WO2024098076A1 - Pelletisation de cellules par billes magnétiques pendant la préparation et/ou le codage à barres de bibliothèque de cellules in situ - Google Patents

Pelletisation de cellules par billes magnétiques pendant la préparation et/ou le codage à barres de bibliothèque de cellules in situ Download PDF

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WO2024098076A1
WO2024098076A1 PCT/US2023/078862 US2023078862W WO2024098076A1 WO 2024098076 A1 WO2024098076 A1 WO 2024098076A1 US 2023078862 W US2023078862 W US 2023078862W WO 2024098076 A1 WO2024098076 A1 WO 2024098076A1
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cells
cell
magnetic beads
conjugated
magnetic
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Hunter RICHARDS
Katie Leigh ZOBECK
John Daniel WELLS
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Factorial Biotechnologies
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Factorial Diagnostics Inc
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    • 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
    • C12N15/1093—General methods of preparing gene libraries, not provided for in other subgroups

Definitions

  • Cell based assays typically use centrifugation to pellet cells for buffer exchanges and washes.
  • centrifugation is used in pelleting cells during library preparation for sequencing and many other methods. Pelleting via centrifugation requires formation of a visible pellet, and/or extreme care to perform the buffer exchanges and washes in a manner that leaves the pellet intact.
  • the centrifugation pelleting step commonly involves centrifugation for a period of time followed by pipetting of all but a few microliters of reagent from the side of the tube opposite the side containing the expected pellet to avoid drawing into the pipette some or all of the pellet itself.
  • the pellet is quite small, such as a pellet containing less than 15 thousand cells, the pellet is difficult to see.
  • the present disclosure describes methods to eliminate or reduce the need of the centrifugation step during buffer exchanges and washes.
  • the present disclosure is directed to methods for conjugating magnetic beads to cells during various types of laboratory methods, such as during in situ library preparation protocols and/or in situ cell barcoding protocols.
  • in situ library preparation protocols such as during in situ library preparation protocols and/or in situ cell barcoding protocols.
  • PCT Patent Application Publication Nos. WO2022036273 and WO2022192603 which are hereby incorporated by reference herein in their entireties.
  • one or more centrifugation pelleting steps occurred.
  • the present disclosure provides a method in which magnetic beads can be conjugated or bioconjugated to cell surfaces by various methods.
  • bioconjugation can be used to form a covalent linkage between a biomolecule on the surface of a cell and an exogenous moiety attached to a magnetic bead.
  • Hassdenteufel and Schuldiner Show your true color: mammalian cell surface staining for tracking cellular identity in multiplexing and beyond.
  • Curr Opin Chem Biol (2021) 66:102102 the disclosure of which is incorporated by reference in its entirety, describes several methods for staining mammalian cell surfaces with fluorophores. These are examples of conjugation methods compatible with cells.
  • magnetic beads are conjugated to an antibody directed at specific cell-surface molecules.
  • magnetic beads with anti-CD45 conjugated onto them can recognize CD45 on the surface of B-Cells. This creates a non-covalent association with the cells. By fixing the cells after antibody binding, covalent linkages will be created between the antigen and its antibody.
  • cell type specific methods for binding beads to cells also include glycan conjugated magnetic beads.
  • methods for binding beads to every cell in a population can include lipid conjugated magnetic beads and universal protein conjugation using NHS-ester or maleimide conjugated beads.
  • magnetic separation provides an easier workflow than centrifugation, and also greatly increases the speed of the separation, since centrifugation takes typically 5-10 minutes whereas magnetic separation can take about 1 minute to pull the bead-conjugated cells to one side of the container for separation.
  • fixation is useful for creating covalent bonds between beads and cells
  • fixation occurring during pellet formation has been shown to cause undesirable crosslinks between multiple cells or beads (e.g., cross-links between two different cells or multiple different cells as opposed to just a cross-link between a single cell and the moiety bound to the bead), creating clusters of cells that cannot be homogenously resuspended.
  • These clumped cells may cause problems in the reaction, such as causing reduced library yield due to issues related to reagent infiltration.
  • the reagents may be unable to reach or may only minimally reach the cells in the center of the clump such that there is uneven reagent distribution.
  • the present disclosure demonstrates that reducing the ability of the formaldehyde to fix proteins to each other, via dilution or quenching can improve the homogenous resuspension of cells after fixation and provide a more uniform library yield.
  • methods of preparing an in situ library for sequencing comprise: (a) mixing a sample comprising live cells with magnetic beads, wherein the magnetic beads are each conjugated to an antibody specific to a cell surface antigen present on at least some of the cells; (b) incubating the mixture, thereby forming a non-covalent bond between the cell surface antigen and the antibody; (c) adding a fixing agent, thereby forming a covalent bond between the cell surface antigen and the antibody that were non-covalently bonded, which fixes the cell to the magnetic bead; (d) washing the cells fixed to the magnetic beads by magnetic pelleting; (e) attaching universal sequences to nucleic acid inside of the cells; and (f) purifying the nucleic acid products for the in situ library.
  • the method further comprises a step of adding a quenching agent after step (c).
  • the quenching agent is Tris-HCL, urea, glycine, an ammonium salt, or a carbamic ester.
  • the ammonium salt is ammonium sulfate or ammonium bicarbonate.
  • the magnetic beads have a diameter of 0.5 pm to 100 pm. In some of these embodiments, the magnetic beads have a diameter of 3 pm to 6 pm. In certain embodiments, the magnetic beads have a diameter of about 4.5 pm.
  • the fixing agent is formaldehyde.
  • the attaching universal sequences is performed through enzymatic fragmentation and ligation.
  • magnetic pelleting is performed during buffer exchanging and washing.
  • the attaching universal sequences is performed through PCR amplification.
  • magnetic pelleting is performed during buffer exchanging and washing.
  • kits for preparing an in situ library for sequencing comprise (a) incubating a sample comprising live cells in the presence of a fixing agent to fix the cells; (b) mixing the fixed cells with magnetic beads, wherein the magnetic beads are conjugated to a moiety that can be physically conjugated to the cell or a molecule attached to the cell; (c) incubating the mixture, thereby forming a covalent bond between proteins of the fixed cell and the moiety, which fixes the cell to the magnetic bead; (d) washing the cells fixed to magnetic beads by magnetic pelleting; (e) attaching universal sequences to nucleic acid inside of the cells; and(f) purifying the nucleic acid products for the in situ library.
  • the magnetic beads are conjugated to maleimide.
  • the method further comprises a step of adding a quenching agent after step (c).
  • the quenching agent comprises free sulfhydryls.
  • the quenching agent is dithiothreitol, glutathione, or cysteine.
  • the magnetic beads are conjugated to an N-hydroxysuccinimide (NHS) ester.
  • the method further comprises a step of adding a quenching agent after step (c).
  • the quenching agent comprises free amine groups.
  • the quenching agent is Tris-HCL or ammonium ions.
  • the magnetic beads have a diameter of 0.5 pm to 100 pm. In some of these embodiments, the magnetic beads have a diameter of 3 pm to 6 pm. In certain embodiments, the magnetic beads have a diameter of about 4.5 pm.
  • the fixing agent is formaldehyde.
  • the attaching universal sequences is performed through enzymatic fragmentation and ligation.
  • magnetic pelleting is performed during buffer exchanging and washing.
  • the attaching universal sequences is performed through PCR amplification.
  • magnetic pelleting is performed during buffer exchanging and washing.
  • methods of conjugating cells with magnetic beads comprise: (a) mixing a sample comprising live cells with magnetic beads, wherein the magnetic beads are each conjugated to an antibody specific to a cell surface antigen present on at least some of the cells; (b) incubating the mixture, thereby forming a non-covalent bond between the cell surface antigen and the antibody; (c) adding a fixing agent, thereby forming a covalent bond between the cell surface antigen and the antibody that were non-covalently bonded, which fixes the cell to the magnetic bead; and (d) washing the cells fixed to the magnetic beads.
  • the method further comprises a step of adding a quenching agent after step (c).
  • the quenching agent is Tris-HCL, urea, glycine, an ammonium salt, or a carbamic ester.
  • the ammonium salt is ammonium sulfate or ammonium bicarbonate.
  • the methods comprise: (a) incubating a sample comprising live cells in the presence of a fixing agent; (b) mixing the fixed cells with magnetic beads, wherein the magnetic beads are conjugated to a moiety that can be physically conjugated to the cell or a molecule attached to the cell; (c) incubating the mixture, thereby forming a covalent bond between proteins of the fixed cell and the moiety, which fixes the cell to the magnetic bead; and (d) washing the cells fixed to the magnetic beads.
  • the magnetic beads are conjugated to maleimide.
  • the method further comprises a step of adding a quenching agent after step (c).
  • the quenching agent comprises free sulfhydryls.
  • the quenching agent is dithiothreitol, glutathione, or cysteine.
  • the magnetic beads are conjugated to an N-hydroxy succinimide (NHS) ester.
  • the method further comprises a step of adding a quenching agent after step (c).
  • the quenching agent comprises free amine groups.
  • the quenching agent is Tris-HCL or ammonium ions.
  • the magnetic beads have a diameter of 0.5 pm to 100 pm. In some of these embodiments, the magnetic beads have a diameter of 3 pm to 6 pm. In certain embodiments, the magnetic beads have a diameter of about 4.5 pm.
  • the fixing agent is formaldehyde.
  • the cells fixed to the magnetic beads are used for in situ library preparation. In certain embodiments, the cells fixed to the magnetic beads are used for flow cytometry.
  • step (d) comprises pelleting by magnetic force. In some embodiments, step (d) comprises pelleting by centrifugation.
  • methods of preparing an in situ library in cells conjugated to magnetic beads comprise: (a) accessing a sample comprising magnetic bead-conjugated cells that have been washed via magnetic pelleting and resuspended; (b) performing a library preparation step comprising one of the following: (i) performing a fragmentation to form nucleic acid fragments within each magnetic-bead conjugated cell and ligating nucleic acid fragments to adapter oligonucleotides in situ to generate a ligated library comprising ligated nucleic acid fragments within each magnetic bead-conjugated cell; or (ii) amplifying a target sequence region of nucleic acids to generate a set of amplicon products within each magnetic bead-conjugated cell; (c) lysing each magnetic bead-conjugated cell to collect the ligated nucleic acid fragments or the amplicon products; and (d) purifying the ligated
  • FIG. 1 provides a schematic of the steps of magnetic bead cell pelleting, according to some embodiments.
  • the steps in this example are: (a) cell suspensions are obtained from cell cultures, liquid biopsies, or tissue samples; (b) cells are incubated with antibody conjugated magnetic beads, such that one or more beads can bind to each cell expressing the target antigen; (c) cells bound to the beads can be magnetically separated from the solution; buffer, reagents and nonbead associated cells can be removed while in contact with or near the magnet; (d) cells can be resuspended in a new buffer after removing from magnet. Fewer, more, or different steps may be included in certain embodiments.
  • FIGs. 2A, 2B, and 2C provide data related to binding cells to beads and performing in situ library preparation.
  • FIG. 2A shows Tapestation analysis of in situ ligation library prepared using standard fixed cells and centrifugation based buffer exchanges (SOP) or conjugation of anti-CD45 antibody magnetic beads with magnetic separation based buffer exchanges (Bead Bound). Bead Bound libraries were analyzed with either 0 or 10 additional PCR cycles after lysis.
  • FIG. 2B shows sequencing data for an SOP control library and the bead bound library with extra PCR cycles. Sequencing reads mapped in proper pairs were identified (Proper Pairs in Run) and deduplication based on start and stop site performed (Unique Proper Pairs in Run). Insert size, average read depth and coverage metrics also provided.
  • FIG. 2C shows plot of chromosome positions (bases) with indicated observed coverage.
  • FIGs. 3A and 3B provide data related to binding cells to beads and performing in situ cell barcoding.
  • FIG. 3A shows Tapestation analysis of in situ cell barcoding library prepared using cells conjugated to anti-CD45 magnetic beads. Two different cell amounts and isothermal enzymes were tested for performance. All buffer exchanges occurred using magnetic separation.
  • FIG. 3B shows sequencing data for two of the samples shown, Bst2.0 with 32K cells and Isopol SD+ with 32K cells. Sequencing reads mapped in proper pairs were identified (Proper Pairs in Run) and deduplication occurred based on start and stop site, and the cell barcode combination was performed (Unique Barcode/Sequence Combinations). Observed metrics regarding barcode complexity (Unique Barcode Combinations) and Clustering metrics are provided. In general, these runs were not sequenced deep enough to provide single cell clusters.
  • FIGs. 4 A and 4B provide data related to optimization of bead binding protocol to improve fragmentation size.
  • FIG. 4A shows Tapestation analysis of in situ ligation library prepared using standard fixed cells and centrifugation based buffer exchanges (SOP) or conjugation of anti-CD45 magnetic beads with magnetic separation based buffer exchanges (Bead Bound). Bead Bound libraries were analyzed with 0 PCR cycles after lysis. The initial protocol was optimized during the fixation step, including a dilution with PBS and/or 250 mM Tris (as indicated) after 1 hour of fixation but before magnetic separation.
  • SOP centrifugation based buffer exchanges
  • Bead Bound libraries were analyzed with 0 PCR cycles after lysis. The initial protocol was optimized during the fixation step, including a dilution with PBS and/or 250 mM Tris (as indicated) after 1 hour of fixation but before magnetic separation.
  • FIG. 4B shows quantification of library concentration at indicated sizes for each sample shown in FIG. 4A.
  • FIG. 5 provides a schematic of conjugation methods with antibody beads and maleimide beads, according to some embodiments.
  • Antibody conjugated beads are used for capturing live cells and then the antigen-antibody interaction is fixed during cell fixation.
  • Maleimide beads are coupled to fixed cells, which allows for disulfide bond reduction without affecting cell state. Quenching can be performed in both cases to prevent conjugation from occurring when the cells are pelleted.
  • FIGs. 6A and 6B provide data on in situ libraries prepared with antibody beads- conjugated cells.
  • FIG. 6A shows a microscopic/fluorescent image of anti-CD45 conjugated magnetic beads.
  • FIG. 6B shows Tapestation analysis of in situ ligation library prepared using standard fixed cells and centrifugation-based buffer exchanges (Un-conjugated) or conjugation of anti-CD45 magnetic beads with magnetic separation-based buffer exchanges (Bead Conjugated). Different number of cells, 16,000 and 32,000 were used for the in situ ligation library preparation.
  • FIG. 6C shows the fragmentation profile of the in situ ligation libraries shown in FIG. 6B.
  • FIGs. 7A, 7B, and 7C provide data on in situ libraries prepared with maleimide beads- conjugated cells.
  • FIG. 7A shows a microscopic/fluorescent image of maleimide conjugated magnetic beads.
  • FIG. 7B shows Tapestation analysis of in situ ligation library prepared using standard fixed cells and centrifugation-based buffer exchanges (Un-conjugated) or conjugation of maleimide magnetic beads with magnetic separation-based buffer exchanges (Bead Conjugated). 16,000 cells were used for the in situ ligation library preparation in each sample.
  • FIG. 7C shows the fragmentation profile of the in situ ligation libraries shown in FIG. 7B.
  • FIGs. 8A and 8B demonstrate the effect of quenching on in situ library prepared with antibody bead conjugated cells.
  • FIG. 8A shows Tapestation analysis of in situ ligation library prepared using standard fixed cells and centrifugation-based buffer exchanges (No Conjugation), or conjugation of antibody magnetic beads with magnetic separation-based buffer exchanges without quenching (BFC No Quench), or conjugation of antibody magnetic beads with magnetic separation-based buffer exchanges with quenching (BFC Quench).
  • FIG. 8B shows the fragmentation profile of the in situ ligation libraries shown in FIG. 8A.
  • FIG. 9 shows Tapestation analysis of in situ ligation library prepared using cells conjugated to maleimide magnetic beads, with different amounts of enzymatic fragmentation and A- tailing enzyme (1.5X, 2X, or 2.5X) and with different lengths of fragmentation reaction (20 min or 40 min).
  • In situ ligation libraries prepared using standard fixed cells and centrifugation-based buffer exchanges were used as control (Un-Conjugated).
  • FIG. 10A, 10B, and 10C provide data on capturing cells with maleimide magnetic beads when starting with either live or fixed cells.
  • FIG. 10A shows the number and percentage of cells not captured by the magnetic bead with either live or fixed cells as starting cells, and using different amounts of magnetic beads.
  • FIG. 10B shows a microscopic/fluorescent image of cells after maleimide magnetic bead conjugation, in which the starting cells were live cells.
  • FIG. 10C shows a microscopic/fluorescent image of cells after maleimide magnetic bead conjugation, in which the starting cells were fixed cells. Green spots identify live cells; red spots identify dead cells.
  • antibody encompasses an immunoglobulin whether natural or partly or wholly synthetically produced, and fragments thereof. The term also covers any protein having a binding domain that is homologous to an immunoglobulin binding domain. “Antibody” further includes a polypeptide comprising a framework region from an immunoglobulin gene or fragments thereof that specifically binds and recognizes an antigen.
  • antibody is meant to include whole antibodies, polyclonal, monoclonal and recombinant antibodies, fragments thereof, and further includes single-chain antibodies, human antibodies, humanized antibodies, murine antibodies, chimeric, mouse-human, mouse-primate, primate-human monoclonal antibodies, anti-idiotype antibodies, antibody fragments, such as, scFv, (scFv)2, Fab, Fab', F(ab')2, Fv, dAb, Nanobody, Fd fragments, diabodies, and antibody-related polypeptides.
  • Antibody includes bispecific antibodies and multispecific antibodies so long as they exhibit the desired biological activity or function.
  • sample refers to a representative part or a small amount from a larger whole that can provide information about the whole that it is taken from.
  • a “sample” includes an aliquot of a sample.
  • in situ library refers to a library of nucleic acid sequences where preparation of the library occurred within a cell.
  • the resulting in situ library after library preparation can include nucleic acids no longer present in situ.
  • adapter refers to a fully or partially double stranded molecule that can be ligated to another molecule.
  • An adapter can include a Y-adapter, hairpin adapter, full double stranded, and the like.
  • the adapter is minimally composed of a common sequence that can be used for sequencing or further amplification of the library.
  • adapter sequence is used to refer to the common sequence added on with adapters or PCR primers.
  • barcode refers to a nucleic acid sequence that is used to identify a single cell, subpopulation of cells, or sample. Barcode sequences can be linked to a target nucleic acid of interest during NGS library preparation and used to trace back the starting DNA, cDNA, or RNA fragment (starting insert) (e.g., products of PCR, tagmentation, ligation, or the like) to the cell or population from which the target nucleic acid originated.
  • starting insert e.g., products of PCR, tagmentation, ligation, or the like
  • a barcode sequence can be added to a target nucleic acid of interest during amplification by carrying out PCR with a barcoding primer that contains a region comprising the barcode sequence and a region that is complementary to the target nucleic acid such that the barcode sequence is incorporated into the final amplified target nucleic acid product (i.e., amplicon).
  • Barcodes can be included in either the forward primer or the reverse primer or both primers used in PCR to amplify a target nucleic acid.
  • a barcode sequence can alternatively be added using a ligation-based technique.
  • a barcode sequence can consist of specific nucleotides, degenerate nucleotides, or partially degenerate nucleotides, or a combination of the above.
  • barcoding oligonucleotide refers to a nucleic acid sequence that includes any one or more of the barcodes (e.g., cellular label(s), sample barcode(s), molecular label(s)) provided herein or known in the art or the reverse complement of any of the barcode (e.g., cellular label(s), sample barcode(s), molecular label(s)) provided herein or known in the art.
  • the barcoding oligonucleotides are amplified using any of the methods described herein to produce one more of a set of barcoding products, including one or more barcoding primers.
  • magnetic beads that can be conjugated to live or fixed cells directly or indirectly.
  • the magnetic beads are paramagnetic beads.
  • the magnetic beads have been directly conjugated to the cells, via direct conjugation of the beads to the target antigen, membrane, or cell surface protein.
  • the primary molecule conjugated to the cells is not directly conjugated to the magnetic bead, but instead relies on another interaction to occur to get the magnetic bead associated. Examples of indirect conjugations include, but are not limited to:
  • the antibody or reactive group is conjugated to biotin
  • the bead is conjugated to streptavidin.
  • the biotin antibody recognizes the antigen on the cell and then the streptavidin binds the biotin moiety for the bead to be bound to the cell.
  • Magnetic beads conjugated to antibodies directed at specific cell-surface molecules are bound to cells expressing the target antigen, this additionally performs positive enrichment of cells containing the antigen within a mixed population of cells or select all cells if the population is uniformly expressing the target antigen.
  • Nonlimiting examples of antibodies include an anti-CD3 antibody, an anti-CD4 antibody, an anti-CD8 antibody, an anti-CD14 antibody, an anti-CD19 antibody, an anti-CD25 antibody, an anti-CD34 antibody, or an anti-CD45 antibody.
  • the magnetic beads are conjugated to an antibody specific to a cell surface antigen present on some of the cells in a biological sample.
  • the magnetic beads are conjugated to an antibody specific to a cell surface antigen present on all the cells in a biological sample.
  • Chemical moieties can be used to conjugate magnetic beads to target cells in a sample.
  • N-hydroxysuccinimide (NHS) esters can be used to conjugate the magnetic beads to cells by creating covalent bonds with primary amine groups present in Lysine and other amino acids.
  • NHS N-hydroxysuccinimide
  • maleimide can be used to conjugate the magnetic beads to cells by creating a covalent bond between a maleimide molecule and a reduced thiol group. Depending on pH, the maleimide will react with either the thiol group in Cysteine (pH >6.5), or the primary amine in Lysine (pH >7.5).
  • the beads can be covalently conjugated to proteins on the cell through the chemical reactions described above.
  • Methods for bioconjugation, or the creation of a covalent link between a biomolecule (i.e. protein) and a exogenous moiety are described, for example, in Koniev and Wagner, Developments and recent advancements in the field of endogenous amino acid selective bond forming reactions for bioconjugation. Chem. Soc. Rev. 2015, 44 (15), 5495-5551, the disclosure of which is incorporated by reference in its entirety.
  • These methods can be used to conjugate appropriately modified magnetic beads to cells. Bead conjugation can occur before, during, after, or instead of cell fixation and/or permeabilization.
  • the magnetic beads are conjugated to a glycan.
  • the magnetic beads are conjugated to a lipid, e.g., cholesterol.
  • the magnetic beads are conjugated to a moiety that can be physically conjugated to the cell or a molecule attached to the cell directly or indirectly. In some embodiments, the magnetic beads are conjugated to a moiety that can formed a chemical bond with a protein or a peptide.
  • the magnetic beads are conjugated to an isocyanate. In certain embodiments, the magnetic beads are conjugated to an isothiocyanate. In some embodiments, the magnetic beads are conjugated to an activated ester. In certain embodiments, the activated ester is N-Hydroxysuccinimide (NHS). In some embodiments, the magnetic beads are conjugated to an aldehyde. In some embodiments, the magnetic beads are conjugated to a sulfonyl halide. In some embodiments, the magnetic beads are conjugated to a sulfonate. In certain embodiment, the magnetic beads are conjugated to a fluorobenzene. In some embodiments, the magnetic beads are conjugated to an imidoester.
  • NHS N-Hydroxysuccinimide
  • the magnetic beads are conjugated to an a- halocarbonyl. In specific embodiments, the magnetic beads are conjugated to a maleimide. In some embodiments, the magnetic beads are conjugated to a vinyl sulfone. In certain embodiments, the magnetic beads are conjugated to a thiol-containing chemical. In some embodiments, the magnetic beads are conjugated to a malondialdehyde. In certain embodiments, the magnetic beads are conjugated to a metallocarbenoid. In some embodiments, the magnetic beads are conjugated to an epoxide. In certain embodiments, the magnetic beads are conjugated to a chemical that comprises transition metal ion. In some embodiments, the magnetic beads are conjugated to a diazonium reagent.
  • the magnetic beads are conjugated to a dicarboxylate. In some embodiments, the magnetic beads are conjugated to a dicarboxamide. In certain embodiments, the magnetic beads are conjugated to a ketene. In some embodiments, the magnetic beads are conjugated to a 2-pyridinecarboxyaldehydes (2PCA).
  • PCA 2-pyridinecarboxyaldehydes
  • the magnetic beads used in the methods described herein can have various sizes.
  • the magnetic beads have a diameter of 0.5 pm to 100 pm, such as 0.5 pm to 1 pm, 0.5 pm to 2 pm, 0.5 pm to 3 pm, 0.5 pm to 4 pm, 0.5 pm to 5 pm, 0.5 pm to 6 pm, 0.5 pm to 8 pm, 0.5 pm to 10 pm, 0.5 pm to 20 pm, 0.5 pm to 50 pm, 1 pm to 2 pm, 1 pm to 3 pm, 1 pm to
  • the magnetic beads have a diameter of 1 pm to 10 pm. In some embodiments, the magnetic beads have a diameter of 2 pm to 8 pm. In some embodiments, the magnetic beads have a diameter of 3 pm to 6 pm.
  • the magnetic beads have a diameter of about 0.5 pm to about 100 pm, such as about 0.5 pm, about 1 pm, about 1.5 pm, about 2 pm, about 2.5 pm, about 3 pm, about 3.5 pm, about 4 pm, about 4.5 pm, about 5 pm, about 5.5 pm, about 6 pm, about 7 pm, about 8 pm, about 9 pm, about 10 pm, about 15 pm, about 20 pm, about 30 pm, about 40 pm, about 50 pm, about 60 pm, about 70 pm, about 80 pm, about 90 pm, or about 100 pm.
  • the magnetic beads have a diameter of about 3 pm.
  • the magnetic beads have a diameter of about 4.5 pm.
  • the magnetic beads have a diameter of about 6 pm.
  • the methods of conjugating cells with magnetic beads in the current disclosure allows the use of a small number of cells as starting cells, for example 15k cells, which is close to the limit of what is possible with centrifugation-based pelleting. Covalent bonds are formed between the magnetic beads and the cells during the fixation or the incubation step.
  • the resulting magnetic bead conjugated cells can go through the downstream library preparation steps without the magnetic beads falling off due to the high temperature of the PCR and ligation reactions.
  • the step of quenching stops the fixation reaction and prevents clumps of cells from forming.
  • the methods comprise: (a) mixing a sample comprising cells with magnetic beads; (b) incubating the mixture; (c) adding a fixing agent; and (d) washing the cells fixed to the magnetic beads.
  • the methods further comprise a step of adding a quenching agent after step (c).
  • the method comprises: (a) mixing a sample comprising live cells with magnetic beads, wherein the magnetic beads are each conjugated to an antibody specific to a cell surface antigen present on at least some of the cells; (b) incubating the mixture, thereby forming a non-covalent bond between the cell surface antigen and the antibody; (c) adding a fixing agent, thereby forming a covalent bond between the cell surface antigen and the antibody that were non- covalently bonded, which fixes the cell to the magnetic bead; and (d) washing the cells fixed to the magnetic beads.
  • the method further comprises a step of adding a quenching agent after step (c).
  • Also provided herein are methods of conjugating cells with magnetic beads comprising: (a) incubating a sample comprising cells in the presence of a fixing agent; (b) mixing the fixed cells with magnetic beads; (c) incubating the mixture; and (d) washing the cells fixed to the magnetic beads.
  • the methods further comprise a step of adding a quenching agent after step (c).
  • the method comprises: (a) incubating a sample comprising live cells in the presence of a fixing agent; (b) mixing the fixed cells with magnetic beads, wherein the magnetic beads are conjugated to a moiety that can be physically conjugated to the cell or a molecule attached to the cell; (c) incubating the mixture, thereby forming a covalent bond between proteins of the fixed cell and the moiety, which fixes the cell to the magnetic bead; and (d) washing the cells fixed to the magnetic beads.
  • the method further comprises a step of adding a quenching agent after step (c).
  • the sample comprising cells are obtained from cell culture, liquid biopsy, tissue sample.
  • the sample comprises live cells.
  • the sample comprises fixed cells.
  • the sample is a liquid biopsy.
  • the sample is a blood sample or a serum sample.
  • the sample is a cell suspension obtained from a liquid biopsy.
  • the sample is a tissue sample.
  • the sample is a cell suspension obtained from a tissue sample.
  • the sample is a cell culture sample.
  • the sample is a cell suspension obtained from a cell culture sample.
  • the cells are from a tumor biopsy.
  • the tumor sample is a solid tumor sample.
  • the tumor biopsy is a liquid tumor sample.
  • a tumor sample can include a heterogenous cell population.
  • the tumor sample is from human tumors such as, but not limited to, tumors from the breast, ovary, lung, prostate, colon, kidney, liver, skin, blood, bone marrow, lymph nodes, spleen, thymus, heart, brain, bladder, adrenal gland, cervix, intestine, pancreas, stomach, smooth muscle, skeletal muscle, thyroid, thymus, endometrium, vulva, etc.
  • cancer cells that can be detected by the methods of the present disclosure include, but are not limited to, cancer cells from hematological cancers, including leukemia, acute lymphoblastic leukemia, acute myeloid leukemia, acute promyelocytic leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, childhood leukemia, lymphoma, Hodgkin lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, Burkitt lymphoma, Waldenstrom’s macroglobulinemia, non-Hodgkin lymphoma, myeloma myelodysplastic syndromes, polycythemia vera, essential thrombocythemia, myelofibrosis, monoclonal gammopathy of undetermined significance, myeloproliferative neoplasms, amyloidosis, and aplastic anemia.
  • hematological cancers including leukemia, acute lymphoblastic leukemia, acute
  • cancer cells that can be detected by the methods of the present disclosure include, but are not limited to, solid cancers, including for example tumors of the brain (glioblastomas, medulloblastoma, astrocytoma, oligodendroglioma, ependymomas, acoustic neuroma, astrocytoma and glioblastoma, craniopharyngioma, embryonal tumors, glioma, hemangioblastoma, lymphoma of the brain or spinal cord, meningioma, pineal region tumors, pituitary tumors, spinal cord tumors, and Vestibular Shwannoma).
  • solid cancers including for example tumors of the brain (glioblastomas, medulloblastoma, astrocytoma, oligodendroglioma, ependymomas, acoustic neuroma, astrocytoma and glioblastoma,
  • cancer cells that can be detected by the methods of the present disclosure include, but are not limited to, carcinomas, e.g. carcinoma of the lung, liver, thyroid, bone, adrenal, spleen, kidney, lymph node, small intestine, pancreas, colon, stomach, breast, endometrium, prostate, testicle, ovary, skin, head and neck, and esophagus.
  • carcinomas e.g. carcinoma of the lung, liver, thyroid, bone, adrenal, spleen, kidney, lymph node, small intestine, pancreas, colon, stomach, breast, endometrium, prostate, testicle, ovary, skin, head and neck, and esophagus.
  • cancer cells that can be detected by the methods of the present disclosure include, but are not limited to, small cell lung cancer, combined small cell carcinoma non-small cell lung cancer, adenocarcinoma, squamous cell cancer, large cell carcinoma, salivary gland type tumors, lung sarcoma, lung lymphoma, lung carcinoid tumors, adenoid cystic carcinomas, mesothelioma, and thymomas.
  • cancer cells that can be detected by the methods of the present disclosure include, but are not limited to, hepatocellular carcinoma, fibrolamellar carcinoma, bile duct cancer, angiosarcoma, and hepatoblastoma.
  • cancer cells that can be detected by the methods of the present disclosure include, but are not limited to, papillary thyroid cancer, follicular thyroid cancer, anaplastic thyroid cancer, medullary thyroid cancer, thyroid lymphoma, and thyroid sarcoma.
  • cancer cells that can be detected by the methods of the present disclosure include, but are not limited to osteosarcoma, Ewing tumor, chondrosarcoma, dedifferentiated chondrosarcoma, mesenchymal chondrosarcoma, clear cell, chondrosarcoma, fibrosarcoma, giant cell tumor, and chordoma.
  • cancer cells that can be detected by the methods of the present disclosure include, but are not limited to, adenoma, adrenocortical carcinoma, neuroblastoma, and pheochromocytoma.
  • cancer cells that can be detected by the methods of the present disclosure include, but are not limited to, Hemangiosarcoma, and littoral cell angiosarcoma.
  • cancer cells that can be detected by the methods of the present disclosure include, but are not limited to, renal cell cancer, renal clear cell cancer, renal papillary cancer, chromophobe renal cell cancer, collecting duct carcinoma, renal medullary carcinoma, sarcomatoid type kidney cancer.
  • cancer cells that can be detected by the methods of the present disclosure include, but are not limited to, adult soft tissue sarcoma, childhood soft tissue sarcoma, neuroendocrine tumors, paraganglioma, intestinal lymphoma, gastrointestinal carcinoid tumors, and gastrointestinal stromal tumors.
  • cancer cells that can be detected by the methods of the present disclosure include, but are not limited to, adenocarcinoma, ductal adenocarcinoma, cystic tumors, cancer of the acinar cells, endocrine pancreatic tumors, pancreatoblastoma, sarcomas of the pancreas, and pancreatic lymphomas.
  • cancer cells that can be detected by the methods of the present disclosure include, but are not limited to, small bowel cancer, colon cancer, rectal cancer, anal cancer, squamous cell bowel cancer, carcinoid bowel tumors, bowel sarcomas, bowel lymphoma, and bowel melanomas.
  • cancer cells that can be detected by the methods of the present disclosure include, but are not limited to, stomach adenocarcinoma, soft tissure stomach sarcomas, gastrointestinal stromal tumors, stomach lymphomas, mucosa associated lymphoid tissue lymphomas, stomach carcinoid tumors.
  • cancer cells that can be detected by the methods of the present disclosure include, but are not limited to, invasive breast cancer, invasive lobular breast cancer, triple negative breast cancer, inflammatory breast cancer, angiosarcoma of the breast, ductal carcinoma in situ, lobular carcinoma in situ, medullary breast cancer, mucinous breast cancer, tubular breast cancer, adenoid cystic carcinoma of the breast, metaplastic breast cancer, lymphoma of the breast, basal type breast cancer, phyllodes, cystosarcoma phyllodes, papillary breast cancer, and Paget’s disease of the breast.
  • cancer cells that can be detected by the methods of the present disclosure include, but are not limited to, endometroid adenocarcinoma, uterine serous carcinoma, and clear cell carcinoma of the endometrium.
  • cancer cells that can be detected by the methods of the present disclosure include, but are not limited to, acinar adenocarcinoma of the prostate, ductal adenocarcinoma of the prostate, transitional cell cancer of the prostate, squamous cell cancer of the prostate, small cell prostate cancer, neuroendocrine tumors of the prostate, and sarcomas of the prostate.
  • cancer cells that can be detected by the methods of the present disclosure include, but are not limited to, seminomas, classic seminomas, spermatocytic seminomas, non-seminomatous germ cell tumors, embryonal carcinoma, yolk sac carcinoma, choriocarcinoma teratomas of the testicles, Leydig cell tumors, and Sertoli cell tumors.
  • cancer cells that can be detected by the methods of the present disclosure include, but are not limited to, epithelial ovarian cancer, germ call ovarian tumors, sex cord stromal tumors, and borderline ovarian tumors.
  • cancer cells that can be detected by the methods of the present disclosure include, but are not limited to, basal cell carcinoma of the skin, melanoma, non-melanoma skin cancer, Merkel cell cancer, cutaneous skin lymphomas, Kaposi sarcoma, skin adnexal tumors, skin sarcomas, and squamous cell carcinoma of the skin.
  • cancer cells that can be detected by the methods of the present disclosure include, but are not limited to, oropharyngeal cancer, hypopharyngeal cancer, laryngeal cancer, lip and oral cavity cancer, nasopharyngeal cancer, paranasai sinus and nasal cavity cancer, salivary gland cancer, squamous cell neck cancer, and soft tissue sarcoma.
  • cancer cells that can be detected by the methods of the present disclosure include, but are not limited to, esophageal adenocarcinoma, esophageal squamous cell carcinomas, esophageal small cell carcinoma.
  • cancer cells that can be detected by the methods of the present disclosure include, but are not limited to, secondary cancers caused by metastasis.
  • Tumor microenvironments contain a heterogenous population of cells. Characterizing the composition and the interaction, dynamics, and function of a heterogenous population of cells at the single-cell resolution are important for fully understanding the biology of tumor heterogeneity, under both normal and diseased conditions. For example, cancer, a disease caused by somatic mutations conferring uncontrolled proliferation and invasiveness, can benefit from advances in single-cell analysis. Cancer cells can manifest resistance to various therapeutic drugs through cellular heterogeneity and plasticity.
  • the tumor microenvironment includes an environment containing tumor cells that cooperate with other tumor cells and host cells in their microenvironment and can adapt and evolve to changing conditions.
  • the heterogeneous population of cells can include, but are not limited to, inflammatory cells, cells that secret cytokines and/or chemokines, cytotoxic immune cells (e.g., natural killer cells, natural killer T cells, and/or CD8+ T cells), immune cells, macrophages (e.g., immunosuppressive macrophages, tumor-associated macrophages, classically activated (Ml) macrophages, alternatively-activated (M4) macrophages, and/or all macrophage subtypes including: M4, Mox, Mhem, M(Hb), M2a/2b/2c, and Ml), antigen-presenting cells, cancer cells, tumor- associated neutrophils, erythrocytes, dendritic cells (e.g., myeloid dendritic cells, plasmacytoid dendritic cells, langerhans cells, and/or interdigitating dendritic cells), eosinophils (e.g.
  • cytotoxic immune cells e.g.,
  • mast cells e.g. mucosal mast cells and connective tissue mast cells
  • T helper cells e.g. CD4+ T cells, Thl cells, Th2 cells, Th3 cells, Th 17 cells, and TFH cells
  • regulatory T cells e.g. natural T regularoty cells and induced regulatory T cells
  • memory T cells e.g. central memory T cells and effector memory T cells
  • B cells e.g. transitional, naive, plasma, and/or memory cells
  • tumor-infiltrated T cells fibroblasts
  • endothelial cells e.g. vascular endothelial cells and/or lymphatic endothelial cells
  • PD1+ T cells e.g. mucosal mast cells and connective tissue mast cells
  • T helper cells e.g. CD4+ T cells, Thl cells, Th2 cells, Th3 cells, Th 17 cells, and TFH cells
  • regulatory T cells e.g. natural T regularoty cells and induced regulatory
  • the cell population comprises neuronal cells.
  • neuronal cells include neurons (e.g. motor neurons, sensory neurons, intermediary nerons, and relay neurons), astrocytes, oligodendrocytes, microglia, ependymal cells, satellite cells, and schwann cells.
  • the cell population comprises cardiac cells.
  • cardiac cells include cardiac fibroblast cells, cardiomyocytes, smooth muscle cells, and endothelial cells.
  • the cell population comprises fibroblast cells.
  • the sample can be from cell lines such as ovarian cancer (e.g. A4, OVCAR3, CAOV3, CAOV4, ES-2, OV-90, TOV-112D, TOV-21G, UWB1.289, UWB1.289+BRCA1, 59M, A2780, A2780CIS, A2780ADR, COLO720E, COV318, COV362, COV362.4, COV413A, COV413B, COV504, COV644, OAW28, OAW42, OV56, OV7, OV17R, PEA1, PEA2, PEO1, PEO4, PEO14, PEO16, PEO23, SKOV3, ECACC, 2774, A2780, HOC7, SKOV3, SKOV6, IGROV1), teratocarcinoma (e.g.
  • ovarian cancer e.g. A4, OVCAR3, CAOV3, CAOV4, ES-2, OV-90, TOV-112D, TOV-21G
  • NT2, P19, F9 colon cancer
  • colon cancer e.g. HT29, CL40, SW1417, CW2
  • prostate e.g. PC3, DU145, LNCaP
  • cervical cancer e.g. C33A, HT-3, ME180
  • kidney cancer e.g. ACHN, A-498, 786-0, Caki-1, Caki-2, 769-P, RCC4, SMKT-R
  • lung cancer e.g. A549, PC9, NCIH-322, SHP-77, CORL23, NCIH727, NCI-H358)
  • skin cancer e.g.
  • glioma e.g. C6, LN229, SNB 19, U87, U251
  • the cell population comprises animal cells (in particular, nonhuman animal cells).
  • the cells include, but are not limited to, non-human mammalian cells, guinea pig cells, rabbit cells, hamster cells, non-human primate cells, dog cells, pig cells, domestic cat cells, sheep cells, mice cells, rat cells, bird cells, amphibian cells, reptile cells, fish cells (e.g. zebra fish cells), cattle cells, chicken cells, goat cells, turkey cells, and horse cells.
  • the cell population comprises invertebrate animal cells, such as insect cells e.g. cells from Drosophila.
  • the cell population comprises primary cells from these animals.
  • the cell population comprises cell lines derived from these animals.
  • Nonlimiting examples of cell lines include cell lines from, Spodoptera frugiperda (e.g. Sf9 cells), Trichoplusia ni (e.g. Tni-FNL cells), Drosophila melanogaster (e.g. S2, S2R+, S2-DGRC, and Kcl67 cells), Heliothis virescens (e.g. BCIRL-Hz-AMl and FB33 cells), the mosquito (e.g. Aag2 and A20), Hamster (e.g. Chinese hamster ovary (CHO) cells), mouse (e.g.
  • Spodoptera frugiperda e.g. Sf9 cells
  • Trichoplusia ni e.g. Tni-FNL cells
  • Drosophila melanogaster e.g. S2, S2R+, S2-DGRC, and Kcl67 cells
  • Rat e.g. 9L and B35 cells
  • Zebrafish e.g. AB9 cells
  • Dog e.g. CMT12 and D17 cells
  • African green monkey e.g. MA-104 and Vero cells
  • Cercopithecus aethiops e.g. Cos-7 cells.
  • the cell population comprises cells which have been genetically modified.
  • the cells have been genetically modified in-vitro.
  • the cells comprise of cells derived from human patients and/or animals who have undergone gene therapy e.g. faulty/inactive gene replacement, and introduction of a new gene to a cell(s).
  • the cells may comprise of genetically modified immune cells.
  • a nonlimiting example of a genetically modified immune cell is a chimeric-antigen receptor T cells (CAR-T cells).
  • the cells have been genetically modified through genome editing technology. Examples of genome editing technology which can be used to genetically modify cells include, but are not limited to, CRISPR (e.g. CRISPR/Cas9), transcription activatorlike effector nucleases (TALENs), zinc-finger nucleases (ZFNs), and homing endonucleases/meganucleases.
  • CRISPR e.g. CRISPR/Cas9
  • TALENs transcription activatorlike effector nuclea
  • the cell population comprises plant cells.
  • the cells comprise of plant cells from the following plant species: Arabidopsis thaliana, Boechera spp, Salaginella moellendorffl, Brachypodium distachyon, Setaria viridis, Lotus japonicus, Lemna gibba, Zea mays, Medicago truncatula, Mimulus guttatus, Nicotania benthamiana, Nicotania tabacum, Oryza sativa, Physcomitrella patens, Marchantia polymorpha, Populus spp, Chlamydomonas reinhardtii, Beta vulgaris, Agrostis canina, Agrostis gigantea, Agrostis stolonifera, Agrostis capillaris, Alopecurus pratensis, Arrhenatherum elatius, Bromus catharticus, Bromus sitchensis, Cynodon d
  • the cell population comprise of the following types of plant cells: parenchyma cells, palisade parenchyma cells, ray parenchyma cells, collenchyma cells, angular collenchyma cells, annular collenchyma cells, lamellar collenchyma cells, lacunar collenchyma cells, sclerenchyma cells, fibre sclerenchyma cells, sclereid sclerenchyma cells, xylem cells, phloem cells, sieve tube member cells, companion cells, sieve cells, meristematic cells, apical meristem cells, lateral meristem cells, intercalary meristem cells, epidermal cells, pavement cells, stomatai guard cells, and trichomes cells.
  • the cell population comprise of prokaryotic organisms.
  • the prokaryotic organisms comprise bacterial cells.
  • the bacterial cells comprise gram negative bacteria or gram positive bacteria.
  • Non-limiting examples of bacterial cells include Enterobacteriaceae, such as Salmonella and Escherichia, Caulobacter, myxococcus, streptomyces, bacillus, Clostridium, Bifidobacterium, Helicobacter pylori, Staphylococcus, and Streptococcus.
  • the cell population comprises bacterial cells.
  • bacterial cells include, but are not limited to, Acetobacter aurantius, Acinetobacter baumannii, Actinomyces israelii, Agrobacterium radiobacter, Agrobacterium tumefaciens, Anaplasma spp, Anaplasma phagocytophilum, Azorhizobium caulinodans, Azotobacter vinelandii, viridans streptococci, Bacillus spp, Bacillus anthracis, Bacillus brevis, Bacillus cereus, Bacillus fusiformis, Bacillus licheniformis, Bacillus megaterium, Bacillus mycoides, Bacillus stearothermophilus, Bacillus subtilis, Bacillus thuringiensis, Bacteroides spp, Bacteroides fragilis, Bacteroides gingivalis, Bacteroides melaninogenicus (now known as Prevotella melan
  • the cells comprise of simple eukaryotic organisms.
  • a simple eukaryotic organisms are yeasts, such as Saccharomyces (e.g. Saccharomyces cerevisae), Schizosaccharomyces Candida, or Pichia; also Euglenophyta, Chlorophyta (green algae), Diatoms, Dinoflagellate Euglenophyta, Chlorophyta, Diatoms, and Dinoflagellate.
  • the eukaryotic organisms are fungi cells.
  • the eukaryotic organisms are plant cells.
  • the eukaryotic organisms are mammalian cells.
  • the cell population comprise non-human cells.
  • the cell population comprise human cells.
  • the cell population comprise rodent cells.
  • the cell is selected from the group consisting of: an archaeal cell, a bacterial cell, a eukaryotic cell, a eukaryotic single-cell organism, a somatic cell, a germ cell, a stem cell, a plant cell, an algal cell, an animal cell, in invertebrate cell, a vertebrate cell, a fish cell, a frog cell, a bird cell, a mammalian cell, a pig cell, a cow cell, a goat cell, a sheep cell, a rodent cell, a rat cell, a mouse cell, a non-human primate cell, and a human cell.
  • the cell population is a mixture of one or more cells selected from: an archaeal cell, a bacterial cell, a eukaryotic cell, a eukaryotic single-cell organism, a somatic cell, a germ cell, a stem cell, a plant cell, an algal cell, an animal cell, in invertebrate cell, a vertebrate cell, a fish cell, a frog cell, a bird cell, a mammalian cell, a pig cell, a cow cell, a goat cell, a sheep cell, a rodent cell, a rat cell, a mouse cell, a non-human primate cell, and a human cell.
  • an archaeal cell a bacterial cell, a eukaryotic cell, a eukaryotic single-cell organism, a somatic cell, a germ cell, a stem cell, a plant cell, an algal cell, an animal cell, in invertebrate cell, a vertebrate cell, a
  • the cell populations within the sample are from mutated/malignant tissue or abnormal blood.
  • the methods of the present disclosure steps are also performed on cell populations within the sample that are from non-mutated/benign tissue or normal blood, which serve as a controls sample.
  • the cell populations within the sample are from both non-mutated tissue or normal blood, which serves as a “tumor-normal” control sample, and mutated/malignant tissue and abnormal blood, which serves as a “target” sample.
  • aspects of the present methods also include performing tumor normal analysis from normal cells within a biopsy, e.g., for example where the “target” sample came from. Such methods allow for detecting and diagnosing cell populations from non-mutated tissue or normal blood to determine if mutations are found in familial germlines that may also develop in other places of the body, or if the mutations are somatic to provide for better treatment options.
  • the sample comprises no more than 100k cells, such as no more than 90k, no more than 80k, no more than 70k, no more than 60k, no more than 50k, no more than 40k, no more than 30k, no more than 20k, no more than 15k, no more than 10k cells, no more than 5k, no more than lk, no more than 500, no more than 200, or no more than 100 cells.
  • the sample comprises about 50k cells.
  • the sample comprises about 50k cells.
  • the sample comprises about 40k cells.
  • the sample comprises about 30k cells.
  • the sample comprises about 20k cells.
  • the sample comprises about 15k cells.
  • the sample comprises about 10k cells. In certain embodiments, the sample comprises about 5k cells. In certain embodiments, the sample comprises about lk cells. In specific embodiments, the sample comprises about 500 cells. In specific embodiments, the sample comprises about 200 cells. In specific embodiments, the sample comprises about 100 cells.
  • Fixation of cells can occur before binding of beads to cells, during binding of beads to cells, or after binding of beads to cells. In some cases, fixation results formation of covalent bonds from non-covalent bonds. In some other cases, fixation of non-covalent bonds is not required. The formation of the covalent bond can also be referred to as bioconjugation.
  • Fixation occurs when cells are incubated in the presence of a fixing agent.
  • Fixing the cellular sample can be performed by any convenient method as desired.
  • Fixing the cellular sample can also include permeabilizing the cell membrane.
  • the cellular sample is fixed according to fixing and permeabilization techniques described in U.S. Patent No.: 10,627,389, which is hereby incorporated by reference in its entirety.
  • fixing the cellular sample includes contacting the sample with a fixation reagent.
  • Fixation reagents of interest are those that fix the cells at a desired time-point. Any convenient fixation reagent may be employed, where suitable fixation reagents include, but are not limited to: glutaraldehyde, formaldehyde, paraformaldehyde, formaldehyde/acetone, methanol/acetone, ethanol, IncellMax (IncellDx, Inc) etc.
  • fixation reagent is a mixture of fixatives.
  • fixative mixtures include, Bouin, Clarke solution, Camoy, and formaldehyde solutions.
  • multiple fixative agents are used consecutively. In some embodiments, multiple fixative agents are used simultaneously.
  • the cells in the sample are permeabilized by contacting the cells with a permeabilizing reagent.
  • Permeabilizing reagents of interest are reagents that allow the labeled biomarker probes, e.g., as described in greater detail below, to access to the intracellular environment. Any convenient permeabilizing reagent may be employed, where suitable reagents include, but are not limited to: mild detergents, such as EDTA, Tris, IDTE (10 mM Tris, 0.1 mM EDTA), Triton X-100, NP-40, saponin, digitonin, leucoperm, Tween-20, etc.; methanol, and the like.
  • the fixing agent will also permeabilize cells. Examples of fixing agent which can also permeabilize cells include, but are not limited to, acetone, methanol, and IncellMax (IncellDx, Inc).
  • the cells are incubated in the presence of a fixing agent for about 5 minutes. In certain embodiments, the cells are incubated in the presence of a fixing agent for about 10 minutes. In certain embodiments, the cells are incubated in the presence of a fixing agent for about 15 minutes. In certain embodiments, the cells are incubated in the presence of a fixing agent for about 20 minutes. In certain embodiments, the cells are incubated in the presence of a fixing agent for about 30 minutes. In certain embodiments, the cells are incubated in the presence of a fixing agent for about 1 hour.
  • the fixing agent is formaldehyde.
  • the cells are incubated in the presence of formaldehyde for about 10 minutes. In certain embodiments, the cells are incubated in the presence of formaldehyde for about 15 minutes. In certain embodiments, the cells are incubated in the presence of formaldehyde for about 20 minutes. In certain embodiments, the cells are incubated in the presence of formaldehyde for about 30 minutes. In certain embodiments, the cells are incubated in the presence of a fixing agent for about 1 hour.
  • the fixing agent is methanol.
  • the cells are incubated in the presence of methanol for about 10 minutes. In certain embodiments, the cells are incubated in the presence of methanol for about 15 minutes. In certain embodiments, the cells are incubated in the presence of methanol for about 20 minutes. In certain embodiments, the cells are incubated in the presence of methanol for about 30 minutes.
  • fixation can occur after incubation of the antibody conjugated magnetic beads with the cells, thereby converting the non- covalent bonds between the antibody and the cell surface antigen to covalent bonds.
  • fixation can occur before incubation of the moiety conjugated magnetic beads with the cells, thereby allowing the formation of a chemical bond between the moiety and a molecule of the cell, such as a protein, a peptide, a lipid, or a glycan.
  • the fixation reaction can be reduced or terminated by quenching or dilution.
  • quenching or dilution can reduce the formation of cell clumps that may be caused by fixation occurring when cells are in close proximity to each other.
  • Quenching or dilution can also facilitate a single cell dispersion and reduce the sedimentation rate of the cells.
  • Chemical conjugation methods can also be reduced or terminated by quenching or dilution, eliminating the extra conjugation sites to reduce the formation of cell clumps that may be caused by conjugation when cells are in close proximity to each other.
  • Dilution of the fixation reaction or the conjugation reaction can be performed with any buffer suitable for downstream protocols, such as PBS, dPBS, or H2O. Dilution can be performed at any suitable ratio, such as at the ratio of 1 :2, 1:5, 1:10, 1 :50, 1 : 100, or 1 :250, etc.
  • Quenching of fixation reaction or the conjugation reaction can be performed with the addition of the quenching agent to the fixation reaction or the conjugation reaction.
  • the type of quenching agent varies depending on the nature of the fixing agent and the magnetic beads. For example, for antibody conjugated magnetic beads, quenching can be performed with Tris-HCI, in final concentration of 10 mM Tris-HCI, 250mM Tris-HCI, 500mM Tris-HCI, or IM Tris-HCI.
  • quenching of fixation reaction or the conjugation reaction can be performed in glycine.
  • alternative quenching agents include ammonium salts (e.g. ammonium sulfate, ammonium bicarbonate, etc.), urea, glycine, carbamic esters, etc.
  • the quenching agent is Tris-HCL, urea, glycine, an ammonium salt, or a carbamic ester.
  • the ammonium salt is ammonium sulfate or ammonium bicarbonate.
  • the quenching agent comprises free sulfhydryls.
  • the quenching agent is dithiothreitol, glutathione, or cysteine.
  • the quenching agent comprises free amine groups.
  • the quenching agent is Tris-HCL or ammonium ions.
  • both dilution are quenching are performed. In some embodiments, dilution and quenching are performed sequentially. In some embodiments, dilution and quenching are performed simultaneously.
  • the cells conjugated to magnetic beads can be washed after the conjugation step, fixation step, or the quenching step.
  • the cells conjugated to antibody magnetic beads are washed after the addition of the fixing agent and the formation of the covalent bond between the cell surface antigen and the antibody.
  • the cells conjugated to antibody magnetic beads are washed after quenching.
  • the cells conjugated to magnetic beads with a chemical moiety, such as maleimide or NHS ester are washed after the addition of the fixing agent and the formation of the covalent bond between the cell surface antigen and the antibody.
  • the cells conjugated to chemical moiety magnetic beads are washed after quenching. Wash buffers can include PBS, dPBS, H2O, ethanol, and the like.
  • the washing step is performed entirely by magnetic pelleting. In some embodiments, the washing step involves some centrifugation-based pelleting.
  • the retention of magnetic beads on cells is usually desirable as it can facilitate the downstream application and make the process more efficient.
  • the magnetic beads can be dissociated from the cells while keeping the cell membrane intact, and this dissociation can be performed at any later point in the processes, whenever it is desirable.
  • the cells are dissociated from the magnetic beads before being used in downstream applications.
  • the dissociated cells are used for in situ library preparation.
  • the dissociated cells are used for in situ cell barcoding.
  • the dissociated cells are used for library partition and droplet-based sequencing.
  • the cells conjugated with magnetic beads can be used for various downstream applications include, but not limited to in situ library preparation, in situ cell barcoding, library partition and droplet-based sequencing, flow cytometry, positive cell selection, or negative cell selection.
  • the cells fixed to the magnetic beads are used for in situ library preparation.
  • the cells fixed to the magnetic beads are used for in situ cell barcoding.
  • the cells fixed to the magnetic beads are used for library partition and droplet-based sequencing.
  • the cells fixed to the magnetic beads are used for flow cytometry.
  • the cells fixed to the magnetic beads are used for positive cell selection.
  • the cells fixed to the magnetic beads are used for negative cell selection.
  • Example methods of using cells conjugated to magnetic beads in the preparation of an in situ library, in the cell barcoding process, and the library partition and droplet-based sequencing are described in detail below. These are just examples, and other types of method steps or protocols may involve the magnetic-bead conjugated cells, such as other library preparation methods using DNA or RNA as starting material.
  • Methods of preparing an in situ library in cells conjugated to magnetic beads [0123]
  • the cells conjugated to magnetic beads generated using the methods described above can be used in the preparation of an in situ library.
  • the methods comprise: (a) accessing a sample comprising magnetic bead-conjugated cells; (b) performing a library preparation step; (c) lysing each magnetic bead-conjugated cell to collect the ligated nucleic acid fragments; and d) purifying the ligated nucleic acid fragments.
  • the use of cells conjugated to magnetic beads in the in situ library preparation has the benefits of allowing small reaction volume and reducing pipetting error, because magnetic pelleting can be performed instead of centrifugation-base pelleting.
  • all centrifugation steps during the in situ library preparation are replaced by magnetic pelleting. In certain other embodiments, some but not all centrifugation steps during the in situ library preparation are replaced by magnetic pelleting. In some embodiments, at least one centrifugation step during the in situ library preparation is replaced by magnetic pelleting.
  • Performing library preparation inside cells in situ allows for one to perform NGS library preparation inside of a multitude of individual cells within one reaction. This is a platform technology with a range of potential applications including cancer diagnostics, prenatal diagnostics, and profiling the microbiome, and it will aid sequencing of rare subpopulations by leveraging the ability to enrich the cell populations after library preparation.
  • the method for preparing a ligation-based library in situ for sequencing includes (a) providing a sample comprising cells; (b) performing, in each cell, an enzymatic fragmentation reaction to form DNA fragments within the cell; (c) ligating, in each cell, the DNA fragments to adapter sequences to create a ligated library comprising ligated DNA fragments; (d) lysing each of the cells to collect the ligated DNA fragments; (e) purifying the ligated DNA fragments; and (f) sequencing the ligated DNA fragments.
  • the method includes contacting the cells with a fragmentation buffer and a fragmentation enzyme to form an enzymatic fragmentation mixture.
  • Performing an enzymatic fragmentation reaction in the present ligation-based method provides for generating smaller sized DNA or RNA fragments containing the target region of interest.
  • Methods for fragmenting DNA or RNA can include mechanical, chemical, or enzyme-based fragmenting. Mechanical shearing methods include acoustic shearing, sonication, hydrodynamic shearing and nebulization.
  • Chemical fragmentation methods include the use of agents which generate hydroxyl radicals for random DNA cleavage or the use of heat with divalent metal cations, while enzymebased methods include transposases, restriction enzymes (e.g.
  • enzyme-based DNA/RNA fragmentation methods include using a mixture of at least two different enzymes e.g. two or more of the enzymes mentioned in the preceding sentence e.g. two or more nucleases, Any standard enzymatic fragmentation buffer and enzymatic fragmentation enzyme can be used for fragmenting the DNA or RNA.
  • the method optionally includes denaturing, by heat, prior to enzymatic fragmentation to improve fragmentation, likely by opening the chromatin structure of DNA or RNA in the cells.
  • the heat denaturation step is not performed prior to enzymatic fragmentation.
  • the enzymatic fragmentation mixture does not include EDTA. In certain embodiments, the enzymatic fragmentation mixture includes EDTA.
  • the fragmentation enzyme is selected from a KAPA fragmentation enzyme, TaKara fragmentation enzyme, NEBNext Ultra enzymatic fragmentation enzyme, biodynamic DNA Fragmentation Enzyme Mix, KAPA Fragmentation Kit for Enzymatic Fragmentation, SureSelect Fragmentation enzyme, Ion ShearTM Plus Enzyme, and the like.
  • the fragmentation enzyme is a Caspase-Activated DNase (CAD).
  • CAD Caspase-Activated DNase
  • a fragmentation enzyme and fragmentation buffer are contacted with the cells in an amount sufficient to perform a fragmentation reaction.
  • the fragmentation buffer is selected from a KAPA fragmentation buffer, TaKara fragmentation buffer, NEBNext Ultra enzymatic fragmentation buffer, biodynamic DNA Fragmentation buffer, KAPA Fragmentation buffer, SureSelect Fragmentation Buffer, Ion ShearTM Plus Reaction Buffer, and the like.
  • any commercially available enzymatic fragmentation buffer can be used for fragmenting the DNA or RNA of the cells.
  • the enzymatic fragmentation mixture comprises a conditioning solution.
  • the volume of conditioning solution added to the enzymatic fragmentation mixture ranges from 1 pl to 20 pl.
  • the conditioning solution is a solution that adjusts the enzymatic fragmentation buffer to handle highly sensitive reagent compositions, and in some cases sequesters EDTA (or other chelators) in the sample.
  • the conditioning solution contains a reagent that binds EDTA in the sample.
  • the conditioning solution contains Magnesium or other cations to bind to EDTA in the cell population.
  • the conditioning solution is a solution that binds to magnesium in the sample.
  • the conditioning solution contains a divalent cation chelator to bind to excess magnesium in the sample.
  • the method includes performing enzymatic fragmentation on the nucleic acids (e.g., DNA or RNA) within the cell to form an enzymatic fragmentation reaction mixture.
  • performing an enzymatic fragmentation reaction on the mixture comprises loading the enzymatic fragmentation mixture into a suitable temperature-control device (although, in some such embodiments: (a) the mixture contains fewer than 15,000 fixed cells, or from 17,000-79,000 fixed cells, or more than 81,000 fixed cells; and/or (b) the temperature-control device maintains the temperature at from 15-36°C or from 38-45°C during the fragmentation reaction; and/or (c) for fewer than 59 minutes).
  • performing an enzymatic fragmentation reaction on the mixture comprises loading the enzymatic fragmentation mixture onto a thermocycler. In some embodiments, performing an enzymatic fragmentation reaction on the mixture comprises loading the enzymatic fragmentation mixture onto a heat block. In some embodiments, performing an enzymatic fragmentation reaction on the mixture comprises loading the enzymatic fragmentation mixture into a water bath. In some embodiments, performing an enzymatic fragmentation reaction on the mixture comprises loading the enzymatic fragmentation mixture into an incubator.
  • the method includes incubating the enzymatic fragmentation mixture in the temperature control device (e.g. thermocycler) for a duration/time period ranging from 1 minute to 120 minutes. In some embodiments, before fragmenting, the method includes a pre-incubation step to allowing the enzymes to enter the cell.
  • the temperature control device e.g. thermocycler
  • performing an enzymatic fragmentation reaction on the mixture comprises loading the mixture onto a temperature control device (e.g. thermocycler) and incubating the mixture at a temperature ranging from 2°C to 80°C.
  • performing an enzymatic fragmentation reaction on the mixture comprises loading the mixture onto a temperaturecontrol device (e.g. thermocycler or heat-block) and incubating the mixture at a temperature of 14- 20°C.
  • performing an enzymatic fragmentation reaction on the mixture comprises loading the mixture onto a temperature-control device (e.g. thermocycler or heat-block) and incubating the mixture at a temperature of 20-30°C.
  • performing an enzymatic fragmentation reaction on the mixture comprises loading the mixture onto a temperaturecontrol device (e.g. thermocycler or heat-block) and incubating the mixture at a temperature 35- 38°C.
  • the method before the ligating step (c) of the ligation-based method, includes performing an end-repair and/or A-tailing reaction on the one or more DNA or RNA fragments.
  • the enzymatic fragmentation enzyme is heat inactivated before end repair and A (ERA) tailing (described below) at a known temperature for inactivating the specific enzyme 65-99.5°C for 5-60 minutes.
  • the End repair and A tailing incubation step also acts as the heat inactivation step for enzymatic fragmentation enzymes.
  • the End-repair and A-tailing reaction and the enzymatic fragmentation reaction occurs in a single reaction, with multiple temperature incubations.
  • the End repair and/or A-tailing reaction can occur during the enzymatic fragmentation reaction in a single reaction.
  • the End repair reaction can occur at a certain temperature.
  • A-tailing reaction can occur at a different temperature following a temperature change.
  • the End repair and/or A-tailing reaction can occur in different, separate reactions.
  • the End-repair and A-tailing reaction and the enzymatic fragmentation reaction are separate reactions.
  • the method includes performing an End-repair and/or A-tailing reaction on the one or more fragmented DNA or RNA within the cell.
  • End Repair and/or A-Tailing are two enzymatic steps configured to blunt the DNA or RNA fragments and, optionally, add an overhanging A nucleotide to the end of the DNA or RNA fragments, for example, to improve ligation efficiency.
  • the end-repair and/or A-tailing reaction is performed before ligating the DNA or RNA fragments.
  • the End Repair (ER) and/or A-tailing can occur in the same reaction as the enzymatic fragmentation reaction described above. In certain embodiments, the end repair and/or A-tailing occurs in the same reaction as the enzymatic fragmentation. In some embodiments, the method comprises mixing the cell with an enzyme fragmentation, ER, and A- tailing enzyme cocktail mixture.
  • the cocktail mixture comprises a concentration that is at least 0.125X, 0.5X, IX, 1.5X, 2X, 2.5X, 3X, 3.5X, 4X, 4.5X, 5X, 5.5X, 6X, 6.5X, 7X, 7.5X, 8X, 8.5X, 9X, 9.5X, or 10X the manufactured recommended enzyme concentrations (Watchmaker Genomics).
  • the enzyme fragmentation, ER, and A-tailing enzyme are included in concentrations suitable for producing appropriate library fragment sizes.
  • performing an end-repair and/or A-tailing reaction comprises contacting the fragmented DNA or RNA within the cell with an End Repair A-tail buffer and an End Repair A-tail enzyme to form an End Repair A-tail mixture.
  • performing an End-repair and A-tailing reaction comprises contacting the fragmented DNA or RNA within the cell in the enzymatic fragmentation reaction mixture with an End Repair A-tail buffer and an End Repair A-tail enzyme to form an End Repair A-tail mixture.
  • contacting the fragmented DNA or RNA within the cell in the enzymatic fragmentation reaction mixture with an End Repair A-tail buffer and an End Repair A-tail enzyme occurs on ice.
  • the temperature may then be increased for enzymatic reactions to occur e.g. to from 25-40°C.
  • the method further comprises running the End Repair A-tail mixture in a thermocycler to form an End Repair A-tail reaction mixture.
  • the End Repair A-tail mixture is incubated in the thermocycler at a temperature ranging from 2°C to 90°C.
  • performing an End Repair A-tail reaction on the End Repair A-tail mixture comprises loading the End Repair A-tail mixture onto a thermocycler and incubating the End Repair A-tail mixture at a temperature ranging from 2°C to 50°C, such as 4°C to 37°C, 4°C to 50°C, or 5°C to 40 °C.
  • the End Repair A-tail mixture is incubated for a duration ranging from 5 minutes to 50 minutes. In some embodiments the End repair and A tail enzymes are heat inactivated before proceeding to ligation at 65-100°C for 5-60 minutes or more.
  • the enzymatic fragmentation step is optimized for the cells conjugated with magnetic beads.
  • enzymatic fragmentation and A-tailing enzymes are added separately to the enzymatic fragmentation reaction.
  • enzymatic fragmentation and A-tailing enzymes are added together to the enzymatic fragmentation reaction.
  • the enzymatic fragmentation reaction is performed for no less than 10 min, such as no less than 15 min, no less than 20 min, no less than 30 min, or no less than 40 min. In specific embodiments, the fragmentation reaction is performed for about 15 min, about 20 min, about 30 min, about 40 min, or about 50 min.
  • the enzymatic fragmentation and A-tailing enzymes used in the enzymatic fragmentation reaction are about 1.5 times, about 2 times, about 2.5 times, or about 3 times of the manufacturer’s recommendation.
  • the ligation step has a reaction volume of 5 pL to 75 pL, such as 5 pL to 50 pL, 5 pL to 30 pL, 5 pL to 25 pL, 5 pL to 20 pL, 5 pL to 15 pL, or 5 pL to 10 pL.
  • the ligated nucleic acid fragments have a size of 100 bp to 1000 bp, such as 200 bp to 800 bp, 200 bp to 600 bp, 200 bp to 400 bp, 200 bp to 300 bp, 300 bp to 800 bp, 300 bp to 600 bp, 300 bp to 400 bp, 400 bp to 800 bp, or 400 bp to 600 bp.
  • the ligated nucleic acid fragments have a size about 100 bp, about 150 bp, about 200 bp, about 250 bp, about 300 bp, about 350 bp, about 400 bp, about 500 bp, about 600 bp, about 800 bp, or about 1000 bp.
  • the present ligation-based method includes ligating, in each cell, the DNA or RNA fragments to adapter sequences in situ to create a ligated library comprising ligated DNA or RNA fragments.
  • Ligation adapter sequences may include modifications such as: methylation, capping, 3'- deoxy-2',5'-DNA, N3' P5' phosphoramidates, 2'-O-alkyl-substituted DNA, 2’-O-methyl DNA, 2’ Fluoro DNA, Locked Nucleic Acids (LNAs) with 2’-O-4’-C methylene bridge, inverted T modifications (e.g. 5’ and 3’), or PNA (with such modifications at one or more nucleotide positions).
  • LNAs Locked Nucleic Acids
  • Ligation adapter sequences may also include known types of modifications, for example, labels which are known in the art, methylation, “caps,” substitution of one or more of the naturally occurring nucleotides with an analog, intemucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), with negatively charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), and with positively charged linkages (e.g., aminoalklyphosphoramidates, aminoalkylphosphotriesters).
  • uncharged linkages e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.
  • negatively charged linkages e.g., phosphorothioates, phosphorodithioates, etc.
  • positively charged linkages e.g., aminoalklyphosphoramidates,
  • ligating includes performing ligase chain reaction (LCR).
  • LCR ligase chain reaction
  • the ligase chain reaction (LCR) is an amplification process that involves a thermostable ligase to join two probes or other molecules together.
  • the thermostable ligase can include, but is not limited to, Pfu ligase, Taq ligase, HiFi Taq DNA ligase, 9°N DNA ligase, Thermostable 5’ AppDNA/RNA ligase, Ampligase® ligase, or a T4 RNA ligase (e.g. T4 RNA ligase 2).
  • the ligated product is then amplified to produce an amplicon product.
  • LCR can be used as an alternative approach to PCR. In other embodiments, PCR can be performed after LCR.
  • Ligating the DNA fragments to the adapter sequences comprises running the DNA fragments and adapter sequences in a thermocycler at a temperature and duration sufficient to ligate the DNA fragmented to the adapter sequences.
  • Ligation reagents and/or enzymes can be used for ligating the DNA or RNA fragments.
  • ligation chain reaction LCR can be used for ligating the DNA or RNA fragments.
  • Ligation of fragments to adapter sequences can also be performed using ligation without LCR (e.g. without the use of thermal cycling).
  • Adapters can be ligated enzymatically, using any suitable DNA/RNA ligase.
  • ligation can use Pfu ligase from Pyrococcus furiosus, Taq ligase from Thermus aquaticus (e.g. HiFi Taq DNA ligase), DNA ligase from Cholorella virus (e.g.
  • PBCV-1 DNA ligase T4 DNA ligase, Quick ligase, Blunt/TA ligase, T3 bacteriophage DNA ligase, T7 bacteriophage DNA ligase, a DNA ligase from Thermococcus (e.g. 9°N DNA ligase), Thermostable 5’ AppDNA/RNA ligase, Ampligase® ligase, Instant Sticky End ligase, T4 RNA ligases (e.g.
  • T4 RNA ligase 1 T4 RNA ligase 2 truncated, T4 RNA ligase truncated K227Q, and T4 RNA ligase 2 truncated KQ), or a RtcB ligase.
  • Ligases which are able to be heat-inactivated are preferred. For example, ligases which can be heat inactivated through heating to 65°C for 10 minutes are preferred.
  • the fragmented DNA or RNA are contacted with adapter sequences to form a ligated library/ligation mixture containing the ligated DNA or RNA fragments.
  • the ligation mixture can include a Ligation Master Mix.
  • the ligation mixture can include a Blunt/TA Ligase Master Mix, or an Instant Sticky End Ligase Master Mix.
  • Adapter Ligation enzymatically combines (e.g., ligates) adapters provided in the reaction to the prepared DNA or RNA fragments.
  • adapter sequences include, but are not limited to, adapter nucleotide sequences that allow high-throughput sequencing of amplified or ligated nucleic acids.
  • the adapter sequences are selected from one or more of: a Y-adapter nucleotide sequence, a hairpin nucleotide sequence, a duplex nucleotide sequence, and the like.
  • the adapter sequences are for paired-end sequencing.
  • the adapter sequences include sequencing read primer sequences (e.g., Rl, R2, i5, i7 etc.). In some embodiments, the adapter sequences include sample barcodes. Adapter sequences can be used in a ligation reaction of the disclosed method for the desired sequencing method used.
  • the ligation mixture includes the End-repair A-tail reaction mixture or enzymatic fragmentation reaction mixture, a set of adapter sequences, and a ligation master mix.
  • ligation mixture includes the End-repair A-tail reaction mixture or enzymatic fragmentation reaction mixture, a set of adapter sequences, nuclease free H2O, and a ligation master mix.
  • the ligation mixture includes a final volume ranging from 10 pl to 200 pl.
  • the method includes ligating the fragmented DNA or RNA to the adapter sequences.
  • ligating the fragmented DNA or RNA to the adapter sequences comprises running the ligation mixture in the thermocycler at a temperature and duration sufficient to ligate the fragmented DNA or RNA to the adapter sequences, such as, but not limited to: barcoding sequences, consensus read regions for sequencing, adapter sequences, or other indexing sequences for the sequencing method being used.
  • the temperature ranges from 4°C to 90°C.
  • the method includes incubating the ligation mixture at a temperature of 20+5 °C. In some embodiments, the method includes incubating the ligation mixture at a temperature of about 20°C. In some embodiments, the duration ranges from 5 minutes to 4 hours.
  • the ligase enzyme is heat inactivated e.g. at a temperature ranging from 65-99.5°C for a duration ranging from 5-60 minutes before proceeding to the next steps. In some embodiments, ligase enzymes do not need to be heat inactivated.
  • the method further comprises amplifying the ligated DNA or RNA fragments to form amplicon products.
  • Amplifying the ligated DNA or RNA fragments allows for to creating more copies of the DNA or RNA fragments, reducing the likelihood of region drop out due to in efficiencies in purification and/or hybridization capture protocols. Additionally, the method allows for adding additional sequences such as adapter sequences, read sequences, full primer sequences with sample barcodes, and the like during amplification.
  • amplifying the ligated DNA or RNA fragments to form amplicon products comprises contacting the ligated DNA or RNA fragments with amplification primers (e.g., primers used to hybridize with sample DNA or RNA that define the region to be amplified, but can also include, barcoding primers, P5/P7 primers, R1/R2 primers, other sequencing primers, and the like).
  • amplification primers e.g., primers used to hybridize with sample DNA or RNA that define the region to be amplified, but can also include, barcoding primers, P5/P7 primers, R1/R2 primers, other sequencing primers, and the like.
  • multiple PCR reactions may be performed, for example, after ligation but before sequencing the ligated DNA or RNA fragments of the cells. Some, none, or all of these additional PCR steps could occur before cell lysis, while some, none, or all of these additional PCR steps could occur after cell lysis. Additional PCR steps can include adding additional components to a PCR reaction, with each addition defined as a “PCR step”. For example, adding targeting primers, followed by adding amplification primers can take place in two PCR reactions, e.g. two PCR steps or one PCR reaction, e.g., one PCR step.
  • one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more distinct PCR reactions can be performed.
  • two PCR reactions are performed between ligation and sequencing steps (e.g., after ligation, but before lysing).
  • three PCR reactions are performed between ligation and sequencing steps (e.g., after ligation, but before lysing).
  • four PCR reactions are performed between ligation and sequencing steps (e.g., after ligation, but before lysing).
  • the PCR reactions are performed after ligation but before the lysing step.
  • the PCR reactions are performed after ligation but before the lysing step.
  • the method includes contacting the ligated library (e.g., adapter ligated DNA or RNA fragments) with primers.
  • the method includes amplifying the ligated library with primers containing minimal sequences (e.g., read 1, read 2 sequences, P5 and/or P7 sequences, etc.).
  • the method includes amplifying the ligated library with primers including sample barcodes.
  • the method includes amplifying the ligated library with primers including the sequencing adapters, such as P5 and P7.
  • Primers may include modifications such as: methylation, capping, 3'-deoxy-2',5'-DNA, N3' P5' phosphoramidates, 2'-O-alkyl-substituted DNA, 2’-O-methyl DNA, 2’ Fluoro DNA, Locked Nucleic Acids (LNAs) with 2’-O-4’-C methylene bridge, inverted T modifications (e.g. 5’ and 3’), or PNA (with such modifications at one or more nucleotide positions).
  • LNAs Locked Nucleic Acids
  • Ligation adapter sequences may also include known types of modifications, for example, labels which are known in the art, methylation, “caps,” substitution of one or more of the naturally occurring nucleotides with an analog, intemucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), with negatively charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), and with positively charged linkages (e.g., aminoalklyphosphoramidates, aminoalkylphosphotriesters).
  • uncharged linkages e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.
  • negatively charged linkages e.g., phosphorothioates, phosphorodithioates, etc.
  • positively charged linkages e.g., aminoalklyphosphoramidates,
  • the method includes amplifying the adapter-ligated fragments (e.g., ligated library) to create more copies before going through hybridization capture and/or sequencing. In some embodiments, the method includes amplifying the adapter-ligated fragments to add full length adapter sequences onto the adapter-ligated fragments, if necessary. [0164] In some embodiments, after the ligating step to produce the ligated library but before sequencing, the method includes contacting the ligated library with an amplification mixture. In some embodiments, the amplification mixture comprises any readily available, standard amplification library mix or one or more components thereof, a set of amplification primers, and the adapter-ligated library.
  • the amplification mixture comprises any readily available, standard amplification library mix or one or more components thereof, a set of amplification primers, and the adapter-ligated library.
  • the amplification mixture comprises a KAPA HiFi Hotstart Ready Mix (2X) or one or more components from the ready mix thereof, a set of amplification primers, and the adapter-ligated library.
  • the amplification mixture comprises a xGen Library Amplification Primer Mix or one or more components from the primer mix thereof, a set of amplification primers, and the adapter-ligated library.
  • the amplification mixture includes a Library Amplification Hot Start Master Mix and a xGen UDI primer Mix (IDT).
  • the amplification mixture comprises a total volume ranging from 10 to 100 pl.
  • the method comprises amplifying the amplification mixture to produce a first set of amplicon products.
  • amplifying is performed using a thermocycler.
  • amplifying is performed using polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • amplifying comprises running the amplification mixture in the thermocycler for a duration ranging from 1 second to 5 minutes.
  • the temperature of incubation of the amplification mixture in the thermocycler ranges from 4°C to 110°C.
  • aspects of the present ligation-based method include lysing each of the cells to collect the ligated and/or amplified DNA or RNA fragments.
  • the lysing step can be accomplished by contacting the DNA or RNA fragments within the cell with a cell lysing agent or physically disrupting the cell structure. In some embodiments, said lysing occurs after the ligation step. In some embodiments, lysing occurs after one or more PCR steps. In some embodiments, lysing occurs after a sorting step. Lysing the cells with a cell lysing agent facilitates purification and isolation of the DNA or RNA fragments for each cell.
  • Lysing the cells breaks open the cells, and in some cases, also breaks down the proteins in the cells leaving the ligated DNA or RNA behind (e.g., ligated DNA or RNA fragments).
  • Non-limiting examples of cell lysing methods include, but are not limited to, an enzyme solution-based method, mechanical based methods, physical manipulation, or chemical methods.
  • the lysis solution includes a proteases or proteinase K, phenol and guanidine isothiocyanate, RNase inhibitors, SDS, sodium hydroxide, potassium acetate, and the like.
  • any known cell lysis buffer may be used to lyse the cells within the one or more cell populations.
  • Mechanical lysis methods include breaking down cell membranes using shear force. Examples of mechanical lysis methods include, but are not limited to, using a High Pressure Homogenizer (HPH) or a bead mill (also known as the bead beating method). Physical methods include thermal lysis, such as repeated freeze thaws, cavitation, or osmotic shock. Chemical denaturation includes use of detergents, chaotropic solutions, alkaline lysis, or hypotonic solutions. Detergents for cell lysis can be ionic (anionic or cationic) or non-ionic detergents, or mixtures thereof.
  • HPH High Pressure Homogenizer
  • bead mill also known as the bead beating method
  • Physical methods include thermal lysis, such as repeated freeze thaws, cavitation, or osmotic shock. Chemical denaturation includes use of detergents, chaotropic solutions, alkaline lysis, or hypotonic solutions.
  • Detergents for cell lysis can be ionic (anionic or
  • non-ionic detergents used for lysis include, but are not limited to, 3-[(3-cholamidopropyl)dimethylammonio]- 1 -propanesulfonate (CHAPS), 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-l- propanesulfonate (CHAPSO), and Triton X-100.
  • a non-limiting example of an ionic detergent used for lysis includes, sodium dodecyl sulfate (SDS).
  • chaotropic agents include, but are not limited to, ethylenediaminetetraacetic acid (EDTA), and urea.
  • lysing includes heating the cells for a period of time sufficient to lyse the cells.
  • the cells can be heated to a temperature of about 25 °C or more , 30°C or more , 35°C or more , 37°C or more, 40°C or more, 45°C or more, 50°C or more, 55°C or more, 60°C or more, 65°C or more, 70°C or more, 80°C or more, 85°C or more, 90°C or more, 96°C or more, 97°C or more, 98 °C or more, or 99°C or more.
  • the cells can be heated to a temperature of about 90°C, 95°C, 96°C, 97°C, 98°C, or 99°C.
  • the present ligation-based methods include some additional steps that can be performed either before or after performing the lysing step, according to the step. Some or all of these steps do not occur in some embodiments.
  • the method includes adding barcoding sequences to the isolated DNA or RNA fragments to create a barcoded indexed library.
  • the set of indexing primers include barcoding sequences.
  • barcode sequences are added to the DNA or RNA fragments to allow for identification of specific cell phenotypes from which amplified nucleic acids originated.
  • barcodes may be added at one or both ends of each DNA or RNA fragment.
  • the barcoding sequence is discontinuous. In some embodiments, the barcoding sequence is split into two different regions of a DNA/RNA fragment.
  • barcode refers to a nucleic acid sequence that is used to identify a single cell or a subpopulation of cells. Barcode sequences can be linked to a target nucleic acid of interest during amplification or ligation and used to trace back the amplicon or ligated DNA or RNA fragment to the cell or population of cells from which the target nucleic acid originated. A barcode sequence can be added to a target nucleic acid of interest during amplification or ligation by carrying out PCR or ligation with a with the barcode sequence such that the barcode sequence is incorporated into the final amplified or ligated target nucleic acid product.
  • the barcoding sequence is 4-20 base pairs in length, or 5-19 base pairs in length, or 6-18 base pairs in length, or 6-17 base pairs in length, or 6-16 base pairs in length, or 6-15 base pairs in length, or 6- 14 base pairs in length, or 6-13 base pairs in length, or 6-12 base pairs in length, or 6-11 base pairs in length, or 6-10 base pairs in length or 6-9 base pairs in length, or 6-8 base pairs in length, or 6-7 base pairs in length.
  • the barcoding sequence is 6-8 base pairs in length.
  • the barcoding sequence can comprise a degenerate sequence.
  • the barcoding sequence is degenerate.
  • the degenerate barcoding sequence is 6-8 base pairs in length.
  • the entire barcoding sequence may be degenerate, where all nucleotides are randomized (e.g. a mixture of oligonucleotides of sequence N6,N?, or Ng).
  • the barcoding sequence may be partially degenerate where one or more nucleotides are randomized.
  • the barcoding sequence may be degenerate at defined nucleotides.
  • the barcoding sequence may contain nucleotide positions which only contain purines.
  • the barcoding sequence may contain nucleotide positions which only contain pyrimidines. These barcoding sequences can act as unique molecular identifiers.
  • the set of barcodes (e.g. being 6-20 nucleotides long, such as 6-10 nucleotides) have a Hamming distance of at least 2 (e.g.
  • the set of barcodes have a Levenshtein distance of at least 2 (e.g. 3, 4, 5 or more).
  • each barcode in the set differs form each other barcode by at least two nucleotides at corresponding sequence positions, to reduce the potential that cross-mutation of one barcode into another member of the set, and so that a single point mutation does not convert any single barcode into any other member of the set.
  • Tools are available to facilitate the design of such barcode sets e.g. BARCOSEL (Somervuo et al. BMC Bioinformatics. 2018; 19: 257), or the scripts described by Bystrykh (PLoS ONE 7(5): e36852).
  • the method after performing the lysing step, includes ligating the DNA or RNA fragments with barcode adapter sequences.
  • the barcode adapter sequences comprise a set of forward and/or reverse barcoding adapter sequences.
  • ligating the forward and/or reverse barcode adapter sequences occurs before sorting, after sorting but before the purifying step, or after the purifying in step.
  • the method after performing the lysing step, includes contacting the DNA or RNA fragments with a set of forward and/or reverse barcoding primers, and amplifying the DNA or RNA fragments to produce a barcoded indexed library.
  • the ligation-based method or amplicon-based method of the present application can include additional steps such as antibody staining and/or cell sorting.
  • contacting the cells with an antibody or detectable molecule recognizing DNA, RNA, protein, or other molecule can occur after the ligation step or after amplification in a amplicon-based method. In some embodiments, contacting the cells with an antibody or detectable molecule recognizing DNA, RNA, protein, or other molecule can occur before the enzymatic fragmentation step. In some embodiments, contacting the cells with an antibody or detectable molecule recognizing DNA, RNA, protein, or other molecule can occur after an in situ PCR step.
  • the ligation-based method includes sorting the cells into subpopulations by phenotypes (i.e. combinations of detectable molecules) to determine target cells and non-target cells.
  • the sorting occurs after the ligation step.
  • the sorting occurs after an in situ PCR step.
  • Cell sorting and/or detectable labels facilitates the differentiation of cells by cell size, granularity, DNA content, morphology, differential protein expression (e.g., presence or absence of protein expression, or an amount of protein expression), calcium flux, and the like.
  • aspects of the amplicon-based library preparation method of the present disclosure include after the first amplification step (e.g., target amplification), and/or after the second amplification step (e.g., adding adapter sequences), the method optionally includes antibody staining and sorting the cell into subpopulations by phenotypes to determine target cells and non-target cells.
  • the first amplification step e.g., target amplification
  • the second amplification step e.g., adding adapter sequences
  • sorting the cells or contacting the cells with one or more detectable label provides for sorting protein-expressing cells, cells that secrete proteins, cells expressing an antigen-specific antibody, and the like.
  • the cell before sorting, the cell is contacted with an antibody being directed against a distinct cell surface molecule on the cell, under conditions effective to allow antibody binding.
  • cell sorting and/or contacting the sample with a detectable label provides for differentiating cells by morphology presence or absence of chromatin (e.g., clumped chromatin), or the absence of conspicuous nucleoli.
  • the cells can be prepared to include a detectable label, e.g., aptamers, cell stains, etc.
  • the cells can be prepared by adding one or more primary and/or secondary antibodies to the sample.
  • Primary antibodies can include antibodies specific for a particular cell type or cell surface molecule on a cell.
  • Secondary antibodies can include detectable labels (e.g., fluorescence label) that bind to the primary antibody.
  • detectable labels include: Haematoxylin and Eosin staining, Acid and Basic Fuchsin Stain, Wright's Stain, antibody staining, cell membrane fluorescent dye, carboxyfluorescein succinimidyl ester (CFSE), DNA stains, cell viability dyes such as DAPI, PI, 7-AAD, fixable compatible dyes, amine dyes, and the like.
  • primary antibodies are added to the sample containing the cells before enzymatic fragmentation. In some embodiments, primary antibodies are added to the sample containing the cells the lysing step of lysing the cells.
  • primary and secondary antibodies are added to the sample before the lysing step of lysing the cells.
  • primary antibodies are added to the sample before the enzymatic fragmentation step, and the secondary antibody or detectable label are added to the sample before the lysing step of lysing the cells.
  • Non-limiting examples of cell sorting techniques that can be used in the present methods include, but are not limited to, flow cytometry, fluorescence activated cell sorting (FACS), in situ hybridization (ISH), fluorescence in situ hybridization, Ramen flow cytometry, fluorescence microscopy, optical tweezers, micro-pipettes, microfluidic magnetic separation devices, and magnetic activated cell sorting, and methods thereof.
  • the sorting step of the methods of the present disclosure includes FACS techniques, where FACS is used to select cells from the population containing a particular surface marker, or the selection step can include the use of magnetically responsive particles as retrievable supports for target cell capture and/or background removal.
  • FACS systems are known in the art and can be used in the methods of the invention (see e.g., PCT Application Publication No.: WO99/54494, US Application No. 20010006787, US Patent No. 10,161,007, each expressly incorporated herein by reference in their entirety).
  • sorting comprises sorting the cells having a plurality of phenotypes into subpopulations by phenotypes to determine target cells and non-target cells within the population.
  • cells are sorted into subpopulations of cells irrespective of phenotypes.
  • the ligation-based method of the present application includes purifying the ligated DNA or RNA fragments of the cells.
  • Purification of the ligated DNA or RNA fragments can be performed after lysing the cells, but before sequencing.
  • the purification step can be performed after any one of the following steps: after ligation and lysing; after ligation, one or more additional PCR steps and lysing; after ligation-based or amplification-based barcoding and lysing; or after ligation, cell sorting, and lysing the cells.
  • purifying ligated DNA or RNA fragments are well-known in the art and include, for example, using size selection based magnetic bead purification reagent (e.g., Solid Phase Reversible Immobilization (SPRI) beads), passing through a column, phenol chloroform and the like.
  • SPRI Solid Phase Reversible Immobilization
  • purifying ligated DNA or RNA fragments can include using magnetic streptavidin beads, for example if the DNA or RNA fragments contain biotin.
  • the bead purification method uses Solid Phase Reversible Immobilisation (SPRI) beads.
  • the purification beads are made from polysterene - magnetite. These beads can be coated with negatively charged carboxyl groups.
  • Bead-based size purification can include a step which involves the addition of an appropriate amount of salt (Na + ) to aid in the precipitation of the DNA/RNA.
  • This bead-based size purification method can also include a size selection step.
  • the bead-based purification method can also include an elution step through the addition of an aqueous solution. Examples of aqueous solutions for elution include, but are not limited to, water, nuclease free water, and Tris-EDTA.
  • the beads are magnetic beads. These beads can bind to the DNA/RNA in a pH dependent manner. The magnetic beads may be positively charged at low pH, and negatively charged at high pH.
  • the pH of the DNA/RNA sample may be controlled to allow DNA/RNA binding to beads or its release from the beads.
  • the column based purification is silica based. This may require the presence of chaotropic salts.
  • An non-limiting example of a chaotropic salt is guanidine hydrochloride.
  • the chaotropic salt may be present in high quantities.
  • the column based purification may involve one or more wash steps with an appropriate buffer. Examples of appropriate buffers include, but are not limited to, salt and/or ethanol solutions.
  • the DNA/RNA can then be eluted in an appropriate elution buffer. Examples of appropriate elution buffers include, but are not limited to, water, nuclease free water, and Tris-EDTA.
  • the phenol-chloroform purification method involves adding the phenol-chlorform mixture to equal volume of the DNA/RNA sample.
  • Phenol-chloroform purification involves the extraction of DNA/RNA through isolation of the aqueous phase.
  • the phenol-chloroform purification procedure can be repeated one or more times to increase the purity of the DNA/RNA.
  • the phenokchloroform ratio in the phenol-chloroform mixture is made close to a 1:1 ratio.
  • the phenol-chloroform mixture also contains alcohol.
  • An example of an alcohol which can be used is isomyl alcohol.
  • the phenol-chloroform purification method may include an additional ethanol precipitation step.
  • the ethanol precipitation step involves isolating the DNA/RNA in a precipitate.
  • purifying the ligated DNA or RNA fragments of the present methods creates an enriched or purified library for sequencing.
  • enriched refers to isolated nucleotide sequences containing the genomic regions of interest (e.g., target regions) using known purification techniques (e.g., hybridization capture, magnetic bead purification techniques, and the like).
  • purified libraries described in the methods herein includes the final purified library before sequencing.
  • the purifying step includes bead purification techniques using one or more of the following techniques: a bead-based size selection (e.g., AMPure, MagJet, Mag-Bind, Promega Beads, and Kapa Pure Beads), column based PCR cleanup (e.g., Qiagen), or a DNA precipitation based technique such as phenol chloroform or ethanol.
  • a bead-based size selection e.g., AMPure, MagJet, Mag-Bind, Promega Beads, and Kapa Pure Beads
  • column based PCR cleanup e.g., Qiagen
  • a DNA precipitation based technique such as phenol chloroform or ethanol.
  • the ligation-based method includes performing additional amplification/PCR and/or ligation steps after purification.
  • aspects of the present methods include sequencing the purified libraries. Sequencing occurs after the purification step or after the purification and additional ligation/PCR steps.
  • DNA sequencing techniques include dideoxy sequencing reactions (Sanger method) using labeled terminators or primers and gel separation in slab or capillary, sequencing by synthesis using reversibly terminated labeled nucleotides, pyrosequencing, 454 sequencing, sequencing by synthesis using allele specific hybridization to a library of labeled clones followed by ligation, real time monitoring of the incorporation of labeled nucleotides during a polymerization step, polony sequencing, SOLID sequencing, and the like.
  • These sequencing approaches can thus be used to sequence target nucleic acids of interest, for example, nucleic acids encoding target genes and other phenotypic markers amplified from the cell.
  • sequencing comprises whole genome sequencing.
  • Certain high-throughput methods of sequencing comprise a step in which individual molecules are spatially isolated on a solid surface where they are sequenced in parallel.
  • Such solid surfaces may include nonporous surfaces (such as in Solexa sequencing, e.g. Bentley et al, Nature, 456: 53-59 (2008) or Complete Genomics sequencing, e.g. Drmanac et al, Science, 327: 78-81 (2010)), arrays of wells, which may include bead- or particle-bound templates (such as with 454, e.g. Margulies et al, Nature, 437: 376-380 (2005) or Ion Torrent sequencing, U.S.
  • micromachined membranes such as with SMRT sequencing, e.g. Eid et al, Science, 323: 133-138 (2009)), or bead arrays (as with SOLID sequencing or polony sequencing, e.g. Kim et al, Science, 316: 1481-1414 (2007)).
  • Such methods may comprise amplifying the isolated molecules either before or after they are spatially isolated on a solid surface.
  • Prior amplification may comprise emulsion-based amplification, such as emulsion PCR, or rolling circle amplification.
  • sequencing may be performed using a flow cell.
  • DNA/RNA fragments which contain adapter molecules on either end, are washed across a flow cell (DNA is first denatured into single stranded DNA).
  • This flow cell contains primers which are complementary to the adapter sequences.
  • the bound DNA/RNA is then amplified repeatedly, using unlabelled nucleotides. This forms clusters of DNA/RNA which help produce an amplified signal during sequencing.
  • primers and 4 different fluorescently labelled (reversible) terminator nucleotides are added. Each time a fluorescently labelled nucleotide is incorporated, the label is excited and the fluorescence detected by a camera.
  • sequencing is performed on the Illumina® MiSeq platform, (see, e.g., Shen et al. (2012) BMC Bioinformatics 13:160; Junemann et al. (2013) Nat. Biotechnol. 31(4):294-296; Glenn (2011) Mol. Ecol. Resour. 11 (5):759-769; Thudi et al. (2012) Brief Funct. Genomics 11 (1):3- 11; herein incorporated by reference in its entirety), NovaSeq, NextSeq, HiSeq, and the like.
  • sequencing may be performed using a nanopore system, in which DNA/RNA molecules pass through a transmembrane protein (e.g. alpha hemolysin or MspA), with different nucleotides providing a different detectable signal as they pass through the channel.
  • a transmembrane protein e.g. alpha hemolysin or MspA
  • sequencing is performed on the Oxford Nanopore platform (see, e.g., Lu et al (2016), Genomics, Proteomics and Bioinformatics 14:5, herein incorporated by reference in its entirety).
  • sequencing may be performed using a solid state nanopore system, in which DNA molecules pass through pores in a metal substrate, with different nucleotides providing a different detectable signal as they pass through the pores.
  • sequencing may be performed through utilising a single circular strand of DNA/RNA. This is created through the ligation of adapters to both ends of a template DNA/RNA molecule. This would then be loaded onto a sequencing unit which provides the smallest available volume for light detection. A single polymerase would be immobilised to the bottom of the base, and replication would begin. The polymerase would use 4 differently labelled nucleotides as a substrate. This would produce a small light pulse with each nucleotide addition which allows identification of the base. This sequencing protocol would produce a movie of light pulses allowing sequencing of the template. In some embodiments, sequencing is performed on the PacBio platform (see, e.g., Rhoads and Au (2015), Genomics, Proteomics and Bioinformatics 13:5, herein incorporated by reference in its entirety).
  • sequencing is performed on any preferred, standard sequencing platform.
  • aspects of the present methods include sequencing target nucleic acids of interest, for example, nucleic acids encoding target genes and other phenotypic markers amplified from the one or more cell populations.
  • aspects of the present disclosure include amplicon-based library preparation methods.
  • the amplicon-based library in situ preparation includes (a) providing a sample comprising a cell population; (b) amplifying, in each cell within the cell population, DNA or RNA with a primer pool set to produce a first set of amplicon products for each cell; (c) lysing each of the cells to isolate DNA or RNA fragments within the first set of amplicon products; (d) purifying the DNA or RNA fragments of the cells; and (e) sequencing the DNA or RNA fragments of the cells.
  • the method includes amplifying, in each cell within the cell, DNA or RNA with a primer pool set to produce a first set of amplicon products for each cell.
  • the primers in the primer pool set are DNA primers. In some embodiments, the primers in the primer pool set are RNA primers. In some embodiments, the primer pool set includes targeting primers for targeting the target sequence region of the DNA or RNA within the cell.
  • Primers may include modifications such as: methylation, capping, 3'-deoxy-2',5'-DNA, N3' P5' phosphoramidates, 2'-O-alkyl-substituted DNA, 2’-O-methyl DNA, 2’ Fluoro DNA, Locked Nucleic Acids (LNAs) with 2’-O-4’-C methylene bridge, inverted T modifications (e.g. 5’ and 3’), or PNA (with such modifications at one or more nucleotide positions).
  • LNAs Locked Nucleic Acids
  • Ligation adapter sequences may also include known types of modifications, for example, labels which are known in the art, methylation, “caps,” substitution of one or more of the naturally occurring nucleotides with an analog, intemucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), with negatively charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), and with positively charged linkages (e.g., aminoalklyphosphoramidates, aminoalkylphosphotriesters).
  • uncharged linkages e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.
  • negatively charged linkages e.g., phosphorothioates, phosphorodithioates, etc.
  • positively charged linkages e.g., aminoalklyphosphoramidates,
  • the first primer pool set of the present disclosure is designed to amplify multiple targets with the use of multiple primer pairs in a PCR experiment (e.g. in 1 or more PCR steps, 2 or more PCR steps, or 3 or more PCR steps).
  • specific target sites are selected (particularly during the ampliconbased library preparation). In some embodiments, 1-10 target loci are selected.
  • the first primer pool set comprises a first forward primer pool. In some embodiments, the first primer pool set comprises a first reverse primer pool. The number of primers within each primer pool set is dependent on the number of targets that will be prepared using the amplicon-based method. In some embodiments, the primers in the primer pool set further comprises indexing primers (e.g. barcoding primers).
  • the primer pool set comprises a first forward primer pool and a reverse primer pool.
  • each forward primer and each reverse primer includes a nucleotide sequence having a length ranging from 10 to 200 nucleotides.
  • each forward and each reverse primer includes a nucleotide sequence having a length of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides.
  • Forward primers within the set of forward primers can have different lengths.
  • reverse primers within the set of reverse primers can have different lengths.
  • forward primers within the set of forward primers can have different lengths but similar Melting Temperature (Tm) and thus can have similar PCR reaction times.
  • Reverse primers within the set of reverse primers can have different lengths but similar Melting Temperature (Tm) and thus can have similar PCR reaction times.
  • each forward primer comprises a nucleotide sequence that hybridize to an anti-sense strand of a nucleotide sequence encoding a target region (e.g., target region of the DNA or RNA) of one or more cells.
  • the nucleotide sequence is a DNA sequence.
  • the nucleotide sequence is an RNA sequence.
  • each primer comprises a unique nucleotide sequence that hybridizes to an anti-sense strand of a nucleotide sequence encoding a different target region (e.g., a different target region of the DNA or RNA) of one or more cells.
  • a forward primer pool can include a plurality of forward primers, where each forward primer hybridizes to a distinct target nucleic acid.
  • each reverse primer comprises a nucleotide sequence that hybridize to a sense strand of a nucleotide sequence encoding a target region of one or more cells.
  • each primer comprises a unique nucleotide sequence that hybridizes to an anti-sense strand of a nucleotide sequence encoding a different target region of one or more cells.
  • a reverse primer pool can include a plurality of reverse primers, where each reverse primer hybridizes to a distinct target nucleic acid.
  • the primers can include a modification that is cleaved off before they are able to polymerize.
  • a first primer pool set can include publicly available primer pool sets of known nucleic target regions of interest.
  • the first primer pool set can include any standard multiplexing primer panel for sequencing.
  • a forward primer pool includes primers selected from a rhAmp PCR Panel, CleanPlex® NGS Panel, and Ampliseq Panel.
  • a reverse primer pool includes primers of a rhAmp PCR Panel, CleanPlex® NGS Panel, and Ampliseq Panel.
  • the primer pool set comprises RNA:DNA hybrids.
  • the panel includes only the target regions of interest.
  • the panel includes both the target region of interest and a common sequence, such that the target region of interest is on the 3’ end of the common sequence.
  • amplicon refers to the amplified nucleic acid product of a PCR reaction or other nucleic acid amplification process (e.g., ligase chain reaction (LGR), nucleic acid sequence-based amplification (NASBA), transcription-mediated amplification (TMA), Q-beta amplification, strand displacement amplification, target mediated amplification, and the like).
  • LGR ligase chain reaction
  • NASBA nucleic acid sequence-based amplification
  • TMA transcription-mediated amplification
  • Q-beta amplification Q-beta amplification
  • strand displacement amplification strand displacement amplification
  • target mediated amplification target mediated amplification
  • Amplicons may comprise RNA or DNA depending on the technique used for amplification. For example, DNA amplicons may be generated by RT-PCR, whereas RNA amplicons may be generated by TMA/NASBA.
  • PCR is a technique for amplifying desired target nucleic acid sequence contained in a nucleic acid molecule or mixture of molecules.
  • a pair of primers is employed in excess to hybridize to the complementary strands of the target nucleic acid.
  • the primers are each extended by a polymerase using the target nucleic acid as a template.
  • the extension products become target sequences themselves after dissociation from the original target strand.
  • New primers are then hybridized and extended by a polymerase, and the cycle is repeated to geometrically increase the number of target sequence molecules.
  • PCR method for amplifying target nucleic acid sequences in a sample is well known in the art and has been described in, e.g., Innis et al. (eds.) PCR Protocols (Academic Press, NY 1990); Taylor (1991) Polymerase chain reaction: basic principles and automation, in PCR: A Practical Approach, McPherson et al. (eds.) IRL Press, Oxford; Saiki et al. (1986) Nature 324:163; as well as in U.S. Pat. Nos. 4,683,195, 4,683,202 and 4,889,818, all incorporated herein by reference in their entireties.
  • the present methods can use PCR for amplification of DNA or RNA fragments in one or more PCR reactions, with one or more of the PCR steps occurring in situ.
  • PCR steps can also be used to create copies of amplicon products containing the DNA or RNA products.
  • multiple PCR reactions are performed between the first amplification step (e.g., target amplification) and the sequencing steps.
  • PCR uses relatively short oligonucleotide primers which flank the target nucleotide sequence to be amplified, oriented such that their 3' ends face each other, each primer extending toward the other.
  • the polynucleotide sample is extracted and denatured, e.g., by heat, and hybridized with first and second primers that are present in molar excess.
  • Polymerization is catalyzed in the presence of the four deoxyribonucleotide triphosphates (dNTPs— dATP, dGTP, dCTP and dTTP) using a primer- and template-dependent polynucleotide polymerizing agent, such as any enzyme capable of producing primer extension products, for example, E.
  • dNTPs deoxyribonucleotide triphosphates
  • thermostable DNA polymerases isolated from Thermus aquaticus (Taq), available from a variety of sources (for example, Perkin Elmer), Thermus thermophilus (United States Biochemicals), Bacillus stereothermophilus (Bio-Rad), or Thermococcus litoralis ("Vent" polymerase, New England Biolabs). This results in two "long products" which contain the respective primers at their 5' ends covalently linked to the newly synthesized complements of the original strands.
  • the reaction mixture is then returned to polymerizing conditions, e.g., by lowering the temperature, inactivating a denaturing agent, or adding more polymerase, and a second cycle is initiated.
  • the second cycle provides the two original strands, the two long products from the first cycle, two new long products replicated from the original strands, and two "short products" replicated from the long products.
  • the short products have the sequence of the target sequence with a primer at each end.
  • an additional two long products are produced, and a number of short products equal to the number of long and short products remaining at the end of the previous cycle.
  • the number of short products containing the target sequence grows exponentially with each cycle.
  • PCR is carried out with a commercially available thermal cycler, e.g., Perkin Elmer, ProFlex PCR system, VeritiPro Thermal Cycler, Automated Thermal Cycler, SimpliAmp Thermal Cycler, MiniAmp thermal Cycler, C100 Touch Thermal Cycler, SI 000 Thermal cycler, or T100 Thermal Cycler.
  • a commercially available thermal cycler e.g., Perkin Elmer, ProFlex PCR system, VeritiPro Thermal Cycler, Automated Thermal Cycler, SimpliAmp Thermal Cycler, MiniAmp thermal Cycler, C100 Touch Thermal Cycler, SI 000 Thermal cycler, or T100 Thermal Cycler.
  • RNA may be amplified by reverse transcribing the RNA into cDNA (RT-PCR) using an RNA dependent DNA polymerase (RT-PCR) with a single targeting primer set to the anti-sense strand of RNA, oligo-dT primers, or random sequences, such as a random hexamer. PCR amplification can then occur with addition targeting primers as described above. Alternatively, a single enzyme may be used for both steps as described in U.S. Pat. No. 5,322,770, incorporated herein by reference in its entirety. RNA may also be reverse transcribed into cDNA, followed by asymmetric gap ligase chain reaction (RT-AGLCR) as described by Marshall et al.
  • RT-AGLCR asymmetric gap ligase chain reaction
  • Suitable DNA polymerases include reverse transcriptases, such as avian myeloblastosis virus (AMV) reverse transcriptase (available from, e.g., Seikagaku America, Inc.), Moloney murine leukemia virus (MMLV) reverse transcriptase (available from, e.g., Bethesda Research Laboratories), HIV reverse transcriptase, and Telomerase reverse transcriptase.
  • AMV avian myeloblastosis virus
  • MMLV Moloney murine leukemia virus
  • HIV reverse transcriptase e.g., Bethesda Research Laboratories
  • Telomerase reverse transcriptase Telomerase reverse transcriptase
  • PCR reaction mixture e.g., used interchangeably herein as “PCR Enzyme Master Mix” and heat-resistant DNA polymerase may be used to produce amplicon products.
  • PCR reaction mixture can include other enzymes that aid in transcription (e.g., such as RNAseH to cleave a modification in primers).
  • Non-limiting examples of a PCR kit includes rhAmpSeq Library Kit (IDT) and rhAmpSeq Library Mix.
  • one or more components of a PCR kit can be used in the PCR reaction mixture, at various concentrations.
  • any buffer known to be usually used for PCR can be used.
  • examples include IDTE (10 mM Tris, 0.1 mM EDTA; Integrated DNA Technologies), Tris-HCl buffer, a Tris-sulfuric acid buffer, a tricine buffer, and the like.
  • examples of heat-resistant polymerases include Taq DNA polymerase (e.g., FastStart Taq DNA Polymerase (Roche), Ex Taq (registered trademark) (Takara), Z-Taq, AccuPrime Taq DNA Polymerase, M-PCR kit (QIAGEN), KOD DNA polymerase, Pfu DNA polymerase, and the like.
  • the heat resistant DNA polymerase has a low error rate and has a high degree of accuracy for DNA replication.
  • the heat resistant DNA polymerase is a high-fidelity polymerase (Hi-Fi).
  • Hi-Fi DNA polymerases include, but are not limited to, Phusion High-Fidelity DNA Polymerase, Phusion Plus DNA polymerase, VWR® HiFi DNA polymerase, ALLinTM HiFi DNA polymerase, and AccuPrime Taq DNA Polymerase.
  • the heat resistant DNA polymerase is modified so that it is unreactive at ambient temperatures. This allows for a reduction of non-specific amplification.
  • the heat resistant DNA polymerase is a hot-start DNA polymerase.
  • hot-start DNA polymerases include, but are not limited to, DreamTaq Hot Start DNA polymerase, Takara Taq DNA polymerase, and KOD Hot Start DNA polymerase.
  • the heat resistant DNA polymerase is capable of amplifying long DNA strands. This could be DNA polymerases that are capable of amplifying fragments of up to 30 Kb in length.
  • the heat resistant DNA polymerase is a long-range DNA polymerase. Examples of long-range DNA polymerases include, but are not limited to, LA Taq DNA polymerase, QIAGEN LongRange PCR kit and Platinum SuperFi II DNA Polymerase.
  • the amounts of the primer and template DNA used, etc., in the present disclosure can be adjusted according to the PCR kit and device used. In some embodiments, about 0.1 to 1 pl of the first primer pool set is added to the in situ PCR reaction mixture.
  • the PCR reaction mixture includes the first primer pool set, the population of cells, and a PCR library mix. Any standard PCR library mix can be used in the PCR reaction mixture.
  • the library mix is a rhAmpSeq Library Mix or components of the rhAmpSeq Library Mix.
  • the PCR library mix contains one or more components of a rhAmpSeq Library mix or one or more components of any standard PCR Library mixture.
  • a forward primer pool of the first primer pool set includes forward primers of a rhArnp PCR Panel.
  • a reverse primer pool of the first primer pool set includes reverse primers of a rhArnp PCR Panel.
  • any standard PCR library mix or PCR Enzyme Master Mix for sequencing can be used.
  • PCR reaction mixture of the present disclosure includes one or more cell populations.
  • the cell population is diluted to a volume of about 0.5 pl to about 20 pl.
  • the PCR cycling conditions are not particularly limited as long as the desired target genes can be amplified.
  • the thermal denaturation temperature can be set to 92 to 100°C., e.g., 94 to 98°C.
  • the thermal denaturation time can be set to, for example, 5 to 180 seconds, e.g., 10 to 130 seconds.
  • the annealing temperature for hybridizing primers can be set to, for example, 55 to 80°C, e.g., 60 to 70°C.
  • the annealing time can be set to, for example, 10 to 60 seconds, e.g., 10 to 20 seconds.
  • the extension reaction temperature can be set to, for example, 55 to 80°C, e.g., 60 to 70°C.
  • the elongation reaction time can be set to, for example, 4 to 15 minutes, e.g., 10 to 20 minutes.
  • the annealing and extension reaction can be performed under the same conditions.
  • the operation of combining thermal denaturation, annealing, and an elongation reaction is defined as one cycle. This cycle can be repeated until the required amounts of amplification products are obtained.
  • the number of cycles can be set to 30 to 40 times, e.g., about 30 to 35 times.
  • the number of cycles can be set to 5 to 10 cycles, 10 to 15 cycles, 15 to 20 cycles, 20 to 25 cycles, 25 to 30 cycles, 35 to 40 cycles, 45 to 50 cycles, or 55 to 60 cycles.
  • the optimal amount for in-solution PCR (IX) of polymerase enzyme is also used in the in situ PCR reaction.
  • an optimal amount of polymerase for in-solution PCR may be from about 0.25 units/50 pl to about 2.5 units/50 pl where one unit is defined as the amount of enzyme needed to catalyze the incorporation of 10 nanomoles of deoxyribonucleotides into acid-insoluble material in 30 minutes at 70°C using herring sperm DNA as a substrate.
  • an optimal amount of polymerase for insolution PCR may be from about 2.5 units/50 pl to about 5 units/50 pl where one unit is defined as the amount of enzyme that will incorporate 10 nmol of dNTP into acid-insoluble products in 30 minutes at 74°C with activated salmon sperm DNA as the template-primer.
  • an optimal amount of polymerase for in-solution PCR may be from about 1 units/50 pl to about 2.5 units/50 pl where one unit is defined as the amount of enzyme which incorporates 10 nmol of deoxyribonucleotide into DNA in 30 minutes at 74°C.
  • an optimal amount of polymerase for in-solution PCR may be from about 2.5 units/50 pl to about 5 units/50 pl where one unit is defined as the amount of enzyme required to catalyze the incorporation of 10 nmol of dNTPs into acid-insoluble material in 30 minutes at 75°C.
  • an increased amount of enzyme is used for the in situ PCR reaction.
  • 2X, 3X, 4X, 5X, 6X, 7X, 8X, 9X, or 10X the concentration of enzyme is used for the in situ PCR reaction.
  • an appropriate concentration of forward and reverse primers are used in the PCR reaction.
  • An appropriate concentration of primers may be from about 0.1 pM to about 1 pM. In some embodiments, a concentration of primer greater than 1 pM is used in the PCR reaction.
  • the “PCR cycling conditions” may include one of, any combination of, or all of the conditions with respect to the temperature and time of each thermal denaturation, annealing, and elongation reaction of PCR and the number of cycles.
  • the touchdown PCR method can be used in terms of inhibiting nonspecific amplification.
  • Touchdown PCR is a technique in which the first annealing temperature is set to a relatively high temperature and the annealing temperature is gradually reduced for each cycle, and, midway and thereafter, PCR is performed in the same manner as general PCR.
  • Shuttle PCR may also be used in terms of inhibiting non-specific amplification.
  • Shuttle PCR is a PCR in which annealing and extension reaction are performed at the same temperature.
  • Nested PCR may also be used for inhibiting non-specific amplification.
  • Nested PCR is when PCR is done with two sets of primers, an inner and outer set. The outer primers are used to generate the DNA products first, followed by the inner primers. The likelihood of the outer primers amplifying the wrong locus followed by the inner primers also amplifying this locus is very small.
  • Multiplex-PCR can be used to amplify multiple targets in a single PCR experiment. This works by using multiple primer sets which have been optimized to work simultaneously in a single reaction.
  • PCR cycling conditions are set in such a manner that the same PCR cycling conditions can be used for different primer pairs and the variation of PCR cycling conditions used to obtain necessary amplification products is minimized.
  • the number of variations of PCR cycling conditions is preferably 10 or less, 5 or less, more preferably 4 or less, still more preferably 3 or less, even more preferably 2 or less, and even still more preferably 1.
  • PCRs using the same PCR cycling conditions can be simultaneously performed using one PCR device. Accordingly, the desired amplification products can be obtained in a short time using smaller amounts of resources.
  • the method of the present disclosure includes, after producing the first set of amplicon products, purifying the first set of amplicon products.
  • Techniques for purifying amplicon products include, for example, using magnetic bead purification reagent, passing through a column, use of ampure beads, phenol chloroform and the like.
  • purifying amplicons include, using size selection based magnetic bead purification reagent (e.g., Solid Phase Reversible Immobilization (SPRI) beads), passing through a column, phenol chloroform and the like.
  • SPRI Solid Phase Reversible Immobilization
  • purifying the ligated DNA or RNA fragments can include using magnetic streptavidin beads, for example if the DNA or RNA fragments contain biotin.
  • the bead purification method uses Solid Phase Reversible Immobilisation (SPRI) beads.
  • the purification beads are made from polysterene - magnetite. These beads can be coated with negatively charged carboxyl groups.
  • Bead-based size purification can include a step which involves the addition of an appropriate amount of salt (Na + ) to aid in the precipitation of the DNA/RNA.
  • the bead-based purification method can also include a size selection step.
  • the bead-based purification method can also include an elution step through the addition of an aqueous solution. Examples of aqueous solutions for elution include, but are not limited to, water, nuclease free water, and Tris-EDTA.
  • the beads are magnetic beads. These beads can bind to DNA/RNA in a pH dependent manner. The magnetic beads may be positively charged at low pH, and negatively charged at high pH.
  • the pH of the DNA/RNA sample may be controlled to allow the DNA/RNA binding to beads or its release from the beads.
  • the column based purification is silica based. This may require the presence of chaotropic salts.
  • An non-limiting example of a chaotropic salt is guanidine hydrochloride.
  • the chaotropic salt may be present in high quantities.
  • the column based purification may involve one or more wash steps with an appropriate buffer. Examples of appropriate buffers include, but are not limited to, salt and/or ethanol solutions.
  • the DNA/RNA can then be eluted in an appropriate elution buffer. Examples of appropriate elution buffers include, but are not limited to, water, nuclease free water, and Tris-EDTA.
  • the phenol-chloroform purification method involves adding the phenol-chlorform mixture to equal volume of the DNA/RNA sample. Phenol-chloroform purification involves the extraction of DNA/RNA through isolation of the aqueous phase. The phenol-chloroform purification procedure can be repeated one or more times to increase the purity of the DNA/RNA.
  • the phenol: chloroform ratio in the phenol-chloroform mixture is made close to a 1 : 1 ratio.
  • the phenol-chloroform mixture also contains alcohol.
  • An non-limiting example of an alcohol which can be used is isomyl alcohol.
  • the phenol-chloroform purification method may include an additional ethanol precipitation step.
  • the ethanol precipitation step involves isolating the DNA/RNA in a precipitate.
  • the amplicon-based method of the present disclosure can include multiple additional PCR steps after the first amplification step and before sequencing.
  • additional PCR steps can be performed before or after lysing or after purification.
  • the method can also include ligation steps to ligate on adapter sequences for subsequent PCR or direct sequencing.
  • Adapter sequences may include modifications such as: methylation, capping, 3'-deoxy- 2',5'-DNA, N3' P5' phosphoramidates, 2'-O-alkyl-substituted DNA, 2’-O-methyl DNA, 2’ Fluoro DNA, Locked Nucleic Acids (LNAs) with 2’-O-4’-C methylene bridge, inverted T modifications (e.g. 5’ and 3’), or PNA (with such modifications at one or more nucleotide positions).
  • LNAs Locked Nucleic Acids
  • Ligation adapter sequences may also include known types of modifications, for example, labels which are known in the art, methylation, “caps,” substitution of one or more of the naturally occurring nucleotides with an analog, intemucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), with negatively charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), and with positively charged linkages (e.g., aminoalklyphosphoramidates, aminoalkylphosphotriesters).
  • uncharged linkages e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.
  • negatively charged linkages e.g., phosphorothioates, phosphorodithioates, etc.
  • positively charged linkages e.g., aminoalklyphosphoramidates,
  • the method further comprises amplifying the first set of amplicon products with primer sequences to produce a set of amplicon products.
  • This step can be performed after the first amplification step and before the lysing step, after the lysing step, or after a second amplification step (e.g., amplification with sample barcoding sequences).
  • the primer sequences include sample barcodes.
  • the method further comprises, after the sorting step or lysing step, contacting the first set of amplicon products with sample barcoding sequences.
  • sample barcoding sequences comprise a set of forward and/or reverse sample barcoding primers, and wherein the method comprises amplifying the first set of amplicon products with the set of forward and/or reverse sample barcoding primers to produce a barcoded indexed library comprising sample barcoded amplicon products.
  • the sample barcoding sequences comprise a set of barcoding adapters, and wherein the method comprises ligating the set of barcode adapters to produce a barcoded indexed library comprising barcoded amplicon products.
  • the method further comprises ligating on adapter sequences.
  • adapter sequences include, but are not limited to, adapter nucleotide sequences that allow high-throughput sequencing of amplified nucleic acids.
  • the adapter sequences are selected from one or more of: a Y-adapter nucleotide sequence, a hairpin nucleotide sequence, a duplex nucleotide sequence, and the like.
  • the adapter sequences are for pair-end sequencing.
  • the adapter sequences include sequencing reads (e.g., Rl, R2, etc.).
  • the adapter sequences include sample barcodes. Adapter sequences can be used in a ligation reaction of the disclosed method for the desired sequencing method used.
  • Barcodes used in amplicon-based library preparation methods can have the same characteristics as described above for ligation-based library preparation e.g. the use of degenerate sequences, etc.
  • ligating includes performing ligase chain reaction (LCR).
  • LCR ligase chain reaction
  • the ligase chain reaction (LCR) is an amplification process that involves a thermostable ligase to join two probes or other molecules together.
  • the ligated product is then amplified to produce a second amplicon product.
  • LCR can be used as an alternative approach to PCR.
  • PCR can be performed after LCR.
  • thermostable ligase can include, but is not limited to, Pfu ligase, Taq ligase, HiFi Taq DNA ligase, 9°N DNA ligase, Thermostable 5’ AppDNA/RNA ligase, Ampligase® ligase, or a T4 RNA ligase (e.g. T4 RNA ligase 2).
  • the method further comprises, after the sorting step or lysing step, contacting the first set of amplicon products with sample barcoding sequences.
  • sample barcoding sequences comprise a set of forward and/or reverse sample barcoding primers
  • the method comprises amplifying the first set of amplicon products with the set of forward and/or reverse sample barcoding primers to produce a barcoded indexed library comprising sample barcoded amplicon products.
  • At least two in situ PCRs are done for amplicon-based library preparation.
  • the first PCR is used to produce a first set of amplicons
  • the second PCR is done after the first PCR to amplify frorn the first set of amplicons.
  • the second PCR step also adds barcoding and/or adapter sequences to the first set of amplicons.
  • aspects of the amplicon-based library preparation method of the present disclosure include after the first amplification step (e.g., target amplification), and/or after the second amplification step (e.g., adding adapter sequences), the method optionally includes antibody staining and sorting the cells into subpopulations by phenotypes to determine target cells and non-target cells.
  • Cell sorting and/or detectable labeling of DNA or RNA fragments that can be performed in the amplicon-based library preparation method is described above under the section titled “Cell Sorting”.
  • Lysins the cells to collect DNA or RNA fragments
  • the amplicon-based library preparation method of the present disclosure includes lysing each of the cells to isolate DNA or RNA fragments within the first set of amplicon products and is described above under the section titled “Lysing the cells to collect ligated DNA or RNA fragments”.
  • the lysing step can be accomplished by contacting the DNA or RNA fragments within the cell with a cell lysing agent. In some embodiments, said lysing occurs after the ligation step. In some embodiments, lysing occurs after a sorting step. In some embodiments, lysing occurs after a PCR step (e.g. reaction). Lysing the cells with a cell lysing agent facilitates purification and isolation of the DNA or RNA fragments for each cell.
  • Non-limiting examples of cell lysing agents include, but are not limited to, an enzyme solution-based methods, mechanical-based methods, and physical methods.
  • the enzyme solution includes a proteases or proteinase K, phenol and guanidine isothiocyanate, RNase inhibitors, SDS, sodium hydroxide, potassium acetate, and the like.
  • any known cell lysis buffer may be used to lyse the cells within the one or more cell populations.
  • Mechanical lysis methods include breaking down cell membranes using shear force. Examples of mechanical lysis methods include, but are not limited to, using a High Pressure Homogenizer (HPH) or a Bead mill (also known as the bead beating method).
  • HPH High Pressure Homogenizer
  • Bead mill also known as the bead beating method.
  • Physical methods include thermal lysis, such as repeated freeze thaws, cavitation, or osmotic shock.
  • Chemical denaturation includes use of detergents, chaotropic solutions, alkaline lysis, or hypotonic solutions.
  • Detergents for cell lysis can be ionic (anionic and cationic), non-ionic detergents or a mixture therof.
  • non-ionic detergents used for lysis include, but are not limited to, 3-[(3-cholamidopropyl)dimethylammonio]-l- propanesulfonate (CHAPS), 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy- 1 - propanesulfonate (CHAPSO), and Triton X-100.
  • a non-limiting example of an ionic detergent used for lysis includes, sodium dodecyl sulphate (SDS).
  • SDS sodium dodecyl sulphate
  • chaotropic agents include, but are not limited to, Ethylenediaminetetraacetic acid (EDTA), and urea.
  • lysing includes heating the cells for a period of time sufficient to lyse the cells.
  • the cells can be heated to a temperature of about 80°C or more, 85°C or more, 90°C or more, 96°C or more, 97°C or more, 98°C or more, or 99°C.
  • the cells can be heated to a temperature of about 90°C, 95°C, 96°C, 97°C, 98°C, or 99°C.
  • the amplicon-based method of the present application includes purifying the amplicon products of the cells.
  • Purification of the amplicon products can be performed after lysing the cells, but before sequencing.
  • the purification step can be performed after any one of the following steps: after amplification and lysing; after amplification, one or more additional PCR or ligation steps and lysing; after ligation-based or amplification-based barcoding and lysing; or after amplification, cell sorting, and lysing the cells.
  • Techniques for purifying amplicon products are well-known in the art and include, for example, using magnetic bead purification reagent, passing through a column, use of arnpure beads, and the like.
  • Techniques for purifying ligated DNA or RNA fragments are well-known in the art and include, for example, using size selection based magnetic bead purification reagent (e.g., Solid Phase Reversible Immobilization (SPRI) beads), passing through a column, phenol chloroform and the like.
  • SPRI Solid Phase Reversible Immobilization
  • purifying ligated DNA or RNA fragments can include using magnetic streptavidin beads, for example if the DNA or RNA fragments contain biotin.
  • the bead purification method uses Solid Phase Reversible Immobilisation (SPRI) beads.
  • the purification beads are made from polysterene - magnetite. These beads can be coated with negatively charged carboxyl groups.
  • Bead-based size purification can include a step which involves the addition of an appropriate amount of salt (Na + ) to aid in the precipitation of DNA/RNA.
  • the bead-based purification method can also include a size selection step.
  • the bead-based purification method can also include an elution step through the addition of an aqueous solution. Examples of aqueous solutions for elution include, but are not limited to, water, nuclease free water, and Tris-EDTA.
  • the beads are magnetic beads. These beads can bind to DNA/RNA in a pH dependent manner.
  • the magnetic beads may be positively charged at low pH, and negatively charged at high pH.
  • the pH of the DNA/RNA sample may be controlled to allow DNA/RNA binding to beads or its release from the beads.
  • the column based purification is silica based. This may require the presence of chaotropic salts.
  • An non-limiting example of a chaotropic salt is guanidine hydrochloride.
  • the chaotropic salt may be present in high quantities.
  • the column based purification may involve one or more wash steps with an appropriate buffer. Examples of appropriate buffers include, but are not limited to, salt and/or ethanol solutions.
  • the DNA/RNA can then be eluted in an appropriate elution buffer.
  • appropriate elution buffers include, but are not limited to, water, nuclease free water, and Tris- EDTA.
  • the DNA/RNA would usually be eluted under low salt conditions.
  • the phenol-chloroform purification method involves adding the phenol-chlorform mixture to equal volume of the DNA/RNA sample. Phenol-chloroform purification involves the extraction of DNA/RNA through isolation of the aqueous phase. The phenol-chloroform purification procedure can be repeated one or more times to increase the purity of the DNA/RNA.
  • the phenokchloroform ratio in the phenol-chloroform mixture is made close to a 1:1 ratio.
  • the phenol-chloroform mixture also contains alcohol.
  • An non-limiting example of an alcohol which can be used is isomyl alcohol.
  • An appropriate amount of isomyl alcohol is added to the phenol-chloroform mixture.
  • the phenol: chloroform: isomyl alcohol ratio can be approximately 25:24:1.
  • the phenol-chloroform mixture is buffered.
  • the phenol-chlorform purification method may include an additional ethanol precipitation step.
  • the ethanol precipitation step involves isolating the DNA/RNA in a precipitate.
  • purifying the amplicon products of the present methods creates an enriched or purified library for sequencing.
  • aspects of the present methods include sequencing the purified libraries. Sequencing occurs after the purification step; after the purification and additional ligation/PCR steps; or after the purification and additional PCR and/or ligation steps. [0254] Any high-throughput technique for sequencing can be used in the practice of the methods described herein.
  • DNA sequencing techniques include dideoxy sequencing reactions (Sanger method) using labeled terminators or primers and gel separation in slab or capillary, sequencing by synthesis using reversibly terminated labeled nucleotides, pyrosequencing, 454 sequencing, sequencing by synthesis using allele specific hybridization to a library of labeled clones followed by ligation, real time monitoring of the incorporation of labeled nucleotides during a polymerization step, polony sequencing, SOLID sequencing, and the like.
  • These sequencing approaches can thus be used to sequence target nucleic acids of interest, for example, nucleic acids encoding target genes and other phenotypic markers amplified from the one or more cell populations.
  • Embodiments of the invention include using Sanger sequencing to sequence libraries, preferably in situations where a selected number of target loci were amplified (e.g. in ampliconbased library preparation). In some embodiments, Sanger sequencing is used when 1-10 target loci were amplified. In some embodiments, Sanger sequencing is used when one target locus was amplified.
  • the cells conjugated to magnetic beads generated using the methods described above can be used to perform in situ cell barcoding.
  • the methods comprise: (a) accessing a sample comprising magnetic bead-conjugated cells; (b) performing a cell barcoding step; (c) lysing each magnetic bead- conjugated cell to collect the amplicon products; and d) purifying the amplicon products.
  • the use of magnetic separation simplifies the process of contact the barcoding oligonucleotides to the individual cells and removing the barcoding oligonucleotides not-introduced to the cells. It also allows optimal isothermal amplification of the barcoding oligonucleotides, and then PCR amplification of the libraries by making the additional changing of polymerase buffers more efficient.
  • all centrifugation steps during the in situ cell barcoding are replaced by magnetic pelleting. In certain other embodiments, some but not all centrifugation steps during the in situ cell barcoding are replaced by magnetic pelleting. In some embodiments, at least one centrifugation step during the in situ cell barcoding is replaced by magnetic pelleting.
  • the cellular barcoding method that is described herein does not rely on or need physical isolation of individual cells for labeling single cell with sets of unique cell identifiers, instead it relies on the natural structure of each cell to provide barriers against the intermingling of nucleic acids (DNA, RNA, cDNA) or intracellular proteins from different cells.
  • This method can be performed by splitting an individual population of cells into separate sub-populations of cells (containing 1 or more cells) and then re-combining the pools after cell barcoding is performed, however, it does not require splitting and re-combining to achieve single cell resolution.
  • one advantage is that it can label DNA/RNA within the cells in a single reaction such that the DNA/RNA can grouped together based on which cell they are from.
  • aspects of the present disclosure include methods for preparing barcoding sequences, such as for cellular barcoding in situ, methods for performing barcoding, such as whole cell barcoding of a cellular population (e.g. heterogeneous cell population) in situ, and methods of detecting disease- associated genetic alterations, such as of single cells within a population that were prepared in situ and sequenced.
  • the methods of the present disclosure include contacting a population, such as a heterogeneous population comprising nucleic acid sequences such as DNA, cDNA, or RNA sequences (e.g., a DNA, cDNA, or RNA insert), with barcoding sequences, for the purpose of extending or bridging cell specific barcoding primers to the ends of the target DNA or RNA sequences within each cell.
  • a population such as a heterogeneous population comprising nucleic acid sequences such as DNA, cDNA, or RNA sequences (e.g., a DNA, cDNA, or RNA insert)
  • barcoding sequences e.g., a DNA, cDNA, or RNA insert
  • DNA inserts can be prepared using a library prep method that maintains cell integrity during the NGS library preparation, and could be performed by amplifying adapter sequence to DNA, RNA or cDNA (generated by reverse transcription of RNA), ligation of adapters to the nucleic acids, or tagmentation to nucleic acids.
  • adapters containing one or more universal sequences e.g., readl sequence, read2 sequence, P5 sequence, and/or P7 sequence
  • a barcode sequence degenerate/partially degenerate, or set of defined sequences
  • An NGS library including fragments with sequencing adapters (e.g., P5 and/or P7 sequences) in which the progeny of each unique molecule may or may not have the same pair of cellular barcodes.
  • sequencing adapters e.g., P5 and/or P7 sequences
  • the first and second barcoding oligonucleotides each include barcode.
  • the barcode is selected from a sample barcode, a molecular barcode, a cellular barcode, a molecular cellular barcode, and a population barcode.
  • the barcodes include a designed sequence.
  • the barcode is a designed sequence similar to sample barcodes (e.g., present 1 version in a set).
  • the barcode is a designed sequence pooled together such that greater than 1 barcode sequence is in a set to greater than 1E6 to greater than 2E20 or more.
  • the barcode is a designed sequence that can be adjusted for hamming distances.
  • the barcode is a degenerate sequence.
  • the barcode is a partially degenerate sequence. In such cases, the partially degenerate sequence is interrupted at specific positions with designed bases.
  • the barcode is a partially degenerate sequence using degenerate bases that only include a subset of ACGT in a position.
  • the barcode (e.g., a molecular cellular label) can include a degenerate sequence, repeat sequence, variable sequence, or a combination of degenerate, repeat, and/or variable sequences that serve as short nucleotide sequences used to tag each molecule from a single cell with one to hundreds to thousands of unique cellular labels.
  • the first barcode (e.g., molecular cellular label) includes 1-50 nucleotides (e.g., such as 1-10, 2-10, 3-10, 4-10, 5-10, 6-10, 7-10, 8-10, 8-20, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, or 45-50).
  • the first barcode (e.g., molecular cellular label) includes 8-50 nucleotides (e.g., such as 8-10, 8-20, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, or 45-50).
  • the first barcode (e.g., molecular cellular label) includes a length of 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, or 20 or more nucleotides.
  • the first barcode (e.g., molecular cellular label) includes 8 nucleotides.
  • the barcode (e.g., molecular cellular label) of the first barcoding oligonucleotide is distinguishable (e.g., has different nucleotide sequences) from the barcode (e.g., molecular cellular label) of the second barcoding oligonucleotide.
  • the second barcode (e.g., molecular cellular label) includes 1-50 nucleotides (e.g., such as 1-10, 2-10, 3-10, 4- 10, 5-10, 6-10, 7-10, 8-10, 8-20, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, or 45-50).
  • the second barcode (e.g., molecular cellular label) includes 8-50 nucleotides (e.g., such as 8-10, 8-20, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, or 45-50).
  • the second barcode (e.g., molecular cellular label) includes a length of 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, or 20 or more nucleotides.
  • the second barcode (e.g., molecular cellular label) includes 8 nucleotides.
  • the barcoding oligonucleotides of the present methods can include degenerate or mismatch bases within its central region to alter the sequence of the DNA, cDNA, or RNA fragment. Non-limiting examples of barcoding oligonucleotides can be found in U.S. Patent No.: 10,155,944, which is hereby incorporated by reference in its entirety.
  • each cell within the heterogeneous cell population of the sample includes less than 10%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% of barcoding oligonucleotides with the same first and second barcodes (e.g., molecular cellular label) as a different cell within the heterogeneous cell population.
  • first and second barcodes e.g., molecular cellular label
  • Combinations of the first barcoding oligonucleotide and second barcoding oligonucleotides are then identified and grouped together in a way to identify what combinations of barcodes existed in each cell.
  • the unique combination of cellular labels within a cell can act as a unique sample index for that cell.
  • the first and second barcoding oligonucleotides each include at least one consensus region. In some embodiments, the first and second oligonucleotides that do not include a barcode include at least one consensus region.
  • the first and second barcoding oligonucleotides each include at least two consensus regions, at least three consensus regions, at least four consensus regions, at least five consensus regions, at least six consensus region, at least seven consensus regions, at least eight consensus regions, at least nine consensus regions, or at least ten consensus regions.
  • the first and second oligonucleotides each include at least one consensus region, at least two consensus regions, at least three consensus regions, at least four consensus regions, at least five consensus regions, at least six consensus regions, at least seven consensus regions, at least eight consensus regions, at least nine consensus regions, or at least ten consensus regions.
  • a consensus region comprises a nucleotide sequence length ranging from 15-50 nucleotides, such as 15-20 nucleotides, 20-35 nucleotides, 15-35 nucleotides, 30-35 nucleotides, 40-50 nucleotides, 30-50 nucleotides, 15-40 nucleotides, and the like).
  • at least one consensus region comprises 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides.
  • the performing a library preparation step comprises amplifying a target sequence region of nucleic acids to generate a set of amplicon products within each magnetic bead-conjugated cell.
  • the amplification is performed with barcoding oligonucleotides.
  • the amplification step has a reaction volume of 5 pL to 75 pL, such as 5 pL to 50 pL, 5 pL to 30 pL, 5 pL to 25 pL, 5 pL to 20 pL, 5 pL to 15 pL, or 5 pL to 10 pL.
  • the amplicon products have a size of 100 bp to 1000 bp, such as 200 bp to 800 bp, 200 bp to 600 bp, 200 bp to 400 bp, 200 bp to 300 bp, 300 bp to 800 bp, 300 bp to 600 bp, 300 bp to 400 bp, 400 bp to 800 bp, or 400 bp to 600 bp.
  • the amplicon products have a size about 100 bp, about 150 bp, about 200 bp, about 250 bp, about 300 bp, about 350 bp, about 400 bp, about 500 bp, about 600 bp, about 800 bp, or about 1000 bp.
  • the present disclosure also includes using the magnetic beads conjugated cells in methods where pre-cursor ligation libraries can be prepared in situ within a bulk reaction before partitioning the cells in a partition. Each cell droplet can then be merged with zero, one, or more additional droplets such that the droplet is capable of labeling the cell with a unique cell barcode or barcode combination.
  • the methods comprise: (a) mixing a sample comprising cells with magnetic beads; (b) incubating the mixture; (c) adding a fixing agent; (d) washing the cells fixed to the magnetic beads by magnetic pelleting; (e) attaching universal sequences to nucleic acid inside of the cells; and (f) purifying the nucleic acid products.
  • the methods further comprise a step of adding a quenching agent after step (c). In various embodiments, adding the quenching agent improves the fragment distribution of the in situ library.
  • Also provided herein are methods of preparing an in situ library for sequencing with magnetic beads comprising: (a) incubating a sample comprising cells in the presence of a fixing agent; (b) mixing the fixed cells with magnetic beads; (c) incubating the mixture; (d) washing the cells fixed to the magnetic beads by magnetic pelleting; (e) attaching universal sequences to nucleic acid inside of the cells; and (f) purifying the nucleic acid products.
  • the methods further comprise a step of adding a quenching agent after step (c). In various embodiments, adding the quenching agent improves the fragment distribution of the in situ library.
  • the universal sequences can be any sequences, oligonucleotides, or primers described above for the preparation of a ligation-based, or amplicon-based in situ library. It can also include barcodes. In some embodiments, the universal sequences are adapter oligonucleotides. In some embodiments, the universal sequences are barcoding oligonucleotides.
  • kits for preparing an in situ library with magnetic beads using the methods described herein comprises the magnetic beads described herein. It can also include one or more components selected from the fixing agent, the dilution/quenching agent, the washing buffer, and optionally the dissociation agent.
  • the kit can also comprise one or more of the primer sets described herein or the adapter sequences described herein.
  • the kit may further comprise one or more reagents, enzymes, and/or buffers described herein used in the amplicon-based or ligation-based in situ library preparation methods.
  • Example 1 In situ library preparation with CD45 antibody conjugated magnetic beads
  • Two million B-cells were aliquoted into a 2 ml tube and pellet by centrifugation for 5 min at 300 x g. The cells were resuspended in 1 ml of PBS (2,000 cells/pL). In a separate tube, 200 pL CD45-Dynabeads were washed and resuspend in 200 pL of PBS by magnetic pelleting. The resuspended beads were transferred to the cell suspension and incubate for 30 min at 4 °C with rotation.
  • the samples were removed from the magnet and flicked repeatedly for the pellet to loosen.
  • the magnetic beads conjugated cells were then resuspended in 200 L of PBS.
  • the cells were counted. 16,000 cells were aliquoted into as many PCR tubes as needed per sample.
  • the initial protocol was optimized after the fixation step, including a dilution with PBS and/or 250 mM Tris at 1:2, 1:10, 1:50, 1:100, or 1:250 after 1 hour of fixation but before magnetic separation.
  • In situ libraries was prepared using the in situ ligation library preparation protocol as described in PCT Patent Application Publication Nos. WO2022036273 and WO2022192603, which are hereby incorporated by reference in their entireties.
  • in situ ligation libraries prepared using anti-CD45 magnetic beads has comparable quality as the situ ligation libraries prepared using standard fixed cells and centrifugation-based buffer exchanges.
  • Example 2 In situ library preparation with CD45 antibody conjugated magnetic beads
  • dPBS washed cells were resuspend at 1 million cells/ml.
  • the antibody-conjugated dynabead slurry (ThermoFisher, cat #11153D) were washed using magnetic pelleting (incubating for 1 minute on the magnet, removing supernatant, and resuspending with 6.25X dPBS than bead volume).
  • Equal volumes of washed beads and washed cells were mixed together by inversion and incubated for 30min at 4 °C with rotation. After incubation, 2X Incell MAX (IncellDx) was added to a final concentration of IX.
  • the antibody bead conjugated cells were fixed by incubation for 1 hour at 25 °C with rotation. The cells were washed via magnetic pelleting by incubating on magnet for 1 min, removing supernatant, and resuspending in dPBS. The washing step was repeated once. The cells were resuspended in dPBS to an estimated concentration of 1 million cells/ml.
  • Bead conjugated cells were used in the in situ ligation library preparation according to the protocol described in WO2022036273 and WO2022192603, which are hereby incorporated by reference in their entireties, with the modification of using magnetic pelleting instead of centrifugation, except for the no-conjugation control.
  • bead conjugated cells were incubated for 1 minute on the magnet, supernatant was removed. 200pL dPBS was added to the cells and removed while on the magnet. 1.5X the amount of Enzymatic Fragmentation and A- Tailing Enzyme as recommended from the Watchmaker protocol (Watchmaker Genomics) was used and the bead conjugated cells were incubated for 20 minutes for the fragmentation reaction.
  • the bead conjugated cells showed library formation with a yield compared to non-conjugated cells.
  • the bead conjugated cells exhibited a different fragmentation profile compared to non-conjugated cells.
  • Conjugated cells have a peak library size closer to 600bp, while the non-conjugated cells are closer to 250bp. Notably the shift in peak size was more pronounced when more cells are used, and overall library yield was diminished.
  • Example 3 In situ library preparation with maleimide conjugated magnetic beads
  • Lyophilized maleimide magnetic bead powder (RayBiotech, cat# 8MM-4.5-508MM-4.5- 50) was resuspended in Coupling Buffer (30 mM MES, 150 mM NaCl, pH 7.0) to 36% w/v to produce a bead slurry containing 200,000 beads/ L.
  • Coupling Buffer (30 mM MES, 150 mM NaCl, pH 7.0)
  • the cells were then resuspended in Coupling Buffer to 350 cells/pL.
  • the cells were added to the maleimide bead slurry at a ratio of 20 beads/cell and were incubate at room temperature for 2 hours with rotation. DTT was then added to the mixture to lOrnM final concentration and incubated at room temperature for 5 minutes with rotation.
  • the maleimide magnetic bead conjugated cells were washed using a magnet by incubating for 1 minute on the magnet, removing supernatant, and resuspending in dPBS. The washing step was repeated once.
  • the maleimide magnetic bead conjugated cells were resuspended in dPBS to experiment specifications.
  • Beads conjugated cells were then used in the in situ ligation library preparation according to the protocol described in WO2022036273 and WO2022192603, which are hereby incorporated by reference in their entireties, with the modification of using magnetic pelleting instead of centrifugation.
  • For a magnetic bead wash bead conjugated cells were incubated for 1 minute on the magnet and the supernatant was then removed. 200pL of dPBS was added to the cells and removed while on the magnet. 1.5X the amount of Enzymatic Fragmentation and A-Tailing Enzyme as recommended from the Watchmaker protocol (Watchmaker Genomics) was used and the bead conjugated cells were incubated for 20 minutes for the fragmentation reaction.
  • FIG. 7A the maleimide beads (black) and cells (red) are in close proximity, which indicates conjugation of the maleimide beads to the cells.
  • FIGs. 7B and 7C illustrates the results of library preparations for maleimide magnetic bead conjugated cells and unconjugated cells.
  • the bead conjugated cells showed library formation with a yield compared to non-conjugated cells.
  • the library distribution is bimodal for the bead conjugated cells.
  • Bead conjugated cells were prepared using the antibody conjugation method and the cells were fixed after conjugation as described in Example 2. Quenching was performed by incubating the cells in Tris-HCI, PH 8.0 at a final concentration of 250 mM for less than 5 minutes at room temperature after fixation and before pelleting via magnet. Cells were then used in the in situ library according to the protocol described in WO2022036273 and WO2022192603, which are hereby incorporated by reference in their entireties, with the modification of using magnetic pelleting instead of centrifugation, except for the no-conjugation control. 1.5X the amount of enzymatic fragmentation and A-tailing enzyme as recommended from the Watchmaker protocol (Watchmaker Genomics) was used and the bead conjugated cells were incubated for 20 minutes for the fragmentation reaction.
  • Watchmaker Genomics Watchmaker Genomics
  • FIGs. 8A and 8B show the fragment distribution after library preparation.
  • a strong peak is present at ⁇ 250bp for the non-conjugation control. This peak is absent in the no quenching sample. With quenching, the smaller peak begins to appear. Therefore, the result demonstrates that quenching improves the fragment distribution of the in situ library preparation using cells conjugated with magnetic beads.
  • Bead conjugate cells were prepared using the maleimide conjugation protocol as described in Example 3. DTT was added to quench the maleimide reaction post-coupling. Cells were then used in the in situ library according to the protocol described in WO2022036273 and WO2022192603, which are hereby incorporated by reference in their entireties, with the modification of using magnetic pelleting instead of centrifugation, except for the no-conjugation control. 1.5X, 2X, or 2.5X amount of enzymatic fragmentation and A-tailing enzyme as indicated from the Watchmaker protocol (Watchmaker Genomics) was used and the bead conjugated cells were incubated for 20 or 40 minutes for the fragmentation reaction.
  • Watchmaker Genomics Watchmaker Genomics
  • FIG. 9 shows and gel image of the in situ ligation libraries prepared using different amounts of enzymatic fragmentation and A-tailing enzyme and with different lengths of fragmentation reaction.
  • the 20 min fragmentation with 1.5X enzyme did not produce the same results as observed with maleimide bead conjugated cells from FIG. 7B. This was due to the addition of DTT, which was included to reduce clumping immediately after the conjugation. DTT appeared to have an inhibitory effect on the enzymatic fragmentation.
  • the in situ library preparation protocol produced fragments of a similar size and yield as the unconjugated control.
  • Bead conjugated cells were prepared using the maleimide conjugation protocol as described in Example 3. Either live cells or fixed cells were used. DTT was not added to the reaction. Conjugated cells were pelleted using a magnet and the supernatant was collected and counted on a cell counter. Pellets were resuspended and imaged on a EVOS microscope system with AOPI dye.
  • Example 7 In situ library preparation in cells conjugated to magnetic beads
  • Magnetic bead conjugated cells were prepared using the antibody conjugation method described above with the following modifications: cells were resuspended in Cell Staining Buffer (BioLegend) and incubated with washed beads at a ratio of 16 beads to 1 cell. After fixation and magnetic pelleting, the beads-conjugated cells were washed and resuspended a ratio of about 1 million cells/ml.
  • SOP cells were prepared by fixing 1 million cells/ml with 2X IncellMAX (IncellDx) to a final concentration of IX IncellMax and 0.5 million cells/ml. Cells were incubated at 25 C for 1 hour with rotation. Cells were then washed via centrifugation for 5 minutes at 1500xg, removal of supernatant, and resuspension in dPBS. The washing step was repeated once. The cells were resuspended in dPBS to an estimated concentration of 1 million cells/ml.
  • Libraries were prepared using the protocol for in situ ligation library preparation, described in WO2022036273, which is hereby incorporated by reference in its entirety, with the modification of using magnetic pelleting during the post-ligation wash/buffer exchange and the post-in situ PCR amplification. 1.5X the amount of Enzymatic Fragmentation and A-Tailing Enzyme as recommended from the Watchmaker protocol (Watchmaker Genomics) was used and the bead conjugated cells were incubated the fragmentation for 20 minutes.
  • Example 8 In situ library preparation with barcoding in cells conjugated to magnetic beads
  • Magnetic bead conjugated cells were prepared using the antibody conjugation method described above with the following modifications: cells were resuspended in Cell Staining Buffer (BioLegend) and incubated with washed beads at a ratio of 16 beads to 1 cell. After fixation and magnetic pelleting, the beads-conjugated cells were washed and resuspended a ratio of about 1 million cells/ml.
  • Barcoding oligos were amplified inside of the cells with 19.2 U of Bst2.0 (NEB) in a 20pL reaction consisting of IX NEB Isothermal Amplification Buffer (NEB) for 30 minutes at 60 °C or 16U of IsoPol SD+ (ArticZyme) in a 20pL reaction consisting of IX IsoPol Buffer (ArticZyme) for 30 minutes at 30 °C.
  • both the Bst2.0 and the IsoPol SD+ methods produced libraries with fragments in the sequenceable size range and with unique barcode combinations.

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Abstract

Des aspects de la présente invention concernent de manière générale des procédés, des compositions et des kits pour conjuguer des cellules à des billes magnétiques. Des aspects de la présente invention comprennent également des procédés, des compositions et des kits d'utilisation des cellules conjuguées à des billes magnétiques pour des processus en aval, tels qu'une préparation de bibliothèque in situ.
PCT/US2023/078862 2022-11-04 2023-11-06 Pelletisation de cellules par billes magnétiques pendant la préparation et/ou le codage à barres de bibliothèque de cellules in situ Ceased WO2024098076A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2025042729A1 (fr) * 2023-08-18 2025-02-27 Life Technologies Corporation Composition de préparation cellulaire, système et procédé de transcriptomique unicellulaire

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6072086A (en) * 1996-04-12 2000-06-06 Intergen Company Method and composition for controlling formaldehyde fixation by delayed quenching
US20220326251A1 (en) * 2019-11-08 2022-10-13 10X Genomics, Inc. Spatially-tagged analyte capture agents for analyte multiplexing

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6072086A (en) * 1996-04-12 2000-06-06 Intergen Company Method and composition for controlling formaldehyde fixation by delayed quenching
US20220326251A1 (en) * 2019-11-08 2022-10-13 10X Genomics, Inc. Spatially-tagged analyte capture agents for analyte multiplexing

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
KRISTIANA INA, LETHORN ARRON; JOLL CYNTHIA; HEITZ ANNA: "To add or not to add: The use of quenching agents for the analysis of disinfection by-products in water samples", WATER RESEARCH, ELSEVIER, AMSTERDAM, NL, vol. 59, 1 August 2014 (2014-08-01), AMSTERDAM, NL, pages 90 - 98, XP093170905, ISSN: 0043-1354, DOI: 10.1016/j.watres.2014.04.006 *
SALIBA: "Microfluidic sorting and multimodal typing of cancer cells in self-assembled magnetic arrays", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE U.S.A., vol. 107, no. 33, 17 August 2010 (2010-08-17), pages 14524 - 14529, XP055873638, DOI: 10.1073/pnas.1001515107 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2025042729A1 (fr) * 2023-08-18 2025-02-27 Life Technologies Corporation Composition de préparation cellulaire, système et procédé de transcriptomique unicellulaire

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