Barcoding of nucleic acids from single cells

Abstract

The present invention provides a method that allows for the separation of different workflow steps for barcoding target nucleic acids, thereby providing optimal reaction conditions for each workflow step, particularly for template-switching reactions. Furthermore, this method provides the opportunity to perform reactions, such as barcoding of two different nucleic acid molecules derived from a single cell, such as RNA molecules and genomic DNA molecules, in a single step. This method includes the steps of: (a) providing a plurality of cells containing target RNA molecules and at least one solid support containing capture oligonucleotides for the target RNA molecules and barcode oligonucleotides; (b) partitioning the plurality of cells and the solid support so that each cell is contained in a separate partition, each partition containing a solid support; (c) lysing the cells, thereby obtaining a mixture of target RNA molecules and non-target RNA molecules; (d) hybridizing the target RNA molecules to capture oligonucleotides for the target RNA molecules, thereby obtaining target RNA molecules attached to the solid support; and (e) disrupting the partitions and dissolving the target RNA molecules attached to the solid support. (f) separating non-target RNA molecules from target RNA molecules; (f) generating double-stranded nucleic acids from the target RNA molecules by nucleic acid synthesis, wherein a capture oligonucleotide serves as a primer and the target RNA molecule serves as a template; and (g) attaching a barcode oligonucleotide to the double-stranded nucleic acid derived from the target RNA molecules, thereby generating barcoded nucleic acids derived from the target RNA molecules, wherein in step a), the plurality of cells further comprise target genomic DNA molecules, and the at least one solid support further comprises a capture oligonucleotide for the target genomic DNA molecule, and the capture oligonucleotide for the target RNA molecule and the capture oligonucleotide for the target genomic DNA molecule are different from each other.

Description

[Technical Field]

[0001] The present invention relates to the fields of single cell analysis and nucleic acid barcoding.

[0002] Background of the Invention Methods for analyzing nucleic acids at the single-cell level are increasingly being utilized in biological and biomedical research, allowing for understanding the heterogeneity of tissues or cell populations and can be used to identify subpopulations of cells involved in disease.

[0003] To facilitate single-cell analysis and enable multiplexing, current state-of-the-art workflows include two essential features: partitioning of each single cell and barcoding of cell-specific target nucleic acids.

[0004] Current methods for analyzing single biological particles, including single cells, use droplets (water-in-oil emulsions) or small containers to partition these particles, then incorporate specific barcodes into target nucleic acid molecules within these partitions. Based on this, barcoded target nucleic acid molecules from one cell can be distinguished from target nucleic acid molecules from another cell during cell analysis processes such as next-generation sequencing. One example is the highly parallel analysis of different mRNA transcripts expressed in individual cells.

[0005] However, current state-of-the-art methods, such as those shown in Figure 1, have certain drawbacks. When using droplet-based methods, it is very difficult to add reagents after droplet formation. Therefore, all reagents for accessing biological particles (e.g., cell lysis), isolating molecules of interest (e.g., mRNA molecules), converting the molecules of interest (e.g., cDNA synthesis of mRNA molecules), and incorporating barcodes must be added simultaneously, and each reaction must be performed in the same reaction mixture. An example of such a workflow is the Chromium Next GEM Single Cell 5' Library & Gel Bead kit v1.1 (PN-100165, 10x Genomics, Pleasanton, CA, USA).

[0006] In contrast, methods utilizing small wells allow the addition of reagents during the process described in the paragraph above. However, the reagent mixture cannot be exchanged. Therefore, reactants from previous steps can still affect subsequent reactions. An example of such a workflow is the Smart-seq2 approach by Picelli et al. (2014).

[0007] In either case, complete buffer exchange between different workflow steps is not possible, and therefore each reaction must be carried out under non-ideal reaction conditions.

[0008] Some methods attempt to circumvent these limitations by using a hybridization-based approach to isolate molecules of interest on barcoded capture molecules bound to beads. The bound target nucleic acid can then be isolated, and barcodes can be incorporated in a subsequent step using ideal reaction conditions. One example of such a workflow is the drop-seq approach by Macosko et al. (2015). However, it has several limitations. For example, when barcoding entire mRNA molecules, this approach can only be used to add barcodes via capture sequences (oligo(dT)), thus resulting in the barcodes being adjacent to the 3' end of the transcript. A further drawback of this method is the difficulty of using target-specific primers to limit reverse transcription to only a subset of mRNA or other RNA species (as opposed to the entire poly(A) RNA when using oligo(dT) primers), since each specific primer must be ligated to a barcode on the surface of the bead.

[0009] Additionally, for some downstream applications, such as T cell receptor sequencing using kits like the 10x Genomics Single Cell Immune Profiling kit, barcode sequences at the 5' end of transcripts are essential. To achieve this, a process called template switching is employed (Zhu et al., 2001). Because the template switching process is transient, it must be performed while mRNA molecules from different cells are still partitioned. As a result, at least two process steps (cell lysis and reverse transcription with template switching) must be performed while cells are partitioned (in droplets or wells). Therefore, both reactions must be performed under non-ideal conditions. The template switching reaction, in particular, requires specific conditions, such as buffer conditions, to work properly and achieve optimal results. This is one of the major limitations of the current state of the art.

[0010] Furthermore, based on these limitations, current state-of-the-art workflows cannot provide a solution for the combined analysis of different target nucleic acid molecules, such as mRNA and genomic DNA, derived from the same cell in a single step. Such a method would represent a significant improvement, since it would, on the one hand, require less cellular material and, on the other hand, facilitate the analysis and direct comparison of mRNA and genomic DNA from the same cell.

[0011] For the reasons mentioned above, performing several reactions in one partition remains a major limitation in sample preparation and nucleic acid barcoding workflows.

[0012] Brief description of the invention Surprisingly, we have discovered a method that allows for the separation of different workflow steps for barcoding target nucleic acids, thereby providing optimal reaction conditions for each workflow step, particularly the template-switching reaction. Furthermore, this method offers the opportunity to perform barcoding reactions for two different nucleic acid molecules, such as RNA molecules and genomic DNA molecules, derived from a single cell in a single step.

[0013] One object of the present invention is a method for generating barcoded target nucleic acids from a plurality of cells, the method comprising: a. Providing a plurality of cells containing target RNA molecules and at least one solid support containing capture oligonucleotides for the target RNA molecules and barcode oligonucleotides (FIG. 2A part A / B); b. partitioning the plurality of cells and the solid support such that each cell is contained in a separate partition and each partition comprises a solid support (FIG. 2A part A / B); c. lysing the cells, thereby obtaining a mixture of target and non-target RNA molecules (Figure 2A part C); d. hybridizing the target RNA molecule to a capture oligonucleotide for the target RNA molecule, thereby obtaining a target RNA molecule attached to the solid support (Figure 2B part D); e. Disrupting the partition and separating non-target RNA molecules from the target RNA molecules attached to the solid support (Figure 2B part E / F); f. Producing a double-stranded nucleic acid from the target RNA molecule by nucleic acid synthesis, wherein the capture oligonucleotide serves as a primer and the target RNA molecule serves as a template (Figure 2B part E / F); g. Attaching a barcode oligonucleotide to a double-stranded nucleic acid derived from the target RNA molecule, thereby generating a barcoded nucleic acid derived from the target RNA molecule (Figure 2B part G) A method comprising: The method is characterized in that in step a), the plurality of cells further contain target genomic DNA molecules, the at least one solid support further contains capture oligonucleotides for the target genomic DNA molecules, and the capture oligonucleotides for the target RNA molecules and the target genomic DNA molecules are different from each other.

[0014] In a first variant of the present invention, steps a-f) are also performed on the target genomic DNA molecule, thereby generating a double-stranded target genomic DNA molecule attached to the same solid support as the double-stranded nucleic acid derived from the target RNA molecule. Optionally, step g) may be performed to obtain a barcoded double-stranded target genomic DNA molecule. In this case, the end result of this method is a barcoded target nucleic acid (RNA and DNA) attached to the same solid support.

[0015] In a second variant, the present invention describes a method for generating barcoded target RNA molecules and simultaneously performing whole genome amplification of target genomic DNA molecules. [Brief explanation of the drawings]

[0016] [Figure 1]A / B: A diagram showing the workflow of a standard single-cell cDNA barcoding system: (1) In the first step, single cells and single beads are compartmentalized into partitions (e.g., droplets or wells). The beads contain oligonucleotides containing unique barcodes (each oligonucleotide on a bead has the same barcode, but other beads / partitions in this experiment do not have the same barcode) and oligo(dT) oligonucleotides (SEQ ID NO: 6). (2) The cells are lysed. (3) The mRNA molecules hybridize to the oligo(dT) oligonucleotides (SEQ ID NO: 6) on the beads via their poly(A) tails (SEQ ID NO: 5). (4) The partitions are disrupted (e.g., in the case of droplet-based approaches, chemicals are used to break the emulsion). The mRNA remains bound to the beads. (5) The bead-bound mRNA is subjected to reverse transcription (bulk). During this process, the barcoded oligonucleotides on the beads are extended to generate barcoded cDNA molecules. [Figure 2] This figure shows the principle of the method of the present invention (example of mRNA analysis). Steps A / B) show a partition containing capture oligonucleotides (for target RNA and target genomic DNA), barcode oligonucleotides attached to the same solid support, and one cell. The cell is then lysed, releasing the nucleic acid molecules and resulting in a mixture of target and non-target nucleic acids (step C). Because the steps for target genomic DNA vary depending on the specific embodiment of the present invention (these embodiments are shown in the following figures), the following steps of the method are illustrated only for target RNA. In step D, the target nucleic acid molecule (RNA) then hybridizes to the capture oligonucleotide. The partition is then disrupted, and nucleic acid synthesis occurs (E / F). A barcode oligonucleotide is then attached, generating a barcoded target nucleic acid (G). Nucleic acids (derived from the target RNA) are attached to both sites on the solid support. [Figure 3]This diagram illustrates the principle of target RNA barcoding by incorporating a barcode via template switching. (For clarity, the capture oligonucleotide for the target genomic DNA is not shown / described.) In this example, a solid support containing a capture oligonucleotide (for the target RNA molecule), a barcode oligonucleotide, and a template-switching oligonucleotide sequence is used. (1) A single cell is locally lysed. The target RNA molecule binds to a specific capture oligonucleotide on the solid support (e.g., an mRNA molecule binds to a capture oligonucleotide containing an oligo(dT) sequence). This disrupts the partitioning. (2) cDNA is synthesized. During cDNA synthesis, the template-switching oligonucleotide contained in the barcode oligonucleotide on the same solid surface is used, thereby incorporating a barcode into the newly synthesized cDNA molecule. [Figure 4]A-C: Examples of using the method of the present invention to barcode cell-specific cDNA using beads as a solid support. (For clarity, capture oligonucleotides for target genomic DNA are not shown / described.) In this example, beads containing a capture oligonucleotide and a barcode oligonucleotide (containing a barcode sequence (BC)) and a barcode oligonucleotide containing a template-switching oligonucleotide (TSO) sequence are used. (1) Single cells and single beads are partitioned, with each partition containing one cell and one single bead. This example shows two different partitions, each containing one cell and one bead. Each bead contains a different barcode sequence (the bead-specific barcodes are labeled BC1 and BC2, respectively). (2) Cells are lysed within the partition, and target mRNA molecules bind to the capture oligonucleotides on the beads. The capture oligonucleotides contain capture sequences, which can be oligo(dT) that bind to poly(A) mRNA molecules or specific sequences that bind to a subset of mRNA molecules. (3) For example, if partitioning is achieved using water-in-oil droplets, all beads are combined into a single compartment by breaking the emulsion. Reverse transcription reactions are performed in a single compartment using a reverse transcriptase capable of template switching. Template switching is achieved using template-switching oligonucleotides on the same bead, so that cDNA molecules generated from the same cell contain the same barcode (the bead-specific barcode becomes the cell-specific barcode). [Figure 5]A-C: Examples of using the method of the present invention to barcode cell-specific cDNA using a patterned surface as a solid support. (For clarity, capture oligonucleotides for target genomic DNA are not shown / described.) In this example, a patterned surface containing capture oligonucleotides (including capture sequence CS), barcode oligonucleotides (including barcode sequence (BC)), and template-switching oligonucleotide sequences (TSO) is used. The surface contains a pattern of these barcode oligonucleotides with specific barcodes, and different pattern elements on the surface contain only a single barcode. (1) Single cells are deposited on the surface. The cells are deposited so that each pattern region does not contain more than one cell. (2) Cells are locally lysed under conditions that minimize diffusion from one region to another. This can be achieved by specific buffer conditions or by creating a temporary barrier between regions (e.g., using a hydrophilic surface and depositing cells in a water droplet surrounded by oil). Target mRNA molecules bind to the capture oligonucleotides on the patterned surface. The capture oligonucleotide contains a capture sequence, which can be an oligo(dT) that binds to poly(A) mRNA molecules or a specific sequence that binds to a subset of mRNA molecules. (3) The reverse transcription reaction is performed using a reverse transcriptase capable of template switching. During reverse transcription and template switching, adjacent barcodes (pattern region-specific barcodes) are incorporated into the cDNA. The pattern element-specific barcodes become cell-specific barcodes. [Figure 6]This figure shows an example of using the method of the present invention to double-barcode mRNA molecules. (For clarity, the capture oligonucleotide for the target genomic DNA is not shown / described.) In this example, a surface containing a capture oligonucleotide (comprising a capture sequence and a barcode sequence) and a barcode oligonucleotide containing a barcode sequence and a template-switching oligonucleotide sequence is used as a solid support. The solid support is partitioned with a single cell. (1) The cell is lysed, and the target mRNA binds to the barcoded capture oligonucleotide. (2) The solid surface is washed to remove lysis buffer, cellular debris, and / or other substances that may interfere with subsequent process steps. (3) A reverse transcription reaction is performed using a reverse transcriptase capable of template-switching. Reverse transcription is primed with the barcoded capture oligonucleotide, and the barcode of the capture oligonucleotide is incorporated into newly synthesized cDNA. During reverse transcription and template switching, an adjacent barcode (pattern region-specific barcode) is incorporated into the cDNA. The barcode (reverse complement) of the template-switching oligonucleotide is also incorporated into the newly synthesized cDNA. Thus, the resulting cDNA contains barcodes at both ends. The barcodes of the capture oligonucleotide and the template-switching oligonucleotide can be identical, thereby incorporating the same barcode (same orientation or reverse complement) into the cDNA molecule. This is a major advantage for library preparation workflows that require fragmentation, as it allows both ends of the cDNA molecule to be used to identify the parent nucleic acid molecule. [Figure 7A]This figure shows an example of simultaneous barcoding of mRNA molecules and genomic DNA molecules. In this example, a solid support is used, which contains a barcoded first capture oligonucleotide with a capture sequence (CS1) that binds to a target genomic DNA molecule and a second barcoded capture oligonucleotide with a capture sequence (CS2) that binds to a target RNA molecule. Additionally, the solid support contains barcode oligonucleotides containing a barcode sequence and a template-switching oligonucleotide sequence. (1) Single cells are partitioned onto the solid support. The cells are lysed, and the target genomic DNA molecules bind to the barcoded capture oligonucleotide containing CS1, and the target RNA molecules bind to the barcoded capture oligonucleotide containing CS2. (2) The solid surface is washed to remove lysis buffer, cellular debris, and / or other substances that may interfere with subsequent process steps. (3) The barcoded oligonucleotide containing CS1 is extended with an enzyme having DNA polymerase activity using the captured target DNA molecule as a template. (4) The barcoded oligonucleotide containing CS2 is extended with an enzyme containing a reverse transcriptase having template-switching activity using the captured target RNA molecule as a template. During reverse transcription, another barcode is incorporated using the barcoded template-switching oligonucleotide as a template. [Figure 7B] 10 shows that step G) can optionally be performed on target genomic DNA molecules and target RNA molecules. Barcode oligonucleotides can be attached to the target genomic DNA by ligation, and the barcodes can be attached to the target RNA by template switching. [Figure 8]Figure 1 shows an example of a method for binding a single cell with a specific barcode. (1) Addition to a specific compartment: A single cell and a barcode-containing bead are compartmentalized into a single partition. The partition can be established by a droplet surrounded by an immiscible fluid or a well. The cell is lysed within the partition, releasing the nucleic acid, and the target nucleic acid molecule binds to the bead-bound capture oligonucleotide. (2) Localized lysis on a surface, including a patterned surface with multiple regions containing different barcodes: Cells are deposited on the surface (ideally, no more than two cells are deposited in each region containing a specific barcode). Next, the cells are lysed under conditions that limit the diffusion of the target molecule, allowing the target molecule to bind locally. In the next step, the specific barcode of each region can be used to convert the target molecule into a barcoded nucleic acid. [Figure 9]This diagram illustrates an example workflow for simultaneously barcoding genomic DNA and RNA. This example uses a solid surface containing (a) whole genome amplification oligonucleotides, (b) capture oligonucleotides containing a capture sequence and a barcode sequence (CS2) specific to the target RNA, and (c) barcode oligonucleotides containing a template-switching oligonucleotide. The solid surface contains multiple oligonucleotides of each type; for simplicity, only a single copy is shown in the diagram. (1) A single cell is deposited onto a partition containing multiple whole genome amplification oligonucleotides (including barcodes), multiple barcoded oligonucleotides with RNA capture sequences, and multiple barcoded oligonucleotides containing template-switching oligonucleotides. The barcodes on the solid surface are specific to the partition. (2) In the first step, the cells within the partition are lysed, releasing the nucleic acid molecules, and the whole genome amplification oligonucleotides are released from the solid surface. The cells contain multiple RNA and DNA molecules; for simplicity, only a single copy is shown in the diagram. (3) The target RNA hybridizes to the capture sequence of the barcoded capture oligonucleotide that remains bound to the beads. (4) The target genomic DNA is amplified by whole genome amplification. Whole genome amplification (MDA) is performed within the droplets using whole genome amplification oligonucleotides (see also Figure 10), thereby generating "free" barcoded amplified target DNA. (5) For example (in the case of droplet-based partitioning), the partitions are removed by removing (disassembling) the oil. The supernatant containing the barcoded amplified genomic DNA is retained and used for downstream applications (e.g., genomic DNA-based single-cell library preparation). (6) The beads are subjected to a reverse transcription reaction with template switching. In this way, barcoded cDNA is generated (barcoded at both the 5' and 3' ends). [Figure 10]This diagram illustrates whole genome amplification (multiple displacement amplification, MDA) using whole genome amplification oligonucleotides. First, whole genome amplification oligonucleotides are bound to genomic DNA. The whole genome amplification oligonucleotides serve as primers for nucleotide synthesis by Phi29 polymerase. Because Phi29 polymerase has strand displacement activity, Phi29 continues to extend beyond the newly synthesized nucleic acid strand, displacing other newly synthesized nucleic acid strands. Additional whole genome amplification oligonucleotides bind to these displaced single-stranded nucleic acid strands, and Phi29 uses these displaced single-stranded nucleic acid strands to generate additional nucleic acid molecules. As a result, multiple copies of genomic DNA are synthesized, each of which has a barcode at the 5' end of the newly synthesized nucleic acid. [Figure 11] FIG. 1 shows the results of Example 1 (cDNA amplification on beads).

[0017] Detailed Description of the Invention In a first aspect, the present invention provides a method for generating barcoded target nucleic acids from a plurality of cells, comprising: a. providing a plurality of cells comprising target RNA molecules and at least one solid support comprising capture oligonucleotides for the target RNA molecules and barcode oligonucleotides; b. partitioning the plurality of cells and the solid support such that each cell is contained in a separate partition and each partition comprises a solid support; c. lysing the cells, thereby obtaining a mixture of target and non-target RNA molecules; d. hybridizing the target RNA molecule to a capture oligonucleotide for the target RNA molecule, thereby obtaining a target RNA molecule attached to the solid support; e. disrupting the partitions and separating the non-target RNA molecules from the target RNA molecules attached to the solid support; f. generating double-stranded nucleic acids from the target RNA molecule by nucleic acid synthesis, wherein the capture oligonucleotide serves as a primer and the target RNA molecule serves as a template; g. Attaching a barcode oligonucleotide to a double-stranded nucleic acid derived from the target RNA molecule, thereby generating a barcoded nucleic acid derived from the target RNA molecule. A method comprising: In step a), the plurality of cells further comprises a target genomic DNA molecule, and the at least one solid support further comprises a capture oligonucleotide for the target genomic DNA molecule, and the capture oligonucleotide for the target RNA molecule and the capture oligonucleotide for the target genomic DNA molecule are different from each other.

[0018] The methods of the present invention are for generating barcoded target nucleic acids from a plurality of cells, which can be used for several downstream applications, such as library preparation, next generation sequencing, or polymerase chain reaction.

[0019] The plurality of cells can be derived from a sample, including a single cell containing the target nucleic acid. Samples that can be used to generate the barcoded target nucleic acids described herein can be derived from any specimen, such as whole animals, organs, tissue slices, cell aggregates, or single cells from invertebrates (e.g., Caenorhabditis elegans, Drosophila melanogaster), vertebrates (e.g., zebrafish, Xenopus laevis), and mammals (e.g., Mus musculus, Homo sapiens). Biological samples can be in the form of tissue slices, cell aggregates, suspension cells, adherent cells, or body fluids.

[0020] The target nucleic acid used to generate the barcoded target nucleic acid can be a polynucleotide chain made of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). More specifically, the target nucleic acid is RNA (such as mRNA) and genomic DNA molecules.

[0021] The target nucleic acid can encode a T cell receptor chain and / or a B cell receptor chain. In one embodiment, the target RNA molecule can encode a T cell receptor chain and / or a B cell receptor chain. In another embodiment, the target genomic DNA molecule can encode a T cell receptor chain and / or a B cell receptor chain. In yet another embodiment of the invention, the target RNA molecule and the target genomic DNA molecule can encode a T cell receptor chain and / or a B cell receptor chain.

[0022] In another embodiment, the target nucleic acid can encode a biomarker suitable for diagnosing or treating a disease. In one embodiment, the target RNA molecule can encode a biomarker suitable for diagnosing or treating a disease. In another embodiment, the target genomic DNA molecule can encode a biomarker suitable for diagnosing or treating a disease. In yet another embodiment of the present invention, the target RNA molecule and the target genomic DNA molecule encode a biomarker suitable for diagnosing or treating a disease.

[0023] In yet another embodiment of the present invention, the target nucleic acid molecule can encode a fusion gene generated by chromosomal rearrangement. In one embodiment, the target RNA molecule can encode a fusion gene generated by chromosomal rearrangement. In another embodiment, the target genomic DNA molecule can encode a fusion gene generated by chromosomal rearrangement. In yet another embodiment of the present invention, the target RNA molecule and the target genomic DNA molecule encode a fusion gene generated by chromosomal rearrangement.

[0024] The first step (a) of the method of the present invention involves providing a plurality of cells containing target nucleic acids (target RNA molecules and target genomic DNA molecules) and at least one solid support containing capture oligonucleotides and barcode oligonucleotides.

[0025] The capture oligonucleotides (for target RNA molecules and target genomic DNA molecules) contain a capture sequence (CS) that is at least partially complementary to the target nucleic acid (target RNA or target genomic DNA molecule). The capture sequence can vary depending on the target RNA molecule or target genomic DNA molecule. The capture oligonucleotides are single-stranded. In one embodiment, the capture sequence can be an oligo-dT sequence that can bind to an mRNA molecule (poly(A) tail). In another embodiment, the capture sequence can include a random sequence that binds complementary to all target nucleic acid molecule (DNA and / or RNA) species. In another embodiment, the capture sequence of the capture oligonucleotide can be complementary to a specific target nucleic acid molecule (DNA and / or RNA), thereby allowing for specific target nucleic acid binding.

[0026] In another embodiment, the capture sequence may be complementary to a specific target sequence, such as the constant region of a T cell receptor or B cell receptor, or a biomarker for the diagnosis or treatment of disease.

[0027] A partition may contain different capture oligonucleotides with different capture sequences to bind to different RNA molecules or genomic DNA molecules, or both. Furthermore, a partition may contain the same capture oligonucleotide with the same capture sequence to bind to the same RNA molecule or genomic DNA molecule. In one embodiment, a partition may contain the same capture oligonucleotide for a target RNA molecule to bind to an RNA molecule with (at least partially) the same sequence, and a partition may further contain the same capture oligonucleotide for a target genomic DNA to bind to a DNA molecule with (at least partially) the same sequence. Some capture oligonucleotides may be specific for RNA and some may be specific for genomic DNA. In one embodiment, the capture sequence of the capture oligonucleotide specific for RNA binding is an oligo d(T) sequence. In contrast, the capture sequence of the capture oligonucleotide specific for genomic DNA may be a random hexamer.

[0028] In another embodiment, different partitions may contain capture oligonucleotides that contain the same or different capture sequences. In a preferred embodiment, the capture oligonucleotides among multiple partitions contain the same capture sequence.

[0029] The capture oligonucleotide may further comprise a barcode sequence (referred to as a barcoded capture oligonucleotide), a primer binding sequence, or both.

[0030] Such oligonucleotides are also referred to as barcoded capture oligonucleotides. The barcode sequence may be a cellular barcode and / or a unique molecular identifier (UMI). In one embodiment of the present invention, the capture oligonucleotide for the target genomic DNA molecule may comprise a barcode sequence (UMI or cellular barcode). Such a barcode may preferentially be a cellular barcode. In another embodiment of the present invention, the capture oligonucleotide for the target RNA and the target genomic DNA may comprise a barcode sequence (UMI or cellular barcode). This barcode sequence may be the same (cellular barcode) or different (UMI). In a preferred embodiment, the barcode sequence may be a cellular barcode. In other words, the barcode sequence may be the same for the capture oligonucleotide for the target RNA and the target genomic DNA.

[0031] The primer binding sequence may contain sequencing and / or amplification primer binding sites to allow for nucleic acid amplification and / or sequencing reactions for downstream applications.

[0032] The barcode oligonucleotide comprises at least one barcode sequence. The barcode sequence may be the same or different within one partition. In addition, the barcode sequence may be the same or different between multiple partitions. In another embodiment, the barcode sequences of the barcode oligonucleotides prepared in step a) are different between partitions, and the barcode sequences of the barcode oligonucleotides contained in the partitions are the same. Based on this, the barcode oligonucleotides prepared in step a) may be different between partitions, and the barcode oligonucleotides contained in the partitions may be the same.

[0033] The barcode sequence may be a cell barcode and / or a unique molecular identifier (UMI). In one partition, the barcode oligonucleotide may contain a sequence encoding the same cell barcode and a sequence encoding a different UMI. The sequence of the cell barcode may differ among multiple partitions. The barcode oligonucleotide may further contain a primer binding sequence. The primer binding sequence may contain a sequencing and / or amplification primer binding site to enable nucleic acid amplification and / or sequencing reactions as downstream applications.

[0034] The barcode oligonucleotide may further comprise a sequence encoding a template-switching oligonucleotide or a sequence suitable for template switching to perform a template-switching reaction. Thus, it can be said that the barcode oligonucleotide can function as a template-switching oligonucleotide (Figures 3 / 4).

[0035] The barcode oligonucleotide may be a single-stranded or double-stranded nucleic acid. In one embodiment of the present invention, the target nucleic acid may be RNA, the barcode oligonucleotide may include a template-switching oligonucleotide, and the barcode oligonucleotide may be single-stranded.

[0036] An important feature of the method of the present invention is a solid support comprising capture oligonucleotides for target RNA molecules and target genomic DNA molecules, and barcode oligonucleotides, which are different from each other.

[0037] The solid support may be a plate or beads. In one embodiment, the solid support may be beads (FIG. 4). A common example of beads is a microbead, which may be color-coded or magnetic, or both. In a preferred embodiment, the beads are color-coded. In one embodiment, a partition comprises at least one bead. In a preferred embodiment, a partition comprises one single bead.

[0038] In another embodiment, the solid support can be a plate (Figure 5). Common examples of plates are nanoplates or microplates. The surface of such plates can be structured or patterned. A microplate can be a flat plate or a well plate containing multiple partitions. Each partition contains multiple capture oligonucleotides (for target RNA molecules and target genomic DNA molecules) and barcode oligonucleotides. Partitioning cells according to the present invention means that each partition within the plate is considered a solid support. Diffusion between partitions can be prevented by the physical walls of the wells or the chemical properties of the plate, such as hydrophilic and hydrophobic regions. Different partitions can be sealed with foil or other barriers. Common examples of such plates are 96-well or 384-well plates, ICELL8 nanowells (Goldstein et al. 2017), or the SCOPE-chip® used by Singleron Biotechnologies (Nanjing, Jiangsu Province, China).

[0039] The capture oligonucleotides (for target RNA molecules and target genomic DNA molecules) and barcode oligonucleotides can be directly (covalently bonded) or indirectly (electrostatic interaction) linked to the solid support. Direct linkage can be achieved through a linker, such as a polyethylene glycol chain or other molecules such as those disclosed in EP 3037821. These linkers may have branches or multiple functional sites to increase the number of oligonucleotides bound to the solid surface. In addition, the linker may be cleavable using specific enzymatic or chemical conditions.

[0040] The link between the solid support and the oligonucleotide may be via electrostatic interaction. A common example is the use of biotinylated oligonucleotides that bind to streptavidin on the surface of the solid support. This link can be released by competitive addition of biotin.

[0041] In another embodiment, the oligonucleotide may contain an enzymatic cleavage site that facilitates release of the oligonucleotide from the bead.

[0042] In addition, other additional oligonucleotides mentioned in subsequent variations of the invention can be linked to the solid support in the same manner.

[0043] In another embodiment, the solid surface can be coated with a polymer that affects the diffusion behavior or movement of nucleic acid molecules bound to the capture oligonucleotides. A typical example is illustratively disclosed in U.S. Patent Application Publication No. 2009 / 0181370.

[0044] According to the present invention, a plurality of cells and the solid support are partitioned. Standard procedures for partitioning include droplet formation or microwells. This can be performed by standard procedures known in the art. Examples of protocols for partitioning cells using droplets or microwells are the Drop-seq approach (Macosco et al., 2015) or the Smart-Seq approach (Ramskold et al., 2012), respectively. In addition, commercially available devices such as the 10x Genomics Chromium Controller (10x Genomics, Pleasanton, CA, USA) can be used to partition components into droplets.

[0045] According to the present invention, a plurality of cells containing target nucleic acids and at least one solid support containing capture oligonucleotides (for target RNA molecules and target genomic DNA molecules) and barcode oligonucleotides are partitioned. This generates a plurality of partitions. A partition includes a cell (single cell) and a solid support containing capture oligonucleotides (for target RNA molecules and target genomic DNA molecules) and barcode oligonucleotides. The term capture oligonucleotides and barcode oligonucleotides can be interpreted as a plurality of capture oligonucleotides and barcode oligonucleotides.

[0046] In one embodiment of the present invention, the partitions can be droplets containing cells and beads (as solid supports) containing capture oligonucleotides and barcode oligonucleotides. The droplets can be generated according to protocols known in the art.

[0047] In another embodiment, the solid support can be a plate containing different compartments / partitions, each containing capture oligonucleotides (for target RNA molecules and target genomic DNA molecules) and barcode oligonucleotides. Cells are deposited onto the plate such that each partition on the plate contains one cell.

[0048] Of the plurality of partitions, more than 1%, more than 10%, more than 50% of the partitions have a given composition. In preferred embodiments, at least 50% of the partitions comprise a solid support and a single cell.

[0049] It is understood that the partitions further comprise reagents required for the reactions of the methods and embodiments thereof. Thus, the partitions may comprise reaction buffers and enzymes, such as enzymes for amplification, ligation, fragmentation, whole genome amplification, etc.

[0050] In the next step (step c) of the method of the present invention, cells are lysed in the partition. As a result, nucleic acid molecules are released into the partition, and a mixture containing target nucleic acid and non-target nucleic acid is obtained. More specifically, it is a mixture of target RNA molecules and non-target RNA molecules. It is understood that the mixture further contains target genomic DNA molecules and non-target genomic DNA molecules.

[0051] Lysis of cells can be carried out enzymatically and / or chemically using specific buffer conditions. Several methods and compositions are known in the art. An example of a lysis buffer suitable for subsequent hybridization of RNA (especially mRNA) to capture oligonucleotides is the Lysis / Binding Buffer for Dynabeads™ mRNA Purification Kit (Cat. No. A33562, ThermoFisher Scientific, Waltham, Massachusetts, USA).

[0052] Then, in step d) of the method of the present invention, the released target RNA molecule is hybridized with capture oligonucleotide (corresponding to the target RNA molecule), thereby obtaining the target RNA molecule attached to solid support.Hybridization is achieved by the complementary or partially complementary binding between capture sequence (contained in capture oligonucleotide) and the complementary sequence in target nucleic acid (here RNA).Hybridization conditions are known in the art.

[0053] Next (step e), the partitions are disrupted, separating the non-target RNA molecules from the target RNA molecules attached to the solid support. The disruption of the partitions can be carried out chemically using specific buffer conditions. After the disruption of the partitions, the non-target nucleic acids, such as the non-target RNA molecules, are in the supernatant of the solution, while the target RNA molecules are attached to the solid support. By removing the supernatant, the non-target nucleic acids, such as the non-target RNA molecules, can be separated from the target RNA molecules. This can be done by precipitating the target RNA molecules attached to the solid support and removing the supernatant containing the non-target nucleic acids, such as the non-target RNA molecules. The supernatant can also be removed by pipetting. Other methods can be used, for example, by applying a magnetic field or other methods known in the art.

[0054] Optionally, step e) is followed by a washing step to remove lysis buffer residues and cell debris, and / or other substances that may interfere with subsequent process steps. Buffer conditions are commonly known in the art. In certain embodiments, such a washing step is preferred.

[0055] In step f), nucleic acid synthesis is carried out, with the capture oligonucleotide for the target RNA molecule serving as a primer and the target RNA molecule serving as a template, thereby generating a double-stranded nucleic acid from the target RNA molecule. Techniques and conditions for nucleic acid synthesis and amplification, such as reverse transcription, linear amplification with enzymes such as Phi29, or polymerase chain reaction, are well known in the art.

[0056] In one embodiment of the present invention, a double-stranded nucleic acid derived from a target RNA is generated in step f) by nucleic acid synthesis, which is a reverse transcription reaction involving template switching, and the barcode oligonucleotide serves as the template-switching oligonucleotide. The generated synthetic nucleic acid is called "cDNA."

[0057] The techniques and conditions for template-switching reverse transcription are well known in the art. Examples of template-switching reverse transcription protocols can be found in Zhu et al., 2001 and Wellenreuther et al., 2004. The key elements are a specific polymerase and a template-switching oligonucleotide. Commonly used polymerases are Moloney murine leukemia virus reverse transcriptase (MMLV-RT) and its derivatives, or thermostable group II intron reverse transcriptase (TIGRT).

[0058] It is understood that additional components such as buffers, nucleotides (dNTPs) and polymerases may be added to carry out such reactions.

[0059] In step g), a barcode oligonucleotide is subsequently attached to the double-stranded nucleic acid derived from the target RNA molecule synthesized in step f), thereby generating a barcoded nucleic acid derived from the target RNA molecule. The barcoded nucleic acid derived from the target RNA molecule comprises a capture oligonucleotide (for the target RNA molecule) and the barcode oligonucleotide, and both ends of the double-stranded nucleic acid are attached to the same solid support. The attachment of the barcode oligonucleotide can be carried out by ligation using a ligase, template switching during reverse transcription, or other reactions well known in the art. Common examples of such ligases include T4 DNA ligase, Taq ligase, or equivalent enzymes. Common examples of enzymes that promote reverse transcription include MMLV reverse transcriptase and its derivatives.

[0060] In one embodiment, the target nucleic acid molecule (e.g., derived from the target RNA and genomic DNA) is released from the solid support after step g). In one embodiment, the capture oligonucleotide and the barcode oligonucleotide may be linked to the solid support by a linker, and the linker is cleaved after step g), thereby releasing the target nucleic acid molecule from the solid support. More specifically, the capture oligonucleotide for the target RNA molecule, the capture oligonucleotide for the target DNA molecule, and the barcode oligonucleotide are linked to the solid support by a linker, and the linker is cleaved after step g), thereby releasing the nucleic acid from the solid support.

[0061] The release reaction can be carried out chemically and / or enzymatically by disputing the bond between the solid support and the capture / barcode oligonucleotide. Common techniques are known in the art, for example, as disclosed in EP 3037821. One example would be the use of a photocleavable linker on the oligonucleotide bound to the solid support. The release can also be mediated by a site-specific endonuclease, such as a restriction enzyme. In this embodiment, the capture oligonucleotide and the barcode oligonucleotide are cleaved by the site-specific endonuclease after step g), thereby releasing the barcoded nucleic acid from the solid support. This allows the release of the nucleic acid by the restriction enzyme based on the recognition site incorporated in each oligonucleotide. Another example is the incorporation into the oligonucleotide of specific bases that can be recognized by DNA glycosylases, for example, one or more uracil bases that can be excised by New England Biolab's USER enzyme or thermolabile USER enzyme (catalog M5508 and M5507, New England Biolabs, Ipswich, Massachusetts, USA), thereby generating a nick in the respective nucleic acid strand, allowing the release of the barcoded target nucleic acid molecule.

[0062] The target nucleic acid can then be used in downstream applications such as nucleic acid amplification and sequencing, based on which gene expression analysis, mutation analysis or copy number variation analysis can be performed.

[0063] Target molecule - genomic DNA In a first variant of the invention, steps a to f of the method described in aspect 1 are also performed on a target genomic DNA molecule (Figure 7). It is understood that all embodiments described in the first aspect of the invention also apply to this variant of the invention.

[0064] In this variant of the invention, the mixture obtained in step c) further comprises target genomic DNA molecules and non-target genomic DNA molecules, so that the complete mixture obtained in step c) comprises target genomic DNA molecules and non-target genomic DNA molecules, target RNA molecules and non-target RNA molecules.

[0065] Furthermore, in step d), the target genomic DNA molecule is hybridized with a capture oligonucleotide corresponding to the target genomic DNA molecule, thereby obtaining the target genomic DNA molecule attached to the solid support.As a result, the final result of step d) is the target genomic DNA molecule and the target RNA molecule attached to the same solid support.

[0066] In addition, step e) further separates non-target genomic DNA molecules from the target genomic DNA molecules attached to the solid support, and as a result, in step e) of this variation of the present invention, non-target genomic DNA molecules and non-target RNA molecules are separated from the target genomic DNA molecules and target RNA molecules attached to the same solid support.

[0067] Finally, in step f), double-stranded nucleic acids are generated from the target genomic DNA molecules by nucleic acid synthesis, with the capture oligonucleotides for the genomic DNA molecules acting as primers and the target genomic DNA molecules acting as templates. As a result, the final result of step f) is a double-stranded target RNA and a double-stranded target genomic DNA attached to the same solid support.

[0068] In one embodiment of this variation of the present invention, the target genomic DNA molecule is further subjected to step g) by attaching a barcode oligonucleotide to the double-stranded nucleic acid derived from the target genomic DNA molecule, thereby generating a barcoded nucleic acid derived from the target genomic DNA molecule. As a result, the end result of this embodiment is a barcoded target RNA molecule and a barcoded genomic DNA molecule.

[0069] The barcode oligonucleotide can be attached by ligation in step (g). Ligation can be performed according to methods known in the art and disclosed herein. For optimal results, the double-stranded nucleic acid derived from the target genomic DNA molecule obtained in step f) can be ligated to the double-stranded barcode oligonucleotide. Alternatively, the double-stranded nucleic acid derived from the target genomic DNA obtained in step f) can be ligated to a single-stranded barcode oligonucleotide.

[0070] In one embodiment of the present invention, genomic DNA molecules can be fragmented and dissociated into single strands prior to hybridization (step d). Several techniques for DNA fragmentation are known in the art. DNA fragmentation can be performed enzymatically. Enzymes commonly used for fragmentation are restriction endonucleases or nonspecific endonucleases. Enzymatic fragmentation can also be mediated by CRISPR / Cas9. Non-enzymatic approaches, such as fragmentation by sonication, can also be used. The preferred length of the genomic DNA fragments is 50 to 10,000 nucleotides, 75 to 1,000 nucleotides, or 100 to 250 nucleotides. In a preferred embodiment, the length of the genomic DNA fragments is 50 to 10,000 nucleotides. In another embodiment, at least 80%, 50%, or 10% of the genomic DNA molecules are fragmented.

[0071] Targeted mRNA and genomic DNA and whole genome amplification This second variant of the present invention allows for combined barcoding of target RNA and genomic DNA molecules (Figures 9 and 10). It is understood that all embodiments described in the previous section also apply to this variant. In this variant of the present invention, the capture oligonucleotide for the target genomic DNA molecule functions as a whole genome amplification oligonucleotide and is attached to the solid support by a releasable linker. Therefore, it can be released from the solid support within the partition. Whole genome amplification of the target genomic DNA is then performed, including the steps of releasing the whole genome amplification oligonucleotide from the solid support and performing whole genome amplification using a polymerase with strand displacement activity, where the whole genome amplification oligonucleotide functions as a primer, thereby obtaining amplified target genomic DNA fragments.

[0072] The whole genome amplification oligonucleotide comprises at least one barcode sequence and a sequence that is a whole genome amplification primer sequence. Based on this, the whole genome amplification oligonucleotide functions as a primer for whole genome amplification. The whole genome amplification primer sequence can be a random hexamer sequence, a degenerate primer that binds only to a subset of the whole genome, or even a specific primer sequence. In one embodiment, the whole genome amplification primer sequence within a partition can be different. In a specific embodiment, the whole genome amplification primer sequence is a random hexamer sequence.

[0073] The barcode sequence of the whole genome amplification oligonucleotide can be a cell barcode and / or a unique molecular identifier. In one partition, the barcode whole genome amplification oligonucleotide may contain a sequence encoding the same cell barcode and a sequence encoding a different UMI. In one embodiment, the whole genome amplification oligonucleotide contains the same cell barcode and a different UMI as the barcode oligonucleotide and / or capture oligonucleotide.

[0074] Additionally, the whole genome amplification oligonucleotide may further comprise at least one primer binding sequence, which may include a sequencing and / or amplification primer binding site to enable downstream nucleic acid amplification and / or sequencing reactions.

[0075] The whole genome amplification oligonucleotides are attached to the same solid support via releasable linkers. In this variation of the present invention, the release mechanisms for the whole genome amplification oligonucleotides (capture oligonucleotides for target genomic DNA molecules) and the capture oligonucleotides for target RNA / barcode oligonucleotides are different. The release of the whole genome amplification oligonucleotides (capture oligonucleotides for target genomic DNA molecules) can be achieved chemically and / or enzymatically by reversing the bond between the solid support and the oligonucleotide. General techniques are known in the art, for example, as disclosed in EP 3037821. In one embodiment, the whole genome amplification oligonucleotides are linked to the solid support via a photocleavable linker, which can be cleaved with light. In one embodiment, the linker contains a disulfide bond, which can be cleaved with a reducing agent. In one embodiment, the linker contains a restriction enzyme binding site, which can be cleaved by a specific endonuclease. In one embodiment, the linker contains a uracil base, which can be cleaved by uracil-N-glycosylase and an endonuclease. In one embodiment, the linker comprises an 8-oxo-G base, and the linker is cleaved by formamidopyrimidine [fapy]-DNA glycosylase (Fpg).

[0076] In one embodiment, the whole genome amplification oligonucleotide (capture oligonucleotide for the target genomic DNA molecule) and the capture oligonucleotide / barcode oligonucleotide for the target RNA can be attached to the solid support by a linker, and the cleavable linkers of the whole genome amplification oligonucleotide (capture oligonucleotide for the target genomic DNA molecule) and the capture oligonucleotide / barcode oligonucleotide for the target RNA can be different. In one embodiment, the whole genome amplification oligonucleotide (capture oligonucleotide for the target genomic DNA molecule) and the capture oligonucleotide / barcode oligonucleotide for the target RNA can contain different enzymatic cleavage sequences. In yet another embodiment, the whole genome amplification oligonucleotide (capture oligonucleotide for the target genomic DNA molecule) can contain an enzymatic cleavage sequence, and the capture oligonucleotide / barcode oligonucleotide for the target RNA can be linked to the solid support by a cleavable linker, or vice versa. In this way, specific release of the whole genome amplification oligonucleotide (capture oligonucleotide for the target genomic DNA molecule) is possible.

[0077] This method generates barcoded RNA and genomic DNA molecules, which then undergo two distinct reactions within the partition: (1) The target RNA binds to the complementary capture sequence of the capture oligonucleotide for the target RNA molecule (Figure 9A; 2). Subsequently, a reverse transcription reaction with template switching in step f) is performed, and the barcode oligonucleotide serves as the template switching oligonucleotide (Figure 9B; 5 / 6).

[0078] (2) The target genomic DNA molecule is barcoded by whole genome amplification (after step b) by releasing the whole genome amplification oligonucleotide (capture oligonucleotide for the target genomic DNA molecule) from the solid support (Figure 9A; 2). Whole genome amplification is then performed within the partition, with the released whole genome amplification oligonucleotide (capture oligonucleotide for the target genomic DNA molecule) functioning as a primer and the polymerase being a polymerase with strand displacement activity. The amplified target genomic DNA fragment is not attached to the solid support (Figure 9B; 4).

[0079] Based on this, in step e), a mixture containing target RNA molecules (RNA) attached to a solid support, target genomic DNA molecules not bound to the solid support, and non-target nucleic acid molecules (non-target RNA molecules and non-target genomic DNA molecules) is obtained. The amplified genomic DNA molecules can be separated from the RNA bound to the solid support in step e). After the partition is destroyed, the target genomic DNA molecules are in the supernatant and can be removed. This can then be used in further workflow steps, such as target amplification using target-specific primers and primers specific to the primer binding sites in the barcoded oligonucleotides.

[0080] Several whole genome amplification methods are known in the art, including multiple displacement amplification (MDA), degenerate oligonucleotide PCR (DOP-PCR), and primer extension preamplification (PEP), which result in amplified target genomic DNA molecules containing the whole genome amplification oligonucleotides.

[0081] Whole genome amplification can be performed using polymerases with strand displacement activity. Common examples include Phi29 polymerase, Bst DNA polymerase (large fragment), T4 DNA polymerase, and T7 DNA polymerase. The preferred length of the amplified genomic DNA fragment is 50 to 10,000 nucleotides, 75 to 1,000 nucleotides, or 100 to 250 nucleotides.

[0082] It is understood that in addition to polymerase, standard amplification components such as dNTPs, buffer conditions, etc. are required.

[0083] All definitions, properties and embodiments defined herein with respect to the first aspect of the invention disclosed herein also apply mutatis mutandis in the context of the other aspects of the invention disclosed herein.

[0084] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0085] As used herein, the terms "comprising" or "comprises" are used in reference to compositions, methods and their respective components that are essential to the method or composition, but which may include non-specified elements, whether essential or not.

[0086] As used herein, the term partitioning means dividing a sample or volume into two or more samples or volumes.

[0087] "Bind" and "hybridize," as well as their grammatical abbreviations (exuviates), can be used interchangeably. Hybridization of two nucleic acid strands occurs when they are complementary to each other. Hybridization can occur under conditions known in the art.

[0088] As used herein, the term "complementary" refers to the ability for precise pairing between two nucleotides via Watson-Crick base pairing. To explain, if a nucleotide at a given position in a nucleic acid strand can form a hydrogen bond with a nucleotide in another nucleic acid strand, the two nucleic acids are considered complementary to each other at that position. Complementarity between two single-stranded nucleic acid molecules can be "partial," where only a portion of the nucleotides bind, or complete, where total complementarity exists between the single-stranded molecules.

[0089] As used herein, a "primer" is a single-stranded oligonucleotide made up of nucleotides capable of binding to a complementary nucleic acid sequence. It is understood that all primers described in this invention can serve as a starting point for nucleic acid synthesis / amplification.

[0090] As used herein, the terms "nucleic acid synthesis" and "nucleic acid amplification" can be used interchangeably. The process of nucleic acid synthesis is well known in the art. Briefly, for nucleic acid synthesis, a template nucleic acid, which may be single-stranded or double-stranded, is prepared. When double-stranded nucleic acid is used initially, the first step is to denature it into a single nucleic acid strand (complement and reverse complement) using techniques known in the art. A denaturation step is not required for single-stranded nucleic acid. The next step is to prepare a primer that binds to the complementary region of the nucleic acid strand. The 3' end of the primer is then extended using a polymerase and filled with complementary nucleotides to generate a complementary strand. As a result, a complementary nucleic acid strand is formed. The result of the nucleic acid synthesis reaction is a double-stranded nucleic acid.

[0091] The term "reverse transcription with template switching" is well known in the art. Briefly, a primer is first hybridized to an RNA molecule. This primer serves as a priming site for cDNA synthesis by an enzyme with reverse transcriptase activity. When the enzyme reaches the 5' end of the RNA template, a subset of reverse transcriptases (such as MMLV reverse transcriptase) can add one or more additional nucleotides (mainly deoxycytidines) to the 3' end of the newly synthesized cDNA. A template-switching oligonucleotide then binds to these deoxycytidines. The reverse transcriptase can then switch templates and continue cDNA synthesis. Examples of protocols using reverse transcription and template switching can be found in Zhu et al., 2001 and Wellenreuther et al., 2004.

[0092] The process of "whole genome amplification" is well known in the art. There are several methods for whole genome amplification in the art, including multiple displacement amplification (MDA), degenerate oligonucleotide PCR (DOP-PCR) and primer extension preamplification (PEP). In short, all methods have in common that random or degenerate oligonucleotides are used to prime nucleotide synthesis. However, it is also possible to use specific primers to amplify only specific targets in the genome.

[0093] The terms "oligonucleotide," "nucleic acid," and "nucleic acid molecule" refer to a biological polymer composed of nucleotide monomers covalently linked in a chain. An amplified nucleic acid is sometimes called an "amplicon." Nucleic acids can be DNA or RNA.

[0094] The oligonucleotide according to the present invention may comprise a barcode sequence. The barcode sequence is a short nucleotide sequence for identification purposes. The barcode may be a cellular barcode. All target nucleic acids derived from the same cell are labeled with the same cellular barcode. In addition, the barcode may be a unique molecular identifier (UMI), which labels all nucleic acids with different barcode sequences. The barcode sequence may comprise both a cellular barcode and a UMI.

[0095] A "random hexamer" sequence may contain all four nucleotides (A, T, G, C) at each position, or only a subset of the four nucleotides at each position. Alternative nucleotides, such as uracil, or nucleotide analogs or derivatives, may also be used. Random hexamer oligonucleotides may also contain backbones modified to alter binding behavior, such as locked nucleic acid (LNA) or minor groove binding (MGB) nucleotides. Instead of random hexamer sequences, random or semi-random sequences of different lengths may also be used.

[0096] As used herein, the term "plurality" of something means two or more.

[0097] Example The following examples are intended to provide a more detailed explanation of the present invention, but the present invention is not limited to these examples.

[0098] Example 1: Bridge cDNA synthesis on a solid surface: Template switching is a common approach for introducing specific sequences into the 5' end of newly synthesized cDNA (Zhu et al., 2001): a specific oligonucleotide is incubated with mRNA in the presence of a reverse transcriptase to initiate reverse transcription using the mRNA as a template. This oligonucleotide can contain a stretch of multiple T nucleotides that bind to the poly(A) tail of the mRNA, or it can be specific (reverse complementary) to a single mRNA sequence. As disclosed in Zhu et al., 2001, certain reverse transcriptases can switch templates when they reach the 5' end of the template mRNA, thereby incorporating nucleotides in a reverse-complementary manner into the template-switching oligonucleotide.

[0099] In current technology protocols, both the oligonucleotide for initiating reverse transcription and the template-switching oligonucleotide are provided in solution (e.g., SMARTer® Stranded RNA-Seq Kit, catalog number 634839, Takara Bio Inc.). Single-cell workflows, such as the Chromium Next GEM Single Cell 5' Library & Gel Bead kit v1.1 (PN-100165, 10x Genomics, Pleasanton, CA, USA), also use both in solution (the bead-bound template-switching oligonucleotide is released by dissolving the gel beads). It has also been shown that the release of barcode-containing oligonucleotides delivered by beads is required for efficient cDNA synthesis in current technology single-cell workflows that deliver barcoded oligonucleotides by beads (Klein et al., 2015).

[0100] Therefore, we evaluated whether this limitation could be overcome by attaching both the oligonucleotides for priming the reverse transcription reaction (capture oligonucleotides according to the present invention) and the oligonucleotides for template switching (barcode oligonucleotides, including template-switching oligonucleotides according to the present invention) to a solid surface.

[0101] As a model system, we used roughly 1 μm-sized beads with both oligonucleotides attached to their surfaces. When held in solution, the distance between the two beads is greater than the length of an mRNA molecule. Therefore, the majority of template switching occurs only with primers on the same surface.

[0102] Oligonucleotide beads were produced by synthesizing the following oligonucleotides having SEQ ID NO: 1 and SEQ ID NO: 2. The oligonucleotides were biotinylated at the 5' end.

[0103] These oligonucleotides were bound to streptavidin-coated beads (Dynabeads™ MyOne™ Streptavidin C1, Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's instructions (note: the last three nucleotides of SEQ02 have an RNA backbone).

[0104] These beads, along with a control (see Figure 4 ), were then used in reverse transcription reactions using two different commercially available reverse transcriptases (Maxima RT, catalog number EP0742, ThermoFisher, Waltham, MA, USA, and NEB template switching RT Enzyme Mix, catalog number M0466S, New England Biolabs, Ipswich, MA, USA).

[0105] Reverse transcription was performed using a custom RT buffer, and the reaction was incubated for 45 minutes at 53° C. After reverse transcription, the newly synthesized cDNA was amplified for 18 cycles using Q5 High-Fidelity polymerase (catalog number M0492, New England Biolabs, Ipswich, MA, USA; amplification primers: SEQ ID NO: 3 and SEQ ID NO: 4).

[0106] The yield of the resulting amplified cDNA was assessed using a ThermoFisher Qubit 4 Fluorometer (ThermoFisher, Waltham, MA, USA), and the size distribution was assessed using an Agilent 4200 TapeStation System with D5000 or High Sensitivity D5000 Screen Tape (catalog numbers 5067-5588 and 5067-5592, Agilent, Santa Clara, CA, USA).

[0107] The results are shown in Figure 11: Condition 1, using a bead-bound oligo(dT) primer (a capture oligonucleotide according to the present invention) and a bead-bound template-switching oligonucleotide (a barcode oligonucleotide comprising a template-switching oligonucleotide according to the present invention), surprisingly showed similar yields compared to the control (condition 5), using both oligonucleotides in solution, strongly suggesting that "bridge cDNA synthesis" is possible when suitable buffer conditions are used.

[0108] References Goldstein, LD, Chen, YJ.J., Dunne, J. et al. Massively parallel nanowell-based single-cell gene expression profiling. BMC Genomics 18, 519 (2017). https: / / doi.org / 10.1186 / s12864-017-3893-1 Klein AM, Mazutis L, Akartuna I, Tallapragada N, Veres A, Li V, Peshkin L, Weitz DA, Kirschner MW. Droplet barcoding for single-cell transcriptomics applied to embryonic stem cells. Cell. 2015 May 21;161(5): 1187-1201. doi: 10.1016 / j.cell.2015.04.044. PMID: 26000487; PMCID: PMC4441768. Macosko EZ, Basu A, Satija R, Nemesh J, Shekhar K, Goldman M, Tirosh I, Bialas AR, Kamitaki N, Martersteck EM, Trombetta JJ, Weitz DA, Sanes JR, Shalek AK, Regev A, McCarroll SA. Highly Parallel Genome-wide Expression Profiling of Individual Cells Using Nanoliter Droplets. Cell. 2015 May 21; 161(5): 1202-1214. doi: 10.1016 / j.cell.2015.05.002. PMID: 26000488; PMCID: PMC4481139. Ramskoeld D, Luo S, Wang YC, Li R, Deng Q, Faridani OR, Daniels GA, Khrebtukova I, Loring JF, Laurent LC, Schroth GP, Sandberg R. Full-length mRNA-Seq from single-cell levels of RNA and individual circulating tumor cells. Nat Biotechnol. 2012 Aug;30(8):777- 82. doi: 10.1038 / nbt.2282. Erratum in: Nat Biotechnol. 2020 Mar;38(3):374. PMID: 22820318; PMCID: PMC3467340. Wellenreuther R, Schupp I, Poustka A, Wiemann S; German cDNA Consortium. SMART amplification combined with cDNA size fractionation in order to obtain large full-length clones. BMC Genomics. 2004 Jun 15;5(1):36. doi: 10.1186 / 1471-2164-5-36. PMID: 15198809; PMCID: PMC436056. Zhu YY, Machleder EM, Chenchik A, Li R, Siebert PD. Reverse transcriptase template switching: a SMART approach for full-length cDNA library construction. Biotechniques. 2001 Apr;30(4):892-7. doi: 10.2144 / 01304pf02. PMID: 11314272.

[0109] Brief description of the drawings continued Figure 1A: 1. Partitioning (e.g., encapsulation into droplets) single cells with beads containing oligonucleotides containing capture and barcode sequences; 2. Lysing cells and releasing mRNA Figure 1B: 3. Target mRNA binds to the capture sequence of the oligonucleotide; 4. Removal of the partition / generation of a single reaction compartment; 5. Reverse transcription → extension of the barcoded oligonucleotide → generation of the barcoded nucleic acid Figure 2A: A / B Partition containing capture oligonucleotides (CS), barcode oligonucleotides (BC) attached to the same solid support, and one cell; C Lysis of an isolated single cell to obtain a mixture of target and non-target nucleic acids Figure 2B: D: The target RNA molecule hybridizes to the capture oligonucleotide for the target RNA molecule; E / F: The partition is disrupted and nucleic acid synthesis occurs; G: The barcode is attached, generating a barcoded target nucleic acid attached to both sites on the solid support. Figure 3: A solid support containing a capture oligonucleotide (containing a CS) and a barcode oligonucleotide containing a BC and a TSO; 1. Cells are locally lysed and specific mRNA molecules bind to the capture sequence of the capture oligonucleotide; 2. cDNA is synthesized and barcodes are incorporated by template switching using adjacent barcoded TSOs on the solid support. Figure 4A: 1. A single cell and a single bead within each partition (containing capture nucleotides and barcode oligonucleotides containing BC and TSO) Figure 4B: 2. Cells are lysed, mRNA molecules hybridize to the capture sequence of the capture oligonucleotide, and mRNA binds to the capture sequence (CS) Figure 4C: 3. Combining beads into a single vessel for nucleic acid synthesis (cDNA synthesis) and template switching Figure 5A: 1. Placing cells on a patterned surface containing capture oligonucleotides and barcode oligonucleotides containing BC and TSO sequences (each element has a different barcode) Figure 5B: 2. Lysing cells under conditions that minimize diffusion between different components and allowing mRNA to bind to the capture sequence (CS) Figure 5C: 3. Nucleic acid synthesis (cDNA synthesis) and template switching Figure 6: 1. Cell lysis and capture of mRNA (hybridization to capture sequences); 2. Washing (removal of lysis buffer, cell debris, etc.); 3. Nucleic acid (cDNA) synthesis with template switching. Figure 7A: Cells are lysed and mRNA and genomic DNA are captured; washed (to remove lysis buffer, cell debris, etc.); genomic DNA is extended; bridge cDNA synthesis with template switching is performed. FIG. 7B: (1) Optionally, step g) may be performed on target genomic DNA molecules and target RNA molecules. Figure 8: In a droplet or compartment; single cell + single bead containing a unique barcode → cell is lysed and all target nucleic acid molecules of the cell hybridize to a single bead with a unique barcode; localized lysis on a surface; surface patterning; each region has a capture oligonucleotide with a region-specific barcode → cell is lysed and target nucleic acid molecules of the cell hybridize to a single region with a unique barcode Figure 9A: Single cells and solid surfaces are deposited within a partition; cells are lysed, releasing whole genome amplification oligonucleotides (e.g., random hexamers) within the partition; target mRNA is hybridized to capture sequences (within the partition) Figure 9B: Perform whole genome amplification (MDS) (within the partition); remove (e.g., disrupt) the partition; process all solid surfaces within a single compartment; collect the supernatant containing the barcoded WGA product; perform reverse transcription with template switching. Figure 10: Barcoded whole genome amplification oligonucleotides (e.g., random hexamers) are bound to genomic DNA; whole genome amplification oligonucleotides (e.g., random hexamers) are extended by Phi29 polymerase; whole genome amplification oligonucleotides (e.g., random hexamers) also bind to newly synthesized DNA and are extended by Phi29 polymerase.

Claims

1. 1. A method for generating barcoded target nucleic acids from a plurality of cells, comprising: a. providing a plurality of cells comprising target RNA molecules and at least one solid support comprising capture oligonucleotides for the target RNA molecules and barcode oligonucleotides; b. partitioning the plurality of cells and the solid support such that each cell is contained in a separate partition and each partition comprises a solid support; c. lysing the cells, thereby obtaining a mixture of target and non-target RNA molecules; d. Hybridizing the target RNA molecule to the capture oligonucleotide for the target RNA molecule, thereby obtaining a target RNA molecule attached to the solid support; e. Disrupting the partitions and separating the non-target RNA molecules from the target RNA molecules attached to the solid support; f. generating a double-stranded nucleic acid from the target RNA molecule by nucleic acid synthesis, wherein the capture oligonucleotide serves as a primer and the target RNA molecule serves as a template; g. Attaching the barcode oligonucleotide to the double-stranded nucleic acid derived from a target RNA molecule, thereby generating a barcoded nucleic acid derived from a target RNA molecule. A method comprising: The method, wherein in step a), the plurality of cells further comprises a target genomic DNA molecule, the at least one solid support further comprises a capture oligonucleotide for the target genomic DNA molecule, and the capture oligonucleotides for the target RNA molecule and the target genomic DNA molecule are different from each other.

2. The method of claim 1, wherein the capture oligonucleotide for the target genomic DNA molecule comprises a barcode sequence.

3. The method according to claim 2, wherein in step f), the double-stranded nucleic acid derived from the target RNA is generated by nucleic acid synthesis, which is a reverse transcription reaction involving template switching, and the barcode oligonucleotide functions as a template-switching oligonucleotide.

4. and further performing steps a to f on the target genomic DNA molecule; - the mixture obtained in step c) further comprises target genomic DNA molecules and non-target genomic DNA molecules; In step d), hybridizing the target genomic DNA molecule to the capture oligonucleotide for the target genomic DNA molecule, thereby obtaining a target genomic DNA molecule attached to the solid support; In step e), further separating the non-target genomic DNA molecules from the target genomic DNA molecules attached to the solid support; In step f), a double-stranded nucleic acid is generated from the target genomic DNA molecule by nucleic acid synthesis, with the capture oligonucleotide for the genomic DNA molecule acting as a primer and the target genomic DNA molecule acting as a template.

4. The method according to claim 1, wherein the first and second electrodes are connected to a first electrode.

5. 5. The method of claim 4, wherein the target genomic DNA molecule is fragmented and dissociated into single strands prior to hybridization (step d).

6. 6. The method of claim 5, wherein the fragmented genomic DNA molecules have a length of 50 to 10,000 nucleotides.

7. Furthermore, for the target genomic DNA molecule, G) attaching the barcode oligonucleotide to the double-stranded nucleic acid derived from the target genomic DNA molecule, thereby generating a barcoded nucleic acid derived from the target genomic DNA molecule.

7. The method according to claim 4, wherein step g) is carried out by

8. The method of claim 7, wherein the barcode oligonucleotide is attached to the target genomic DNA molecule by ligation in step (g).

9. 9. The method according to claim 1, wherein the capture oligonucleotide for the target RNA molecule, the capture oligonucleotide for the target DNA molecule, and the barcode oligonucleotide are linked to the solid support by a linker, and the linker is cleaved after step g), thereby releasing the nucleic acids from the solid support.

10. The method of claim 9, wherein the linker is enzymatically cleaved.

11. 11. The method of claim 9 or 10, wherein the capture oligonucleotide for the target RNA molecule, the capture oligonucleotide for the target DNA molecule, and the barcode oligonucleotide are cleaved by a site-specific endonuclease after step g), thereby releasing the nucleic acids from the solid support.

12. the capture oligonucleotides for the target genomic DNA molecules function as whole genome amplification oligonucleotides and are attached to the solid support by releasable linkers; After step c), a. Releasing whole genome amplification oligonucleotides from the solid support; b. Performing whole genome amplification using a polymerase with strand displacement activity, wherein the whole genome amplification oligonucleotides function as primers, thereby obtaining amplified target genomic DNA fragments. performing whole genome amplification of target genomic DNA molecules, 4. The method according to claim 1, wherein the first and second electrodes are connected to a first electrode.

13. 13. The method according to any one of claims 1 to 12, characterized in that the target nucleic acid is selected from the group consisting of nucleic acids encoding a T cell receptor chain or a B cell receptor chain.

14. 14. The method according to any one of claims 1 to 13, characterized in that at least 50% of the partitions contain a solid support and one cell.

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