Single-cell nucleic acid sequence analysis

Abstract

To provide a method and a composition for gene expression analysis of multiplexed single cell.SOLUTION: Some methods and compositions involve use of droplets and / or beads that retain a unique bar code, e.g., a unique molecular bar code (UMI).SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] (Related Applications) This application claims priority to U.S. Provisional Patent Application 62 / 211,597, filed Aug. 28, 2015, and is a continuation-in-part of PCT Application PCT / US15 / 28062, filed Apr. 28, 2015, which claims priority to U.S. Provisional Patent Applications 61 / 985,983, filed Apr. 29, 2014, and 61 / 987,433, filed May 1, 2014. Each of these earlier applications is hereby incorporated by reference in its entirety. (Government Support) This invention was made with government support under National Institutes of Health (NIH) Grant No. MH098977, awarded by the Public Health Service (PHS). The government has certain rights in this invention.

Background Art

[0002] Determination of the content of mRNA in cells or tissues (i.e., “gene expression profiling”) provides a method for the functional analysis of normal as well as diseased tissues and organs. Typically, gene expression profiling is performed by isolating mRNA from a tissue sample and subjecting the mRNA to microarray hybridization. However, such methods only enable the analysis of known genes and cannot be used for the analysis of alternative splicing, promoters, and polyadenylation signals. In addition, microarrays have two major drawbacks. Namely, microarrays are linked to known genes, and microarrays have limitations in sensitivity and dynamic range. The use of direct sequencing of all or part of the content of an organization's mRNA has been increasing. However, conventional methods of analyzing the content of a cell's mRNA by direct sequencing typically require analyzing large amounts of mRNA obtained from tissue samples containing millions of cells. This means that much of the functional information present in a single cell is lost or becomes ambiguous when gene expression is analyzed with large amounts of mRNA. In addition, it is impossible to observe dynamic processes (such as the cell cycle) as the average of a population. Similarly, distinct cell types in complex tissues (such as the brain) can only be examined when the cells are analyzed individually. Suitable cell surface markers for isolating the single cell to be examined often do not exist, and even when markers exist, they are not sufficient to capture the natural range of gene expression in a small number of single cells. What is needed is a method for preparing a cDNA library that can be used for analyzing gene expression in multiple single cells.

[0003] (Sequence Listing) This application has been filed with an electronic format sequence listing. The sequence listing is provided as a file entitled IP-1388-US_SequenceListing.txt (created on September 15, 2015, 4 Kb). The entire electronic format information of the sequence listing is incorporated herein by reference.

Summary of the Invention

[0004] Methods and compositions for the analysis of nucleic acids derived from single cells and / or nucleic acids derived from the nuclei and organelles of single cells are presented herein. Some methods and compositions can be used for multiplexed single cell gene expression analysis. Some methods and compositions include the use of droplets and / or beads that carry unique barcodes (such as unique molecular barcodes (UMIs)). In one embodiment, methods and compositions for analyzing nucleic acids derived from a single cell are presented herein. In some embodiments, the nucleic acid sequencing methods and compositions can be used to prepare a sequencing library from nucleic acids derived from a single cell organelle. In some embodiments, the organelle may be a nucleus derived from a single cell. In some embodiments, the organelle is obtained from a single cell. Other exemplary organelles include, but are not limited to, mitochondria and ribosomes.

[0005] In certain embodiments, the method includes the step of releasing nuclei from a plurality of cells to provide a plurality of nuclei, where each nucleus is derived from one single cell. The plurality of nuclei are spatially separated from each other, and thus one nucleus is present in a spatially isolated compartment. The first strand of cDNA is synthesized from the mRNA of each individual mRNA sample by a first strand synthesis primer. In some embodiments, the first strand synthesis primer is an oligo dT primer, which further includes a first amplification primer binding site. In some embodiments, the first strand synthesis primer is a randomer. In some embodiments, the first strand synthesis primer is a randomer, which further includes a first amplification primer binding site. In some embodiments, the first strand synthesis primer is a mixture of an oligo dT primer and a randomer, each of which further includes a first amplification primer binding site. In some embodiments, the method further includes the step of incorporating a template switching oligonucleotide primer (TSO primer) together with a mixture of oligo dT and randomers, where each of the oligo dT primer and the randomer further includes a first amplification primer binding site. In some embodiments, the TSO primer further includes a second amplification primer binding site. In some embodiments, the first strand synthesis primer is extended beyond the mRNA template and further copies the TSO primer strand. In some embodiments, the second strand of cDNA is synthesized using the TSO primer. In some embodiments, the second strand of cDNA is synthesized using a second amplification primer complementary to the first strand of cDNA, which is extended beyond the mRNA template and includes a complementary TSO strand. In some embodiments, the double-stranded cDNA is amplified by the first and second amplification primers. In some embodiments, the first, second, and both first and second amplification primers are immobilized on a solid support. Exemplary solid supports include beads, flow cells, and microwells.

[0006] In some embodiments, the double-stranded cDNA is subjected to a tagging reaction, and thus a barcode can be introduced into the double-stranded cDNA. Exemplary methods of tagging are disclosed in U.S. Patent Nos. 9,115,396, 9,080,211, 9,040,256, and U.S. Patent Application Publication No. 2014 / 0194324. Each of the foregoing documents is hereby incorporated by reference in its entirety. In some embodiments, the barcode can be used to determine the flanking sequence information. In some embodiments, the barcode can be used as a source identifier. In some embodiments, the method includes the step of incorporating a tag into the cDNA to provide a plurality of tagged cDNA samples, wherein the cDNA of each tagged cDNA sample is complementary to mRNA derived from one single cell. In certain embodiments, the tag includes a cell-specific identifier sequence and a unique molecular identifier (UMI) sequence. In some embodiments, the first-strand synthesis primer includes a tag. In some embodiments, the TSO primer includes a tag. In some embodiments, the tagged cDNA derived from the nuclei of a plurality of single cells can be pooled and optionally amplified. In some embodiments, the method includes preparing a sequencing library from microRNA (miRNA), small interfering RNA (siRNA), ribosomal RNA, or mitochondrial DNA derived from a single cell. The method includes releasing a plurality of organelles from a single cell to provide the plurality of organelles. The plurality of organelles are spatially separated from each other, such that one organelle is present in a spatially isolated compartment. The first strand of DNA is synthesized by a first strand synthesis primer from microRNA (miRNA), small interfering RNA (siRNA), ribosomal RNA, or the first strand synthesis primer. In some embodiments, the first strand synthesis primer is a randomer. In some embodiments, the first strand synthesis primer is a randomer and further includes a primer binding site. In some embodiments, the first strand synthesis primer is a mixture of a first strand specific synthesis primer and a randomer. In some embodiments, the first strand synthesis primer is a mixture of a first strand specific synthesis primer and a randomer, each further including a first amplification primer binding site.

[0007] In some embodiments, the primer that binds to the first primer binding site is an amplification primer. In some embodiments, the method further includes incorporating a template switching oligonucleotide primer (TSO primer) together with a mixture of a first strand specific synthesis primer and a randomer, wherein the first strand specific synthesis primer and the randomer further include the first primer binding site. In some embodiments, the TSO primer further includes a second primer binding site. In some embodiments, the primer that binds to the second primer binding site is an amplification primer. In some embodiments, the first strand synthesis primer extends beyond a microRNA (miRNA), small interfering RNA (siRNA), ribosomal RNA, or mitochondrial DNA template and further copies the TSO primer strand. In some embodiments, the second strand of DNA is synthesized using the TSO primer. In some embodiments, the second strand of DNA is synthesized using a second primer complementary to the first strand of DNA, which extends beyond the template RNA or DNA to include a complementary TSO strand. In some embodiments, the double-stranded DNA is amplified by the first and second primers. In certain embodiments, multiple organelles (e.g., nuclei, mitochondria, ribosomes) are spatially separated by fluorescence-activated cell sorting (FACS), and each organelle is sorted into a spatially isolated compartment (e.g., a single microwell or a Fluidigm C1 chip). In some embodiments, each organelle is spatially separated in a spatially isolated compartment by being immobilized on a solid surface. For example, this is carried out via an antibody, where the antibody specifically binds to the organelle and the antibody is immobilized on the solid surface. In some embodiments, the solid surface is a flow cell or beads.

[0008] In some embodiments, the randomer comprises one or more quasi-random primers selected from the group consisting of: an AT-rich set of random amplification primers; a set of random amplification primers containing an AT-rich 5' end; a set of length-variable random amplification primers (wherein each primer contains a random 3' portion and a degenerate 5' end, and the degenerate 5' end may be proportional in length to the A / T content of the random 3' portion of the primer); a Tm-normalized random primer set (wherein each primer in the set contains one or more base analogs capable of normalizing the Tm of each primer relative to the Tm of the other primers in the primer set); a set of random primers (wherein each primer contains a random 3' portion and a constant 5' priming portion); a set of random amplification primers (wherein each primer contains a random 3' portion and a constant 5' priming portion, and further the random 3' portion contains RNA); a set of random amplification primers (wherein each primer contains a random 3' portion and a constant 5' priming portion, and further the random 3' portion contains at least one unnatural base selected from the group consisting of nucleic acids containing: 2'-deoxy-2-thiothymidine (2-thio-dT), 2-aminopurine-2'-deoxyriboside (2-amino-dA), N4-ethyl-2'-deoxycytidine (N4-Et-dC), N4-methyldeoxycytidine (N4-Me-dC), 2'-deoxyinosine, 7-deazaguanine (7-deaza-G), 7-iodo-7-deazaguanine (I-deaza G), 7-methyl-7-deazaguanine (MecG), 7-ethyl-7-deazaguanine (EtcG)); and any combination of the aforementioned primer sets. The quasi-random primers shown above are described in more detail herein. In some embodiments described herein, the quasi-random primers are provided in pairs or sets.

[0009] One embodiment presented herein is a method for preparing a cDNA library from a plurality of single cells. The method includes the following steps: releasing mRNA from each single cell to provide a plurality of samples of individual mRNAs (wherein each mRNA of the individual mRNA samples is derived from one single cell); synthesizing the first strand of cDNA from each mRNA of the individual mRNA samples with a first strand synthesis primer and further incorporating a tag into the cDNA to provide a plurality of tagged cDNA samples (wherein the cDNA of each tagged cDNA sample is complementary to the mRNA derived from one single cell). In one embodiment, the tag includes a cell-specific identifier sequence and a unique molecular identifier (UMI) sequence. In some embodiments, the tag includes a cell-specific identifier sequence without a UMI. The method further includes the following steps: pooling the tagged cDNA samples; optionally amplifying the pooled cDNA samples to generate a cDNA library containing double-stranded cDNA; and performing a tagmentation reaction while simultaneously cleaving each cDNA and incorporating an adapter into each strand of the cDNA, thereby generating a plurality of tagged cDNA fragments. In some embodiments, a sufficient number of single cells are present and amplification of the cDNA can be avoided. The second strand of the cDNA is synthesized using a template switching oligonucleotide primer (TSO primer), followed by symmetric Nextera. In certain embodiments, the method further includes the step of amplifying the tagged cDNA fragments to generate amplified tagged cDNA fragments. In some features, the amplification step includes the step of adding additional sequences to the 5' end of the amplification product. In some features, the additional sequences include primer binding sequences for amplification on a solid support. In some features, the additional sequences include additional index sequences. In certain embodiments, the method further includes the step of amplifying the amplified tagged cDNA fragments on a solid support. In certain embodiments, the method further includes the step of sequencing the amplification product on a solid support.

[0010] In some features, the tagging reaction includes contacting the double-stranded cDNA with a transposase mixture that includes an adapter sequence not found in the first-strand synthesis primer. In some features, the transposase mixture consists essentially of transposomes having one type of adapter sequence. In certain embodiments, the method further includes sequencing the tagged cDNA fragments. In some features, the sequencing step includes measuring the 3' tag. In some features, the sequencing step includes analyzing the entire transcriptome. In some features, the first-strand synthesis is performed using a mixture of random primers, and the random primers further include tags. In some features, the first-strand synthesis primer includes a double-stranded portion. In some embodiments, the first-strand synthesis primer including a double-stranded portion further includes one single-stranded loop at one end. In some features, the first-strand synthesis primer includes a region capable of forming a hairpin. In some features, the first-strand synthesis primer reduces ligation by-products as compared to a single-stranded first-strand synthesis primer. In some features, the first-strand synthesis primer includes an RNA region. In some features, the first-strand synthesis primer hybridizes with a complementary oligonucleotide, thereby forming a double-stranded portion. Furthermore, a plurality of beads are presented herein, where each bead includes a plurality of oligonucleotides, and each oligonucleotide includes the following: (a) a linker, (b) an amplification primer binding site, (c) optionally a unique molecular identifier (different for each oligonucleotide), (d) a bead-specific sequence (the same for each oligonucleotide of the bead but different for other beads), and (e) a capture sequence that captures mRNA and primes reverse transcription. In some features, the capture sequence includes oligo dT. In some features, each bead is present in a separate droplet isolated from other beads. Details of one or more embodiments are described in the accompanying drawings and the description below. Other features, objects, and advantages will be apparent from the description and drawings and from the claims.

Brief Description of the Drawings

[0011]

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Mode for Carrying Out the Invention

[0012] Methods and compositions for multiplexed single-cell gene expression analysis are presented herein. Some methods and compositions include the use of droplets and / or beads that carry unique barcodes (e.g., unique molecular barcodes (UMIs)). To date, the most commonly used method for single-cell RNA-Seq has been based on the CLONTECH TM SMART-SEQ TM technology or its derivative technologies. Briefly, an oligo(dT) primer primes the first-strand cDNA synthesis reaction. When the reverse transcriptase (SMARTSCRIBE TM ) reaches the 5' end of the mRNA, the terminal transferase activity of the enzyme adds several additional non-template nucleotides to the 3' end of the cDNA. A template switch oligo (designed to form base pairs with this non-template nucleotide stretch) anneals and extends to create a template, allowing the RT to continue replicating to the end of the oligonucleotide (Figure 1). The methods presented in this specification can include, for example, methods of making cDNA with sample-specific tags as described in the disclosure of US2012 / 0010091 (which is hereby incorporated by reference in its entirety). As used herein, the terms single cell tagging reverse transcription and STRT refer to methods disclosed in materials incorporated herein by reference to US2012 / 0010091. In some embodiments, double-stranded cDNA is not degraded by DNase in the STRT method. Instead, double-stranded cDNA is tagged with transposase (e.g., standard Nextera). The double-stranded cDNA can then be, for example, NEXTREA TM or TRUSEQ TM for use in, for example, whole transcriptome RNA-Seq, or for 5' end sequencing, enzymatically digested, e.g., with DNase I or Fragmentase, followed by adapter ligation to be converted into a sequencing library. There are pros and cons to both methods. That is, in the former, sample barcodes can only be multiplexed after being introduced during library preparation, while in the latter, barcodes can be introduced during the first strand synthesis step so multiplexing can be done after cDNA synthesis. Thus, the higher the throughput per sample and the lower the cost, the more advantageous the latter is. However, the information obtained by both methods has different applications. That is, the former enables sequencing of the whole transcriptome, while the latter interrogates only gene expression levels.

[0013] Disclosed herein is a rapid gene expression library preparation that can be applied at the single cell input level and further enables high levels of sample multiplexing early in the protocol. In some embodiments, first strand synthesis is primed with (immobilized) oligo dT (optionally with one or a combination of two randomizers), to which a sample barcode (BC), an amplification primer binding site, and optionally a template switch (TS) primer sequence are attached. In some embodiments, the amplification primer binding site is a transposase adapter sequence, such as the Nextera adapter sequence V2.A14 or V2.B15. The barcode may be either after or before the molecular barcode (unique molecular identifier or “UMI” which will allow detection of PCR duplicates). When the reverse transcriptase reaches the 5’ end of the mRNA, a template switch occurs as described herein and in the incorporated materials of US2012 / 0010091. The template switch incorporates the complement of the TS primer sequence into the first strand cDNA. Since the sample barcode is incorporated into the first strand cDNA, different samples can be pooled at this point. Subsequently, the first strand pool is made double-stranded and further optionally amplified by PCR with the TS primer (Figure 2A). Due to the fact that both ends of the cDNA contain complementary sequences, hairpin formation will result in suppression of amplification of smaller fragments (such as artifacts).

[0014] In some embodiments, as shown in Figures 2A and 3A, first strand synthesis is primed with oligo dT, to which a sample barcode (BC), a copy of the transposase adapter sequence (“Nextera V2.A14 sequence”), optionally a molecular barcode (UMI), and a template switch (TS) primer sequence are attached. The template switch at the 3’ end of the cDNA strand incorporates the TS primer sequence at the other end of the first strand. cDNA from different samples can be pooled at this point. In some embodiments, as shown in Figures 2B and 3B, the cDNA is amplified with a TS oligo. This single primer PCR suppresses small amplicons (such as primer dimers). The cDNA pool is treated with a transposase (such as “NEXTERA TM”) can be tagged. The transposase contains only one adapter instead of the typical two adapters. In the examples shown in FIGS. 2B and 3B, the transposase is loaded with the V2.B15 oligo. After tagging, PCR with the p5-V2.A14 and p7-V2.B15 amplification primers preferentially amplifies the 3′-end fragment of the cDNA. Fragments generated by two tagging events (“symmetric fragments”) are suppressed during PCR and will not generate sequenceable fragments. For example, as shown in FIGS. 2B and 3B, the amplification products resulting from the symmetric fragments present the P7 primer sequence at both the 5′ and 3′ ends of the amplification product and will not form sequenceable clusters on a standard Illumina flow cell that holds the P5 and P7 amplification primers. In addition, they will be suppressed during PCR due to their complementary ends. In contrast, the 3′-end fragments retain the P5 and P7 primer binding sites after amplification and can form sequenceable clusters on an Illumina flow cell. Paired-end sequencing provides the BC and UMI of the sample in read 1 and the cDNA sequence in read 2.

[0015] Thus, in certain embodiments presented herein, instead of performing sequencing of the entire transcript, a digital gene expression assay type that relies on the measurement of 3′ tags is performed. The methods presented herein provide the ability to individually barcode cells in the first-strand synthesis step. In addition, barcoding of the cDNA 3′ end with inexpensive oligo dT primers or random primers provides a significant cost-saving advantage. Furthermore, using random primers for cDNA synthesis is advantageous because it makes the method similar to whole RNA sequencing protocols in addition to the 3′ tag measurement assay. Subsequent pooling, cleanup, single-primer cDNA PCR amplification, tagmentation, and sequencing library preparation can be performed in a single tube for 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more than 100 cells. Thus, the methods and compositions described herein are highly amenable to multiplexing. As an example, as shown in FIG. 5, the methods provided herein enable multiplexing at the cell level (e.g., 96 samples per 96-well plate). Furthermore, the methods also enable further multiplexing using uniquely barcoded plates (e.g., performing tagmentation to incorporate a barcode that identifies a 96-well plate). These methods are highly amenable to automation and provide a marked reduction in cost and time. The order of the sample barcode and UMI in the first-strand synthesis primer can be varied. For example, in some embodiments, the sample barcode is placed 3' to the UNI. In some embodiments, the sample barcode is placed 5' to the UNI. In some embodiments, the sample barcode is directly contiguous with the UNI. In some embodiments, the sample barcode (BC) is separated from the UMI by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 or more nucleotides. In some embodiments, the sample barcode (BC) overlaps with the UMI by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 or more nucleotides.

[0016] In some embodiments, tagging is performed using a transposase mixture (the transposase mixture contains an adapter sequence not found in the first strand synthesis primer). In this way, tagging fragments are generated that carry different adapter sequences compared to the sequences incorporated into the first strand synthesis primer. For example, in some embodiments, the transposase mixture contains transposomes having only one type of adapter sequence. Fragments generated by this type of mixture are referred to herein as "symmetric fragments" and do not produce clusters that can be sequenced on an Illumina flow cell. In some embodiments, the transposase mixture can contain some amount of the sequence incorporated into the first strand synthesis primer, in which case the amount is sufficiently small to still allow all or substantially all of the 3' cDNA fragments to be amplified and sequenced. For example, the adapter sequence of the transposome mixture can contain less than 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, or less than 25%, 30%, 35%, 40%, 45%, or less than 50% of the sequence incorporated into the first strand synthesis primer. It will be understood that when typically two transposase adapters are used, either one of the two adapters can be incorporated into the first strand synthesis primer and the other adapter can be used during the tagging event. For example, in the exemplary embodiments shown in FIGS. 3A, 3B, 4A, and 4B, when one adapter sequence is incorporated into the first strand synthesis primer, that adapter is not used in the tagging mixture. FIGS. 3A and 3B show one embodiment where adapter V2.A14 is incorporated into the first strand synthesis primer and adapter V2.B15 is used as the transposome adapter during the tagging step. FIGS. 4A and 4B show another embodiment, in which case adapter V2.B15 is incorporated into the first strand synthesis primer and adapter V2.A14 is used as the transposome adapter during the tagging step.In both cases, the resulting taggedmentation fragments are divided into two categories. Namely, symmetric fragments (which cannot be amplified and / or sequenced) and asymmetric fragments (which can be amplified using the V2.A14 and V2.B15 primer sets).

[0017] In some embodiments, the number of single cells that can be multiplexed is significantly increased by incorporating an index into the transposome adapter sequence. As shown in Figure 5, each individual cell can be identified using a cell-specific barcode, and furthermore, each set of multiple cells can be contacted with a taggedmentation mixture incorporating a plate-specific barcode into the transposome adapter sequence. In the example shown in Figure 5, the cDNAs derived from each of the 96 cells of a plate are pooled prior to taggedmentation, and subsequently the taggedmentation implementation samples are pooled prior to multiplexed sequencing. It will be understood that the ability to pool any number of cells prior to taggedmentation and the use of 96-well plates are merely one of a variety of embodiments. For example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100 cells, or more than 100, 200, 300, 400, 500, 600, 700, 800, 900 cells, or more than 1000 cells, and any set of cells in between can be pooled for taggedmentation. First-strand synthesis can be performed in any single well or multi-well container (e.g., multi-well plate, chip, microfluidic device, emulsion, bead mixture, or any other suitable format for multiplex manipulation of multiple cells). As shown in Fig. 7 (multiple single cells), when gene expression was analyzed using either symmetric tagging (NextEra V2.B15 version) as compared to asymmetric tagging (NextEra V2.B15 / V2.A14 version), a significant number of genes were detected and additional metrics were obtained. Fig. 8 shows that when tagging was performed using only one transposase adapter (V2.B15) as opposed to two transposase adapters (V2.A14 and V2.B15), the transcription coverage region is almost completely biased towards the 3’ end of the transcript.

[0018] In some embodiments, sample and UMI barcoding is performed by isolating individual cells into droplets. In some embodiments, the droplets are isolated from each other in an emulsion. In some embodiments, the droplets are formed and / or manipulated using droplet actuators. In certain embodiments, one or more droplets contain various sets of barcode-containing first-strand synthesis primers. In some embodiments, each droplet contains a number of first-strand synthesis primers, each of these primers having the same sequence containing the same barcode, and further, the barcode of one droplet is different from the barcode of another droplet, but the remaining portion of the first-strand synthesis primer remains the same between droplets. Thus, in these embodiments, the barcode acts as an identifier for the droplet and also as an identifier for the single cell contained within the droplet. In certain embodiments, one or more droplets contain various sets of UMI-containing first-strand synthesis primers. Thus, each of the individual cells lysed in each droplet will be distinguishable by the barcode in each droplet. As shown in FIG. 11, barcode assignment by droplets can be performed by mixing droplets containing single cells with other droplets containing a unique set of barcodes. This mode allows for additional multiplexing beyond what is available in the multi-well mode. First-strand synthesis and template switching are performed within each of the individual droplets. In some embodiments, two or more droplets can be mixed prior to PCR. Additionally or alternatively, in some embodiments, the droplets can be mixed prior to tagmentation. Additionally or alternatively, in some embodiments, the droplets can be mixed after PCR and prior to tagmentation. For example, in some embodiments, after performing first-strand synthesis in individual droplets, the tagged cDNAs can be mixed, and thus the cDNAs can be pooled. Similarly, in some embodiments, sample and UMI barcoding can be performed by isolating individual cells with beads that carry UMIs and / or barcode tags for first strand synthesis. In some embodiments, the beads are isolated into droplets in an emulsion. In some embodiments, the beads are isolated and / or manipulated using droplet actuators. As shown in FIG. 12, barcoding with beads can be performed by creating a set of beads (each bead carrying one or more unique barcode sets).

[0019] Sequencing of the entire transcriptome In some embodiments, whole transcriptome sequencing can be performed using the methods provided herein. In such embodiments, first strand synthesis is extended using random primers. As shown in FIG. 9A, random priming extends a window of fragments that can be sequenced anywhere along the full length of a transcript where the random primer can hybridize and prime first strand synthesis. The random primer can include the same combination of sample barcode and unique molecular identifier in addition to other adapter and primer binding sites (e.g., transposome adapter sequences and / or template switch (TS) primers). Template switching and second strand synthesis can be performed as described above for oligo dT primed cDNA synthesis. The resulting double-stranded cDNA can subsequently be subjected to a tagging reaction as described above. The difference in using a random primer versus an oligo dT primer is that a complete or substantially complete sequence of the transcript can be obtained rather than just the 3’ portion of the transcript. As shown in FIG. 6 (100 pg of RNA), when gene expression was analyzed using either oligo dT, a significant number of genes were detected and additional metrics were obtained as compared to a mixture of oligo dT and random primer for first strand synthesis. One type of by-product that can occur when using randomizers for priming first-strand synthesis is a ligation by-product. In particular, in some situations shown in Figure 9B, randomizers can hybridize with other random primers and outcompete annealing to the RNA transcript. The resulting cDNA products can also be further subjected to random priming, and a chain of template-switching events can occur. This chain can lead to the formation of ligation by-products (which may depend on the presence of the template-switching oligonucleotide).

[0020] To reduce and / or minimize the formation of such by-products, various primer designs that reduce the by-product formation probability are presented herein. Exemplary designs are shown in Figure 10, although it will be understood that the range of primer compositions shown herein extends beyond the examples shown in that figure. In certain embodiments, the primer is configured to form a hairpin, which can prevent or minimize randomizer priming for any portion of the barcode, adapter, or amplification primer binding region. The double-stranded portion formed by the primer itself can thus outcompete random hybridization. In some embodiments, the double-stranded portion of the hairpin includes the mosaic end (ME) sequence of the transposase adapter. Separately or in addition, in some embodiments, the double-stranded portion can include some or all of the transposase adapter sequence beyond the mosaic end sequence. In some embodiments, the adapter sequence is completely replaced with a short RNA sequence, thus shortening the length of the primer and minimizing the potential for randomizers to hybridize to the primer. In some embodiments, the complement of the short RNA sequence is provided at or near the 5' end of the primer, thus enabling hairpin formation. In some embodiments, a double-stranded region is formed by annealing a complementary oligonucleotide to the adapter and / or primer portion, thus preventing or minimizing randomizer priming for any portion of the barcode, adapter, or amplification primer binding region, thereby reducing or avoiding ligation by-products.

[0021] Randomers or random primers : As used herein, the terms "randomer" and "random primer" are used interchangeably. The term "randomer" refers to a random primer that can exhibit four-fold degeneracy at each position. In some embodiments, a randomer is any nucleic acid primer having a variety of random sequence lengths known in the art. For example, a randomer can include a random sequence having a length of 3, 4, 5, 6, 7, 8, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20 nucleotides. In certain embodiments, a plurality of random primers can include randomers having a variety of lengths. In certain embodiments, a plurality of randomers can include randomers having equal lengths. In certain embodiments, a plurality of randomers can include a random sequence that is about 5 to about 18 nucleotides in length. In some embodiments, a plurality of randomers includes random hexamers. Random primers (and in particular random hexamers) are commercially available and widely used in amplification reactions (e.g., multiple displacement amplification (MDA)) (MDA is embodied in the REPLI-g whole genome amplification kit (QIAGEN, Valencia, CA)). It will be understood that randomers of any suitable length can be used in the methods and compositions presented herein. An exemplary randomer sequence including a 3' random sequence portion and a 5' specified sequence portion is shown below: GTGTAGATCT CGGTGGTCGC CGTATCATTN NNNN GTGTAGATCT CGGTGGTCGC CGTATCATTN NNNNN GTGTAGATCT CGGTGGTCGC CGTATCATTN NNNNNN GTGTAGATCT CGGTGGTCGC CGTATCATTN NNNNNNN GTGTAGATCT CGGTGGTCGC CGTATCATTN NNNNNNNN ATCTCGTATG CCGTCTTCTG CTTGNNNNN ATCTCGTATG CCGTCTTCTG CTTGNNNNNN ATCTCGTATG CCGTCTTCTG CTTGNNNNNNN ATCTCGTATG CCGTCTTCTG CTTGNNNNNNNN ATCTCGTATG CCGTCTTCTG CTTGNNNNNNNNN As used herein, the term "SMART-Seq Plus" refers to a method for preparing cDNA from RNA, the method using a first-strand synthesis primer (including a first amplification primer binding site), a randomer (including a first amplification primer binding site), and an oligonucleotide switching oligonucleotide (partially complementary to the first strand of cDNA and further including a second amplification primer binding site). In some embodiments, the first-strand synthesis primer includes an oligo(dT) portion.

[0022] Barcodes and UMI : As used herein, "barcode" or "BC" refers to a nucleic acid tag that can be used to identify a sample or the source of a nucleic acid material. Thus, when a nucleic acid sample is derived from multiple sources, the nucleic acids of each nucleic acid sample are tagged with different nucleic acid tags and thus the source of the sample can be identified. Barcodes (commonly referred to as indexes, tags, etc.) are well known to those of skill in the art. Any suitable barcode or barcode set known in the art and exemplified in the disclosures of U.S. Patent 8,053,192 and PCT Publication WO05 / 068656 (the entire disclosures of which are incorporated herein by reference) can be used. Barcoding of single cells can be performed as described in the disclosure of, for example, U.S. 2013 / 0274117 (the entire disclosure of which is incorporated herein by reference). Nucleic acids from one or more sources can incorporate diverse tag sequences. The tag sequences are preferably up to 100 nucleotides (base pairs in the case of double-stranded molecules) in length, preferably from 1 to 10 nucleotides in length, most preferably 4, 5, or 6 nucleotides in length, and further include combinations of nucleotides. For example, in one embodiment, if 6 base pairs are selected for tag formation and permutations of 4 different nucleotides are used, a total of 4096 nucleic acid anchors (e.g., hairpins), each having a unique 6-base tag, can be created. As used herein, the terms UMI, unique identifier, and unique molecular identifier refer to unique nucleic acid sequences attached to each of a plurality of nucleic acid molecules. For example, when incorporated into a nucleic acid molecule during first-strand synthesis, the UMI can be used to correct for subsequent amplification bias. Correction of the bias is performed by directly measuring the unique molecular identifier (UMI) that is sequenced after amplification. The design, incorporation, and application of UMIs can be carried out as known in the art, for example, as exemplified by the disclosures of the following documents: WO 2012 / 142213; Islam et al. Nat. Methods (2014) 11:163 - 166; and Kivioja, T. et al. Nat. Methods (2012) 9: 72 - 74 (each of the foregoing documents is incorporated herein by reference in its entirety).

[0023] Tagmentation : As used herein, the term "tagmentation" refers to the modification of DNA by a transpososome complex, which complex includes a transposase enzyme complexed with an adapter containing transposon end sequences. Tagmentation results in the simultaneous fragmentation of DNA and the ligation of adapters to the 5' ends of both strands of the duplex fragment. Following a purification step to remove the transposase enzyme, additional sequences can be added to the ends of the modified fragment, for example, by PCR, ligation, or other suitable methods known to those skilled in the art. The method of the present invention can accept transposase end sequences to fragment target nucleic acids and can utilize any transposase that attaches transfer ends but not non-transfer ends. A "transposome" is composed of at least one transposase enzyme and a transposase recognition site. In some such systems called "transposomes", the transposase can form a functional complex with the transposon recognition site, which can catalyze the transfer reaction. A transposase or integrase can bind to the transposase recognition site and insert the transposase recognition site into the target nucleic acid. This process is sometimes referred to as "tagmentation". In some such insertion events, one strand of the transposase recognition site can be inserted into the target nucleic acid. In standard sample preparation methods, each template contains adapters at either end of the insert, and often multiple steps are required for both the modification of DNA or RNA and the purification of the desired product of the modification reaction. These steps are carried out in solution before attaching the modified fragments to the flow cell, where the fragments are ligated to the surface by a primer extension reaction (the extension reaction copies the fragment hybridized to the end of the primer covalently bound to the surface). Subsequently, these "seed" templates result in monoclonal clusters of templates copied by several amplification cycles.

[0024] The number of steps required to convert DNA into adapter-modified templates in solution so that they can be used for cluster formation and sequencing can be minimized by the use of transposase-mediated fragmentation and tagging. In some embodiments, transposon-based technologies can be utilized for DNA fragmentation. Such technologies include, for example, NEXTERA TMEmbodied in a workflow for a DNA sample preparation kit (Illumina, Inc.), where genomic DNA can be fragmented by an engineered transposon (the engineered transposon simultaneously fragments and tags the input DNA (“tagmentation”), thereby creating a population of fragmented nucleic acid molecules containing unique adapter sequences at the ends of the fragments). Some embodiments include the following uses: hyperactive Tn5 transposase and Tn5 transposase recognition sites (Goryshin and Reznikoff, J. Biol. Chem., 273:7367, 1998), or MuA transposase and Mu transposase recognition sites containing R1 and R2 end sequences (Mizuuchi, K., Cell, 35: 785, 1983; Savilahti, H, et al., EMBO J., 14: 4893, 1995). Exemplary transposase recognition sites that form a complex with hyperactive Tn5 transposase are, for example, EZ-Tn5 TM transposase (Epicentre Biotechnologies, Madison, Wis.). Further examples of transfer systems that can be used in certain embodiments provided herein include: Staphylococcus aureus Tn552 (Colegio et al., J. Bacteriol., 183: 2384-8, 2001; Kirby C et al., Mol. Microbiol., 43: 173-86, 2002), Ty1 (Devine & Boeke, Nucleic Acids Res., 22: 3765-72, 1994; and International Patent Publication WO 95 / 23875), transposon Tn7 (Craig, N L, Science. 271: 1512, 1996; Craig, N L, Review in: Curr Top Microbiol Immunol., 204: 27-48, 1996), Tn / O and IS10 (Kleckner N, et al., Curr Top Microbiol Immunol., 204: 49-82, 1996), Mariner transposase (Lampe D J, et al., EMBO J., 15: 5470-9, 1996), Tc1 (Plasterk R H, Curr. Topics Microbiol. Immunol., 204: 125-43, 1996), P element (Gloor, G B, Methods Mol. Biol., 260: 97-114, 2004), Tn3 (Ichikawa & Ohtsubo, J Biol. Chem. 265: 18829-32, 1990), bacterial insertion sequences (Ohtsubo & Sekine, Curr. Top. Microbiol. Immunol. 204: 1-26, 1996), retroviruses (Brown, et al., Proc Natl Acad Sci USA, 86: 2525-9, 1989), and yeast retrotransposons (Boeke & Corces, Annu Rev Microbiol. 43: 403-34, 1989).Further examples include: IS5, Tn10, Tn903, IS911, and engineered versions of transposase family enzymes (Zhang et al., (2009) PLoS Genet. 5:e1000689. Epub 2009 Oct. 16; Wilson C. et al (2007) J. Microbiol. Methods 71:332-5).

[0025] Briefly, a "transposition reaction" is a reaction in which one or more transposons are inserted into random or near-random sites of a target nucleic acid. The essential components of the transposition reaction are a transposase and a DNA oligonucleotide, which presents the transposon sequence and contains, together with the transferred transposon sequence and its complement (i.e., the non-transferred transposon end sequence), other components necessary for functional transposition or the formation of a transpososome complex. The DNA oligonucleotide can further contain additional sequences that are necessary or desired (e.g., adapter or primer sequences). Briefly, in vitro transposition can be initiated by contacting a transpososome complex with a target DNA. Exemplary transposition procedures and systems that can be readily adapted for use with the transposases of the present disclosure are described, for example, in WO 10 / 048605; US 2012 / 0301925; US 2013 / 0143774 (each of the foregoing documents is incorporated herein by reference in its entirety). The adapters added to the 5’ and / or 3’ ends of nucleic acids can contain universal sequences. A universal sequence is a region of nucleotide sequence that is common (i.e., shared) among two or more nucleic acid molecules. Optionally, the two or more nucleic acid molecules also have regions of sequence difference. Thus, for example, the 5’ adapter can contain an identical or universal nucleic acid sequence, and the 3’ adapter can contain an identical or universal sequence. The universal sequences that can be present in various members of a plurality of nucleic acid molecules can enable the replication or amplification of many different sequences using only one universal primer (complementary to the universal sequence). Some of the universal primer sequences used in the examples presented herein include V2.A14 and V2.B15 NEXTERA TM sequences are included. However, it will be understood that any suitable adapter sequences can be utilized in the methods and compositions presented herein. For example, Tn5 Mosaic End Sequence A14 (Tn5MEA) and / or Tn5 Mosaic End Sequence B15 (Tn5MEB) (including the complementary non-transfer sequences (NTS) shown below) can be used in the methods provided herein. Tn5MEA: 5’-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-3’ (SEQ ID NO: 1) Tn5MEB: 5’-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-3’ (SEQ ID NO: 2) Tn5 NTS: 5’-CTGTCTCTTATACACATCT-3’ (SEQ ID NO: 3)

[0026] Droplet barcodes and UMI In some embodiments, primers that carry sample barcodes can be present in solution. Additionally or alternatively, primers that carry UMI sequences can be present in solution. For example, the solid support can be one or more droplets. Thus, in certain embodiments, multiple droplets can be present, in which case each of the multiple droplets carries a unique sample barcode and / or UMI sequence (each of which is unique to the molecule). Thus, in some embodiments, it will be understood by those skilled in the art that the barcode is unique to the droplet and the UMI is unique to the molecule and thus the UMI is repeated many times within the droplet collection. In some embodiments, to identify individual cells, droplets having a unique set of sample barcodes and / or UMI sequences are contacted with the individual cells. In some embodiments, to identify lysates of individual cells, droplets having a unique set of sample barcodes and / or UMI sequences are contacted with the lysates of the individual cells. In some embodiments, to identify purified nucleic acids of individual cells, droplets having a unique set of sample barcodes and / or UMI sequences are contacted with the purified nucleic acids of the individual cells. In the embodiments presented herein where each of the multiple droplets carries a unique set of sample barcodes and / or UMI sequences, any suitable system can be used to form and manipulate the droplets.

[0027] "Droplet actuator" means a device for manipulating droplets. For examples of droplet actuators, see, for example: U.S. Patent No. 6,911,132 (Pamula et al.), title of invention "Apparatus for Manipulating Droplets by Electrowetting-Based Techniques" (issued June 28, 2005); U.S. Patent Publication No. 20060194331 (Pamula et al.), title of invention "Apparatuses and Methods for Manipulating Droplets on a Printed Circuit Board" (published August 31, 2006); International Patent Publication WO / 2007 / 120241 (Pollack et al.), title of invention "Droplet-Based Biochemistry" (published October 25, 2007); U.S. Patent No. 6,773,566 (Shenderov), title of invention: "Electrostatic Actuators for Microfluidics and Methods for Using Same" (issued August 10, 2004); U.S. Patent No. 6,565,727 (Shenderov), title of invention: "Actuators for Microfluidics Without Moving Parts" (issued May 20, 2003); U.S. Patent Publication No. 20030205632 (Kim et al.), title of invention: "Electrowetting-driven Micropumping" (published November 6, 2003); U.S. Patent Publication No. 20060164490 (Kim et al.)、Title of the Invention "Method and Apparatus for Promoting the Complete Transfer of Liquid Drops from a Nozzle" (published on July 27, 2006); US Patent Publication No. 20070023292 (Kim et al.), Title of the Invention "Small Object Moving on Printed Circuit Board" (published on February 1, 2007); US Patent Publication No. 20090283407 (Shah et al.), Title of the Invention "Method for Using Magnetic Particles in Droplet Microfluidics" (published on November 19, 2009); US Patent Publication No. 20100096266 (Kim et al.), Title of the Invention "Method and Apparatus for Real-time Feedback Control of Electrical Manipulation of Droplets on Chip" (published on April 22, 2010); US Patent No. 7,547,380 (Velev), Title of the Invention "Droplet Transportation Devices and Methods Having a Fluid Surface" (issued on June 16, 2009); US Patent No. 7,163,612 (Sterling et al.), Title of the Invention "Method, Apparatus and Article for Microfluidic Control via Electrowetting, for Chemical, Biochemical and Biological Assays and the Like" (issued on January 16, 2007); US Patent No. 7,641,779 (Becker et al.) Title of the Invention: "Method and Apparatus for Programmable Fluidic Processing" (issued on January 5, 2010); U.S. Patent No. 6,977,033 (Becker et al.), Title of the Invention: "Method and Apparatus for Programmable Fluidic Processing" (issued on December 20, 2005); U.S. Patent No. 7,328,979 (Decre et al.), Title of the Invention: "System for Manipulation of a Body of Fluid" (issued on February 12, 2008); U.S. Patent Publication No. 20060039823 (Yamakawa et al.), Title of the Invention: "Chemical Analysis Apparatus" (published on February 23, 2006); U.S. Patent Publication No. 20110048951 (Wu), Title of the Invention: "Digital Microfluidics Based Apparatus for Heat-exchanging Chemical Processes" (published on March 3, 2011); U.S. Patent Publication No. 20090192044 (Fouillet et al.), Title of the Invention: "Electrode Addressing Method" (published on July 30, 2009); U.S. Patent No. 7,052,244 (Fouillet et al.), Title of the Invention: "Device for Displacement of Small Liquid Volumes Along a Micro-catenary Line by Electrostatic Forces" (issued on May 30, 2006); U.S. Patent Publication No. 20080124252 (Marchand et al.), Title of the Invention: "Droplet Microreactor" (published on May 29, 2008); U.S. Patent Publication No.20090321262 (Adachi et al.), titled "Liquid Transfer Device" (published on December 31, 2009); US Patent Publication No. 20050179746 (Roux et al.), titled "Device for Controlling the Displacement of a Drop Between Two or Several Solid Substrates" (published on August 18, 2005); and the paper by Dhindsa et al. (Dhindsa et al., "Virtual Electrowetting Channels: Electronic Liquid Transport with Continuous Channel Functionality," Lab Chip, 10:832 - 836, 2010) (the entire disclosure of the above documents is incorporated herein by reference).

[0028] Certain droplet actuators will include one or more substrates disposed with a droplet processing gap therebetween, and electrodes disposed in close relation to (overlying, attached to, and / or embedded within) the one or more substrates to perform the operation of one or more droplets. For example, certain droplet actuators will include a base (or bottom) substrate, droplet processing electrodes coupled to the substrate, one or more dielectric layers over the top of the substrate and / or electrodes, and optionally one or more hydrophobic layers over the top of the substrate, a dielectric layer and / or electrodes forming a droplet processing surface. An upper substrate is also provided, which is separated from the droplet processing surface by a gap (commonly referred to as the droplet processing gap). Various electrode arrangements on the upper and / or bottom substrates are considered in the patents and patent applications referenced above, and certain novel electrode arrangements are considered in the description of the present disclosure. During droplet processing, it is preferred that the droplet maintain continuous or frequent contact with an outer electrode or a reference electrode. The outer or reference electrode can be in close relation within the gap with the upper substrate facing the gap and the bottom substrate facing the gap. When electrodes are provided on both substrates, the electrical contacts connecting the electrodes to the droplet actuator device for control or monitoring of the electrodes can be in close relation with one or both plates. In some cases, the electrodes on one substrate are electrically connected to the other substrate such that only one substrate contacts the droplet actuator. In one embodiment, a conductive material (e.g., an epoxy, e.g., MASTER BOND TM polymer-based EP79 (Master Bond, Inc., Hackensack, NJ)) provides electrical communication between the electrodes of one substrate and the electrical path of the other substrate. For example, the outer electrode of the upper substrate can be connected to the electrical path of the bottom substrate by such a conductive material.

[0029] When multiple substrates are used, spacers can be provided between the substrates to determine the height of the gap between the substrates and define the on-actuator distribution tank. The height of the spacer can be, for example, at least about 5 μm, 100 μm, 200 μm, 250 μm, 275 μm or can exceed the above. Separately or in addition to the above, the height of the spacer can be at most about 600 μm, 400 μm, 350 μm, 300 μm or less than the above. The spacer can be formed, for example, of a single layer of protrusions from the upper or bottom substrate and / or a substance inserted between the upper substrate and the bottom substrate. One or more openings can be provided in one or more substrates to form a fluid path through which a fluid can be delivered to the droplet processing gap. In some cases, one or more openings can be arranged in a line for interaction with one or more electrodes. For example, the fluid flowing through the opening can be arranged in a line so as to be sufficiently close to one or more droplet processing electrodes and droplet processing can be performed by the droplet processing electrodes using the fluid. In some cases, the base (or bottom) and the upper substrate can be formed as one complete component. One reference electrode can be provided on and / or in the gap between the base (or bottom) and / or the upper substrate. Examples of the arrangement of the reference electrode are provided in the above-referenced patents and patent applications. In various embodiments, the manipulation of droplets by the droplet actuator can be mediated by electrodes. For example, electrowetting mediation, or dielectrophoresis mediation, or Coulomb force mediation.Examples of other techniques for droplet processing control that can be used in the droplet actuator of the present disclosure include the use of devices that induce hydrodynamic fluid pressure, the devices being, for example, devices that operate based on the following principles: mechanical principles (such as external syringe pumps, pneumatic diaphragm pumps, vibrating diaphragm pumps, vacuum devices, centrifugal forces, piezoelectric / ultrasonic pumps, and acoustic forces); electrical or magnetic principles (such as electroosmotic flow, electrokinetic pumps, magnetic fluid plugs, electrohydrodynamic pumps, suction or repulsion using magnetic forces, and magnetohydrodynamic pumps); thermodynamic principles (such as bubble generation / phase change-induced volume expansion); other types of surface wetting principles (such as chemically, thermally, structurally, and radioactively induced surface tension gradients together with electrowetting and optoelectrowetting); gravity; surface tension (such as capillary action); electrostatic forces (such as electroosmotic flow); centrifugal flow (placing the substrate on a compact disc and rotating it); magnetic forces (such as vibrating ions causing a flow); magnetohydrodynamic forces; and vacuum or pressure differences.

[0030] In certain embodiments, two or more combinations of the foregoing techniques can be utilized to perform droplet processing with the droplet actuators of the present disclosure. Similarly, one or more of the foregoing techniques can be used to deliver a fluid to the droplet processing gap from, for example, a water reservoir of another device or a water reservoir external to the droplet actuator (e.g., a water reservoir coupled to a droplet actuator substrate and a flow path from the water reservoir to the droplet processing gap). The droplet processing surfaces of certain droplet actuators of the present disclosure can be made of a hydrophobic material or can be coated or treated to make those surfaces hydrophobic. For example, in some cases, some or all of the droplet processing surface can be induced by depositing a low surface energy material or chemical reaction with, for example, a polyfluorinated or perfluorinated compound, or by utilizing in situ synthesis using the same as a solution or polymerizable monomer. Examples of the foregoing include: TEFLON® AF (available from DuPont, Wilmington, DE), members of the cytop family of materials, coating with FLUOROPEL™ of hydrophobic and superhydrophobic coatings (available from Cytonix Corporation, Beltsville, MD), silane coatings, fluorosilane coatings, hydrophobic phosphonate derivatives (e.g., those sold by Aculon, Inc), and NOVEC TM electronic coatings (available from 3M Company, St. Paul, MN), other fluorinated monomers for plasma enhanced chemical vapor deposition (PECVD), and organosiloxanes (e.g., SiOC) for PECVD. In some cases, the droplet processing surface can include a hydrophobic coating having a thickness in the range of about 10 nm to about 1,000 nm.

[0031] Furthermore, in some embodiments, the top substrate of the droplet actuator includes a conductive organic polymer, which is subsequently coated with a hydrophobic coating or otherwise treated to make the droplet processing surface hydrophobic. For example, the conductive organic polymer deposited on a plastic substrate can be poly(3,4-ethylenedioxythiophene) poly(styrenesulfonate) (PEDOT:PSS). Other examples of conductive organic polymers and alternative conductive layers are described below: International Patent Publication WO / 2011 / 002957 (Pollack et al.), titled "Droplet Actuator Devices and Methods" (published January 6, 2011) (the entire disclosure of which is incorporated herein by reference). One or both substrates can be fabricated using a printed circuit board (PCB), glass, indium tin oxide (ITO)-coated glass, and / or a semiconductor material as the substrate. When the substrate is ITO-coated glass, the ITO coating preferably has a thickness of at least about 20 nm, 50 nm, 75 nm, 100 nm or more. Separately or in addition to the above, the thickness can be at most about 200 nm, 150 nm, 125 nm or less. In some cases, the top and / or bottom substrate includes a PCB substrate coated with a dielectric (e.g., a polyimide dielectric). In some cases, the same is also coated or treated to make the droplet processing surface hydrophobic. When the substrate includes a PCB, the following materials are examples of suitable materials: MITSUI TM BN-300 (available from Mitsui Chemicals America, Inc., San Jose CA); ARLON TM 11N (available from Arlon, Inc, Santa Ana, CA); NELCO (trademark) N4000-6 and N5000-30 / 32 (available from Park Electrochemical Corp., Melville, NY); ISOLA TMFR406 (available from Isola Group, Chandler, AZ), in particular IS620; fluoropolymer family (suitable for fluorescence detection as it has low background fluorescence); polyimide family; polyester; polyethylene naphthalate; polycarbonate; polyetheretherketone; liquid crystal polymer; cycloolefin copolymer (COC); cycloolefin polymer (COP); aramid; THERMOUNT (trademark) non-woven aramid reinforcement (available from DuPont, Wilmington, DE); NOMEX (trademark) brand fiber (available from DuPont, Wilmington, DE); and paper.

[0032] A variety of substances are also suitable for use as the dielectric component of the substrate. Examples include: vapor deposited dielectrics such as PARYLENE TM C (especially on glass), PARYLENE TM N and PARYLENE TM HT (for high temperatures, about 300 °C) (available from Parylene Coating Services, Inc., Katy, TX); TEFLON (registered trademark) AF coating; Cytop; solder mask, such as a flowable photoimageable solder mask (e.g., on a PCB), such as TAIYO TM PSR4000 series, TAIYO TM PSR and AUS series (available from Taiyo America, Inc., Carson City, NV) (having good thermal properties for applications requiring thermal management), and PROBIMER TM8165 (available from Huntsman Advanced Materials Americas Inc., Los Angeles, CA); having good thermal properties for applications requiring thermal management); dry film solder masks, such as those of the VACREL (trademark) dry film solder mask system (available from DuPont, Wilmington, DE); film dielectrics, such as polyimide films (e.g., KAPTON (trademark) polyimide films (available from DuPont, Wilmington, DE)), polyethylene, and fluoropolymers (e.g., PEP), polytetrafluoroethylene; polyesters; polyethylene naphthalate; cycloolefin copolymers (COC); cycloolefin polymers (COP); any other PCB substrate materials listed above; black matrix resins; polypropylene; and black flexible circuit materials, such as DuPont TM Pyralux (trademark) HXC and DuPont TM Kapton (trademark) MBC (available from DuPont, Wilmington, DE).

[0033] The droplet transfer voltage and frequency can be selected according to the performance of the reagents used in the specific assay protocol. The design parameters are variable. For example, the number and arrangement of the on-actuator reservoirs, the number of independent electrode connections, the sizes (volumes) of the various reservoirs, the arrangement of the magnet / bead washing zone, the electrode size, the pitch between electrodes, and the height of the gap (between the upper and bottom substrates) can vary depending on the use of specific reagents, protocols, droplet volumes, etc. In some cases, the substrate of the present disclosure can be induced by depositing a low surface energy substance or chemical reaction, for example, a polyfluorinated or perfluorinated compound, or by utilizing in situ synthesis using the same in solution or a polymerizable monomer. Examples include: TEFLON (R) AF coating and FLUOROPEL (TM) coating for dip or spray coating, other fluorinated monomers for plasma enhanced chemical vapor deposition (PECVD), and organosiloxanes (e.g., SiOC) for PECVD. Additionally, in some cases, some or all of the droplet processing surface can be coated with a substance that reduces background noise (e.g., background fluorescence) from the PCB substrate. For example, the noise reduction coating can include a black matrix resin (e.g., a black matrix resin available from Toray Industries, Inc. (Japan)). The electrodes of the droplet actuator are typically controlled by a controller or processor, which itself is provided as part of the system and can include data and software storage as well as input / output capabilities along with processing functions. The reagents can be provided to the droplet actuator in the droplet processing gap or reservoir (fluidly connected to the droplet processing gap). The reagents can exist in liquid form (e.g., droplets), or they can be provided in a form that can be reconstituted in the droplet processing gap or a reservoir fluidly connected to the droplet processing gap. Reconstitutable reagents can typically be combined with a liquid for reconstitution.Examples of reconfigurable reagents suitable for use with the methods and apparatus shown herein include those described below: U.S. Patent No. 7,727,466 (Meathrel et al.), entitled "Disintegratable Films for Diagnostic Devices" (issued June 1, 2010) (the entire disclosure of which is incorporated herein by reference).

[0034] "Activating" with respect to one or more electrodes means affecting a change in the electrical state of the one or more electrodes, which results in droplet manipulation in the presence of droplets. Activation of the electrodes can be achieved using alternating current (AC) or direct current (DC). Any suitable voltage can be used. For example, the electrodes can be activated using a voltage greater than about 150 V, or greater than about 200 V, or greater than about 250 V, or from about 275 V to about 1000 V, or about 300 V. When an AC signal is used, any suitable frequency can be used. For example, the electrodes can be activated using an AC signal having a frequency from about 1 zH to about 10 MHz, or from about 10 Hz to about 60 Hz, or from about 20 Hz to about 40 Hz, or about 30 Hz. With respect to the beads of the droplet actuator, "beads" means any beads or particles that can interact with droplets on or proximate to the droplet actuator. The beads can be of any wide range of shapes (e.g., spherical, roughly spherical, oval, disc-shaped, cubic, amorphous, and other three-dimensional shapes). The beads can, for example, have the ability to undergo droplet processing as droplets on the droplet actuator, or otherwise the beads can be configured with respect to the droplet actuator in such a way that droplets on the droplet actuator can contact beads on and / or away from the droplet actuator. The beads can be provided within the droplet, within the droplet processing gap, or on the droplet processing surface. The beads can be provided in a reservoir that is outside of the droplet processing gap or located away from the droplet processing surface, and the reservoir can be coupled to a flow path that enables droplets containing the beads to reach the droplet processing gap or contact the droplet processing surface. The beads can be manufactured using a wide range of materials (including, for example, resins and polymers). The beads can be of any suitable size and can include, for example, microbeads, microparticles, nanobeads, and nanoparticles. In some cases, the beads are magnetically responsive, and in other cases, the beads are not significantly magnetically responsive. For magnetically responsive beads, a magnetically responsive material can substantially constitute all of the beads, a portion of the beads, or just one component of the beads. The remaining portion of the beads can include, among other things, a polymeric material, a coating, and a portion that enables attachment of assay reagents.

[0035] Examples of suitable beads include the following: flow cytometry microbeads, polystyrene microparticles and nanoparticles, functionalized polystyrene microparticles and nanoparticles, coated polystyrene microparticles and nanoparticles, silica microbeads, fluorescent microspheres and nanospheres, functionalized fluorescent microspheres and nanospheres, coated microspheres and nanospheres, colored microparticles and nanoparticles, magnetic microparticles and nanoparticles, superparamagnetic microparticles and nanoparticles (e.g., DYNABEADS™ particles, available from Invitrogen Group, Carlsbad, CA), fluorescent microparticles and nanoparticles, coated magnetic microparticles and nanoparticles, ferromagnetic microparticles and nanoparticles, coated ferromagnetic microparticles and nanoparticles, and those described below: U.S. Patent Publication No. 20050260686 (Watkins et al.), titled "Multiplex Flow Assays Preferably with Magnetic Particles as Solid Phase" (published Nov. 24, 2005); U.S. Patent Publication No. 20030132538 (Chandler), titled "Encapsulation of Discrete Quanta of Fluorescent Particles" (published Jul. 17, 2003); U.S. Patent Publication No. 20050118574 (Chandler et al.), titled "Multiplexed Analysis of Clinical Specimens Apparatus and Method" (published Jun. 2, 2005); U.S. Patent Publication No. 20050277197 (Chandler et al.), titled "Microparticles with Multiple Fluorescent Signals and Methods of Using Same" (published Dec. 15, 2005); and U.S. Patent Publication No. 20060159962 (Chandler et al.)、Title of the Invention "Magnetic Microspheres for use in Fluorescence-based Applications" (published on July 20, 2006) (For the teachings of the said document regarding beads and magnetic-responsive substances and beads, the entire disclosure content of the said document is incorporated herein by reference).

[0036] Beads can be pre-linked with biomolecules or other molecules that can bind to biomolecules to form a complex. Beads can be pre-linked with antibodies, proteins or antigens, DNA / RNA probes, or any other molecule having an affinity for a desired target. Examples of droplet actuator technologies for immobilizing magnetic-responsive beads and / or non-magnetic-responsive beads and / or for performing droplet processing protocols using beads are described below: U.S. Patent Publication No. 20080053205 (Pollack et al.), titled "Droplet-Based Particle Sorting" (published on March 6, 2008); U.S. Patent Application No. 61 / 039,183, titled "Multiplexing Bead Detection in a Single Droplet" (filed on March 25, 2008); U.S. Patent Application No. 61 / 047,789 (Pamula et al.), titled "Droplet Actuator Devices and Droplet Operations Using Beads" (filed on April 25, 2008); U.S. Patent Application No. 61 / 086,18, titled "Droplet Actuator Devices and Methods for Manipulating Beads" (filed on August 5, 2008); International Patent Publication WO / 2008 / 098236 (Eckhardt et al.), titled "Droplet Actuator Devices and Methods Employing Magnetic Beads" (filed on August 14, 2008); International Patent Publication WO / 2008 / 134153 (Grichko et al.), titled "Bead-based Multiplexed Analytical Methods and Instrumentation" (published on November 6, 2008); International Patent Publication WO / 2008 / 116221 (Eckhardt et al.)、Title of the Invention "Bead Sorting on a Droplet Actuator" (filed on September 25, 2008); International Patent Publication WO / 2007 / 120241 (Eckhardt et al.), Title of the Invention "Droplet-based Biochemistry" (published on October 25, 2007) (the entire disclosure of the foregoing document is incorporated herein by reference). The characteristics of the beads can be utilized with the multiplexed characteristics of the present disclosure. Examples of beads that are suitable for multiplexing and have characteristics suitable for methods of detecting and analyzing signals emitted from such beads can be found below: U.S. Patent Publication No. 20080305481 (Whitman et al.), titled "Systems and Methods for Multiplex Analysis of PCR in Real Time" (published December 11, 2008); U.S. Patent Publication No. 20080151240 (Roth), titled "Methods and Systems for Dynamic Range Expansion" (published June 26, 2008); U.S. Patent Publication No. 20070207513 (Sorensen et al.), titled "Methods, Products, and Kits for Identifying an Analyte in a Sample" (published September 6, 2007); U.S. Patent Publication No. 20070064990 (Roth), titled "Methods and Systems for Image Data Processing" (published March 22, 2007); U.S. Patent Publication No. 20060159962 (Chandler et al.), titled "Magnetic Microspheres for use in Fluorescence-based Applications" (published July 20, 2006); U.S. Patent Publication No. 20050277197 (Chandler et al.), titled "Microparticles with Multiple Fluorescent Signals and Methods of Using Same" (published December 15, 2005); and U.S. Patent Publication No. 20050118574 (Chandler et al.)、Title of the Invention "Multiplexed Analysis of Clinical Specimens Apparatus and Method" (published on June 2, 2005) (the entire disclosure of the foregoing document is incorporated herein by reference).

[0037] "Droplet" means a large amount of liquid on a droplet actuator. Typically, the droplet is at least partially in contact with the boundary of the filling fluid. For example, the droplet can be completely surrounded by the filling fluid or can contact one or more surfaces of the filling fluid and the droplet actuator. As another example, the droplet can contact the filling fluid, one or more surfaces of the droplet actuator, and / or the atmosphere. As yet another example, the droplet can contact the filling fluid and the atmosphere. The droplet can be, for example, aqueous or non-aqueous, or a mixture or emulsion containing aqueous and non-aqueous components. The droplet can have various shapes, and non-limiting examples generally include: disk-shaped, slug-shaped, truncated sphere, ellipse, spherical, partially compressed sphere, hemispherical, oval, cylindrical, combinations of such shapes, and various shapes formed during droplet processing, such as shapes that appear, split, or are formed as a result of contact between such a shape and one or more surfaces of the droplet actuator. For examples of droplet liquids that can be subjected to droplet processing using the approach of the present disclosure, see: International Patent Publication WO / 2007 / 120241 (Eckhardt et al.), Title of the Invention "Droplet-Based Biochemistry" (published on October 25, 2007) (the entire disclosure of the foregoing document is incorporated herein by reference).

[0038] In various embodiments, the droplets can include a biological sample, such as whole blood, lymphatic fluid, serum, plasma, sweat, tear fluid, saliva, sputum, cerebrospinal fluid, amniotic fluid, vaginal secretion, serous fluid, myeloid fluid, pericardial fluid, peritoneal fluid, pleural effusion, transudate, exudate, cyst fluid, bile, urine, gastric juice, intestinal fluid, fecal sample, a fluid containing single or multiple cells, a fluid containing cell organelles, a liquefied tissue, a liquefied organ, a fluid containing a multicellular organism, a biological swab, and a biological cleaning solution. Additionally, the droplets can include a reagent (such as water, deionized water, aqueous salt solution, acidic solution, basic solution, detergent solution, and / or buffer solution). The droplets can include the following: nucleic acids, such as DNA, genomic DNA, RNA, mRNA, or an analog thereof; nucleotides, such as deoxyribonucleotides, ribonucleotides, or an analog thereof, such as an analog having a terminator moiety (such as those described below: the paper by Bentley et al. (Bentley et al., Nature 456:53-59, 2008); International Patent Publication WO / 2013 / 131962 (Gormley et al.), title of the invention "Improved Methods of Nucleic Acid Sequencing" (published on September 12, 2013); U.S. Patent No. 7,057,026 (Barnes et al.), title of the invention "Labelled Nucleotides" (issued on June 6, 2006); International Patent Publication WO / 2008 / 042067 (Kozlov et al.), title of the invention "Compositions and Methods for Nucleotide Sequencing" (published on April 10, 2008); International Patent Publication WO / 2013 / 117595 (Rigatti et al.), title of the invention "Targeted Enrichment and Amplification of Nucleic Acids on a Support" (published on August 15, 2013); U.S. Patent No. 7,329,492 (Hardin et al.)、Title of the Invention “Methods for Real-Time Single Molecule Sequence Determination (Methods for Real-Time Single Molecule Sequencing)” (issued on February 12, 2008); U.S. Patent No. 7,211,414 (Hardin et al.), Title of the Invention “Enzymatic Nucleic Acid Synthesis: Compositions and Methods for Altering Monomer Incorporation Fidelity (Enzymatic Nucleic Acid Synthesis: Compositions and Methods for Modifying Monomer Incorporation Fidelity)” (issued on May 1, 2007); U.S. Patent No. 7,315,019 (Turner et al.), Title of the Invention “Arrays of Optical Confinements and Uses Thereof (Arrays of Optical Confinements and Their Uses)” (issued on January 1, 2008); U.S. Patent No. 7,405,281 (Xu et al.), Title of the Invention “Fluorescent Nucleotide Analogs and Uses Therefor (Fluorescent Nucleotide Analogs and Their Uses)” (issued on July 29, 2008); and U.S. Patent Publication No. 20080108082 (Ranke et al.), Title of the Invention “Polymerase Enzymes and Reagents for Enhanced Nucleic Acid Sequencing (Polymerase Enzymes and Reagents for Enhanced Nucleic Acid Sequencing)” (published on May 8, 2008) (the entire disclosures of the foregoing documents are incorporated herein by reference)); enzymes, such as polymerases, ligases, recombinases, or transposases; binding partners, such as antibodies, epitopes, streptavidin, biotin, lecithin, or carbohydrates; or other biochemically active molecules. Other examples of the contents of the droplets include reagents, such as reagents for biochemical protocols (e.g., nucleic acid amplification protocols, affinity-based assay protocols, enzyme assay protocols, sequencing protocols, and / or biological fluid analysis protocols). The droplets can contain one or more beads.

[0039] "Droplet processing" means any manipulation of droplets on a droplet actuator. Droplet processing includes, for example: loading droplets onto a droplet actuator, dispensing one or more droplets from a droplet source, splitting, separating or dividing one droplet into two or more droplets, transporting a droplet from one location to another location in any direction, combining or joining one or more droplets into one single droplet, diluting a droplet, mixing droplets, agitating droplets, deforming droplets, maintaining a droplet in a proper position, incubating a droplet, heating a droplet, vaporizing a droplet, cooling a droplet, disposing of a droplet, transporting a droplet out of the droplet actuator, other droplet processing described herein, and / or combinations of the foregoing. The terms "combining", "combination", "joining", "join", etc. are used to refer to producing one droplet from two or more droplets. When such terms are used with respect to two or more droplets, it should be understood that any combination of droplet processing sufficient to effect the joining of two or more droplets into one droplet is possible. For example, "combination of droplet A with droplet B" can be achieved by: transporting droplet A and contacting it with stationary droplet B; transporting droplet B and contacting it with stationary droplet A; or transporting droplets A and B and contacting them with each other. The terms "splitting", "separating" and "dividing" are not intended to suggest any particular result with respect to the volume of the resulting droplets (i.e., the volume of the resulting droplets may be the same or different) or the number of the resulting droplets (the number of the resulting droplets may be 2, 3, 4, 5, or more). The term "mixing" refers to droplet processing that results in a more homogeneous distribution of one or more components within a droplet. Examples of "loading" for droplet processing include microdialysis, pressure-assisted loading, automated loading, passive loading, and pipette loading. Droplet processing can be mediated by electrodes. In some instances, droplet processing is further facilitated by the use of hydrophilic and / or hydrophobic regions on a surface and / or by physical barriers. For droplet processing, reference is made to the patents and patent applications cited above under the definition of "droplet actuator".

[0040] To detect or confirm the results of droplet processing, impedance or capacitance detection or imaging techniques can sometimes be used. Examples of such techniques are described in U.S. Patent Publication No. 20100194408 (Sturmer et al.), titled "Capacitance Detection in a Droplet Actuator" (published August 5, 2010) (the entire disclosure of which is incorporated herein by reference). Generally speaking, detection or imaging techniques can be used to confirm the presence or absence of droplets at individual electrodes. For example, if a droplet is present at the electrode at the target location following droplet dispensing processing, it is confirmed that the droplet dispensing processing was effective. Similarly, if a droplet is present at the detection location, which is an appropriate step in the assay protocol, it can be confirmed that the previous series of droplet processing successfully produced droplets for detection. The droplet transfer time is extremely fast. For example, in various embodiments, the transfer of a droplet from one electrode to the next can exceed about 1 second, or about 0.1 second, or about 0.01 second, or about 0.001 second. In one embodiment, the electrodes are operated in AC mode but switched to DC mode for imaging. It is beneficial to perform droplet processing such that the footprint area of the droplet is similar to the electrowetting area. In other words, 1x-, 2x-, 3x-droplets are effectively controlled by operating with 1, 2, and 3 electrodes, respectively. When the droplet footprint is larger than the number of electrodes available for performing droplet processing at a given time, the difference between the droplet size and the number of electrodes should typically not exceed 1. In other words, 2x droplets are effectively controlled using one electrode, and 3x droplets are effectively controlled using two electrodes. When the droplet contains beads, it is useful for the droplet size to be equal to the number of electrodes that control the droplet (e.g., transfer the droplet).

[0041] "Filling fluid" means a fluid having a close relationship with the droplet processing substrate of the droplet actuator, and the fluid is extremely immiscible with the droplet phase and facilitates electrode-mediated droplet processing of the droplet phase. For example, the droplet processing gap of the droplet actuator is typically filled with a filling fluid. The filling fluid is, for example, a low-viscosity oil (such as silicone oil) or a hexadecane filling fluid or includes the foregoing. The filling fluid can be a halogenated oil (such as a fluorinated or perfluorinated oil) or include the foregoing. The filling fluid can fill the entire gap of the droplet actuator or coat one or more surfaces of the droplet actuator. The filling fluid is conductive or non-conductive. The filling fluid can be selected to achieve the following: improving droplet processing and / or reducing the loss of droplet reagents or target substances, improving the formation of microdroplets, reducing cross-contamination between droplets, reducing contamination of the droplet actuator surface, reducing the decomposition of the droplet actuator material, etc. For example, the filling fluid can be selected for compatibility with the droplet actuator material. As an example, a fluorinated filling fluid can be advantageously used with a fluorinated surface coating. Fluorinated filling fluids are useful for reducing the disappearance of lipophilic compounds. Lipophilic compounds are, for example, umbelliferone substrates, such as 6-hexadecanoyl amide-4-methylumbelliferone substrates (used, for example, in assays for Krabb disease, Niemann-Pick disease or other assays), and other umbelliferone substrates are described below: U.S. Patent Publication No. 20110118132 (Winger et al.), title of the invention "Enzymatic Assays Using Umbelliferone Substrates with Cyclodextrins in Droplets of Oil" (published May 19, 2011) (the entire disclosure of the foregoing document is incorporated herein by reference).Examples of suitable fluorinated oils include the following: Galden series, such as Galden HT170 (bp = 170 °C, viscosity = 1.8 cSt, density = 1.77), Galden HT200 (bp = 200 °C, viscosity = 2.4 cSt, density = 1.79), Galden HT230 (bp = 230 °C, viscosity = 4.4 cSt, density = 1.82) (all from Solvay Solexis); Novec series, such as Novec 7500 (bp = 128 °C, viscosity = 0.8 cSt, density = 1.61), Fluorinert FC-40 (bp = 155 °C, viscosity = 1.8 cSt, density = 1.85), Fluorinert FC-43 (bp = 174 °C, viscosity = 2.5 cSt, density = 1.86) (both from 3M).

[0042] Generally, the selection of the perfluorinated filling fluid is based on kinematic viscosity (<7 cSt is preferred but not a requirement) and boiling point (>150 °C is preferred but not a requirement for DNA / RNA-based applications (such as PCR)). The filling fluid can be doped with, for example, surfactants or other additives. For example, additives can be selected to achieve the following: improve droplet handling and / or reduce loss of reagent or target substance in droplets, improve formation of microdroplets, reduce cross-contamination between droplets, reduce contamination of the droplet actuator surface, reduce degradation of the droplet actuator material, etc. The composition of the filling fluid (including surfactant doping) can be selected for the performance of the reagents used in specific assay protocols and for effective interaction or non-interaction with the droplet actuator material. Examples of suitable filling fluids and filling fluid formulations for use with the methods and devices shown herein are provided below: International Patent Publication WO / 2010 / 027894 (Srinivasan et al.), titled "Droplet Actuators, Modified Fluids and Methods" (published June 3, 2010); International Patent Publication WO / 2009 / 021173 (Srinivasan et al.), titled "Use of Additives for Enhancing Droplet Operations" (published February 12, 2009); International Patent Publication WO / 2008 / 098236 (Sista et al.), titled "Droplet Actuator Devices and Methods Employing Magnetic Beads" (published January 15, 2009); U.S. Patent Publication No. 20080283414 (Monroe et al.), titled "Electrowetting Devices" (published November 20, 2008) (the entire disclosures of the foregoing documents are incorporated herein by reference together with other patents and patent applications cited herein).In some cases, the fluorinated oil is doped with a fluorinated surfactant (e.g., Zonyl FSO-100 (Sigma-Aldrich) and / or others). The filling fluid is typically a liquid. In some embodiments, a filling gas can be used instead of the liquid.

[0043] "Fixing" with respect to the magnetic-responsive beads means that the beads are positionally substantially constrained by the droplets on the droplet actuator or by the filling fluid. For example, in one embodiment, the fixed beads are positionally sufficiently constrained by the droplets to enable the performance of the droplet splitting process, resulting in one droplet having substantially all of the beads and one droplet substantially lacking beads. "Magnetic-responsive" means responsive to a magnetic field. "Magnetic-responsive beads" contain or are composed of a magnetic-responsive material. Examples of magnetic-responsive materials include paramagnetic materials, ferromagnetic materials, ferrimagnetic materials, and metamagnetic materials. Examples of suitable paramagnetic materials include metal oxides together with iron, nickel, and cobalt, such as Fe3O4, BaFe 12 O 19 , CoO, NiO, Mn2O3, Cr2O3, and CoMnP. "Water reservoir" means an enclosure or partial enclosure having a structure for holding, storing, or supplying a liquid. The droplet actuator system of the present disclosure can include an on-cartridge water reservoir and / or an off-cartridge water reservoir. An on-cartridge water reservoir can be (1) an on-actuator water reservoir (a water reservoir within or on the droplet processing gap surface); (2) an off-actuator water reservoir (a water reservoir on the droplet actuator cartridge but outside the droplet processing gap and not in contact with the droplet processing surface); or (3) a hybrid water reservoir (having an on-actuator region and an off-actuator region). An example of an off-actuator water reservoir is a water reservoir within an upper substrate. The off-actuator water reservoir typically has fluid communication by way of an opening or a flow path, which is arranged to flow liquid from the off-actuator water reservoir into the droplet processing gap (e.g., into an on-actuator water reservoir). An off-cartridge water reservoir can be a water reservoir that is not part of the droplet actuator cartridge at all but that flows liquid to some part of the droplet actuator cartridge. For example, the off-cartridge water reservoir can be a part of the system or a docking station to which the droplet actuator cartridge is connected during processing. Similarly, the off-cartridge water reservoir can be a reagent storage container or a syringe, the latter being used to force fluid into an on-cartridge water reservoir or into the droplet processing gap. A system using an off-cartridge water reservoir typically includes fluid transfer means by which liquid can be transferred from the off-cartridge water reservoir into an on-cartridge water reservoir or into the droplet processing gap.

[0044] As used herein, "transfer to the magnetic field of a magnet", "transfer towards a magnet", etc. refer to droplets and / or magnetic responsive beads within the droplets, and are intended to refer to transfer into a magnetic field region that can substantially attract the magnetic responsive beads within the droplets. Similarly, "remove from a magnet or magnetic field", "transfer out of the magnetic field of a magnet", etc. as used herein refer to droplets and / or magnetic responsive beads within the droplets, and are intended to refer to transfer out of a magnetic field region that can substantially attract the magnetic responsive beads within the droplets, regardless of whether the droplets or magnetic responsive beads are completely removed from the magnetic field. In any of the examples described herein, it will be understood that the droplets can be transferred towards or out of a desired magnetic field region, and / or that the desired magnetic field region can be moved towards or away from the droplets. When it is said that an electrode, droplet, or magnetic responsive bead is within or present in a magnetic field, it is intended to refer to a situation where the electrode is arranged in a manner that enables the electrode to transfer the droplet within and / or out of a desired magnetic field region, or a situation where the droplet or magnetic responsive bead is arranged in a desired magnetic field region, and in each case, the magnetic field in the desired region has the ability to substantially attract all of the magnetic responsive beads within the droplet. Similarly, when it is said that an electrode, droplet, or magnetic responsive bead is present "outside" a magnetic field or "away from" a magnetic field, it is intended to refer to a situation where the electrode is arranged in a manner that enables the electrode to transfer the droplet out of a certain magnetic field region, or a situation where the droplet or magnetic responsive bead is arranged away from a certain magnetic field region, and in each case, the magnetic field in such a region either has no ability to substantially attract all of the magnetic responsive beads within the droplet or any residual attractive force does not preclude the effectiveness of the droplet processing carried out within the region. In various features of the present disclosure, the system, droplet actuator, or another component of the system includes a magnet (e.g., one or more paramagnetic magnets (e.g., a single cylindrical or rod-shaped magnet, or an array of such magnets (e.g., a Halbach array)), or an electromagnet or electromagnet array) and can form a magnetic field to interact with magnetic responsive beads or other components on the chip.Such interactions include, for example, substantially fixing or constraining the movement or flow of magnetic responsive beads in droplets during storage or droplet processing, or pulling magnetic responsive beads out of droplets by magnetic force.

[0045] With respect to bead washing, "washing" means reducing the amount and / or concentration of one or more substances that have contacted or been exposed to the beads from the droplets in contact with the beads. The reduction in the amount and / or concentration of the substance can be partial, substantially complete, or completely complete. The substance can be any of a very wide variety of substances. Examples include target substances for further analysis and unwanted substances (e.g., components of a sample, contaminants, and / or excess reagents). In some embodiments, the washing process begins with a starting droplet in contact with the magnetic responsive beads, where the droplet contains an initial amount and an initial concentration of the substance. The washing process can be carried out using a variety of droplet processes. The washing process can result in a droplet containing magnetic responsive beads having a total amount and / or concentration lower than the initial amount and / or concentration of the substance. Suitable washing techniques are described below: U.S. Patent No. 7,439,014 (Pamula et al.), title of the invention "Droplet-Based Surface Modification and Washing" (issued October 21, 2008) (the entire disclosure of the foregoing document is incorporated herein by reference). The terms "upper", "lower", "above", "below", and "on" are used through descriptions of the relative positions of the components of the droplet actuator, for example, the relative positions of the upper and lower substrates of the droplet actuator. It will be understood that the droplet actuator functions regardless of its spatial orientation. When it is described that a liquid of any shape (e.g., a droplet or a continuum whether moving or stationary) is "on," "at," or "above" an electrode, an array, a matrix, or a surface, such a liquid can be in direct contact with the electrode / array / matrix / surface or can be in contact with one or more layers or films (inserted between the liquid and the electrode / array / matrix / surface). In certain embodiments, a filling fluid can be regarded as a film between such a liquid and the electrode / array / matrix / surface. When a droplet is described as being "present on" or "loaded onto" a droplet actuator, it is to be understood that the droplet is positioned on the droplet actuator in a manner that facilitates performance of one or more droplet processes on the droplet using the droplet actuator, and / or in a manner that facilitates detection of properties of a signal arising from the droplet or detection of a signal, and / or the droplet is being subjected to droplet processing on the droplet actuator.

[0046] Barcodes and UMI on beads In some embodiments, primers that carry sample barcodes can be immobilized on a solid support. Additionally or alternatively, primers that carry UMI sequences can be immobilized on a solid support. For example, the solid support can be one or more beads. Thus, in certain embodiments, a plurality of beads can be presented, where each of the plurality of beads carries a unique sample barcode and / or UMI sequence. In some embodiments, individual cells are contacted with one or more beads that have a unique set of sample barcodes and / or UMI sequences for identification of the individual cells. In some embodiments, lysates of individual cells are contacted with one or more beads that have a unique set of sample barcodes and / or UMI sequences for identification of the individual cell lysates. In some embodiments, purified nucleic acids of individual cells are contacted with one or more beads that have a unique set of sample barcodes and / or UMI sequences for identification of the purified nucleic acids of the individual cells. The beads can be manipulated in any suitable manner known in the art, for example, using the droplet actuators described above. The terms “solid surface,” “solid support,” and other grammatical equivalents herein refer to any material that can be adapted or is adaptable to serve as an attachment for the primers, barcodes, and sequences described herein. As will be understood by those of skill in the art, the number of possible substrates is very large. Possible substrates include, but are not limited to: glass and modified or functionalized glass, plastics (including acrylic, polystyrene, and copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethane, Teflon TM ), polysaccharides, nylon or nitrocellulose, ceramics, resins, silica or silica-based materials (including silicon and modified silicon), carbon, metals, inorganic glasses, plastics, bundles of optical fibers, and other diverse polymers. Solid supports and solid surfaces that are particularly useful for some embodiments are present in flow cell devices. Exemplary flow cells are shown in more detail below.

[0047] In some embodiments, the solid support has a patterned surface suitable for immobilizing the primers, barcodes, and sequences described herein in a specified pattern. "Patterned surface" refers to the arrangement of various regions in or on the exposed layer of the solid support. For example, the one or more regions can be artifices where one or more transpososome complexes are present. The artifices can be separated by intervening regions (where no transpososome complexes are present). In some embodiments, the artifices are in an x-y format and can form rows and columns. In some embodiments, the pattern can be an arrangement where the artifices and / or intervening regions are repeated. In some embodiments, the pattern can be a random arrangement of the artifices and / or intervening regions. In some embodiments, the transpososome complexes are randomly distributed on the solid support. In some embodiments, the transpososome complexes are distributed on the patterned surface. Exemplary patterned surfaces that can be used in the methods and compositions shown herein are described in US Ser.No.13 / 661,524 or US Pat.App.Publ.No.2012 / 0316086 A1 (each of the foregoing documents is incorporated herein by reference). In some embodiments, the solid support includes an array of wells or depressions on the surface. The foregoing can be fabricated using a variety of techniques that are generally known in the art. Such techniques include, but are not limited to, for example, photolithography, stamping techniques, molding techniques, and microetching techniques. As will be understood by those skilled in the art, the techniques used will depend on the composition and shape of the array substrate.

[0048] The composition and geometric structure of the solid support can vary depending on its use. In some embodiments, the solid support is a flat structure, such as a slide, chip, microchip, and / or array. Thus, the surface of the substrate can be in the form of a flat layer. In some embodiments, the solid support includes one or more surfaces of a flow cell. As used herein, the term "flow cell" refers to a chamber that includes a solid surface and through which one or more flowing reagents can flow. Examples of flow cells and related fluid systems and detection platforms that can be readily used in the methods of the present disclosure are described, for example, in: Bentley et al., Nature 456:53-59, 2008; WO 04 / 018497; US 7,057,026; WO 91 / 06678; WO 07 / 123744; US 7,329,492; US 7,211,414; US 7,315,019; US 7,405,281; and US 2008 / 0108082 (each of the foregoing documents is incorporated herein by reference). In some embodiments, the solid support or its surface is non-flat, such as the inner or outer surface of a tube or container. In some embodiments, the solid support includes microspheres or beads. "Microspheres" or "beads" or "particles" or other grammatical equivalents herein mean small discrete particles. Suitable bead compositions include, but are not limited to: plastics, ceramics, glass, polystyrene, methylstyrene, acrylic polymers, paramagnetic materials, triazoles, carbon graphite, titanium dioxide, latex, or cross-linked dextran, such as sepharose, cellulose, nylon, cross-linked micelles, and Teflon®. Any of the other materials outlined herein for the solid support can likewise be used. The following guidebook ("Microsphere Detection Guide", Bangs Laboratories (Fishers Ind.)) is a useful reference. The beads do not have to be spherical, and irregular particles can be used. Separately or in addition to the above, the beads may be porous. The size of the beads ranges from nanometers (i.e., 100 nm) to millimeters (i.e., 1 mm), beads of about 0.2 microns to about 200 microns are preferred, and beads of about 0.5 to about 5 microns are particularly preferred, provided that in some embodiments, smaller or larger beads can be used. Throughout this application, various publications, patents and / or patent applications are referenced. The entire disclosure of these publications is incorporated herein by reference. The term "comprising" is intended to be unrestrictive in this specification and includes not only the recited components but also any additional components. Numerous embodiments have been described. Nevertheless, it will be understood that various modifications can be made. Accordingly, other embodiments are within the scope of the following claims. This disclosure includes the following embodiments. <1> A method for preparing a cDNA library from a plurality of single cells, comprising the following steps: Releasing mRNA from each single cell to provide a plurality of samples of individual mRNAs, wherein each mRNA of each individual mRNA sample is derived from one single cell; Synthesizing the first strand of cDNA from each mRNA of each individual mRNA sample with a first strand synthesis primer, and further incorporating a tag into the cDNA to provide a plurality of tagged cDNA samples, wherein the cDNA of each tagged cDNA sample is complementary to the mRNA derived from one single cell, and further wherein the tag comprises a cell-specific identifier sequence and optionally a unique molecular identifier (UMI) sequence; Pooling the tagged cDNA samples; Optionally, amplifying the pooled cDNA samples to generate a cDNA library containing double-stranded cDNA; and Performing a tagmentation reaction to simultaneously cleave each cDNA and incorporate an adapter into each strand of the cDNA, thereby generating a plurality of tagged cDNA fragments. <2> The method according to <1>, further comprising amplifying the tagged cDNA fragment to generate an amplified tagged cDNA fragment. <3> The method according to <2>, wherein the amplification step comprises adding an additional sequence to the 5' end of the amplification product. <4> The method according to <3>, wherein the additional sequence comprises a primer binding sequence for amplification on a solid support. <5> The method according to <4>, further comprising amplifying the amplified tagged cDNA fragment on a solid support. <6> The method according to <5>, further comprising sequencing the amplification product on a solid support. <7> The method according to <1>, wherein the tagmentation reaction comprises contacting the double-stranded cDNA with a transposase mixture comprising an adapter sequence not found in the first strand synthesis primer. <8> The method according to <7>, wherein the transposase mixture consists essentially of transposomes having one type of adapter sequence. <9> The method according to <1>, further comprising the step of sequencing the tagged cDNA fragment. <10> The method according to <9>, wherein the sequencing step includes measurement of the 3' tag. <11> The method according to <9>, wherein the sequencing step includes analysis of the entire transcriptome. <12> The method according to <1>, wherein the first-strand synthesis is carried out using a mixture of random primers, and the random primers further contain tags. <13> The method according to <12>, wherein the first-strand synthesis primer contains a double-stranded portion. <14> The method according to <13>, wherein the first-strand synthesis primer reduces ligation by-products as compared to a single-stranded first-strand synthesis primer. <15> The method according to <13>, wherein the first-strand synthesis primer contains a region capable of forming a hairpin. <16> The method according to <13>, wherein the first-strand synthesis primer contains an RNA region. <17> The method according to <13>, wherein the first-strand synthesis primer hybridizes with a complementary oligonucleotide, thereby forming a double-stranded portion. <18> A plurality of beads, each bead containing a plurality of oligonucleotides, each oligonucleotide comprising (a) a linker, (b) an amplification primer binding site, (c) optionally, a unique molecular identifier that differs for each oligonucleotide, (d) a bead-specific sequence that is the same for each oligonucleotide of the bead but differs for other beads, and (e) a capture sequence that captures mRNA and primes reverse transcription, wherein the plurality of beads are as described above. <19> The plurality of beads according to <18>, wherein the capture sequence contains oligo dT. <20> The plurality of beads according to <18>, wherein each bead is present in a separate droplet isolated from other beads.

Claims

**Claim 1** A method for preparing a cDNA library from a plurality of single cells, comprising the following steps: A step of releasing mRNA from each single cell to provide a plurality of samples of individual mRNAs, wherein the mRNA of each individual mRNA sample is derived from one single cell; A step of synthesizing the first strand of cDNA from the mRNA of each individual mRNA sample using a first strand synthesis primer containing a tag, and further incorporating the tag into the cDNA to provide a plurality of tagged cDNA samples, wherein the first strand synthesis primer is a mixture of an oligo dT primer and a random primer, the random primer contains a 5' amplification primer binding site and a 3' random sequence portion, the cDNA of each tagged cDNA sample is complementary to the mRNA derived from one single cell, and further the tag contains a cell-specific identifier sequence and a unique molecular identifier (UMI) sequence; A step of pooling the tagged cDNA samples derived from a plurality of single cells; A step of amplifying the pooled tagged cDNA to generate a pooled tagged double-stranded cDNA; and A step of performing a tagmentation reaction on the pooled tagged double-stranded cDNA sample and simultaneously cleaving each cDNA to incorporate an adapter into each strand of the cDNA, thereby generating a cDNA library derived from a plurality of single cells, wherein the tagmentation reaction includes contacting a transposase mixture with the double-stranded cDNA. **Claim 2** The method according to claim 1, wherein the transposase mixture contains an adapter sequence not found in the first strand synthesis primer. **Claim 3** The method according to claim 1 or claim 2, wherein the oligo dT primer further contains an amplification primer binding site. **Claim 4** A method for preparing a cDNA library from a plurality of single cells, comprising: A step of releasing mRNA from each single cell to provide a plurality of samples of individual mRNAs, wherein the mRNA of each individual mRNA sample is derived from one single cell; A step of synthesizing the first strand of cDNA from the mRNA of each individual mRNA sample using a first strand synthesis primer containing a tag, thereby incorporating the tag into the cDNA to provide a plurality of tagged cDNA samples, wherein the first strand synthesis primer is a mixture of an oligo dT primer and a random primer, the random primer contains a 5' amplification primer binding site and a 3' random sequence portion, the cDNA of each tagged cDNA sample is complementary to the mRNA derived from one single cell, and the tag contains a cell-specific identifier sequence, a first read sequencing adapter sequence, and a different second portion; A step of pooling the tagged cDNA samples; A step of amplifying the pooled tagged cDNA to generate a pooled tagged double-stranded cDNA; A step of generating a cDNA library by performing a tagmentation reaction on the pooled tagged double-stranded cDNA by contacting the pooled tagged cDNA with a plurality of transposome complexes each having a transposase complexed with a transposon containing a second read sequencing adapter sequence, wherein the second read sequencing adapter sequence is different from the first read sequencing adapter sequence, and the cDNA library contains a plurality of tagged cDNA fragments having the first read sequencing adapter sequence on the first strand and the second read sequencing adapter sequence on the second strand; A method comprising the above steps.

5. The method according to claim 4, wherein the different second portion contains a UMI sequence.

6. The method according to claim 4 or claim 5, wherein the oligo dT primer further contains an amplification primer binding site.

7. A method for preparing a cDNA library from a plurality of single cell organelles, comprising: A step of spatially separating single cell organelles; A step of releasing mRNA, microRNA, small interfering RNA, ribosomal RNA, and / or mitochondrial RNA from each single cell organelle to provide a plurality of samples of individual RNAs, wherein the RNA of each individual RNA sample is derived from one single cell organelle; A step of synthesizing the first strand of cDNA from the RNA of each individual RNA sample with a first strand synthesis primer containing a tag, thereby incorporating the tag into the cDNA to provide a plurality of tagged cDNA samples, wherein the first strand synthesis primer is a mixture of an oligo dT primer and a random primer, the random primer contains a 5' amplification primer binding site and a 3' random sequence portion, the cDNA of each tagged cDNA sample is complementary to the RNA derived from one single cell organelle, and the tag contains an organelle-specific identifier sequence and a first read sequencing adapter sequence; A step of pooling the tagged cDNA samples derived from a plurality of single cell organelles; A step of amplifying the pooled tagged cDNA to generate a pooled tagged double-stranded cDNA; and A step of generating a cDNA library by performing a tagmentation reaction on the pooled tagged double-stranded cDNA sample by contacting the pooled tagged cDNA with a plurality of transposome complexes each having a transposase complexed with a transposon containing a second read sequencing adapter sequence, wherein the second read sequencing adapter sequence is different from the first read sequencing adapter sequence, and the cDNA library contains a plurality of tagged cDNA fragments having the first read sequencing adapter sequence on the first strand and the second read sequencing adapter sequence on the second strand; A method comprising the above steps.

8. The method according to claim 7, wherein the oligo dT primer further contains an amplification primer binding site.

9. The method according to claim 5, wherein the tag further contains a unique molecular identifier (UMI) sequence.

10. The method according to any one of claims 1 to 9, wherein the tagmentation reaction comprises a step of contacting the double-stranded cDNA with a transposase mixture containing Tn5 transposase.

11. The method according to any one of claims 1 to 10, wherein the oligo dT primer and / or the random primer contains a primer containing a double-stranded site.

12. (a)The oligo dT primer and / or the random primer reduces ligation by-products compared to a single-stranded first strand synthesis primer. (b) the oligo dT primer and / or the random primer includes a primer containing a region capable of forming a hairpin, (c) the oligo dT primer and / or the random primer includes a primer containing an RNA region, and / or (d) the oligo dT primer and / or the random primer includes a primer that hybridizes with a complementary oligonucleotide, thereby forming a double-stranded portion, The method according to claim 11.

13. The method according to any one of claims 1 to 12, wherein the random primer has a length of 3, 4, 5, 6, 7, 8, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more than 20 nucleotides.

14. The method according to any one of claims 1 to 13, wherein the random primer includes primers having different lengths.

15. The method according to any one of claims 1 to 13, wherein the random primer includes primers having equal lengths.

16. The method according to any one of claims 1 to 15, wherein the 3' random sequence portion has a length of 5 to 18 nucleotides.

17. The 5' amplification primer binding site and the 3' random sequence portion are GTGTAGATCT CGGGTGGTCGC CGATCATTN NNNN; GTGTAGATCT CGGGTGGTCGC CGATCATTN NNNNN; GTGTAGATCT CGGGTGGTCGC CGATCATTN NNNNNN; GTGTAGATCT CGGGTGGTCGC CGATCATTN NNNNNNN; GTGTAGATCT CGGGTGGTCGC CGATCATTN NNNNNNNN; ATCTCGTATG CCGTCTTCTG CTTCNNNNNN; ATCTCGTATG CCGTCTTCTG CTTCNNNNNNN; ATCTCGTATG CCGTCTTCTG CTTCNNNNNNNN; ATCTCGTATG CCGTCTTCTG CTTCNNNNNNNNN; or ATCTCGTATG CCGTCTTCTG CTTCNNNNNNNNNN The method according to any one of claims 1 to 16, comprising.

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