chemical or physical. For example, the oligonucleotide tag may be covalently bonded to the particle via a biotin-steptavidin linkage, an amino linkage, or an acrylic phosphoramidite linkage. See, e.g., Fig. 13 for an example of an acrylic phosphoramidite linkage. In another set of embodiments, the oligonucleotide may be incorporated into the particle, e.g., physically, where the oligonucleotide may be released by altering the particle. Thus, in some cases, the oligonucleotide need not have a cleavable linkage. For instance, in one set of embodiments, an oligonucleotide may be incorporated into particle, such as an agarose particle, upon formation of the particle. Upon degradation of the particle (for example, by heating the particle until it begins to soften, degrade, or liquefy), the oligonucleotide may be released from the particle.

The particle is a microparticle in certain aspects of the invention. The particle may be of any of a wide variety of types; as discussed, the particle may be used to introduce a particular oligonucleotide tag into a droplet, and any suitable particle to which

oligonucleotide tags can associate with (e.g., physically or chemically) may be used. The exact form of the particle is not critical. The particle may be spherical or non- spherical, and may be formed of any suitable material. In some cases, a plurality of particles is used, which have substantially the same composition and/or substantially the same average diameter. The "average diameter" of a plurality or series of particles is the arithmetic average of the average diameters of each of the particles. Those of ordinary skill in the art will be able to determine the average diameter (or other characteristic dimension) of a plurality or series of particles, for example, using laser light scattering, microscopic examination, or other known techniques. The average diameter of a single particle, in a non- spherical particle, is the diameter of a perfect sphere having the same volume as the non-spherical particle. The average diameter of a particle (and/or of a plurality or series of particles) may be, for example, less than about 1 mm, less than about 500 micrometers, less than about 200 micrometers, less than about 100 micrometers, less than about 75 micrometers, less than about 50 micrometers, less than about 25 micrometers, less than about 10 micrometers, or less than about 5 micrometers in some cases. The average diameter may also be at least about 1 micrometer, at least about 2 micrometers, at least about 3 micrometers, at least about 5 micrometers, at least about 10 micrometers, at least about 15 micrometers, or at least about 20 micrometers in certain cases. The particle may be, in one set of embodiments, a hydrogel particle. See, e.g., Int. Pat. Apl. Pub. No. WO 2008/109176, entitled "Assay and other reactions involving droplets" (incorporated herein by reference) for examples of hydrogel particles, including hydrogel particles containing DNA. Examples of hydrogels include, but are not limited to agarose or acrylamide -based gels, such as polyacrylamide, poly-N-isopropylacrylamide, or poly N- isopropylpolyacrylamide. For example, an aqueous solution of a monomer may be dispersed in a droplet, and then polymerized, e.g., to form a gel. Another example is a hydrogel, such as alginic acid that can be gelled by the addition of calcium ions. In some cases, gelation initiators (ammonium persulfate and TEMED for acrylamide, or Ca²⁺ for alginate) can be added to a droplet, for example, by co-flow with the aqueous phase, by co-flow through the oil phase, or by coalescence of two different drops, e.g., as discussed in U.S. Patent

Application Serial No. 11/360,845, filed February 23, 2006, entitled "Electronic Control of Fluidic Species," by Link, et ah, published as U.S. Patent Application Publication No.

2007/000342 on January 4, 2007; or in U.S. Patent Application Serial No. 11/698,298, filed January 24, 2007, entitled "Fluidic Droplet Coalescence," by Ahn, et al. ; each incorporated herein by reference in their entireties.

In another set of embodiments, the particles may comprise one or more polymers. Exemplary polymers include, but are not limited to, polystyrene (PS), polycaprolactone (PCL), polyisoprene (PIP), poly(lactic acid), polyethylene, polypropylene, polyacrylonitrile, polyimide, polyamide, and/or mixtures and/or co-polymers of these and/or other polymers. In addition, in some cases, the particles may be magnetic, which could allow for the magnetic manipulation of the particles. For example, the particles may comprise iron or other magnetic materials. The particles could also be functionalized so that they could have other molecules attached, such as proteins, nucleic acids or small molecules. Thus, some embodiments of the present invention are directed to a set of particles defining a library of, for example, nucleic acids, proteins, small molecules, or other species such as those described herein. In some embodiments, the particle may be fluorescent.

In one set of embodiments, droplets are formed containing a cell or other source of nucleic acid, and a particle, e.g., comprising an oligonucleotide tag as described above. Any suitable method may be chosen to create droplets, and a wide variety of different techniques for forming droplets will be known to those of ordinary skill in the art. For example, a junction of channels may be used to create the droplets. The junction may be, for instance, a T-junction, a Y-junction, a channel- within-a-channel junction (e.g., in a coaxial arrangement, or comprising an inner channel and an outer channel surrounding at least a portion of the inner channel), a cross (or "X") junction, a flow-focusing junction, or any other suitable junction for creating droplets. See, for example, International Patent Application No.

PCT/US2004/010903, filed April 9, 2004, entitled "Formation and Control of Fluidic Species," by Link, et al, published as WO 2004/091763 on October 28, 2004, or

International Patent Application No. PCT/US2003/020542, filed June 30, 2003, entitled "Method and Apparatus for Fluid Dispersion," by Stone, et al., published as WO

2004/002627 on January 8, 2004, each of which is incorporated herein by reference in its entirety. In some embodiments, the junction may be configured and arranged to produce substantially monodisperse droplets. The droplets may also be created on the fluidic device, and/or the droplets may be created separately then brought to the device.

If cells are used, the cells may arise from any suitable source. For instance, the cells may be any cells for which nucleic acid from the cells is desired to be studied or sequenced, etc., and may include one, or more than one, cell type. The cells may be for example, from a specific population of cells, such as from a certain organ or tissue (e.g., cardiac cells, immune cells, muscle cells, cancer cells, etc.), cells from a specific individual or species (e.g., human cells, mouse cells, bacteria, etc.), cells from different organisms, cells from a naturally- occurring sample (e.g., pond water, soil, etc.), or the like. In some cases, the cells may be dissociated from tissue.

In certain embodiments, the cells may be cells that have been fixed and/or

permeabilized. For instance, the cells may be fixed, where DNA and/or RNA is crosslinked to DNA-and/or RNA -binding proteins and/or permeabilized. Optionally, nuclei can be isolated from the cells. In some embodiments, identity-coded antibodies can be incubated with the cells (or isolated nuclei), optionally washed, and encapsulated at less than 1 cell (nucleus) per droplet (or other densities such as those described herein), and in some cases, with a bar-coding gel or bead, etc. as discussed herein. In some embodiments, the gel- delivered barcodes may be attached to antibody- affixed DNA and/or to the cellular nuclei acids of interest. Drops can be heated in some embodiments, to reverse crosslinks, e.g., to improve nucleic acid manipulations such as reverse transcription, restriction enzyme digest or ligation etc.

The use of fixation and permeabilization may allow, in some embodiments, antibody access to the cell interior, or to proteins that are bound to nucleic acids. In some

embodiments, fixation conditions may be optimized for particular classes of protein, e.g., DNA-binding, RNA-binding, cytoskeletal, etc. In addition, certain embodiments of the invention involve the use of other discrete compartments, for example, microwells of a microwell plate, individual spots on a slide or other surface, or the like. In some cases, each of the compartments may be in a specific location that will not be accidentally mixed with other compartments. The compartments may be relatively small in some cases, for example, each compartment may have a volume of less than about 1 ml, less than about 300 microliters, less than about 100 microliters, less than about 30 microliters, less than about 10 microliters, less than about 3 microliters, less than about 1 microliter, less than about 500 nl, less than about 300 nl, less than about 100 nl, less than about 50 nl, less than about 30 nl, or less than about 10 nl.

In one set of embodiments, the droplets (or other compartments) are loaded such that, on the average, each droplet has less than 1 particle in it. For example, the average loading rate may be less than about 1 particle/droplet, less than about 0.9 particles/droplet, less than about 0.8 particles/droplet, less than about 0.7 particles/droplet, less than about 0.6 particles/droplet, less than about 0.5 particles/droplet, less than about 0.4 particles/droplet, less than about 0.3 particles/droplet, less than about 0.2 particles/droplet, less than about 0.1 particles/droplet, less than about 0.05 particles/droplet, less than about 0.03 particles/droplet, less than about 0.02 particles/droplet, or less than about 0.01 particles/droplet. In some cases, lower particle loading rates may be chosen to minimize the probability that a droplet will be produced having two or more particles in it. Thus, for example, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, or at least about 99% of the droplets may contain either no particle or only one particle.

Similarly, in some embodiments, the droplets (or other compartments) are loaded such that, on the average, each droplet has less than 1 cell in it. For example, the average loading rate may be less than about 1 cell/droplet, less than about 0.9 cells/droplet, less than about 0.8 cells/droplet, less than about 0.7 cells/droplet, less than about 0.6 cells/droplet, less than about 0.5 cells/droplet, less than about 0.4 cells/droplet, less than about 0.3 cells/droplet, less than about 0.2 cells/droplet, less than about 0.1 cells/droplet, less than about 0.05 cells/droplet, less than about 0.03 cells/droplet, less than about 0.02 cells/droplet, or less than about 0.01 cells/droplet. In some cases, lower cell loading rates may be chosen to minimize the probability that a droplet will be produced having two or more cells in it. Thus, for example, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, or at least about 99% of the droplets may contain either no cell or only one cell. In addition, it should be noted that the average rate of particle loading and the average rate of cell loading within the droplets may the same or different.

In some cases, a relatively large number of droplets may be created, e.g., at least about 10, at least about 30, at least about 50, at least about 100, at least about 300, at least about 500, at least about 1,000, at least about 3,000, at least about 5,000, at least about

10,000, at least about 30,000, at least about 50,000, at least about 100,000 droplets, etc. In some cases, as previously discussed, some or all of the droplets may be distinguishable, e.g., on the basis of the oligonucleotide tags present in at least some of the droplets (e.g., which may comprise one or more unique sequences or barcodes). In some cases, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, or at least about 99% of the droplets may be distinguishable.

In one embodiment, the cell may be an antibody- secreting cell. As an example, in some cases, antibody secreted by an encapsulated antibody-secreting cell (ASC) may inhibit viral infection of a co-encapsulated target cell. Non-limiting examples of antibody- secreting cells include hybridoma cells, B-cells, plasma cells, or the like. Inhibition of viral infection may be determined by survival of the target cell. In some cases, target cells can be genetically modified to provide a fluorescent indicator of viral infection. In some

embodiments, droplets in which target cells remain non-fluorescent are likely to contain an ASC that secretes an antibody that inhibits viral infection. Droplets in which viral infection is inhibited may be selected by microfluidic sorting so that the sequence of the neutralizing antibodies can be determined. For example, referring to Fig. 5A, a target cell, a virus, and an antibody- secreting cell may be contained within a droplet. Droplets where the antibody- secreting cells are able to at least partially neutralize the virus may be separated from those droplets where the antibody- secreting cells did not neutralize the virus. In some

embodiments, controls containing irrelevant cells (e.g., irrelevant antibody- secreting cells) may also be used to show neutralization of the viruses is due to the antibodies produced by the antibody-secreting cells.

After loading of the particles and cells into droplets, the oligonucleotide tags may be released or cleaved from the particles, in accordance with certain aspects of the invention. As noted above, any suitable technique may be used to release the oligonucleotide tags from the droplets, such as light (e.g., if the oligonucleotide tag includes a photocleavable linker), a chemical, or an enzyme, etc. If a chemical or an enzyme is used, the chemical or enzyme may be introduced into the droplet after formation of the droplet, e.g., through picoinjection or other methods such as those discussed in Int. Pat. Apl. Pub. No. WO 2010/151776, entitled "Fluid Injection" (incorporated herein by reference), through fusion of the droplets with droplets containing the chemical or enzyme, or through other techniques known to those of ordinary skill in the art.

As discussed, in certain aspects, the droplets may contain nucleic acid. The nucleic acid may arise from a cell, or from other suitable sources. In one set of embodiments, if cells are present, the cells may be lysed within the droplets, e.g., to release DNA and/or RNA from the cell, and/or to produce a cell lysate within the droplet. For instance, the cells may be lysed via exposure to a lysing chemical or a cell lysis reagent (e.g., a surfactant such as Triton-X or SDS, an enzyme such as lysozyme, lysostaphin, zymolase, cellulase,

mutanolysin, glycanases, proteases, mannase, proteinase K, etc.), or a physical condition (e.g., ultrasound, ultraviolet light, mechanical agitation, etc.). If a lysing chemical is used, the lysing chemical may be introduced into the droplet after formation of the droplet, e.g., through picoinjection or other methods such as those discussed in U.S. Pat. Apl. Ser. No. 13/379,782, filed December 21, 2011, entitled "Fluid Injection," published as U.S. Pat. Apl. Pub. No. 2012/0132288 on May 31, 2012, incorporated herein by reference in its entirety, through fusion of the droplets with droplets containing the chemical or enzyme, or through other techniques known to those of ordinary skill in the art. Lysing of the cells may occur before, during, or after release of the oligonucleotide tags from the particles. In some cases, lysing a cell will cause the cell to release its contents, e.g., cellular nucleic acids, proteins, enzymes, sugars, etc. In some embodiments, some of the cellular nucleic acids may also be joined to one or more oligonucleotide tags contained within the droplet, e.g., as discussed herein. For example, in one set of embodiments, RNA transcripts typically produced within the cells may be released and then joined to the nucleic acid tags.

In some embodiments, once released, the released nucleic acids from the cell (e.g.,

DNA and/or RNA) may be bonded to the oligonucleotide tags, e.g., covalently, through primer extension, through ligation, or the like. Any of a wide variety of different techniques may be used, and those of ordinary skill in the art will be aware of many such techniques. The exact joining technique used is not necessarily critical, and can vary between

embodiments.

For instance, in certain embodiments, the nucleic acids may be joined with the oligonucleotide tags using ligases. Non-limiting examples of ligases include DNA ligases such as DNA Ligase I, DNA Ligase II, DNA Ligase III, DNA Ligase IV, T4 DNA ligase, T7 DNA ligase, T3 DNA Ligase, E. coli DNA Ligase, Taq DNA Ligase, or the like. Many such ligases may be purchased commercially. As additional examples, in some embodiments, two or more nucleic acids may be ligated together using annealing or a primer extension method. In yet another example, the linkage may be performed using a topoisomerase.

In yet another set of embodiments, the nucleic acids may be joined with the oligonucleotide tags and/or amplified using PCR (polymerase chain reaction) or other suitable amplification techniques, including any of those recited herein. Typically, in PCR reactions, the nucleic acids are heated to cause dissociation of the nucleic acids into single strands, and a heat-stable DNA polymerase (such as Taq polymerase) is used to amplify the nucleic acid. This process is often repeated multiple times to amplify the nucleic acids.

In one set of embodiments, PCR or nucleic acid amplification may be performed within the droplets. For example, the droplets may contain a polymerase (such as Taq polymerase), and DNA nucleotides, and the droplets may be processed (e.g., via repeated heated and cooling) to amplify the nucleic acid within the droplets. The polymerase and nucleotides may be added at any suitable point, e.g., before, during, or after various nucleic acids encoding various conditions are added to the droplets. For instance, a droplet may contain polymerase and DNA nucleotides, which is fused to the droplet to allow

amplification to occur. Those of ordinary skill in the art will be aware of suitable PCR techniques and variations, such as assembly PCR or polymerase cycling assembly, which may be used in some embodiments to produce an amplified nucleic acid. Non-limiting examples of such procedures are also discussed below. In addition, in some cases, suitable primers may be used to initiate polymerization, e.g., P5 and P7, or other primers known to those of ordinary skill in the art. In some embodiments, primers may be added to the droplets, or the primers may be present on one or more of the nucleic acids within the droplets. Those of ordinary skill in the art will be aware of suitable primers, many of which can be readily obtained commercially.

In some cases, the droplets may be burst, broken, or otherwise disrupted. A wide variety of methods for "breaking" or "bursting" droplets are available to those of ordinary skill in the art, and the exact method chosen is not critical. For example, droplets contained in a carrying fluid may be disrupted using techniques such as mechanical disruption or ultrasound. Droplets may also be disrupted using chemical agents or surfactants, for example, 1H, lH,2H,2H-perfluorooctanol.

Nucleic acids (labeled with oligonucleotide tags) from different droplets may then be pooled or combined together or analyzed, e.g., sequenced, amplified, etc. The nucleic acids from different droplets, may however, remain distinguishable due to the presence of different oligonucleotide tags (e.g., containing different barcodes) that were present in each droplet prior to disruption.

For example, the nucleic acids may be amplified using PCR (polymerase chain reaction) or other amplification techniques. Typically, in PCR reactions, the nucleic acids are heated to cause dissociation of the nucleic acids into single strands, and a heat-stable DNA polymerase (such as Taq polymerase) is used to amplify the nucleic acid. This process is often repeated multiple times to amplify the nucleic acids.

In one set of embodiments, the PCR may be used to amplify the nucleic acids. Those of ordinary skill in the art will be aware of suitable PCR techniques and variations, such as assembly PCR or polymerase cycling assembly, which may be used in some embodiments to produce an amplified nucleic acid. Non-limiting examples of such procedures are also discussed below. In addition, in some cases, suitable primers may be used to initiate polymerization, e.g., P5 and P7, or other primers known to those of ordinary skill in the art. Those of ordinary skill in the art will be aware of suitable primers, many of which can be readily obtained commercially.

Other non-limiting examples of amplification methods known to those of ordinary skill in the art that may be used include, but are not limited to, reverse transcriptase (RT) PCR amplification, in vitro transcription amplification (IVT), multiple displacement amplification (MDA), or quantitative real-time PCR (qPCR).

In some embodiments, the nucleic acids may be sequenced using a variety of techniques and instruments, many of which are readily available commercially. Examples of such techniques include, but are not limited to, chain-termination sequencing, sequencing-by- hybridization, Maxam-Gilbert sequencing, dye-terminator sequencing, chain-termination methods, Massively Parallel Signature Sequencing (Lynx Therapeutics), polony sequencing, pyrosequencing, sequencing by ligation, ion semiconductor sequencing, DNA nanoball sequencing, single-molecule real-time sequencing, nanopore sequencing, microfluidic Sanger sequencing, digital RNA sequencing ("digital RNA-seq"), etc. The exact sequencing method chosen is not critical.

In addition, in some cases, the droplets may also contain one or more DNA-tagged antibodies, e.g., to determine proteins in the cell, e.g., by suitable tagging with DNA. Thus, for example, a protein may be detected in a plurality of cells as discussed herein, using DNA- tagged antibodies specific for the protein.

Additional details regarding systems and methods for manipulating droplets in a microfluidic system follow, e.g., for determining droplets (or species within droplets), sorting droplets, etc. For example, various systems and methods for screening and/or sorting droplets are described in U.S. Patent Application Serial No. 11/360,845, filed February 23, 2006, entitled "Electronic Control of Fluidic Species," by Link, et al., published as U.S.

Patent Application Publication No. 2007/000342 on January 4, 2007, incorporated herein by reference. As a non-limiting example, by applying (or removing) a first electric field (or a portion thereof), a droplet may be directed to a first region or channel; by applying (or removing) a second electric field to the device (or a portion thereof), the droplet may be directed to a second region or channel; by applying a third electric field to the device (or a portion thereof), the droplet may be directed to a third region or channel; etc., where the electric fields may differ in some way, for example, in intensity, direction, frequency, duration, etc.

In some aspects, antibodies may be used. Antibodies are generally proteins or glycoproteins having one or more polypeptides substantially encoded by immunoglobulin genes or fragments of immunoglobulin genes. The antibody may be complete or a fraction of an antibody, e.g., a Fab or Fc fragment. The antibody may be recombinant in some cases. In some cases, the antibody may be attached to or associated with one or more nucleic acids, e.g., an oligonucleotide sequence or tag such as those discussed herein. For instance, the oligonucleotide may comprise a barcode or other sequences including those discussed herein. In some cases, the oligonucleotide may comprise an overhang or unpaired portion that can be used to associate the antibody with another oligonucleotide, e.g., one attached to a particle

(e.g., through annealing or other suitable techniques). For instance, the overhang may have 3, 4, 5, 6, 7, 8, or any other number of unpaired bases, and the other oligonucleotide may have a sequence that is substantially or fully complementary to the overhang portion. In some cases, a linker may be used to attach the nucleic acid to the antibody. The oligonucleotide may also have one or more amplification sequences.

Examples of recognized immunoglobulin genes for antibodies include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively. A typical

immunoglobulin (antibody) structural unit is known to comprise a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" (about 25 kD) and one "heavy" chain (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms variable light chain (VL) and variable heavy chain (VH) refer to these light and heavy chains respectively. Antibodies exist as intact immunoglobulins or as a number of well characterized fragments produced by digestion with various peptidases.

Thus, for example, pepsin digests an antibody below (i.e. toward the Fc domain) the disulfide linkages in the hinge region to produce F(ab)'2, a dimer of Fab which itself is a light chain joined to V_H-C_H1 by a disulfide bond. The F(ab)'2 may be reduced under mild conditions to break the disulfide linkage in the hinge region thereby converting the (Fab')2 dimer into an Fab' monomer. The Fab' monomer is essentially a Fab with part of the hinge region. While various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such fragments may be synthesized de novo either chemically, by utilizing recombinant DNA methodology, or by "phage display" methods. Examples of antibodies include single chain antibodies, e.g., single chain Fv (scFv) antibodies in which a variable heavy and a variable light chain are joined together (directly or through a peptide linker) to form a continuous polypeptide.

In certain embodiments of the invention, sensors are provided that can sense and/or determine one or more characteristics of the fluidic droplets, and/or a characteristic of a portion of the fluidic system containing the fluidic droplet (e.g., the liquid surrounding the fluidic droplet) in such a manner as to allow the determination of one or more characteristics of the fluidic droplets. Characteristics determinable with respect to the droplet and usable in the invention can be identified by those of ordinary skill in the art. Non-limiting examples of such characteristics include fluorescence, spectroscopy (e.g., optical, infrared, ultraviolet, etc.), radioactivity, mass, volume, density, temperature, viscosity, pH, concentration of a substance, such as a biological substance (e.g., a protein, a nucleic acid, etc.), or the like. In another set of embodiments, an enzymatic assay may be used, e.g., including those discussed in U.S. Pat. Apl. Ser. No. 62/008,341, entitled "Protein analysis assay system"; U.S. Pat. Apl. Ser. No. 62/054,263, entitled "Systems and methods of cell-free protein synthesis in droplets and other compartments"; or U.S. Pat. Apl. Ser. No. 62/121,743, entitled "Protein synthesis in cell-free systems," each of which is incorporated herein by reference.

In some cases, the sensor may be connected to a processor, which in turn, cause an operation to be performed on the fluidic droplet, for example, by sorting the droplet, adding or removing electric charge from the droplet, fusing the droplet with another droplet, splitting the droplet, causing mixing to occur within the droplet, etc., for example, as previously described. For instance, in response to a sensor measurement of a fluidic droplet, a processor may cause the fluidic droplet to be split, merged with a second fluidic droplet, etc. One or more sensors and/or processors may be positioned to be in sensing communication with the fluidic droplet. "Sensing communication," as used herein, means that the sensor may be positioned anywhere such that the fluidic droplet within the fluidic system (e.g., within a channel), and/or a portion of the fluidic system containing the fluidic droplet may be sensed and/or determined in some fashion. For example, the sensor may be in sensing communication with the fluidic droplet and/or the portion of the fluidic system containing the fluidic droplet fluidly, optically or visually, thermally, pneumatically, electronically, or the like. The sensor can be positioned proximate the fluidic system, for example, embedded within or integrally connected to a wall of a channel, or positioned separately from the fluidic system but with physical, electrical, and/or optical communication with the fluidic system so as to be able to sense and/or determine the fluidic droplet and/or a portion of the fluidic system containing the fluidic droplet (e.g., a channel or a microchannel, a liquid containing the fluidic droplet, etc.). For example, a sensor may be free of any physical connection with a channel containing a droplet, but may be positioned so as to detect electromagnetic radiation arising from the droplet or the fluidic system, such as infrared, ultraviolet, or visible light. The electromagnetic radiation may be produced by the droplet, and/or may arise from other portions of the fluidic system (or externally of the fluidic system) and interact with the fluidic droplet and/or the portion of the fluidic system containing the fluidic droplet in such as a manner as to indicate one or more characteristics of the fluidic droplet, for example, through absorption, reflection, diffraction, refraction, fluorescence, phosphorescence, changes in polarity, phase changes, changes with respect to time, etc. As an example, a laser may be directed towards the fluidic droplet and/or the liquid surrounding the fluidic droplet, and the fluorescence of the fluidic droplet and/or the surrounding liquid may be determined. "Sensing communication," as used herein may also be direct or indirect. As an example, light from the fluidic droplet may be directed to a sensor, or directed first through a fiber optic system, a waveguide, etc., before being directed to a sensor.

Non-limiting examples of sensors useful in the invention include optical or electromagnetically-based systems. For example, the sensor may be a fluorescence sensor (e.g., stimulated by a laser), a microscopy system (which may include a camera or other recording device), or the like. As another example, the sensor may be an electronic sensor, e.g., a sensor able to determine an electric field or other electrical characteristic. For example, the sensor may detect capacitance, inductance, etc., of a fluidic droplet and/or the portion of the fluidic system containing the fluidic droplet. As used herein, a "processor" or a "microprocessor" is any component or device able to receive a signal from one or more sensors, store the signal, and/or direct one or more responses (e.g., as described above), for example, by using a mathematical formula or an electronic or computational circuit. The signal may be any suitable signal indicative of the environmental factor determined by the sensor, for example a pneumatic signal, an electronic signal, an optical signal, a mechanical signal, etc.

In one set of embodiments, a fluidic droplet may be directed by creating an electric charge and/or an electric dipole on the droplet, and steering the droplet using an applied electric field, which may be an AC field, a DC field, etc. As an example, an electric field may be selectively applied and removed (or a different electric field may be applied, e.g., a reversed electric field) as needed to direct the fluidic droplet to a particular region. The electric field may be selectively applied and removed as needed, in some embodiments, without substantially altering the flow of the liquid containing the fluidic droplet. For example, a liquid may flow on a substantially steady-state basis (i.e., the average flowrate of the liquid containing the fluidic droplet deviates by less than 20% or less than 15% of the steady- state flow or the expected value of the flow of liquid with respect to time, and in some cases, the average flowrate may deviate less than 10% or less than 5%) or other

predetermined basis through a fluidic system of the invention (e.g., through a channel or a microchannel), and fluidic droplets contained within the liquid may be directed to various regions, e.g., using an electric field, without substantially altering the flow of the liquid through the fluidic system.

In some embodiments, the fluidic droplets may be screened or sorted within a fluidic system of the invention by altering the flow of the liquid containing the droplets. For instance, in one set of embodiments, a fluidic droplet may be steered or sorted by directing the liquid surrounding the fluidic droplet into a first channel, a second channel, etc.

Techniques useful for sorting droplets include, for example, flow cytometry techniques or microfluidic techniques known to those of ordinary skill in the art.

In another set of embodiments, pressure within a fluidic system, for example, within different channels or within different portions of a channel, can be controlled to direct the flow of fluidic droplets. For example, a droplet can be directed toward a channel junction including multiple options for further direction of flow (e.g., directed toward a branch, or fork, in a channel defining optional downstream flow channels). Pressure within one or more of the optional downstream flow channels can be controlled to direct the droplet selectively into one of the channels, and changes in pressure can be effected on the order of the time required for successive droplets to reach the junction, such that the downstream flow path of each successive droplet can be independently controlled. In one arrangement, the expansion and/or contraction of liquid reservoirs may be used to steer or sort a fluidic droplet into a channel, e.g., by causing directed movement of the liquid containing the fluidic droplet. The liquid reservoirs may be positioned such that, when activated, the movement of liquid caused by the activated reservoirs causes the liquid to flow in a preferred direction, carrying the fluidic droplet in that preferred direction. For instance, the expansion of a liquid reservoir may cause a flow of liquid towards the reservoir, while the contraction of a liquid reservoir may cause a flow of liquid away from the reservoir. In some cases, the expansion and/or contraction of the liquid reservoir may be combined with other flow-controlling devices and methods, e.g., as described herein. Non-limiting examples of devices able to cause the expansion and/or contraction of a liquid reservoir include pistons and piezoelectric components. In some cases, piezoelectric components may be particularly useful due to their relatively rapid response times, e.g., in response to an electrical signal. In some

embodiments, the fluidic droplets may be sorted into more than two channels.

As mentioned, certain embodiments are generally directed to systems and methods for sorting fluidic droplets in a liquid, and in some cases, at relatively high rates. For example, a property of a droplet may be sensed and/or determined in some fashion (e.g., as further described herein), then the droplet may be directed towards a particular region of the device, such as a microfluidic channel, for example, for sorting purposes. In some cases, high sorting speeds may be achievable using certain systems and methods of the invention. For instance, at least about 10 droplets per second may be determined and/or sorted in some cases, and in other cases, at least about 20 droplets per second, at least about 30 droplets per second, at least about 100 droplets per second, at least about 200 droplets per second, at least about 300 droplets per second, at least about 500 droplets per second, at least about 750 droplets per second, at least about 1,000 droplets per second, at least about 1,500 droplets per second, at least about 2,000 droplets per second, at least about 3,000 droplets per second, at least about 5,000 droplets per second, at least about 7,500 droplets per second, at least about 10,000 droplets per second, at least about 15,000 droplets per second, at least about 20,000 droplets per second, at least about 30,000 droplets per second, at least about 50,000 droplets per second, at least about 75,000 droplets per second, at least about 100,000 droplets per second, at least about 150,000 droplets per second, at least about 200,000 droplets per second, at least about 300,000 droplets per second, at least about 500,000 droplets per second, at least about 750,000 droplets per second, at least about 1,000,000 droplets per second, at least about 1,500,000 droplets per second, at least about 2,000,000 or more droplets per second, or at least about 3,000,000 or more droplets per second may be determined and/or sorted.

In some aspects, a population of relatively small droplets may be used. In certain embodiments, as non-limiting examples, the average diameter of the droplets may be less than about 1 mm, less than about 500 micrometers, less than about 300 micrometers, less than about 200 micrometers, less than about 100 micrometers, less than about 75

micrometers, less than about 50 micrometers, less than about 30 micrometers, less than about 25 micrometers, less than about 20 micrometers, less than about 15 micrometers, less than about 10 micrometers, less than about 5 micrometers, less than about 3 micrometers, less than about 2 micrometers, less than about 1 micrometer, less than about 500 nm, less than about 300 nm, less than about 100 nm, or less than about 50 nm. The average diameter of the droplets may also be at least about 30 nm, at least about 50 nm, at least about 100 nm, at least about 300 nm, at least about 500 nm, at least about 1 micrometer, at least about 2

micrometers, at least about 3 micrometers, at least about 5 micrometers, at least about 10 micrometers, at least about 15 micrometers, or at least about 20 micrometers in certain cases. The "average diameter" of a population of droplets is the arithmetic average of the diameters of the droplets.

In some embodiments, the droplets may be of substantially the same shape and/or size (i.e., "monodisperse"), or of different shapes and/or sizes, depending on the particular application. In some cases, the droplets may have a homogenous distribution of cross- sectional diameters, i.e., the droplets may have a distribution of diameters such that no more than about 5%, no more than about 2%, or no more than about 1% of the droplets have a diameter less than about 90% (or less than about 95%, or less than about 99%) and/or greater than about 110% (or greater than about 105%, or greater than about 101%) of the overall average diameter of the plurality of droplets. Some techniques for producing homogenous distributions of cross-sectional diameters of droplets are disclosed in International Patent Application No. PCT/US2004/010903, filed April 9, 2004, entitled "Formation and Control of Fluidic Species," by Link et al., published as WO 2004/091763 on October 28, 2004, incorporated herein by reference.

Those of ordinary skill in the art will be able to determine the average diameter of a population of droplets, for example, using laser light scattering or other known techniques. The droplets so formed can be spherical, or non-spherical in certain cases. The diameter of a droplet, in a non- spherical droplet, may be taken as the diameter of a perfect mathematical sphere having the same volume as the non-spherical droplet. In some embodiments, one or more droplets may be created within a channel by creating an electric charge on a fluid surrounded by a liquid, which may cause the fluid to separate into individual droplets within the liquid. In some embodiments, an electric field may be applied to the fluid to cause droplet formation to occur. The fluid can be present as a series of individual charged and/or electrically inducible droplets within the liquid. Electric charge may be created in the fluid within the liquid using any suitable technique, for example, by placing the fluid within an electric field (which may be AC, DC, etc.), and/or causing a reaction to occur that causes the fluid to have an electric charge.

The electric field, in some embodiments, is generated from an electric field generator, i.e., a device or system able to create an electric field that can be applied to the fluid. The electric field generator may produce an AC field (i.e., one that varies periodically with respect to time, for example, sinusoidally, sawtooth, square, etc.), a DC field (i.e., one that is constant with respect to time), a pulsed field, etc. Techniques for producing a suitable electric field (which may be AC, DC, etc.) are known to those of ordinary skill in the art. For example, in one embodiment, an electric field is produced by applying voltage across a pair of electrodes, which may be positioned proximate a channel such that at least a portion of the electric field interacts with the channel. The electrodes can be fashioned from any suitable electrode material or materials known to those of ordinary skill in the art, including, but not limited to, silver, gold, copper, carbon, platinum, copper, tungsten, tin, cadmium, nickel, indium tin oxide ("ITO"), liquid salt electrodes, etc., as well as combinations thereof.

In another set of embodiments, droplets of fluid can be created from a fluid surrounded by a liquid within a channel by altering the channel dimensions in a manner that is able to induce the fluid to form individual droplets. The channel may, for example, be a channel that expands relative to the direction of flow, e.g., such that the fluid does not adhere to the channel walls and forms individual droplets instead, or a channel that narrows relative to the direction of flow, e.g., such that the fluid is forced to coalesce into individual droplets. In some cases, the channel dimensions may be altered with respect to time (for example, mechanically or electromechanically, pneumatically, etc.) in such a manner as to cause the formation of individual droplets to occur. For example, the channel may be mechanically contracted ("squeezed") to cause droplet formation, or a fluid stream may be mechanically disrupted to cause droplet formation, for example, through the use of moving baffles, rotating blades, or the like. Other techniques of creating droplets include, for example mixing or vortexing of a fluid. For example, a container containing two fluids may be vortexed to produce droplets of a first fluid contained within a second fluid. Certain embodiments are generally directed to systems and methods for splitting a droplet into two or more droplets. For example, a droplet can be split using an applied electric field. The droplet may have a greater electrical conductivity than the surrounding liquid, and, in some cases, the droplet may be neutrally charged. In certain embodiments, in an applied electric field, electric charge may be urged to migrate from the interior of the droplet to the surface to be distributed thereon, which may thereby cancel the electric field experienced in the interior of the droplet. In some embodiments, the electric charge on the surface of the droplet may also experience a force due to the applied electric field, which causes charges having opposite polarities to migrate in opposite directions. The charge migration may, in some cases, cause the drop to be pulled apart into two separate droplets.

Some embodiments of the invention generally relate to systems and methods for fusing or coalescing two or more droplets into one droplet, e.g., where the two or more droplets ordinarily are unable to fuse or coalesce, for example, due to composition, surface tension, droplet size, the presence or absence of surfactants, etc. In certain cases, the surface tension of the droplets, relative to the size of the droplets, may also prevent fusion or coalescence of the droplets from occurring.

As a non-limiting example, two droplets can be given opposite electric charges (i.e., positive and negative charges, not necessarily of the same magnitude), which can increase the electrical interaction of the two droplets such that fusion or coalescence of the droplets can occur due to their opposite electric charges. For instance, an electric field may be applied to the droplets, the droplets may be passed through a capacitor, a chemical reaction may cause the droplets to become charged, etc. The droplets, in some cases, may not be able to fuse even if a surfactant is applied to lower the surface tension of the droplets. However, if the droplets are electrically charged with opposite charges (which can be, but are not necessarily of, the same magnitude), the droplets may be able to fuse or coalesce. As another example, the droplets may not necessarily be given opposite electric charges (and, in some cases, may not be given any electric charge), and are fused through the use of dipoles induced in the droplets that causes the droplets to coalesce. Also, the two or more droplets allowed to coalesce are not necessarily required to meet "head-on." Any angle of contact, so long as at least some fusion of the droplets initially occurs, is sufficient. See also, e.g., U.S. Patent Application Serial No. 1 1/698,298, filed January 24, 2007, entitled "Fluidic Droplet

Coalescence," by Ahn, et ah , published as U.S. Patent Application Publication No.

2007/0195127 on August 23, 2007, incorporated herein by reference in its entirety. In one set of embodiments, a fluid may be injected into a droplet. The fluid may be microinjected into the droplet in some cases, e.g., using a microneedle or other such device. In other cases, the fluid may be injected directly into a droplet using a fluidic channel as the droplet comes into contact with the fluidic channel. Other techniques of fluid injection are disclosed in, e.g., International Patent Application No. PCT/US 2010/040006, filed June 25, 2010, entitled "Fluid Injection," by Weitz, et al, published as WO 2010/151776 on

December 29, 2010; or International Patent Application No. PCT/US2009/006649, filed December 18, 2009, entitled "Particle-Assisted Nucleic Acid Sequencing," by Weitz, et al., published as WO 2010/080134 on July 15, 2010, each incorporated herein by reference in its entirety.

Some aspects of the invention are generally directed to double or higher multiple emulsion droplets. The droplet may be any of the ones discussed herein. Generally, in a double emulsion, a first (or inner) fluidic droplet comprising a first fluid is surrounded by a second (or middle) fluidic droplet comprising a second fluid, which in turn is contained within a continuous or carrying third fluid. Typically, a fluid is substantially immiscible with an adjacent fluid, although fluids that are not adjacent need not be immiscible, and may be miscible (or even identical) in some cases. Thus, for example, the first fluid may be immiscible with the second fluid, but may be miscible or immiscible with the third fluid. However, it should be understood that immiscibility is not necessarily required in all embodiments; in some cases, two adjacent fluids are not immiscible, but may retain separation in other ways, e.g., kinetically or through short exposure times.

Thus, as a non-limiting example, in a double emulsion droplet, the first fluid

(innermost fluid) may be an aqueous or hydrophilic fluid (a "water" phase), the second fluid (middle fluid) may be a lipophilic or hydrophobic or "oil" phase that is substantially immiscible with the aqueous fluid, and the third fluid (or outer fluid) may be an aqueous fluid (a "water" fluid) that is substantially immiscible with the second fluid. This is sometimes generally referred to as a W/O/W double emulsion droplet (for water/oil/water), although it should be understand that this is mainly for the sake of convenience; for instance, the first fluid can be any suitable aqueous fluid, and it need not be pure water. For example, the aqueous fluid may be water, saline, an aqueous solution, ethanol, or the like, or any other fluid miscible in water. The oil, in contrast, may be immiscible in water, at least when left undisturbed under ambient conditions. In similar fashion, an 0/W/O double emulsion droplet may be similarly defined. Furthermore, these principles may be extended to higher-order multiple emulsions droplets. For example, a triple emulsion droplet may comprise a first fluid, surrounded by a second fluid, surrounded by a third fluid, contained in a fourth fluid; a quadruple emulsion droplet may comprise a first fluid, surrounded by a second fluid, surrounded by a third fluid, surrounded by a fourth fluid, contained in a fifth fluid, etc. In addition, it should be understood that other arrangements are also possible. For example, in one embodiment, the first fluid, the second fluid, and the third fluid may be all mutually immiscible.

As used herein, two fluids are immiscible, or not miscible, with each other when one is not soluble in the other to a level of at least 10% by weight at the temperature and under the conditions at which the emulsion is produced. For instance, two fluids may be selected to be immiscible within the time frame of the formation of the fluidic droplets. In some embodiments, two fluids (e.g., the carrying fluid and the inner droplet fluid of a multiple emulsion) are compatible, or miscible, while the outer droplet fluid is incompatible or immiscible with one or both of the carrying and inner droplet fluids. In other embodiments, however, all three (or more) fluids may be mutually immiscible, and in certain cases, all of the fluids do not all necessarily have to be water soluble. In still other embodiments, as mentioned, additional third, fourth, fifth, sixth, etc. fluids may be added to produce increasingly complex droplets within droplets, e.g., a carrying fluid may surround a first fluid, which may in turn surround a second fluid, which may in turn surround a third fluid, which in turn surround a fourth fluid, etc. In addition, the physical properties of each nesting layer of fluidic droplets may each be independently controlled, e.g., by control over the composition of each nesting level.

In addition, in some aspects of the invention, at least a portion of a double or other multiple emulsion droplet may be solidified to form a particle or a capsule, for example, containing an inner fluid and/or a species as discussed herein. A fluid, e.g., within an outermost layer of a multiple emulsion droplet, can be solidified using any suitable method. For example, in some embodiments, the fluid may be dried, gelled, and/or polymerized, and/or otherwise solidified, e.g., to form a solid, or at least a semi-solid. The solid that is formed may be rigid in some embodiments, although in other cases, the solid may be elastic, rubbery, deformable, etc. In some cases, for example, an outermost layer of fluid may be solidified to form a solid shell at least partially containing an interior containing a fluid and/or a species. Any technique able to solidify at least a portion of a fluidic droplet can be used. For example, in some embodiments, a fluid within a fluidic droplet may be removed to leave behind a material (e.g., a polymer) capable of forming a solid shell. In other embodiments, a fluidic droplet may be cooled to a temperature below the melting point or glass transition temperature of a fluid within the fluidic droplet, a chemical reaction may be induced that causes at least a portion of the fluidic droplet to solidify (for example, a polymerization reaction, a reaction between two fluids that produces a solid product, etc.), or the like. Other examples include pH-responsive or molecular-recognizable polymers, e.g., materials that gel upon exposure to a certain pH, or to a certain species. In some

embodiments, a fluidic droplet is solidified by increasing the temperature of the fluidic droplet. For instance, a rise in temperature may drive out a material from the fluidic droplet (e.g., within the outermost layer of a multiple emulsion droplet) and leave behind another material that forms a solid. Thus, in some cases, an outermost layer of a multiple emulsion droplet may be solidified to form a solid shell that encapsulates one or more fluids and/or species.

A variety of materials and methods, according to certain aspects of the invention, can be used to form articles or components such as those described herein, e.g., channels such as microfluidic channels, chambers, etc. For example, various articles or components can be formed from solid materials, in which the channels can be formed via micromachining, film deposition processes such as spin coating and chemical vapor deposition, laser fabrication, photolithographic techniques, etching methods including wet chemical or plasma processes, and the like. See, for example, Scientific American, 248:44-55, 1983 (Angell, et al).

In one set of embodiments, various structures or components of the articles described herein can be formed of a polymer, for example, an elastomeric polymer such as

polydimethylsiloxane ("PDMS"), polytetrafluoroethylene ("PTFE" or Teflon^®), or the like. For instance, according to one embodiment, a microfluidic channel may be implemented by fabricating the fluidic system separately using PDMS or other soft lithography techniques (details of soft lithography techniques suitable for this embodiment are discussed in the references entitled "Soft Lithography," by Younan Xia and George M. Whitesides, published in the Annual Review of Material Science, 1998, Vol. 28, pages 153-184, and "Soft

Lithography in Biology and Biochemistry," by George M. Whitesides, Emanuele Ostuni, Shuichi Takayama, Xingyu Jiang and Donald E. Ingber, published in the Annual Review of Biomedical Engineering, 2001, Vol. 3, pages 335-373; each of these references is

incorporated herein by reference).

Other examples of potentially suitable polymers include, but are not limited to, polyethylene terephthalate (PET), polyacrylate, polymethacrylate, polycarbonate,

polystyrene, polyethylene, polypropylene, polyvinylchloride, cyclic olefin copolymer (COC), polytetrafluoroethylene, a fluorinated polymer, a silicone such as polydimethylsiloxane, polyvinylidene chloride, bis-benzocyclobutene ("BCB"), a polyimide, a fluorinated derivative of a polyimide, or the like. Combinations, copolymers, or blends involving polymers including those described above are also envisioned. The device may also be formed from composite materials, for example, a composite of a polymer and a

semiconductor material.

In some embodiments, various structures or components of the article are fabricated from polymeric and/or flexible and/or elastomeric materials, and can be conveniently formed of a hardenable fluid, facilitating fabrication via molding (e.g. replica molding, injection molding, cast molding, etc.). The hardenable fluid can be essentially any fluid that can be induced to solidify, or that spontaneously solidifies, into a solid capable of containing and/or transporting fluids contemplated for use in and with the fluidic network. In one embodiment, the hardenable fluid comprises a polymeric liquid or a liquid polymeric precursor (i.e. a "prepolymer"). Suitable polymeric liquids can include, for example, thermoplastic polymers, thermoset polymers, waxes, metals, or mixtures or composites thereof heated above their melting point. As another example, a suitable polymeric liquid may include a solution of one or more polymers in a suitable solvent, which solution forms a solid polymeric material upon removal of the solvent, for example, by evaporation. Such polymeric materials, which can be solidified from, for example, a melt state or by solvent evaporation, are well known to those of ordinary skill in the art. A variety of polymeric materials, many of which are elastomeric, are suitable, and are also suitable for forming molds or mold masters, for embodiments where one or both of the mold masters is composed of an elastomeric material. A non-limiting list of examples of such polymers includes polymers of the general classes of silicone polymers, epoxy polymers, and acrylate polymers. Epoxy polymers are characterized by the presence of a three-membered cyclic ether group commonly referred to as an epoxy group, 1,2- epoxide, or oxirane. For example, diglycidyl ethers of bisphenol A can be used, in addition to compounds based on aromatic amine, triazine, and cycloaliphatic backbones. Another example includes the well-known Novolac polymers. Non-limiting examples of silicone elastomers suitable for use according to the invention include those formed from precursors including the chlorosilanes such as methylchlorosilanes, ethylchlorosilanes,

phenylchlorosilanes, dodecyltrichlorosilanes, etc.

Silicone polymers are used in certain embodiments, for example, the silicone elastomer polydimethylsiloxane. Non-limiting examples of PDMS polymers include those sold under the trademark Sylgard by Dow Chemical Co., Midland, MI, and particularly Sylgard 182, Sylgard 184, and Sylgard 186. Silicone polymers including PDMS have several beneficial properties simplifying fabrication of various structures of the invention. For instance, such materials are inexpensive, readily available, and can be solidified from a prepolymeric liquid via curing with heat. For example, PDMSs are typically curable by exposure of the prepolymeric liquid to temperatures of about, for example, about 65 °C to about 75 °C for exposure times of, for example, about an hour. Also, silicone polymers, such as PDMS, can be elastomeric and thus may be useful for forming very small features with relatively high aspect ratios, necessary in certain embodiments of the invention. Flexible (e.g., elastomeric) molds or masters can be advantageous in this regard.

One advantage of forming structures such as microfluidic structures or channels from silicone polymers, such as PDMS, is the ability of such polymers to be oxidized, for example by exposure to an oxygen-containing plasma such as an air plasma, so that the oxidized structures contain, at their surface, chemical groups capable of cross-linking to other oxidized silicone polymer surfaces or to the oxidized surfaces of a variety of other polymeric and non- polymeric materials. Thus, structures can be fabricated and then oxidized and essentially irreversibly sealed to other silicone polymer surfaces, or to the surfaces of other substrates reactive with the oxidized silicone polymer surfaces, without the need for separate adhesives or other sealing means. In most cases, sealing can be completed simply by contacting an oxidized silicone surface to another surface without the need to apply auxiliary pressure to form the seal. That is, the pre-oxidized silicone surface acts as a contact adhesive against suitable mating surfaces. Specifically, in addition to being irreversibly sealable to itself, oxidized silicone such as oxidized PDMS can also be sealed irreversibly to a range of oxidized materials other than itself including, for example, glass, silicon, silicon oxide, quartz, silicon nitride, polyethylene, polystyrene, glassy carbon, and epoxy polymers, which have been oxidized in a similar fashion to the PDMS surface (for example, via exposure to an oxygen-containing plasma). Oxidation and sealing methods useful in the context of the present invention, as well as overall molding techniques, are described in the art, for example, in an article entitled "Rapid Prototyping of Microfluidic Systems and Polydimethylsiloxane," Anal. Chem., 70:474-480, 1998 (Duffy et ah), incorporated herein by reference.

Thus, in certain embodiments, the design and/or fabrication of the article may be relatively simple, e.g., by using relatively well-known soft lithography and other techniques such as those described herein. In addition, in some embodiments, rapid and/or customized design of the article is possible, for example, in terms of geometry. In one set of

embodiments, the article may be produced to be disposable, for example, in embodiments where the article is used with substances that are radioactive, toxic, poisonous, reactive, biohazardous, etc., and/or where the profile of the substance (e.g., the toxicology profile, the radioactivity profile, etc.) is unknown. Another advantage to forming channels or other structures (or interior, fluid-contacting surfaces) from oxidized silicone polymers is that these surfaces can be much more hydrophilic than the surfaces of typical elastomeric polymers (where a hydrophilic interior surface is desired). Such hydrophilic channel surfaces can thus be more easily filled and wetted with aqueous solutions than can structures comprised of typical, unoxidized elastomeric polymers or other hydrophobic materials.

The following documents are incorporated herein by reference in their entirety for all purposes: U.S. Pat. Apl. Ser. No. 61/980,541, entitled "Methods and Systems for Droplet Tagging and Amplification," by Weitz, et al; U.S. Pat. Apl. Ser. No. 61/981,123, entitled

"Systems and Methods for Droplet Tagging," by Bernstein, et al; Int. Pat. Apl. Pub. No. WO 2004/091763, entitled "Formation and Control of Fluidic Species," by Link et al ; Int. Pat. Apl. Pub. No. WO 2004/002627, entitled "Method and Apparatus for Fluid Dispersion," by Stone et al; Int. Pat. Apl. Pub. No. WO 2006/096571, entitled "Method and Apparatus for Forming Multiple Emulsions," by Weitz et al; Int. Pat. Apl. Pub. No. WO 2005/021151, entitled "Electronic Control of Fluidic Species," by Link et al; Int. Pat. Apl. Pub. No. WO 2011/056546, entitled "Droplet Creation Techniques," by Weitz, et al; Int. Pat. Apl. Pub. No. WO 2010/033200, entitled "Creation of Libraries of Droplets and Related Species," by Weitz, et al; U.S. Pat. Apl. Pub. No. 2012-0132288, entitled "Fluid Injection," by Weitz, et al; Int. Pat. Apl. Pub. No. WO 2008/109176, entitled "Assay And Other Reactions Involving Droplets," by Agresti, et al ; and Int. Pat. Apl. Pub. No. WO 2010/151776, entitled "Fluid Injection," by Weitz, et al.

Also incorporated herein by reference are U.S. Prov. Pat. Apl. Ser. No. No.

61/982,001, filed April 21, 2014; U.S. Prov. Pat. Apl. Ser. No. No. 62/065,348, filed October 17, 2014; U.S. Prov. Pat. Apl. Ser. No. No. 62/066,188, filed October 20, 2014; and U.S. Prov. Pat. Apl. Ser. No. No. 62/072,944, filed October 30, 2014.

In addition, the following are incorporated herein by reference in their entireties: U.S. Pat. Apl. Ser. No. 61/981,123 filed April 17, 2014; a PCT application filed April 17, 2015, entitled "Systems and Methods for Droplet Tagging" (PCT/US 15/26338); U.S. Pat. Apl. Ser. No. 61/981,108 filed April 17, 2014; a PCT application filed on April 17, 2015, entitled

"Methods and Systems for Droplet Tagging and Amplification" (PCT/US 15/26422); a U.S. patent application filed on April 17, 2015, entitled "Immobilization-Based Systems and Methods for Genetic Analysis and Other Applications" (U.S. Ser. No. 62/149,372); a U.S. patent application filed on April 17, 2015, entitled "Barcoding Systems and Methods for Gene Sequencing and Other Applications" (U.S. Ser. No. 62/149,361); U.S. Pat. Apl. Ser. No. 62/072,944, filed October 30, 2014; and Int. Pat. Apl. No. PCT/US 15/26443, entitled "Systems and Methods for Barcoding Nucleic Acids," filed April 17, 2015.

Also, U.S. Provisional Patent Application Serial No. 62/366,991, filed July 26, 2016, entitled "Barcoded Systems with Multiple Information," by Weitz, et al. is incorporated herein by reference in its entirety.

The following examples are intended to illustrate certain embodiments of the present invention, but do not exemplify the full scope of the invention.

EXAMPLE 1

This example presents methods to create and use genetic labels that contain at least two types of information. They indicate the identity of the molecule and they indicate the location history of the molecule. This example describes one assay that takes advantage of this method: In-droplet simultaneous analysis of protein and mRNA levels.

There are numerous other applications. A simpler assay for single-cell protein analysis without mRNA profiling will be useful; and the labeled antibodies can for the basis for a single-cell chromatin immunoprecipitation method.

The example method also allows novel and sensitive multiplex protein analysis of patient samples, not necessarily individual cells. For example, a sample-barcoding bead can be combined with coded antibodies and a tissue sample in a well. After sample is washed, RT-PCR is performed to reveal the amount of antibody bound. This could be useful in a hospital setting, or for diagnostics.

In addition, as is described below, the barcoded antibodies do not need to be encapsulated with a bead. If the sample, for example cells, can be washed, then the sample can be incubated with the barcoded antibodies, washed, and then encapsulated along with a bead. This can greatly simplify experiments in some cases. It may also greatly increases the number of antibodies that can be used in a single experiment in some embodiments.

This example illustrates in-droplet simultaneous analysis of protein and mRNA levels. Single-cell analysis of protein and mRNA levels may be important to understanding cell state and function, and in determining the roles of cellular proteins. This example presents a rapid in-droplet method to simultaneously measure protein and mRNA levels in many individual single cells.

This example couples an in-droplet sandwich-type assay with reverse-transcriptase PCR to create a rapid method for single-cell protein and mRNA analysis. In Fig. 1, the assay is described for one cell; however, the system also allows for analysis of many individual cells in other instances. For clarity, Fig. 1 is greatly simplified. In practice, a single bead barcoding bead (BCB) may bear several different identity-coded antibody species, each represented many times (e.g., hundreds, thousands) on the bead. The bead may also bear several mRNA capture molecules corresponding to the identity-coded antibodies. In this way the assay simultaneously quantifies numerous proteins and the corresponding mRNA molecules.

This example presents methods to affix genetic codes to individual nucleic-acid and non-nucleic acid molecules. These codes can provide two types of information: the identity of the molecule and the location history and/or source of the molecule. This may be used to barcode antibodies that are directed against protein targets of interest.

Genetic identity-coding of antibodies may link the specificity of antibody-based detection to the enormous throughput of DNA sequencing. The identity-coded antibodies described in this example are generally useful for sensitive multiplex protein analysis. This may allow novel and sensitive multiplex protein analysis of patient samples, not necessarily individual cells. For example, a sample-barcoding bead can be combined with coded antibodies and a tissue sample and in a well. After sample is washed, RT-PCR is performed to reveal the amount of antibody bound. This could be useful, for example, in hospital setting, or for diagnostics.

In addition, the identity-coded antibodies may be suited for Chromatin

Immunoprecipitation in some embodiments. In one embodiment, identity-coded antibodies are linked (ligated) directly to the sequence to which they bind. DNA sequencing of ChiP samples would then simultaneously reveal the DNA targets and the antibodies bound to these targets.

This example uses droplet-based microfluidics to perform barcoding using hydrogels. A typical microfluidic device is shown in Fig. 1. Use of this device for the barcoding scheme is described in Fig. 2. This type of device is also used for the schemes described below.

Briefly, a sample of closely packed hydrogels, each bearing barcoded antibodies and barcoded gene-specific reverse transcription primers, is loaded into the device through channel 1. Cell-lysis reagents and capture beads are loaded into channel 2. Cells are loaded into channel 3; and the continuous phase (e.g., HFE7500 engineered fluid with surfactant) is loaded into channel 4. Droplets are formed as the combined aqueous streams pass through the HFE7500 stream. After drop formation, the drops are treated with UV light to release the barcoded antibodies. Transcripts are free to anneal to capture oligos, and the released cell proteins can be bound by the capture- and detection-antibody pairs. After incubation to allow binding, the antibody-coated beads and the gene- specific-primer-coated beads are collected and washed. The primer-captured RNA is reverse-transcribed into cDNA, and then the cDNA and the target-bound antibody barcodes are amplified and products are prepared for next generation sequencing.

Fig.lA shows an example microfluidic device for creating barcoding droplets.

Aqueous stream 1 introduces barcode-bearing hydrogels into the microfluidic device;

Aqueous stream 2 introduces cells; Aqueous stream 3 introduces the reagents to lyse the cell and to perform reverse-transcription. The combined aqueous stream is cut into three nanoliter droplets by the oil stream (4). In the droplet, cells are lysed and transcripts are captured by the oligo-dT-containing barcoding primers delivered by the hydrogel bead. The flow rates are set to ensure that >90% of droplets contain one single sequencing hydrogel. Cells can be loaded at 0.1 cells per droplet to ensure very few droplets contain >1 cell. Fig. IB shows a schematic of microfluidic device showing channel dimensions. It should be noted that these dimensions are by way of example only and that in other embodiments, other dimensions are also possible.

Fig. 2 shows a schematic of assay for simultaneous analysis of protein and mRNA levels. Assay captures and quantifies mRNA molecules of interest and their encoded proteins.

In this figure, in step 1, a droplet is formed containing a cell, an antibody-coated capture bead ("ACB"), and a barcoding bead ("BCB") that bears coded antibody and gene- specific capture/reverse-transcription oligos. Buffers for cell lysis and mRNA capture are also included.

Step 2: Treat with UV to release antibody from barcoding bead. The antibodies and the gene-specific capture oligos bind to their mRNA and protein targets released from lysed cell.

Step 3: Break drops and isolate the beads. mRNA and proteins of interest are captured on beads and retained. The amount of identity-coded antibody retained is determined by the amount of protein-of-interest captured on the antibody-coated bead. Uncaptured mRNA and unbound identity-coded antibody is discarded. Sample is prepared for DNA sequencing to quantify the DNA tags retained.

EXAMPLE 2

This example shows an overview of an example of a method to create beads that each provide two types of barcodes. This example describes methods to prepare cell/droplet barcoding beads that can accept both identity-coded antibodies and capture/reverse transcription primers. For clarity, the method is divided into several steps within this example, for illustration purposes only. Step 1 describes the generation of droplet-barcoding beads. Step 2 details the creation of identity-coded antibodies. In Step 3, the identity-coded antibodies are linked to the barcoding beads. In Step 4, the capture/reverse transcription primers are linked to the barcoding beads. In some cases, Steps 3 and 4 can be performed simultaneously.

Cell-barcoding beads: These beads contain genetic elements that enable barcoding of the material contained in one droplet. These beads are used one-bead per droplet.

Identity-coded antibodies: These are antibodies that each contain one single DNA molecule. For a given antibody species, for example a population of antibody molecules from the same hybridoma, each antibody molecule contains the same identifying DNA sequence. It may be necessary to attach one single DNA fragment to each antibody molecule, and it may be technically difficult to attach a single DNA molecule to a complete antibody IgG molecule. To overcome this, recombinant antibodies, or Fab fragments, might be used instead of full-length IgG.

In this example, all the cell-barcoding beads contain DNA fragments with the same overhanging end: 5'-TTTC, although this is not a requirement. The identity-coded antibodies and the RT-PCR primer oligos are designed to anneal to this sequence. However, two overhangs, one specific for identity-coded antibodies and one specific for the RT-PCR primer oligos, might be used. In principle, more than two overhangs can also be used so that more than two different species can be specifically attached to the beads.

In this step (Step A), "droplet barcoding" beads are created that each can be ligated to a set of antibodies and to a corresponding set of gene-specific RT -primers. See Fig. 3.

Step 1: Prepare "stubbed" hydrogel beads. There are two types of stubs on each bead. Type I: A single-stranded nucleic acid comprised of a moiety that links to the bead (linker), a photocleavable element (PCE), a common amplification sequence (CAS), and "Annealing Bases" (in this example, GAGAGG) that will anneal to the first Barcoding Oligo (BC1). Type II stub does not contain a PCE and may have annealing bases GAGAGG. See notes for discussion of these sequences.

Step 2: Dispense stubbed hydrogel beads into 384 well plate. Each of the 384 recipient wells contains many copies of two single-stranded DNA oligos. One oligo, BC1 in bold, is unique to each well and is comprised of four elements: 1) Sequence complementary to the Annealing Bases, 2) BarCode One (BC1 - NNNNNN in bold in this example), 3) Unique molecule identifier sequence (UMI), and 4) Sequence that encodes a restriction site followed by 8 bases to ensure efficient cutting (RS+8). The second oligo, in bold and italicized, is complementary to RS+8 (cRS+8).

Step 3: Perform a fill-in reaction that uses cRS+8 as a primer to generate sequence complementary to the UMI and BC1 sequence.

Step 4: Pool beads, perform restriction digest to leave an overhanging end, clean up the reaction. Distribute beads into 384 recipient wells.

Step 5: Each of the 384 recipient wells contains many copies of an oligo that is unique to each well and encodes a second barcode (BC2 - NNNNNN). The oligo anneals to the overhanging end created in step 4. The sequence that remains single-stranded after annealing is filled in to create a double-stranded sequence that encodes a restriction site.

Step 6: Pool beads, perform restriction digest to leave an overhanging end, clean up the

reaction. These beads are now ready to be linked to antibody and/or to gene-specific RT primers.

Next, in Step B, identity-coded antibodies are created. The antibody molecule contains a single DNA fragment. The sequence of this DNA fragment is common to all molecules within an antibody species. See Fig. 4.

Antibody species: A collection of antibody molecules that all recognize the same epitope. For example, IgG molecules from the same monoclonal antibody hybridoma represent one species.

DNA species: A collection of DNA molecules, all with the same antibody-identifying sequence (Antibody Barcoding Sequence, ABC).

Linker: Molecule that allows DNA attachment to an antibody or to a modified antibody or to a recombinant antibody.

Step 1: Many molecules of single antibody species are placed in a tube (or other suitable container) along with many molecules of a DNA species.

Step 2: Linking and clean-up is performed so that DNA molecules are attached to the antibody molecules, and each antibody is affixed to precisely one DNA molecule.

If one DNA per antibody does not allow consistent detection due to low signal or inconsistent amplification, methods to affix more than one identifying DNA per antibody molecule may be used. This may allow the attaching of a single antibody molecule to more than one cell-barcoding bead,

In Step C (see Fig. 5), the identity-coded antibodies are ligated to cell/droplet- encoding beads to create antibodies that are droplet- and identity- bar-coded. Step 1: Barcoded beads are mixed with pooled bar-coded antibodies. The beads and antibody molecules contain complementary single- stranded DNA sequence. These regions anneal, affixing the mixture of antibodies to the beads. Each bead contains millions/billions of barcodes, each with an overhanging end that is complementary to the overhanging on the antibodies. In this way, each bead is affixed to a mixture of the barcoded antibodies.

Step 2: The annealed DNA barcodes may be covalently ligated together though chemical or enzymatic (e.g., ligase- or topoisomerase-mediated) methods. This creates a robust connection between the antibody- and cell barcodes.

Step 3 (optional): A fill-in reaction can be performed to make the antibody barcode and amplification sequence double-stranded.

In Step D (Fig. 6), gene-specific capture/reverse-transcriptase primers (GSRTP) are ligated to droplet-encoding beads to create reverse-transcription primers that will add cell barcoding sequences to cellular mRNA.

In step 1, barcoded beads are mixed with reverse-transcription primers that correspond to the proteins of interest. For example, if an antibody to detect TNF is used in the assay, then a primer with sequence complementary to the TNF transcript is included. The primer also contains sequence complementary to the 3' overhang of the bead barcode.

In step 2, the oligo is double-stranded for some of its length so that the bottom strand, containing a 5' phosphate, can be ligated to the bottom strand of the linker shown at right. Lower arrow indicates the site of ligation. At the heavier arrow, the "T" base is not covalently linked to the "A" base.

In step 3, the beads are washed. This can be performed at elevated temperature so that the top oligo (sequence AAAG-(GSRTP) is melted off the bottom strand to expose the reverse-transcriptase primer. Various other methods, including enzymatic and chemical means, may be used to expose the reverse-transcriptase primer.

For clarity, beads are shown without antibody in this figure. However, the beads may be ligated to antibody in Step 3. In some cases, Steps 3 and 4 will be performed

simultaneously. In this figure at the bottom, GSRTP-containing oligo is not covalently attached to the bead. Additional steps to make a covalent linkage may be performed.

Fig. 7 shows the final product of Steps A-D. Beads that perform two functions. Each bead will barcode transcripts within a drop and it will also deliver bar-coded antibodies to that drop.

EXAMPLE 3 This example illustrates barcoded antibody methods as alternative to flow cytometry to measure cellular protein levels.

In the previous examples, cells of interest are encapsulated, lysed, and then the released proteins are bound by co-encapsulated antibodies. The droplet contains the lysed- cell material so that contents from one cell are associated with only one barcode.

It is also possible to incubate cells with identity-coded antibodies, wash, and then encapsulate the cells. However, the cells must remain intact throughout the incubation, wash, and encapsulation steps.

The cells may remain intact during interrogation with antibodies directed against surface proteins, as demonstrated by standard Flow Cytometry methods. In some cases, the barcoding methods described herein may be adapted for incubation and washing prior to encapsulation. This use of antibody-barcoding may have several advantages over standard flow cytometry. Because the antibody-binding is performed in bulk, there is no volume limitation, and a larger number of different interrogation antibodies can be used. The assay may be performed with only one antibody per target, rather than the two required for the bead-based sandwich assay.

Because there is a wash step, a very large number of identity-barcoded antibodies can be used simultaneously. This is especially true when cells are incubated with an antibody mixture prior to encapsulation into drops. For example, 10,000 well-characterized antibodies can be obtained and a unique identity-encoding nucleic acid sequence may be affixed to each. These identity-encoding nucleic acid sequences will contain free ends for ligation to the bead-affixed barcodes. Cells with these antibodies may be incubated and then unbound antibodies may be washed away. The washed cells can be encapsulated into droplets along with barcoding beads. Cells will be lysed and cell transcripts and the captured antibody-free ends will be affixed to the bead-delivered bar codes. In this way, the transcripts from a single cell, and the antibodies that bound to that cell, will receive the same cell-identity barcode.

Fig. 8 shows a barcoded antibody alternative to fluorescent-antibody flow cytometry, without analysis of cellular nucleic acids. These may be prepared as follows: Mix barcoded proteins with cell, incubate, wash. Encapsulate antibody-incubated cells into drops along with a barcoding bead, cell-lysis reagents, and reagents to attach the antibody-identifier to the bead-delivered barcode (e.g., annealing buffer, possibly including ligase or a polymerase). Droplets can now be broken and sequencing library prepared from the barcoded nucleic-acid contents. Optionally, a PCE (photocleavable element) can be used to release the bead-affixed barcodes so that they are more accessible to join to the nucleic acid-based antibody identifiers (not shown).

Fig. 9 shows barcoded antibody alternative to fluorescent-antibody flow cytometry, with analysis of cellular nucleic acids. No photocleavage is required. These may be prepared as follows. Mix barcoded proteins with cell, then incubate and wash the proteins.

Encapsulate antibody-incubated cells into drops along with a barcoding bead and cell-lysis and reverse transcriptase reagents (reverse transcription may be performed in bulk after contents are released from drops). Cells are lysed and barcodes may anneal to transcripts of interest and to the antibody- affixed DNA. Release droplet contents and wash beads. The nucleic acids are then prepared for next generation sequencing. The contents of each drop can be identified by the bead-delivered barcode.

Fig. 10 shows barcoded antibody alternative to fluorescent-antibody flow cytometry, with analysis of cellular nucleic acids with photocleavage. These can be prepared as follows. Mix barcoded proteins with cell, then incubate and wash. Encapsulate antibody-incubated cells into drops along with a barcoding bead and cell-lysis and reverse transcriptase reagents. Phototreat to release the bar-bound barcodes. The barcodes can anneal to transcripts of interest and to the antibody- affixed DNA. The droplets now contains lysed cells, released mRNA and proteins, photo-released barcodes, and the hydrogel bead from which the barcodes have been released. Antibodies can be attached to antibody-accepting barcodes and the mRNA anneals to gene- specific capture barcodes. Reverse transcription converts RNA to cDNA. The drops can now be broken and the nucleic acids prepared for sequencing.

EXAMPLE 4

This example illustrates use of oligo-dT primer for transcriptome profiling. This example describes using the bead-based barcoding to simultaneously identify transcription and protein levels for a defined number of targets. For example, the transcripts and corresponding proteins from ten different genes. However, the methods can be used in different formats to analyze different target sets.

For example, beads containing an oligo-dT primer for transcript are used with a "target-focused" subset of antibodies. In this case, the entire transcriptional profile of each cell is obtained, along with a defined protein signature.

EXAMPLE 5

This example describes alternative methods to create barcodes on beads.

Pool-and- split methods. PCT/US 15/26443, entitled "Systems and Methods for Barcoding Nucleic Acids," incorporated herein by reference in its entirety, describes a pool- and-split method to create nucleic acid barcodes on the beads. Droplets are not necessarily used to create these barcodes. Briefly, a set of half-barcode oligos is attached to beads in separate wells. These beads are pooled and split into wells containing the second half of the barcode. The oligos contain sequences to enable annealing and extension, which links the second oligo to the first. However, variety of molecular biology techniques can be used to create barcodes on beads.

For example, pool-and- split using ligation may be used. The oligo set will contain overhangs to allow annealing and ligation to the second set of oligos. This method may require a shorter annealing region than the annealing method and thus the overall barcoding nucleic acid length may be shorter, resulting in more economical use of sequencing space. Ligation-based strategies might use nickases, which cut only one strand of DNA, to create ligation- appropriate overhangs.

The use of overhanging oligos at the free end of the bead-affixed barcodes may allow the attachment of a single barcode species to numerous probes, either nucleic acids (RNA or DNA oligos) or proteins (antibodies). It provides a general method to deliver barcodes to barcoding targets.

For convenience in the figures, a single overhang sequence is shown joined to two or more targets. For example, in Fig. 2 and in several of the other drawings, the overhang - TTTC-3' is joined to the antibody- affixed DNA and to gene-specific RT-PCR primers.

In addition, it is also possible to use more than one overhanging sequence. For example, the barcoding beads can have a defined mixture of overhanging ends, each end a match for a specific probe. In this way, the composition of probes can be defined precisely in some embodiments.

Alternatively, chemically active groups, rather than overhanging ends, may be placed at the free ends of the bar-affixed nucleic acids. These ends will allow a variety of conjugation schemes, greatly expanding the repertoire of barcode-compatible probes.

While several embodiments of the present invention have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and/or structures for performing the functions and/or obtaining the results and/or one or more of the advantages described herein, and each of such variations and/or modifications is deemed to be within the scope of the present invention. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or configurations will depend upon the specific application or applications for which the teachings of the present invention is/are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, the invention may be practiced otherwise than as specifically described and claimed. The present invention is directed to each individual feature, system, article, material, kit, and/or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and/or methods, if such features, systems, articles, materials, kits, and/or methods are not mutually inconsistent, is included within the scope of the present invention.

All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms.

The indefinite articles "a" and "an," as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean "at least one."

The phrase "and/or," as used herein in the specification and in the claims, should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with "and/or" should be construed in the same fashion, i.e., "one or more" of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the "and/or" clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to "A and/or B", when used in conjunction with open-ended language such as "comprising" can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

As used herein in the specification and in the claims, "or" should be understood to have the same meaning as "and/or" as defined above. For example, when separating items in a list, "or" or "and/or" shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as "only one of or "exactly one of," or, when used in the claims, "consisting of," will refer to the inclusion of exactly one element of a number or list of elements. In general, the term "or" as used herein shall only be interpreted as indicating exclusive alternatives (i.e. "one or the other but not both") when preceded by terms of exclusivity, such as "either," "one of," "only one of," or "exactly one of." "Consisting essentially of," when used in the claims, shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase "at least one," in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase "at least one" refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B," or, equivalently "at least one of A and/or B") can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another

embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

When the word "about" is used herein in reference to a number, it should be understood that still another embodiment of the invention includes that number not modified by the presence of the word "about."

It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

In the claims, as well as in the specification above, all transitional phrases such as "comprising," "including," "carrying," "having," "containing," "involving," "holding," "composed of," and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases "consisting of and "consisting essentially of shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.

Claims

CLAIMS What is claimed is:

1. A composition, comprising:

a particle contained within a microfluidic droplet, the particle containing a first oligonucleotide sequence encoding a primer and a second oligonucleotide sequence attached to an antibody.

2. The composition of claim 1, wherein the second oligonucleotide sequence further comprises a first barcode sequence.

3. The composition of claim 2, wherein the first oligonucleotide sequence further

comprises a second barcode sequence.

4. The composition of claim 3, wherein the first barcode sequence and the second

barcode sequence are substantially identical.

5. The composition of any one of claims 2-4, wherein the first barcode sequence is

selected from a pool of barcode sequences.

6. The composition of claim 5, wherein the pool of barcode sequences comprises at least 10,000 barcode sequences.

7. The composition of claim 6, wherein the pool of barcode sequences comprises at least 100,000 barcode sequences.

8. The composition of any one of claims 1-7, wherein at least some of the

oligonucleotide sequences comprise at least two barcode sequences.

9. The composition of any one of claims 1-8, wherein the second oligonucleotide

sequence further comprises a cleavable linker.

10. The composition of claim 9, wherein at least some of the cleavable linkers are

chemically cleavable linkers.

11. The composition of any one of claims 9 or 10, wherein at least some of the cleavable linkers are enzymatically cleavable linkers.

12. The composition of any one of claims 1-11, wherein the second oligonucleotide sequence further comprises an amplification sequence.

13. The composition of any one of claims 1-12, wherein the first oligonucleotide

sequence further comprises an amplification sequence.

14. The composition of any one of claims 1-13, wherein the first oligonucleotide

sequence comprises an overhang portion.

15. The composition of any one of claims 1-14, wherein the first oligonucleotide

sequence comprises a gene-specific reverses transcription primer.

16. An article, comprising:

a plurality of microfluidic droplets containing particles, at least some of the particles comprising a first oligonucleotide sequence comprising a first barcode encoding a gene- specific reverse transcription primer and a second oligonucleotide sequence comprising a second barcode encoding an antibody.

17. The article of claim 16, wherein the first barcodes contained within a droplet are distinguishable from the first barcodes contained within other droplets of the plurality of droplets.

18. The article of any one of claims 16 or 17, wherein the second barcodes contained within a droplet are distinguishable from the second barcodes contained within other droplets of the plurality of droplets.

19. The article of any one of claims 16-18, wherein at least about 90% of the plurality of microfluidic droplets contains only one particle.

20. The article of any one of claims 16-19, wherein the first oligonucleotide sequence comprises a third barcode sequence and the second oligonucleotide sequence comprises a substantially identical third barcode sequence, wherein third barcodes contained within a droplet are distinguishable from the third barcodes contained within other droplets of the plurality of droplets.

21. The article of any one of claims 16-20, wherein the gene-specific reverse transcription primer contained within a droplet are distinguishable from the gene-specific reverse transcription primers contained within other droplets of the plurality of droplets.

22. The article of any one of claims 16-21, wherein the antibody contained within a

droplet are distinguishable from the antibodies contained within other droplets of the plurality of droplets.

23. A method, comprising:

encapsulating a plurality of cells and a plurality of particles within a plurality of droplets, at least some of the particles comprising a first oligonucleotide sequence comprising a gene-specific reverse transcription primer and a first barcode encoding the gene- specific reverse transcription primer and a second oligonucleotide sequence attached to an antibody and comprising a second barcode encoding the antibody, such that the first barcode and/or the second barcode contained within a droplet is distinguishable from the first barcodes and/or the second barcodes contained in other droplets of the plurality of droplets;

releasing the antibody from the particle internally of the droplet; and lysing at least some of the cells within the droplets to release nucleic acid from the cell internally of the droplet.

24. The method of claim 23, wherein releasing the antibody from the particle internally of the droplet comprises cleaving at least a portion of the second oligonucleotide sequence.

25. The method of claim 24, comprising photocleaving at least a portion of the second oligonucleotide sequence.

26. The method of any one of claims 23-25, further comprising bursting at least some of the droplets.

27. The method of claim 26, further comprising collecting the particles.

28. The method of any one of claims 23-27, wherein a protein released by the lysed cell binds to the antibody.

29. The method of claim 28, further comprising determining the protein.

30. The method of any one of claims 28 or 29, further comprising sequencing the second barcode to determine the antibody.

31. The method of any one of claims 23-30, wherein a nucleic acid released by the lysed cell binds to the first oligonucleotide.

32. The method of claim 31, further comprising sequencing the nucleic acid released by the lysed cell.

33. The method of claim 32, further comprising sequencing the first barcode.

34. The method of any one of claims 31-33, wherein the nucleic acid released by the lysed cell is RNA.

35. The method of claim 34, further comprising reverse transcribing the RNA to produce DNA.

36. The method of any one of claims 34 or 35, wherein the RNA is mRNA.

37. The method of any one of claims 23-36, comprising encapsulating the plurality of cells in the plurality of microfluidic droplets such that at least about 90% of the microfluidic droplets contain one cell or no cell.

38. The method of any one of claims 23-37, wherein the particles are encapsulated within the droplets at no more than about 1 particle/droplet.

39. The method of any one of claims 23-38, wherein the particles are encapsulated within the droplets at no more than about 0.1 particles/droplet.

40. The method of any one of claims 23-39, wherein the particles are encapsulated within the droplets at no more than about 0.01 particles/droplet.

41. The method of any one of claims 23-40, wherein the cells are encapsulated within the droplets at no more than about 1 cell/droplet.

42. The method of any one of claims 23-41, wherein the cells are encapsulated within the droplets at no more than about 0.1 cell/droplet.

43. The method of any one of claims 23-42, wherein at least some of the particles are hydrogel particles.

44. The method of any one of claims 23-43, wherein at least some of the particles are polymeric particles.

45. The method of any one of claims 23-44, wherein at least some of the particles are microparticles.

46. The method of any one of claims 23-45, wherein at least some of the particles

comprise polyacrylamide.

47. The method of any one of claims 23-46, wherein at least some of the particles

comprise agarose.

48. The method of any one of claims 23-47, wherein at least some of the particles

comprise polystyrene.

49. The method of any one of claims 23-48, wherein at least some of the particles

comprise poly-N-isopropylacrylamide.

50. The method of any one of claims 23-49, wherein at least some of the particles are magnetic.

51. The method of any one of claims 23-50, wherein the plurality of particles have an average diameter of no more than about 500 micrometers.

52. The method of any one of claims 23-51, wherein the plurality of particles have an average diameter of at least about 1 micrometer.

53. The method of any one of claims 23-52, wherein at least some of the oligonucleotide sequences are covalently bonded to the particles via an acrylic phosphoramidite linkage.

54. The method of any one of claims 23-53, wherein at least some of the oligonucleotide sequences are covalently bonded to the particles via an amino linkage.

55. The method of any one of claims 23-54, wherein at least some of the oligonucleotide sequences are covalently bonded to the particles via a biotin-steptavidin linkage.

56. The method of any one of claims 23-55, comprising covalently bonding the released nucleic acids and the oligonucleotide sequences within at least some of the droplets.

57. The method of any one of claims 23-56, comprising bonding the released nucleic acids and the oligonucleotide sequences within at least some of the droplets using an enzyme.

58. The method of any one of claims 23-57, wherein at least some of the antibodies are IgG antibodies.

59. The method of any one of claims 23-58, wherein at least some of the antibodies are recombinant antibodies.

60. The method of any one of claims 23-59, wherein at least some of the antibodies are Fab fragments.