WO2022268932A1 - Systems and methods for single cell analysis - Google Patents

Systems and methods for single cell analysis Download PDF

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Publication number
WO2022268932A1
WO2022268932A1 PCT/EP2022/067121 EP2022067121W WO2022268932A1 WO 2022268932 A1 WO2022268932 A1 WO 2022268932A1 EP 2022067121 W EP2022067121 W EP 2022067121W WO 2022268932 A1 WO2022268932 A1 WO 2022268932A1
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Prior art keywords
cell
bead
well
chip
wells
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PCT/EP2022/067121
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French (fr)
Inventor
Phillip KUHN
Remigijus Skirgaila
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Thermo Fisher Scientific Geneart Gmbh
Thermo Fisher Scientific Baltics Uab
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Publication of WO2022268932A1 publication Critical patent/WO2022268932A1/en

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/508—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above
    • B01L3/5085—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above for multiple samples, e.g. microtitration plates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1065—Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0647—Handling flowable solids, e.g. microscopic beads, cells, particles
    • B01L2200/0668—Trapping microscopic beads
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/06—Auxiliary integrated devices, integrated components
    • B01L2300/0627—Sensor or part of a sensor is integrated
    • B01L2300/0636—Integrated biosensor, microarrays
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/06—Auxiliary integrated devices, integrated components
    • B01L2300/0627—Sensor or part of a sensor is integrated
    • B01L2300/0645—Electrodes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0809—Geometry, shape and general structure rectangular shaped
    • B01L2300/0819—Microarrays; Biochips
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
    • B01L2400/0442—Moving fluids with specific forces or mechanical means specific forces thermal energy, e.g. vaporisation, bubble jet

Definitions

  • cells are analysed in the platform of the present disclosure to identify features (e.g. optically or electrochemically) such as cell surface markers and only retrieve cells of interest for subsequent analysis, for example, for next generation sequencing (NGS).
  • features e.g. optically or electrochemically
  • NGS next generation sequencing
  • the identified features may be linked to a specific well position in the chip by using a cell barcode unique to the well.
  • step d) the single cell is lysed while using a constant or pulsing electrical field to redirect charged molecules towards the bottom of the well. 10.
  • step f) or after step c) the content of each well is characterized, and the well location (x-y position) is recorded.
  • step g) the bead is lifted by gas bubble ejection.
  • FIG. 10 Graphical representation of alternative chip designs.
  • An exemplary workflow for the alternative chip design is illustrated in Fig. 11.
  • Reference signs refer to, a platform (“P”) as claimed, e.g., of design 1 (“PI”) or design 2 (“P2”), a cell (“C”), e.g. 12 pm cell, and a bead (“B”), e.g. a 30 pm bead coated with a plurality of oligonucleotide linkers (“Bl”), or a 10 pm bead (“B2”).
  • PI design 1
  • P2 design 2
  • B bead
  • exemplary “design 1” cell loading to wells may be limited by the Poisson distribution (i.e. to less than 10,000 cells), whereas exemplary “design 2” may allow for loading of 30,000 cells.
  • CMOS complementary metal-oxide-semiconductor
  • a CMOS chip contains an integrated circuit with high density electrode pattern and high voltage electronics.
  • individually controllable electrode refers to electrodes that may be deposited on or connected to the CMOS part of a microfluidic chip (CMOS layer).
  • the individually controllable electrodes located at the bottom of each well allow for selective pulsing bubble out single beads carrying, for example, target nucleic acid molecules, and collecting the beads for further processing.
  • a lifting buffer may comprise less than 50% water (such as e.g. up to 2%, up to 5%, up to 10%, up to 20%, up to 30% or up to 40% water) and more than 50% methanol (such as e.g. up to 95%, up to 90%, up to 80%, up to 70%, up to 60% methanol).
  • other organic solvents such as, for example, acetonitrile may be used instead of methanol.
  • the monodisperse bead may be further defined by its surface area.
  • the surface area of a bead may be within a range of 10 to 1000 m 2 /g, between 100 and 500 m 2 /g, between 200 and 400 m 2 /g such as, for example, around 380 m 2 /g.
  • the surface area of porous monodisperse beads can, for example, be determined according to a method developed by Brunauer, Emmett and Teller referred to as the BET method which is based on the physical adsorption of a vapor or gas onto the surface of a solid ([14]: Brunauer, S., Emmett, P. and Teller, E., J. Amer. Chem. Soc.
  • a dimethoxytrityl (DMT) cation assay may be performed to compare linker loading capacity of different types of beads by determining trityl group concentration which corresponds to the concentration of the oligonucleotide linkers on the bead.
  • an acid-based deprotection step could be performed to remove the terminal hydroxyl protecting DMT group which can be spectrometrically measured.
  • DNA synthesizers or synthesis chips as described herein may be configured to automatically collect, with the aid of a fraction collector, the solution containing the DMT cation during the detritylation step of the synthesis cycle.
  • modified or synthetic nucleobases include 3-methyluracil, 5,6-dihydrouracil, 4-thiouracil, 5-bromouracil, 5-thorouracil, 5-iodouracil, 6-dimethyl aminopurine, 6-methyl amino purine, 2-amino purine, 2,6-diamino purine, 6-amino-8- bromopurine, inosine, 5-methyl cytosine, 7-deazaadenine, and 7-deaza guanosine.
  • modified RNA nucleotides include locked nucleic acids (LNA). Oligonucleotides may further comprise additional molecules that have been joined, either covalently or non-covalently.
  • the coupling moiety will be inert to all the reagents used during the present methods, but cleavable under specific conditions.
  • One coupling moiety commonly used in nucleic acid molecule synthesis is the succinyl coupling moiety.
  • universal coupling moieties may be used.
  • a universal coupling moiety is a coupling moiety that allows for the synthesis of nucleic acid molecules regardless of the nature of the terminal base of the first nucleotide that is to be coupled.
  • the bead carries a universal coupling moiety such as a UNYLINKERTM.
  • Different coupling moieties with different properties are known to those skilled in the art and can be selected by the skilled person depending on the type of synthesis (i.e. 5’-3’ or 3’-5’) and downstream process requirements.
  • one or more wells may be selected (e.g. based on cell labelling), the content of the well may be released and collected for further processing.
  • collected cDNA from one or more selected wells may be purified and/or concentrated by magnetic bead-based clean-up, PAGE-based clean-up, column based clean-up or other methods known by the person skilled in the art. Magnetic bead-based purification is the method of choice, although other methods are possible which the skilled artisan would be able to identify.
  • the oligonucleotide-tethered nucleotides may be used as reagents in cDNA synthesis reactions such that the nucleotide with oligonucleotide tether may be incorporated into the cDNA formed from the synthesis reaction (e.g., extension reaction, amplification reaction).
  • the coupling moiety when present, may be a chemical entity that attaches the 3'-0 of the nucleic acid molecule to the bead (e.g., a functional group on a bead).
  • the coupling moiety when present, may have a structure such that it allows for attachment of other functionalities in addition to the 3’-0.
  • Such coupling moiety structures are disclosed, for example, in [18]: U.S. Patent No. 7,202,264. In most cases, the coupling moiety will be inert to all the reagents used during nucleic acid molecule synthesis, but cleavable under specific conditions at the end of the synthesis process.
  • beads prederivatized with a base e.g., dU, dA, dT, dC, dG, etc.
  • a base e.g., dU, dA, dT, dC, dG, etc.
  • a synthesis chip with multiple (e.g., four) loading regions e.g., reagent flow zones
  • multiple synthesis runs could be made in which the starting base is different in each run.
  • the first run may be made with nucleic acid molecules that begin with dA, followed by dC, then dT, then dG.
  • thermocycler was preheated to 55 °C.
  • Each cDNA sample was transferred to a fresh 0.2 ml PCR tube, i.e. 600 pg of purified cDNA were dissolved in nuclease- free water (total volume of 5 pi).
  • the wash step was repeated for a total of two washes. After washing, sample were briefly spun down and the remaining EtOH was collected by placing the tubes back on the magnetic rack. On the magnetic rack, samples were air dried for 2 min or until all of the ethanol had evaporated. Each pellet was resuspended in 12 m ⁇ of nuclease-free water by vortexing. Samples were incubated for 5 min at room temperature (not on the magnetic rack). Tubes were placed back on the magnetic rack for 2 min or until a solid bead pellet had formed. The supernatant was transferred to new tubes.

Abstract

Provided herein are systems and methods for single cell analysis, using CMOS chips, with individually controllable electroIdes. The systems and methods provided herein are useful for genomic, transcriptomic (e.g., scRNA-seq), or other single cell analyses.

Description

SYSTEMS AND METHODS FOR SINGLE CELL ANALYSIS
SEQUENCE LISTING
[0000] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on May 24, 2022, is named LT01550PCT_SL.txt and is 1,248 bytes in size.
TECHNICAL FIELD
[0001] The disclosure relates to systems and methods in the field of single cell analysis.
BACKGROUND
[0002] To identify and characterize the specific function of a single cell within a complex heterogenous cell population, it is an essential prerequisite that single cell-derived molecules such as nucleic acids and proteins can be detected and quantified. In recent years, several technologies to study for example the transcriptome of single cells have been developed using plate-based approaches (see for example [1]: Wang etal. , bioRxiv preprint 2019, doi: 10.1101/541433), bead- based approaches (see for example [2]: Macosko etal. , Cell 2015, doi: 10.1016/j.cell.2015.05.002; [3]: Klein et al, Cell 2015, doi: 10.1016/j.cell.2015.04.044,), or combinatorial barcoding approaches wherein cells themselves are used as compartments (see [4]: Rosenberg etal. , Science 2018, doi: 10.1126/science. aam8999). These approaches and platforms differ in their sensitivity, specificity, throughput, and other parameters (for comparison see [5]: Ding et al. , Nature Biotechnology 2020, doi: 10.1038/s41587-020-0465-8; [1]: Wang et al, bioRxiv preprint 2019, doi: 10.1101/541433).
[0003] In plate-based approaches, single cells are loaded to individual wells, for example by using fluorescence-activated cell sorting (FACS) and submitted to further processing including cell lysis, library preparation and sequencing. By using FACS, labelled cells of interest can be sorted out and unwanted cells that would otherwise dilute the data can be removed. However, one limitation is the diffusion of cell-derived nucleic acid molecules between wells during and after cell lysis resulting in a limited nucleic acid capture efficiency and cell-to-cell cross-contamination. One aspect of the current disclosure is to address this limitation.
[0004] To overcome this limitation, platforms are described wherein wells are covered by laminar flow oil (see [6]: Bose et al, Genome Biology 2015, doi: 10.1186/sl3059-015-0684-3), or by reversible attachment of a semi-permeable polycarbonate membrane to trap cell-derived molecules (see [7]: Gierahn et al. , Nature Methods 2017, doi: 10.1038/nmeth.4179). Furthermore, buffer additives are mentioned to increase the density and viscosity of the buffers during cell lysis and thus, to reduce cross-contamination (see [8]: WO 2016/118915 Al).
[0005] Bead-based approaches rely on microfluidic devices wherein each cell is encapsulated in an individual droplet together with a bead. The bead allows to associate cell-derived molecules with a cell barcode unique to that cell (see [2]: Macosko et al. , Cell 2015, doi: 10.1016/j .cell.2015.05.002; [3]: Klein et al., Cell 2015, doi: 10.1016/j cell.2015.04.044). Furthermore, combinations have been described, wherein cells and beads are loaded into a well plate (see for example [8]: WO 2016/118915 Al). In bead-based approaches, compartments (i.e. individual droplets or wells) may also contain two or multiple cells sticking together. These co encapsulated cells are assigned one cell barcode. As a result, the cell barcode is not unique to a single cell and single cell resolution is lost. Another aspect of the current disclosure is to address this limitation.
[0006] To overcome such limitations, the use of image analysis was described to identify wells containing more than one cell and to optically identify for each well the unique cell barcode attached to the bead using sequential fluorescence hybridization. Cells of interest and unwanted cells were both processed further since selection could only be performed after library generation and sequencing (see [9]: Yuan et al, Genome Biology 2018, doi: 10.1186/sl3059-018-1607-x). As alternative approach, it was mentioned to retrieve beads after image analysis from selected well (i.e. containing a single cell) by physical removal using micropipettes, optical tweezers or miniaturized magnetic probes, or to destruct beads after image analysis in the selected well (i.e. containing more than one cell) using laser photoablation (see [8]: WO 2016/118915 Al). [0007] In the context of de novo nucleic acid synthesis, Life Technologies et al. describe a mechanism for bead removal from wells by electrolysis. Specifically, a voltage is applied between a first electrode located at the bottom of the well and a second electrode to cause the fluid in a fluid-filled well to undergo electrolysis producing one or more bubbles in the fluid to rise to the top of the fluid-filled well along with the bead (see [10]: WO 2016/094512 Al).
[0008] However, there remains a need for a new method and system allowing for high-throughput single cell analysis, high nucleic acid capture efficiency, low cell-to-cell cross-contamination, and selection of cells of interest.
SUMMARY OF THE INVENTION
[0009] The present invention meets the above needs and solves the above problems in the art by providing the embodiments described below:
[0010] The invention provides a platform for single cell analysis comprising a chip having a plurality of wells, wherein a complementary metal-oxide-semiconductor (CMOS) layer is provided to connect individually controllable electrodes located at the bottom of each well allowing for generation of an electrical field in one or more selected well to retain or reject the well’s content such as a bead, a cell and/or cell-derived molecules. The rejected content may be collected for further processing.
[0011] In some embodiments, cells are analysed in the platform of the present disclosure to identify features (e.g. optically or electrochemically) such as cell surface markers and only retrieve cells of interest for subsequent analysis, for example, for next generation sequencing (NGS). The identified features may be linked to a specific well position in the chip by using a cell barcode unique to the well.
[0012] Specific embodiments include systems and methods for attracting nucleic acids from lysed cells to the well surface, methods of linker synthesis and attachment to a bead and alternative platform designs, e.g., having chip geometries for improved bead to cell distribution and capture and the like. In some embodiments, each well has a first chamber and a second chamber connected with the first chamber. In some embodiments, the platform may further comprise an electrode layer which is electrically independent from the CMOS layer provided to connect the individually controllable electrodes.
[0013] Thus, the present disclosure provides improved means for single cell analysis by providing the preferred embodiments described in the following list of representative items:
1. A method for single cell analysis comprising the following steps: a) providing a chip comprising a plurality of wells, wherein a complementary metal-oxide- semiconductor (CMOS) layer is provided to connect individually controllable electrodes located at the bottom of each well, and wherein each well can accommodate a microscale bead and a eukaryotic or prokaryotic cell, b) providing single beads coated with a plurality of linkers to at least two wells of the chip, c) loading at least two wells containing single beads with a single eukaryotic or prokaryotic cell, d) lysing the single cell in the well while addressing the individually controllable electrode in the well to retain cell-derived molecules within the well, e) contacting cell-derived molecules with the plurality of linkers on the bead, f) selecting one or more well, and g) releasing the bead from the one or more well by addressing the individually controllable electrode in the selected well.
2. The method according to item 1, wherein the genetic, epigenetic, spatial, and/or the proteomic data of a single cell is analysed.
3. The method according to one or more of items 1 to 2, wherein the transcriptome, genome, DNA modifications, chromatin accessibility, and/or chromosome conformation is analysed.
4. The method according to one or more of items 1 to 3, wherein in step b), each linker or a portion thereof is attached to and/or synthesized on the single bead prior to loading to the chip.
4. (a) The method according to item 4, wherein the portion of the linker is selected from at least one of the following sequence segments: a spacer, a PCR handle, a cell barcode, a unique molecular identifier (UMI) and/or nucleic acid binding sequences (e.g., an RNA binding sequence or a poly (dT) sequence).
5. The method according to one or more of items 1 to 3, wherein in step b), each linker or a portion thereof is attached to and/or synthesized on the single bead after loading to the chip.
5. (a) The method according to item 5, wherein the portion of the linker is selected from at least one of the following sequence segments: a spacer, a PCR handle, a cell barcode, a unique molecular identifier (UMI) and/or nucleic acid binding sequences (e.g., an RNA binding sequence or a poly (dT) sequence).
6. The method according to item 5, wherein the well location (x-y position) of said linkers or a portion thereof is recorded.
7. The method according to one or more of items 1 to 6, wherein said single cell is a eukaryotic, mammalian or human cell.
7. (a) The method according to item 7, wherein said single cell is a mammalian cell.
7.(b) The method according to item 7, wherein said single cell is derived from a part of a mammal.
7.(c) The method according to item 7, wherein said single cell is derived from pathological situations like abnormal organ development, autoimmune diseases, chronic diseases, infectious disease or in cancer.
7.(d) The method according to item 7, wherein said single cell is isolated from a multicellular organism by fluorescence-activated cell sorting (FACS).
8. The method according to one or more of items 1 to 7, wherein prior to step d) the wells are covered by oil or a semi-permeable membrane.
9. The method according to one or more of items 1 to 8, wherein in step d) the single cell is lysed while using a constant or pulsing electrical field to redirect charged molecules towards the bottom of the well. 10. The method according to one or more of items 1 to 9, wherein prior to step f) or after step c) the content of each well is characterized, and the well location (x-y position) is recorded.
11. The method according to one or more of items 1 to 10, wherein prior to step f) or after step c) the content of one or more wells is characterized using microscopy on the chip, imaging and analysis.
12. The method according to one or more of items 10 to 11, wherein the content comprises the bead and/or the eukaryotic or prokaryotic cell.
12. (a) The method according to items 10 to 12, wherein the content comprises features of the eukaryotic or prokaryotic cell.
12. (b) The method according to items 10 to 12, wherein the content comprises at least two features of the eukaryotic or prokaryotic cell.
13. The method according to one or more of items 10 to 12, wherein the content comprises cell surface markers.
13. (a) The method according to items 10 to 12, wherein the content comprises the viability status of the eukaryotic or prokaryotic cell.
14. The method according to one or more of items 1 to 13, wherein the environment of the well is changed, wherein the environment comprises composition, viscosity, pH, temperature and/or conductivity of a fluid in the well, wherein the environment further comprises voltage and/or current of the individually controllable electrode in the well.
15. The method according to one or more of items 1 to 14, wherein in step g), the bead is lifted by gas bubble ejection.
15. (a) The method according to item 15, wherein after step g), the beads are collected for further processing of the cell-derived molecules. 15. (b) The method according to item 15, wherein after step g), the beads are collected for further processing in a filter material characterized in that its pore sizes are less than the bead diameter.
16. The method according to one or more of items 1 to 15, wherein in step d), the cell-derived molecules are nucleic acid molecules or proteins.
17. The method according to one or more of items 1 to 16, wherein the transcriptome of a single cell is analysed, and wherein in step d), the cell-derived molecules are nucleic acid molecules.
18. The method according to one or more of items 1 to 17, wherein in step b), the plurality of linkers are nucleic acid molecules, and optionally wherein the 3’ ends of the nucleic acid molecules are deprotected.
19. The method according to item 18, wherein each of said nucleic acid molecules comprises the following sequence segments: a spacer, a PCR handle, a cell barcode, a unique molecular identifier (UMI) and/or a binding sequence for cell-derived nucleic acid molecules.
19. (a) The method according to item 19, wherein the binding sequence is a random or specific nucleotide sequence.
19. (a) The method according to item 19, wherein the binding sequence is a DNA binding sequence, an RNA binding sequence or poly(dT) sequence.
20. The method according to one or more of items 17 to 19, wherein the contacting in step e) comprises hybridizing or binding the cell-derived nucleic acid molecules to the plurality of linkers on the bead.
21. The method according to one or more of items 17 to 20, wherein after step e), cDNA is synthesized.
21. (a) The method according to one or more of items 17 to 20, wherein after step e), cDNA synthesis by reverse transcription is performed.
21. (b) The method according to item 21(a), wherein a template switch oligo (TSO) is added to generate cDNA. 22. The method according to one or more of items 1 to 21, wherein after step g), the released bead is collected for further processing.
23. The method according to one or more of items 17 to 22, wherein after step g), the released bead is collected for further processing to generate a single cell RNA-sequencing library.
24. The method according to item 1, wherein the transcriptome of a single cell is analysed, comprising the following steps: a) providing a chip comprising a plurality of wells, wherein a complementary metal-oxide- semiconductor (CMOS) layer is provided to connect individually controllable electrodes located at the bottom of each well, and wherein each well can accommodate a microscale bead and a eukaryotic or prokaryotic cell, b) providing single beads coated with a plurality of linkers to at least two wells of the chip, wherein the plurality of linkers are nucleic acid molecules, c) loading at least two wells containing single beads with a single eukaryotic or prokaryotic cell, d) lysing the single cells in the well while the individually controllable electrode in the well is positively charged to retain negatively charged cell-derived nucleic acid molecules within the well, e) contacting cell-derived nucleic acid molecules with the plurality of linkers on the bead, f) selecting one or more well, and g) releasing one or more beads by addressing the individually controllable electrode in the selected well.
25. The method according to item 24, wherein in step b), the 3’ ends of the nucleic acid molecules are deprotected.
26. The method of any previous item, wherein the single eukaryotic or prokaryotic cell is labelled.
26. (a) The method of item 26, wherein the cell is labelled with a protein, aptamer, dye or combination thereof.
26. (b) The method of item 26, wherein the cell is labelled with a photoswitchable or photoconvertible fluorescent molecule. 26. (c) The method of item 26, wherein the cell is labelled with at least one fluorescent protein, optionally wherein the fluorescent protein is a fluorescently labelled antibody directed to a cell surface marker.
26. (d) The method of item 26, wherein the cell is labelled with a dye, optionally wherein the dye is a cytoplasmic dye, nuclear dye, membrane dye and/or combinations thereof.
27. A platform for single cell analysis comprising a chip with a plurality of wells, wherein a complementary metal-oxide-semiconductor (CMOS) layer is provided to connect individually controllable electrodes located at the bottom of each well, and wherein each well can accommodate a microscale bead and a eukaryotic or prokaryotic cell.
28. The platform according to item 27, wherein a plurality of wells accommodates a microscale bead coated with a plurality of linkers.
29. The platform according to item 28, wherein the linker comprises a cell barcode having a pre defined sequence for each bead.
29. (a) The platform according to item 28, wherein the microscale bead is coated with a plurality of linkers.
29. (b) The platform according to item 29(a), wherein each linker of the plurality of linkers comprises a nucleotide sequence.
29. (c) The platform according to item 29(b), wherein the nucleotide sequence comprises at least the following sequence segments: a unique molecular identifier (UMI) and a cell barcode; wherein a specific bead is designed to contain a plurality of linkers having the same nucleotide sequence segment to provide the cell barcode and a different nucleotide sequence segment as compared to the other linkers on the specific bead to provide the UMI, and wherein the cell barcode on each bead is different to the other beads.
29. (d) The platform according to item 28, wherein each well comprises a bead with a plurality of linkers having a cell barcode which is unique to the bead. 30. The platform according to one or more of items 27 to 29, wherein each well has at least a first chamber and a second chamber connected with the first chamber, and wherein the first chamber can accommodate a single microscale bead and the second chamber can accommodate a single eukaryotic or prokaryotic cell.
31. The platform according to item 30, wherein the individually controllable electrode is provided at the bottom of only the first chamber.
32. The platform according to one or more of items 30 to 31, further comprising an electrode layer which is controllable for the entire chip and which is not electrically connected with the segment comprising the individually controllable electrodes.
33. The platform according to item 32, wherein the individually controllable electrode is provided at the bottom of the first chamber, and the electrode layer which is controllable for the entire chip is provided at the bottom of the second chamber.
34. The platform according to one or more of items 32 to 33, wherein the electrode layer is a platinum layer.
35. The platform according to item 30, wherein two individually controllable electrodes are provided at two not electrically connected segments of the bottom of each well, wherein one segment is provided at the bottom of the first chamber and the other segment is provided at the bottom of the second chamber.
36. The platform according to one or more of items 27 to 35, wherein the chip geometry allows for a 1 : 1 bead to cell distribution and capture in each well.
37. The platform according to one or more of items 27 to 36, wherein the bead is a monodisperse, optionally porous microparticle.
38. The platform according to one or more of items 27 to 37, wherein the bead has a size of between 10 pm and 100 pm. 39. The platform according to one or more of items 27 to 38, further comprising an imaging system, optionally wherein the imaging system comprises a microscope.
40. An array of beads coated with a plurality of linkers, wherein the plurality of linkers comprise nucleic acid molecules, each nucleic acid molecule having at least the following sequence segments: a PCR handle, a unique molecular identifier (UMI) and a cell barcode, wherein each linker on a specific bead is designed to contain the same nucleotide sequence segment to provide a cell barcode and a different nucleotide sequence segment as compared to the other linkers on the specific bead to provide a unique molecular identifier (UMI), and wherein the cell barcode on each bead is different.
41. The array of beads of item 40, wherein each linker further comprises a poly(dT) or RNA binding sequence.
42. The array of beads of item 40 or item 41, wherein each bead is located in a well of a chip or a multiwell plate.
DESCRIPTION OF THE DRAWINGS
[0014] Figure 1: Graphical representation of the general workflow comprising single bead loading, single cell loading and imaging, cell lysis, cDNA synthesis and subset selection by bead lifting.
[0015] Figure 2: Graphical representation of an exemplary single cell RNA-sequencing (scRNA-seq) workflow as described in the Examples, comprising steps of chip surface blocking, bead loading, cell loading, pre-RT buffer, reverse transcription, bead lifting and collecting, Exo I treatment, bead counting, PCR, tagmentation and sequencing.
[0016] Figure 3: Graphical representation of an exemplary scRNA-seq workflow comprising detailed steps of a) cell preparation including, e.g., cell counting, labelling, filtering and diluting; b) chip preparation using, e.g., argon plasma, ethanol priming and BSA coating; c) bead filling including, e.g., centrifugation and optionally imaging-based control of bead loading; d) cell loading including, e.g., automatic loading, washing to remove excess cells and optionally, e) imaging-based control of cell loading; f) cell lysis including, e.g., adding Pre-RT buffer, applying voltage, adding reverse transcription (RT) buffer and optionally imaging-based control of cell lysis; g) cDNA synthesis including, e.g., incubation at room temperature (RT) for 1 h; h) bead lifting including, e.g., adding bead lifting solution, applying current and optionally, imaging- based control and bead counting; i) bead post-processing including, e.g., washing beads, resuspending and washing in tube; j) Exo I treatment to remove single-stranded DNA, e.g., at 37 °C for 45 min; k) PCR and clean-up (e.g. with magnetic beads) and h) analysis of cDNA, e.g., using BioAnalyzer.
[0017] Figure 4: Graphical representation of a bead oligonucleotide structure. The oligonucleotide may comprise a PCR handle, a cell barcode (CB), a unique molecular identifier (UMI), an oligo(dT)30 sequence (SEQ ID NO: 3) which may capture the polyA-tail of an RNA molecule; a template switch oligo (TSO) may be added during reverse transcription to generate full-length double-stranded cDNA.
[0018] Figure 5A: Graphical representation of the principle of nucleic acid oligonucleotides (“oligos”) retention in a well. Wells’ electrodes are activated and charged positively. Negatively charged covalently 6-carboxyfluorescein (FAM) labelled oligonucleotides are attracted to the positively charged electrodes located beneath a plurality of wells of the microfluidic chip, causing a signal accumulation in the wells.
[0019] Figure 5B: Images show the accumulation of oligonucleotide signal in the wells. Specifically, 0.1 nmol HPLC purified (dT)60-FAM labelled oligonucleotide ("(dT)60" disclosed as SEQ ID NO: 4) in 200 mΐ 5 mM Tris pH 11 in water. The electrodes in the selected square pattern of electrodes located beneath a plurality of wells of the microfluidic chip are charged with 1.5 V at a current limit of 4 mA. Pulses of 100 ms on, 200 ms off were applied.
[0020] Figure 6A: Graphical representation of the cell lysis and nucleic acid manipulation workflow comprising steps of loading chip with cells, washing cells, cell lysis and oligonucleotide capture. Exemplary buffers and applied voltage are shown. [0021] Figure 6B-C: Images show fluorescence over time [s] for GFP-labelled cells and cyanine dye labelled oligonucleotides, i.e. (dT)60-Cy5-labeled oligonucleotides ("(dT)60" disclosed as SEQ ID NO: 4). Images were analyzed with ImageJ ([26]: https://imagej.nih.gov/ij/). Areas of interest (positions, “Pos”, 1-4) were defined in the image stacks of the respective fluorescent channel (6B). Pos 1+2 were chosen around single cells in the GFP channel, Pos 3 at an area outside of the activated area in the Cy5 channel, and Pos 4 in the region where electrodes within wells were activated. Mean grey values from these areas were normalized and plotted (6C).
[0022] Figure 7: Well selection. The procedure comprises steps of finding coordinates of calibration points, imaging chip and selecting cells in image (top image prior to bead lifting: selected well highlighted by circles), running custom Python script, uploading bmp into chip software, bubbling beads out (bottom image after bead lifting: selected well highlighted by circles).
[0023] Figure 8: General set-up of bead/cell pairing allowing for multiple parallel reactions (for example 1,000s in parallel) based on the defined linker segments such as a PCR handle, cell barcode, UMI and oligo(dT) sequence to capture the poly(A)-tail of the RNA.
[0024] Figure 9: Graphical representation of direct synthesis of cell barcodes on chip comprising exemplary steps of pre-synthesizing an oligonucleotide comprising a PCR handle and UMI, and subsequent synthesis of cell barcode and poly(dT) on CMOS chip.
[0025] Figure 10: Graphical representation of alternative chip designs. An exemplary workflow for the alternative chip design is illustrated in Fig. 11. Reference signs refer to, a platform (“P”) as claimed, e.g., of design 1 (“PI”) or design 2 (“P2”), a cell (“C”), e.g. 12 pm cell, and a bead (“B”), e.g. a 30 pm bead coated with a plurality of oligonucleotide linkers (“Bl”), or a 10 pm bead (“B2”). In exemplary “design 1”, cell loading to wells may be limited by the Poisson distribution (i.e. to less than 10,000 cells), whereas exemplary “design 2” may allow for loading of 30,000 cells. Starting from design 1, the chip which contains a CMOS layer connected to individually controllable electrodes located at the bottom of each well may be further designed (arrow, “D”) to include, e.g., an additional electrode layer(“E”) which is not electrically connected with the segment comprising the individually controllable electrodes, e.g., to remove the content of wells (or chambers of wells) before cell loading.
[0026] Figure 11: Graphical representation of workflow with alternative chip designs with wells having a first chamber and a second chamber, an individually controllable electrode layer connected to a CMOS layer is provided at the bottom of the first chamber (labelled as “CMOS”), and an electrode layer which is controllable for the entire chip is provided at the bottom of the second chamber (labelled as “electrode layer”). The chambers of a well a connected, e g. by a permeable structure. In the first step (top, arrow 1), all well chambers are filled with a bead coated with a plurality of linkers. Each linker comprises tethered oligonucleotides with defined nucleotide sequence segments, for example, PCR handle, cell barcode, UMI and poly(dT) sequence. Next (top, arrow 2), beads are removed from second chambers by applying a negative electrical force, e.g. -8 V, to the electrode layer at the bottom of the second chamber to create space for single cell trapping. Then (top, arrow 3), cells are flushed into the chip to occupy free second chambers Optionally, wells may be covered, e.g. with an oil layer. After having cells and beads combined in a well (bottom, arrow 1), the cells may be lysed (e.g. with a surfactant, or electrochemically), while applying a positive electrical force, e.g. +1.5 V or +2 V, to the electrode connected to the CMOS layer at the bottom of the first chamber containing the bead and negatively charged nucleic acid molecules, e.g. RNA molecules, diffuse or migrate under an electric field to the bead (e.g directly or through pores of permeable structure such as a membrane) where the nucleic acid molecules hybridize to a nucleic acid binding segment of a linker, e.g. the poly (dT) sequence of a linker to capture poly(A) RNA. In some configurations, RNA may be reverse transcribed into cDNA. Next (bottom, arrow 2), selected beads may be released from the well by addressing the individually controllable electrodes connected to the CMOS layer in the first chamber, by applying a negative electrical force, e.g. -8V.
[0027] Figure 12A-B: Quality of sequencing data and influence of voltage on cross-contamination [%] . RNA from two different cell lines was captured on beads in wells of a chip, converted into cDNA and sequenced. The shown sequencing data demonstrate a clear positive effect of voltage applied during RNA capture on sequence cross-contamination level. Whereas “no voltage” capture resulted in 21.05% of cross-contamination, applying voltage during cell lysis resulted in 3.72% cross-contamination level, which demonstrates efficient RNA immobilization on the bead using electrical fields.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0028] The following definitions are provided to assist the reader. Unless otherwise indicated, all terms of art, notations, and other scientific or medical terms or terminology used herein are intended to have the meanings commonly understood by those skilled in the arts. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not be construed as representing a substantial difference over the definition of the term as generally understood in the art.
[0029] All publications, patents and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.
[0030] The articles “a”, “an” and “the” are used herein to refer to one or to more than one (i.e. to at least one) of the grammatical object of the article unless otherwise clearly indicated by contrast. By way of example, “an element” means one element or more than one element. The term “or” is used herein to mean, and is used interchangeably with, the term “and/or,” unless context clearly indicates otherwise. The term “comprising” includes, as one embodiment, the meaning “consisting of’.
[0031] In some embodiments, the term “about” refers to a deviation of ± 10 % from the recited value. When the word “about” is used herein in reference to a number, it should be understood that still another embodiment of the invention includes that number not modified by the presence of the word “about”. In the absence of the term “about” and unless the context dictates otherwise, generally accepted rounding rules apply to the specified values. [0032] The term “ single cell analysis ” as used herein refers to the study of single cell “-omics” such as genomics, transcriptomics, proteomics and metabolomics as well as cell-cell interactions at the single cell level. For example, single cell RNA-sequencing (scRNA-seq) is a very powerful tool in early diagnostic and cell population analysis.
[0033] The term “ single celF as used herein refers to an individual cell of any cell type either alive or dead, specifically a prokaryotic or eukaryotic cell. The cell may be derived from a multicellular organism or parts thereof. Each cell is characterized by its molecular data.
[0034] The term “ molecular data of a celF as used herein refers to the molecular composition of a cell comprising, e.g. its genetic, epigenetic, spatial, and/or the proteomic data.
[0035] The term “ labelled celF as used herein refers to the identification, characterization and/or tracking of a eukaryotic or prokaryotic cell by microscopy. Labelling facilitates to identify all cells (non-specific staining) or a specific subset of cells such as a cell type, cell cycle phase or live/dead cells (viability). A cell may be stained with photoswitchable or photoconvertible fluorescent proteins (e.g. a fluorescently labelled antibody directed to a cell surface marker), aptamers or dyes (e.g. cytoplasmid and nuclear dyes, membrane dyes, and/or viability dyes). An overview of fluorescent labelling techniques that may be used herein are disclosed in [27] (Takeshi Suzuki et al. 2007). In some cases, multi-color labelling strategies may be used for labelling as known in the art. In addition or as an alternative to ex vivo labelling, genetic manipulation of a cell leading to expression of at least one fluorescent reporter (e.g. GFP) may be used as a labelling method to visualize cells.
[0036] The term “cell derived molecule ” as used herein refers to organic molecules such as nucleic acids, peptides and proteins, carbohydrates and lipids.
[0037] The term “ chip ” as used herein refers to an electronic computer microchip comprising at least two wells. The chip is part of a platform further comprising, for example, a lid operable to be formed on a top surface of the microchip and operable to provide a fluid flow path into and out of the well. The chip may be a complementation metal-oxide-semiconductor (CMOS) chip. [0038] The term “ chip geometry ” as used herein refers to the overall three-dimensional shape of the chip (length, width and thickness), smaller-scale structural features of the chip (such as well and chamber shape) and the organisation of the smaller-scale structural features (such as arrangement of wells in a rectangular matrix or connections between wells).
[0039] The term “ weir or “ microweir as used herein refers to a structural feature of the chip. The microwell is characterized by a three-dimensional shape allowing the microwell to function as a reaction vessel for processing and/or analysis of a cell of interest.
[0040] The term “ chamber ” as used herein refers to a structural feature of a well. The well may have at least one chamber wherein the chamber provides a reaction vessel. The environment, content and dimension or size of the chamber may vary. In some embodiments, a well comprises a first chamber and a second chamber which are “ connected' to allow for buffer and/or cell-derived molecule exchange between the two chambers. For example, the first chamber may accommodate a single microscale bead and the second chamber may accommodate a single eukaryotic or prokaryotic cell. Upon cell lysis in the second chamber, the connection between the two chambers allows cell-derived molecules to migrate to the bead in the first chamber, e.g. by diffusion or electrostatic attraction.
[0041] The term “ environment ” as used herein refers to the characteristics of the material and the surrounding area of the chip, well and/or chamber. The material may be characterized by surface roughness, conductivity and/or charge. The surrounding area of the chip, well and/or chamber may be characterised by its composition, viscosity, pH, temperature and/or conductivity. The term “ composition ” as used in said context encompasses chemical components, i.e. buffer formulation (concentration of ingredients etc.).
[0042] The term “ complementary metal-oxide-semiconductor (CMOS)” as used herein refers to both, a particular style of digital circuitry design and the family of processes used to implement that circuitry on integrated circuits (i.e. chips). A CMOS chip contains an integrated circuit with high density electrode pattern and high voltage electronics. [0043] The term “ individually controllable electrode ” as used herein refers to electrodes that may be deposited on or connected to the CMOS part of a microfluidic chip (CMOS layer). The individually controllable electrode may be located at the bottom (or a segment of the bottom) or along one or more side wall of a well (or a chamber of a well) of a microfluidic chip and configured to deliver current to the individual well (or the individual chamber of a well) that is associated with it. Each electrode communicates with a current controller which regulates current to the electrode. The CMOS layer acts like a “cable” that can be configured where it goes and is used to charge the individually controllable electrode.
[0044] The term “ electrode layer ” as used herein refers to a metal layer that may be part of a microfluidic chip. The electrode layer may be controllable for the entire chip and is not electrically connected with the segment comprising the individually controllable electrodes. The electrode layer may be located at the bottom (or a segment of the bottom), along one or more side wall or beneath a plurality of wells (or chambers of wells) of a microfluidic chip and configured to deliver current to the wells (or chambers of wells) that is associated with it. The electrode layer provides a single counter electrode on the chip connecting all wells (or chambers of wells) to allow, for example, simultaneous pulsing to lyse all cells or to bubble out all cells or beads. The counter electrode communicates with a current controller which regulates current to the electrode. In some instances, the electrodes of an electrode layer may be made of platinum.
[0045] The term “ content of a weir as used herein refers to the amount of bead(s), cell(s) per well and/or cell-derived molecules. It may further relate to features of the cell such as detectable cell surface markers and the viability status (dead or alive) of the cell.
[0046] The term “ cross-contamination ” as used herein refers to well-to-well contamination on a chip comprising a plurality of wells, e.g. contamination of cell-derived molecules of wells in close proximity (e.g., neighbouring wells). In some instances, cross-contamination with molecules from other cells may be measured by mixture experiments as known in the art (see for example [2]: Macosko et al. , Cell 2015, doi: 10.1016/j.cell.2015.05.002; [5]: Ding et al, Nature Biotechnology 2020, doi: 10.1038/s41587-020-0465-8). [0047] The term “bead” as used herein refers to a substantially spherical, optionally monodisperse and optionally porous particle that can be accommodated in a microwell of the chip. The term “ porous ” as used herein refers to a particle containing pores which may be of non-uniform or uniform diameters. The term “ monodisperse ” as used herein is used to characterize a population of beads with low heterogeneity and a homogenous size distribution.
[0048] The term “ gas bubble ejection ” as used herein refers to the electrochemical production of gas bubbles at the bottom of a selected well allowing for efficient lifting and removing of the content out of the well. The content from selected wells is flushed out and may be collected for further processing.
[0049] The term “ linker ” or “ oligonucleotide linker ” as used herein refers to molecules coated onto the beads which allow contacting of cell-derived molecules to the bead (e.g. ligation, hybridization, annealing, binding). A linker may comprise one or more defined sequence elements. In the context of a linker used for contacting cell-derived nucleic acid molecules, the linker may comprise nucleic acid sequence elements such as a spacer, a PCR handle, barcodes (i.e. a cell barcode and/or a UMI) and/or a poly(dT) sequence as further described below. These sequence elements or sequence segments may be in a specific order (5’ to 3’ direction), e.g., as shown in Fig. 8: a PCR handle, a cell barcode, a UMI, a poly(dT)30 sequence (SEQ ID NO: 3). The number of linker molecules coated on a bead depends on bead loading capacity as described elsewhere herein and is typically optimized for high capture efficiency of cell derived molecules (such as RNA molecules) on a bead. In some instances, the number of linkers (e.g., plurality of linkers) on a single bead matches at least the number of cell derived target molecules (e.g., 105-106 mRNA per mammalian cell).
[0050] The term “ oligonucleotide ” as used herein refers to nucleotide multimers such as ribonucleotides (RNA) and/or deoxyribonucleotides (DNA) and may be composed of natural or non-natural nucleotides or a combination of both. [0051] The term “ hybridize ” or “ hybridization ” as used herein refers to the pairing of substantially complementary nucleotide sequences (strands of nucleic acid) to form a duplex or heteroduplex through formation of hydrogen bonds between complementary base pairs. It is a specific, i.e., non- random, interaction between two complementary polynucleotides. Hybridization and the strength of hybridization (i.e., the strength of the association between the nucleic acids) is influenced by such factors as the degree of complementary between the nucleic acids, stringency of the conditions involved, and the Tm of the formed hybrid. In the context of the present disclosure, hybridize" or “ hybridization ” can refer to binding of cell-derived nucleic acid molecules to bead- bound nucleic acid molecules (e.g. linkers) to capture the cell-derived nucleic acid molecules and/or to prime the cell-derived nucleic acid molecules for cDNA synthesis. Hybridization is an example of “ contacting’ ’ cell-derived molecules with the linker on the bead. In the context of nucleic acid molecules, hybridization conditions are not particularly limited and suitable buffers and the reaction temperature could be optimized in many different ways as known in the art by taking into account, e.g., GC content, secondary structure, and degree of homology to the target.
[0052] The term “ barcode ” as used herein generally refers to a nucleic acid segment that may be used as a component of a nucleic acid molecule to identify this nucleic acid molecule. For example, a single-stranded or double-stranded nucleic acid molecule of 50 nucleotides (nt) in length may have a 10 nucleotides region that identifies it as coming from a particular sample (e.g. a particular cell). Such barcodes may be added to nucleic acid molecules from different samples, where nucleic acid molecules in each sample share a common barcode or may be added to individual molecules within a sample such that each nucleic acid molecule receives a different barcode. These barcoded samples or nucleic acid molecules may then be mixed (e.g. during a sequencing step), with each nucleic acid molecule in the new mixture being traceable back to the sample (or nucleic acid) that it came from (sample origin tracing).
[0053] In some instances, barcodes may be used to identify individual nucleic acid molecules that result in the generation of specific amplification products after, for example, multiple rounds of amplification (molecule origin tracing). In such instances, barcodes may be used, for example, to determine the effects of “PCR heterogeneity”. Typically, individual nucleic acid molecules to be analyzed are connected (e.g., by using methods described below) to a library of diverse (e.g., degenerate) barcodes. This connection is typically done in a manner such that each nucleic acid molecule in a sample (e.g. derived from a single cell) is statistically connected to a different barcode that remains associated with it during amplification processes. The origin of amplified nucleic acid molecules may then be traced back to starting nucleic acid molecules or samples (such as a specific cell). This is typically done by sequencing of nucleic acid molecules present after amplification.
[0054] Barcodes may be of any number of lengths but they will typically be long enough such that they can be readily identified (e.g., by hybridization, by sequencing, etc.) but short enough so that they do not interfere with processes related to the workflows they are used in. For example, a two hundred nucleotide barcode would typically not be used to identify nucleic acid molecules when identification is done by DNA sequencing using platforms with limited read length. Barcodes will typically be from about 5 nucleotides (or base pairs) to about 50 nucleotides (or base pairs) (e.g., from about 7 to about 50, from about 10 to about 50, from about 7 to about 45, from about 7 to about 40, from about 5 to about 35, from about 5 to about 30, from about 5 to about 25, from about 5 to about 15, from about 7 to about 15, etc. nucleotides). In many instances, a barcode may have a length that is sufficient to allow for specific binding of a barcode-specific primer such as, e.g., between about 10 and about 30 nucleotides (e.g., 10, 12, 15, 18, 20, 25 or 30 nucleotides or between 18 and 25 nucleotides etc.). Generally, the length of the barcode may be chosen by taking the read length of the selected sequencing platform into account.
[0055] Where barcodes are used to identify desired nucleic acid molecules (e.g., nucleic acid molecules associated with a cell of interest), the nucleotide composition of each barcode within a barcode library may be designed to allow for sufficient distinction between all barcodes even if sequencing errors occur (e.g., where a sequencing-based mutation in a first barcode would result in a sequence that is identical with the sequence of a second barcode). In some instances, sufficiently distinct barcodes may be designed using “ edit distance’ ’ or “ Levenshtein distance” between two sequences (see, e.g., [11]: “Introduction to Algorithms”, Third Edition (2009) by Cormen et ah, The MIT Press Cambridge, Massachusetts London, England) or “ Hamming distance ” (as described, e.g., in [12]: Bystrykhor “Generalized DNA Barcode Design Based on Hamming Codes”, PLoS One, 2012, Vol. 7, issue 5, e36852) . The “ Levenshtein distance ” is a string metric for measuring the difference between two sequences. For example, the Levenshtein distance between two barcodes is the minimum number of single nucleotide changes (insertions, deletions or substitutions) required to change one barcode into another barcode. In some instances, a barcode library used in methods described herein may be designed with a Levenshtein distance of an overall minimum of 2, 3, 4, 5, 6, 7, 8, 9 or 10 (i.e., requiring a minimum of 2, 3, 4, 5, 6, 7, 8, 9 or 10 changes to convert into another barcode). The Levenshtein distance may vary over the length of the barcode molecules or only a portion of the barcode. For example, the Levenshtein distance at the 3’ end of a barcode may be different than the Levenshtein distance set for the 5’end of the barcode. Barcodes and barcode libraries may comprise UMI and cell barcodes as further defined below.
[0056] The term “ UMF or “ unique molecular identifier ” as used herein refers to a type of molecular barcoding that provides error correction and increased accuracy during sequencing. These molecular barcodes are short nucleic acid sequences used to uniquely tag each cell-derived molecule. In some examples, a UMI may comprise a sequence of random nucleotides (e.g., 2n, 4n, 6n, 8n, 10h or more) as described above. Preferably, each linker has a unique UMI sequence (with one nucleotide position being represented by 4 different bases). The number of random nucleotide positions n in a UMI may be adjusted according to the expected number of target molecules to be analysed. For example, 2 random nucleotide positions will result in 42 = 16 different sequences, 3 random nucleotide positions will result in 43 = 64 different sequences 10 random nucleotide positions will result in 410 = le6 different sequences etc.). The UMI will be used for data analysis to confirm unique cDNA synthesis event and be designed to remove PCR-based amplification biases. In a preferred setting, all linkers coated on an individual bead will have a different UMI barcode sequence.
[0057] The term “ce// barcode ” as used herein refers to a type of molecular barcoding that provides a unique nucleic acid sequence to identify nucleic acid molecules that originate from the same cell and to distinguish them from nucleic acid molecules of other cells. A cell barcode may comprise a random or semi-random or defined sequence as described above. In a preferred setting, all linkers coated on an individual bead will have the same cell barcode such that each retrieved cDNA containing the respective cell barcode sequence can be traced back to an individual cell. In some instances, a cell barcode may be synthesized using a split-and-pool synthesis as described e.g. in [2]: Macosko et al., Cell 2015, doi: 10.1016/j .cell.2015.05.002. To generate the cell barcode, a pool of beads may be repeatedly split into four equally sized synthesis reactions, to which one of the four dNTPs is added, and then pooled together after each cycle. The barcode synthesized on any individual bead reflects that bead’s unique path through the series of synthesis reactions. For example, a cell barcode comprising 12 bases would undergo a total of 12 split-pool cycles. In that case the result would be a pool of beads, each possessing one of 412 (16,777,216) possible sequences on its entire complement of linkers.
[0058] The term “ spacer ” as used herein refers to a short segment of the linker positioned adjacent to the bead. For example, in the context of a nucleic acid linker, a spacer allows for sufficient space for the reverse transcriptase to bind to a linker on the bead for subsequent cDNA synthesis. A spacer may be composed of different residues. In some examples, a spacer may comprise a string of nucleotides (i.e. a nucleic acid sequence). In other examples, a spacer may comprise -CFF alkyl chains.
[0059] The term “ PCR handle" or “ PCR handles ” as used herein provides a universal binding site for PCR-based amplification of a target nucleic acid molecule, such as a cell-derived nucleic acid molecule. A PCR handle will typically comprise a defined (i.e. non-random) sequence and be designed to allow for efficient and specific binding of a primer for downstream application. In a preferred setting, all linkers coated on beads comprise a universal PCR handle to allow simultaneous amplification of all nucleic acid molecules captured by the beads.
[0060] The term “array of beads” as used herein refers to a three-dimensional structure consisting of a collection of elements (beads) wherein each position of a specific bead in the array of beads is associated with a unique cell barcode. The cell barcode is comprised in a nucleotide sequence segment of a linker. A specific bead is coated with a plurality of linkers. In addition to the cell barcode each linker comprises a nucleotide sequence segment to provide a UMI which has a different nucleotide sequence as compared to the UMIs (as described elsewhere herein) of other linkers coated on the specific bead. In some instances, a microchip with wells or a multiwell plate are used to position the beads. A specific bead with a unique cell barcode in an array of beads is addressable. In some instances, the array of beads may be used as a microarray and adopted to various applications, e.g., in large-scale genetic, epigenetic, and expression studies.
The platform
[0061] The disclosure relates to a platform comprising a chip with a plurality of wells (e.g. microwells), wherein a complementary metal-oxide-semiconductor (CMOS) layer is provided to connect individually controllable electrodes located at the bottom of each well (or at least at one segment of the bottom of each well), wherein the electrodes are individually controllable for each well, and wherein each well is configured to accommodate a microscale bead and a eukaryotic or prokaryotic cell. In some instances, the bead may be coated with a plurality of linkers.
[0062] In some instances, an open end of the wells will be located at an upper surface of the chip that comprises the plurality of wells.
[0063] The shape (and size) of wells will be determined based on the types of cells and/or beads to be trapped within the microwells.
[0064] In some instances, the shape of a well and/or the shape of a chamber of a well (as described elsewhere herein) will be a non-cylindrical shape and may be a pyramid, cone or quadratic shape. In some instances, the wells or chambers may be in the shape of a reverse, truncated cone. In other instances, the microwells may comprise a shape that combines two or more geometries (i.e. each of which is referred to herein as a chamber). For example, it may include two side-by-side cylinders, one of larger diameter (for example, corresponding roughly to the diameter of a cell) than the other (for example, corresponding roughly to the diameter of a bead), that are connected. In some instances, the closed end (or bottom) of the microwells will be flat, but curved surfaces are also possible.
[0065] Distances between wells may be within a range of between 10% to 90% of well diameter, such as e.g. between 20% and 50%, between 30% and 70%, between 50% and 90% or between
60% and 80% such, as, e.g., 75%. Furthermore, the depth of a well may be within a range of about
100% to about 200% of the diameter of the well, such as e.g. between about 100% to about 130%, between about 110% to about 150%, between about 120% to about 170%, between about 150% to about 200%, between about 120% to about 130%, such as, e.g., about 125%. In instances, where a well is configured to accommodate a single bead, the bead diameter may be within a range of between about 55% to about 99% of the well diameter, such as, e.g., between about 60% and about 95%, between about 65% and about 85%, between about 75% and about 90%, such as e.g. about 87%. In other instances, the microchip may be configured to accommodate two or more beads of smaller sizes in a single well.
[0066] The chip used in the present platform may comprise a number of wells. In any event, the number of wells may be in number, for example, between 10 and 10,000,000, between 10 and 5,000,000, between 10 and 2,000,000, between 10 and 1,000,000, between 10 and 800,000, between 10 and 650,000, between 10 and 500,000, between 500 and 500,000, between 500 and 200,000, between 10 and 50,000, between 1,000 and 500,000, between 10,000 and 500,000, between 20,000 and 500,000, between 1,000 and 50,000, between 10 and 50,000, between 10 and 25,000, between 10 and 10,000, between 10 and 5,000, between 10 and 2,500, between 100 and 50,000, between 100 and 25,000, between 100 and 10,000, between 100 and 5,000, between 100 and 2,500, between 350 and 50,000, between 350 and 25,000, between 350 and 10,000, between 350 and 5,000, between 350 and 2,500, between 1000 and 50,000, between 1000 and 25,000, between 1000 and 10,000, between 1000 and 5,000, between 1000 and 2,500, between 1,500 and 50,000, between 1,500 and 25,000, between 1,500 and 10,000, between 1,500 and 5,000, between 1500 and 2,500, between 20,000 and 50,000, between 20,000 and 40,000, between 30,000 and 40,000, or about 35,000. In certain instances, the number of wells may be, for example, 35,440.
[0067] The size of a well and/or the size of a chamber of a well is determined by bead size and cell size. For example, in some instances, well or chamber sizes may be designed to accommodate single prokaryotic cells having typical sizes between about 0.1 and about 5 pm. In other instances, the well or chamber sizes may be designed to accommodate single eukaryotic cells having typical sizes between about 10 and about 100 pm. Thus, well or chamber sizes may be ranging from about 0.1 pm to about 5 pm or from about 1 pm to about 30 pm or from about 10 to about 100 pm. In principle, all shapes of wells or chambers are possible as long as it allows for co-placement of a single bead and a single cell. [0068] The total volume of each well is another item which may vary and may be, for example, between 1.0 x 109 mΐ and 50 mΐ, between 1.0 x 109 mΐ and 10 mΐ, between 1.0 x 109 mΐ and 1.0 mΐ, between 1.0 x 109 mΐ and 0.1 mΐ, between 1.0 x 109 mΐ and 1.0 x 102 mΐ, between 1.0 x 109 mΐ and 1.0 x 103 mΐ, between 1.0 x 109 mΐ and 1.0 x 104 mΐ, between 1.0 x 109 mΐ and 50 mΐ, between 1.0 x 105 mΐ and 1.0 x 106 mΐ, between 1.0 x 109 mΐ and 1.0 x 107 mΐ, between 2.5 x 109 mΐ and 1.0 x 102 mΐ, between 2.5 x 109 mΐ and 1.0 x 103 mΐ, between 2.5 x 109 mΐ and 1.0 x 104 mΐ, between 2.5 x 109 mΐ and 1.0 x 105 mΐ, between 2.5 x 109 mΐ and 1.0 x 106 mΐ, between 1.0 x 108 mΐ and 1.0 x 106 mΐ, between 1.0 x 108 mΐ and 1.0 x 105 mΐ, between 1.0 x 107 mΐ and 1.0 x 105 mΐ, between 1.0 x 107 mΐ and 1.0 x 104 mΐ, between 1.0 x 107 mΐ and 1.0 x 103 mΐ, between 1.0 x 107 mΐ and 1.0 x 102 mΐ, between 0.1 mΐ and 50 mΐ, between 0.01 mΐ and 50 mΐ, between 0.01 mΐ and 25 mΐ, between 0.01 mΐ and 15 mΐ, between 0.01 mΐ and 10 mΐ, between 0.001 mΐ and 50 mΐ, between 0.001 mΐ and 5 mΐ, between 0.001 mΐ and 1 mΐ, between 0.001 mΐ and 0.01 mΐ, between 0.001 mΐ and 1 mΐ, between 1 mΐ and 50 mΐ, between 1 mΐ and 25 mΐ, between 1 mΐ and 10 mΐ, between 10 mΐ and 50 mΐ, between 10 mΐ and 25 mΐ, or between 15 mΐ and 25 mΐ. In certain exemplary instances, the total volume of each well may be between 1 x 106 mΐ and 1 x 104 mΐ, between 1 x 105 mΐ and 1 x 104 mΐ, such as about 6.3 x 105 mΐ.
[0069] The number and size of wells will be determined by the overall configuration of the chip. In certain instances, the configuration of a chip may be adapted by the variation of certain parameters such as chip size and number or size of wells and chambers. Exemplary configurations (design 1 or 2) showing possible variations of well or chamber geometry and electrode design are illustrated in Fig. 10.
[0070] Conventional microfabrication (such as Micro-Electro-Mechanical Systems; MEMS) technology may be used for forming wells of desired shape or size on a CMOS chip. In some instances, wells may be formed by MEMS processes, e.g. by laminating an epoxy based dry film resist, after completing the CMOS chip. Electrodes may be fabricated with wells or separately.
[0071] The individually controllable electrodes located at the bottom of each well (or at least at one segment of the bottom of each well) allow for selective pulsing bubble out single beads carrying, for example, target nucleic acid molecules, and collecting the beads for further processing.
[0072] In some instances, the content is released by flushing bead lifting solution into chip, applying voltage and generating gas bubble. Next, the content is lifted by gas bubble ejection.
[0073] The generation of gas bubbles in a microwell of the chip can be used as a method to remove beads from the respective well. Gas bubbles can be produced electrochemically in aqueous or non- aqueous buffers, e.g., water, NaCl dissolved in water and more complex non-aqueous buffers. The inherent properties of the buffer used can have significant implications on the efficiency or performance of the system. For example, the composition of the buffer can influence the surface tension of the bubbles produced. The surface tension is critical for the bubble movement in the well (retention potential). For an efficient removal of beads from a well it is desirable that both, beads and bubbles escape from the well (compared to the bubbles remaining in the well and disturbing the fluidic flow of the system). If the surface tension is too low, the generated bubbles will escape through the gap between well and bead without lifting the bead. If, however, the surface tension is too high, gas bubbles will stick tightly to the walls of the well, which may require additional treatment to remove the gas bubbles such as longer rinsing or rinsing with low surface tension solvents (such as e.g. methanol). Further, high surface tension may also result in beads sticking to gas bubble and not being released into the fluid stream. A favourable surface tension can be achieved by mixing organic solvents (e.g., acetonitrile, isopropanol) and aqueous solutions, preferably 50-90% organic solvents, more preferably, 60-70% organic solvent. The buffer can also be selected to avoid negative effects on certain steps of the workflow, such as e.g. on-chip synthesis of linkers or parts thereof. For example, acidic environment has the potential to damage the nascent oligonucleotide chain, and basic condition can promote premature cleavage of the linker from the bead. Therefore, a buffered system can be used to avoid the generation of such an undesired condition.
[0074] Generally, a lifting buffer may comprise water, a solvent, a salt as an electrolyte and methanol. In certain instances, a lifting buffer may comprise water, an ammonium salt, acetonitrile and methanol. Such buffers have a high conductivity required for electrochemical generation of gas bubbles while its surface tension allows for efficient bead lifting and removal of bubbles from wells. Using such buffer, bead lifting can be achieved at a potential of >4.5 V. In other instances, a lifting buffer may comprise an inorganic salt (e.g. a lithium salt), water and methanol. In an exemplary embodiment a lifting buffer comprises 0.05 M L1CIO4, 20% water and 80% methanol. Using this buffer, bead lifting can be achieved at a potential of 8.5 V and 6 mA current limit. In other instances, other salts may be used. For example, organic or inorganic salts may be added at a concentration of 0.01 M, 0.05 M, 0.1 M, 0.25 M, 0.4 M, 0.5 M, 0.7 M, 0.8 M, 0.9 M or 1.0 M. In certain instances a lifting buffer may comprise less than 50% water (such as e.g. up to 2%, up to 5%, up to 10%, up to 20%, up to 30% or up to 40% water) and more than 50% methanol (such as e.g. up to 95%, up to 90%, up to 80%, up to 70%, up to 60% methanol). In some instances, other organic solvents (such as, for example, acetonitrile) may be used instead of methanol.
[0075] Generally, potentials of > 0 V and up to 10 V may be applied to allow bead lifting. In some instances, a higher potential may be used for optimal bead lifting. For example, 8.5 V may be used without limiting the current. In other instances, current on the chip may be limited during bead lifting. For example, current may be limited by only pulsing a portion of the chip’s individually controllable electrodes simultaneously. For purposes of illustration only, bead lifting using a microfluidic chip with about 35,440 wells may be achieved by pulsing individually controllable electrodes in 400 wells simultaneously in subsequent steps such that not all individually controllable electrodes will be activated at the same time thereby limiting current on the chip.
[0076] In some instances, the content of all or selected wells (or a chamber of a well) (for example, beads) may be lifted from the well by electro-ejection and collected for further processing. Electro ejection refers to efficient lifting and removing the content of a well by reversing the current of all or selected wells (or a chamber of a well). The content from all or selected wells is flushed out and may be collected (e.g. in a collection plate or vessel) for further processing.
[0077] In some instances, the platform comprises a chip wherein each well has two connected chambers (e.g., a first and a second chamber) of a size which can accommodate at least a microscale bead or a single cell. Each chamber may be connected to a CMOS layer by individually controllable electrodes provided at the bottom or a segment of the bottom of the chamber. In some instances, a first and a second chamber are not electrically connected. For example, a first chamber has an individually controllable electrode (connected to a CMOS layer) and a second chamber is characterized in that an electrode layer is provided at its bottom which is controllable for the entire chip (electrode layer). The electrode layer provides a single counter electrode on the chip connecting all wells (or chambers of wells, e.g. all second chambers) to allow, for example, simultaneous pulsing to lyse all cells or to bubble out all cells or beads. In some instances, a counter electrode may be placed in the chip instead of the lid to allow for pulsing if wells are covered by oil.
[0078] Beads may be monodisperse and optionally porous particles. The bead is a support matrix for linkers. In some instances, substantially each well of the chip comprises a bead.
[0079] Monodisperse beads have a coefficient of variation of their diameters of less than 20%, for example less than 15%, typically of less than 10% and optionally of less than 8%, e.g. less than 5%. Monodisperse beads are characterized in low heterogeneity and a homogenous size distribution. The size distribution of a bead may be defined by the coefficient of variation (percentage CV) which may be determined by methods known in the art (for example, as described in [13]: WO2017/211913A1). CV is defined as 100 times (standard deviation) divided by average where "average" is mean particle diameter and standard deviation is standard deviation in particle size. The CV for a plurality of beads may for example be within a range of 50 to 100%. For example, a monodisperse bead population may have more than 90%, preferably more than 95% of the particles with sizes within their mean diameter of ± 5 %.
[0080] Porous beads are characterized by a specific pore volume, for example, 1 ml of pore volume per 1 gram of polymer is equal to 50% porosity. For example, a bead with a pore volume of 2.2 ml/g polymer has a porosity of 70%. Bead porosity depends on the polymer used and may be an important factor in achieving synthesis of linkers of a certain length. In certain instances, the pore volume of a bead may be within a range of 0.1 to 2.5 ml/g polymer.
[0081] The monodisperse bead may be further defined by its surface area. For example, the surface area of a bead may be within a range of 10 to 1000 m2/g, between 100 and 500 m2/g, between 200 and 400 m2/g such as, for example, around 380 m2/g. The surface area of porous monodisperse beads can, for example, be determined according to a method developed by Brunauer, Emmett and Teller referred to as the BET method which is based on the physical adsorption of a vapor or gas onto the surface of a solid ([14]: Brunauer, S., Emmett, P. and Teller, E., J. Amer. Chem. Soc. 60 (1938), p. 309). This method uses dry beads for testing so, for accurate measurement, the pores should be of stable volume when exposed to solvents as compared to when dry. In certain instances the surface area may be determined for beads dried at 60°C for 2 hours from tetrahydrofuran or methanol.
[0082] Beads may be produced by methods known in the art (see in [13]: WO2017/211913A1 or [15]: US6,335,438) including, for example, emulsion polymerisation and dispersion polymerisation. For example, by including an appropriate porogen into a polymerisation mixture, porous materials in form of beads can be produced. Porogens are compounds that separate out and form pores when the polymer is formed. Porogens may be in forms of liquids, solids or gases and shall be removed after polymerisation. The polymerisation material may be a natural or synthetic polymer. The polymer may be crosslinked.
[0083] In one example, the bead may be a polystyrene bead coated with reactive groups such as amine groups or hydroxyl groups, and wherein said bead comprises: a diameter of between 10 and 100 pm or between 20 and 40 pm with a coefficient of variation of less than 10% or less than 5%, a surface area ([14]: Brunauer, S., Emmett, P. and Teller, E., J. Amer. Chem. Soc. 60 (1938)) within a range of between 100 and 500 m2/g or within a range of between 150 and 300 m2/g, a porosity within a range of 60% to 80%, optionally, an amine content of between about 2% and about 8% or between about 3% and about 5%, or less than 3%, a linker loading capacity of between about 15 pmol/g to about 100 pmol/g, or between about 35 pmol/g to about 70 pmol/g, optionally, wherein said bead carries a plurality of linkers as described elsewhere herein. [0084] Bead sizes used in the present platform and methods may vary widely but include beads with diameters between 0.1 pm and 100 pm, 0.05 pm and 100 pm, 0.05 pm and 10 pm, 0.1 pm and 100 pm, 0.1 pm and 1,000 pm, between 1.0 pm and 2.0 pm, between 1.0 pm and 100 pm, between 2.0 pm and 100 pm, between 3.0 pm and 100 pm, between 0.5 pm and 50 pm, between 0.5 pm and 20 pm, between 1.0 pm and 10 pm, between 1.0 pm and 20 pm, between 1.0 pm and 30 pm, between 10 pm and 40 pm, between 10 pm and 60 pm, between 10 pm and 80 pm, or between 0.5 pm and 10 pm. In certain instances, the beads may have a diameter between 30 pm and 40 pm, such as a diameter of 31 pm or 32 pm or 33 pm or 34 pm or 35 pm. Where specific bead sizes are disclosed herein the exact bead size may not be so limiting in that context but may represent a broader range (e.g. a 35 pm bead may encompass beads sizes of between about 30 and about 40 pm). In a preferred instance, the bead size may be chosen depending on the size of the well to allow only one single bead to occupy a well.
[0085] A bead provides the solid support and the bead’s surface is functionalized for cell-derived molecule capture by chemical groups, i.e. a linker. For example, linker molecules coated onto the beads allow reversible coupling of cell-derived molecules to the bead. Generally, a linker molecule is designed to be biologically inert and should not comprise any bulky side chains to avoid steric hindrance effects on coupled molecules. In some instances, linkers may have lengths between about 1 and about 10 nm.
[0086] The bead may be further functionalized or coated with reactive groups which may affect the linker loading capacity. To achieve efficient coating of the bead with a plurality of linkers, bead-specific parameters (such as bead surface area, pore volume, composition) may be important parameters for coating efficiency and after coating for “ linker loading capacity" . The higher the linker loading capacity of a bead, the higher the overall yield of cell-derived molecules will be.
[0087] The “linker loading capacity ” of a bead defines the amounts of linker molecules (such as oligonucleotides) that can be loaded onto the bead and is therefore a determinant of the yields of linkers that can be loaded per gram bead. The loading capacity may depend on the structure and composition of the bead (i.e. number of functional groups available for linking the molecules as described below) as well as the length of the loaded linker (steric hindrance effects). [0088] A dimethoxytrityl (DMT) cation assay may be performed to compare linker loading capacity of different types of beads by determining trityl group concentration which corresponds to the concentration of the oligonucleotide linkers on the bead. E.g. after a base has been coupled to a bead, an acid-based deprotection step could be performed to remove the terminal hydroxyl protecting DMT group which can be spectrometrically measured. DNA synthesizers or synthesis chips as described herein may be configured to automatically collect, with the aid of a fraction collector, the solution containing the DMT cation during the detritylation step of the synthesis cycle. The DMT group is completely ionized in the acidic solution in which it was cleaved. The yield can be accurately measured (± 0.5%) at 498 nm. This assay is valid only when used in conjunction with a synthesis cycle utilizing a capping procedure that quantitatively blocks unreacted oligonucleotide chains. If these chains are not capped, the assay will give incorrectly high yield data because of the coupling and subsequent detritylation of the n- sequences.
[0089] Beads that may be used in methods or systems described herein may have a linker loading capacity within a range of 10 to 500 pmol/g, within a range of 20 to 200 pmol /g or within a range of 40 to 400 pmol /g or within a range of 50 to 100 pmol/g. In certain instances, the bead may be characterized by a high loading capacity, e.g. a loading capacity of at least 80 pmol/g, at least 100 pmol/g, at least 150 pmol/g, at least 200 pmol /g, at least 250 pmol /g, or at least 300 pmol/g.
[0090] The yield of oligonucleotide linkers synthesized on a bead (e.g. in wells of a chip) may be defined as the percentage of linker molecules generated per well/bead having the desired length or the amount of full-length oligonucleotides generated per well/bead. For example, a 35 pm bead with a linker loading capacity of 300 femtomole may carry 30 to 50% of full-length oligonucleotide linkers resulting in an amount of 90 to 150 femtomole of oligonucleotide linkers having the desired length. Alternatively, the amount of full-length oligonucleotide linkers synthesized in a well/on a bead may be between 90 to 150 femtomole, between 100 and 200 femtomole, between 150 and 300 femtomole, more than 300 femtomole, etc.
[0091] The monodisperse bead may be further functionalized or coated with reactive groups which may affect the linker loading capacity. A bead functionalized for oligonucleotide capture, attachment and/or synthesis may, for example, carry amine groups and may be defined by its amine content. The amine content of a bead may be expressed by weight % nitrogen per gram of beads and may be within a range of 0.01% and 5%, 0.1% and 3%, 0.15% and 0.5%, 2% and 5%, 0.5% and 1.5%, 1.5% and 2%. Methods for elemental analysis to determine the weight percentage (weight %) nitrogen and calculate amine content of solid supports (mol amine per gram of support) are known in the art and may, for example, be calculated according to methods described by [16]: Dumas A. (1826): Annales de chimie, 33,342 or as further set forth by [17]: the US Environmental Protection Agency in method 440.0: Determination of Carbon and Nitrogen in Sediments and Particulates of Estuarine/Coastal Waters Using Elemental Analysis. In certain instances, the amine content may be about 1.8%, about 1.5%, about 1.2%, about 1.0%, about 0.8%, about 0.5%, about 0.25%, about 3%, about 3.5%, about 4% or about 5%.
[0092] The skilled person will understand that the amine content of a bead depends on the amine- containing compound or monomer used for polymerization. The amine content of a bead may thus be adapted by using different amounts of an amine-containing monomer. For example, lower amounts of aminostyrene such as less than 10 weight % or less than 5 weight % per gram of the total amount of monomers used in a polymerization mixture may be used to generate beads with a lower amine content. Alternatively, the amine content may be modified based on the selection of a different amine containing monomer or a monomer containing a functionalizable group. For example, monomers such as, vinylbenzyl-chloride may be used in the polymerization of a bead. Thus, in one example the amine content of a monosized bead is determined by the amount of vinylbenzyl-chloride used in polymerization.
[0093] In some instances, an oligonucleotide linker may comprise one or more defined sequence elements (as defined above) such as a barcode sequence. A barcode sequence allows each nucleic acid molecule being traceable back to the sample it came from (sample origin tracing), e.g. a cell barcode is unique to a cell (see for example, [2]: Macosko et al ., Cell 2015, doi: 10.1016/j .cell.2015.05.002).
[0094] Barcodes and barcode libraries may comprise UMIs and/or cell barcodes. Barcodes may be made and designed in a number of ways. For example, individual barcodes of a barcode set may be degenerate (also referred to as “random”) at one of more positions (e.g., between 2 and 10, or between 4 and 20), meaning that two or more bases can be present at specific positions. In some instances, such random barcodes can be made in a single synthesis run using, for example, “dirty bottle” synthesis where mixtures are used that result in the incorporation of two or more bases at specific location. Typically, the incorporation of the different bases will be designed to be fully random but concentrations of “building blocks” may be adjusted so that one base is present in a higher or lower ratio than one or more other base. Barcodes may also be designed individually to have specific sequences. In many such instances, each barcode will be produced in a separate nucleic acid synthesis run. Like oligonucleotides, barcodes may be synthesized on a microchip or array in microscale amounts using standard chemistry, electrochemistry or enzymatic synthesis as disclosed elsewhere herein.
[0095] In some examples, barcodes as used herein may be derived from a barcode library. A barcode library may be designed such that each barcode is sufficiently different from another barcode in the library. One exemplary approach to design a barcode library may rely on a method comprising the following steps: (a) designing, in silico a set of candidate barcode sequences having desired sequence properties (e.g., a pre-determined GC content, length, melting temperature, or other properties known to be desirable for primer sequence function); (b) picking, in silico one candidate barcode of the set of candidate barcode sequences and (c) removing, in silico all candidate barcode sequences from the set of candidate barcode sequences that are “too similar” to the picked candidate barcode sequence, where similarity is determined by identifying the number of deletions, insertions or mutations necessary to transform one candidate barcode sequence to another candidate barcode sequence, a parameter known as the “ edit distance’ ’ or “ Levenshtein distance ” between two sequences (see, e.g., [11]: “Introduction to Algorithms”, Third Edition (2009) by Cormen et ah, The MIT Press Cambridge, Massachusetts London, England). Alternatively, “ Hamming distance ” (as described, e.g., in [12]: Bystrykhor “Generalized DNA Barcode Design Based on Hamming Codes”, PLoS One, 2012, Vol. 7, issue 5, e36852) or other appropriate distances may be used to remove barcode sequences of high similarity. The method may further comprise the step d) of iteratively picking further candidate barcode sequences until no candidate sequences of high similarity remain. The resulting pool of picked barcode sequences forms the barcode library with barcodes sufficiently different from each other. In some examples, the candidate barcode picked in step (b) may be chosen randomly or by using a “greedy” approach such that a plurality of barcode libraries with varying barcode numbers may be obtained from which the largest one may be chosen.
[0096] In some instances, each nucleic acid molecule within a population of nucleic acid molecules may receive an individual barcode (e.g. a UMI). This may be achieved by using a barcode library with a diversity that exceeds the diversity of nucleic acid molecules in the population. In other instances, it may not be required to tag each molecule with an individual barcode. For example, the diversity of the barcode library may be from about 1,000 to about 100,000, from about 10,000 to about 1,000,000 or more different molecules. In some examples, the diversity of the library may be up to 10,000 different barcodes. In other examples, the diversity of the library may be up to about 100,000 different barcodes. In yet other examples, the diversity may be about 1,000,000 different barcodes. In yet other examples the diversity of the library may be up to about 10,000,000 different barcodes. In some instances, the diversity of a barcode library may be within a range of 1,000 to 1.1 x 1012. Such diversity may be achieved by using barcodes with degenerate nucleotide positions (e.g., a 20N degenerate barcode).
[0097] An oligonucleotide linker or certain segments thereof may comprise an oligonucleotide of either ribonucleotides (RNA) or deoxyribonucleotides (DNA), incorporating natural and non natural nucleotides and may be composed of natural or synthetic nucleobases, or a combination of both. The backbone of the oligonucleotide can be composed entirely of native phosphodiester linkages, or it may contain one or more modified linkages such as one or more phosphothioate, phosphoramidite or other modified linkages. Examples of modified or synthetic nucleobases include 3-methyluracil, 5,6-dihydrouracil, 4-thiouracil, 5-bromouracil, 5-thorouracil, 5-iodouracil, 6-dimethyl aminopurine, 6-methyl amino purine, 2-amino purine, 2,6-diamino purine, 6-amino-8- bromopurine, inosine, 5-methyl cytosine, 7-deazaadenine, and 7-deaza guanosine. Examples of modified RNA nucleotides include locked nucleic acids (LNA). Oligonucleotides may further comprise additional molecules that have been joined, either covalently or non-covalently. These additional molecules may be attached to any site on the oligonucleotide depending on steric hindrance and/or downstream applications. [0098] The oligonucleotide linker may have a length ranging from at least 20 to about 200 nucleotides. In some instances, the oligonucleotide linker may be immobilized on a support (e.g. a bead) via its 5’- or 3’-terminal ends.
[0099] In some instances, the oligonucleotide linker may be cleavable for retrieving nucleic acids such as cDNA linked to the bead or may comprise a cleavable coupling moiety adjacent to the bead as described below. The linker or cleavable coupling moiety may be e.g. photocleavable, or base-cleavable. For example, photocleavable linkers or moieties may be cleaved by generating light waves from a light source and cleaving the nucleic acid from the bead with the light waves. In other examples, nucleic acids linked to beads may be cleaved by reductive reactions comprising generating an electrochemically reduced compound and cleaving the nucleic acid from the solid support with the electrochemically reduced compound.
[0100] To make a bead suitable for an oligonucleotide linker, non-nucleosidic coupling moieties or nucleoside succinates may be covalently attached to reactive amino groups. If necessary, however, other surface functions such as carboxyl, hydroxyl, or thiol, for example, could be used to attach a coupling moiety carrying a hydroxyl group or alternatively a 3 '-attached nucleotide to the bead. These molecules incorporated into the bead or coated onto the bead’s surface allow physical attachment to or synthesis of an oligonucleotide linker on the bead and are referred to as “ coupling moiety" .
[0101] In many instances, an oligonucleotide (e.g. a linker) synthesized on a bead, may be physically coupled to the bead by a coupling moiety. In certain exemplary embodiments, the coupling moiety, when present, may be a chemical entity that attaches the 3'-0 of the oligonucleotide to the bead (e.g., a functional group on a bead). In other exemplary embodiments, the coupling moiety, when present, may have a structure such that it allows for attachment of other functionalities in addition to the 3’-0. Such coupling moiety structures are disclosed, for example, in [18]: U.S. Patent No. 7,202,264. In most cases, the coupling moiety will be inert to all the reagents used during the present methods, but cleavable under specific conditions. One coupling moiety commonly used in nucleic acid molecule synthesis is the succinyl coupling moiety. In some instances, universal coupling moieties may be used. A universal coupling moiety is a coupling moiety that allows for the synthesis of nucleic acid molecules regardless of the nature of the terminal base of the first nucleotide that is to be coupled. In certain instances, the bead carries a universal coupling moiety such as a UNYLINKER™. Different coupling moieties with different properties are known to those skilled in the art and can be selected by the skilled person depending on the type of synthesis (i.e. 5’-3’ or 3’-5’) and downstream process requirements.
[0102] The cells may be living on their own such as bacteria and protozoa or be part of a multicellular organism. Multicellular organisms may be plants, animals or human beings. In some instances, the cells are animal cells. In some instances, the cells are invertebrates, for example, flies such as Drosophila, beetles, nematodes such as Caenorhabditis elegans or worms. In some instances, the cells are vertebrates such as chicken, rat, mouse, bats, naked-mole rat, monkey or zebrafish. The cells may be mammalian cells. In some instances, cells may be derived from a part of a mammal such as skin, brain, heart, spleen, lungs, stomach, lymph node, bladder, intestines, kidneys or muscle. In some instances, the cells may be human cells, for example, stem cells, bone cells, blood cells, muscle cells, fat cells, or nerve cells. Cells may be T, B, NK, dendritic, myeloid, epithelial and stromal cells.
[0103] In some instances, the cells may be of the same origin (e.g. human or mouse), from the same tissue or organ of an organism, or be a mixture of different species (e.g. non-human and human cells) and/or of different tissues. In some instances, cell lines are mixed at a 1:1 ratio. In other instances, the cells may be different cell types derived from the same sample (e.g. a tissue). In many instances, populations of the same cell type or originating from the same sample or source will be analyzed.
[0104] A cell may be characterized by its age, developmental stage and physiological or disease state. In some instances, the cell may be derived from a healthy organism to analyse the physiological functions related to natural organ function, homeostasis, aging, or regeneration. Said functions may also be analysed upon changes in the environment of the cell. For example, the cell may be derived from an area of interest after injury to analyse its temporal or permanent changes of its molecular data upon wounding, fibrosis or scar formation. Cells may also be derived from vaccinated individuals and compared with cells of non-vaccinated or infected individuals. In other instances, the cell may also be derived from pathological situations like abnormal organ development, autoimmune diseases, chronic diseases, infectious disease or in cancer.
[0105] The cells may be isolated from multicellular organisms by methods used in the art for single cell isolation such as fluorescence-activated cell sorting (FACS), enzymatic digestion, manual picking, microfluidics, serial dilution or hydrodynamic traps. Individual cells may be analysed by the present platform and methods, or subpopulations of cells may be selected prior to analysis. In some instances, the cells may be sorted prior to associating a cell with a bead. For example, the cells can be sorted by fluorescence-activated cell sorting or magnetic-activated cell sorting, or more generally by flow cytometry. In some instances, the cells may be filtered by size or based on surface marker expression.
[0106] The platform has the ability to the control its environment. The environment comprises, for example, the composition, viscosity, pH, temperature and/or conductivity of a fluid in the well. The environment further comprises voltage and/or current of an individually controllable electrode (connected to the CMOS layer) and/or an electrode layer in the well (or a chamber or the well). For example, chemical reagents, temperature, pH of the reaction environment can be controlled in the platform. In some instances, the platform may contain chemical reagents associated with bead loading, cell loading, cell lysis, nucleic acid synthesis, bead ejection or mixtures thereof.
[0107] A platform comprises electrodes, for example, within wells (or chambers of wells) of a chip. In some instances, at least one counter-electrode may be provided. For example, in a first instance a counter-electrode may be placed at the bottom or along one or more sidewall of each well. In other instances, the chip may comprise a cover or lid containing a counter-electrode. Depending on the type of activation/step in the workflow, voltages applied to these electrodes may be in the range from about 0.1 V to about 10 V. In some instances, voltage applied for nucleic acid attraction (such as RNA molecules) may be between about 0.5 V and about 2 V (such as about 1.7 V, about 1.5 V, about 1 V or lower). In some instances, voltage applied for bead lifting may be between about 2 V and about 10 V (such as about 5 V, about 7 V or about 8.5 V). [0108] In some instances, the current of the individually controllable electrodes (CMOS layer) within a well (or chamber of a well) and/or an electrode layer across a chip may be restricted and may be kept in the range of between about 1 mA to about 20 mA, preferably from about 3 mA to about 10 mA.
[0109] In some instances, a platform for single cell analysis comprises a chip (e.g. a microchip) with a plurality of wells, wherein each well has at least a first chamber and a second chamber, and wherein the first chamber can accommodate at least one microscale bead, and the second chamber can accommodate at least one single eukaryotic or prokaryotic cell; wherein a CMOS layer connecting individually controllable electrodes is provided at the bottom of each well or at least at one segment of the bottom of each well. In some instances, the chip geometry allows for a 1 : 1 bead to cell distribution and capture in each well (e.g. a single bead in the first chamber and a single cell in the second chamber). In some instances, the individually controllable electrodes (connected to a CMOS layer) is provided at the bottom of only the first chamber. In some instances, the chip further comprises an electrode layer which is controllable for the entire chip and which is not electrically connected with the segment comprising the individually controllable electrodes.
[0110] Specific instances include alternative chip geometries for improved bead to cell distribution and capture and the like.
[0111] The chip is an electronic computer chip on which oligonucleotide synthesis on a solid support (for example, a bead) and/or cDNA synthesis can occur. The chip may comprise at least two wells, optionally a lid operable to be formed on a top surface of the chip and operable to provide a fluid flow path into and out of each of the wells (or chambers of wells). Exemplary chips suitable for use in the present platform and methods are described in [10]: WO 2016/094512 A1 and [19]: GB 2012261.0.
[0112] To arrive at an optimal chip configuration, the impact of the variation of certain parameters needs to be considered. Whereas certain parameters (such as, e.g., the number of wells or active area) may provide means for scale-up, other parameters may impact the performance or suitability of the chip for certain uses. For example, a microchip too small in size may not allow for fluidics applications and may therefore have a minimum size of not less than about 10 mm2 whereas a chip too large in size (i.e., larger than about 20 to 25 mm2) may not be compatible with high-yield manufacturing when CMOS technology is used. Likewise, the size and dimension of a well and/or a chamber should be such that a bead and a cell can be accommodated, or if a well has at least two chambers (as shown in design 2 of Fig. 10) it should be such that at least a bead or a cell can be accommodated per chamber. For example, chambers with a diameter of less than about 10 pm may be suboptimal, whereas chambers with a diameter of more than about 90 pm to about 110 pm may cause too long diffusion times for reagents, which may be problematic in certain instances. In some instances, the chip is between about 15 to about 25 mm2 in size. In a preferred embodiment, the chip is 18 mm2 in size.
[0113] An exemplary chip may have about 35,000 wells and be suitable for the analysis of 3,000 to 10,000 single cells.
[0114] In some instances, the platform comprises a chip with a single ~35 pm bead per well. The wells may be designed as cylindrical holes or chambers that are about 45 pm deep and about 40 pm in diameter. There may be about 30 pm spacing between wells. In certain instances, an 18 mm2 chip can accommodate about 35,440 individually addressable wells.
[0115] In some instances, the platform comprises a chip comprising 35,440 wells, wherein a CMOS layer is provided at the bottom of each well which is individually controllable for each well, and wherein each well can accommodate a bead and a eukaryotic or prokaryotic cell, wherein the size of the bead is chosen depending on the size of the well to allow only one bead to occupy a well.
[0116] In some instances, the platform comprises an epoxy based dry film resist (TMMF) CMOS chip containing about 35,000 wells of 45 pm depth. Wells may be loaded with aminostyrene beads with a size of 35 pm (as described in [15]: U.S. Patent No. 6,335,438).
[0117] In some instances, the platform further comprises an imaging system (e.g. a microscope) configured to capture and process images of all or a portion of the wells of the chip and well content (e.g. labelled cells), wherein the imaging system further comprises an illumination subsystem, an imaging subsystem, and a processor. An imaging system may be configured to perform bright-field, dark-field, fluorescence, or quantitative phase imaging. In some instances, the imaging system is configured to provide real-time image analysis capability, wherein the distribution of cells or beads across the wells of the chip can be analysed. An imaging system may further comprise a selection mechanism, wherein information derived from the processed images is used to identify a subset of cells (e.g. based on a label) exhibiting one or more specified characteristics, and the selection mechanism is configured to either include or exclude the subset of cells from subsequent data analysis or processing. A selection mechanism may comprise e.g. physical removal of beads co-localized with cells of the identified subset of cells from the wells as described elsewhere herein.
Methods
[0118] The present disclosure relates to methods for single cell analysis wherein the molecular data of a single cell is analysed in a platform as described elsewhere herein (see section “the platform”). Specifically, the genetic, epigenetic, spatial, and/or the proteomic data may be analysed by the present platform and methods, for example, by cell lysis and further processing of cell-derived molecules.
[0119] The present methods allow for analysis of a specific single cell by compartmentalization within certain reaction compartments. Cell-derived molecules cross-contamination is excluded or at least minimized in the experimental workflow. Where single cells are localized in microwells of a microfluidic chip, CMOS technology can be used to generate an electrical field within the chip to retain charged molecules released from lysed cells within a specific microwell thereby preventing cross-contamination of cell-derived molecules between different wells which provides an advantage over existing well-based systems.
[0120] In some instances, methods for single cell analysis which may comprise the following steps: a) providing a chip comprising a plurality of wells, wherein a complementary metal-oxide- semiconductor (CMOS) layer is provided to connect individually controllable electrodes located at the bottom of each well which is individually controllable for each well, and wherein each well can accommodate a microscale bead and a eukaryotic or prokaryotic cell, b) providing single beads coated with a plurality of linkers to at least two wells of the chip, c) loading at least two wells containing single beads with a single eukaryotic or prokaryotic cell, d) lysing the single cells in the well while addressing the individually controllable electrodes at the bottom of the well to retain cell-derived molecules within the well, e) contacting cell-derived molecules with the plurality of linkers on the bead, f) selecting one or more well, and g) releasing the bead by addressing the individually controllable electrode of the selected well.
[0121] Wells which have been co-occupied by two or more cells (i.e. yielding non-single-cell data) can be excluded from further analysis. For example, in some instances, cells are counted and diluted to a known concentration prior to loading to the platform. After loading to the platform, occupancy may be assessed using microscopy. In other words, wells of interest can be selected for further processing such as wells being occupied by a bead and a single cell or a single cell having specific cell surface markers. This gives the possibility to fill the more than 10% of the wells on a chip of the present platform with cells (which is according to Poisson the maximum where doublets are still rare and single occupancies are high). This can be pushed to up to 30% of the wells on a chip, where 30% are empty, 30% are two or more, and 30% are single cells, which can then be further analysed and processed. Another possibility to improve Poisson distribution of single cells is by using alternative chip designs as shown in Figs. 10 and 11.
[0122] While the present platform and methods will be described with primary reference to systems and methods for single cell analysis of molecules derived from a single cell such as nucleic acid molecules (RNA and/or DNA), proteins and/or metabolites, the present technology may also be applied in other fields.
[0123] Methods for single cell transcriptome or genome analysis wherein the molecular data of a single cell is analysed in a platform as described elsewhere herein (see section “the platform”). For example, expression levels, DNA or RNA modifications, chromatin accessibility, and/or chromosome conformation may be analysed.
[0124] An exemplary workflow is depicted in Fig. 1. The method may comprise single bead loading to a chip having multiple wells, wherein well sizes are configured to accommodate a single bead per well. A single labelled cell (e.g. stained with a fluorescently labelled antibody directed to a cell surface marker, nuclear, and/or viability dyes) may then be loaded to the wells of said chip followed by optical analysis of loaded cells with a microscope, imaging and saving x-y position of cells of interest as shown in Fig. 7, which allows to calculate well number on chip. Alternatively, reagents for cell staining could also be flushed into the chip after cell loading, or stimuli (e.g. chemicals) could be flushed over the cells to observe temporal changes within the cell. An overview of fluorescent labelling techniques that may be used herein are disclosed in [27] (Takeshi Suzuki et al. 2007). Following cell lysis, negatively charged nucleic acid molecules are directed to the bottom of the well (or chamber of the well) by a positively charged electrode to prevent cross contamination. cDNA synthesis is then performed on the bead, wherein RNA molecules released from lysed cells hybridize to linker molecules attached to the bead and are reverse transcribed into cDNA including the cell barcode provided by the linker (see Fig. 8). After adding bead lifting solution into the chip, a voltage is applied to pre-selected wells (e.g. selection according to specific cell surface markers) comprising molecules obtained from cells of interest to generate gas bubble in said wells and flushing out beads linked to molecules obtained from cells of interest. Nucleic acids retrieved from collected beads may then be processed for NGS analysis.
[0125] In some instances, methods for single cell analysis and may comprise the following steps: a) providing a chip comprising a plurality of wells, wherein a complementary metal-oxide- semiconductor (CMOS) layer is provided to connect individually controllable electrodes located at the bottom of each well or at least at one segment of the bottom of each well, and wherein each well can accommodate a microscale bead and a eukaryotic or prokaryotic cell, b) providing single beads coated with a plurality of linkers to at least two wells of the chip, c) loading at least two wells containing single beads with a single eukaryotic or prokaryotic cell, d) lysing the single cells in the well while addressing the individually controllable electrodes at the bottom of the well to retain cell-derived molecules within the well, e) contacting cell-derived molecules with the plurality of linkers on the bead, f) selecting one or more well, and g) releasing the bead by addressing the individually controllable electrode of the selected well.
[0126] Step a) of the methods for single cell analysis relates to providing a chip comprising a plurality of wells, wherein a CMOS layer is provided to connect individually controllable electrodes located at the bottom of each well, and wherein each well can accommodate a microscale bead and a eukaryotic or prokaryotic cell. The chip is provided by the present platform as described in detail elsewhere herein (see section “the platform”).
[0127] Step b) of the methods for single cell analysis relates to providing single beads coated with a plurality of linkers to at least two wells of the chip.
[0128] In some instances, linkers or portions thereof are attached to and/or synthesized on the single bead prior to loading to the chip. The linkers may be used for single cell molecular barcoding and may comprise oligonucleotides, for example, deoxyribonucleotides, ribonucleotides or modified nucleotides such as bridged nucleic acid (BNA) polymers, 2’-0-methyl-substituted RNA and the like. In some instances of step b), the linkers are nucleic acid molecules, optionally wherein each of said nucleic acid molecules comprises the following sequence segments: a cell barcode, a spacer, a PCR handle and/or a unique molecular identifier (UMI). Further, the 3 ’ end of the nucleic acid molecules may be deprotected.
[0129] In some instances, each linker or a portion thereof is attached to and/or synthesized on the single bead after loading to the chip. After synthesis of the linker or the portion thereof, the well location (x-y position) of said linker or portion thereof is recorded as described in Fig. 7.
[0130] Step c) of the methods for single cell analysis relates to loading at least two wells containing single beads with a single eukaryotic or prokaryotic cell. [0131] When loading cells, the concentration of the cell suspension (i.e. the number of cells per mL) may be adjusted so that the probability of having more than one cell settle into a given microwell is very small. In some instances, the concentration of the cell suspension may be adjusted so that the volume of cell suspension used to load contains approximately the number of cells as the number of wells in the microwell array.
[0132] In some instances, steps c) and b) of the methods for single cell analysis could also be used in reverse order such that cells are provided first, and beads coated with a plurality of linkers are loaded subsequently.
[0133] In some instances, prior to step d) of the methods for single cell analysis, the wells are covered by oil or semi-permeable membranes. Cell lysis and subsequent step e) of the present methods may be performed with such additional cover.
[0134] Step d) of the methods for single cell analysis relates to lysing the single cells in the well while addressing the CMOS layer within the well to retain cell-derived molecules within the well (as shown in Fig. 5). In some instances, the single cell is lysed using a constant or pulsing electrical field to redirect charged molecules towards the bottom of wells.
[0135] Step e) of the methods for single cell analysis relates to contacting cell-derived molecules with the plurality of linkers on the bead. In some instances of step e), cell-derived nucleic acid molecules are kept at the bottom of the microwell by electrical field to enhance hybridization of the nucleic acid molecules to oligonucleotide linkers coupled on the bead.
[0136] A linker may comprise a poly(dT) sequence segment to capture poly(A)-tailed RNA, an anchored poly(dT) sequence segment, a random sequence segment to capture RNA or a specific sequence segment (such as a sequence complementary to a bacterial RNA or partially degraded RNA). In some instances, the linker may capture nucleic acid molecules by ligation, hence, said linker may comprise an adapter to stabilize the linker-nucleic acid complex upon the ligation reaction. In the context of scRNA-seq, an RNA molecule hybridized or ligated to a linker molecule can be converted to cDNA.
[0137] Prior to step f) of the methods for single cell analysis, the content of each well may be characterized, and the well location (x-y position) may be recorded as described in Fig. 7. The content of each well may be characterized using microscopy on the chip, imaging and analysis, optionally wherein the content comprises the bead and/or the eukaryotic or prokaryotic cell, further optionally wherein the content comprises cell surface markers.
[0138] Step f) of the methods for single cell analysis relates to selecting one or more well. Selection may be based on optical analysis of the content of the wells (such as e.g. labelled cells). For example, the transcriptome of cells having specific cell surface markers may be studied. Selection may also be based on specific linker sequences or parts thereof, i.e. if the linker or parts thereof were synthesized after loading of the bead and its location was recorded.
[0139] Step g) of the methods for single cell analysis relates to releasing the bead by addressing the CMOS layer of the selected well. In some instances, applying a voltage and/or current to the electrode may create air bubbles in a fluid comprised by the selected well, which may advantageously lift the bead.
[0140] In certain instances, the displacement of one or more beads in step g) from the chip is controlled and programmed by an operator, for example, by computer-directed automation. The computer with pre-set parameters may then choose to take actions such as directing displacement of one or more beads only in wells which comprised cells of interest.
[0141] An operator, for example, computer-directed automation, may add a quality control step prior to each step of the methods for single cell analysis to maintain or reject the content of the chip for further processing. In some instances, quality control is performed only prior to step b) and/or after step g) of the present methods. As an example, if bead filling, chemical synthesis of the linker on the bead or cell filing does not result in sufficient quality for downstream processes (for example, a chip having more empty wells or wells filled with several cells and/or beads than wells filled with a single cell and a single bead) the chip may be rejected and the content of the wells is not submitted to further processing.
[0142] The released beads may be collected for further processing after step g). For example, beads containing synthesized cDNA molecules may be released and collected in one or more wells of a filter plate as described in [10]: WO 2016/094512 A1 and [19]: GB 2012261.0. Generally, the filter or material for collecting the beads should be selected to be compatible with the used reagents and be configured to retain the bead, e.g. during a washing step. The filter material should therefore have pore sizes less than the bead diameter. In the context of nucleic acid molecules, further processing may comprise generating a single cell RNA-sequencing (scRNA-seq) library. Methods of generating a single cell RNA-sequencing library are known in the art and described e.g. in [2]: Macosko et ah, Cell 2015 and [3]: Klein et al., Cell 2015.
[0143] Further processing steps in the methods for single cell analysis may relate to processing according to single cell “-omics” assays known in the art such as library preparation for next or further generation sequencing wherein cell-derived nucleic acid molecules may be sequenced directly or by conversion. If the cell-derived molecules are RNA molecules it can be analysed directly or converted to cDNA for next generation sequencing as known in the art.
[0144] The present methods for single cell analysis may further comprise post-processing of the collected beads and coupled cDNA which may comprise amplifying the cDNA in a subsequent PCR reaction on the bead by adding terminal primers. Alternatively, cDNA may be cleaved off the bead, e.g. by cleaving a cleavable moiety included in or adjacent to the linker. In other words, the method may comprise destroying the connection of the nucleic acid to the respective bead. In some instances, the collected beads comprising the cDNA may be washed (e.g. using a buffer as described in the examples) and may then be removed from the collection device (e.g. a filter plate), for example, by using a Tris buffer followed by thorough mixing and pipetting. The supernatant containing the beads may then be separated from the collection plate for further processing such as PCR amplification of the cDNA. [0145] In some instances, the methods for transcriptome analysis of single cells comprises the following steps: step 1) providing a chip comprising a plurality of wells, wherein a complementary metal-oxide- semiconductor (CMOS) layer is provided to connect individually controllable electrodes located at the bottom of each well, and wherein each well can accommodate a microscale bead and a eukaryotic or prokaryotic cell, step 2) providing single beads coated with a plurality of oligonucleotide linkers to at least two wells, step 3) loading at least two wells containing single beads with a single eukaryotic or prokaryotic cell, step 4) optically analysing said cells, and recording location of cells of interest (i.e. x-y position of the well on the chip), step 5) lysing the single cell in the well while addressing the individually controllable electrode in the well (e.g. at the bottom of the well or a segment of the bottom of the well or a chamber of a well) to retain negatively charged nucleic acid molecules within the well, step 6) hybridizing cell-derived nucleic acid molecules with a plurality of oligonucleotide linkers on the bead and synthesizing cDNA, step 7) selecting one or more well by using the location of cells recorded in step 4); and step 8) releasing the bead by addressing the individually controllable electrode of the selected well.
[0146] In some instances, the methods for transcriptome analysis of single cells can be combined with fluidics as disclosed in [19]: GB 2012261.0 such as bead lifting with rotation valves and repeated lifting for several times while flushing beads into different wells of a collection unit. These wells can later be prepared differently by adding, for example, different sequencing barcodes, and thus keeping their phenotypic information. For example, the third round of bead lifting may address beads from cells that were labelled with cyanine dye labelled (Cy5 and/or Cy3) antibodies.
[0147] To allow for analysis of individual genetic information of a specific cell, individual cells must be compartmentalized within certain reaction compartments (i.e. wells). In the present methods, genetic material cross-contamination is minimized in the experimental workflow. Specifically, where single cells are localized in wells of a microfluidic chip, CMOS technology is used to generate an electrical field within the well to retain negatively charged nucleic acids released from lysed cells thereby preventing cross-contamination of genetic material between different wells which provides an advantage over existing well-based systems such as the BD Rhapsody™ Single-Cell Analysis System (see [8]: WO 2016/118915 Al).
[0148] Step 2 of said method for transcriptome analysis of single cells provides single beads coated with oligonucleotide linkers to at least two wells.
[0149] Step 5 of said method for transcriptome analysis of single cells comprises lysing the single cell in the well while addressing the CMOS layer at the bottom of the well to retain negatively charged nucleic acid molecules within the well. The CMOS layer may be addressed by generating a constant or pulsing electrical field. Retaining negatively charged nucleic acid molecules within the well allows to minimize or prevent cell-to-cell cross-contamination of, for example, genetic material between neighbouring wells.
[0150] Step 6 of said method for transcriptome analysis of single cells comprises hybridizing of cell-derived nucleic acid molecules to a plurality of oligonucleotide linkers and synthesizing cDNA. Buffers may comprise buffering salts and ionic salts to regulate the pH and osmolarity upon cell lysis and hybridization of cell-derived molecules to the linker molecules. Sometimes detergents are added to the buffer. For example, a buffer may comprise L-histidine (His) in an aqueous buffer solution, hydrophilic polysaccharides (such as Ficoll) and a nonionic, non denaturing detergent (such as IGEPAL). Buffer conditions are not particularly limited and suitable buffers (and the reaction temperature) could be optimized in many different ways as known in the art by taking into account, e.g., GC content, secondary structure, and degree of homology to the target.
[0151] For synthesis of cDNA in step 6 of said method for transcriptome analysis of single cells, a template switch oligonucleotide (TSO) such as
5 ’ - AAGC AGT GGT AT C AACGC AGAGT GAAT ggg-3 ’ (SEQ ID NO: 1) may be added in the present methods (Fig. 4). The TSO is an oligonucleotide that hybridizes to untemplated C nucleotides added by the reverse transcriptase (RT) during cDNA synthesis. The TSO acts as an additional template for cDNA elongation, which results in addition of a common 5' sequence to full length cDNA that is used for downstream cDNA amplification (see [25]: Zhu et al. 2001). A TSO preferably has a ggg at the 3‘ end (can be ribo or deoxyribo nucleotides) such that the complementarity between these consecutive g bases and the 3' dC extension of the cDNA molecule enables the subsequent template switching of the RT from cellular RNA to the TSO sequence. This results in generation of a full-length cDNA and allows for incorporation of additional universal sequences by the TSO used for downstream processing (see [25]: Zhu et al. 2001).
[0152] After step 6 of said method for transcriptome analysis of single cells, one or more wells may be selected (e.g. based on cell labelling), the content of the well may be released and collected for further processing. For example, collected cDNA from one or more selected wells may be purified and/or concentrated by magnetic bead-based clean-up, PAGE-based clean-up, column based clean-up or other methods known by the person skilled in the art. Magnetic bead-based purification is the method of choice, although other methods are possible which the skilled artisan would be able to identify.
[0153] In some instances, the method for transcriptome analysis of 3,000 to 10,000 single cells comprises the following steps: step 1) providing an 18 mm2 chip having about 35,440 wells (e.g. with about 30 pm spacing between wells), wherein each well is designed as cylindrical hole or chambers having a depth of about 50 pm and a diameter about 40 pm, wherein a CMOS layer is provided to connect individually controllable electrodes located at the bottom of each well (or a segment of the bottom of each well), and wherein each well can accommodate at least a 35 pm bead and a mammalian cell, step 2) providing single 35 pm beads coated with a plurality of oligonucleotide linkers to at least 90% of the wells, step 3) loading at least 3,000 wells containing single 35 pm beads with a single mammalian cell, step 4) optically analysing said mammalian cells, and recording location of cells of interest (i.e. x-y position of the well on the chip), step 5) lysing the single mammalian cell in the well while addressing the individually controllable electrodes at the bottom of the well (or the segment of the bottom of each well) to retain negatively charged nucleic acid molecules within the well, step 6) hybridizing cell-derived RNA molecules with the plurality of oligonucleotide linkers on the bead and synthesizing cDNA, step 7) selecting one or more well by using the location of cells of interest recorded in step 4); and step 8) releasing one or more of the 35 pm beads by addressing the individually controllable electrodes of the selected wells.
[0154] In some instances, the method for transcriptome analysis of at least 10,000 single cells comprises the following steps: step 1) providing an 18 mm2 chip with a plurality of wells, wherein each well is designed as two connected chambers having a depth of about 50 pm, wherein a CMOS layer is provided to connect individually controllable electrodes at the bottom (or a segment of the bottom) of the first chamber and an electrode layer which is controllable for the entire chip is provided at the bottom (or a segment of the bottom) of the second chamber connected thereto, and wherein each chamber can accommodate a single bead ora single mammalian cell. step 2) providing single beads (e.g. having a diameter of 10 pm) coated with a plurality of oligonucleotide linkers to at least 90% of the wells, step 3) loading at least 3,000 wells containing single beads with a single mammalian cell (e.g. the first chamber contains the single bead and the second chamber contains the single cell), step 4) optically analysing said mammalian cells, and recording location of cells of interest (i.e. x-y position of the well or chamber of the well on the chip), step 5) lysing the single mammalian cell in the well while addressing the individually controllable electrode and/or the electrode layer at the bottom of the chambers to retain negatively charged nucleic acid molecules within the well, step 6) hybridizing cell-derived RNA molecules with a plurality of oligonucleotide linkers on the bead and synthesizing cDNA, step 7) selecting one or more wells (or chambers of wells) by using the location of cells of interest recorded in step 4); and step 8) releasing one or more of the beads by addressing the individually controllable electrode of the selected wells.
[0155] In some instances, the method for transcriptome analysis at least 3,000 single cells and comprises the following steps: step 1) providing a dry film resist (TMMF) CMOS chip containing about 35,000 wells (e.g., each of the wells having a 45 pm depth), wherein a CMOS layer is provided to connect individually controllable electrodes located at the bottom of each well, and wherein each well can accommodate an aminostyrene bead with a size of about 35 pm (as described in [15]: U.S. patent No. 6,335,438) and a mammalian cell, step 2) providing single aminostyrene beads coated with a plurality of oligonucleotide linkers to at least 90% of the wells on the chip, step 3) loading at least 3,000 wells containing single aminostyrene beads with a single mammalian cell, step 4) optically analysing said mammalian cells (e.g. by labelling with fluorescent markers), and recording location of cells of interest (i.e. x-y position of the well on the chip), step 5) lysing the single mammalian cell in the well while addressing the individually controllable electrode at the bottom of the well (or a segment of the bottom of the well, e.g. a chamber) to retain negatively charged nucleic acid molecules within the well, step 6) hybridizing cell-derived RNA molecules with the plurality of oligonucleotide linkers on the bead and synthesizing cDNA, step 7) selecting one or more well by using the location of cells of interest recorded in step 4); and step 8) releasing one or more of the aminostyrene beads by addressing the CMOS layer of the selected wells; and collecting released aminostyrene beads for further processing.
[0156] Specific instances include methods of linker synthesis and attachment to a bead using the chip of the present platform and methods. [0157] In some instances, present methods may be performed using different chip geometries like or similar to the designs shown in Fig. 10. For example, if a chip of design 2 is used, a procedure as shown in Fig. 11 may be applied. This has the advantage that the poisson distribution of single cells pairing with single beads can be improved. When cells are flushed over the chip, typically 20-25% of the wells will be occupied by single cells, whereas the remaining wells will be empty or may receive multiple cells. Using alternative chip design as disclosed herein the number of wells occupied with single cells can be extended to >80% without changing the CMOS part of the chip.
[0158] In the present platform and methods, single beads coated with a plurality of linkers are provided. Said linkers or parts thereof (e.g. sequence segments such as a cell barcode) may be synthesized prior to loading to the chip or after loading to the chip.
[0159] For a single-cell RNA sequencing analysis, a single cell must be paired with an oligonucleotide containing a unique cell barcode as described elsewhere herein. By tagging each of the cell-derived nucleic acid with said unique cell barcode (with one bead only carrying one specific cell barcode), one can later use said cell barcode to retrieve information about nucleic acid molecules of said cell even if nucleic acid molecules are mixed together with other cells for downstream processes. In some instances, the plurality of oligonucleotide linker (or linker nucleic acid molecule) on a specific bead is designed to contain the same cell barcode (i.e. having an identical nucleotide sequence) and each linker of the plurality of oligonucleotide linkers on the specific bead contains a different UMI (i.e. having a different nucleotide sequence as compared to the other linkers on the specific bead), and wherein the cell barcode on each bead has a different nucleotide sequence as compared to the other beads.
[0160] An oligonucleotide linker including a cell barcode can be synthesized on the beads chemically or enzymatically or can be added by an emulsion PCR reaction. Bead functionalization and reverse direction phosphoramidite synthesis (5’ to 3’) may be performed as described in the Supplemental Experimental Procedures in [2] Macosko et al. 2015, in [28] Bhardwaj et al., 2019, or as described in Example 4 herein. [0161] An advantage of pre-synthesizing oligonucleotide linkers on beads is that beads are easy to handle, can be prepared in advance and can be used in microfluidics systems.
[0162] A limitation of pre-synthesizing oligonucleotide linkers on beads prior to loading onto the chip may be that the information which bead carries which unique cell barcode sequence is lost once the beads are removed from the chip and optionally combined for subsequent processing. A further limitation may be that it cannot be controlled which sequences are added since random nucleotide sequences (including barcodes) are generated on the beads. If barcode sequences on different beads are highly similar, there is a risk that single cell resolution might be lost. Specifically, the introduction of sequence errors (e.g. introduced during barcode synthesis, through amplification or sequencing) may result in combining sequencing reads from nucleic acid molecules carrying two (or even more) highly similar “unique” barcodes in the computational analysis. This limits error correction in the next generation sequencing (NGS) analysis (see, for example, [20]: Mitchell et al. (2020) doi: 10.1186/sl3059-020-01988-3 and [21]: U.S. Patent No. 8,865,410). For example, a comparison of input barcode sequences and sequenced barcode sequences (output) is not possible.
[0163] If one could design the sequences by controlled synthesis on defined positions of the chip (i.e. well specific), one may exclusively use cell barcode sequences having diverse sequences which can easily be distinguished in NGS analysis, even when they contain synthesis and/or sequencing errors.
[0164] One embodiment of the present platform designs and methods is to address such limitations by synthesising oligonucleotide linkers or elements thereof on beads in wells of a chip and recording the location on the chip (i.e. x-y-position of the well comprising said linker or linker element) as shown in Fig. 9.
[0165] In some instances, synthesising oligonucleotide linkers on beads in wells of a chip, and recording the location on the chip (i.e. x-y-position of the well) is combined with providing a single cell and analysing its molecular data. For downstream NGS data analysis, the sequence information of the oligonucleotide linker comprising the cell barcode may be linked to molecular data of the cell such as its transcriptome.
[0166] In the context of scRNA-seq, linkers with oligo(dT) sequences may be used. Such oligo(dT) sequences may be provided (a) in bulk or (b) individually into a well or specific x-y-z- position of the chip. In approach (a), the information on the cell (e.g. morphology analysed by microscopy and recorded per position on the chip) cannot be combined with the sequence information of a particular cell barcode (see, for example, Fig. 8). In approach (b), both is retained, i.e. the information on the cell and the barcode sequence for said cell.
[0167] In some instances, the present methods for single cell analysis comprises steps a) to g), wherein in step b), the linkers or a part thereof is attached to and/or synthesized on the single bead after loading to the chip. Specifically, said step comprises loading single beads to at least two wells of the chip and synthesizing linkers or a part thereof at the surface of the bead. After synthesis, the oligonucleotides would be prepared for efficient capture of cell-derived molecules.
[0168] In some instances, after loading beads to at least two wells of the chip, an oligonucleotide linker or part of an oligonucleotide linker (for example, a cell barcode and optionally a poly(dT) sequence) is synthesized on the surface of the bead and its sequence and location on the chip (i.e. x-y position of the well) is recorded. Next, oligonucleotides are deprotected at the 3 ’ end to remove the dimethoxytrityl (DMT) protecting group (e.g., via detritylation using, for example, an electrochemically generated acid, trichloroacetic acid in methylene chloride or dichloroacetic acid in toluene, as described in [10]: WO 2016/094512 Al) and beads coated with said linkers are ready to use for a single-cell RNA-seq experiment. Specifically, in step c), a single eukaryotic or prokaryotic cell is loaded to least two wells containing single beads coated with a plurality of oligonucleotide linkers and cells are optically and/or electrochemically analysed. This phenotypic information may be correlated with the unique cell barcode synthesised in each well of the chip. For example, in the NGS sequencing analysis, the transcriptome of a cell (i.e. identified by a unique cell barcode) could be correlated with the phenotypic information by combining the location of the cell barcode and the location of the cell prior to processing. [0169] In some instances, different oligonucleotide sequences are synthesized on beads on the microfluidic chip such as described in [10]: WO 2016/094512 Al.
[0170] In some instances, split-pool synthesis (as described e.g. in [22]: U.S. Patent No. 6,799,120 and references therein) may be used on synthesis beads which facilitates the generation of barcode sequences (e.g. UMIs) having random or semi-random sequences (i.e. combinatorial libraries having degenerate sequence positions). In contrast to the standard synthesis procedure, the oligonucleotide is not cleaved after synthesis and reverse amidites are used (see e.g. ref [28] Bhardwaj et al, 2019). In other instances, the oligonucleotide synthesis is performed on standard oligonucleotide synthesizers (ABI).
[0171] In some instances, methods for single cell analysis comprise steps a) to g), wherein in step b), the following steps are performed:
(pre)-synthesis of oligonucleotide linker comprising a spacer sequence, a PCR handle sequence and optionally a UMI sequence on the bead, adding beads coated with pre-synthesized oligonucleotide linker to at least two wells of the chip, continuing synthesis of oligonucleotide linker by synthesizing a cell barcode unique to each well of the chip and synthesizing sequence for RNA capture (for example, poly (dT) for capture of polyadenylated RNA), and deprotecting oligonucleotides.
[0172] (Pre-)synthesis of an oligonucleotide may be performed based on a chemical reaction of phosphate, pentose sugar, and a nitrogenous base which may comprise ribonucleotides (RNA) or deoxyribonucleotides (DNA), natural and non-natural nucleotides, or a combination thereof. Alternatively, pre-synthesis of an oligonucleotide may be performed by enzymatic synthesis as described e.g. in [23]: WO 2014/165864 A2). In instances where both, a UMI sequence and a cell barcode are pre-synthesized on the bead prior to loading on the chip, the cell barcode may be synthesized first by standard synthesis followed by a split-pool procedure to synthesize the UMI resulting in the order of sequence elements as illustrated in Fig. 4. However, a cell barcode and a UMI may also be provided in reverse order. [0173] A cell barcode must be unique to each well of the chip (or the single bead loaded to said well). Hence, the required length of the barcode may depend on the number of wells or beads present. Generally, a barcode needs to be longer if a chip comprises more wells. For example, if a chip comprising 35,440 wells, a unique cell barcode may comprise at least 8 bases, yielding 66,536 different possible barcodes. A benefit is that the skilled person may exclude certain sequences, for examples stretches of the same nucleotide such as poly (dT) or sequences that may result in high similarity as discussed elsewhere herein.
[0174] In some instances, barcodes of less than 6 bases, for example, 5-6 nucleotides, may be synthesized in certain areas of the chip comprising several wells. Upon well selection, only the content of only one well of said area may be lifted (for example, as described in [10]: WO 2016/094512 Al) and collected for further processing.
[0175] A UMI sequence segment may be added prior to or during cDNA synthesis or amplification (e.g., during or after step 6 of a method for transcriptome analysis described herein). For example, a UMI sequence segment may be added during the contacting or hybridizing of cell-derived nucleic acid molecules with a plurality of oligonucleotide linkers on the bead or may be added during the cDNA synthesis step. In some instances, a UMI may be added to a nucleic acid molecule (e.g., linker with hybridized cell-derived nucleic acid molecule) using “oligonucleotide tethered ddNTPs” as described in [24]: WO 2020/257797 Al. Such method advantageously allows tethering of an oligonucleotide to any nucleotide and its later incorporation into a nucleic acid sequence while performing strand synthesis with nucleic acid polymerase. Thus, adding UMIs by using oligonucleotide tethered ddNTPs allows the attachment of an oligonucleotide to any final (i.e., terminal, such as for example 3’ terminal) nucleotide of any nucleic acid sequence composition, which allows to fully cover the sequence information at the 3’ end of captured nucleic acid molecules. The oligonucleotide-tethered nucleotides may be used as reagents in cDNA synthesis reactions such that the nucleotide with oligonucleotide tether may be incorporated into the cDNA formed from the synthesis reaction (e.g., extension reaction, amplification reaction). This allows for the incorporation of various types of functional sequences (e.g., sequencing adapters, barcodes, UMIs, PCR handle sequences, and the like) either directly, i.e., when the oligonucleotide of the oligonucleotide-tethered nucleotide provides the functional sequence, or indirectly, e.g., by providing sequences that enable the addition of various functional sequences thereby improving traditional workflows for preparing NGS libraries by greatly simplifying workflows.
[0176] A method for transcriptome analysis of a single cell comprises steps of synthesizing nucleic acids such as a plurality of linkers attached to a bead and cDNA after hybridization of cell-derived nucleic acid molecules.
[0177] An oligonucleotide linker (nucleic acid molecule linker) comprises at least the following sequence segments: a PCR handle, a cell barcode, a unique molecular identifier (UMI), an oligonucleotide sequence for cell-derived nucleic acid capture such as a poly (dT) sequence as defined elsewhere herein. In some instances, a linker may comprise a spacer. Exemplary linkers are shown in Figs. 4 and 8. The linker may be connected to the bead by a coupling moiety as described elsewhere herein.
[0178] For purpose of illustration only, an exemplary PCR handle may be 5’-TTTTTTTAAGCAGTGGTATCAACGCAGAGTACGT-3’ (SEQ ID NO: 2).
[0179] A step of synthesising nucleic acids may comprise synthesising on a bead nucleic acid molecules (e.g. an oligonucleotide linker or part thereof). Each molecule may comprise 10 to 500 bases, preferably 20 to 250 bases, more preferably 30 to 100 bases. In other words, the method may comprise a step of synthesising oligonucleotide linkers in certain areas of the chip comprising several wells.
[0180] To make the solid support material (i.e. bead) suitable for nucleic acid molecule synthesis, non-nucleosidic coupling moieties or nucleoside succinates may be covalently attached to reactive amino groups. If necessary, however, other surface functions such as carboxyl, hydroxyl, or thiol, for example, could be used to attach a coupling moiety carrying a hydroxyl group or alternatively a 3 '-attached nucleotide. [0181] In many instances, a nucleic acid molecule synthesized on a bead in certain areas of the chip comprising several wells, may be physically coupled to the bead by a coupling moiety. In certain exemplary aspects, the coupling moiety, when present, may be a chemical entity that attaches the 3'-0 of the nucleic acid molecule to the bead (e.g., a functional group on a bead). In other exemplary aspects, the coupling moiety, when present, may have a structure such that it allows for attachment of other functionalities in addition to the 3’-0. Such coupling moiety structures are disclosed, for example, in [18]: U.S. Patent No. 7,202,264. In most cases, the coupling moiety will be inert to all the reagents used during nucleic acid molecule synthesis, but cleavable under specific conditions at the end of the synthesis process. One coupling moiety commonly used in nucleic acid molecule synthesis is the succinyl linker. Additionally, universal coupling moieties may be used for nucleic acid molecule synthesis according as disclosed elsewhere herein. A universal coupling moiety is a coupling moiety that allows for the synthesis of nucleic acid molecules regardless of the nature of the 3’ -terminal base of the first nucleotide that is to be sequenced. Different coupling moieties with different properties are known to those skilled in the art and can be selected by the skilled person depending on the downstream process requirements.
[0182] Nucleosidic solid supports (e.g., support prederivatized with base) are widely used in nucleic acid molecule synthesis. One example of such a support is one where the 3'-hydroxy group of the 3'-terminal nucleoside residue is attached to the solid support via a 3’-0-succinyl arm. The use of nucleosidic solid supports requires usage of beads prederivatized with different types of bases (one for each base). However, the fact that a nucleosidic solid support has to be selected in a sequence-specific manner (according to the first base required for each nucleic acid molecule) reduces the throughput of the entire synthesis process due to laborious pre-selection and distribution of beads attached to a specific starter base to individual microwells.
[0183] A more convenient method for synthesis starts with a universal support where a non- nucleosidic linker is attached to the solid support material. An advantage of this approach is that the same solid support may be used irrespectively of the sequence of the nucleic acid molecule to be synthesized. One example of a universal support that can be used is described in ref: [18]: U.S. Patent No. 7,202,264, the disclosure of which is incorporated herein by reference. However, other universal coupling moieties known by the skilled in the art may be equally appropriate to carry out nucleic acid synthesis.
[0184] In some instances, beads prederivatized with a base (e.g., dU, dA, dT, dC, dG, etc.) present may be employed. For example, a synthesis chip with multiple (e.g., four) loading regions (e.g., reagent flow zones) may be used so that beads prederivatized with the same base can be loaded in the same region. Also, multiple synthesis runs could be made in which the starting base is different in each run. Thus, for example, the first run may be made with nucleic acid molecules that begin with dA, followed by dC, then dT, then dG. Another possibility would be to synthesize nucleic acid segments, wherein all of the nucleic acid molecules being synthesized begin with the same base. For example, synthesis start points could be chosen that begin with a dG, with initial dGs chosen as start points being positioned so that suitable sequence complementarity regions are generated to allow for assembly of a final product nucleic acid molecule.
[0185] A number of methods for synthesizing nucleic acid are known. Many of these methods follow a series of basic steps, such as, for example, the following, with appropriate washing steps using, for example, acetonitrile, ethylacetate or other washing reagents suitable for practicing the present methods.
[0186] An exemplary method for synthesizing nucleic acid molecules may comprise the following steps: a) the first nucleotide, which has been protected at the 5' position (or, in certain instances wherein synthesis proceeds in the 5’ to 3’ direction, the first nucleotide may be protected at the 3’ position), is derivatized to a solid support, such as a polystyrene bead or controlled pore glass, or is obtained pre-derivatized) the sugar group of the first nucleotide is deprotected (e.g., via detritylation) (a process often referred to as “deprotection”), using, for example, an EGA, trichloroacetic acid in methylene chloride or dichloroacetic acid in toluene, which results in a colored product which may be monitored for reaction progress; b) the second nucleotide, which has the phosphorus, sugar and base groups protected, is added to the growing chain, usually in the presence of a catalyst, such as, for example, tetrazole or 4,5-dicyanoimidazole (a process often referred to as “coupling”); c) unreacted first nucleotide is capped to avoid accumulation of deletions, using, for example, acetic anhydride and N-methylimidazole (a process often referred to as “capping”); d) the phosphite triester is oxidized to form the more stable phosphate triester, usually using any suitable compound, for example, iodine reagents (a process often referred to “oxidizing”); e) the process is repeated as needed depending on the desired length of the nucleic acid molecule; and f) cleavage from the solid support is done, usually using aqueous or gaseous ammonia at elevated temperatures.
[0187] The skilled in the art will recognize that in certain instances the order of steps may vary or some of the steps including the washing steps may be repeated as appropriate according to the used protocol.
[0188] An array of beads may be coated with a plurality of linkers, wherein the plurality of linkers comprise nucleic acid molecules, each nucleic acid molecule having at least the following sequence segments: a PCR handle, a UMI and a cell barcode, wherein each linker nucleic acid molecule on a specific bead is designed to contain the same nucleotide sequence segment to provide a cell barcode and a different nucleotide sequence segment as compared to the other linkers on the specific bead to provide a UMI, and wherein the cell barcode on each bead is different.
[0189] Preferably, each position in the array of beads is associated with a specific cell barcode sequence.
[0190] In some instances, a specific bead of an array of beads is coated with a plurality of linkers. Each linker comprises several nucleotide sequence segments, e.g. a UMI and a cell barcode. The nucleotide sequence segment providing the cell barcode is unique to a specific bead, i.e. the plurality of linkers has an identical cell barcode. The nucleotide sequence segment providing the UMI is unique to the linker, i.e. the plurality of linkers on a specific bead has different UMIs. The combination of identical cell barcode and UMI per linker allows to track, distinguish and count identical cell-derived molecules derived from a single cell. [0191] In some instances, the array of beads coated with a plurality of linkers comprises nucleic acid molecule linkers having the following sequence segments: a UMI, a cell barcode, and optionally, a poly(dT) or RNA binding sequence.
[0192] In some instances, the array of beads coated with a plurality of linkers as defined elsewhere herein has a defined three-dimensional structure, wherein each bead is located in specific position (e.g., a well of a chip or a multiwell plate).
[0193] The present disclosure will now be described in further detail, by way of example only, with reference to the following Examples and related Figures.
EXPERIMENTAL SECTION
[0194] The following are examples of systems and methods disclosed herein. It is understood that various other aspects may be practiced, given the general and detailed descriptions provided elsewhere herein.
[0195] The following Examples illustrate workflows shown in Figs. 2 and 3.
Example 1: transcriptome analysis of single cells on microfluidic chip (Fig. 3)
Preparation of cells
[0196] Cultured human embryonic kidney 293 (HEK293) cells (Expi293F™, Gibco™) and Chinese hamster ovary (CHO) cells (CHO-S Cells, Gibco™) were centrifuged at 1,000 rpm for 1 min and the supernatant was removed.
[0197] Cells were gently resuspended in pre-warmed labelling solution containing 1 mM CellTracker™ Green CMFDA Dye (Invitrogen) dissolved in DMSO and incubated at 37°C for 30 mins before they were pelleted at 1,000 rpm for 1 min and the supernatant was removed. Cells were then washed three times using 1 mL of warmed solution of lx PBS with 0.5% Pluronic®
F-108 (Sigma Aldrich) and filtered into a falcon tube through a 40 pm filter to remove aggregates. [0198] Cells were diluted with lx PBS/0.5% Pluronic® F-108 to a concentration of 100,000 cells/mL. Both cell lines were then mixed at a 1:1 ratio.
Preparation of a chip
[0199] A TMMF CMOS chip containing about 35,000 wells of 45 pm depth was used. The chip was prepared by plasma cleaning prior to use for 5 min at 100% argon and 300 W to remove organic residues on the chip surface.
[0200] The chip was first primed with ethanol to remove air from wells. The ethanol was then exchanged with 300 to 500 pi of lx PBS and the chip was centrifuged at 500 rpm for 1 min before the PBS was exchanged with approx. 300 pi of bovine serum albumin (BSA) (10% in PBS) to block the hydrophobic surface of the chip, and prevent cells from sticking to it. The chip was then centrifuged at 500 rpm for 1 min and incubated overnight. On the following day, the BSA solution was removed from the chip.
Providing beads
[0201] Aminostyrene beads as described in [15]: U.S. patent No. 6,335,438 (about 35 pm) were provided with pre-synthesized oligonucleotide linkers (as described elsewhere herein) comprising poly(dT) sequences. A bead solution was prepared in TE/Tween solution (10 mM Tris HC1 pH 8.0, ImM EDTA pH 8.0, 0.01% Tween 20).
[0202] The dry chip was filled with the bead solution containing the beads with a plurality of oligonucleotide linkers and was centrifuged three times at 500 rpm for 1 min. The bead solution was removed and re-introduced with a syringe between centrifugation steps to ensure homogenous loading of all wells.
[0203] Quantitative bead loading was controlled using a microscope (Axio Imager. M2m, Carl Zeiss). The chip was then slowly rinsed with 200 pL of TE/Tween solution to remove any beads located outside the wells. Loading cells
[0204] The solution was removed from the chip by using a syringe. A 10 mL Nemesys syringe was filled with 6 mL of lx PBS/0.5% Pluronic® F-108 solution. The chip holder was then loaded with the solution at 100 pL/min
[0205] Cells were then loaded by slowly sucking the cell solution into the chip at 150 pL/min for 1 min. The flow was then stopped for 30 s to let the cells settle into the wells of the chip.
[0206] Cell loading was controlled under a microscope and repeated if necessary.
[0207] The chip was then washed with lx PBS/0.5% Pluronic® F-108 solution before chip tubings were closed for imaging.
Characterizing content of wells by imaging
[0208] Images of the chip were taken with a microscope to document final cell and bead loading, including assessment of the total number of cells in the chip, the ratio of beads to cells, and percentage of wells with single bead and single cell were determined. Well positions containing single bead-cell pairs were determined and saved by using Axio Vision software by Zeiss (see Fig. 7).
Lysis and cDA synthesis of the cells
[0209] Single cells in wells were flushed with Pre-RT buffer (50 mM L-Histidine/NFP pH 8.7; 3 mM MgCL, 10 mM DTT) at a flow rate of 100 pL/min for 2 to 3 min. At the same time, voltage was applied to the electrodes of the chip (connected to the CMOS layer) at the following settings: 1.7 V, Pulse Width Modulation (PWM) on for 100 ms, off for 200 ms, for the time needed to exchange the buffer to the lysis buffer (a few minutes).
[0210] The Pre-RT buffer was then exchanged with Lysis/RT -buffer containing 2.5 pM template switch oligonucleotide (TSO) (5 ’ - AAGC AGT GGT AT C AACGC AGAGT GAAT ggg-3 ’ ; SEQ ID NO: 1 provided by Metabion), 50 mM L-Histidine (ReagentPlus® Sigma Aldrich) pH 8.7, 3 mM MgCh, 0.3% IGEPAL CA630 (SIGMA Aldrich), 0.5 mM dNTPs (final cone. 0.5 mM; Thermo Fisher Scientific), 4% Ficoll PM-400 (Sigma Aldrich), 20 U/mI Super Script IV Reverse Transcriptase, 1 U/mI RiboLock™ RNase Inhibitor (Thermo Fisher Scientific) in nuclease free water. At the same time, voltage was applied to the electrodes of the chip as described above for 60 min to attract the released nucleic acids to the electrode at the bottom of the well. The chip was incubated with the Lysis/RT -buffer at RT for 1 hour to allow for cDNA synthesis.
Bead lifting and collection
[0211] Following cDNA synthesis, bead lifting solution (0.05M LiClCE in 20% water, 80% methanol) was applied to the chip. Beads from selected wells (based on x-y position recorded during imaging analysis of the cells in the same well) were collected by flushing the chip at 500 mΐ/min for 11 mins. Beads were removed by repeated generation of gas bubbles in the selected wells (200 ms pulses at 8.5 V, 6 mA current limit) and flushed into a 5 pm pore filter plate. Vacuum was applied to remove buffer solution while beads were collected and concentrated in the filter plate.
Bead post-processing
[0212] The collected beads were washed twice with 1 ml TE/SDS buffer (10 mM Tris HC1 pH 8.0, ImM EDTA pH 8.0, 0.5% SDS) and then once with 1 ml TE/Tween buffer using vacuum.
[0213] Subsequently, the beads were removed from the filter plate by adding 4 x 0.1 ml of 10 mM Tris pH 8.0 to the bead pellets in each well and thorough mixing by pipetting up and down 5 times. The beads solution was then collected from the wells into respective centrifuge 1.5 ml tubes. The tubes were then centrifuged (6,600 rpm or l,500g for 1 min) and the supernatants carefully removed from the bead pellets.
[0214] Each bead pellet was then resuspended in 100 mΐ Exonuclease I solution (containing 1 U/pl Exonuclease 1 and lx Exonuclease I buffer (Thermo Fisher Scientific) in nuclease free water) to remove excess bead primers that did not capture an RNA molecule and incubated at 37°C for 45 min while shaking at 1,400 rpm in a thermocycler. [0215] Beads were then pelleted at 6,600 rpm (or 1,500 g) for 1 min and supernatants carefully removed. The bead pellets were then washed with 200 mΐ of TE/SDS buffer and twice with 200 mΐ of TE/Tween buffer by centrifuging each time at 6,600 rpm (or 1,500 g) for 1 min to remove Exonuclease enzyme. Finally, beads were washed with 200 mΐ of 10 mM Tris pH 8.0, and centrifuged at 6,600 rpm or l,500g for 1 min. The beads containing the cDNA were then carefully resuspended in PCR reaction mixture of Table 1. a) and transferred to PCR tubes.
[0216] 50-m1 PCR reactions containing lx KAPA HiFi mix (Fisher Scientific) as indicated in Table l.a were prepared and PCR samples were cycled using the amplification reaction conditions indicated in Table l.b.
[0217] Table 1. a) PCR KAPA HiFi mix
Figure imgf000068_0001
[0218] Table 1. b) PCR amplification reaction
Figure imgf000068_0002
[0219] The amplified cDNA was then cleaned-up by adding 40 mΐ of Dynabeads™ MyOne carboxylic acid beads (Thermo Fisher Scientific) to each of the 50 mΐ PCR reactions. Reaction samples were mixed and incubated at room temperature for 5 min before magnetic beads were collected on a magnetic stand and supernatants removed. Beads were washed twice with 200 mΐ of 80% ethanol. Residual ethanol was removed after centrifugation of samples for a couple of seconds and beads were air-dried for about 1 min. Subsequently, 12 mΐ of nuclease-free water were added and incubated at room temperature for 5 min to elute the captured cDNA.
[0220] PCR amplification products were then quantified using the Agilent 2100 BioAnalyzer and Agilent High Sensitivity DNA Kit (Agilent) according to the manufacturer’s instructions.
Example 2: cell lysis and nucleic acid manipulation (Fig. 6)
[0221] An exemplary workflow is shown in Fig. 6A.
[0222] BSA-blocked microchips were primed with ethanol and then washed with IX PBS. HEK 293 cells, dyed with Cell Tracker Green and diluted to 100,000 cells/mL in IX PBS, were loaded onto the chip automatically using neMESYS syringe pumps (Cetoni, Korbussen) at a flowrate of 150 pL/min for 1 min. The chip was then washed with an excess volume of IX PBS at the same flowrate to get rid of excess cells.
[0223] Pre-lysis buffer (50mM L-His + 4% Ficoll in water, pH=7.87) was flushed into the chip at 150 pL/min.
[0224] The oligonucleotide manipulation was launched by activating electrodes within wells using the following settings: 1.5 V, pulses: 100ms on, 200 ms off. A square pattern, with an empty area inside, of 176 electrodes was used, as it is easily distinguishable from the background. A lysis buffer (50 mM L-His + 4% Ficoll + 0.3% IGEPAL) with added (dT)60-Cy5-labelled oligonucleotides ("(dT)60" disclosed as SEQ ID NO: 4) was then loaded onto the chip at 150 pL/min, while the electrode activation was initiated and image taking was launched at the same time (Zeiss Imager M.2m with Axio Vision; GFP and Cy5 fluorescence, 1 fps). [0225] Images were analyzed with ImageJ: areas of interest (Pos 1-4) were defined in the image stacks of the respective fluorescent channel (see Fig. 6B). Pos 1+2 were chosen around single cells in the GFP channel, Pos 3 at an area outside of the activated area in the Cy5 channel, and Pos 4 in the region where electrodes within wells were activated. Mean grey values from these areas were normalized and plotted (see Fig. 6C). The fluorescent values of Pos3 show when fluorescent oligonucleotides (and therefore at the same time lysis agent) was introduced in the field of view. Pos4 shows that trapping of oligonucleotides immediately started, while cell lysis also happened (drop in Posl and Pos2).
[0226] Cell lysis and accumulation of oligonucleotides are shown to occur at the same time, suggesting that negatively charged RNA released from the cells will also be directed to the bottom of the well.
Example 3: post-processing to prepare sequencing library
[0227] The following reagents were provided: Qubit dsDNA HS Assay Kit, Nextera XT Kit, Nuclease-free H2O, 10 mM PCR hybrid oligonucleotide, 10 pM indexing oligonucleotides (e.g. Nextera N7XX oligonucleotides), Dynabeads™ MyOne™ Carboxylic acid beads, 80 % EtOH, Invitrogen Collibri Library Quantification Kit.
[0228] The cDNA concentrations of samples obtained from Example 1 were determined using Qubit dsDNA HS Assay Kit.
Tagmentation
[0229] For tagmentation, a thermocycler was preheated to 55 °C. Each cDNA sample was transferred to a fresh 0.2 ml PCR tube, i.e. 600 pg of purified cDNA were dissolved in nuclease- free water (total volume of 5 pi).
[0230] To each sample 10 pi of Tagment DNA buffer (TD) and 5 pi of Amplicon Tagment Mix (ATM) were added for a total volume of 20 pi, mixed thoroughly by pipetting and spun down to collect all liquid within the tube. [0231] Samples were incubated in the preheated thermocycler at 55 °C for 5 min.
[0232] To stop the reaction, 5 mΐ of Neutralization Buffer (NT) was added to each sample, mixed thoroughly by pipetting and briefly spun down to collect all liquid within the tube.
[0233] Samples were incubated at room temperature for 5 min.
[0234] The Tagmentation PCR mix was prepared. To each sample, the following solutions were added: 15 mΐ Nextera PCR Master Mix (NPM), 8 mΐ Nuclease-free H2O, 1 mΐ 10 mM PCR hybrid oligonucleotide and 1 mΐ Nextera N7XX oligonucleotide (different in each reaction).
[0235] The PCR was performed using the following settings:
[0236] Table 2. PCR reaction
Figure imgf000071_0001
PCR purification and quality control (QC)
[0237] Dynabeads™ MyOne™ Carboxylic acid beads were equilibrated to room temperature prior use. In a 0.8:1 bead to sample ratio, Dynabeads™ MyOne™ Carboxylic acid beads (40 pL) were added to each PCR tube, mixed by pipetting or carefully vortexing, and briefly spun down to collect all droplets. Samples were incubated at room temperature for 5 min. Tubes were placed onto a magnetic rack for 2 min or until a solid pellet had formed. The supernatant was removed without disturbing the pellet. On the magnetic rack, each sample was washed with 200 mΐ of 80 % ethanol (incubation for 30 s and removing the supernatant without disturbing the pellet). The wash step was repeated for a total of two washes. After washing, sample were briefly spun down and the remaining EtOH was collected by placing the tubes back on the magnetic rack. On the magnetic rack, samples were air dried for 2 min or until all of the ethanol had evaporated. Each pellet was resuspended in 12 mΐ of nuclease-free water by vortexing. Samples were incubated for 5 min at room temperature (not on the magnetic rack). Tubes were placed back on the magnetic rack for 2 min or until a solid bead pellet had formed. The supernatant was transferred to new tubes.
[0238] For quality control (QC), Agilent High Sensitivity DNA Assay was performed according to the manufacturer’s instructions.
[0239] DNA sample quantification was performed using Invitrogen Collibri Library Quantification Kit according to the manufacturer’s instructions.
Sequencing
[0240] DNA libraries were prepared at a concentration of 4 nM.
[0241] Libraries for Illumina sequencing were prepared for sequencing by denaturing and diluting the pooled libraries according to Illumina recommendations. The final concentration was 10 pM.
[0242] Sequencing was performed on the Illumina MiSeq system.
Example 4: direct synthesis of cell barcode on microfluidic chip (Fig. 9)
[0243] Bead functionalization and reverse direction phosphoramidite synthesis (5’ to 3’) was performed on Toyopearl HW-65S resin (~30 micron mean particle diameter) from Tosoh Biosciences (catalog #19815, Tosoh Bioscience) or on custom-made aminostyrene beads as described in [15]: US patent No. 6335438 for a more homogeneous size distribution, after surface hydroxyls were reacted with a PEG derivative to generate an 18-carbon long, flexible-chain linker ([2]: Macosko et ah, Cell 2015).
[0244] The functionalized bead was then used as a solid support for reverse-direction phosphoramidite synthesis (5’ to 3’) on an Expedite 8909 DNA/RNA synthesizer using DNA Synthesis at 10 micromole scale and a coupling time of 3 minutes. The following amidites were used: N 6 -Benzoyl-3’-0-DMT-2’- deoxyadenosine-5’-cyanoethyl-N,N- diisopropylphosphoramidite (dA-N 6 -Bz-CEP); N 4- Acetyl -3 ’ -O-DMT -2’ -deoxy cyti di ne-5 ’ - cyanoethyl-N,Ndiisopropyl-phosphoramidite (dC-N 4 -Ac-CEP); N 2 -DMF-3’-0-DMT-2’- deoxyguanosine-5’- cyanoethyl-N,N-diisopropyl-phosphoramidite (dG-N 2 -DMF-CEP); and 3’-0-DMT-2’- deoxythymidine5’-cyanoethyl-N,N-diisopropyl-phosphoramidite (T-CEP). Acetic anhydride and N-methylimidazole were used in the capping step; ethylthio-tetrazole was used in the activation step; oxidizer (0.1 M iodide in tetrahydrofuran/water/pyridine) was used in the oxidation step, and dichloroacetic acid was used in the deblocking step.
[0245] After the synthesis of some spacer amidites, a PCR handle (5’-TTTTTTTAAGCAGTGGTATCAACGCAGAGTACGT-3’; SEQ ID NO: 2) and the UMI sequence (8 bases of equimolarly mixed bases “N”: 5,-NISίNISίNISίNN-3,), the beads were removed from the column, resuspended in methanol and loaded onto a CMOS microchip with synthesis capabilities as described in [10]: WO 2016/094512 A1 by centrifugation. After excess beads were removed from the chip by washing gently with methanol, leaving only beads trapped inside the wells, the chip was connected to a custom-built chip-synthesis instrument. The instrument, which is usually used to synthesize oligonucleotides from the 3’ end to the 5’ end, can be as well equipped with above mentioned amidites and all other chemicals to continue the synthesis in the reverse direction. Electronically generated acid was used to locally determine different sequences on each bead in each of the 35,440 wells (as described in [10]: WO 2016/094512 Al). For example, an 8 bases oligonucleotide synthesis can lead to 66,536 barcode sequence possibilities (which gives some possibility to de-select some sequences which are less preferred, such as for example homopolymers).
[0246] After the cell-barcode synthesis, a poly(dT) part was synthesized at each oligonucleotide either by using electrochemically generated acid that was flushed over the whole chip (as all oligonucleotides on all beads need to be deprotected at the same time). Alternatively, an acid such as e.g. trichloroacetic acid (TCA) in acetonitrile may be used.
[0247] Finally, after the final deblocking step, AMA (freshly prepared 1:1 mixture of 40% methylamine in water with 35% ammonia) was flushed into the chip and incubated for 16 h at room temperature (shorter incubation at elevated temperatures is also possible), thereby removing the side-protection groups of the oligonucleotides, which remain coupled to the beads.
[0248] After removing the AMA solution, washing thoroughly with acetonitrile, and drying the chip, the chip was ready for a single cell RNA-seq experiment with known cell barcode locations as described elsewhere herein.
Example 5: quality of sequencing data (Fig. 12A-B)
[0249] Quality of sequencing data and influence of voltage on cross-contamination [%] can be measured by mixture experiments.
[0250] RNA from two different cell lines (human and hamster) as described in Example 1 was captured on beads in wells of a chip, converted into cDNA and sequenced.
[0251 ] To calculate the fraction of cross contaminated reads the two genomes used in this example were first tagged and then merged into a single reference genome. This was applied for both genome and annotations files. Next, the generated reference genome was indexed and sequenced reads were aligned using STAR aligner (v2.6.0). Then, for each cell barcode (indicated on x-axis) reads mapping to either of the tagged genomes were calculated and depicted on the graph as shown m Eig. 12A-B (with y-axis showing portion of origin organism). Finally, overall cross contamination was calculated as a percentage of reads assigned to the unexpected organism. The calculation of cross-contamination was performed as described in [2]: Macosco et al 2015 with some minor modifications.
[0252] The shown sequencing data demonstrate a clear positive effect of voltage applied during RNA capture on sequence cross-contamination level. Whereas “no voltage” capture resulted in 21.05% of cross-contamination, applying voltage during cell lysis resulted in 3.72% cross contamination level, which demonstrates efficient RNA immobilization on the bead using electrical fields and more than 5-fold reduction of cross-contamination. List of references
[1] Wang et al., bioRxiv preprint 2019, doi: 10.1101/541433
[2] Macosko et al., Cell 2015, doi: 10.1016/j cell.2015.05.002
[3] Klein et al., Cell 2015, doi: 10.1016/j cell.2015.04.044
[4] Rosenberg et al., Science 2018, doi: 10.1126/science.aam8999
[5] Ding et al., Nature Biotechnology 2020, doi: 10.1038/s41587-020-0465-8
[6] Bose et al., Genome Biology 2015, doi: 10.1186/sl 3059-015-0684-3
[7] Gierahn et al., Nature Methods 2017, doi: 10.1038/nmeth.4179
[8] WO 2016/118915 Al
[9] Yuan et al., Genome Biology 2018, doi: 10.1186/sl3059-018-1607-x
[10] WO 2016/094512 Al
[11] “Introduction to Algorithms”, Third Edition (2009) by Cormen et al., The MIT Press Cambridge, Massachusetts London, England
[12] Bystrykhor “Generalized DNA Barcode Design Based on Hamming Codes”, PLoS One, 2012, Vol. 7, issue 5, e36852
[13] WO 2017/211913 Al
[14] Brunauer, S., Emmett, P. and Teller, E., J. Amer. Chem. Soc. 60 (1938), p. 309
[15] U.S. Patent No. 6,335,438
[16] Dumas A. (1826): Annales de chimie, 33,342
[17] US Environmental Protection Agency in method 440.0: Determination of Carbon and Nitrogen in Sediments and Particulates of Estuarine/Coastal Waters Using Elemental Analysis
[18] U.S. Patent No. 7,202,264
[19] GB 2012261.0
[20] Mitchell et al. (2020) doi: 10.1186/sl3059-020-01988-3
[21] U.S. Patent No. 8,865,410
[22] U.S. Patent No. 6,799,120 and references therein
[23] WO 2014/165864 A2
[24] WO 2020/257797 Al
[25] Zhu YY, Machleder EM, et al. (2001) Reverse transcriptase template switching: a SMART approach for fulMength cDNA library construction Biotechniques, 30(4): 892-897. [26] Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA, https://imagej.nih.gov/ij/, 1997-2018
[27] Takeshi Suzuki et al., 2007 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2156041/pdf/ahc-40-131.pdf
[28] Bhardwaj et al., 2019; https://www.mdpi.eom/2076-3417/9/7/1357

Claims

1. A method for single cell analysis comprising the following steps: a) providing a chip comprising a plurality of wells, wherein a complementary metal-oxide- semiconductor (CMOS) layer is provided to connect individually controllable electrodes located at the bottom of each well, and wherein each well can accommodate a microscale bead and a eukaryotic or prokaryotic cell, b) providing single beads coated with a plurality of linkers to at least two wells of the chip, c) loading at least two wells containing single beads with a single eukaryotic or prokaryotic cell, d) lysing the single cell in the well while addressing the individually controllable electrode in the well to retain cell-derived molecules within the well, e) contacting cell-derived molecules with the plurality of linkers on the bead, f) selecting one or more well, and g) releasing the bead from the one or more well by addressing the individually controllable electrode in the selected well.
2. The method according to claim 1, wherein the genetic, epigenetic, spatial, and/or the proteomic data of a single cell is analysed.
3. The method according to one or more of claims 1 to 2, wherein the transcriptome, genome, DNA modifications, chromatin accessibility, and/or chromosome conformation is analysed.
4. The method according to one or more of claims 1 to 3, wherein in step b), each linker or a portion thereof is attached to and/or synthesized on the single bead prior to loading to the chip.
5. The method according to one or more of claims 1 to 3, wherein in step b), each linker or a portion thereof is attached to and/or synthesized on the single bead after loading to the chip.
6. The method according to claim 5, wherein the well location (x-y position) of said linkers or a portion thereof is recorded.
7. The method according to one or more of claims 1 to 6, wherein said single cell is a eukaryotic, mammalian or human cell.
8. The method according to one or more of claims 1 to 7, wherein prior to step d) the wells are covered by oil or a semi-permeable membrane.
9. The method according to one or more of claims 1 to 8, wherein in step d) the single cell is lysed while using a constant or pulsing electrical field to redirect charged molecules towards the bottom of the well.
10. The method according to one or more of claims 1 to 9, wherein prior to step f) or after step c) the content of each well is characterized, and the well location (x-y position) is recorded.
11. The method according to one or more of claims 1 to 10, wherein prior to step f) or after step c) the content of one or more wells is characterized using microscopy on the chip, imaging and analysis.
12. The method according to one or more of claims 10 to 11, wherein the content comprises the bead and/or the eukaryotic or prokaryotic cell.
13. The method according to one or more of claims 10 to 12, wherein the content comprises cell surface markers of the eukaryotic or prokaryotic cell.
14. The method according to one or more of claims 1 to 13, wherein the environment of the well is changed, wherein the environment comprises composition, viscosity, pH, temperature and/or conductivity of a fluid in the well, wherein the environment further comprises voltage and/or current of the individually controllable electrode in the well.
15. The method according to one or more of claims 1 to 14, wherein in step g), the bead is lifted by gas bubble ejection.
16. The method according to one or more of claims 1 to 15, wherein in step d), the cell-derived molecules are nucleic acid molecules or proteins.
17. The method according to one or more of claims 1 to 16, wherein the transcriptome of a single cell is analysed, and wherein in step d), the cell-derived molecules are nucleic acid molecules.
18. The method according to one or more of claims 1 to 17, wherein in step b), the plurality of linkers are nucleic acid molecules, and optionally wherein the 3’ ends of the nucleic acid molecules are deprotected.
19. The method according to claim 18, wherein each of said nucleic acid molecules comprises the following sequence segments: a spacer, a PCR handle, a cell barcode, a unique molecular identifier and/or a poly (dT) sequence.
20. The method according to one or more of claims 17 to 19, wherein the contacting in step e) comprises hybridizing or binding the cell-derived nucleic acid molecules to the plurality of linkers on the bead.
21. The method according to one or more of claims 17 to 20, wherein after step e), cDNA is synthesized.
22. The method according to one or more of claims 1 to 21, wherein after step g), the released bead is collected for further processing.
23. The method according to one or more of claims 17 to 22, wherein after step g), the released bead is collected for further processing to generate a single cell RNA-sequencing library.
24. The method according to claim 1, wherein the transcriptome of a single cell is analysed, comprising the following steps: a) providing a chip comprising a plurality of wells, wherein a complementary metal-oxide- semiconductor (CMOS) layer is provided to connect individually controllable electrodes located at the bottom of each well, and wherein each well can accommodate a microscale bead and a eukaryotic or prokaryotic cell, b) providing single beads coated with a plurality of linkers to at least two wells of the chip, wherein the plurality of linkers are nucleic acid molecules, c) loading at least two wells containing single beads with a single eukaryotic or prokaryotic cell, d) lysing the single cells in the well while the individually controllable electrode in the well is positively charged to retain negatively charged cell-derived nucleic acid molecules within the well, e) contacting cell-derived nucleic acid molecules with the plurality of linkers on the bead, f) selecting one or more well, and g) releasing one or more beads by addressing the individually controllable electrode in the selected well.
25. The method according to claim 24, wherein in step b), the 3’ ends of the nucleic acid molecules are deprotected.
26. The method of any previous claim wherein the single eukaryotic or prokaryotic cell is labelled.
27. A platform for single cell analysis comprising a chip with a plurality of wells, wherein a complementary metal-oxide-semiconductor (CMOS) layer is provided to connect individually controllable electrodes located at the bottom of each well, and wherein each well can accommodate a microscale bead and a eukaryotic or prokaryotic cell.
28. The platform according to claim 27, wherein a plurality of wells accommodates a microscale bead coated with a plurality of linkers.
29. The platform according to claim 28, wherein the linker comprises a cell barcode having a pre-defmed sequence for each bead and optionally, wherein each well comprises a bead with a different cell barcode.
30. The platform according to one or more of claims 27 to 29, wherein each well has at least a first chamber and a second chamber connected with the first chamber, and wherein the first chamber can accommodate a single microscale bead and the second chamber can accommodate a single eukaryotic or prokaryotic cell.
31. The platform according to claim 30, wherein the individually controllable electrode is provided at the bottom of only the first chamber.
32. The platform according to one or more of claims 30 to 31, further comprising an electrode layer which is controllable for the entire chip and which is not electrically connected with the segment comprising the individually controllable electrodes.
33. The platform according to claim 32, wherein the individually controllable electrode is provided at the bottom of the first chamber, and the electrode layer which is controllable for the entire chip is provided at the bottom of the second chamber.
34. The platform according to one or more of claims 32 to 33, wherein the electrode layer is a platinum layer.
35. The platform according to claim 30, wherein two individually controllable electrodes are provided at two not electrically connected segments of the bottom of each well, wherein one segment is provided at the bottom of the first chamber and the other segment is provided at the bottom of the second chamber.
36. The platform according to one or more of claims 27 to 35, wherein the chip geometry allows for a 1 : 1 bead to cell distribution and capture in each well.
37. The platform according to one or more of claims 27 to 36, wherein the bead is a monodisperse optionally porous microparticle.
38. The platform according to one or more of claims 27 to 37, wherein the bead has a size of between 10 pm and 100 pm.
39. The platform according to one or more of claims 27 to 38, further comprising an imaging system, optionally wherein the imaging system comprises a microscope.
40. An array of beads coated with a plurality of linkers, wherein the plurality of linkers comprise nucleic acid molecules, each nucleic acid molecule having at least the following sequence segments: a PCR handle, a UMI and a cell barcode, wherein each linker on a specific bead is designed to contain the same nucleotide sequence segment to provide a cell barcode and a different nucleotide sequence segment as compared to the other linkers on the specific bead to provide a UMI, and wherein the cell barcode on each bead is different.
41. The array of beads of claim 40, wherein each linker further comprises a poly(dT) or RNA binding sequence.
42. The array of beads of claim 40 or claim 41, wherein each bead is located in a well of a chip or a multiwell plate.
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