Barcoding of nucleic acids from single cells

Abstract

The present invention provides a method that makes it possible to separate different workflow steps for barcoding of a target nucleic acid and thus provide optimal reaction conditions for each workflow step, especially a template conversion reaction. Furthermore, the method provides an opportunity to perform a reaction, such as a barcoding reaction, of two different nucleic acid molecules, such as RNA and genomic DNA molecules, from one cell in a single step. The method comprises the steps of: (a) providing a plurality of cells comprising a target RNA molecule and at least one solid support comprising a capture oligonucleotide and a barcode oligonucleotide for the target RNA molecule; (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 RNA molecules and non-target RNA molecules; (d) hybridizing the target RNA molecule to a capture oligonucleotide of the target RNA molecule, thereby obtaining a target RNA molecule attached to the solid support; (e) disrupting the partitions and separating 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 is used as a primer and the target RNA molecule is used as a template; (g) attaching a barcoded oligonucleotide to the double stranded nucleic acid from the target RNA molecule, thereby producing a barcoded nucleic acid from the target RNA molecule; characterized in that, in step a), the plurality of cells additionally comprise a target genomic DNA molecule and the at least one solid support additionally comprises a capture oligonucleotide of the target genomic DNA molecule, and in that the target RNA and the capture oligonucleotide of the target genomic DNA molecule are different.

Description

Technical Field

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

[0002] Methods for analyzing nucleic acids at the single-cell level are increasingly being exploited in biological and biomedical research. Such methods enable understanding of the heterogeneity of tissues or cell populations and can be used to identify cell subpopulations implicated in disease.

[0003] To facilitate single-cell analysis and achieve multiplexing, existing 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 and then incorporate specific barcodes into target nucleic acid molecules within these partitions. Based on this, during cell analysis steps (such as next-generation sequencing), barcoded target nucleic acid molecules from one cell can be distinguished from target nucleic acid molecules from another cell. An example is the analysis of different mRNA transcripts expressed in individual cells in a highly parallel manner.

[0005] However, as shown in FIG1 , the prior art methods have certain disadvantages. When using a method using droplets, it is difficult to add reagents once the droplets are formed. Therefore, all reagents for accessing biological particles (e.g., lysing cells), isolating target molecules (e.g., mRNA molecules), converting target molecules (e.g., cDNA synthesis of mRNA molecules), and incorporating barcodes must be added simultaneously, and the corresponding reactions 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, 10× Genomics, Pleasanton, CA, USA).

[0006] In contrast, methods using small holes enable the addition of reagents during the methods described in the above paragraphs. However, reagent mixtures cannot be exchanged. Therefore, reactants from previous steps may still affect subsequent reactions. An example of such a workflow is the Smart-seq2 method of Picelli et al. (2014).

[0007] In both cases, it was not possible to perform a complete buffer exchange between the different workflow steps and therefore the reactions had to be performed under non-ideal reaction conditions.

[0008] Some methods attempt to circumvent these limitations by using hybridization-based methods to isolate target molecules on barcoded capture molecules coupled to beads. The bound target nucleic acid can then be isolated and the barcode can be incorporated in a subsequent step using ideal reaction conditions. An example of such a workflow is the drop-seq method of Macosko et al. (2015). However, there are several limitations, such as in the case of barcoding all mRNA molecules, this method can only be used to add barcodes via capture sequences (oligo(dT)), so the barcode will be adjacent to the 3' end of the transcript. Another disadvantage of this method is the difficulty in using target-specific primers, which limits reverse transcription to only a subset of mRNA or other RNA species (as opposed to all poly(A) RNA when using oligo(dT) primers), because each specific primer must be attached to the barcode on the bead surface.

[0009] However, for several downstream applications, barcode sequences at the 5' end of the transcript are necessary, such as T cell receptor sequencing using kits such as 10×Genomics single cell immune profile analysis kit. To achieve this, a method called template switching is used (Zhu et al., 2001). The template switching process is an instantaneous process, and therefore must be performed when the mRNA molecules of different cells are still partitioned. Therefore, when the cells are partitioned (in droplets or wells), at least two process steps (cell lysis and reverse transcription with template switching) must be performed. Both reactions must therefore be performed under non-ideal conditions. In particular, for template switching reactions, specific conditions (such as buffer conditions) are required to work properly and produce optimal results. This is one of the main limitations in the prior art.

[0010] Based on these limitations, the workflow of the prior art also cannot provide a solution for combining the analysis of different target nucleic acid molecules such as mRNA and genomic DNA from the same cell in a single step. Such a method would provide a great improvement because on the one hand less cell material would be required, and on the other hand, the method would facilitate the analysis and direct comparison of mRNA and genomic DNA from the same cell.

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

[0012] Our researchers unexpectedly found a method that enables to separate the different workflow steps for barcoding of target nucleic acids and thus provide optimal reaction conditions for each workflow step, especially the template switching reaction. Furthermore, the method provides the opportunity to perform reactions, such as barcoding reactions, of two different nucleic acid molecules, such as RNA and genomic DNA molecules, from one cell in a single step.

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

[0014] In a first variant of the invention, steps af) are also performed on the target genomic DNA molecules, thereby producing double-stranded target genomic DNA molecules attached to the same solid support as the double-stranded nucleic acid from the target RNA molecule. Optionally, step g) can be performed to obtain barcoded double-stranded target genomic DNA molecules. The final result of the method is thus a barcoded target nucleic acid (RNA and DNA) attached to the same solid support with a site.

[0015] In a second variation, the invention describes a method for generating barcoded target RNA molecules and simultaneously performing whole genome amplification of target genomic DNA molecules. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1A / B: Standard single-cell cDNA barcoding workflow: (1) In the first step, a single cell and a single bead are compartmentalized in a partition (e.g., a droplet or well). The bead contains an oligonucleotide containing a unique barcode (each oligonucleotide on the bead has the same barcode, but no other bead / partition will have the same barcode in this experiment) and an oligo(dT) oligonucleotide (SEQ ID NO:6). (2) The cell is lysed. (3) mRNA molecules hybridize to the oligo(dT) oligonucleotide (SEQ ID NO:6) on the bead via their poly A tails (SEQ ID NO:5). (4) The partition is disrupted (e.g., in the case of a droplet-based approach, chemicals are used to disrupt the emulsion). The mRNA remains bound to the bead. (5) A reverse transcription reaction is performed on the mRNA bound to the bead (batch). During this period, barcoded cDNA molecules are produced by extending the barcoded oligonucleotide on the bead.

[0017] Fig. 2: Principle of the method of the present invention (exemplary mRNA analysis). Step A / B) shows a partition comprising a capture oligonucleotide (capture oligonucleotide of target RNA and target genomic DNA), a barcode oligonucleotide and a cell attached to the same solid support. The cell is then lysed, thereby releasing the nucleic acid molecules and obtaining a mixture of target nucleic acid and non-target nucleic acid (step C). The following steps of the method are only depicted for target RNA, because the steps for target genomic DNA depend on the specific embodiments of the present invention (these embodiments are shown in the following figures). In D, the target nucleic acid molecule (RNA) is then hybridized with the capture oligonucleotide. The partition is then destroyed and nucleic acid synthesis is performed (E / F). The barcode oligonucleotide is then attached to produce a barcoded target nucleic acid (G). Nucleic acid (from target RNA) is attached to two sites on the solid support.

[0018] Figure 3 : This figure illustrates the principle of target RNA barcoding by incorporating a barcode via template switching. (For clarity, the capture oligonucleotide of the target genomic DNA is not shown / illustrated.) In this example, a solid support comprising a capture oligonucleotide (capture oligonucleotide of the target RNA molecule) and a barcode oligonucleotide and a template switching oligonucleotide sequence is used. (1) Local lysis of a single cell. The target RNA molecule will bind to a specific capture oligonucleotide on the solid support (e.g., an mRNA molecule binds to a capture oligonucleotide comprising a capture sequence containing an oligo(dT) sequence). The partition is destroyed. (2) cDNA is synthesized. During cDNA synthesis, a template switching oligonucleotide contained in the barcode oligonucleotide on the same solid surface is used, thereby incorporating the barcode into the newly synthesized cDNA molecule.

[0019] Figure 4A -C: An example of barcoding cell-specific cDNA using beads as a solid support using the method of the present invention. (For clarity, the capture oligonucleotide of the target genomic DNA is not shown / illustrated.) This example uses beads comprising a capture oligonucleotide and a barcode oligonucleotide (comprising a barcode sequence (BC)) and a template switching oligonucleotide (TSO) sequence. (1) Single cells and single beads are partitioned, each partition containing one cell and one single bead. This example shows two different partitions, each with one cell and one bead. Each bead contains a different barcode sequence (bead-specific barcodes denoted as BC1 and BC2, respectively). (2) Cells are lysed within the partitions, and target mRNA molecules bind to the capture oligonucleotides on the beads. The capture oligonucleotide contains a capture sequence, which can be an oligo (dT) that binds to a poly(A) mRNA molecule or a specific sequence that binds to a subset of mRNA molecules. (3) All beads are merged into a single compartment, for example by breaking an emulsion in the case where partitioning is achieved by oil-in-water droplets. In a single compartment, a reverse transcription reaction is performed using a reverse transcriptase capable of template switching. Template switching will be performed using a template switching oligonucleotide on the same bead. Thus, cDNA molecules produced by the same cell will contain the same barcode (bead-specific barcodes will become cell-specific barcodes).

[0020] Figure 5A-C: An example of barcoding cell-specific cDNA using a patterned surface as a solid support using the method of the present invention. (For clarity, the capture oligonucleotides of the target genomic DNA are not shown / illustrated.) This example uses a patterned surface comprising a capture oligonucleotide (containing a capture sequence CS) and a barcode oligonucleotide (containing a barcode sequence (BC)) and a template switching oligonucleotide sequence (TSO). The surface contains a pattern of these barcode oligonucleotides with specific barcodes, wherein different pattern elements of the surface contain only a single barcode. (1) A single cell is deposited on the surface. The cells are deposited in such a way that each pattern area will contain no more than one cell. (2) The cells are locally lysed under conditions that minimize diffusion from one area to another. This can be achieved by specific buffer conditions, or by creating a temporary barrier between areas (for example, by using a hydrophilic surface and depositing the cells in an aqueous droplet surrounded by oil). The target mRNA molecules will 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 a poly(A) mRNA molecule or a specific sequence that binds to a subset of mRNA molecules. (3) A 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 barcode will become a cell-specific barcode.

[0021] Figure 6: An example of dual barcoding of mRNA molecules using the method of the present invention. (For clarity, the capture oligonucleotide of the target genomic DNA is not shown / illustrated.) This example uses a surface comprising a capture oligonucleotide (containing a capture sequence and a barcode sequence) and a barcode oligonucleotide as a solid support, wherein the barcode oligonucleotide comprises a barcode sequence and a template switching oligonucleotide sequence. The solid support is partitioned with a single cell. (1) The cell is lysed and the target mRNA is bound to the barcoded capture oligonucleotide. (2) The solid surface is washed to remove lysis buffer, cell debris and / or other substances that interfere with subsequent process steps. (3) A reverse transcription reaction is performed using a reverse transcriptase capable of performing a template switching reaction. Reverse transcription is initiated by the barcoded capture oligonucleotide, thereby incorporating the barcode of the capture oligonucleotide into the newly synthesized cDNA. During reverse transcription and template switching, adjacent barcodes (pattern region-specific barcodes) are incorporated into the cDNA. Thus, the barcode (reverse complement) of the template switching oligonucleotide is also incorporated into the newly synthesized cDNA. The resulting cDNA will therefore contain barcodes at both ends. The barcodes of the capture oligonucleotide and the template switching oligonucleotide can be the same, thereby incorporating the same barcode (same orientation or reverse complement) into the cDNA molecule. This is very advantageous for library preparation workflows that require fragmentation because it enables the use of both ends of the cDNA molecule to identify the parental nucleic acid molecule.

[0022] Fig. 7A :An example of barcoding mRNA and genomic DNA molecules at the same time. This example uses a solid surface as a solid support, wherein the solid surface comprises a barcoded first capture oligonucleotide having a capture sequence (CS1) that binds to a target genomic DNA molecule, and a second barcoded capture oligonucleotide having a capture sequence (CS2) that binds to a target RNA molecule. In addition, the solid support comprises a barcode oligonucleotide, wherein the barcode oligonucleotide comprises a barcode sequence and a template switching oligonucleotide sequence. (1) A single cell is partitioned to the solid support. The cells are lysed, and the target genomic DNA molecule binds to the barcoded capture oligonucleotide comprising CS1, and the target RNA molecule binds to the barcoded capture oligonucleotide comprising CS2. (2) The solid surface is washed to remove lysis buffer, cell debris and / or other substances that interfere with subsequent process steps. (3) Using the captured target DNA molecule as a template, the barcoded oligonucleotide comprising CS1 is extended with an enzyme having DNA polymerase activity. (4) Using the captured target RNA molecule as a template, the barcoded oligonucleotide containing CS2 is enzymatically extended using a reverse transcriptase with template switching activity. During reverse transcription, another barcode is incorporated using the barcoded template switching oligonucleotide as a template.

[0023] Figure 7BOptionally, step g) can be performed on target genomic DNA and target RNA molecules. The barcode oligonucleotide can be attached to the target genomic DNA by ligation, while the barcode can be attached to the target RNA by template switching.

[0024] Figure 8 : Examples of methods for associating single cells with specific barcodes. (1) Addition in specific compartments: Single cells and beads containing barcodes are divided into individual partitions. Partitions can be established via droplets surrounded by immiscible fluids or by holes. In the partitions, cells are lysed, nucleic acids are released, and target nucleic acid molecules bind to capture oligonucleotides bound to beads. (2) Local lysis on a surface comprising a patterned surface with multiple areas containing different barcodes: Cells are deposited on the surface (ideally, no more than one cell on each area containing a specific barcode). Next, the cells are lysed under conditions that limit the diffusion of the target molecules, and the target molecules are locally bound. In the next step, the target molecules can be converted into barcoded nucleic acids using the specific barcodes of the corresponding areas.

[0025] Figure 9: Example of a workflow for barcoding genomic DNA and RNA simultaneously. In this example, a solid surface is used, which contains (a) whole genome amplification oligonucleotides, (b) capture oligonucleotides (CS2) that are specific for the target RNA and contain a capture sequence and a barcode sequence, and (c) barcode oligonucleotides that contain template switching oligonucleotides. The solid surface contains multiple oligonucleotides of each type - for simplicity, only a single copy is shown in the figure. (1) A single cell is deposited in a partition, which contains a solid surface with multiple whole genome amplification oligonucleotides (containing 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 partitions. (2) In a first step, the cells in the partitions are lysed, the nucleic acid molecules are released, 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 figure. (3) The target RNA hybridizes to the capture sequence of the barcoded capture oligonucleotide that is still bound to the beads. (4) Amplifying the target genomic DNA by whole genome amplification. Whole genome amplification (MDA) is performed in droplets using whole genome amplification oligonucleotides (see also Fig.10), thereby generating "free" barcoded amplified target DNA. (5) The partitions are removed, for example (in the case of droplet-based partitioning) by removing oil (disruption). The supernatant containing the barcoded amplified genomic DNA is retained and used for downstream applications (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).

[0026] Fig.10 :Whole genome amplification (multiple displacement amplification, MDA) using whole genome amplification oligonucleotides. First, whole genome amplification oligonucleotides are combined with genomic DNA. Whole genome amplification oligonucleotides are used as primers for nucleotide synthesis by Phi 29 polymerase. Phi 29 polymerase has strand displacement activity, so Phi 29 will continue its extension beyond the newly synthesized nucleic acid chain, thereby displacing other newly synthesized nucleic acid chains. Additional whole genome amplification oligonucleotides will be combined with these displaced single-stranded nucleic acid chains, and use those displaced single-stranded nucleic acid chains to produce additional nucleic acid molecules by Phi29. As a result, multiple copies of genomic DNA will be synthesized, and each of these copies will have a barcode at the 5' end of the newly synthesized nucleic acid.

[0027] Fig.11 : shows the results of Example 1 (cDNA amplification on beads). DETAILED DESCRIPTION

[0028] In a first aspect, the present invention provides a method for generating barcoded target nucleic acids from a plurality of cells, the method comprising the steps of: a. providing a plurality of cells comprising a target RNA molecule and at least one solid support, wherein the solid support comprises a capture oligonucleotide and a barcode oligonucleotide of the target RNA molecule; 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 to obtain a mixture of target RNA molecules and non-target RNA molecules; d. hybridizing the target RNA molecule with the capture oligonucleotide of the target RNA molecule, thereby obtaining the target RNA molecule attached to the solid support; e. disrupting the partition 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 a target RNA molecule by nucleic acid synthesis, wherein the capture oligonucleotide is used as a primer and the target RNA molecule is used as a template; g. attaching a barcode oligonucleotide to the double-stranded nucleic acid from the target RNA molecule, thereby generating a barcoded nucleic acid from the target RNA molecule; Characterized in that in step a), the multiple cells further contain target genomic DNA molecules, and the at least one solid support further contains capture oligonucleotides of target genomic DNA molecules, and characterized in that the capture oligonucleotides of target RNA and target genomic DNA molecules are different.

[0029] The methods of the present invention are used to generate barcoded target nucleic acids from multiple cells. The barcoded target nucleic acids can be used in several downstream applications, such as library preparation, next generation sequencing or polymerase chain reaction.

[0030] A plurality of cells may be derived from a sample and include a single cell containing a target nucleic acid. Samples that can be used to generate barcoded target nucleic acids as described herein may be derived from any specimen, such as whole animals, organs, tissue sections, cell aggregates or single cells of invertebrates (e.g., Caenorhabditis elegans, Drosophila melanogaster), vertebrates (e.g., zebrafish, Xenopus laevis) and mammals (e.g., Mus musculus, Homo sapiens). Biological samples may be in the form of tissue sections, cell aggregates, suspended cells, adherent cells or body fluids.

[0031] The target nucleic acid used to generate the barcoded target nucleic acid can be a polynucleotide chain composed of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). More specifically, the target nucleic acid is RNA (such as mRNA) and genomic DNA molecules.

[0032] The target nucleic acid can encode T cell receptor chains and / or B cell receptor chains. In one embodiment, the target RNA molecule can encode T cell receptor chains and / or B cell receptor chains. In another embodiment, the target genomic DNA molecule can encode T cell receptor chains and / or B cell receptor chains. In yet another embodiment of the present invention, the target RNA and target genomic DNA molecules can encode T cell receptor chains and / or B cell receptor chains.

[0033] In another embodiment, the target nucleic acid can encode a biomarker suitable for diagnosis or treatment of a disease. In one embodiment, the target RNA molecule can encode a biomarker suitable for diagnosis or treatment of a disease. In another embodiment, the target genomic DNA molecule can encode a biomarker suitable for diagnosis or treatment of a disease. In yet another embodiment of the present invention, the target RNA and target genomic DNA molecules encode biomarkers suitable for diagnosis or treatment of a disease.

[0034] In yet another embodiment of the present invention, the target nucleic acid molecule can encode a fusion gene produced by chromosomal rearrangement. In one embodiment, the target RNA molecule can encode a fusion gene produced by chromosomal rearrangement. In another embodiment, the target genomic DNA molecule can encode a fusion gene produced by chromosomal rearrangement. In yet another embodiment of the present invention, the target RNA and target genomic DNA molecules encode a fusion gene produced by chromosomal rearrangement.

[0035] In the first step (a) of the method of the present invention, a plurality of cells comprising target nucleic acids (target RNA and target genomic DNA molecules) and at least one solid support comprising capture oligonucleotides and barcode oligonucleotides are provided.

[0036] Capture oligonucleotide (capture oligonucleotide of target RNA and target genomic DNA molecule) comprises capture sequence (CS), and it is complementary to target nucleic acid (target RNA or target genomic DNA molecule) at least in part. Capture sequence can vary according to target RNA or target genomic DNA molecule. Capture oligonucleotide is single-stranded. In one embodiment, capture sequence can be oligo dT sequence, it can bind to mRNA molecule (poly (A) tail). In another embodiment, capture sequence can contain random sequence, it complementarily binds to all target nucleic acid molecule (DNA and / or RNA) species. In another embodiment, the capture sequence of capture oligonucleotide can be complementary to specific target nucleic acid molecule (DNA and / or RNA), thereby enabling to bind to specific target nucleic acid.

[0037] In another embodiment, the capture sequence can be complementary to a specific target sequence, such as the constant region of a T cell or B cell receptor, or a biomarker for diagnosis or treatment of a disease.

[0038] A partition can include different capture oligonucleotides with different capture sequences, in order to combine different RNA or genomic DNA molecules or both. In addition, a partition can include the same capture oligonucleotide with the same capture sequence, in order to combine the same RNA or genomic DNA molecules. In one embodiment, a partition can include (identical) capture oligonucleotides of target RNA molecules, in order to combine (at least in part) with RNA molecules of the same sequence, and the partition can additionally include (identical) capture oligonucleotides of target genomic DNA, in order to combine (at least in part) with DNA molecules of the same sequence. Some capture oligonucleotides can be specific to RNA, and some capture oligonucleotides can be specific to genomic DNA. In one embodiment, the capture sequence of the capture oligonucleotide with specificity to RNA is an oligomeric d (T) sequence. In contrast, the capture sequence of the capture oligonucleotide with specificity to genomic DNA can be a random hexamer.

[0039] In another embodiment, different partitions may contain capture oligonucleotides containing the same or different capture sequences. In a preferred embodiment, the capture oligonucleotides in multiple partitions contain the same capture sequence.

[0040] The capture oligonucleotide can further comprise a barcode sequence (referred to as a barcoded capture oligonucleotide), a primer binding sequence, or both.

[0041] Such oligonucleotides are also referred to as barcoded capture oligonucleotides. The barcode sequence may be a cell barcode and / or a unique molecular identifier (UMI). In one embodiment of the invention, the capture oligonucleotide of the target genomic DNA molecule may comprise a barcode sequence (UMI or cell barcode). Such barcodes may preferably be cell barcodes. In another embodiment of the invention, the capture oligonucleotides of the target RNA and target genomic DNA may comprise a barcode sequence (UMI or cell barcode). The barcode sequence may be the same (cell barcode) or different (UMI). In a preferred embodiment, the barcode sequence may be a cell barcode. In other words, the barcode sequence may be the same for the capture oligonucleotides of the target RNA and target genomic DNA.

[0042] The primer binding sequence may comprise a sequencing and / or amplification primer binding site to enable nucleic acid amplification and / or sequencing reactions as downstream applications.

[0043] The barcode oligonucleotide comprises at least one barcode sequence. The barcode sequence may be the same or different within a partition. In addition, the barcode sequence may be the same or different between multiple partitions. In another embodiment, the barcode sequence of the barcode oligonucleotide provided in step a) is different between partitions, and the barcode sequence of the barcode oligonucleotides contained in the partitions is the same. Based on this, the barcode oligonucleotide provided in step a) may be different between partitions, and the barcode oligonucleotides contained in the partitions may be the same.

[0044] The barcode sequence may be a cell barcode and / or a unique molecular identifier (UMI). In one partition, the barcode oligonucleotide may comprise a sequence encoding the same cell barcode and a sequence encoding a different UMI. The sequence of the cell barcode may be different between multiple partitions. The barcode oligonucleotide may further comprise a primer binding sequence. The primer binding sequence may comprise a sequencing and / or amplification primer binding site to enable nucleic acid amplification and / or sequencing reactions as downstream applications.

[0045] The barcode oligonucleotide may further comprise a sequence encoding a template switching oligonucleotide or a sequence suitable for template switching so as to perform a template switching reaction. Therefore, it can be said that the barcode oligonucleotide can be used as a template switching oligonucleotide ( Figure 3 / 4).

[0046] The barcode oligonucleotide may be a single-stranded or double-stranded nucleic acid. In one embodiment of the invention, the target nucleic acid may be RNA, and the barcode oligonucleotide may comprise a template-switching oligonucleotide, wherein the barcode oligonucleotide may be single-stranded.

[0047] A key feature of the method of the present invention is that the solid support includes capture oligonucleotides and barcode oligonucleotides for target RNA and target genomic DNA molecules. The capture oligonucleotides for the target RNA and target genomic DNA molecules are different.

[0048] The solid support can be a plate or a bead. In one embodiment, the solid support can be a bead (Fig. 4). Common examples of beads are microbeads, which can be color-coded or magnetic or both. In a preferred embodiment, the bead is color-coded. In one embodiment, the partition comprises at least one bead. In a preferred embodiment, the partition comprises a single bead.

[0049] 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 comprising a plurality of partitions. Each partition comprises a plurality of capture oligonucleotides (capture oligonucleotides of target RNA and target genomic DNA molecules) and a barcode oligonucleotide. Partitioning cells according to the present invention means that each partition within the plate is counted as a solid support. Diffusion between partitions can be prevented by the physical walls of the wells or by the chemical properties of the plate (e.g., by hydrophilic and hydrophobic regions). Different partitions can be sealed by foil or another barrier. Common examples of such plates are 96-well or 384-well plates, ICELL8 nanopores (Goldstein et al., 2017) or SCOPE- used by Singleron Biotechnologies (Nanjing, Jiangsu, China).

[0050] Capture oligonucleotide (capture oligonucleotide of target RNA and target genomic DNA molecule) and barcode oligonucleotide can be directly (covalently) or indirectly (electrostatic interaction) connected to solid support. Direct connection can be realized by joint such as polyethylene glycol chain or other molecules (such as disclosed in EP3037821A1). These joints can have side chains or multiple functional sites for increasing the number of oligonucleotides attached to the surface of solid surface. In addition, joints can be cut using specific enzymatic or chemical conditions.

[0051] The connection between the solid support and the oligonucleotide can be through electrostatic interactions. A common example is the use of a biotinylated oligonucleotide bound to streptavidin on the surface of the solid support. The connection can be released by competitive addition of biotin.

[0052] In another embodiment, the oligonucleotide may contain an enzyme cleavage site that facilitates release of the oligonucleotide from the beads.

[0053] Besides this, other additional oligonucleotides mentioned in the subsequent variants of the present invention can be attached to the solid support in the same manner.

[0054] In another embodiment, a solid surface can be coated with a polymer to affect the diffusion behavior or movement of nucleic acid molecules bound to the capture oligonucleotide. Common examples are exemplarily disclosed in US 2009 / 0181370 A1.

[0055] According to the present invention, a plurality of cells and the solid support are partitioned. Standard procedures for partitioning are, for example, droplet formation or microwells. This can be accomplished by standard procedures known in the art. Exemplary schemes for partitioning cells using droplets or microwells are the Drop-seq method (Macosco et al., 2015) or the Smart-Seq method (Ramskold et al., 2012), respectively. In addition, commercially available devices such as the 10×Genomics Chromium Controller (10×Genomics, Pleasanton, CA, USA) can be used to partition components into droplets.

[0056] According to the present invention, multiple cells comprising target nucleic acid and at least one solid support comprising capture oligonucleotides (capture oligonucleotides of target RNA and target genomic DNA molecules) and barcode oligonucleotides are partitioned. Thus multiple partitions are produced. The partition comprises cells (single cells) and a solid support, and the solid support comprises capture oligonucleotides (capture oligonucleotides of target RNA and target genomic DNA molecules) and barcode oligonucleotides. The wording capture oligonucleotides and barcode oligonucleotides can be interpreted as multiple capture oligonucleotides and barcode oligonucleotides.

[0057] In one embodiment of the invention, the partitions may be droplets comprising cells and beads containing capture oligonucleotides and barcode oligonucleotides as solid supports. Droplets may be generated according to protocols known in the art.

[0058] In another embodiment, the solid support can be a plate comprising different compartments / partitions, each compartment / partition comprising capture oligonucleotides (capture oligonucleotides of target RNA and target genomic DNA molecules) and barcode oligonucleotides. Cells are deposited on the plate in a manner that each partition on the plate comprises one cell.

[0059] In the plurality of partitions, more than 1%, more than 10%, more than 50% of the partitions have a given composition. In a preferred embodiment, at least 50% of the partitions comprise a solid support and one cell.

[0060] It should be understood that the partition further comprises the reagents required for the reactions of the method and embodiments thereof. The partition may thus comprise reaction buffers and enzymes, such as enzymes for amplification, ligation, fragmentation, whole genome amplification.

[0061] In the next step (step c) of the method of the present invention, cells are lysed in the partitions. Therefore, nucleic acid molecules are released into the partitions, and a mixture comprising target nucleic acid and non-target nucleic acid is obtained. More specifically, a mixture of target RNA and non-target RNA molecules. It is to be understood that the mixture further comprises target genomic DNA and non-target genomic DNA molecules.

[0062] Lysis of cells can be performed enzymatically and / or chemically using specific buffer conditions. Several methods and compositions are known in the art. Examples of lysis buffers compatible with subsequent hybridization of RNA (especially mRNA) to capture oligonucleotides are Dynabeads TM Lysis / binding buffer of the mRNA purification kit (Cat. No. A33562, ThermoFisher Scientific, Waltham, MA, USA).

[0063] Subsequently, in step d) of the method of the present invention, the released target RNA molecule is hybridized with a capture oligonucleotide (capture oligonucleotide of the target RNA molecule), thereby obtaining a target RNA molecule attached to a solid support. Hybridization is achieved by combining the complementary or partially complementary sequence of the capture sequence (contained in the capture oligonucleotide) and the target nucleic acid (here, RNA). Hybridization conditions are known in the art.

[0064] Subsequently (step e), the partition is destroyed, and the non-target RNA molecules are separated from the target RNA molecules attached to the solid support. The destruction of the partition can be carried out using specific buffer conditions chemistry. After the partition is destroyed, non-target nucleic acids such as non-target RNA molecules are in the supernatant of the solution, and the target RNA molecules are attached to the solid support. By removing the supernatant, non-target nucleic acids such as non-target RNA molecules can be separated from the target RNA molecules. This can be accomplished by precipitating the target RNA molecules attached to the solid support and removing the supernatant comprising non-target nucleic acids such as non-target RNA molecules. The supernatant can be removed by pipetting. Other methods can be, for example, applying a magnetic field or other methods known in the art.

[0065] Optionally, a washing step is performed after step e) to remove residues of the lysis buffer and cell debris and / or other substances that interfere with subsequent process steps. Buffer conditions are well known in the art. In a particular embodiment, such washing steps are preferred.

[0066] In step f), nucleic acid synthesis is performed, wherein the capture oligonucleotide of the target RNA molecule is used as a primer, and the target RNA molecule is used 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 Phi 29, or polymerase chain reaction are well known in the art.

[0067] In one embodiment of the invention, double-stranded nucleic acid from the target RNA is generated in step f) by nucleic acid synthesis, which is a reverse transcription reaction with template switching, wherein the barcode oligonucleotide is used as a template switching oligonucleotide. The synthetic nucleic acid generated is referred to as "cDNA".

[0068] Techniques and conditions for reverse transcription reactions with template switching are well known in the art. Examples of protocols for reverse transcription with template switching can be found in Zhu et al., 2001 and Wellenreuther et al., 2004. The key elements are specific polymerases and template switching oligonucleotides. Commonly used polymerases are Moloney murine leukemia virus reverse transcriptase (MMLV-RT) and its derivatives, or thermostable group II intron reverse transcriptase (TIGRT).

[0069] It will be appreciated that additional components may be added in order to conduct such reactions, such as buffers, nucleotides (dNTPs), and polymerases.

[0070] In step g), the next step is to attach the barcode oligonucleotide to the double-stranded nucleic acid from the target RNA molecule synthesized in step f), thereby producing the barcoded nucleic acid from the target RNA molecule. The barcoded nucleic acid from the target RNA molecule comprises a capture oligonucleotide (capture oligonucleotide of the target RNA molecule) and the barcode oligonucleotide, wherein the double-stranded nucleic acid is attached to the same solid support with two ends. The attachment of the barcode oligonucleotide can be completed by template switching during the connection, reverse transcription reaction, or other reactions known in the art by a ligase. Common examples of such ligases are, for example, T4 DNA ligase, Taq ligase, or equivalent enzymes. Common examples of enzymes that promote reverse transcription are MMLV reverse transcriptase and derivatives thereof.

[0071] In one embodiment, the target nucleic acid molecules (e.g., target nucleic acid molecules from target RNA and genomic DNA) are released from the solid support after step g). In one embodiment, the capture oligonucleotide and the barcode oligonucleotide can be connected to the solid support via a connector, wherein the connector is cut after step g), thereby releasing the target nucleic acid molecules from the solid support. More specifically, the capture oligonucleotide of the target RNA molecule, the capture oligonucleotide of the target DNA molecule, and the barcode oligonucleotide are connected to the solid support via a connector, wherein the connector is cut after step g), thereby releasing the nucleic acid from the solid support.

[0072] The release reaction can be carried out chemically and / or enzymatically by destroying the combination between the solid support and the capture / barcode oligonucleotide. Common techniques are known in the art, such as disclosed in EP3037821A1. An example can be to use a photocleavable joint in the oligonucleotide coupled to the solid support. The release can also be mediated by site-specific endonucleases such as restriction enzymes. In this embodiment, the capture oligonucleotide and the barcode oligonucleotide are cut by site-specific endonucleases after step g), thereby releasing the barcoded nucleic acid from the solid support. This is based on the recognition site incorporated into the corresponding oligonucleotide, which enables the nucleic acid to be released by restriction enzymes. Another example is to incorporate specific bases into the oligonucleotide that can be recognized by a DNA glycosylase, such as one or more uracil bases that can be cleaved by USER enzyme or thermolabile USER enzyme from New England Biolabs (Catalog Nos. M5508 and M5507, New England Biolabs, Ipswich, MA, USA), thereby creating a nick in the corresponding nucleic acid chain, thereby enabling the release of a barcoded target nucleic acid molecule.

[0073] The target nucleic acid can then be used for downstream applications such as nucleic acid amplification and sequencing. Based on this, gene expression analysis, mutation analysis or copy number variation analysis can be performed.

[0074] Target molecule - genomic DNA In the first variant of the invention, the target genomic DNA molecules are also subjected to steps af of the method described in aspect 1 (Figure 7). It is to be understood that all embodiments described in the first aspect of the invention also apply to this variant of the invention.

[0075] In this variant of the invention, the mixture obtained in step c) further comprises target genomic DNA and non-target genomic DNA molecules. Therefore, the complete mixture obtained in step c) comprises target genomic DNA and non-target genomic DNA molecules, target RNA and non-target RNA molecules.

[0076] In addition, in step d), the target genomic DNA molecule is hybridized with the capture oligonucleotide of the target genomic DNA molecule, thereby obtaining the target genomic DNA molecule attached to the solid support. Therefore, the final result of step d) is the target genomic DNA molecule and the target RNA molecule attached to the same solid support.

[0077] Besides this, in step e), non-target genomic DNA molecules are additionally separated from the target genomic DNA molecules attached to the solid support.

[0078] Thus, in step e) of this variant of the invention, non-target genomic DNA molecules and non-target RNA molecules are separated from target genomic DNA and target RNA molecules attached to the same solid support.

[0079] Finally, in step f), double-stranded nucleic acids from the target genomic DNA molecules are generated by nucleic acid synthesis, wherein the capture oligonucleotides of the genomic DNA molecules are used as primers and the target genomic DNA molecules are used as templates. Therefore, the final result of step f) is double-stranded target RNA and double-stranded target genomic DNA attached to the same solid support.

[0080] In one embodiment of this variant of the invention, the target genomic DNA molecule is additionally subjected to step g) by attaching a barcode oligonucleotide to a double-stranded nucleic acid from the target genomic DNA molecule, thereby producing a barcoded nucleic acid from the target genomic DNA molecule. Thus, the final result of this embodiment is a barcoded target RNA and a barcoded genomic DNA molecule.

[0081] The barcode oligonucleotide can be attached by connection in step g). Connection can be performed according to methods known in the art and as disclosed herein. For best results, the double-stranded nucleic acid from the target genomic DNA molecule obtained in step f) can be connected to the double-stranded barcode oligonucleotide. However, the double-stranded nucleic acid from the target genomic DNA obtained in step f) can also be connected to a single-stranded barcode oligonucleotide.

[0082] In one embodiment of the present invention, the genomic DNA molecules can be fragmented and dissociated into single strands before hybridization (step d). Several techniques for DNA fragmentation are known in the art. DNA fragmentation can be performed enzymatically. Common enzymes used for fragmentation are restriction endonucleases or non-specific endonucleases. Enzymatic fragmentation can also be mediated by CRISPR / Cas9. Non-enzymatic methods, such as fragmentation by ultrasonic treatment, can also be used. The preferred length of the genomic DNA fragment is 50-10,000 nucleotides, 75-1000 nucleotides, or 100 to 250 nucleotides. In a preferred embodiment, the length of the genomic DNA fragment is 50-10,000 nucleotides. In another embodiment, at least 80%, 50% or 10% of the genomic DNA molecules are fragmented.

[0083] Target mRNA and genomic DNA and whole genome amplification This second variant of the present invention enables combined barcoding of target RNA and genomic DNA molecules (Figures 9, 10). It is to be understood that all embodiments described in the previous sections also apply to this variant. In this variant of the present invention, the capture oligonucleotide of the target genomic DNA molecule is used as a whole genome amplification oligonucleotide and is attached to a solid support by a releasable joint. Therefore, it can be released from the solid support in the partition. The whole genome amplification of the target genomic DNA is then performed, comprising the following steps: releasing the whole genome amplification oligonucleotide from the solid support; performing whole genome amplification using a polymerase with strand displacement activity, wherein the whole genome amplification oligonucleotide is used as a primer, thereby obtaining the amplified target genomic DNA fragment.

[0084] The whole genome amplification oligonucleotide comprises at least one barcode sequence and a sequence as a whole genome amplification primer sequence. Based on this, the whole genome amplification oligonucleotide is used as a primer for whole genome amplification. The whole genome amplification primer sequence can be a random hexamer sequence, a degenerate primer that is only combined with a subset of the whole genome, or even a specific primer sequence. In one embodiment, the whole genome amplification primer sequence in a partition can be different. In a specific embodiment, the whole genome amplification primer sequence is a random hexamer sequence.

[0085] The barcode sequence of the whole genome amplification oligonucleotide can be a cellular barcode and / or a unique molecular identifier. In one partition, the barcode whole genome amplification oligonucleotide can include a sequence encoding the same cellular barcode and a sequence encoding a different UMI. In one embodiment, the whole genome amplification oligonucleotide includes the same cellular barcode as the barcode and / or capture oligonucleotide and a different UMI.

[0086] In addition, the whole genome amplification oligonucleotide may further comprise at least one primer binding sequence. The primer binding sequence may comprise a sequencing and / or amplification primer binding site to enable nucleic acid amplification and / or sequencing reactions as downstream applications.

[0087] The whole genome amplification oligonucleotide is attached to the same solid support by a releasable joint. In this variant of the present invention, the release mechanism of the whole genome amplification oligonucleotide (capture oligonucleotide of target genome DNA molecule) and the capture oligonucleotide / barcode oligonucleotide of target RNA is different. The release of the whole genome amplification oligonucleotide (capture oligonucleotide of target genome DNA molecule) can be carried out chemically and / or enzymatically by destroying the combination between the solid support and the oligonucleotide. Common techniques are known in the art, such as disclosed in EP3037821A1. In one embodiment, the whole genome amplification oligonucleotide is connected to the solid support via a photocleavable joint, and the joint can be cut with light. In one embodiment, the joint contains a disulfide bond, and the joint can be cut with a reducing agent. In one embodiment, the joint contains a restriction enzyme binding site, and the joint is cut by a specific endonuclease. In one embodiment, the joint contains a uracil base, and the joint is cut by uracil-N-glycosylase and endonuclease. In one embodiment, the linker contains an 8-oxo-G base and the linker is cleaved by formamidopyrimidine [fapy]-DNA glycosylase (Fpg).

[0088] In one embodiment, the whole genome amplification oligonucleotide (capture oligonucleotide of target genome DNA molecule) and the capture oligonucleotide / barcode oligonucleotide of target RNA can be attached to the solid support by a joint, wherein the whole genome amplification oligonucleotide (capture oligonucleotide of target genome DNA molecule) and the capture oligonucleotide / barcode oligonucleotide of target RNA are different. In one embodiment, the whole genome amplification oligonucleotide (capture oligonucleotide of target genome DNA molecule) and the capture oligonucleotide / barcode oligonucleotide of target RNA can include enzyme cleavage sequences, which are different. In another embodiment, the whole genome amplification oligonucleotide whole genome amplification oligonucleotide (capture oligonucleotide of target genome DNA molecule) can include enzyme cleavage sequences, and the capture oligonucleotide / barcode oligonucleotide of target RNA can be connected to a solid support by a joint, and the joint can be cut, or vice versa. Thus enabling specific release of the whole genome amplification oligonucleotide (capture oligonucleotide of target genome DNA molecule).

[0089] In this method, barcoded RNA and genomic DNA molecules are generated. Based on this, two different reactions are performed within the partition: (1) The target RNA is complementary to the capture sequence of the capture oligonucleotide of the target RNA molecule ( Fig.9A ; 2). This is followed by a reverse transcription reaction with template switching in step f), wherein the barcode oligonucleotide is used as the template switching oligonucleotide ( Fig. 9B ; 5 / 6).

[0090] (2) The target genomic DNA molecules will be barcoded via whole genome amplification (after step b) by releasing whole genome amplification oligonucleotides (capture oligonucleotides of target genomic DNA molecules) from the solid support ( Fig.9A ; 2). Whole genome amplification is then performed within the partition, wherein the released whole genome amplification oligonucleotide (capture oligonucleotide of the target genomic DNA molecule) is used as a primer, and wherein the polymerase is a polymerase with strand displacement activity. The amplified target genomic DNA fragments are not attached to the solid support ( Fig. 9B ; 4).

[0091] Based on this, in step e), a mixture is obtained, the mixture comprising: 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 and non-target genomic DNA molecules). 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. They can then be used for further workflow steps, such as targeted amplification using target-specific primers and primers specific to the primer binding sites in the barcoded oligonucleotides.

[0092] Several whole genome amplification methods are known in the art, including multiple displacement amplification (MDA), degenerate oligonucleotide PCR (DOP-PCR) and primer extension pre-amplification (PEP). As a result, amplified target genomic DNA molecules are obtained, which contain the whole genome amplification oligonucleotides.

[0093] Whole genome amplification can be performed using a polymerase with strand displacement activity. Common examples are Phi29 polymerase, Bst DNA polymerase (large fragment), T4 DNA polymerase, and T7 DNA polymerase. The preferred length of the amplified genomic DNA fragment is 50-10,000 nucleotides, 75-1000 nucleotides, or 100 to 250 nucleotides.

[0094] It is to be understood that in addition to the polymerase, standard amplification components such as dNTPs, buffer conditions are also required.

[0095] All definitions, features and embodiments defined herein in relation to the first aspect of the invention as disclosed herein also apply mutatis mutandis in the context of the other aspects of the invention as disclosed herein.

[0096] definition Unless defined otherwise, 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.

[0097] As used herein, the terms "comprising" or "comprises" are used to refer to compositions, methods, and their respective components, which are essential to the method or composition, but are open to including unspecified elements, whether essential or not.

[0098] The term partitioning as used herein means dividing a sample or volume into two or more samples or volumes.

[0099] The words "bind" and "hybridize" and their grammatical variations can be used interchangeably. Hybridization of two nucleic acid strands occurs if the two nucleic acid strands are complementary to each other. Hybridization can occur under conditions known in the art.

[0100] The term "complementarity" as used herein refers to the ability to precisely pair between two nucleotides via Watson and Crick base pairing. In interpretation, if a nucleotide at a given position of a nucleic acid strand is able to form a hydrogen bond with a nucleotide of another nucleic acid strand, the two nucleic acids are considered to be complementary to each other at that position. Complementarity between two single-stranded nucleic acid molecules can be "partial", in which only some nucleotides bind, or it can be complete when there is total complementarity between single-stranded molecules.

[0101] As used herein, a "primer" is a single-stranded oligonucleotide composed of nucleotides that is capable of binding to a complementary nucleic acid sequence. It is to be understood that all primers described in the present invention can be used as a starting point for nucleic acid synthesis / amplification.

[0102] 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. In short: for nucleic acid synthesis, a template nucleic acid is provided, which can be single-stranded or double-stranded. In the case of initially using double-stranded nucleic acids, the first step is to denature into single nucleic acid strands (complements and reverse complements) using techniques known in the art. Single-stranded nucleic acids do not require a denaturation step.

[0103] In the next step, a primer is provided that binds to the complementary region of the nucleic acid chain. The 3' end of the primer is then extended using a polymerase, and a complementary chain is produced by filling with complementary nucleotides. As a result, a complementary nucleic acid chain is formed. The result of the nucleic acid synthesis reaction is a double-stranded nucleic acid.

[0104] The term "reverse transcription with template switching" is well known in the art. In short: first, a primer hybridizes with an RNA molecule. The primer is used as a priming site for synthesizing cDNA by an enzyme with reverse transcriptase activity. Once the enzyme reaches the 5' end of the RNA template, a subclass of reverse transcriptase (such as MMLV reverse transcriptase) can add one or more additional nucleotides to the 3' end (mainly deoxycytidine) of the newly synthesized cDNA. This enables the template switching oligonucleotide to bind to these deoxycytidines. Subsequently, the reverse transcriptase can switch templates and continue cDNA synthesis. Examples of schemes using reverse transcription and template switching can be found in Zhu et al., 2001 and Wellenreuther et al., 2004.

[0105] The method 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 pre-amplification (PEP). In short, all methods have in common that random or degenerate oligonucleotides are used to initiate nucleotide synthesis. However, specific primers can also be used to amplify only specific targets in the genome.

[0106] The terms "oligonucleotide," "nucleic acid," and "nucleic acid molecule" refer to a biopolymer composed of nucleotide monomers covalently bonded in a chain. An amplified nucleic acid may be referred to as an "amplicon." The nucleic acid may be DNA or RNA.

[0107] The oligonucleotides according to the present invention may comprise a barcode sequence. A barcode sequence is a short nucleotide sequence used for identification purposes. The barcode may be a cellular barcode. Here, all target nucleic acids from the same cell are labeled with the same cellular barcode. In addition, the barcode may be a unique molecular identifier (UMI), labeling all nucleic acids with different barcode sequences. The barcode sequence may include both cellular barcodes and UMIs.

[0108] "Random hexamer" sequences may contain all four nucleotides (A, T, G, C) in each position, or only a subset of the four nucleotides in each position. Alternative nucleotides such as uracil, or nucleotide analogs or derivatives may also be used. Random hexamer oligonucleotides may also contain modified backbones to modify binding behavior, such as locked nucleic acids (LNA) or minor groove binding (MGB) nucleotides. Random or semi-random sequences of varying lengths may also be used in place of random hexamer sequences.

[0109] As used herein, the term "plurality" of something means two or more.

[0110] Example

[0111] The following examples are intended to explain the present invention in more detail without limiting the present invention to these examples.

[0112] Example 1: Bridge cDNA Synthesis on Solid Surface: Template switching is a common method for introducing a specific sequence at the 5' end of a newly synthesized cDNA (Zhu et al., 2001): In order to initiate reverse transcription using mRNA as a template, a specific oligonucleotide is incubated with the mRNA in the presence of a reverse transcriptase. The oligonucleotide may contain a stretch of multiple T nucleotides to bind to the poly(A) tail of the mRNA, or may be specific for a single mRNA sequence (reverse complement). Once the reverse transcriptase reaches the 5' end of the template mRNA, some reverse transcriptases are able to switch the template, thereby incorporating nucleotides that are reverse complementary to the template switching oligonucleotide, as disclosed by Zhu et al., 2001.

[0113] Prior art solutions provide both oligonucleotides for initiating reverse transcription and template switching oligonucleotides in solution (e.g. Stranded RNA-Seq Kit, catalog number 634839, Takara). Even single-cell workflows such as the Chromium Next GEM Single Cell 5' Library & Gel Bead Kit v1.1 (PN-100165, 10× Genomics, Pleasanton, CA, USA) use both in solution (the bead-bound template-switching oligonucleotide is released by dissolving the gel beads). It has even been shown that releasing the barcode-containing oligonucleotide delivered via the beads is required for efficient cDNA synthesis in the prior art single-cell workflow that delivers barcoded oligonucleotides via beads (Klein et al., 2015).

[0114] We therefore evaluated whether we could overcome this limitation by coupling two oligonucleotides to the solid surface: an oligonucleotide that initiates the reverse transcription reaction (according to the invention: a capture oligonucleotide) and an oligonucleotide for template switching (according to the invention, a barcode oligonucleotide comprising a template switching oligonucleotide).

[0115] As a model system, we used beads of about 1 μm size and coupled two oligonucleotides to their surface. When kept in solution, the distance between the two beads is greater than the length of the mRNA molecule. Therefore, the vast majority of template switching will occur only in the case of primers on the same surface.

[0116] Oligonucleotide beads were generated by synthesizing the following oligonucleotides having SEQ ID NO: 1 and SEQ ID NO: 2. The oligonucleotides were biotinylated on the 5' end.

[0117] These oligonucleotides were attached to streptavidin-coated beads (Dynabeads TM MyOne TM Streptavidin Cl, Thermo Fisher Scientific, Waltham, MA, USA). Note: The last three nucleotides of SEQ02 have the RNA backbone.

[0118] We next used these beads as well as controls in reverse transcription reactions using two different commercially available reverse transcriptases (Maxima RT, catalog number EP0742, ThermoFisher, Waltham, MA, USA and NEB Template Switch RT Enzyme Mix, catalog number M0466S, New England Biolabs, Ipswich, MA, USA) (see FIG4 ).

[0119] Reverse transcription was performed with 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 (Cat. No. M0492, New England Biolabs, Ipswich, MA, USA; amplification primers: SEQ ID NO: 3 and SEQ ID NO: 4).

[0120] The yield of amplified cDNA obtained was assessed using a ThermoFisher Qubit 4 Fluorometer (ThermoFisher, Waltham, MA, USA), and the size distribution was evaluated using an Agilent 4200 TapeStation system using D5000 or High Sensitivity D5000 Screen Tapes (Cat. Nos. 5067-5588 and 5067-5592, Agilent, Santa Clara, CA, USA).

[0121] The results are shown in Fig.11 Middle: Condition 1 using a bead-bound oligo(dT) primer (according to the invention: capture oligonucleotide) and a bead-bound template-switching oligonucleotide (according to the invention: barcode oligonucleotide comprising the template-switching oligonucleotide) unexpectedly showed similar yields compared to the control using both oligonucleotides in solution (Condition 5), strongly suggesting that "bridge cDNA synthesis" is possible when appropriate buffer conditions are used.

[0122] References BMC Genomics 18,519(2017).https: / / doi.org / 10.1186 / sl2864-017-3893-1 Klein AM,Mazutis L,Akartuna.BMC Genomics 18,519(2017). I,Tallapragada N,Veres A,Li V,Peshkin L,Weitz DA,Kirschner MW.Droplet barcodingfor single-cell transcriptomics applied to embryonic stem cells 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.Using Highly Parallel Genome-wide Expression Profiling ofIndividual Cells Nanoliter Droplets.Cell.2015May21:161(5):1202-1214.doi:10.1016 / j.cell.2015.05.002.PMID:26000488;PMCID:PMC4481139. 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.NatBiotechnol.2012Aug;30(8):777-82.doi:10.1038 / nbt.2282.Erratumin:NatBiotechnol.2020Mar;38(3):374.PMID:22820318;PMCID:PMC3467340.Wellenreuther R, Schupp I, Poustka A, Wiemann S; German cDNA Consortium.SMART amplificationcombined with cDNA size fractionation in order to obtain large full-lengthclones.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 transcriptasetemplate switching: a SMART approach for full-length cDNA library construction. Biotechniques. 2001 Apr;30(4):892-7.doi:10.2144 / 01304pf02.PMID:11314272. Description of the Figures – continued Figure 1A : 1. Partition (e.g., encapsulate in droplets) single cells and beads containing oligonucleotides containing capture sequences and barcode sequences; 2. Lyse cells and release mRNA.

[0123] Figure 1B: 3. Binding of target mRNA to capture sequence of oligonucleotide; 4. Removal of partitions / creation of single reaction compartments; 5. Reverse transcription → extension of barcoded oligonucleotides → generation of barcoded nucleic acids Figure 2A : A / B partition comprising a capture oligonucleotide (containing CS), a barcode oligonucleotide (containing BC), and a cell attached to the same solid support; C lyses the isolated single cells, thereby obtaining a mixture of target nucleic acid and non-target nucleic acid.

[0124] Figure 2B : D hybridizes the target RNA molecule to the capture oligonucleotide of the target RNA molecule; E / F disrupts the partition and performs nucleic acid synthesis; G attaches the barcode and generates a barcoded target nucleic acid attached to two sites on the solid support.

[0125] Figure 3 : A solid support comprising a capture oligonucleotide (containing CS) and a barcode oligonucleotide containing BC and TSO; 1. Locally lyse cells and specific mRNA molecules bind to the capture sequence of the capture oligonucleotide; 2. Synthesize cDNA and incorporate the barcode by template switching using the adjacent barcoded TSO on the solid support.

[0126] Figure 4A : 1. Single cell and single bead (containing capture oligonucleotide and barcode oligonucleotide with BC and TSO) in each partition.

[0127] Figure 4B : 2. Lyse the cells and allow the mRNA molecules to hybridize with the capture sequence of the capture oligonucleotide, and the mRNA will bind to the capture sequence (CS).

[0128] Figure 4C : 3. The beads are combined into a single container and nucleic acid synthesis (cDNA synthesis) and template switching are performed.

[0129] Figure 5A : 1. Distribute cells onto a patterned surface containing capture oligonucleotides and barcode oligonucleotides containing BC and TSO sequences (each element has a different barcode).

[0130] Figure 5B : 2. Lyse the cells under conditions that minimize diffusion between the different elements and the mRNA will bind to the capture sequence (CS).

[0131] Figure 5C :3. Perform nucleic acid synthesis (cDNA synthesis) and template switching Figure 6:1. Lyse cells and capture mRNA (hybridize with capture sequence); 2. Wash (remove lysis buffer, cell debris, etc.); 3. Perform nucleic acid (cDNA) synthesis with template switching.

[0132] Fig. 7A : Lyse cells and capture mRNA and genomic DNA; wash (remove lysis buffer, cell debris, etc.); extend genomic DNA; perform bridge cDNA synthesis with template switching Figure 7B : (1) Optionally, step g) can be performed on the target genomic DNA molecule and the target RNA molecule Figure 8 : In droplets or compartments; Single cell + single bead with unique barcode → Cell is lysed and all target nucleic acid molecules of the cell are hybridized to the single bead with unique barcode; Localized lysis on a surface; Patterned surface; Each region has a capture oligonucleotide with region-specific barcode → Cell is lysed and target nucleic acid molecules of the cell are hybridized to a single region with unique barcode Fig.9A : Deposition of single cells and solid surface in partitions; Lysis of cells and release of whole genome amplification oligonucleotides (exemplary random hexamers) within partitions; Target mRNA hybridizes to capture sequence (within partitions) Fig. 9B : Perform whole genome amplification (MDS) (within partitions); remove partitions (e.g., disrupt); all solid surfaces are treated in a single compartment; collect supernatant containing barcoded WGA products; perform reverse transcription with template switching. Fig.10 : Barcoded whole genome amplification oligonucleotides (exemplary random hexamers) bind to genomic DNA; whole genome amplification oligonucleotides (exemplary random hexamers) are extended by Phi29 polymerase; whole genome amplification oligonucleotides (exemplary random hexamers) are also bound to newly synthesized DNA and extended by Phi29 polymerase.

Claims

1. A method for generating barcoded target nucleic acids from a plurality of cells, the method comprising the steps of: a. providing a plurality of cells comprising a target RNA molecule and at least one solid support, wherein the solid support comprises a capture oligonucleotide and a barcode oligonucleotide of the target RNA molecule; 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 to obtain a mixture of target RNA molecules and non-target RNA molecules; d. hybridizing the target RNA molecule with the capture oligonucleotide of the target RNA molecule, thereby obtaining the target RNA molecule attached to the solid support; e. disrupting the partition 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 is used as a primer and the target RNA molecule is used as a template; g. attaching the barcode oligonucleotide to the double-stranded nucleic acid from the target RNA molecule, thereby generating a barcoded nucleic acid from the target RNA molecule; Characterized in that in step a), the multiple cells further contain target genomic DNA molecules, and the at least one solid support further contains capture oligonucleotides of target genomic DNA molecules, and characterized in that the capture oligonucleotides of the target RNA and target genomic DNA molecules are different.

2. The method according to claim 1, characterized in that The capture oligonucleotides of the target genomic DNA molecules contain a barcode sequence.

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

4. The method according to any one of claims 1 to 3, characterized in that The target genomic DNA molecules are additionally subjected to steps af, wherein: The mixture obtained in step c) further comprises target genomic DNA and non-target genomic DNA molecules; In step d), the target genomic DNA molecule is hybridized with the capture oligonucleotide of the target genomic DNA molecule, thereby obtaining the target genomic DNA molecule attached to the solid support; In step e), the non-target genomic DNA molecules are additionally separated from the target genomic DNA molecules attached to the solid support; - In step f), a double-stranded nucleic acid from the target genomic DNA molecule is generated by nucleic acid synthesis, wherein the capture oligonucleotide of the genomic DNA molecule is used as a primer and the target genomic DNA molecule is used as a template.

5. The method according to claim 4, characterized in that The target genomic DNA molecules are fragmented and dissociated into single strands prior to hybridization (step d).

6. The method according to claim 5, characterized in that The fragmented genomic DNA molecules have a length of 50-10 000 nucleotides.

7. The method according to claims 4-6, characterized in that The target genomic DNA molecules are additionally subjected to step g) by: G) attaching the barcode oligonucleotide to the double-stranded nucleic acid from the target genomic DNA molecule, thereby generating a barcoded nucleic acid from the target genomic DNA molecule.

8. The method according to claim 7, characterized in that For the target genomic DNA molecule, the barcode oligonucleotide is attached in step g) by ligation.

9. The method according to any one of claims 1 to 8, characterized in that The capture oligonucleotides of the target RNA molecules, the capture oligonucleotides of the target DNA molecules and the barcode oligonucleotides are linked to the solid support via a linker, wherein the linker is cleaved after step g), thereby releasing the nucleic acids from the solid support.

10. The method according to claim 9, characterized in that The linker is enzymatically cleaved.

11. The method according to any one of claims 9 to 10, characterized in that The capture oligonucleotides of the target RNA molecules, the capture oligonucleotides of the target DNA molecules and the barcode oligonucleotides are cleaved by a site-specific endonuclease after step g), thereby releasing the nucleic acids from the solid support.

12. The method according to any one of claims 1 to 3, characterized in that The capture oligonucleotide of the target genomic DNA molecule is used as a whole genome amplification oligonucleotide and is attached to the solid support via a releasable linker, and is characterized in that whole genome amplification of the target genomic DNA molecule is performed after step c), comprising the following steps: a. releasing whole genome amplification oligonucleotides from the solid support; b. performing whole genome amplification using a polymerase having strand displacement activity, wherein the whole genome amplification oligonucleotide is used as a primer, thereby obtaining an amplified target genomic DNA fragment.

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

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

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