HK40087699A - Nucleic acid sequence analysis from single cells - Google Patents

Description

Single-cell nucleic acid sequence analysis

This application is a divisional application of the invention patent application filed on August 26, 2016, with international application number PCT/US2016/049046, national application number 201680064504.7, and entitled "Single-cell nucleic acid sequence analysis".

Related applications

This application claims priority to U.S. Provisional Application No. 62/211,597, filed August 28, 2015, and is a continuation-in-part of PCT Application No. PCT/US15/28062, filed April 28, 2015, which claims priority to U.S. Provisional Application No. 61/985,983, filed April 29, 2014, and U.S. Provisional Application No. 61/987,433, filed May 1, 2014, each of which is incorporated herein by reference in its entirety.

Government support

This invention was made with government support from the National Institutes of Health (NIH) under grant number MH098977, granted by the Public Health Service (PHS). The government has certain rights in this invention.

Background Technology

Determining the mRNA content of cells or tissues (i.e., "gene expression profiling") provides a method for functional analysis of normal and diseased tissues and organs. Gene expression profiling is typically performed by isolating mRNA from a tissue sample and subjecting that mRNA to microarray hybridization. However, such methods only allow analysis of previously known genes and cannot be used to analyze alternative splicing, promoters, and polyadenylation signals. Furthermore, microarrays have two main drawbacks: they are linked to known genes, and they have limited sensitivity and dynamic range.

Direct sequencing of all or part of the mRNA content in tissues is increasingly being adopted. However, current methods for analyzing cellular mRNA content through direct sequencing rely on analyzing large amounts of mRNA obtained from tissue samples typically containing millions of cells. This means that when analyzing gene expression within a large amount of mRNA, much functional information presented in individual cells is lost or obscured. Furthermore, dynamic processes such as the cell cycle cannot be observed as a whole average. Similarly, only by analyzing these cells individually can the unique cell types in complex tissues (e.g., the brain) be studied.

Typically, suitable cell surface markers are lacking for isolating single cells for research, and even if suitable markers exist, a small number of single cells is insufficient to capture the range of natural variation in gene expression. A method for preparing cDNA libraries is needed that can be used to analyze gene expression in multiple single cells.

sequence list

This application is submitted together with an electronic sequence list. The sequence list is provided as an ASCII file of size 6742 bytes, created on October 12, 2015, and named IP-1388-US_SL.txt. The information in the electronic sequence list is incorporated herein by reference in its entirety.

Summary of the Invention

This article provides methods and compositions for single-cell nucleic acid analysis and/or nucleic acid analysis from single-cell nuclei and organelles. Some methods and compositions can be used for multiplex single-cell gene expression analysis. Some methods and compositions involve the use of droplets and/or beads having unique barcodes such as unique molecular barcodes (UMIs).

In one embodiment, this document provides methods and compositions for single-cell nucleic acid sequence analysis. In some embodiments, the methods and compositions for nucleic acid sequence analysis can be used to prepare sequencing libraries from nucleic acids derived from a single organelle. In some embodiments, the organelle may be the nucleus of a single cell. In some embodiments, the single organelle is obtained from a single cell. Other exemplary organelles include, but are not limited to, mitochondria and ribosomes.

In one embodiment, the method includes releasing a cell nucleus from a cell to provide a plurality of cell nuclei, each cell nucleus originating from a single cell. The cell nuclei are spatially spaced from each other, such that one cell nucleus resides in a spatial compartment. A first strand of cDNA is synthesized from mRNA in each mRNA sample using a first-strand synthesis primer. In some embodiments, the first-strand synthesis primer is an oligo-dT primer that further includes a first amplification primer binding site. In some embodiments, the first-strand synthesis primer is a random compound. In some embodiments, the first-strand synthesis primer is a random compound that 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 random compound, wherein the oligo-dT primer and the random compound each further include a first amplification primer binding site. In some embodiments, the method further includes incorporating a template-converting oligonucleotide primer (TSO primer) together with the mixture of the oligo-dT primer and the random compound, each of the oligo-dT primer and the random compound further including 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 extends beyond the mRNA template and further replicates 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, the first strand extending beyond the mRNA template to include the complementary TSO strand. In some embodiments, double-stranded cDNA is amplified using a first amplification primer and a second amplification primer. In some embodiments, the first amplification primer, the second amplification primer, or both the first amplification primer and the second amplification primer are immobilized on a solid support. Exemplary solid supports include beads, flow cells, and micropores.

In some embodiments, a tagging reaction is performed on double-stranded cDNA to introduce a barcode into the double-stranded cDNA. Exemplary methods of tagging are disclosed in U.S. Patents 9,115,396, 9,080,211, 9,040,256 and U.S. Patent Application Publication 2014/0194324. Each of the foregoing patents is incorporated herein by reference in its entirety. In some embodiments, the barcode can be used to determine sequence contiguity information. In some embodiments, the barcode can be used as a source identifier.

In some embodiments, the method includes incorporating a tag into cDNA to provide a plurality of labeled cDNA samples, wherein the cDNA in each labeled cDNA sample is complementary to mRNA from a single cell. In one embodiment, the tag includes a cell-specific identifier sequence and a unique molecular identifier (UMI) sequence. In some embodiments, a first-strand synthetic primer includes the tag. In some embodiments, a TSO primer includes the tag. In some embodiments, labeled cDNA from the nucleus of a single cell 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 from a single cell. The method includes releasing organelles from a single cell to provide a plurality of organelles. The organelles are spatially spaced from each other, such that one organelle resides in one spatial compartment. A first strand of DNA is synthesized from the microRNA (miRNA), small interfering RNA (siRNA), ribosomal RNA, or mitochondrial DNA using first-strand synthesis primers. In some embodiments, the first-strand synthesis primers are random. In some embodiments, the first-strand synthesis primers are random and also include a primer binding site. In some embodiments, the first-strand synthesis primers are a mixture of first-strand-specific synthesis primers and random. In some embodiments, the first-strand synthesis primers are a mixture of first-strand-specific synthesis primers and random, wherein each of the first-strand-specific synthesis primers and the random further includes a first amplification primer binding site.

In some embodiments, the primer binding to the first primer binding site is an amplification primer. In some embodiments, the method further includes incorporating a template-converting oligonucleotide primer (TSO primer) along with a mixture of a first-strand-specific synthetic primer and a random substance, each of the first-strand-specific synthetic primer and the random substance further comprising a first primer binding site. In some embodiments, the TSO primer further comprises a second primer binding site. In some embodiments, the primer binding to the second primer binding site is an amplification primer. In some embodiments, the first-strand synthetic primer extends beyond a microRNA (miRNA), small interfering RNA (siRNA), ribosomal RNA, or mitochondrial DNA template and further replicates the TSO primer strand. In some embodiments, a 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, the first strand of which extends beyond the template RNA or DNA to include the complementary TSO strand. In some embodiments, double-stranded DNA is amplified using both the first and second primers.

In one embodiment, organelles such as the nucleus, mitochondria, and ribosomes are spatially separated by fluorescence-activated cell sorting (FACS), with each organelle sorted into a spatial compartment, such as a single microwell on a Fluidigm C1 chip. In some embodiments, each organelle is spatially separated in a spatial compartment by being immobilized on a solid surface. For example, immobilization is performed using an antibody that specifically binds to the organelle and is immobilized on a solid surface. In some embodiments, the solid surface is a flow cell or a bead.

In some embodiments, the random primer includes one or more quasi-random primers selected from the group consisting of: AT-enriched random amplification primer sets; random amplification primer sets including AT-enriched 5' ends; variable-length random amplification primer sets, wherein each primer includes a random 3' portion and a degenerate 5' end, the length of which may be proportional to the A/T content of the random 3' portion of the primer; Tm-normalized random primer sets, wherein each primer in the group includes one or more base analogues that can normalize the Tm of each primer to the Tm of the other primers in the primer set; random primer sets, wherein each primer includes a random 3' portion and a constant 5' initiation portion; and random amplification primer sets, wherein each primer includes a random 3' portion and a constant 5' initiation portion, and its The random 3' portion comprises RNA; a random amplification primer set, wherein each primer comprises a random 3' portion and a constant 5' initiation portion, and wherein the random 3' portion comprises at least one non-natural base selected from the group consisting of any combination of nucleic acids and the aforementioned primer set, wherein the nucleic acid is selected from 2'-deoxy-2-thiothymidine (2-thio-dT), 2-aminopurine-2'-deoxyribonucleoside (2-amino-dA), N4-ethyl-2'-deoxycytidine (N4-Et-dC), N4-methyldeoxycytidine (N4-Me-dC), 2'-deoxyinosine, 7-denitroguanine (7-denitro-G), 7-iodo-7-denitroguanine (I-denitro-G), 7-methyl-7-denitroguanine (MecG), and 7-ethyl-7-denitroguanine (EtcG). The aforementioned quasi-random primers are further described in detail herein. In some embodiments described herein, the quasi-random primers are provided in pairs or groups.

One embodiment of this document provides a method for preparing a cDNA library from multiple single cells, the method comprising the steps of: releasing mRNA from each single cell to provide multiple individual mRNA samples, wherein the mRNA in each individual mRNA sample originates from a single cell; synthesizing a first strand of cDNA from the mRNA in each individual mRNA sample using a first-strand synthesis primer, and incorporating a tag into the cDNA to provide multiple labeled cDNA samples, wherein the cDNA in each labeled cDNA sample is complementary to the mRNA from the 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 comprises pooling the labeled cDNA samples; optionally amplifying the pooled cDNA samples to generate a cDNA library comprising double-stranded cDNA; and performing a tagging reaction to simultaneously cleave each cDNA and incorporate an adaptor into each strand of the cDNA, thereby generating multiple labeled cDNA fragments. In some embodiments, a sufficient number of single cells are present to avoid cDNA amplification. The second strand of cDNA was synthesized using template-converting oligonucleotide primers (TSO primers) followed by symmetric Nextera.

In some embodiments, the method further includes amplifying the labeled cDNA fragment to generate an amplified labeled cDNA fragment. In some aspects, the amplification includes adding an additional sequence to the 5' end of the amplification product.

In some respects, the additional sequence includes primer-binding sequences for amplification on a solid support. In other respects, the additional sequence includes additional index sequences.

In some embodiments, the method further includes amplifying the amplified labeled cDNA fragment on a solid support.

In some embodiments, the method further includes sequencing the amplification products on a solid support.

In some respects, the tagging reaction involves contacting double-stranded cDNA with a mixture of transposases that include an adaptor sequence not found in the first-strand synthetic primer.

In some respects, transposase mixtures consist primarily of transposons with one type of adaptor sequence.

In some embodiments, the method further includes sequencing the labeled cDNA fragment.

In some aspects, sequencing includes 3' tag counting.

In some respects, sequencing includes whole transcriptome analysis.

In some aspects, a mixture of random primers is used for first-strand synthesis, the random primers further comprising tags.

In some aspects, the first-strand synthetic primer includes a double-stranded portion. In some embodiments, the first-strand synthetic primer including the double-stranded portion further includes a single-stranded loop at one end. In some aspects, the first-strand synthetic primer includes a region capable of forming a hairpin.

In some respects, this first-strand synthesis primer reduces tandem byproducts compared to single-strand first-strand synthesis primers.

In some respects, the first-strand synthesis primer contains an RNA region.

In some cases, the first-strand synthetic primer is hybridized to a complementary oligonucleotide, thereby forming a double-stranded portion.

This document also provides multiple beads, each of which includes multiple oligonucleotides, each oligonucleotide including: (a) a linker; (b) an amplification primer binding site; (c) an optional unique molecular identifier, which is different for each oligonucleotide; (d) a bead-specific sequence, which is the same on each oligonucleotide of the bead but different on other beads; and (e) a capture sequence for capturing mRNA and initiating reverse transcription.

In some respects, the capture sequence includes oligo-dT.

In some respects, each bead is in a separate droplet, separate from the other beads.

Details of one or more embodiments are set forth in the following drawings and description. Other features, objectives, and advantages will become apparent from this description and the drawings, and from the claims.

Attached Figure Description

Figure 1 is an exemplary schematic diagram illustrating the synthesis of cDNA using the SMARTer method.

Figure 2A is an exemplary schematic diagram illustrating the synthesis of a first strand according to one embodiment, the synthesis of which is initiated by an oligo-dT with an optional molecular barcode (UMI), a sample barcode (BC), an amplification primer binding sequence (V2.A14), and a template transition (TS) primer sequence, followed by template transition, sample pooling, and single-primer PCR. The figures, in order of appearance, disclose SEQ ID NOS 14, 14-15, 14-17, and 16.

Figure 2B is an exemplary schematic diagram illustrating the tagging of pooled amplification products using symmetrical Nextera primers, followed by amplification using different forward (V2.B15) and reverse (V2.A14) PCR primers, and subsequent paired-end sequencing. The figures are disclosed in order of appearance as SEQ ID NOS 17, 16, 17, 16, 17, 16, 17, and 16.

Figure 3A is an exemplary schematic diagram illustrating the synthesis of a first strand according to one embodiment, the synthesis of which is initiated by an oligo-dT with an appended sample barcode (BC), an optional molecular barcode (UMI), an amplification primer binding sequence (V2.A14), and a template transition (TS) primer sequence, followed by template transition, sample pooling, and single-primer PCR. The optional UMI is located at the 5' end of the BC. SEQ ID NOS 14, 14-15, and 14-16 are disclosed in order of appearance.

Figure 3B is an exemplary schematic diagram illustrating the tagging, amplification, and sequencing of pooled amplification products according to one embodiment. The figures disclose SEQ ID NOS 16, 18, 16, 18, 16, 18, 16, 18, 16, 18, and 16, respectively, in order of appearance.

Figure 4A is an exemplary schematic diagram illustrating the synthesis of a first chain according to one embodiment. The figures disclose SEQ ID NOS 14, 14-15, and 14-16 in order of appearance.

Figure 4B is an exemplary schematic diagram illustrating the tagging, amplification, and sequencing of pooled amplification products according to one embodiment. The figures disclose SEQ ID NOS 16, 18, 16, 18, 16, 18, 16, 18, 16, 18, and 16, respectively, in order of appearance.

Figure 5 is an exemplary schematic diagram illustrating a pooling and sequencing method for multiple samples according to one embodiment.

Figure 6 is a table comparing methods for gene expression analysis using 100 pg human brain reference RNA.

Figure 7 is a table comparing methods for high-throughput single-cell gene expression analysis using single cells.

Figure 8 shows a graph comparing transcript coverage using only one transposase adaptor (V2.B15) with that using two transposase adaptors (V2.A14 and V2.B15) with standard/asymmetric tagging.

Figure 9A is a schematic diagram illustrating a whole transcriptome analysis using random material for first-strand synthesis according to one embodiment. The figures are disclosed in order of appearance as SEQ ID NOS 14 and 19, respectively.

Figure 9B is a schematic diagram showing the tandem byproducts that may occur when a random substance is used for the first chain synthesis.

Figure 10 shows various primer designs used to reduce or avoid tandem byproducts.

Figure 11 is a schematic diagram illustrating droplet-based barcode encoding according to one embodiment.

Figure 12 is a schematic diagram illustrating bead-based barcode encoding according to one embodiment. The figures are disclosed in order of appearance as SEQ ID NOS 16, 20, 16 and 21.

Figure 13 is a schematic diagram illustrating the workflow of single-nucleus RNA sequencing.

Figure 14 illustrates the steps involved in synthesizing cDNA from a single cell nucleus.

Figure 15 is a schematic diagram illustrating the synthesis of cDNA using the template conversion SMARTer method. SEQ ID NOS 14, 14, 22, 14, and 22 are disclosed in the order of appearance.

Figure 16 is a schematic diagram of cDNA synthesis in the presence of random primers and oligo-dT primers. SEQ ID NOS 14 and 22 are disclosed in the diagram in the order of appearance.

Figure 17 illustrates the advantages of the SMART-Plus assay method. Figure 17A shows the assay performance of SMART-Plus assays compiled from more than 1000 high-quality individual cell nuclei. Figure 17B shows transcript coverage data.

Figure 18 shows the improved test sensitivity of SMART-Seq Plus compared to the SMART-Seq method.

Detailed Implementation

This article provides methods and compositions for multiplex single-cell gene expression analysis. Some methods and compositions involve the use of droplets and/or beads having unique barcodes such as unique molecular barcodes (UMIs).

Currently, the most commonly used method for single-cell RNA-Seq is based on CLONTECH ^™ SMART-SEQ ^™ technology or its derivatives. In short, oligomeric (dT) primers initiate the first-strand cDNA synthesis reaction. When reverse transcriptase (SMARTSCRIBE ^™ ) reaches the 5' end of the mRNA, the enzyme's terminal transferase activity adds some additional non-template nucleotides to the 3' end of the cDNA. The template-converting oligonucleotide, designed to pair with the elongating bases of this non-template nucleotide, anneals and creates an extended template, allowing RT to continue replicating to the end of the oligonucleotide (Figure 1).

The methods provided herein may include methods for generating labeled cDNA using sample-specific tags, as described in the disclosure of U.S. 2012/0010091, the entire contents of which are incorporated herein by reference. As used herein, the terms single-cell labeled reverse transcription and STRT refer to, for example, the methods disclosed in the incorporated material U.S. 2012/0010091. In some embodiments, in the STRT method, the double-stranded cDNA is not degraded by DNase. Instead, the double-stranded cDNA is tagged with a transposase (e.g., standard Nextera).

The double-stranded cDNA can then be converted into a sequencing library in the following ways: using, for example, NEXTERA ^™ or TRUSEQ ^™ (ILLUMINA, Illumina, Inc.) for whole transcriptome RNA sequencing; or by degradation with enzymes such as DNase I or fragmentation enzymes, followed by adaptor ligation for 5' end sequencing. Both methods have their advantages and disadvantages: the former can only be multiplicative after sample barcoding is introduced during library preparation, while the latter, because barcoding can be introduced during the first-strand synthesis step, can be multiplicative after cDNA synthesis. Therefore, the latter is more advantageous for achieving higher throughput and lower cost per sample. However, the information obtained using the two methods has different applications: the former allows for whole transcriptome sequencing, while the latter only probes gene expression levels.

This article provides a rapid gene expression library preparation method that can be applied at the single-cell input level and allows for high-level sample multiplexing to begin early in the protocol.

In some embodiments, first-strand synthesis is initiated with (anchored) oligomeric dT primers (or possibly randomized primers or a combination of both) appended with a sample barcode (BC), an amplification primer binding site, and optionally a template transition (TS) primer sequence. In some embodiments, the amplification primer binding site is a transposase adaptor sequence, such as the Nextera adaptor sequence V2.A14 or V2.B15. The barcode may precede or follow a molecular barcode (unique molecular identifier or “UMI”) that allows detection of the PCR replica. Template transition occurs when the reverse transcriptase reaches the 5’ end of the mRNA, as described above and in the incorporated material U.S. 2012/0010091. This incorporates the complement of the TS primer sequence into the first cDNA strand. Since the sample barcode has been introduced into the first-strand cDNA, different sample pools can be pooled at this time. The first-strand pool is then converted to double strands and optionally amplified using TS primers in a PCR reaction (Figure 2A). Because of the fact that cDNA contains complementary sequences at both ends, the formation of hairpin structures will lead to the inhibition of the amplification of smaller fragments (such as orthopedic crops).

In some embodiments, as illustrated in Figures 2A and 3A, the first-strand synthesis employs oligo-dT initiation with an appended sample barcode (BC), a copy of the transposase adaptor sequence (“Nextera V2.A14 sequence”), optionally an appended molecular barcode (UMI), and a template transition (TS) primer sequence. Template transition at the 3’ end of the cDNA strand is incorporated into the TS’ primer sequence at the other end of the first strand. This allows for pooling of cDNA from different samples at this stage.

In some embodiments, as illustrated in Figures 2B and 3B, TS oligonucleotides are used to amplify cDNA. This single-primer PCR will suppress small amplicons such as primer dimers. The cDNA pool can be tagged using, for example, a transposase containing only one adaptor sequence instead of the typical two adaptors (“NEXTERA ^™ ”). In the example shown in Figures 2B and 3B, the transposase is loaded with the V2.B15 oligonucleotide. After tagging, PCR performed with p5-V2.A14 and p7-V2.B15 amplification primers preferentially amplifies the 3' end fragment of the cDNA. Fragments generated by the two tagging events (“symmetric fragments”) will be suppressed during PCR and will not generate sequenceable fragments. For example, as shown in Figures 2B and 3B, the amplification products generated from the symmetric fragments will 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 NEXTERA flow pool with P5 and p7 amplification primers. Furthermore, they will be suppressed during PCR due to their complementary ends. In contrast, after amplification, the 3' end fragment will have P5 and P7 primer binding sites and can form a sequenceable cluster on the Yimingda flow cell. Paired end sequencing will produce sample BC and UMI sequences during read 1 and cDNA sequences during read 2.

Therefore, in some embodiments provided herein, instead of sequencing the entire transcript, a digital gene expression assay based on 3' tag counting is performed. The method provided herein offers the ability to barcode cells individually during the first-strand synthesis step. Furthermore, the use of inexpensive oligo-dT primers or random materials to barcode the 3' end of cDNA provides a significant cost advantage. Moreover, the use of random materials for cDNA synthesis is advantageous because it makes the method more similar to total RNA sequencing protocols other than 3' tag counting assays.

Subsequently, pooling, clearing, single-primer cDNA PCR amplification, tagging, and sequencing library preparation 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 cells can be performed in a single tube. Therefore, the methods and compositions described herein are highly suitable for multiplexing. As an example, the method provided herein, illustrated in Figure 5, enables cell-level multiplexing (e.g., 96 samples per 96-well plate), and further, the method enables plate-level multiplexing using uniquely barcoded plates (e.g., tagging to incorporate a barcode identifying the 96-well plate). These methods are suitable for automation and offer significant cost and time savings.

It will be understood that the order of the sample barcode and UMI on the first-strand synthesis primer can be changed. For example, in some embodiments, the sample barcode (BC) is located at the 3' of the UMI. In some embodiments, the sample barcode (BC) is located at the 5' of the UMI. In some embodiments, the sample barcode (BC) is directly linked to the UMI. In some embodiments, the sample barcode (BC) is separated from the UMI by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 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 more nucleotides.

In some embodiments, tagging is performed using a transposase mixture containing an adaptor sequence not found in the first-strand synthetic primers. This produces tagged fragments with different adaptor sequences compared to those incorporated into the first-strand synthetic primers. For example, in some embodiments, the transposase mixture exclusively comprises transposases having one type of adaptor sequence. Fragments generated by this type of mixture are referred to herein as “symmetric fragments” and do not produce sequenceable clusters on a flow cell. In some embodiments, the transposase mixture may contain a number of sequences incorporated into the first-strand synthetic primers, said number being low enough to still allow all or substantially all of the 3’ cDNA fragment to be amplified and sequenced. For example, the adaptor sequence in the transposase mixture may include 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 synthetic primer. It will be understood that in cases where two transposase adaptors are typically used, either of the two adaptors may be incorporated into the first-strand synthetic primer, and the other adaptor may be used during the tagging event. For example, in the exemplary embodiments illustrated in Figures 3A, 3B, 4A, and 4B, in the case where an adaptor sequence is incorporated into the first-strand synthetic primer, that adaptor is no longer used in the tagging mixture. Figures 3A and 3B illustrate one embodiment in which, during the tagging step, adaptor V2.A14 is incorporated into the first-strand synthetic primer, and adaptor V2.B15 is used as the transposon adaptor. Figures 4A and 4B illustrate an alternative embodiment in which, during the tagging step, adaptor V2.B15 is incorporated into the first-strand synthetic primer, and adaptor V2.A14 is used as the transposon adaptor. In both examples, the resulting tagged fragments fall into two categories: symmetric fragments (which cannot be amplified and/or sequenced), and asymmetric fragments that can be amplified using a set of primers V2.A14 and V2.B15.

In some embodiments, the number of single cells that can be multiplexed can be significantly increased by incorporating an index into the transposon adaptor sequence. As illustrated in Figure 5, each individual cell can be identified using a cell-specific barcode, and each group of cells (e.g., a plate of 96 cells) can be contacted with a tagged mixture having a plate-specific barcode incorporated into the transposon adaptor sequence. In the example shown in Figure 5, cDNA from each of the 96 cells on the plate is pooled prior to tagging, and then the tagged sample is pooled for multiplexed sequencing. It will be understood that any number of cells can be pooled prior to tagging, and the use of a 96-well plate is just one of many embodiments. For example, a set of numbers can be represented as 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, 6. Cell pooling for tagging is possible in quantities of 0, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, or more than 100, 200, 300, 400, 500, 600, 700, 800, 900, or more than 1000, as well as any intermediate number of cells. First-chain synthesis can be performed in any single-well or multi-well container, such as multi-well plates, chips, microfluidic devices, emulsions, bead mixtures, or any other suitable form for multiplexing multiple cells.

As shown in Figure 7 (single cell), when gene expression was analyzed using either symmetric tagging (Nextera form V2.B15) compared to asymmetric tagging (Nextera form V2.B15A/2.A14), a considerable number of genes were detected and other metrics were obtained. Figure 8 shows a comparison of tagging using two transposase adaptors (V2.A14 and V2.B15). When only one transposase adaptor (V2.B15) was used for tagging, transcript coverage was almost entirely biased towards the 3' end of the transcript.

In some embodiments, sampled and UMI barcoded samples can be achieved by separating individual cells into droplets. In some embodiments, droplets are separated from each other in an emulsion. In some embodiments, droplet actuators are used to form and/or manipulate droplets. In a particular embodiment, one or more droplets comprise a different set of first-strand synthetic primers containing barcodes. In some embodiments, each droplet comprises a large number of first-strand synthetic primers, each having the same sequence including the same barcode, and the barcode from one droplet differs from the barcode from another droplet, while the remainder of the first-strand synthetic primers remains the same between these droplets. Thus, in these embodiments, the barcode acts as an identifier for the droplet and the single cell contained within it. In a particular embodiment, one or more droplets comprise a different set of first-strand synthetic primers containing UMIs. Thus, each individual cell lysed in each droplet will be identified by the barcode in each droplet. As illustrated in Figure 11, droplet-based barcoding can be performed by fusing droplets containing single cells with other droplets that include a unique set of barcodes. This format allows for additional multiplexing beyond that available in the multi-wall format. First-strand synthesis and template conversion occur within each individual droplet. In some embodiments, two or more droplets may be fused prior to PCR. Additionally or alternatively, in some embodiments, droplets may be fused prior to tagging. Additionally or alternatively, in some embodiments, droplets may be fused after PCR and prior to tagging. For example, in some embodiments, labeled cDNA may be fused after first-strand synthesis in individual droplets, thereby pooling the cDNA.

Similarly, in some embodiments, sampling and UMI barcoding can be performed by separating individual cells with beads containing primers for first-chain synthesis and/or barcode markings. In some embodiments, the beads are separated into droplets in an emulsion. In some embodiments, droplet actuators are used to separate and manipulate the beads. As shown in Figure 12, bead-based barcoding can be performed by creating a set of beads, each bead having one or more unique barcodes.

Whole transcriptome sequencing

In some embodiments, the methods provided herein can be used for whole transcriptome sequencing. In such embodiments, a random agent is used to extend first-strand synthesis. As shown in Figure 9A, random priming extends a window of sequenceable fragments to any point along the transcript length where the random agent hybridizes and initiates first-strand synthesis. The random agent may include the same combination of sample barcodes and unique molecular identifiers, such as transposon adaptor sequences and/or template conversion (TS) primers, among other adaptor and primer binding sites. Template conversion and second-strand synthesis can be performed as described above for oligo-dT-primed cDNA synthesis. The resulting double-stranded cDNA can then be subjected to a tagging reaction as described above. The difference between using a random agent and oligo-dT primers is that a complete or substantially complete transcript sequence can be obtained, rather than the 3' portion of the transcript. As shown in Figure 6 (100 pg RNA), a considerably larger number of genes were detected and additional metrics were obtained when analyzing gene expression using either oligo-dT, compared to a mixture of oligo-dT and random agent used for first-strand synthesis.

One type of byproduct that can occur when a random agent is used to initiate first-strand synthesis is a tandem byproduct. Specifically, in some cases, as shown in Figure 9B, the random agent can hybridize to other random primers, thereby winning the competition for annealing with the RNA transcript. This allows the resulting cDNA product to undergo a cascade of random initiation and template switching events. This cascade can lead to the formation of tandem byproducts, which can depend on the presence of template-switching oligonucleotides.

To reduce and/or minimize the formation of such byproducts, this document provides various primer designs that reduce the likelihood of byproduct formation. Exemplary designs are illustrated in Figure 10, but it will be understood that the scope of primer compositions illustrated herein extends beyond the examples illustrated in the figures. In one embodiment, primers may be configured to form hairpins to prevent or minimize random hybridization to any portion of the barcode, adaptor, or amplification primer binding region. Thus, the double-stranded portion formed within the primer itself can compete for victory in random hybridization. In some embodiments, the double-stranded portion of the hairpin includes a chimeric end (ME) sequence of a transposase adaptor. Alternatively or additionally, in some embodiments, the double-stranded portion may include some or all of the transposase adaptor sequence that crosses the chimeric end sequence. In some embodiments, the adaptor sequence may be completely replaced by a short RNA sequence, thereby reducing primer length and minimizing the likelihood of random hybridization to the primer. In some embodiments, a complement to a short RNA sequence is provided at or near the 5' end of the primer to form the hairpin. In some embodiments, a double-stranded region is formed by annealing an oligonucleotide complementary to the adapter and/or primer portion, thereby preventing or minimizing random material initiation to any portion of the barcode, adapter, or amplification primer binding region, and thereby reducing or avoiding tandem byproducts.

random objects or random primers

As used herein, the terms “random object” and “random primer” are used interchangeably. The term random object means a random primer that can present fourfold degeneracy at each position.

In some embodiments, the random material comprises any nucleic acid primers known in the art having various random sequence lengths. For example, the random material may comprise a random sequence of 3, 4, 5, 6, 7, 8, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleotides in length. In some embodiments, multiple random primers may comprise random materials of different lengths. In some embodiments, multiple random materials may comprise random materials of equal length. In some embodiments, multiple random materials may comprise a random sequence of about 5 to about 18 nucleotides in length. In some embodiments, multiple random materials comprise random hexamers. Random primers, specifically random hexamers, are commercially available and widely used in amplification reactions, such as multiple substitution amplification (MDA), for example, the REPLI-g whole genome amplification kit (QIAGEN, Valencia, CA). It is understood that any random material of suitable length may be used in the methods and compositions described herein.

An exemplary random object sequence including a 3'-random sequence portion and a 5'-determined sequence portion is shown below:

GTGTAGATCT CGGTGGTCGC CGTATCATTN NNNN(SEQ ID NO:6)

GTGTAGATCT CGGTGGTCGC CGTATCATTN NNNNN(SEQ ID NO:4)

GTGTAGATCT CGGTGGTCGC CGTATCATTN NNNNNN(SEQ ID NO:7)

GTGTAGATCT CGGTGGTCGC CGTATCATTN NNNNNNN(SEQ ID NO:8)

GTGTAGATCT CGGTGGTCGC CGTATCATTN NNNNNNNN(SEQ ID NO:9)

ATCTCGTATG CCGTCTTCTG CTTGNNNNN(SEQ ID NO:10)

ATCTCGTATG CCGTCTTCTG CTTGNNNNNN(SEQ ID NO:5)

ATCTCGTATG CCGTCTTCTG CTTGNNNNNNN(SEQ ID NO:11)

ATCTCGTATG CCGTCTTCTG CTTGNNNNNNNN(SEQ ID NO:12)

ATCTCGTATG CCGTCTTCTG CTTGNNNNNNNNN(SEQ ID NO:13)

As used herein, the term "SMART-Seq Plus" refers to a method for preparing cDNA from RNA using a first-strand synthetic primer including a first amplification primer binding site, a random substance including the first amplification primer binding site, and an oligonucleotide-converting oligonucleotide that is partially complementary to the first strand of cDNA and includes a second amplification primer binding site. In some embodiments, the first-strand synthetic primer includes an oligomeric (dT) portion.

Barcodes and UMI

As used herein, the term "barcode" or "BC" refers to a nucleic acid tag that can be used to identify a sample or source of nucleic acid material. Therefore, in cases where nucleic acid samples are derived from multiple sources, different nucleic acid tags can be used to label the nucleic acids in each nucleic acid sample, allowing the source of the sample to be identified. Barcodes (also commonly referred to as indexes, tags, etc.) are well known to those skilled in the art. As is known in the art and illustrated in U.S. Patent No. 8,053,192 and PCT Publication WO 05/068656, any suitable barcode or barcode group can be used, and the entirety of the references are incorporated herein by reference. Single-cell barcoding can be performed, for example, as described in the disclosure of U.S. 2013/0274117, and the entirety of the references are incorporated herein by reference.

Nucleic acids from more than one source can be incorporated into the variable tag sequence. The tag sequence can be up to 100 nucleotides long (base pairs if a double-stranded molecule is involved), preferably 1 to 10 nucleotides long, most preferably 4, 5, or 6 nucleotides long, and includes combinations of nucleotides. For example, in one embodiment, if six base pairs are chosen to form the tag and four different nucleotide arrangements are used, a total of 4096 nucleic acid anchors (e.g., hairpins) can be formed, each with a unique 6-base tag.

As used herein, the terms UMI, unique identifier, and unique molecular identifier refer to a unique nucleic acid sequence attached to each of a plurality of nucleic acid molecules. When incorporated into a nucleic acid molecule, such as during first-strand cDNA synthesis, a UMI can be used to correct for subsequent amplification bias by directly counting the unique molecular identifiers (UMIs) sequenced after amplification. UMIs can be designed, incorporated, and applied as is known in the art, for example, by way of the publications of WO 2012/142213, Nat. Methods (2014) 11:163-166 by Islam et al., and Nat. Methods (2012) 9:72-74 by Kivioja, T. et al., each of which is incorporated herein by reference in its entirety.

Tagging

As used herein, the term "tag" refers to the modification of DNA by means of a transposon complex comprising a transposase, which is complexed with an adaptor comprising a transposon terminal sequence. Tagging results in the fragmentation of DNA and the simultaneous attachment of the adaptor to the 5' ends of both strands of the double-stranded fragment. Following a purification step to remove the transposase, additional sequences may be added to the ends of the adapted fragment, for example by PCR, ligation, or any other suitable method known to those skilled in the art.

The method of the present invention can use any transposase that accepts transposase terminal sequences and fragments the target nucleic acid, wherein the transposase is attached to the transferred end rather than the untransferred end. A “transposome” consists of at least a transposase and a transposase recognition site. In some such systems referred to as “transpossomes,” the transposase can form a functional complex together with a transposon recognition site capable of catalyzing a transposition reaction. In a process sometimes referred to as “tagmentation,” a transposase or integrase can bind to the transposase recognition site and insert the transposase recognition site into the target nucleic acid. In some such insertion events, one strand of the transposase recognition site can be transferred into the target nucleic acid.

In standard sample preparation methods, each template contains an adaptor located at either end of the insert, and multiple steps are typically required to modify the DNA or RNA and purify the desired product of the modification reaction. These steps are performed in solution before adding the adapted fragments to the flow cell, where they are coupled to the surface via a primer extension reaction that copies the hybridized fragments to the primer ends covalently attached to the surface. These "inoculated" templates then generate monoclonal clusters of the copied template through several amplification cycles.

In preparing solutions for cluster formation and sequencing, the number of steps required to convert DNA into adaptor-modified templates can be minimized by using transposase-mediated fragmentation and labeling.

In some embodiments, transposon-based techniques can be used to fragment DNA, as illustrated in the workflow of the Nextera ^™ DNA Sample Preparation Kit (ILLUMINA), where genomic DNA can be fragmented by engineered transposons that simultaneously fragment and label (“tagged” the input DNA, thereby creating a population of fragmented nucleic acid molecules that include unique adaptor sequences at the ends of the fragments.

Some embodiments may include the use of an overactive Tn5 transposase and a Tn5-type transposase recognition site (Goryshin and Reznikoff, *Journal of Biochemistry*, 273:7367 (1998)), or a MuA transposase and a Mu transposase recognition site including R1 and R2 terminal sequences (Mizuuchi, K., *Cell*, 35:785, 1983; Savilahti, H et al., *EMBO J.*, 14:4893, 1995). Exemplary transposase recognition sites form conjugates of overactive Tn5 transposases (e.g., EZ-Tn5 ^™ transposase, Epicentre Biotechnology, Madison, Wisconsin).

Further examples of transposable systems that may be used in some of the embodiments provided herein include Staphylococcus aureus Tn552 (Colegio et al., *Journal of Bacteriology*, 183:2384-8, 2001; Kirby C et al., *Molecular Microbiology*, 43:173-86, 2002), tylosin (Tyl) (Devine and Boeke, *Nucleic Acids Res.*, 22:3765-72, 1994 and International Publication WO 95/23875), and transposon Tn7 (C Craig, N L, Science, 271:1512, 1996; Craig, N L, a 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 DJ) et al., *European Journal of Molecular Biology* (EMBO J.), 15:5470-9, 1996; Tel (Plasterk R H, *Curr. Topics Microbiol. Immunol.*), 204:125-43, 1996); P factor (Gloor, G B, *Methods of Molecular Biology* (Mol. Biol.), 260:97-114, 2004); Tn3 (Ichikawa and Ohtsubo, *Journal of Biochemistry* (J Biol. Chem.), 265: 18829-32, 1990), bacterial insertion sequences (Ohtsubo and Sekine, Current Issues in Microbiology and Immunology, 204:1-26, 1996), retroviruses (Brown et al., Proc Natl Acad Sci USA, 86:2525-9, 1989), and yeast retrotransposons (Boeke and Corces, Annu Rev Microbiol, 43:403-34, 1989). Further examples include engineered forms of IS5, Tnl0, Tn903, IS911, and transposase family enzymes (Zhang et al., (2009) PLoS Genet. 5: el000689. E-published on October 16, 2009; Wilson C. et al. (2007) Journal of Microbiology Methods 71: 332-5).

In brief, a “transposition reaction” is a reaction in which one or more transposons are inserted into a target nucleic acid at random or nearly random sites. Essential components of a transposition reaction are a transposase and a DNA oligonucleotide exhibiting the transposon nucleotide sequence, including the transferred transposon sequence and its constituent parts (i.e., the non-transferred transposon terminal sequence), as well as other components required to form a functional transposon or transposome complex. The DNA oligonucleotide may further include desired or anticipated additional sequences (e.g., adaptor sequences or primer sequences). Briefly, in vitro transposition can be initiated by contacting the transposome complex with the target DNA. Exemplary transposition procedures and systems readily adaptable for use with the transposases of this disclosure are described in, for example, WO 10/048605; US 2012/0301925; US 2013/0143774, each of which is incorporated herein by reference in its entirety.

The adaptor added to the 5' and/or 3' end of a nucleic acid may include a universal sequence. A universal sequence is a nucleotide sequence region common (i.e., shared) by two or more nucleic acid molecules. Optionally, the two or more nucleic acid molecules may also have sequence-dissimilar regions. Thus, for example, the 5' adaptor may include the same or universal nucleic acid sequence, and the 3' adaptor may include the same or universal sequence. A universal sequence that can be present in different members of multiple nucleic acid molecules allows for the replication or amplification of multiple different sequences using a single universal primer complementary to the universal sequence. Some universal primer sequences used in the examples provided herein include the V2.A14 and V2.B15 Nextera ^™ sequences. However, it will be readily understood that any suitable adaptor sequence may be used in the methods and compositions provided herein. For example, the Tn5 chimeric end sequence A14 (Tn5MEA) and/or the Tn5 chimeric end sequence B15 (Tn5MEB) (including the complementary nontransfer sequence (NTS) set forth below) may 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)

The barcode and UMI in the droplet

In some embodiments, primers having a sample barcode may be in solution. Additionally or alternatively, primers having a UMI sequence may be in solution. For example, the solid support may be one or more droplets. Thus, in some embodiments, multiple droplets may be present, each of which has a unique sample barcode and/or UMI sequence, each of which is unique to the molecule. Therefore, those skilled in the art will understand that in some embodiments, the barcode is unique to the droplet and the UMI is unique to the molecule, such that the UMI is repeated many times in a batch of droplets. In some embodiments, individual cells are contacted with droplets having a set of unique sample barcodes and/or UMI sequences to identify the individual cells. In some embodiments, lysates from individual cells are contacted with droplets having a set of unique sample barcodes and/or UMI sequences to identify the individual cell lysates. In some embodiments, purified nucleic acids from individual cells are contacted with droplets having a set of unique sample barcodes and/or UMI sequences to identify the purified nucleic acids from the individual cells.

Any suitable system for forming and manipulating droplets can be used in the embodiments provided herein, wherein each of the plurality of droplets has a set of unique sample barcodes and/or UMI sequences. For example, a droplet actuator can be used.

"Droplet actuator" refers to a device for manipulating droplets. Examples of droplet actuators can be found in U.S. Patent No. 6,911,132, issued June 28, 2005, by Pamula et al., entitled "Apparatus for Manipulating Droplets by Electrowetting-Based Techniques"; and U.S. Patent Publication No. 20060194331, published August 31, 2006, by Pamula et al., entitled "Apparatuses and Methods for Manipulating Droplets on a Printed Circuit". The following patents are cited: Pollack et al.'s International Patent Publication No. WO/2007/120241, published October 25, 2007, entitled "Droplet-Based Biochemistry";Shenderov's U.S. Patent No. 6,773,566, granted August 10, 2004, entitled "Electrostatic Actuators for Microfluidics and Methods for Using Same";Shenderov's U.S. Patent No. 6,565,727, granted May 20, 2003, entitled "Actuators for Microfluidics Without Moving Parts"; and Kim et al.'s U.S. Patent Publication No. 20030205632, published November 6, 2003, entitled "Electrowetting-driven Micropumping". Micropumping); U.S. Patent Publication No. 20060164490, published by Kim et al. on July 27, 2006, entitled "Method and Apparatus for Promoting the Complete Transfer of Liquid Drops from a Nozzle"; U.S. Patent Publication No. 20070023292, published by Kim et al. on February 1, 2007, entitled "Small Object Moving on Printed Circuit Board"; U.S. Patent Publication No. 20090283407, published by Shah et al. on November 19, 2009, entitled "Method for Using Magnetic Particles in Droplet Microfluidics". The text includes several patents and related information, such as U.S. Patent Publication No. 20100096266 (published April 22, 2010 by Kim et al.), titled "Method and Apparatus for Real-time Feedback Control of Electrical Manipulation of Droplets on Chip"; U.S. Patent No. 7,547,380 (granted June 16, 2009 by Velev), titled "Droplet Transportation Devices and Methods Having a Fluid Surface"; and U.S. Patent No. 7,163,612 (granted January 16, 2007 by Sterling et al.), titled "Method, Apparatus and Article for Microfluidic Control via Electrowetting, for Chemical, Biochemical and Biological Assays and the...". Like); US Patent No. 7,641,779, issued January 5, 2010, by Becker et al., entitled "Method and Apparatus for Programmable Fluidic Processing"; US Patent No. 6,977,033, issued December 20, 2005, by Becker et al., entitled "Method and Apparatus for Programmable Fluidic Processing"; US Patent No. 7,328,979, issued February 12, 2008, by Deere et al., entitled "System for Manipulation of a Body of Fluid"; US Patent No. 20060039823, published February 23, 2006, by Yamakaawa et al., entitled "Chemical Analysis Apparatus". Apparatus); Wu's U.S. Patent Publication No. 20110048951, published March 3, 2011, entitled "Digital Microfluidics Based Apparatus for Heat-exchanging Chemical Processes"; Fouillet et al.'s U.S. Patent Publication No. 20090192044, published July 30, 2009, entitled "Electrode Addressing Method"; Fouillet et al.'s U.S. Patent No. 7,052,244, granted May 30, 2006, entitled "Device for Displacement of Small Liquid Volumes Along a Micro-catenary Line by Electrostatic Force". The patents published on May 29, 2008, by Marchand et al., entitled "Droplet Microreactor"; on December 31, 2009, by Adachi et al., entitled "Liquid Transfer Device"; on August 18, 2005, by Roux et al., entitled "Device for Controlling the Displacement of a Drop Between Two or Several Solid Substrates"; and on December 31, 2009, by Dhindsa et al., entitled "Virtual Electrowetting Channels: Electronic Liquid Transport with Continuous Channel Functionality," published in *Lab on a Chip*. Chip), 10:832-836 (2010), the entire disclosure of which is incorporated herein by reference. Some droplet actuators will include one or more substrates with droplet operating gaps arranged between the substrates, and a plurality of electrodes associated with (e.g., stacked on, attached to, and/or embedded in) the one or more substrates and arranged to perform one or more droplet operations. For example, some droplet actuators include a base (or bottom) substrate, droplet operating electrodes associated with the substrates, one or more dielectric layers on top of the substrates and/or electrodes, and optionally one or more hydrophobic layers on top of the substrate, dielectric layers, and/or electrodes forming the droplet operating surface. A top substrate may also be provided, separated from the droplet operating surface by a gap commonly referred to as the droplet operating gap. Various electrode arrangements on the top and/or bottom substrates have been discussed in the patents and applications cited above, and certain novel electrode arrangements are discussed in the description of this disclosure. During droplet operation, it is preferable that the droplet maintains continuous or frequent contact with the ground electrode or reference electrode. The ground electrode or reference electrode may be associated with a top substrate facing the gap and a bottom substrate facing the gap. In cases where electrodes are provided on two substrates, electrical contacts for coupling the electrodes to droplet actuator instruments used for controlling or monitoring the electrodes may be associated with one or both plates. In some cases, electrodes on one substrate are electrically coupled to the other substrates such that only one substrate is in contact with the droplet actuator. In one embodiment, a conductive material (e.g., an epoxy resin, such as MASTER BOND ^™ polymer system EP79, available from Master Bond, Inc., Hackensack, NJ) provides an electrical connection between an electrode on one substrate and an electrical path on another substrate; for example, a ground electrode on the top substrate may be coupled to an electrical path on the bottom substrate using such a conductive material. In cases using multiple substrates, spacers may be provided between these substrates to define the height of the gap between the substrates and to define the dispensing reservoir of the in-line actuator. The height of the spacer may be, for example, at least about 5 μm, 100 μm, 200 μm, 250 μm, 275 μm or greater. Alternatively or additionally, the height of the spacer may be at most about 600 μm, 400 μm, 350 μm, 300 μm or less. The spacer may be formed, for example, from a layer forming a protrusion of a top or bottom substrate and/or from a material inserted between the top and bottom substrates. One or more openings may be provided in one or more substrates to form fluid passages through which liquid can be delivered into the droplet operation gap. In some cases, one or more openings may be aligned for interaction with one or more electrodes, for example, aligned such that liquid flowing through the openings will be sufficiently close to one or more droplet operation electrodes to allow droplet operation using liquid via the droplet operation electrodes. In some cases, the substrate (or bottom) and the top substrate may be formed as a single integral component. One or more reference electrodes may be provided in the substrate (or bottom) and/or the top substrate and/or in the gap. Examples of reference electrode arrangements are provided in the aforementioned cited patents and patent applications. In various embodiments, the manipulation of the droplets by droplet actuators can be electrode-mediated, for example, electrowetting-mediated, dielectrophoresis-mediated, or Coulomb force-mediated. Examples of other techniques that can be used in the droplet actuators of this disclosure for controlling droplet operation include devices that induce hydrodynamic fluid pressure, such as those that operate based on the following principles: mechanical principles (e.g., external injection pumps, pneumatic diaphragm pumps, vibrating diaphragm pumps, vacuum devices, centrifugal force, piezoelectric/ultrasonic pumps, and acoustic force); electrical or magnetic principles (e.g., electroosmosis, electric pumps, ferrohydrodynamic plugs, electrofluidic pumps, attractive or repulsive forces using magnetic forces, and magnetohydrodynamic pumps); thermodynamic principles (e.g., volume expansion induced by bubble formation/phase change); other types of surface wetting principles (e.g., electrowetting and photowetting, and chemically, thermally, structurally, and radioactively induced surface tension gradients); gravity; surface tension (e.g., capillary action); electrostatic forces (e.g., electroosmosis); centrifugal flow (a substrate disposed on an optical disc and rotating); magnetic forces (e.g., flow induced by oscillating ions); magnetohydrodynamics; and vacuum or pressure difference. In some embodiments, combinations of two or more of the foregoing techniques can be used to perform droplet operation in the droplet actuators of this disclosure. Similarly, one or more of the foregoing methods can be used, for example, to deliver liquid into the droplet operating gap from a reservoir of another device or from an external reservoir of the droplet actuator (e.g., a reservoir associated with the droplet actuator substrate and the flow path from the reservoir into the droplet operating gap). The droplet operating surfaces of certain droplet actuators of this disclosure may be made of hydrophobic materials or may be coated or treated to make them hydrophobic. For example, in some cases, a portion or all of the droplet operating surface may be derivatized using low surface energy materials or chemicals, for example, by deposition or in-situ synthesis using compounds such as polyfluorinated or perfluorinated compounds in solution form or polymerizable monomers. Examples include Teflon AF (available from DuPont, Wilmington, Delaware), members of the cytop materials family, coatings from families of hydrophobic and superhydrophobic coatings (available from Cytonix, Beltsville, Maryland), silane coatings, fluorosilane coatings, hydrophobic phosphate derivatives (e.g., those marketed by Aculon), and NOVEC ^™ electronic coatings (available from 3M, St. Paul, Minnesota), other fluorinated monomers for plasma-enhanced chemical vapor deposition (PECVD), and organosiloxanes for PECVD (e.g., SiOC). In some cases, the droplet operating surface may include a hydrophobic coating having a thickness ranging from about 10 nm to about 1,000 nm. Furthermore, in some embodiments, the top substrate of the droplet actuator comprises a conductive organic polymer, which is then coated with a hydrophobic coating or otherwise treated to make the droplet operating surface hydrophobic. For example, the conductive organic polymer deposited on a plastic substrate can be poly(3,4-ethylenedioxythiophene)poly(styrene sulfonic acid) (PEDOT:PSS). Other examples of conductive organic polymers and alternative conductive layers are described in International Patent Publication No. WO/2011/002957 entitled "Droplet Actuator Devices and Methods" published by Pollack et al. on January 6, 2011, the entire disclosure of which is incorporated herein by reference. One or two substrates can be fabricated using printed circuit boards (PCBs), glass, indium tin oxide (ITO)-coated glass, and/or semiconductor materials as substrates. 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 greater. Alternatively or additionally, the thickness may be at most about 200 nm, 150 nm, 125 nm, or less. In some cases, the top and/or bottom substrates comprise a PCB substrate coated with a dielectric (such as a polyimide dielectric), and in some cases, it may be coated or otherwise treated to make the droplet-operating surface hydrophobic. When the substrate comprises a PCB, the following materials are examples of suitable materials: MITSUI ^™ BN-300 (available from MITSUI Chemicals America, Inc., San Jose, California); ARLON ^™ 11N (available from Arlon, Inc., Santa Ana, California); N4000-6 and N5000-30/32 (available from Park Electrochemical Corp., Melville, New York); ISOLA ^™ FR406 (available from Isola Group, Chandler, Arizona). The following materials are available: (Group) (especially IS620); fluoropolymers (suitable for fluorescence detection due to their low background fluorescence); polyimide family; polyesters; polyethylene naphthalate; polycarbonate; polyether ether ketone; liquid crystal polymers; cyclic olefin copolymers (COC); cyclic olefin polymers (COP); aromatic polyamides; nonwoven aromatic polyamide reinforced materials (available from DuPont, Wilmington, Delaware); branded fibers (available from DuPont, Wilmington, Delaware); and paper. Many materials are also suitable as dielectric components of substrates. Examples include: vapor-deposited dielectrics such as PARYLENE ^™ C (especially on glass), PARYLENE ^™ N, and PARYLENE ^™ HT (for high temperatures, ~300°C) (available from Parylene Coating Services, Inc., Katie, Texas); AF coatings; cytoplasm; solder masks such as liquid photosensitive solder masks (e.g., on PCBs), like the TAIYO ^™ PSR4000 series, TAIYO ^™ PSR and AUS series (available from Taiyo America, Inc., Carson City, Nevada) (good thermal properties for applications involving thermal control) and PROBIMER ^™ 8165 (good thermal properties for applications involving thermal control) (available from Huntsman Advanced Materials Americas, Los Angeles, California). (Available from Americas Inc.); dry film solder masks, such as those in the dry film solder mask system (available from DuPont, Wilmington, Delaware); film dielectrics, such as polyimide films (e.g., polyimide films available from DuPont, Wilmington, Delaware), polyethylene and fluoropolymers (e.g., FEP), polytetrafluoroethylene; polyesters; polyethylene naphthalate; cyclic olefin copolymers (COC); cyclic olefin polymers (COP); any other PCB substrate materials listed above; black matrix resins; polypropylene; and black flexible circuit materials, such as DuPont ^™ HXC and DuPont ^™ MBC (available from DuPont, Wilmington, Delaware). The droplet delivery voltage and frequency can be selected to suit the performance of the reagents used in a specific assay. Design parameters can be varied, such as the number and placement of inline actuator reservoirs, the number of independent electrode connectors, the size (volume) of different reservoirs, the placement of the magnet/bead washing zone, electrode dimensions, inter-electrode spacing, and the gap height (between the top and bottom substrates) can be varied for use with specific reagents, assays, droplet volumes, etc. In some cases, the substrate of this disclosure can be derivatized using low surface energy materials or chemicals, e.g. For example, in-situ synthesis of polyfluorinated or perfluorinated compounds or polymerizable monomers using deposition or solution forms. Examples include AF coatings and paints for impregnation or spraying, other fluorinated monomers for plasma-enhanced chemical vapor deposition (PECVD), and organosiloxanes (e.g., SiOC) for PECVD. Furthermore, in some cases, substances can be used to coat portions or the entire surface of droplet-operated surfaces to reduce background noise, such as background fluorescence from the PCB substrate. For example, noise-reducing coatings may include black matrix resins, such as those available from Toray Industries, Inc. The black matrix resin obtained by industries, Inc. The electrodes of the droplet actuator are typically controlled by a controller or processor, which is itself provided as part of the system and may include processing functions as well as data and software storage and input and output capabilities. Reagents may be provided on the droplet actuator, in the droplet operating gap, or in a reservoir fluidly coupled to the droplet operating gap. In the droplet operating gap or in a reservoir fluidly coupled to the droplet operating gap, the reagent may be in liquid form (e.g., droplets) or may be provided in a reconfigurable form. Typically, reconfigurable reagents may be combined with a liquid for reconfiguration. Examples of reconfigurable reagents suitable for use with the methods and apparatus described herein include those described in U.S. Patent No. 7,727,466, issued June 1, 2010, by Meathrel et al., entitled “Disintegratable Films for Diagnostic Devices,” the entire disclosure of which is incorporated herein by reference.

The term "activate" in relation to one or more electrodes means a change in the electrical state of one or more electrodes that causes droplet operation in the presence of the droplet. Electrode activation can be accomplished using alternating current (AC) or direct current (DC). Any suitable voltage can be used. For example, voltages greater than about 150V, or greater than about 200V, or greater than about 250V, or from about 275V to about 1000V, or about 300V can be used to activate the electrodes. In the case of using an AC signal, any suitable frequency can be employed. For example, AC signals with frequencies from about 1Hz to about 10MHz, or from about 10Hz to about 60Hz, or from about 20Hz to about 40Hz, or about 30Hz can be used to activate the electrodes.

The term "bead" in relation to beads on a droplet actuator means any bead or particle capable of interacting with a droplet on or near the droplet actuator. Beads can be any of a variety of shapes, such as spherical, substantially spherical, egg-shaped, disc-shaped, cubic, amorphous, and other three-dimensional shapes. Beads, for example, can be subjected to droplet operation within a droplet on the droplet actuator or configured relative to the droplet actuator in a manner that allows a droplet on the droplet actuator to contact and/or exit the droplet actuator with a bead on the droplet actuator. Beads can be provided in a droplet, in a droplet operation gap, or on a droplet operation surface. Beads can be provided in a reservoir located outside the droplet operation gap or away from the droplet operation surface, and the reservoir can be associated with a flow path that allows a droplet including the bead to be carried into the droplet operation gap or into contact with the droplet operation surface. Beads can be manufactured using a variety of materials, including, for example, resins and polymers. The beads can have any suitable size, including, for example, microbeads, microparticles, nanobeads, and nanoparticles. In some cases, the beads are magnetically responsive; in others, they are not significantly magnetically responsive. For magnetically responsive beads, the magnetically responsive material may constitute substantially all of the bead, a part of the bead, or only one component of the bead. In addition, the remaining portion of the bead may include polymeric materials, coatings, and portions that allow for the attachment of assay reagents. Examples of suitable beads include 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 fluorescent microspheres and nanospheres, pigment-stained microparticles and nanoparticles, magnetic microparticles and nanoparticles, superparamagnetic microparticles and nanoparticles (e.g., DYNABEADS particles, available from the Invitrogen Group in Carlsbad, California), fluorescent microparticles and nanoparticles, coated magnetic microparticles and nanoparticles, ferromagnetic microparticles and nanoparticles, and coated iron microparticles. Magnetic microparticles and nanoparticles, as well as those described in the following literature: U.S. Patent Publication No. 20050260686, published November 24, 2005, by Watkins et al., entitled "Multiplex Flow Assays Preferably with Magnetic Particles as Solid Phase"; and U.S. Patent Publication No. 20030132538, published July 17, 2003, by Chandler, entitled "Encapsulation of Discrete Quantum Fluorescent Particles". The patents mentioned include: "Crete Quanta of Fluorescent Particles"; US Patent Publication No. 20050118574, published June 2, 2005 by Chandler et al., entitled "Multiplexed Analysis of Clinical Specimens Apparatus and Method"; and US Patent Publication No. 20050277197, published December 15, 2005 by Chandler et al., entitled "Microparticles with Multiple Fluorescent Signals and Methods of Using Them Thereof". The entire disclosures of the aforementioned documents, including “magnetic microspheres for use in fluorescence-based applications”, “magnetic microspheres with multiple fluorescent signals and methods of using the same”, and U.S. Patent Publication No. 20060159962, published July 20, 2006, by Chandler et al., entitled “Magnetic Microspheres for use in Fluorescence-based Applications”, are incorporated herein by reference. Beads may be pre-linked to biomolecules or other substances capable of binding to biomolecules and forming complexes. Beads may be pre-linked to antibodies, proteins or antigens, DNA/RNA probes, or any other molecules with affinity for a desired target. Examples of droplet actuator technologies for immobilizing magnetically responsive beads and/or non-magnetically responsive beads and/or using beads for droplet manipulation schemes are described in U.S. Patent Publication No. 20080053205, published March 6, 2008, by Pollack et al., entitled "Droplet-Based Particle Sorting"; and U.S. Patent Application No. 61/039,183, filed March 25, 2008, entitled "Multiplexing Bead Detection in a Single Droplet"; Pa U.S. Patent Application No. 61/047,789, filed April 25, 2008, by Mula et al., entitled "Droplet Actuator Devices and Droplet Operations Using Beads"; and U.S. Patent Application No. 61/086,183, filed August 5, 2008, entitled "Droplet Actuator Devices and Methods for Manipulating Beads". International Patent Publication No. WO/2008/098236, published August 14, 2008 by Eckhardt et al., entitled "Droplet Actuator Devices and Methods Employing Magnetic Beads"; International Patent Publication No. WO/2008/134153, published November 6, 2008 by Grichko et al., entitled "Bead-based Multiplexed Analytical Method". The following patents are cited in their entirety: “Bead Sorting on a Droplet Actuator”; International Patent Publication No. WO/2008/116221, published September 25, 2008, by Eckhardt et al.; and International Patent Publication No. WO/2007/120241, published October 25, 2007, by Eckhardt et al., entitled “Droplet-based Biochemistry”. The bead characteristics can be used in the multiplexing aspects of this disclosure. Examples of beads with features suitable for multiplexing and methods for detecting and analyzing signals emitted from such beads can be found in the following literature: U.S. Patent Publication No. 20080305481, published December 11, 2008, by Whitman et al., entitled "Systems and Methods for Multiplex Analysis of PCR in Real Time"; and U.S. Patent Publication No. 20080151240, published June 26, 2008, by Roth, entitled "Methods and Systems for Dynamic Range Extension (M... The patents mentioned include: U.S. Patent Publication No. 20070207513, published September 6, 2007 by Sorensen et al., entitled "Methods, Products, and Kits for Identifying an Analyte in a Sample"; and U.S. Patent Publication No. 20070064990, published March 22, 2007 by Roth, entitled "Methods for Image Data Processing". Methods and Systems for Image Data Processing; US Patent Publication No. 20060159962, published July 20, 2006, by Chandler et al., entitled "Magnetic Microspheres for use in Fluorescence-based Applications"; US Patent Publication No. 20050277197, published December 15, 2005, by Chandler et al., entitled "Multiple Fluorescent Signals..." The entire publications of the following documents are incorporated herein by reference: “Microparticles with Multiple Fluorescent Signals and Methods of Using Same”; and U.S. Patent Publication No. 20050118574, published June 2, 2005, by Chandler et al., entitled “Multiplexed Analysis of Clinical Specimens Apparatus and Method”.

The term "droplet" refers to the volume of liquid on a droplet actuator. Typically, the droplet is at least partially confined by a packing fluid. For example, the droplet may be completely surrounded by the packing fluid or confined by the packing fluid and one or more surfaces of the droplet actuator. As another example, the droplet may be confined by the packing fluid, one or more surfaces of the droplet actuator, and/or the atmosphere. Yet another example, the droplet may be confined by both the packing fluid and the atmosphere. The droplet may be, for example, aqueous or non-aqueous, or may be a mixture or emulsion comprising both aqueous and non-aqueous components. Droplets can take on a wide variety of shapes; non-limiting examples include generally disc-shaped, block-shaped, truncated spherical, ellipsoidal, spherical, partially compressed spherical, hemispherical, oval, cylindrical, combinations of such shapes, and various shapes formed during droplet operation (such as fusion or separation) or resulting from contact between such shapes and one or more surfaces of the droplet actuator. For examples of droplet fluids that can be subjected to droplet manipulation using the methods disclosed herein, see International Patent Publication No. WO/2007/120241, published October 25, 2007, by Eckhardt et al., entitled “Droplet-based Biochemistry,” the entire disclosure of which is incorporated herein by reference.

In various embodiments, the droplets may include biological samples such as whole blood, lymph, serum, plasma, sweat, tears, saliva, sputum, cerebrospinal fluid, amniotic fluid, semen, vaginal secretions, serous fluid, synovial fluid, pericardial fluid, peritoneal fluid, pleural fluid, transudate, exudate, cystic fluid, bile, urine, gastric juice, intestinal juice, fecal samples, liquids containing single or multiple cells, liquids containing organelles, fluidized tissues, fluidized organisms, liquids containing multicellular organisms, biological swabs, and biological flushing solutions. Furthermore, the droplets may include reagents such as water, deionized water, saline solutions, acidic solutions, alkaline solutions, detergent solutions, and/or buffer solutions. Droplets may include nucleic acids, such as DNA, genomic DNA, RNA, mRNA, or analogs thereof; nucleotides (such as deoxyribonucleotides, ribonucleotides) or analogs thereof (such as analogs having a terminator motif), such as those described in the following literature: Bentley et al., Nature 456:53-59 (2008); Gormley et al., International Patent Publication No. WO/2013/131962, published September 12, 2013, entitled “Improved Methods of Nucleic Acid Sequencing”; Barries et al., U.S. Patent No. 7,057,026, granted June 6, 2006, entitled “Labeled Nucleotides”; Kozlov et al., 2008 International Patent Publication No. WO/2008/042067, published on April 10, entitled "Compositions and Methods for Nucleotide Sequencing"; International Patent Publication No. WO/2013/117595, published by Rigatti et al. on August 15, 2013, entitled "Targeted Enrichment and Amplification of Nucleic Acids on a Support"; and U.S. Patent No. 7,329,492, granted on February 12, 2008, by Hardin et al., entitled "Methods for Real-Time Single-Molecular Sequencing". "Ingle Molecule Sequence Determination"; Hardin et al.'s U.S. Patent No. 7,211,414, issued May 1, 2007, entitled "Enzymatic Nucleic Acid Synthesis: Compositions and Methods for Altering Monomer Incorporation Fidelity"; Turner et al.'s U.S. Patent No. 7,315,019, issued January 1, 2008, entitled "Arrays of Optical Confinements and Uses Thereof"; Xu et al.'s 2008 U.S. Patent No. 7,405,281, granted on July 29, entitled "Fluorescent Nucleotide Analogs and Uses Therefor"; and U.S. Patent Publication No. 20080108082, published on May 8, 2008 by Ranket et al., entitled "Polymerase Enzymes and Reagents for Enhanced Nucleic Acid Sequencing," the entire disclosures of which are incorporated herein by reference; enzymes, such as polymerases, ligases, recombinases, or transposases; binding chaperones, such as antibodies, epitopes, streptavidin, avidin, biotin, lectins, or carbohydrates; or other biochemically active molecules. Other examples of droplet contents include reagents, such as reagents for biochemical protocols, including, for example, nucleic acid amplification protocols, affinity-based assay protocols, enzyme assay protocols, sequencing protocols, and/or protocols for biofluid analysis. A droplet may comprise one or more beads.

"Droplet operation" means any manipulation of a droplet on a droplet actuator. Droplet operations may include, for example,: loading a droplet into a droplet actuator; dispensing one or more droplets from a droplet source; splitting, separating, or dividing a droplet into two or more droplets; transporting a droplet from one location to another in any direction; fusing or merging two or more droplets into a single droplet; diluting a droplet; mixing a droplet; agitating a droplet; deforming a droplet; holding a droplet in place; incubating a droplet; heating a droplet; evaporating a droplet; cooling a droplet; handling a droplet; transporting a droplet away from a droplet actuator; other droplet operations described herein; and/or any combination of the foregoing. The terms "merge," "merging," "combine," "combining," etc., are used to describe creating a droplet from two or more droplets. It should be understood that when such terms are used with respect to two or more droplets, any combination of droplet operations sufficient to result in the combination of two or more droplets into one droplet can be used. For example, “fusion of droplet A and droplet B” can be achieved by bringing droplet A into contact with a stationary droplet B, bringing droplet B into contact with a stationary droplet A, or bringing droplets A and B into contact with each other. The terms “splitting,” “separating,” and “dividing” are not intended to imply any particular result regarding the volume of the resulting droplets (i.e., the volumes of the resulting droplets may be the same or different) or any particular result regarding the number of resulting droplets (the number of resulting droplets may be 2, 3, 4, 5, or more). The term “mixing” refers to a droplet operation that produces a more uniform distribution of one or more components within a droplet. Examples of “loading” droplet operations include microdialysis loading, pressure-assisted loading, autoloading, passive loading, and pipette loading. Droplet operations can be electrode-mediated. In some cases, droplet operations are further facilitated by using hydrophilic and/or hydrophobic regions on the surface and/or by physical barriers. For examples of droplet operations, see the patents and patent applications cited above under the definition of "droplet actuator." Impedance or capacitance sensing techniques or imaging techniques can sometimes be used to determine or confirm the results of droplet operations. Examples of such techniques are described in U.S. Patent Publication No. 20100194408, published August 5, 2010, by Sturmer et al., entitled "Capacitance Detection in a Droplet Actuator," the entire disclosure of which is incorporated herein by reference. Generally, sensing or imaging techniques can be used to confirm the presence or absence of a droplet at a specific electrode. For example, the presence of a dispensed droplet at the target electrode after a droplet dispensing operation confirms that the droplet dispensing operation was effective. Similarly, the presence of a droplet at a detection point in an appropriate step of a determination protocol confirms that the preceding set of droplet operations has successfully generated the droplet for detection. Droplet delivery time can be very fast. For example, in various embodiments, the delivery time of a droplet from one electrode to the next may exceed approximately 1 second, or approximately 0.1 seconds, or approximately 0.01 seconds, or approximately 0.001 seconds. In one embodiment, the electrodes are operated in AC mode, but switched to DC mode for imaging. This facilitates droplet operation over a footprint similar to that of the electrowetting region; in other words, 1x-, 2x-, and 3x- droplets can be effectively controlled using 1, 2, and 3 electrodes, respectively. If the droplet footprint is larger than the number of electrodes available for droplet operation at a given time, the difference between the droplet size and the number of electrodes should generally not exceed 1; in other words, 2x droplets can be effectively controlled using 1 electrode, and 3x droplets can be effectively controlled using 2 electrodes. When the droplet contains beads, it is useful for the droplet size to be equal to the number of electrodes controlling the droplet (e.g., delivering the droplet).

"Filling fluid" refers to a fluid associated with the droplet operating substrate of a droplet actuator, which cannot be sufficiently mixed with the droplet phase to allow the droplet phase to undergo electrode-mediated droplet operation. For example, a filling fluid is typically used to fill the droplet operating gap of a droplet actuator. The filling fluid can be, for example, or include low-viscosity oils, such as silicone oils or hexadecane filling fluids. The filling fluid can be, for example, or include halogenated oils, such as fluorinated oils or perfluorinated oils. The filling fluid can fill the entire gap of the droplet actuator or cover one or more surfaces of the droplet actuator. The filling fluid can be conductive or non-conductive. Filling fluids can be selected to improve droplet operation and/or reduce the loss of reagents or target substances in the droplets, improve droplet formation, reduce cross-contamination between droplets, reduce contamination of the droplet actuator surface, and reduce degradation of the droplet actuator material, etc. For example, the filling fluid can be selected based on compatibility with the droplet actuator material. As an example, fluorinated filling fluids can be used effectively with fluorinated surface coatings. Fluorinated filler fluids can be used to reduce the loss of lipophilic compounds, such as umbelliferone substrates like 6-hexadecamido-4-methylumbelliferone substrates (e.g., for use in Krabbe, Niemann-Pick, or other assays); other umbelliferone substrates are described in U.S. Patent Publication No. 20110118132, published May 19, 2011, by Winger et al., entitled “Enzymatic Assays Using Umbelliferone Substrates with Cyclodextrins in Droplets of Oil,” the entire disclosure of which is incorporated herein by reference. Examples of suitable fluorinated oils include those in the 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, d = 1.79), and Galden HT230 (bp = 230°C, viscosity = 4.4 cSt, d = 1.82) (all from Solvay Solexis); and those in the Novec series, such as Novec 7500 (bp = 128°C, viscosity = 0.8 cSt, d = 1.61), Fluorinert FC-40 (bp = 155°C, viscosity = 1.8 cSt, d = 1.85), and Fluorinert FC-43 (bp = 174°C, viscosity = 2.5 cSt, d = 1.86) (both from 3M). Typically, the selection of perfluorinated filler fluids is based on kinematic viscosity (<7 cSt is preferred, but not required) and boiling point (>150 °C is preferred, but not required for use in DNA/RNA-based applications (PCR, etc.)). The filler fluid may, for example, be doped with surfactants or other additives. For example, additives may be selected to improve droplet handling and/or reduce reagent or target substance loss in the droplets, droplet formation, cross-contamination between droplets, contamination of droplet actuator surfaces, degradation of droplet actuator materials, etc. Compositions including surfactant-doped filler fluids can be selected based on the performance of the reagents used in a particular assay and the effective or non-interacting nature of their interaction with the droplet actuator material. Examples of filler fluids and filler fluid formulations applicable to the methods and apparatus described herein are provided in International Patent Publication No. WO/2010/027894, published June 3, 2010, by Srinivasan et al., entitled "Droplet Actuators, Modified Fluids and Methods"; and in International Patent Publication No. WO/2009/021173, published February 12, 2009, by Srinivasan et al., entitled "Use of Additives for Enhancing Droplet Operation". The entire disclosures of the aforementioned documents, along with other patents and patent applications cited therein, are incorporated herein by reference. These include: International Patent Publication No. WO/2008/098236, published January 15, 2009, by Sista et al., entitled "Droplet Actuator Devices and Methods Employing Magnetic Beads"; and U.S. Patent Publication No. 20080283414, published November 20, 2008, by Monroe et al., entitled "Electrowetting Devices". In some cases, the fluorinated oil may be doped with fluorinated surfactants, such as Zonyl FSO-100 (Sigma-Aldrich) and/or others. The filler fluid is typically a liquid. In some embodiments, a filler gas may be used instead of a liquid.

The term "fixed" in relation to magnetically responsive beads means that the beads are substantially confined in a suitable location within a droplet or in the packing fluid on a droplet actuator. For example, in one embodiment, the fixed beads are sufficiently confined in a location within a droplet to allow for droplet splitting operations, resulting in a droplet having substantially all the beads and a droplet having substantially no beads.

"Magnetic response" refers to a response to a magnetic field. "Magnetic-responsive beads" include or are composed of magnetically responsive materials. Examples of magnetically responsive materials include paramagnetic, ferromagnetic, ferrimagnetic, and variability-magnetic materials. Suitable examples of paramagnetic materials include iron, nickel, and cobalt, as well as metal oxides such as Fe3O4, BaFe12O19, CoO, NiO, Mn2O3, Cr2O3, and CoMnP.

"Reservoirs" means an enclosure or partial enclosure configured to hold, store, or supply liquid. The droplet actuator system of this disclosure may include on-cartridge reservoirs and/or off-cartridge reservoirs. An on-cartridge reservoir may be (1) an on-actuator reservoir, which is a reservoir in the droplet operating gap or on the droplet operating surface; (2) an off-cartridge reservoir, which is a reservoir on the droplet actuator cartridge but outside the droplet operating gap and not in contact with the droplet operating surface; or (3) a hybrid reservoir having both an on-actuator region and an off-actuator region. An example of an off-actuator reservoir is a reservoir in a top substrate. Off-actuator reservoirs are typically in fluid communication with arranged openings or flow passages for allowing liquid from the off-actuator reservoir to flow into the droplet operating gap, for example, into an on-actuator reservoir. An offline cartridge reservoir can be a reservoir that is not part of the droplet actuator cartridge at all, but allows liquid to flow to a portion of the droplet actuator cartridge. For example, an offline cartridge reservoir can be part of a system or plug-in station to which the droplet actuator cartridge is connected during operation. Similarly, an offline cartridge reservoir can be a reagent storage container or syringe used to force fluid into an online cartridge reservoir or into the droplet operating gap. Systems employing offline cartridge reservoirs typically include fluid channel devices through which liquid can be transferred from the offline cartridge reservoir to an online cartridge reservoir or into the droplet operating gap.

As used herein, "delivered into the magnetic field of a magnet," "delivered toward a magnet," etc., refer to the delivery of the droplet and/or the magnetically responsive beads within the droplet to a region of magnetic field capable of substantially attracting the magnetically responsive beads within the droplet. Similarly, as used herein, "delivered away from a magnet or magnetic field," "delivered away from the magnetic field of a magnet," etc., refer to the delivery of the droplet and/or the magnetically responsive beads within the droplet away from a region of magnetic field capable of substantially attracting the magnetically responsive beads within the droplet, regardless of whether the droplet or the magnetically responsive beads are completely removed from the magnetic field. It will be understood that in any of these situations described herein, the droplet may be delivered toward or away from a desired region of the magnetic field, and/or the desired region of the magnetic field may be moved toward or away from the droplet. Regarding electrodes, droplets, or magnetically responsive beads "within" or "in" a magnetic field, this is intended to describe a situation where the electrode is positioned in a manner that allows the electrode to transport the droplet into and/or out of a desired region of the magnetic field, or where the droplet or magnetically responsive bead is located in a desired region of the magnetic field, in each case, the magnetic field in the desired region is capable of substantially attracting any magnetically responsive beads in the droplet. Similarly, regarding electrodes, droplets, or magnetically responsive beads "outside" or "away from" a magnetic field, this is intended to describe a situation where the electrode is positioned in a manner that allows the electrode to transport the droplet away from a region of the magnetic field, or where the droplet or magnetically responsive bead is located away from a region of the magnetic field, in each case, the magnetic field in such region is not capable of substantially attracting any magnetically responsive beads in the droplet, or any remaining attraction therein does not negate the effectiveness of droplet operations performed in that region. In various aspects of this disclosure, the system, droplet actuator, or another component of the system may include a magnet, such as one or more permanent magnets (e.g., a single cylindrical or bar magnet or an array of such magnets, such as a Halbach array) or an electromagnet or an array of electromagnets, to form a magnetic field for interacting with the magnetically responsive beads or other components on the chip. Such interaction may, for example, include substantially fixing or restricting movement or flow of the magnetically responsive beads in the droplet during storage or droplet operation, or attracting the magnetically responsive beads away from the droplet.

The term "washing" in relation to washing beads means reducing the amount and/or concentration of one or more substances in a droplet that is in contact with or exposed to the beads. This reduction in amount and/or concentration can be partial, substantially complete, or even complete. Substances can be any of a wide variety of substances; examples include target substances for further analysis and unwanted substances such as sample components, contaminants, and/or excess reagents. In some embodiments, the washing operation begins with an initial droplet in contact with the magnetically responsive beads, wherein the droplet comprises an initial amount and initial concentration of the substance. Various droplet operations can be used for the washing operation. The washing operation can produce droplets containing magnetically responsive beads, wherein the droplets have a total amount and/or concentration of the substance less than the initial amount and/or concentration of the substance. Examples of suitable washing techniques are described in U.S. Patent No. 7,439,014 to Pamula et al., issued October 21, 2008, entitled “Droplet-Based Surface Modification and Washing,” the entire disclosure of which is incorporated herein by reference.

Referring to the relative positions of the droplet actuator components (e.g., the relative positions of the top and bottom substrates of the droplet actuator), the terms "top," "bottom," "above," "below," and "above" are used throughout the specification. It will be understood that the droplet actuator is functional regardless of its spatial orientation.

When a liquid of any form (e.g., a droplet or continuum, whether moving or stationary) is described as being "above," "on," or "above" an electrode, array, matrix, or surface, such a liquid may be in direct contact with the electrode/array/matrix/surface, or may be in contact with one or more layers or membranes interposed between the liquid and the electrode/array/matrix/surface. In one instance, a filler fluid may be considered as a membrane between such a liquid and the electrode/array/matrix/surface.

When a droplet is described as being "on top of" or "loaded on top of" a droplet actuator, it should be understood that the droplet is arranged on top of the droplet actuator in a manner that facilitates one or more droplet operations on the droplet using the droplet actuator, the droplet is arranged on the droplet actuator in a manner that facilitates sensing the characteristics of the droplet or signals from the droplet, and/or the droplet has been subjected to droplet operations on the droplet actuator.

Barcodes and UMI on the beads

In some embodiments, primers having a sample barcode may be immobilized onto a solid support. Additionally or alternatively, primers having a UMI sequence may be immobilized onto a solid support. For example, the solid support may be one or more beads. Thus, in some embodiments, multiple beads may be present, each of which has a unique sample barcode and/or UMI sequence. In some embodiments, an individual cell is contacted with one or more beads having a set of unique sample barcodes and/or UMI sequences to identify the individual cell. In some embodiments, lysates from an individual cell are contacted with one or more beads having a set of unique sample barcodes and/or UMI sequences to identify the individual cell lysate. In some embodiments, purified nucleic acids from an individual cell are contacted with one or more beads having a set of unique sample barcodes and/or UMI sequences to identify the purified nucleic acids from the individual cell. The beads may be manipulated in any suitable manner known in the art, for example, using the droplet actuator described above.

The terms “solid surface,” “solid support,” and other grammatical equivalents herein refer to any material that is suitable for or can be modified to be suitable for the primers, barcodes, and sequences described herein. As will be understood by those skilled in the art, the number of possible substrates is enormous. 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, polybutene, polyurethane, Teflon ^™ , etc.), polysaccharides, nylon or nitrocellulose, ceramics, resins, silica or silica-based materials (including silicone and modified silicone), carbon, metals, inorganic glasses, plastics, optical fiber bundles, and a variety of other polymers. For some embodiments, particularly useful solid supports and solid surfaces are located within a flow cell. Exemplary flow cells are described in further detail below.

In some embodiments, the solid support includes a patterned surface suitable for fixing primers, barcodes, and sequences described herein in an ordered pattern. A “patterned surface” refers to an arrangement of different regions within or on the exposed layer of the solid support. For example, in the presence of one or more transposon complexes, one or more of the regions may have multiple features. In the absence of transposon complexes, the features may be separated by gap regions. In some embodiments, the pattern may be features in an x-y form of rows and columns. In some embodiments, the pattern may be a repeating arrangement of features and/or gap regions. In some embodiments, the pattern may be a random arrangement of features and/or gap regions. In some embodiments, the transposon complex is randomly distributed on the solid support. In some embodiments, the transposon complex is distributed on the patterned surface. Exemplary patterned surfaces that can be used in the methods and compositions set forth herein are described in U.S. Patent Application Publication No. 2012/0316086A1, U.S. Patent Application Serial No. 13/661,524, each of which is incorporated herein by reference.

In some embodiments, the solid support comprises an array of holes or recesses located in the surface. This can be manufactured using a variety of techniques, including but not limited to photolithography, stamping, forming, and micro-etching, as will be understood by those skilled in the art. The techniques used will depend on the composition and shape of the array substrate.

The composition and geometry of a solid support can vary depending on its intended use. In some embodiments, the solid support is a planar structure, such as a glass slide, chip, microchip, and/or array. Therefore, the surface of the substrate can be in the form of a planar 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 comprising a solid surface across which one or more fluid reagents can flow. Examples of flow cells and associated fluid systems and detection platforms that can be readily used in the methods of this disclosure are described, for example, in Nature et al. 456:53-59 (2008), WO 04/018497; US 7,057,026; WO91/06678; WO 07/123744; US 7,329,492; US 7,211,414; US 7,315,019; US 7,405,281 and US2008/0108082, each of which is incorporated herein by reference.

In some embodiments, the solid support or its surface is non-planar, such as the inner or outer surface of a tube or container. In some embodiments, the solid support comprises microspheres or beads. The terms "microspheres," "beads," or "parcitiles," or their grammatical equivalents herein, refer to small, discrete particles. Suitable bead compositions include, but are not limited to, plastics, ceramics, glass, polystyrene, methylstyrene, acrylic polymers, paramagnetic materials, thorium oxide sol, carbon graphite, titanium dioxide, latex or cross-linked dextran (such as agarose), cellulose, nylon, cross-linked micelles, and Teflon, and any other materials outlined herein for solid supports may be used. The "Microsphere Detection Guide" from Bangs Laboratories, Fishers Ind. is a useful guide. In some embodiments, the microspheres are magnetic microspheres or beads.

The beads do not need to be spherical; irregular particles can be used. Alternatively or additionally, the beads can be porous. The bead size can range from nanometers (i.e., 100 nm) to millimeters (i.e., 1 mm), with beads from about 0.2 micrometers to about 200 micrometers being preferred, and from about 0.5 micrometers to about 5 micrometers being particularly preferred, but smaller or larger beads can be used in some embodiments.

Throughout this application, various publications, patents, and/or patent applications have been cited. The disclosures of these publications, in their entirety, are incorporated herein by reference.

The terminology used in this document is intended to be open-ended, including not only the listed elements but also any additional elements.

Several embodiments have been described. However, it will be understood that various modifications can be made. Therefore, other embodiments are within the scope of the following claims.

Claims (75)

1. A method for preparing a cDNA library from multiple single cells, comprising: mRNA is released from each single cell to provide multiple individual mRNA samples, wherein the mRNA in each individual mRNA sample is derived from a single cell; A first strand of cDNA is synthesized from the mRNA in each individual mRNA sample using a first-strand synthesis primer and a tag is incorporated into the cDNA to provide multiple labeled cDNA samples, wherein the cDNA in each labeled cDNA sample is complementary to the mRNA from a single cell, and wherein the tag includes a cell-specific identifier sequence, a first read sequencing adaptor sequence, and a unique molecular identifier (UMI) sequence. Pool the labeled cDNA samples; Amplify the pooled cDNA sample to generate pooled labeled double-stranded cDNA; and Tagging of the pooled labeled double-stranded cDNA with a transposase involves contacting the pooled labeled cDNA with multiple transposon complexes, each of the multiple transposon complexes comprising a transposase complexed with a transposon, the transposon comprising a second readout sequencing adaptor sequence, wherein the second readout sequencing adaptor sequence is different from the first readout sequencing adaptor sequence, to generate the cDNA library, wherein the cDNA library comprises multiple labeled cDNA fragments having the first readout sequencing adaptor sequence on a first strand and the second readout sequencing adaptor sequence on a second strand. 2. The method of claim 1, further comprising amplifying the labeled cDNA fragment of the cDNA library to generate an amplified labeled cDNA fragment. 3. The method of claim 2, wherein amplifying the labeled cDNA fragment comprises adding an additional sequence to the 5' end of the amplification product. 4. The method of claim 3, wherein the additional sequence comprises a primer-binding sequence for hybridizing with a complementary primer-binding sequence on a solid support and for amplifying the labeled cDNA fragment on the solid support. 5. The method of claim 4, further comprising hybridizing the amplified labeled cDNA fragment to the complementary primer-binding sequence on the solid support, and amplifying the amplified labeled cDNA fragment on the solid support. 6. The method of claim 5, further comprising sequencing the amplification products on the solid support. 7. The method of any one of claims 1-6, wherein the tagging reaction comprises contacting the double-stranded cDNA with a transposase mixture comprising Tn5 transposase. 8. The method of claim 7, wherein the plurality of transposon complexes are primarily composed of transposons having transposons, the transposons including the second readout sequencing adaptor sequence. 9. The method of any one of claims 1-8, the method further comprising sequencing the labeled cDNA fragment of the cDNA library. 10. The method of claim 9, wherein sequencing includes 3' tag counting. 11. The method of claim 9, wherein sequencing comprises whole transcriptome analysis. 12. The method of any one of claims 1-11, wherein the first strand synthesis is performed using a mixture of random primers, wherein the random primers include the tag. 13. The method of any one of claims 1-12, wherein the first-strand synthetic primer comprises a double-stranded portion. 14. The method of claim 13, wherein the first-strand synthesis primer reduces tandem byproducts compared to a single-strand first-strand synthesis primer. 15. The method of claim 13, wherein the first-strand synthetic primer includes a region capable of forming a hairpin. 16. The method of claim 13, wherein the first-strand synthetic primer comprises an RNA region. 17. The method of claim 13, wherein the first-strand synthetic primer is hybridized to a complementary oligonucleotide, thereby forming a double-stranded portion. 18. The method of any one of claims 1-17, wherein the first-strand synthesis primer is attached to the bead, and the synthesis comprises synthesizing a labeled cDNA sample on the bead. 19. The method of claim 18, comprising encapsulating each single cell in a droplet having the beads before releasing mRNA from each single cell. 20. The method of claim 19, wherein the droplets are generated using a droplet actuator. 21. The method of claim 20, wherein the release and synthesis of each labeled cDNA sample are carried out in the droplet. 22. The method of claim 18 or 21, wherein pooling the labeled cDNA sample comprises pooling beads having the attached labeled cDNA sample. 23. The method of any one of claims 1-22, wherein the first-strand synthesis primer is a mixture of oligo-dT primers and random primers. 24. The method of any one of claims 1-23, wherein releasing mRNA from each single cell comprises releasing mRNA from the nucleus of each single cell to provide the plurality of individual mRNA samples, wherein the mRNA in each individual mRNA sample originates from a single cell nucleus. 25. A method for preparing a cDNA sequencing library from multiple single-cell organelles, comprising: Spatially separate single-celled organelles; mRNA, microRNA, small interfering RNA, ribosomal RNA and/or mitochondrial RNA are released from each single organelle to provide multiple separate RNA samples, wherein the RNA in each separate RNA sample is derived from a single organelle; A first strand of cDNA is synthesized from RNA in each individual RNA sample using a first-strand synthesis primer, the first-strand synthesis primer including a tag, thereby incorporating the tag into the cDNA to provide multiple labeled cDNA samples, wherein the cDNA in each labeled cDNA sample is complementary to RNA from a single organelle, and wherein the tag includes an organelle-specific identifier sequence. Pool the labeled cDNA samples from the plurality of single organelles; Amplify the pooled cDNA sample to generate a pooled labeled double-stranded cDNA sample; and Tagging is performed on the pooled labeled double-stranded cDNA sample by contacting the pooled labeled cDNA with multiple transposon complexes to simultaneously cleave each cDNA and incorporate the adaptor into each strand of the cDNA, thereby generating a cDNA library from the multiple single organelles. 26. The method of claim 25, wherein the tag further comprises a unique molecular identifier (UMI) sequence. 27. The method of claim 25 or 26, wherein the first-strand synthesis primer is a mixture of oligo-dT primers and random primers. 28. The method of any one of claims 25-27, wherein the tag comprises a first read sequencing adapter sequence. 29. The method of claim 28, wherein: Each of the plurality of transposon complexes includes a transposon complexed with a transposon, the transposon including a second readout sequencing adaptor sequence, wherein the second readout sequencing adaptor sequence is different from the first readout sequencing adaptor sequence, to generate the cDNA library, wherein the cDNA library includes a plurality of labeled cDNA fragments having the first readout sequencing adaptor sequence on a first strand and the second readout sequencing adaptor sequence on a second strand. 30. The method of claim 29, further comprising amplifying the labeled cDNA fragment of the cDNA library to generate an amplified labeled cDNA fragment. 31. The method of claim 30, wherein amplifying the labeled cDNA fragment of the cDNA library comprises adding an additional sequence to the 5' end of the amplification product. 32. The method of claim 31, wherein the additional sequence comprises a primer-binding sequence for hybridizing with a complementary primer-binding sequence on a solid support and for amplifying the labeled cDNA fragment on the solid support. 33. The method of claim 32, further comprising hybridizing the amplified labeled cDNA fragment to the complementary primer-binding sequence on the solid support, and amplifying the amplified labeled cDNA fragment on the solid support. 34. The method of claim 33, further comprising sequencing the amplification products on the solid support. 35. The method of any one of claims 25-34, wherein the tagging reaction comprises contacting the pooled labeled double-stranded cDNA sample with a transposase mixture comprising Tn5 transposase. 36. The method of claim 35, wherein the plurality of transposon complexes are primarily composed of transposons having transposons, the transposons including a second readout sequencing adaptor sequence. 37. The method of any one of claims 25-36, the method further comprising sequencing the labeled cDNA fragment of the cDNA library. 38. The method of claim 37, wherein sequencing includes 3' tag counting. 39. The method of claim 37, wherein sequencing comprises whole transcriptome analysis. 40. The method of any one of claims 25-39, wherein the first-strand synthesis primer comprises a primer containing a double-stranded portion. 41. The method of claim 40, wherein the first-strand synthesis primer reduces tandem byproducts compared to a single-strand first-strand synthesis primer. 42. The method of claim 40, wherein the first-strand synthetic primer comprises a primer containing a region capable of forming a hairpin. 43. The method of claim 42, wherein the first-strand synthetic primer comprises a primer containing an RNA region. 44. The method of claim 40, wherein the first-strand synthesis primer comprises a primer that hybridizes to a complementary oligonucleotide, thereby forming a double-stranded portion. 45. The method of any one of claims 25-44, wherein the first-strand synthesis primer is attached to the bead, and the synthesis comprises synthesizing a labeled cDNA sample on the bead. 46. The method of claim 45, comprising encapsulating each single cell in a droplet having beads before releasing mRNA from each single cell. 47. The method of claim 46, wherein the release and synthesis of each labeled cDNA sample are performed in the droplet. 48. The method of claim 47, wherein pooling the labeled cDNA sample comprises pooling beads having the attached labeled cDNA sample. 49. The method of any one of claims 46 and 48, wherein the droplets are generated using a droplet actuator. 50. The method of any one of claims 25-49, wherein spatially separating the single-cell organelles comprises separating the organelles into spatial compartments prior to releasing mRNA, microRNA, small interfering RNA, ribosomal RNA and/or mitochondrial RNA from each single-cell organelle. 51. The method of claim 50, wherein the organelles are separated into spatial compartments by fluorescence-activated cell sorting (FACS). 52. The method of claim 51, wherein the organelles are separated in spatial compartments by being fixed to a solid surface. 53. The method of claim 52, wherein the organelle is immobilized on the solid surface by an antibody specifically bound to the organelle, wherein the antibody is immobilized on the solid surface. 54. The method of claim 53, wherein the solid surface is one of a flow pool and beads. 55. The method of any one of claims 25-54, wherein the organelle is selected from the nucleus, mitochondria, and ribosomes. 56. A method for preparing a cDNA library from multiple single cells, the method comprising the following steps: mRNA is released from each single cell to provide multiple individual mRNA samples, wherein the mRNA in each individual mRNA sample is derived from a single cell; A first strand of cDNA is synthesized from the mRNA in each individual mRNA sample using a mixture comprising (i) a first strand synthesis primer including a tag, and incorporating the tag into the cDNA to provide a plurality of labeled cDNA samples, wherein the cDNA in each labeled cDNA sample is complementary to the mRNA from a single cell, and wherein the tag includes a cell-specific identifier sequence and a unique molecular identifier (UMI) sequence, and (ii) a template-converting oligonucleotide (TSO) primer. Pool the labeled cDNA samples from the plurality of single cells; Amplify the pooled labeled cDNA sample to generate pooled labeled double-stranded cDNA; and A tagging reaction is performed on the pooled labeled double-stranded cDNA sample to simultaneously cleave each cDNA and incorporate the adaptor into each strand of the cDNA, thereby generating a cDNA library from the plurality of single cells. 57. The method of claim 56, wherein the tagging reaction comprises contacting the double-stranded cDNA with a mixture of transposases including an adaptor sequence not found in the first strand synthesis primer. 58. The method of claim 56 or claim 57, wherein the first-strand synthetic primer further comprises an amplification primer binding site. 59. A method for preparing a cDNA library from multiple single cells, comprising: mRNA is released from each single cell to provide multiple individual mRNA samples, wherein the mRNA in each individual mRNA sample is derived from a single cell; A first strand of cDNA is synthesized from the mRNA in each individual mRNA sample using a mixture comprising (i) a first-strand synthesis primer comprising a tag, wherein the first-strand synthesis primer is a mixture of oligodT primers and random primers, each of the oligodT primers and the random primers further comprising a first amplification primer binding site, thereby incorporating the tag into the cDNA to provide multiple labeled cDNA samples, wherein the cDNA in each labeled cDNA sample is complementary to the mRNA from a single cell, and wherein the tag comprises a cell-specific identifier sequence, a first read sequencing adaptor sequence and a distinct second portion, and (ii) a template-converting oligonucleotide (TSO) primer; Pool the labeled cDNA samples; Amplify the pooled labeled cDNA to generate pooled labeled double-stranded cDNA; and Tagging is performed on pooled labeled double-stranded cDNA by contacting pooled labeled cDNA with multiple transposon complexes, each of which includes a transposase complexed with a transposon, the transposon including a second readout sequencing adaptor sequence, wherein the second readout sequencing adaptor sequence is different from the first readout sequencing adaptor sequence, to generate the cDNA library, wherein the cDNA library includes multiple labeled cDNA fragments having the first readout sequencing adaptor sequence on a first strand and the second readout sequencing adaptor sequence on a second strand. 60. The method of claim 59, wherein the distinct second portion comprises a unique molecular identifier (UMI) sequence. 61. The method of claim 59 or 60, wherein each oligodT primer and random primer further includes an amplification primer binding site. 62. The method of any one of claims 56-61, wherein the first-strand synthetic primer includes a first amplification primer binding site, and the template-converting oligonucleotide (TSO) primer includes a second amplification primer binding site. 63. The method of any one of claims 56-62, wherein the first-strand synthetic primer extends beyond the mRNA and further replicates the template-converting oligonucleotide (TSO) primer strand to prepare a complementary TSO strand. 64. The method of claim 63, wherein the second strand of cDNA is synthesized using a second amplification primer complementary to the first strand of cDNA, the first strand of cDNA is extended beyond the mRNA template to include a complementary TSO strand, and a pooled labeled double-stranded cDNA is prepared. 65. The method of claim 64, wherein the pooled labeled double-stranded cDNA is amplified by a first amplification primer bound to a first amplification primer binding site and a second amplification primer bound to a second amplification primer binding site. 66. The method of claim 65, wherein the first amplification primer, the second amplification primer, or both the first amplification primer and the second amplification primer are fixed on a solid support. 67. The method of claim 66, wherein the solid support is a bead, a flow cell, or a micropore. 68. The method of claim 66 or 67, further comprising sequencing the amplification product on the solid support. 69. The method of any one of claims 56-68, wherein the tagging reaction comprises contacting the double-stranded cDNA with a transposase mixture comprising Tn5 transposase. 70. The method of claim 69, wherein the plurality of transposon complexes comprises transposons having transposons comprising the second readout sequencing adaptor sequence. 71. The method of any one of claims 56-70, wherein the first-strand synthesis primer comprises a primer containing a double-stranded portion. 72. The method of claim 71, wherein: (a) The first-strand synthesis primer reduces tandem byproducts compared to the single-strand first-strand synthesis primer; (b) The first-strand synthetic primer includes a primer containing a region capable of forming a hairpin; (c) The first-strand synthetic primer includes a primer containing an RNA region; and/or (d) The first-strand synthesis primer includes a primer that hybridizes to a complementary oligonucleotide, thereby forming a double-stranded portion. 73. Multiple beads, wherein each bead comprises multiple oligonucleotides, each oligonucleotide comprising: (a) Connector; (b) Amplify primer binding sites; (c) Optional unique molecular identifier (UMI), which is different for each oligonucleotide; (d) A bead-specific sequence, which is identical on each oligonucleotide of the bead but different on other beads; and (e) A capture sequence for capturing mRNA and initiating reverse transcription. 74. The plurality of beads as claimed in claim 73, wherein the trapping sequence comprises oligo-dT. 75. The plurality of beads as claimed in claim 73 or 74, wherein each bead is in a separate droplet separated from the other beads.