WO2018067792A1

WO2018067792A1 - Sequencing of bacteria or other species

Info

Publication number: WO2018067792A1
Application number: PCT/US2017/055291
Authority: WO
Inventors: David A. Weitz; Huidan ZHANG; Nai Wen CUI; Yamei CAI; Peter Riad Girguis; Frank Stewart; Neha SARODE; Beate KRAFT
Original assignee: President And Fellows Of Harvard College; Georgia Tech Research Corporation
Priority date: 2016-10-07
Filing date: 2017-10-05
Publication date: 2018-04-12

Abstract

The present invention generally relates to sequencing of cells such as bacteria or archeons. In one aspect, cells are lysed to release RNA, which is then polyadenylated and exposed to a sequencing having an identification portion and a poly-T portion, which associates with the RNA. These are then reverse transcribed to produce cDNA, which can then be studied, amplified, etc. In some cases, such reactions may be performed within droplets. The droplets can then be broken to release the cDNAs, which may be combined together for further sequencing, analysis, etc.

Description

SEQUENCING OF BACTERIA OR OTHER SPECIES

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Patent Application Serial No. 62/405,775, filed October 7, 2016, entitled "Sequencing of Bacteria or Other Species," by Weitz, et ah, incorporated herein by reference in its entirety.

GOVERNMENT FUNDING

This invention was made with government support under Grant No. OCE-0939564, Sub-Award No. 41940192 awarded by the National Science Foundation. The government has certain rights in the invention.

FIELD

The present invention generally relates to sequencing of cells such as bacteria or archeons.

BACKGROUND

Recent studies show that even isogenic populations of exponentially growing microorganisms exhibit substantial cell-to-cell heterogeneities at both cellular and molecular levels at an order of magnitude greater than previously thought. Heterogeneity in isogenic cell populations could arise from the intrinsically stochastic processes of the expression of individual genes and such stochasticity, once amplified to a certain amplitude threshold, could result in heterogeneity at the cellular level, eventually leading to different fates for a microbial population. It is increasingly recognized that by using averaged molecular or phenotypic measurements of a whole population to describe cell behaviors, conclusions could be biased by the expression profiles of outliers; meanwhile, these unique patterns could reflect distinctive functional behaviors at a given place and time and would therefore be important to analyze and describe.

Another major drive to pursue single-cell analysis stems from the fact that more than 99% of microbes from environmental samples are ' 'unculturable' ' in the laboratory and thus cannot be studied using conventional microbiological methods. Meanwhile, many of these unculturable microbes represent new genotypes (phylotypes), families and divisions in the domains of Bacteria and Archaea, and could be of direct relevance to valuable biological processes involved in bioremediation, global warming, alternative energy, and the basic sciences of global cycling of carbon, nitrogen and metals.

Current methods for single bacterium RNA sequencing involve loading a single cell into PCR tubes manually, linearly amplifying RNA from the single bacterium, preparing a cDNA library, and then next-generation sequencing of the resulting cDNA. However, throughput using this method is limited by the number of bacteria that can be processed simultaneously considering the cost of reagents, consumables, personnel, and time.

Accordingly, improvements are needed.

SUMMARY

The present invention generally relates to sequencing of cells such as bacteria or archeons. 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 method comprising encapsulating a single-cell prokaryote in a microfluidic droplet; releasing RNA from the prokaryote; adding a poly-A portion to the RNA released from the cell; exposing the RNA sequence to a oligonucleotide comprising an identification portion and a poly-T portion, wherein the poly-A portion and the poly-T portion are able to associate; reverse transcribing the RNA sequence and the oligonucleotide to produce cDNA; removing the cDNA from the droplet; and sequencing the cDNA.

In another aspect, the present invention is generally directed to a method comprising releasing RNA from a single-cell prokaryote contained within a microfluidic droplet; adding a poly-A portion to the RNA released from the cell; exposing the RNA sequence to a oligonucleotide comprising an identification portion and a poly-T portion, wherein the poly- A portion and the poly-T portion are able to associate; and reverse transcribing the RNA sequence and the oligonucleotide to produce cDNA within the droplet.

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. 1 A-1B generally illustrate adding poly-A to an RNA, in one embodiment of the invention;

Fig. 2 illustrates cDNA, in accordance with another embodiment of the invention; Figs. 3A-3B illustrate single-bacteria sequencing results in accordance with one embodiment of the invention; and

Figs. 4A-4B illustrate single-bacteria sequencing results in accordance with another embodiment of the invention.

DETAILED DESCRIPTION

Certain aspects of the invention are generally directed to the sequencing of nucleic acids such as DNA or RNA from single-cell prokaryotes. Unlike eukaryotes, many prokaryotes produce mRNA without poly-A tails. However, in certain embodiments of the invention, the RNA from single-cell prokaryotes may be reacted to add a poly-A portion (or other suitable portion) to the RNA, for instance, using a suitable polymerase. Afterwards, the poly-A portion may be associated to a sequence comprising a poly-T portion (or other suitable sequence), which may also contain other sequences useful for sequencing or identification. This combination may then be reverse transcribed to produce cDNA, which can then be separated, analyzed, sequenced, etc., as desired.

In certain embodiments, such reactions may be performed using droplets, such as microfluidic droplets. For instance, in one set of embodiments, single-cell prokaryotes may be encapsulated in droplets, e.g., at a density of 1 cell/droplet, or less, on the average. By doing so, each of the prokaryotes may be manipulated (for example, lysed to release nucleic acid) without worrying about contamination from other prokaryotes. In such fashion, even a population of different single-cell prokaryotes (e.g., from different species), and/or populations with other contaminates (e.g., dirt, eukaryotic cells, etc.) can be studied. The prokaryotes within the droplets may be lysed to release their nucleic acids (e.g., DNA and/or RNA), which can then be reacted to add a poly- A portion, or other suitable portions, to the nucleic acids. For example, the droplets may contain an enzyme such as a poly-A polymerase that is able to add adenosines onto RNA. Such enzymes may be present initially when the droplet is created, or subsequently added (for example, via picoinjection or droplet merger techniques, see, e.g., Int. Pat. Apl. Pub. Nos. WO 2004/002627 ("Method and Apparatus for Fluid Dispersion"), WO 2004/091763 ("Formation and Control of Fluidic Species"), WO 2005/021151 ("Electronic Control of Fluidic Species"), WO 2010/151776 ("Fluid Injection"), or WO 2015/200616 ("Fluid Injection Using Acoustic Waves"), each incorporated herein by reference in its entirety), and they may be added before and/or after lysis of the prokaryotes. In addition, the droplets may contain other materials to facilitate this reaction, such as RNAse inhibitors (e.g., to inhibit RNA degradation), cell lysis reagents, protease inhibitors, DNAses, etc.

After addition of a poly-A portion (or other suitable sequence) to the nucleic acids released from the prokaryotes, the nucleic acids may be associated to an oligonucleotide comprising a poly-T portion (or other suitable sequence), which may also contain other portions useful for sequencing or identification, such as an identification portion, a primer, a sequencing portion, or the like. More than one may also be present in some cases. The association may be created through complementary interaction of the poly-A portion of the nucleic acids to the poly-T portion of the oligonucleotide. If other portions are present (i.e., in addition or instead of a poly-A portion), the other sequences may similarly be

complementary to each other. The oligonucleotide may be present initially (e.g., when the droplet is formed), or added after formation of the droplet (e.g., added by picoinjection or droplet merger techniques. See, e.g., Int. Pat. Apl. Pub. Nos. WO 2004/002627 ("Method and Apparatus for Fluid Dispersion"), WO 2004/091763 ("Formation and Control of Fluidic Species"), WO 2005/021151 ("Electronic Control of Fluidic Species"), WO 2010/151776 ("Fluid Injection"), or WO 2015/200616 ("Fluid Injection Using Acoustic Waves"), each incorporated herein by reference. These may be added at any suitable time (e.g., before or after lysis of the prokaryote, added with enzymes such as polymerases, or added before or afterwards, etc.).

In one set of embodiments, the oligonucleotides may be added by way of particles. For example, a plurality of particles may be created, containing oligonucleotides such that each particle contains only one uniquely identified oligonucleotide, e.g., on its surface (although multiple copies of the oligonucleotide may be present). For instance, the oligonucleotides may be substantially identical except differing in an identification portion or "barcode," that is different for each particle. In some cases, the identification portion may be relatively short (e.g., less than 20 nt, less than 10 nt, etc.), and/or there may be more than one such identification portion present within the oligonucleotide. The particles may be then be added to droplets at a relatively low density, e.g., at 1 particle/droplet on the average, or less. By doing so, at least some of the droplets will contain only one prokaryote and only one particle, which may allow for unique identification of the prokaryote, as discussed herein. Once contained within the droplets, the oligonucleotides may be released from the particle into the droplet, e.g., by cleaving the oligonucleotide from the droplet (e.g., through photocleavage), reaction or destruction of the particle (e.g., if the particles contain the oligonucleotides internally, e.g., in an internal compartment or fluid within the particle), or the like. Delivery into the droplets may be accomplished, for example, through injection or droplet merger techniques, such as those discussed in Int. Pat. Apl. Pub. Nos. WO

2004/002627 ("Method and Apparatus for Fluid Dispersion"), WO 2004/091763 ("Formation and Control of Fluidic Species"), WO 2005/021151 ("Electronic Control of Fluidic

Species"), WO 2010/151776 ("Fluid Injection"), or WO 2015/200616 ("Fluid Injection Using Acoustic Waves"), each incorporated herein by reference in their entireties.

After association, the nucleic acids may be reverse transcribed to produce DNA (i.e., cDNA), using a suitable reverse transcriptase. Many reverse transcriptases are available commercially. Such reverse transcriptases (along with suitable reagents that may allow reverse transcription to occur, such as deoxyribonucleotides) may be present within the droplet at formation or added afterwards. As with the above, these may be added at any suitable point, e.g., before, during, or after the above reagents, and may be added via any suitable technique, such as via picoinjection or droplet merging techniques, for instance, those discussed in Int. Pat. Apl. Pub. Nos. WO 2004/002627 ("Method and Apparatus for Fluid Dispersion"), WO 2004/091763 ("Formation and Control of Fluidic Species"), WO 2005/021151 ("Electronic Control of Fluidic Species"), WO 2010/151776 ("Fluid

Injection"), or WO 2015/200616 ("Fluid Injection Using Acoustic Waves"), each

incorporated herein by reference.

In some embodiments, the cDNA may be removed from the droplets for subsequent processing or analysis. For instance, the droplets may be burst or broken to release their contents, e.g., by exposure to mechanical disruption, ultrasound, chemical agents or surfactants, or the like. The cDNA from different droplets may be collected together and analyzed or sequenced together. Due to the presence of the identification portions as discussed above within the cDNA, the cDNA from different droplets may be combined together for sequencing or analysis, as their different origins can nonetheless be determined or tracked via the identification portions, even after combination. In this way, more conventional sequencing or amplification techniques may be used to analyze the cDNA. Thus, for instance, the cDNA may be amplified using PCR techniques, purified using gels, and/or sequenced using techniques such as chain-termination sequencing, sequencing-by- hybridization, sequencing-by-synthesis, etc.

The above discussion is a non-limiting example of one embodiment of the present invention that can be used to study or sequence prokaryotes. However, other embodiments are also possible. Accordingly, more generally, various aspects of the invention are directed to various systems and methods for sequencing cells such as bacteria, archeons, or other single-cell prokaryotes, or other cells (e.g., eukaryotes). In some cases, such cells do not generally produce mRNA without poly-A tails.

As mentioned, in one aspect, the present invention is generally directed to the detection of prokaryotic cells (prokaryotes), such as bacteria or archeons. Such cells are typcially single-celled organisms, and some produce RNA without a polyadenylated tail (i.e., a poly-A tail), or produce a polyadenylated tail that is relatively short, e.g., as compared to eukaryotes. Examples of prokaryotes include, but are not limited to, various organisms of the archea domain (i.e., archeons) or the bacteria domain (i.e., bacteria).

In some cases, cells such as prokaryotes may be encapsulated within a droplet, such as a microfluidic droplet. In some embodiments, the droplets can be 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. Those of ordinary skill in the art will be aware of suitable techniques for loading a cell into a droplet, e.g., when the droplet is created, or afterwards. In addition, in certain embodiments, it may be desired to encapsulate cells at higher rates, e.g., greater than 1 cell/droplet, greater than 2 cells/droplets, etc.

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.

In one set of embodiments, 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, through fusion of the droplets with droplets containing the chemical or enzyme, through other techniques known to those of ordinary skill in the art, etc., such as those discussed herein. In some cases, lysing a cell will cause the cell to release its contents, e.g., genomic DNA, RNA, etc.

In some embodiments, compounds such as RNAse inhibitors, DNAse inhibitors, proteinase inhibitors, etc., may be used within the droplets, e.g., to prevent or reduce degradation of RNA, DNA, proteins, and the like from being too degraded upon release from the cells. In some cases, RNA or DNA may be selectively favored (for example, by using RNAse inhibitors and DNAse to favor RNA relative to RNA, or by using DNAse inhibitors and RNAse to favor DNA relative to RNA). Many such compounds can be commercially obtained, and may be present during formation of the droplets, and/or added afterwards. Techniques for adding a reagent to droplets are discussed herein, e.g., picoinjection, droplet fusion, etc.

As mentioned, in some embodiments of the invention, the nucleic acids (e.g., RNA, DNA, etc.) released from the cells may be reacted to add a poly-A portion, or other suitable portion. This may allow other nucleic acid portions to be added to the released nucleic acids, thereby allowing subsequent manipulations to occur, e.g., to add portions to the nucleic acids for sequencing, synthesis, amplification, or the like. Such reagents may be added to a droplet at formation of the droplet, and/or afterwards, e.g., through techniques such as picoinjection, droplet fusion, etc., as discussed herein. Additional reagents for the addition may be present as well, such as adenosine triphosphates, cofactors, and the like.

In certain embodiments, the nucleic acids may be reacted to add a poly-A portion using a suitable enzyme, such as a polymerase. For instance, polyadenylation polymerases such as E. coli poly-A polymerases or GLD-2 (Caenorhabditis elegans) can be used to add a plurality of A's to a nucleic acid to form a poly-A portion, e.g., by sequentially adding adenosine phosphates onto the RNA. In some cases, the poly-A portion is added to one end of the nucleic acid, thereby forming a poly-A "tail." The poly-A portion, or binding portion, may be of suitable length, e.g., a length of at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 75, or at least 100 nt, and may consist only of A residues within the poly-A portion, or may also contain other residues interspersed with the A residues, e.g., such that at least 70%, at least 80%, at least 90%, or at least 95% of the residues within the poly-A portion are A residues. In addition, it should be understood that different nucleic acids present within a droplet need not all have poly-A portions of the same length, e.g., if added through polymerase reactions. Many such polymerases are available commercially.

However, it should be understood that the invention is not limited to only poly-A polymerase. Other polymerases, or other portions, may be used in other cases. In addition, it should be understood that the invention is not limited to only to poly-A portions. For instance, the nucleic acids may be added to a binding portion that can associate with a complementary binding portion. In some cases, for example, the binding portion has a nonsense array of nucleotides, which can be recognized by a complementary binding portion, and may the same or different between different droplets.

Besides polymerases, other techniques may be used to add such portions to a nucleic acid, for example, by introducing suitable nucleic acids strands (e.g., poly-A sequences, or other sequences) and ligating or otherwise joining the nucleic acid strands to the nucleic acids. 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.

The poly-A portion may associate with a sequence comprising a poly-T portion (or other suitable sequence), which may also contain other sequences useful for sequencing or identification, as discussed herein. The association may be, for instance, through noncovalent interaction, e.g., between the poly-A portion and the poly-T portion, or between a binding portion that can associate with a complementary binding sequence. The poly-T portion or complementary binding portion may have the same or different lengths than the poly-A or binding portion. The poly-T portion may be of suitable length, e.g., a length of at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 75, or at least 100 nt, and may consist only of T residues within the poly-T portion, or may also contain other residues interspersed with the T residues, e.g., such that at least 70%, at least 80%, at least 90%, or at least 95% of the residues within the poly-T portion are T residues.

This portion may be part of an oligonucleotide that can also optionally contain other portions useful for sequencing or identification, such as an identification portion, a primer, a sequencing portion, or the like. For instance, in one set of embodiments, the oligonucleotide may contain a unique identification or tag sequence that allows different droplets to be distinguished based, at least in part, on the presence of the unique identification or tag sequences. In this way, as discussed herein, nucleic acids from different droplets may be subsequently combined or pooled together and analyzed, while being able to distinguish different nucleic acids as having arisen from different droplets. One non-limiting example of such a technique is to label the nucleic acids with unique oligonucleotides or "barcodes" prior to their release from the droplets.

In some embodiments, the oligonucleotide may comprise a "barcode" or a unique sequence, which may be selected such that some or all of the oligonucleotides have the unique sequence (or combination of sequences that is unique), but other oligonucleotides

(e.g., in other 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, or released after the droplets are broken or dispersed.

The oligonucleotide may be of any suitable length. The length of the oligonucleotide sequence is not critical, and may be of any length sufficient to distinguish the oligonucleotide sequence from other oligonucleotide sequences. One, two, or more such distinguishing "barcode" sequence may be present in an oligonucleotide, as discussed above. A barcode sequence can have, for instance, 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 oligonucleotide or barcode sequences may be taken from a "pool" of potential sequences. If more than one barcode sequence is present in an oligonucleotide, 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 embodiments of the present invention, the oligonucleotides may be attached to particles or microspheres, e.g., for delivery to droplets. For example, one set of embodiments is generally directed to particles or microspheres carrying oligonucleotides (e.g., encoding a barcode, a primer, and/or other sequences possibly used for capture, amplification and/or sequencing of nucleic acids). Microspheres may include a hydrogel particle (polyacrylamide, agarose, etc.), or a colloidal particle (polystyrene, magnetic or polymer particle, etc.), having 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.

The preparation of 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 384²=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 xi 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 X1X2X3 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.

The oligonucleotide may be of any suitable length or comprise any suitable number of nucleotides. The oligonucleotide 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 is single stranded, although it may be double stranded in other cases. For example, the oligonucleotide 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 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 may be between about 10 nt and about 100 nt. The length of the oligonucleotide is not critical, and a variety of lengths may be used in various embodiments.

The oligonucleotide may also contain a variety of sequences. For example, the oligonucleotide may contain one or more primer sequences, one or more unique or "barcode" sequences as discussed herein, one or more promoter sequences, one or more spacer sequences, or the like. The oligonucleotide may also contain, in some embodiments one or more cleavable spacers, e.g., photocleavable linker. The oligonucleotide may in some embodiments 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 oligonucleotides can be removed from the particle via cleavage. Other examples include portions that may be used to increase the bulk (or length) of the oligonucleotides (e.g., using specific sequences or nonsense sequences), to facilitate handling (for example, an oligonucleotide may include a poly-T 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. In some cases, the

oligonucleotide may contain one or more promoter sequences, e.g., to allow for production of the oligonucleotide, to allow for enzymatic amplification, or the like. In some cases, the oligonucleotide may contain nonsense or random sequences, e.g., to increase the mass or size of the oligonucleotide. 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 may comprise one or more primer sequences, e.g., to facilitate reverse transcription, sequencing, or other reactions including those described herein. Non-limiting examples of primers include the p5 primer (5' AAT GAT ACG GCG ACC ACC GA 3') and P7 primer (5' CAA GCA GAA GAC GGC ATA CGA 3'). Other examples of sequencing adapters include those commercially produced by Illumina, such as the Nextera™ sequences.

In some cases, the oligonucleotide may comprise one or more sequences able to specifically bind a gene or other entity. For example, in one set of embodiments, the oligonucleotide 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 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. In some cases, for example, a plurality of particles (containing oligonucleotides 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 oligonucleotides may be cleaved from the particles, e.g., using light or other suitable cleavage techniques, to allow the oligonucleotides to become present in solution, i.e., within the interior of the droplet. In such fashion, oligonucleotides can be easily loaded into droplets by loading of the particles into the droplets, then cleaved off to allow the oligonucleotides to be in solution, e.g., to interact with nucleotides or other species, such as is discussed herein.

A variety of techniques may be used for preparing oligonucleotides such as those discussed herein. These may be prepared in bulk and/or in one or more droplets, such as microfluidic droplets. In some cases, the oligonucleotides may be prepared in droplets, e.g., to ensure that the barcodes and/or oligonucleotides within each droplet are unique. In addition, in some embodiments, particles may be prepared containing oligonucleotides with various barcodes in separate droplets, and the particles may then be given or sold to a user who then adds the nucleic acids to the oligonucleotides, e.g., as described above. In some cases, an oligonucleotide comprising DNA and/or other nucleic acids may be attached to particles and delivered to the droplets. In some cases, the oligonucleotides 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 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 oligonucleotides that are the same or different.

In some embodiments, the oligonucleotides introduced into droplets 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.

Any suitable method may be used to attach the oligonucleotide to the particle. The exact method of attachment is not critical, and may be, for instance, chemical or physical. For example, the oligonucleotide may be covalently bonded to the particle via a biotin- steptavidin linkage, an amino linkage, or an acrylic phosphor amidite 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 embodiments. The particle may be of any of a wide variety of types; as discussed, the particle may be used to introduce a particular oligonucleotide into a droplet, and any suitable particle to which oligonucleotides 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. 1 1/360,845, 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; 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 some cases, particles such as those discussed herein containing oligonucleotides may be contained within a droplet and the oligonucleotides released from the particle into the interior of the droplet. The droplet may also contain nucleic acids (e.g., produced by lysing a cell), which can be bound to or recognized by the oligonucleotides, as discussed above. The particles and the cells may be introduced within the droplets during and/or after formation of the droplets, and may be added simultaneously or sequentially (in any suitable order).

Suitable techniques include, but are not limited to, picoinjection, droplet fusion, etc., as discussed herein. As mentioned, in some embodiments, the particles and the cells may be placed within droplets such that the droplets typically would contain, on average, no more than one particle and no more than one cell.

After association of the nucleic acids and the oligonucleotides, they may be reverse transcribed to produce DNA (i.e., cDNA), using a suitable reverse transcriptase. Many reverse transcriptases can be readily obtained commercially. Non-limiting examples include Avian Myeloblastosis Virus (AMV) Reverse Transcriptase and Moloney Murine Leukemia Virus (M-MuLV, MMLV) Reverse Transcriptase; besides naturally-occurring reverse transcriptases, several engineered reverse transcriptases are also available commercially. In some cases, the reverse transcriptases, and any suitable reagents to facilitate transcription, such as deoxyribonucleotides, cofactors, etc., may be present within a droplet at its formation, or subsequently added, using techniques such as picoinjection, droplet fusion, or the like, e.g., as discussed herein. In some cases, multiple copies of the cDNA strands may be produced, e.g., from the nucleic acids associated with the oligonucleotides. The cDNAs, for example, may contain identification sequences such as those described above, which may be useful for later identification, sequencing, etc.

In certain embodiments, the cDNA may be amplified, e.g., within the droplets, for example, by including suitable reagents. 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). See also U.S. Pat. Apl. Ser. Nos. 61/981,108, 62/072,944, or 62/133,140, or U.S. Pat. Apl. Pub. Nos. 2010/0136544, 2014/0199730, or 2014/0199731, each incorporated by reference in its entirety.

In one set of embodiments, for example, PCR or nucleic acid amplification may be performed within the droplets. For example, the droplets may contain a primer (such as those discussed herein), 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, primers, and nucleotides may be added at any suitable point, and may be added sequentially and/or simultaneously, using any suitable technique (e.g., using droplet fusion or injection techniques). 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.

In some cases, the droplets may be burst, broken, or otherwise disrupted, e.g., to release or remove the various nucleic acids contained therein, such as the cDNA described above. This may be useful, for example, for subsequent study of the nucleic acids, e.g., via sequencing or other techniques. A wide variety of methods for "breaking" or "bursting" droplets are available to those of ordinary skill in the art. 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 oligonucleotides) 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 oligonucleotides (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.

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 in accordance with various aspects of the invention 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 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 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.

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"), 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.

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. In some embodiments, this may be used, for example, to add material to a droplet, e.g., by fusing the droplet with another droplet.

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. Pat. Apl. Pub. No. 2007/0195127 or U.S. Pat Nos. 7,708,949, 8,765,485, or 9,038,919, each 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 ah, 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 ah, published as WO 2010/080134 on July 15, 2010, each incorporated herein by reference in its entirety.

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 ah).

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 Micro fluidic Systems and Polydimethylsiloxane," Anal. Chem., 70:474-480, 1998 (Duffy et al), 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 each incorporated herein by reference in its 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 ; Int. Pat. Apl. Pub. No. WO 2015/164212, entitled "Systems and Methods for Barcoding Nucleic Acids," by Weitz, et al; Int. Pa. Apl. Pub. No. WO 2015/161223, entitled "Methods and Systems for Droplet Tagging and Amplification"; and Int. Pat. Apl. Pub. No. WO 2015/16117, entitled "Systems and Methods for Droplet Tagging." In addition, U.S. Provisional Patent Application Serial No. 62/405,775, filed October 7, 2016, entitled "Sequencing of Bacteria or Other Species," by Weitz, et al. is also incorporated herein by reference in its entirety for all purposes.

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 illustrates a drop-based high-throughput single bacterium RNA sequencing method that yields transcriptome data for thousands of individual cells in a rapid and cost-effective way, thereby enabling much higher resolution of population-level heterogeneity in gene expression.

As one example, the method may include:

i) Encapsulating a cell such as a bacterium or archeon in a droplet, which may include a lysis buffer or other constituents to ensure cell lysis, protein inactivity if needed, and nucleic acid stability;

ii) Injecting reagents that can add polymerize adenosine monophosphates (i.e. a "poly(A) tail") onto all ribonucleic acids (RNA) (Fig. 1A);

iii) Injecting deoxyribonucleic acids (DNA) barcoded beads and the reagents needed for reverse transcription (RT), thus allowing reverse transcription and labeling of the cDNA with unique DNA barcodes, shown as Fig. IB;

iv) Retrieving cDNA in droplets, for instance, by adding lH, lH,2H,2H-perfluoro- octanol to break the drops and/or by purifying with Spri beads;

v) Amplifying the cDNA, e.g., using 20-cycle PCR, which may also include purification of the amplicons with Spri beads;

vi) Performing another round of 10-cycle PCR using primers containing Illumina sequencing technology-specific adaptors and sample- specific indices, such that the libraries can be multiplexed for sequencing;

vii) Conducting DNA electrophoresis and/orgel purification;

viii) Sequencing cDNA and analyzing the data.

One non-limiting example of a cDNA fragment that can be generated using one embodiment of the invention is shown in Fig. 2.

EXAMPLE 2 This example illustrates an example of data analysis in accordance with one embodiment of the invention. The transcripts per cell may be separated. Forward reads (readl) of all transcripts derived from the same cell may contain the same unique combination of 8bp cell barcode- 1 and barcode-2 separated by the linker cassette (although in other cases, other encoding systems may be used for identification). The reads may be split into sets of transcripts-per-cell by matching the barcodes against a known list of

predetermined barcode combinations (n=9216) and extracting the UMI (Unique Molecular Identifiers, e.g., barcodes) sequences (identified here as N10, shown in Fig. 2) per read. During this screening process, a stringent criteria of one base substitution error is tolerated, while any reads with insertion, deletion or more than 1 bp substitution error are discarded. The barcode based split files are then used for UMI based filtering and read quality processing. The reverse reads (read 2) of paired filtered readl are trimmed for low quality bases (Q25), Illumina adapter sequences and poly-T tails. The processed reads are then aligned to the corresponding reference bacterial genome using Bowtie2. Mapped reads are first filtered for UMI duplications using UMI-tools and then counts per gene are determined. The count information can then be used for visualization of transcripts per cell, sequencing saturation curves etc.

In this method, poly- A tails are added to the RNA at the 3' end. This can be performed, for example, using commercially available polymerases such as E. coli poly-A polymerases (New England BioLabs, Inc.). This can simplify library preparation and decreases cost because barcoded beads are directly used for single mammalian cell RNA-seq that contain poly-T capture oligonucleotides at the 3 ' end, thereby eliminating the need to order new sequences for barcode synthesis. In addition, the reaction for adding poly-A tails can be easily deactivated by heating the temperature to 70 °C for 10 minutes, so it will not interfere with any subsequent RT reactions. Also, no reagents are washed away after adding the poly-A tails.

Using this drop-based bead barcoding method, transcriptomic profiles from 564 cells were obtained, as shown in Figure 3A. Complexity analysis shows the counts/cell was similar to the number of genes/cell, which indicates that the complexity was very high (Fig. 3B).

Fig. 3 shows single -bacteria sequencing result using drop-based microfluidics. Fig. 3A is a boxplot of total transcript counts/cell. Fig. 3B shows a complexity plot using transcript counts in Fig. 3A, representing the number of genes/cell. The more cells there are, the wider the shaded area is. The protein coding RNA counts from these cells was also analyzed. Although the number of the protein coding RNA was smaller, which was expected, downstream analysis could still be performed (Figs. 4A-4B). In addition, protein coding RNA counts/cell was similar to protein coding genes/cell.

Fig. 4 shows single-bacteria sequencing result using drop-based microfluidics. Fig.

4A shows a box plot of protein coding RNA counts/cell. Fig. 4B shows a complexity plot using protein coding RNA counts in Fig. 4A, representing protein coding genes/cell. The more cells there are, the wider the shaded area is.

EXAMPLE 3

This example illustrates materials and methods useful with some embodiments of the invention.

Microfluidic device fabrication. Polydimethylsiloxane (PDMS) microfluidic devices were fabricated using standard soft lithographic methods. The microfluidic channel walls were rendered hydrophobic by treating them with Aquapel (PPG, Pittsburgh, PA). In the PDMS device, electrodes were designed as channels. These channels were filled with

Indalloy 19 (51In, 32.5 Bi, 16.5 Sn; 0.020 inch diameter), a low melting point metal alloy (Indium, Clinton, NY), by pushing the wire into the microfluidics channel designed for electrodes on an 80 °C hot plate. Electrical connections were made using eight-pin terminal blocks (Phoenix Contact, Middletown, PA).

Synthesis of DNA-barcoded hydrogel beads. These were synthesized according to established protocols.

Encapsulate single bacteria and lysis buffer into drops. Single bacteria and lysis buffer were co-encapsulated into 50 micrometer water-in-oil drops using a standard co-flow microfluidics device. The 100 microliter lysis buffer contained 1 microliter 1 U/microliter lysostaphin, 2 microliters 10% Tween 20, 2 microliters 10 mg/mL BSA, 2 microliters 2U/microliter DNase I (NEB, Ipswich, MA), 2 microliters 40U/microliter RNaseOut (Thermo Fisher, Waltham, MA), and 93 microliter xTraxtor buffer (Clonteq, Mountain View, CA). The temperature program for bacteria lysis was 25 °C for 30 min, 37 °C for 10 min, 75 °C for 15 min, and then on ice.

Inject Poly(A) tailing reagent into drops. A microfluidic pico-injector was used to inject Poly(A) tailing reagent (Applied Biosystems, Foster City, CA) into the drops containing lysed bacteria. The device had three inlets. Two inlets at the upstream side were used for injecting spacing oil and drops containing lysed bacteria, and the other inlet on the downstream side was used for injecting Poly A tailing reagent. The 50 microliter Poly(A) tailing reagent contained 20 microliters 5 x E-PAP, 10 microliters 25 mM MgCl₂, 10 microliters 10 mM ATP, 4 microliters E-PAP, 4 microliters 10% Tween 20 and 2 microliters 10 mg/mL BSA. The flow rates for all three inlets were set to ensure an acceptable pairing efficiency for drops and barcoded beads. Typical flow rates were 300 microliters/hr for, 80 microliters hr for drops, and 80 microliters/hr for RT reagent.

Load barcoded hydrogel beads and reverse transcription (RT) reagent into drops. A modified microfluidic pico-injector was used to inject barcoded beads and RT reagent into the drops containing bacteria. The device had four inlets. The two inlets on the upstream side were used for injecting spacing oil and drops containing bacteria, and the other two inlets on the downstream side were used for injecting beads and RT reagent. The flow rates for all four inlets were set to ensure an acceptable pairing efficiency for drops and barcoded beads. The 34.6 microliters RT reagent contained 16 microliters of 5x RT buffer, 8 microliters of dNTPs, 1.6 microliters RNaseOUT, 1.6 microliters 10% Tween 20, 1.6 microliters BSA, 0.8 microliters of the template switching oligo (100 micromolar) and 5 microliters of Maxima H Minus Reverse Transcriptase (Thermo Fisher, Waltham, MA). Typical flow rates were 300 microliters/hr for oil, 100 microliters/hr for drops, 60 microliters hr for barcoded beads, and 60 microliters/hr for RT reagent. The drops were collected and exposed to UV to cleave the barcodes from of beads, followed by incubation at 42 °C for 2 hours. During RT, the unique barcodes on a barcoded bead were incorporated into the final cDNA, such that the amplicons could be pooled by breaking the drops without losing the discrimination of individual cells.

Break drops and Purify cDNA. To break drops and retrieve the cDNA,

lH,lH,2H,2H-perfluoro-l-octanol (PFO; Sigma-Aldrich, St. Louis, MO) was added into drops at a 1:4 ratio, centrifuged, and the liquid phase removed. 80% (v/v) Spri beads (Life Technologies, Grand Island, NY) were used to purify the cDNA, where was eluted into 18 microliters of 0.1 M Tris-HCl.

Exonuclease I treatment. 2 microliters of 10X reaction buffer and 1 microliter of Exonuclease I (NEB, Ipswich, MA) was added to the cDNAs, and then incubated at 37 °C for 30 minutes, then at 80 °C for 20 minutes to inactivate the exonuclease I.

Full length cDNA amplification. The 50 microliter amplification mixture contained 5 microliters of lOx Advantage 2 PCR buffer 1 microliter of dNTPs, 1 microliter of the amplification primer (10 micromolar), 1 microliter of Advantage 2 Polymerase Mix

(Clontech, Mountain View, CA), 2 microliters of 0.1M Tris-HCl, and 20 microliters of sample. The cycling process was 95 °C for 1 min, 18 cycles of 95 °C for 15 sec, 65 °C for 30 sec, 68 °C for 6 min, 72 °C for 10 min, and then at 4 °C indefinitely. Library preparation and next-gen sequencing. The amplicons were purified using 80 % AMPure XP Beads and eluted into 10 microliters of 0.1M Tris-HCl. From the purified full-length cDNA, 1 ng of cDNA was engaged in the Nextera XT library preparation according to Illumina protocols with the exception that only the i7 primer was used to barcode different samples while the forward primer, 5 micromolar, was used as second primer during the library amplification.

The sequencing library was purified with 30 microliters of AMPure XP magnetic beads and eluted in 20 microliters. The entire library was run on an E-Gel EX Gel, 2 % (Life Technologies,) and the band corresponding to a size range of 300 to 800 bp was excised, purified using the QIAquick Gel Extraction Kit (Qiagen,) and eluted in 15 microliters. The library was quantified on a Qubit 2.0 Flurometer using a dsDNA HS Assay. Sequencing can be performed on any Illumina HiSeq or MiSeq using standard Illumina sequencing kit. Libraries were run on paired-end flow cells by running 60 cycles on the first end, then 8 cycles to decode the Nextera barcode, and finally 82 cycles.

Data analysis. The first step of data analysis was to separate out transcripts per cell.

Forward reads (readl) of all transcripts derived from the same cell should contain the same unique combination of 8 bp cell barcode- 1 and barcode-2 separated by the linker cassette. The first step included splitting of reads into sets of transcripts-per-cell by matching the barcodes against the known list of predetermined barcode combinations (n=9216) and extracting the UMI sequences (N10, shown in Figure 2) per read. During this screening process, a stringent criteria of one base substitution error was tolerated, while any reads with insertion, deletion or more than 1 bp substitution error was discarded. The barcode based split files are then used for UMI based filtering and read quality processing. The reverse reads (read 2) of paired filtered readl were trimmed for low quality bases (Q25), Illumina adapter sequences and poly T tails. The processed reads were then aligned to the corresponding reference bacterial genome using Bowtie2. Mapped reads were first filtered for UMI duplications using UMI-tools (bioRxiv 051755), and then counts per gene calculated using featureCounts . The count information was then used for visualization of transcripts per cell, sequencing saturation curves, etc. using R packages like Seurat.

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

claimed is:

A method, comprising:

encapsulating a single-cell prokaryote in a microfluidic droplet;

releasing RNA from the prokaryote;

adding a poly-A portion to the RNA released from the cell;

exposing the RNA sequence to a oligonucleotide comprising an identification portion and a poly-T portion, wherein the poly-A portion and the poly-T portion are able to associate;

reverse transcribing the RNA sequence and the oligonucleotide to produce cDNA;

removing the cDNA from the droplet; and

sequencing the cDNA.

The method of claim 1 , wherein the single-cell prokaryote is a bacterium.

The method of any one of claims 1 or 2, wherein the single-cell prokaryote is an archeon.

The method of any one of claims 1-3, wherein releasing RNA from the prokaryote comprises lysing the prokaryote within the droplet.

The method of any one of claims 1-4, wherein the droplet contains an RNAse inhibitor.

The method of any one of claims 1-5, wherein the droplet contains a cell lysis reagent.

The method of any one of claims 1-6, wherein the droplet contains a DNAse.

The method of any one of claims 1-7, wherein releasing RNA from the prokaryote comprises adding, to the droplet, a cell lysis reagent.

9. The method of any one of claims 1-8, wherein the droplet contains a protease inhibitor.

10. The method of any one of claims 1-9, wherein adding a poly-A portion to the RNA comprises exposing the RNA to an enzyme able to add adenosine phosphates onto

RNA.

11. The method of any one of claims 1-10, wherein adding a poly-A portion to the RNA comprises exposing the RNA to a poly-A polymerase.

12. The method of claim 11, wherein the droplet further contains adenosine triphosphate.

13. The method of any one of claims 1-12, wherein adding a poly-A portion to the RNA released from the cell comprises adding, to the droplet, an enzyme able to add adenosine phosphates onto RNA.

14. The method of any one of claims 1-13, wherein adding a poly-A portion to the RNA released from the cell comprises injecting, into the droplet, an enzyme able to add adenosine phosphates onto RNA.

15. The method of any one of claims 1-14, wherein the poly-A portion comprises at least 10 sequential A residues.

16. The method of any one of claims 1-15, wherein the droplets contains a particle

containing the oligonucleotide attached thereto.

17. The method of claim 16, further comprising cleaving the oligonucleotide from the particle. 18. The method of any one of claims 1-17, wherein the identification portion comprises at least 4 nucleotides.

19. The method of any one of claims 1-18, wherein the identification portion comprises at least 8 nucleotides.

20. The method of any one of claims 1-19, wherein the oligonucleotide comprises at least two identification portions. 21. The method of any one of claims 1-20, wherein the oligonucleotide further comprises a sequencing adapter portion.

22. The method of any one of claims 1-21, wherein the poly-T portion comprises at least 10 sequential T residues.

23. The method of any one of claims 1-22, wherein reverse transcribing the RNA

sequence and the oligonucleotide comprises adding, to the droplet, a reverse transcriptase. 24. The method of any one of claims 1-23, wherein reverse transcribing the RNA

sequence and the oligonucleotide comprises injecting, into the droplet, a reverse transcriptase.

25. The method of any one of claims 1-24, wherein reverse transcribing the RNA

sequence and the oligonucleotide comprises adding, to the droplet, a plurality of deoxyribonucleo tides .

26. The method of any one of claims 1-25, further comprising amplifying the cDNA within the droplet.

27. The method of any one of claims 1-26, comprising amplifying the cDNA within the droplet using PCR.

28. The method of any one of claims 1-27, wherein removing the cDNA from the droplet comprises breaking the droplet.

29. The method of claim 28, wherein breaking the droplet comprises exposing the

droplets to a chemical agent able to break the droplet. The method of any one of claims 28 or 29, wherein breaking the droplet comprises exposing the droplets to lH,lH,2H,2H-perfluorooctanol.

The method of any one of claims 28-30, wherein breaking the droplet comprises exposing the droplets to ultrasound.

The method of any one of claims 1-31, wherein sequencing the cDNA comprises sequencing the cDNA using sequencing by synthesis.

The method of any one of claims 1-32, further comprising purifying the cDNA prior to sequencing.

The method of any one of claims 1-33, wherein a plurality of cDNAs from different single-cell prokaryotes are combined and sequenced together.

A method, comprising:

releasing RNA from a single-cell prokaryote contained within a microfluidic droplet;

adding a poly-A portion to the RNA released from the cell;

exposing the RNA sequence to a oligonucleotide comprising an identification portion and a poly-T portion, wherein the poly-A portion and the poly-T portion are able to associate; and

reverse transcribing the RNA sequence and the oligonucleotide to produce cDNA within the droplet.