Digital measurement of cell fusion events using DNA barcodes

Abstract

Disclosed are compositions and methods for estimating the number of cell fusion events occurring in liquid cultures using multiplexed oligonucleotide molecular barcodes and next generation sequencing.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 304,380, filed January 28, 2022, the contents of which are incorporated herein by reference in their entirety.

[0002] Federally funded research or development This invention was made with Government support under Contract No. 1950992 awarded by the National Science Foundation. The Government has certain rights in this invention.

[0003] FIELD OF THEINVENTION The present disclosure relates to the use of multiplexed DNA barcodes to quantitate cell fusion events in liquid culture, which can be used, for example, to improve the accuracy of high-throughput assays for identifying and measuring protein-protein interactions. [Background technology]

[0004] background Identifying and quantifying the strength of protein-protein interactions (PPIs) between protein binding partners and characterizing complex PPI networks are central goals for biomedical research and development, and high-throughput methods for characterizing PPI networks can be useful for drug discovery, protein engineering, and characterizing receptor-ligand binding kinetics, among other applications. Protein binding partners can include, for example, ligands and their receptors, antibodies and their antigens, and E3 ubiquitin ligases and their substrates, among many other examples of protein binding partners. A variety of high-throughput methods have been developed to interrogate PPI networks, including yeast two-hybrid screening, affinity purification coupled with mass spectrometry, phage and yeast surface display methods, among others. In all these methods, accurately identifying protein binding partners and quantifying binding affinities between protein binding partners while increasing throughput and reducing the cost of the assay are desirable properties that can be optimized.

[0005] Another approach based on synthetic yeast aggregation relies on reprogramming yeast sexual aggregation (naturally occurring protein-protein interactions) to link protein-protein interaction strength to mating efficiency between a-type and α-type recombinant haploid yeast cells in liquid culture (see, e.g., U.S. Pat. No. 11,136,573). For synthetic yeast aggregation-based PPI screening platforms, mating efficiency, as represented by the number of diploid yeast cells formed in turbulent liquid culture, is a surrogate property for PPI affinity. Thus, the accuracy of the PPI screening platform depends on accurately reconstructing the number of diploid yeast cells formed during the course of a liquid culture-based assay from the end-point readout. Summary of the Invention [Means for solving the problem]

[0006] overview The compositions and methods disclosed herein are based at least in part on the discovery that a multiplexed oligonucleotide molecule barcoding approach can be used to more accurately estimate the number of cell-cell fusion events in liquid culture. For example, a multiplexed barcoding approach can be used to estimate the number of diploid formation events in liquid cultures of haploid yeast cells. A multiplexed barcoding approach can also be used to estimate the number of diploid formation events in PPI screening platforms based on yeast synthetic aggregation in liquid cultures. In the case of library sequential screening of PPIs, a library of proteins of interest (POIs) or variants thereof can be screened for interaction against another library of POIs or variants thereof based on the synthetic yeast aggregation compositions and methods disclosed herein. The compositions and methods described herein provide improved accuracy in detecting diploid formation events for PPI screening platforms based on synthetic yeast aggregation.

[0007] The pairing of protein binding partners is referred to herein as a POI involving proteins expressed by a-type recombinant haploid yeast cells and α-type recombinant haploid yeast cells, respectively. A Instead of assigning a single unique oligonucleotide molecular barcode to a particular POI during construction of the POI library, the applicants use a pair of POIs in the subsequent aggregation assay. A We have discovered that each POI can be multiplexed with a sufficient number of unique oligonucleotide molecular barcodes such that a substantial majority of -POIα diploid formation events each contain a unique barcode-barcode combination.

[0008] Regardless of experimental variations introduced in subsequent steps of the PPI screening platform, AThe observed number of unique barcode-barcode combinations with any sequencing support information from the -POIα interaction is A When compared with the number of possible barcode-barcode combinations from -POIα interactions, this can be used to provide a highly accurate estimation of the number of diploid formation events that occurred during the liquid culture yeast synthetic aggregation assay.

[0009] A method for quantifying unique cell fusion events is described herein. The method includes providing a first amount of cells, each cell of the first amount of cells comprising an exogenous nucleic acid vector of a first library of exogenous nucleic acid vectors, each of the exogenous nucleic acid vectors in the first library comprising a first open reading frame (ORF) linked to an oligonucleotide molecular barcode sequence selected from a first plurality of oligonucleotide molecular barcode sequences. The method further includes providing a second amount of cells, each cell of the second amount of cells comprising an exogenous nucleic acid vector of a second library of exogenous nucleic acid vectors, each of the exogenous nucleic acid vectors in the second library comprising a second ORF linked to an oligonucleotide molecular barcode sequence selected from a second plurality of oligonucleotide molecular barcode sequences.

[0010] The method further includes combining the first amount of cells and the second amount of cells in a liquid medium to produce a culture. The method further includes growing the culture for a time and under conditions sufficient to allow a fusion event to occur between cells of the first amount of cells and cells of the second amount of cells to produce a plurality of fused cells, wherein a recombination event occurs between the first exogenous nucleic acid vector and the second exogenous nucleic acid vector in the fused cells to produce combined oligonucleotide molecular barcode sequences.

[0011] The method further includes sequencing the combined oligonucleotide molecular barcode sequences from the culture; for each pair of the first and second ORFs, determining a first number of unique pairs of first and second oligonucleotide molecular barcode sequences in the combined oligonucleotide molecular barcodes observed in the culture; for each pair of the first and second ORFs, determining a second number of possible combined oligonucleotide molecular barcode sequences; and calculating an estimated number of unique fusion events in the culture based on the first and second numbers.

[0012] In some embodiments, the first quantity of cells and the second quantity of cells are yeast cells. In some embodiments, the first quantity of cells comprises a-type haploid yeast cells, and the second quantity of cells comprises α-type haploid yeast cells. In some embodiments, the first ORF encodes a protein of interest "a" (POIa), and the second ORF encodes a protein of interest "α" (POIα).

[0013] In other embodiments, each ORF encoding POIa is operably linked to an oligonucleotide molecular barcode sequence selected from a first plurality of oligonucleotide molecular barcode sequences, and each ORF encoding POIα is operably linked to an oligonucleotide molecular barcode sequence selected from a second plurality of oligonucleotide molecular barcode sequences.

[0014] In some embodiments, each POIa is expressed on the cell surface of a first quantity of cells and each POIa is expressed on the cell surface of a second quantity of cells, In some embodiments, at least one of the first quantity of cells or the second quantity of cells is rendered incapable of mating according to any naturally occurring sexual aggregation process, such that the first quantity of recombinant haploid yeast cells and the second quantity of recombinant haploid yeast cells are incapable of mating according to any naturally occurring sexual aggregation process.

[0015] In some embodiments, each POIa and each POIα is a synthetic adhesion protein (SAP). In certain embodiments, each POIa and each POIα is either i) a fusion protein bound to a cell wall glycosylphosphatidylinositol (GPI)-anchored protein present on the surface of the first amount of recombinant haploid yeast cells or a portion of the second amount of haploid yeast cells, or ii) a glycosylphosphatidylinositol (GPI)-anchored fusion protein present on the surface of the first amount of haploid yeast cells or a portion of the second amount of haploid yeast cells.

[0016] In other embodiments, the first plurality of oligonucleotide molecular barcode sequences comprises 3 or more unique oligonucleotide molecular barcode sequences, and / or the second plurality of oligonucleotide molecular barcode sequences comprises 3 or more oligonucleotide molecular barcode sequences. In some embodiments, the first plurality of oligonucleotide molecular barcode sequences comprises 10 or more unique oligonucleotide molecular barcode sequences, and / or the second plurality of oligonucleotide molecular barcode sequences comprises 10 or more oligonucleotide molecular barcode sequences. In other embodiments, the first plurality of oligonucleotide molecular barcode sequences comprises 100 or more unique oligonucleotide molecular barcode sequences, and / or the second plurality of oligonucleotide molecular barcode sequences comprises 100 or more oligonucleotide molecular barcode sequences. In other embodiments, the first plurality of oligonucleotide molecular barcode sequences comprises 1000 or more unique oligonucleotide molecular barcode sequences, and / or the second plurality of oligonucleotide molecular barcode sequences comprises 1000 or more oligonucleotide molecular barcode sequences.

[0017] In some embodiments, the second number of possible oligonucleotide molecular barcode pairs is 7, 8, 9, 10, or more. In other embodiments, the second number of possible oligonucleotide molecular barcode pairs is 100 or more. In other embodiments, the second number of possible oligonucleotide molecular barcode pairs is 10,000 or more.

[0018] In other embodiments, the POI a The library has more than 10 POIs a and / or the library of POIα comprises 10 or more POIα. a The library has over 100 POIs a and / or the library of POIα comprises 100 or more POIα. a The library contains over 1000 POIs a and / or the library of POIα comprises 1000 or more POIα. a The library contains over 10,000 POIs a and / or the library of POIα comprises more than 10,000 POIα.

[0019] In some embodiments, the first exogenous nucleic acid vector and the second exogenous nucleic acid vector each further comprise a unique primer binding site, a recombination site, and a selectable marker. In some embodiments, each cell of the first amount of cells and each cell of the second amount of cells further comprises an exogenous recombinase. In some embodiments, the exogenous recombinase mediates the recombination event.

[0020] In some embodiments, a portion of the first oligonucleotide molecular barcode sequence and a portion of the second oligonucleotide molecular barcode sequence are sequenced to obtain a plurality of sequencing reads, each sequencing read including a portion of the first oligonucleotide molecular barcode sequence and a portion of the second oligonucleotide molecular barcode sequence.

[0021] In some embodiments, i) each cell of the first amount of cells lacks either a functional Aga1 or a functional Aga2 protein, and / or ii) each cell of the second amount of cells lacks a functional Sag1 protein.

[0022] The term "complementary nucleotides," as used herein, refers to Watson-Crick base pairing between nucleotides, particularly nucleotides that are hydrogen bonded to each other, such as a thymine or uracil residue linked to an adenine residue by two hydrogen bonds, and a cytosine and a guanine residue linked by three hydrogen bonds. Generally, nucleic acids include nucleotide sequences that are described as having a "percent complementarity" to a particular second nucleotide sequence. For example, a nucleotide sequence may have 80%, 90%, or 100% complementarity to a particular second nucleotide sequence, indicating that 8 of 10, 9 of 10, or 10 of 10 nucleotides of the sequence are complementary to the particular second nucleotide sequence. For example, the nucleotide sequence 3'-TCGA-5' is 100% complementary to the nucleotide sequence 5'-AGCT-3', which in turn is 100% complementary to the region of the nucleotide sequence 5'-TTAGCTGG-3'.

[0023] The terms "homology", "identity" or "similarity" as used herein with respect to sequences refer to sequence similarity between two strands of amino acids, e.g., peptides or proteins, or nucleotides or bases, e.g., strands of nucleic acid molecules. The terms "homologous region" or "homology arm" refer to a region on the donor DNA that has a degree of homology with the target genomic DNA sequence. Homology can be determined by comparing positions in each sequence that are aligned for comparison purposes. When a position in the compared sequences is occupied by the same base or amino acid, the molecules are homologous at that position. The degree of homology between sequences depends on the number of matching or homologous positions shared between the sequences.

[0024] "Operably linked" as used herein refers to the arrangement of elements, such as barcode sequences, gene expression cassettes, coding sequences, promoters, enhancers, transcription factor binding sites, such that the components so described are configured to perform their normal functions. Thus, a control sequence operably linked to a coding sequence has the ability to effect transcription, and in some cases translation, of the coding sequence. A control sequence need not be contiguous with a coding sequence, so long as it functions to direct the expression of the coding sequence. Thus, for example, an intermediate sequence that is transcribed but not yet translated can exist between the promoter sequence and the coding sequence, and the promoter sequence can still be considered to be operably linked to the coding sequence. Indeed, such sequences need not be present on the same continuous DNA molecule (i.e., chromosome), and can still have an interaction that causes a change in control.

[0025] As used herein, the term "selectable marker" refers to a gene that is introduced into a cell and confers a characteristic suitable for artificial selection. The general use of selectable markers is well known to those skilled in the art. Drug selectable markers such as ampicillin / carbenicillin, kanamycin, chloramphenicol, erythromycin, tetracycline, gentamicin, bleomycin, streptomycin, puromycin, hygromycin, blasticidin, and G418 may be employed. The selectable marker may be an auxotrophic selectable marker, where the selected cell line carries a mutation that renders it unable to synthesize an essential nutrient. Such a strain will only grow if the missing essential nutrient is provided in the growth medium. For example, essential amino acid auxotrophic selection of yeast mutant strains is common and well known in the art. "Selective medium," as used herein, refers to cell growth medium to which a chemical or biological moiety has been added that selects for or excludes a selectable marker, or a medium that is deficient in an essential nutrient and excludes auxotrophic strains.

[0026] As used herein, the term "vector" refers to any of a variety of nucleic acids that contain a desired sequence or sequences to be delivered to and / or expressed in a cell. Although vectors are generally composed of DNA, RNA vectors are also available. Vectors include, but are not limited to, plasmids, fosmids, phagemids, viral genomes, bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC), P1-derived artificial chromosomes (PAC), and synthetic chromosomes, among others.

[0027] As used herein, "affinity" is the strength of the binding interaction between a biomolecule and its ligand, or binding partner. Affinity is determined by the equilibrium dissociation constant, K D It is usually measured and described using the K D The lower the value, the higher the binding affinity. Affinity can be influenced by hydrogen bonding, electrostatic interactions, hydrophobic and van der Waals forces between the binding partners, or by the presence of other molecules, e.g., binding agonists or antagonists.

[0028] In some implementations, affinity may be described using arbitrary units, where a particular binding affinity in an assay, e.g., between two wild-type protein binding partners, or a wild-type species of a first protein binding partner and a wild-type species of a second protein binding partner, is set to 1.0 arbitrary units, and the binding affinity for another pair of protein binding partners, e.g., a mutant species of the first protein binding partner and a mutant species of the second protein binding partner, is measured relative to the particular binding affinity.

[0029] As used herein, "site-saturation mutagenesis" (SSM) refers to a mutagenesis technique used in protein engineering and molecular biology in which a codon or set of codons is substituted with most or all possible amino acids at a position within a polypeptide. SSM can be performed for one codon, several codons, or for every position within a polypeptide. Substitutions can be made for all possible alternative amino acids or omitted for selected amino acids. For example, substitutions for cysteine ​​are often omitted due to deleterious effects on yeast surface expression and protein folding. The result is a library of mutant proteins representing multiple single residue amino acid substitutions at one, several, or all amino acid positions within a polypeptide.

[0030] As used herein, "user-directed mutagenesis" refers to any process in which a user modifies the amino acid sequence of a polypeptide encoded by a polynucleotide (nucleic acid molecule) by modifying the polynucleotide sequence. A polypeptide sequence can be modified by user-directed mutagenesis of a polynucleotide sequence encoding the polypeptide. A polypeptide can be modified in a defined manner at one or more amino acid residues, e.g., an alanine residue can be changed to an arginine residue, or the polypeptide can be modified in a randomized manner, i.e., by modifying the polynucleotide sequence encoding the polypeptide using degenerate primers and randomized PCR amplification. A polypeptide can be modified by user-directed mutagenesis at one amino acid residue, or at many amino acid residues. A polypeptide can be modified by user-directed mutagenesis such that an amino acid residue at a given position is modified to one of a subset of possible amino acid substitutions at that position, e.g., by conservative amino acid substitutions as known in the art, or by substitutions for all possible amino acids except cysteine. A polypeptide may be modified by user-directed mutagenesis of a polynucleotide sequence encoding the polypeptide to include an insertion and / or deletion of one or more amino acid residues, or the polypeptide sequence may be truncated by user-directed mutagenesis. A polypeptide may be modified by user-directed mutagenesis to include an insertion or substitution with a natural or unnatural amino acid.

[0031] As used herein, the term "protein of interest" ("POI") refers to a polypeptide molecule whose biochemical properties are the subject of investigation by the compositions and methods disclosed herein. The POI can be a full-length protein, a truncated protein, a fusion protein, or a functionally tagged protein, among other protein species and variants. In some implementations, a library of a first POI or variants thereof is screened for binding affinity to a library of a second POI or variants thereof. In some implementations, the first POI is expressed by a-type haploid yeast cells and is referred to as "POI A " and a second POI is expressed by an α-type haploid yeast cell and may be referred to as "POIα." In some embodiments, the interaction is mediated by the POI α-type haploid yeast cell using the compositions and methods disclosed herein. A and POIα, the first POI and the second POI are called “POI A The pair may be referred to as the "POIα-POI pair".

[0032] As used herein, "protein-protein interaction" ("PPI") refers to a highly specific physical contact established between two or more proteins as a result of biochemical events driven, for example, by electrostatic forces, including hydrophobic effects. Many protein-protein interactions are physical contacts between the surfaces of the proteins, involving molecular association between specific domains of the proteins that occur within a cell or organism in the context of a particular biomolecule. In some implementations, the protein-protein interaction is strong enough to replace the function of a native sexual aggregation protein. For example, it is possible to correlate the mating efficiency with the interaction strength of a particular protein-protein interaction. In certain embodiments, the assay can characterize or determine protein-protein interactions between synthetic adhesion proteins. In certain embodiments, the protein-protein interaction is modulated, enhanced or inhibited by a third chemical entity, which may be a small molecule, a polypeptide, or a polynucleotide, among others.

[0033] As used herein, "synthetic adhesion protein" ("SAP") refers to any protein or polypeptide that is assayed for binding to or interaction with any other protein or polypeptide. The protein may be expressed heterologously or exogenously. Synthetic adhesion proteins are so named because they are generally not associated with the adhesion required for aggregation as seen in wild-type sexual aggregation proteins.

[0034] As used herein, "mediate" means to facilitate or catalyze a process, for example, recombinases can mediate recombination between double-stranded or single-stranded polynucleotides. As another example, sexual aggregation proteins expressed on the surface of yeast cells can mediate aggregation and subsequent cell fusion between haploid yeast cells with opposite mating types.

[0035] The compositions and methods disclosed herein offer several advantages. For a PPI screening platform based on yeast synthetic aggregation in liquid culture, the key event to be detected is the POI expressed on the surface of a-type recombinant haploid yeast cells. A The formation of diploid yeast cells is mediated by the interaction of POIα expressed on the surface of recombinant α-type haploid yeast cells with the POIα. The number of diploid formation events, i.e. the mating efficiency between a-type haploids and α-type haploids, is determined by the interaction of POIα with the POIα expressed on the surface of α-type recombinant haploid yeast cells. A In fact, the mating efficiency and POI A -POIα is K D(See Younger et al., “High-throughput characterization of protein-protein interactions by reprogramming yeast mating,” PNAS USA, 14;114(46):12166-12171 (2017)). However, after the diploid formation event occurs over time in liquid culture, several subsequent processes contribute stochastic or systematic variation to the final quantitative output and may be important for determining the relative abundance of a given POI. A The quantitative accuracy of affinity estimation for the -POIα pair is compromised. For example, expression of some proteins in yeast cells may cause more metabolic burden than other proteins, causing diploid yeast cells expressing such proteins to grow slower than diploid yeast cells expressing other proteins. Stochastic or systematic differences in diploid yeast cells contribute to variability in quantifying the number of fusion events in an assay.

[0036] Sources of stochastic or systematic variation can include (1) the time it takes for cell fusion to occur over the course of a long assay period of >90 minutes, (2) differences in the growth rates of diploid yeast cells in liquid culture over the course of an assay period of >90 minutes, (3) amplification bias or stochastic variation in amplification rates during PCR amplification of unique recombinant barcode-barcode pairs, and / or (4) preparation of next generation sequencing (NGS) libraries of PCR amplified barcode-barcode pairs. The output of such a process provides NGS sequencing reads for a given barcode-barcode pair, the abundance of which can be determined by the corresponding POI. A This quantitative readout is, however, prone to the above-mentioned sources of variability.

[0037] The compositions and methods disclosed herein, i.e., utilizing multiple unique oligonucleotide molecular barcodes per POI rather than a single barcode per POI, eliminate the sources of stochastic and systematic variation discussed above and provide a method for determining the relative abundance of a measured POI. A This substantially improves the quantitative accuracy of the estimation of PPI affinity for the POIα-POIα interaction. The result is, in effect, a "digital" read, such that detection of a unique barcode-barcode sequence in the platform's NGS reads represents a unique diploid formation event, regardless of the abundance of the sequencing read corresponding to that unique barcode-barcode combination. A -POIα pairs and associated sequencing read abundance, but not POI A The number of unique barcode-barcode sequences detected for a POIα-POIα pair represents the number of diploid formation events that occurred during the assay, and the .... A -used to infer PPI affinity for POIα interactions.

[0038] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which such compositions and methods belong. Although compositions and methods similar or equivalent to those described herein can be used in practicing or testing the compositions and methods disclosed herein, suitable compositions and methods are described below. All publications, patent applications, patents, and other references described herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting.

[0039] Unless the context clearly requires otherwise, throughout the specification and claims, the words "comprise," "comprising," and the like are to be construed as having an inclusive sense, i.e., "including but not limited to," as opposed to an exclusive or exhaustive sense. Words using the singular or plural also include the plural and singular, respectively, unless expressly limited. Furthermore, the words "herein," "above," and "below," and words of similar import, when used in this application, shall refer to this application as a whole and not to any particular portions of this application.

[0040] Although the disclosure has been specifically shown and described herein with reference to various alternative embodiments, it will be understood by those skilled in the art that various changes in form and details can be made herein without departing from the spirit and scope of the invention. The description of the embodiments of the present disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. Specific embodiments of, and examples thereof, of the present disclosure are described herein for illustrative purposes, but various equivalent modifications are possible within the scope of the present disclosure, as those skilled in the art will recognize.

[0041] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims. [Brief description of the drawings]

[0042] [Figure 1] FIG. 1 shows a schematic of natural and synthetic yeast aggregation in S. cerevisiae. [Figure 2A] FIG. 1 shows a schematic of recombination between SAP expression cassettes mediated by exogenous Cre recombinase. [Figure 2B] FIG. 1 shows a more detailed outline of the recombination between SAP expression cassettes mediated by exogenous Cre recombinase. [Figure 2C]FIG. 1 shows a schematic of exogenous Cre recombinase-mediated recombination between SAP expression cassettes, showing the PCR amplification of unique barcode-barcode pairs resulting from the diploid formation event and subsequent recombination of the SAP expression cassettes. [Diagram 3] FIG. 1 shows a schematic of a yeast synthetic agglutination assay for POIA-POIα, where each POI is linked to a single oligonucleotide barcode species. [Figure 4] FIG. 1 shows a schematic of a yeast synthetic agglutination assay for POIA-POIα, where each POI is linked to multiple oligonucleotide molecular barcode species. [Figure 5A] FIG. 1 shows a schematic of a portion of a nucleic acid construct in which an ORF encoding a POI is synthesized with multiple oligonucleotide molecular barcode sequences, with each ORF linked to a different unique oligonucleotide molecular barcode sequence. [Figure 5B] FIG. 1 shows a schematic of portions of a nucleic acid construct in which a library of oligonucleotide molecular barcode sequences are individually synthesized and assembled with an ORF encoding a POI by isothermal in vitro assembly, resulting in a plurality of nucleic acid constructs, each nucleic acid construct comprising an ORF encoding a POI, with each ORF linked to a different unique oligonucleotide molecular barcode sequence. [Figure 6] FIG. 1 shows a histogram plot of the frequency of "potential" and "observed" barcode-barcode combinations for the POIA-POIα pair. [Figure 7] FIG. 1 shows a histogram plot of the distribution of sequencing reads for the POIA-POIα pair, where 10 diploid yeast cells were formed during the synthetic aggregation assay. [Figure 8] FIG. 13 shows a graph of the distribution of predicted diploids for a POIA-POIα pair with 10 predicted diploid formation events during a synthetic aggregation assay compared to the Poisson distribution of expected values. [Figure 9]FIG. 1 shows plots comparing confidence interval calibrations performed with and without multiplexed barcoding across POIA-POIα networks of various sizes. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0043] Like reference symbols in the drawings represent like elements.

[0044] Detailed Description Synthetic yeast aggregation using multiplexed barcodes The present disclosure provides a method for highly accurate estimation of PPI affinity by improving the accuracy of sequencing read depth as a proxy for protein-protein interaction PPI strength by replacing read depth with an estimate of the number of diploids formed. Synthetic yeast aggregation is based on reprogramming yeast sexual aggregation (naturally occurring protein-protein interaction) to relate protein-protein interaction strength to mating efficiency between a-type recombinant haploid yeast cells and α-type recombinant haploid yeast cells in liquid culture. For synthetic yeast aggregation-based PPI screening platform, mating efficiency (represented by the number of diploid yeast cells formed in turbulent liquid culture) is a surrogate for PPI affinity. Thus, the accuracy of the PPI screening platform depends on accurately reconstructing the number of diploid yeast cells formed during the course of the liquid culture-based assay from the endpoint reads. The compositions and methods disclosed herein provide a significant increase in accuracy in detecting diploid formation events for synthetic yeast aggregation-based PPI screening platform.

[0045] As discussed in more detail below, for each POI in the library of POIs, multiple unique oligonucleotide molecular barcodes are assigned to a single open reading frame (ORF) encoding the POI in the library of POIs. Even in the case of strong PPIs where many diploid formation events are predicted, a sufficient number of unique barcodes are assigned to each POI so that the number of possible barcode-barcode combinations substantially exceeds the expected number of diploids formed in a given assay. A substantial majority of diploid formation events form unique barcode combinations that can be identified by sequencing. A single POI A Rather than quantifying diploid formation based on the sequencing read depth of a single barcode-barcode combination representing a pair of POIα, the method presented herein A The number of unique barcode combinations observed is quantified to represent the number of diploids formed for the -POIα pair. This quantity is minimally affected by yeast cell growth conditions, PCR amplification, or NGS library preparation, and therefore provides a better estimate of diploid formation events than could be derived from sequencing read depth alone.

[0046] For example, when a diploid formation event occurs in liquid culture at hour 7 of a 16-hour synthetic aggregation assay, the resulting barcode combinations are limited to and equivalently quantified as barcodes resulting from diploid formation events occurring at hour 1, despite the fact that the sequencing reads of barcodes at hour 1 may be significantly outnumbered by those at hour 7. Given that the doubling rate of yeast haploid and diploid cells is approximately 90 minutes, it is expected that in the 6 hours between hours 1 and 7, the diploid cells formed by the fusion event at hour 1 will undergo four doublings, resulting in 2^4 cells or 16 cells. Without the multiplexed barcoding method disclosed herein, the diploid formation events at hour 1 would be counted approximately 16 times more than the diploid formation events at hour 7. The multiplexed barcoding methods disclosed herein provide a more accurate estimate of the number of fusion events by controlling for this source of variability and counting fusion events by the presence or absence of unique barcode-barcode pairs formed in cell-cell fusion events.

[0047] In prior methods, such as those disclosed in U.S. Patents 10,988,759 and 11,136,573, each POI is assigned a unique oligonucleotide molecular barcode, and following a diploid formation event, these protein-specific barcodes are recombined and sequenced to identify the individual synthetic adhesion proteins (SAPs) that mediated the corresponding diploid formation event. Quantifying the sequencing reads of unique barcode-barcode combinations serves as a surrogate indicator of the number of diploid formation events, and thus PPI affinity.

[0048] Replacement of native aggregated proteins with multiple barcoded POIs FIG. 1 shows a schematic of natural and synthetic sexual aggregation in S. cerevisiae. On the left, MATa and MATα haploids are shown at the top and bottom, respectively. The cell walls of each haploid cell are shown in grey. In turbulent liquid culture, MATa and MATα haploid cells stick to each other due to the binding sexual agglutinin proteins, allowing mating. The natural sexual agglutinin proteins are composed of Aga1 and Aga2 expressed by MATa cells and Sag1 expressed by MATα cells. Aga1 and Sag1 form glycosylphosphatidylinositol (GPI) anchors with the cell wall and extend to the outside of the cell wall with a glycosylated stalk (see the left frame in the inset). Aga2 is secreted by MATa cells and forms a disulfide bond with Aga1. The interaction between Aga2 and Sag1 is essential for wild-type sexual aggregation. The native sexual agglutinin interaction can be replaced by an engineered one by expressing Aga1 in both mating types and fusing a complementary binder to Aga2 (see center frame of inset). Instead of direct agglutination, it may be possible to express binders to multivalent targets such that agglutination and mating occurs only in the presence of the target (see right frame of inset).

[0049] Figure 2A shows an outline of the Cre recombinase transposition scheme for high-throughput analysis of display pair interactions. Here, a mating between a single recombinant MATa yeast strain and a single recombinant MATα yeast strain is shown. However, for batch mating assays, a library of displayer cells of each mating type is used (each containing a library of SAPs fused to Aga2). Each MATa and MATα haploid cell contains SAPs fused to Aga2 integrated into the target chromosome (e.g., chromosome III). Upon mating, both copies of the target chromosome are present in the same diploid cell.

[0050] In addition to the SAP / Aga2 cassette, each copy of the target chromosome has a unique primer binding site, one of multiple unique oligonucleotide barcodes operably linked to a particular SAP, and a lox recombination site. Multiple oligonucleotide barcodes can be synthesized and assembled using a library of SAP expression cassettes, such that a single SAP species is operably linked to multiple unique oligonucleotide barcodes. When Cre recombinase is expressed, a chromosomal translocation occurs at the lox site, causing juxtaposition of the primer binding site and barcodes on the same copy of the target chromosome. PCR is then performed to amplify the region of the chromosome containing the barcodes from both SAPs, such that sequences containing unique barcode-barcode pairs (each representing a diploid formation event) are amplified.

[0051] In batch mating, the result is a pool of fragments each containing a unique barcode-barcode pair associated with the two SAPs involved in a single diploid formation event. Paired-end next-generation sequencing is then used to match the barcodes and determine the number of diploid formation events mediated by that SAP pair.

[0052] FIG. 2B shows another schematic of the Cre recombinase transposition scheme for high-throughput analysis of display pair interactions. To eliminate native aggregation, the a-agglutinin, Sag1, is knocked out in MATa cells. MATa and MATalpha cells can synthesize lysine or leucine, respectively. Diploids can then be selected in medium lacking both amino acids. MATa cells express the βE-inducible transcription factor ZEV4, which activates Cre recombinase expression in diploid cells. MATa and MATalpha cells express mCherry and mTurquoise, respectively, for strain type identification using flow cytometry. MATa and MATalpha cells constitutively express Aga1 together with a uniquely barcoded SAP fused to Aga2. When Cre recombinase expression is induced in diploids with βE, a chromosomal translocation at the lox site results in the integration of both SAP-Aga2 fusion expression cassettes on the same chromosome. A single fragment containing the unique barcode-barcode sequence associated with the diploid formation event is then identified as P f and P r (primers specific to primers derived from the first and second nucleic acid constructs integrated into the genomic target site) are amplified by PCR and sequenced to quantify the number of diploid formation events and identify the interacting SAP pairs.

[0053] Figure 2C shows an outline of a CRE recombinase transposition scheme for high-throughput analysis of interactions between SAPs derived from library sequential screening. When CRE recombinase expression is induced in diploids with βE, both SAP-Aga2 expression cassettes are integrated on the same chromosome by chromosomal translocation at the lox sites. A single fragment containing the unique barcode-barcode sequence associated with the diploid formation event is then amplified by PCR using primers annealing to the primer binding sites from each of the first and second nucleic acid constructs, and sequenced (e.g., using paired-end analysis of next-generation sequencing) to quantify the number of diploid formation events and identify the pairs of interacting SAPs.

[0054] FIG. 3 illustrates a method for identifying POIs that do not use multiplexed barcoding, i.e., each POI is linked to a single oligonucleotide barcode species. A 3 is a schematic of a yeast synthetic aggregation assay for a -POIα pair. Yeast cell population 300 is a population of a-type recombinant haploid yeast cells containing a first library of proteins of interest or mutant variants thereof. In FIG. 3, for illustrative purposes, one POI species is represented by a single-sided arrow. In this assay, many of the individual cells may each contain the same species of POI linked to the same molecular barcode. Yeast cell population 302 is a population of α-type recombinant haploid yeast cells containing a second library of proteins of interest or mutant variants thereof. Yeast cell population 300 and population 302 are combined in liquid culture according to the methods discussed above, interactions between SAPs promote mating between haploid cells to produce diploid yeast cell population 304, and recombination between SAP expression cassettes results in barcode-barcode combinations, represented as double-sided arrows in FIG. 3.

[0055] DNA isolation, PCR amplification, and next-generation sequencing yielded 306 sequencing reads, the abundance of which was consistent with that of the POI. AThe information available to estimate the strength of the interaction is A - the total number of sequencing reads observed for the POIα pair.

[0056] As an example of the new methods and compositions described herein, FIG. 4 shows a POI using multiplexed barcoding, i.e., each POI is linked to multiple unique oligonucleotide barcode species. A 4 is a schematic of an example of a yeast synthetic aggregation assay for a -POIα pair. Yeast cell population 400 is a population of a-type recombinant haploid yeast cells containing a first library of proteins of interest or mutant variants thereof. In FIG. 4, one POI species is represented by a single-sided arrow. In this assay, many of the individual cells may each contain the same POI species, but each cell containing the POI species should have a unique molecular barcode linked to the POI. Yeast cell population 402 is a population of α-type recombinant haploid yeast cells containing a second library of proteins of interest or mutant variants thereof. Yeast cell population 400 and population 402 are combined in liquid culture according to the methods discussed above, interactions between SAPs promote mating between haploid cells to produce diploid yeast cell population 404, and recombination between SAP expression cassettes results in barcode-barcode combinations, represented as double-sided arrows in FIG. 4.

[0057] As a result of multiplexed barcoding as described herein, each cell in the diploid yeast cell population 404 contains a unique barcode-barcode combination. DNA isolation, PCR amplification, and next generation sequencing yield sequencing reads 406, and the number of unique barcode-barcode combinations detected represents the number of diploid formation events that occurred during the assay. AThe binding affinity of the -POIα pair can be accurately inferred from the number of unique barcode-barcode combinations detected. It is important to point out that, as shown in Figure 4, due to variability in assay conditions (i.e., yeast growth rate, PCR amplification, NGS library preparation), each unique barcode-barcode combination may be detected by a different number of sequencing reads.

[0058] However, the useful data in this method is not the abundance of sequencing reads detected for each barcode-barcode combination, but rather the number of unique barcode-barcode combination species detected. A The information available to infer the strength of the -POIα interaction is the total number of unique barcode-barcode combinations detected (representing the number of diploid formation events). Rather than attempting to quantify diploid formation events based on the total number of sequencing reads and sequencing reads 306 as shown in FIG. 3, the number of unique barcode-barcode combinations with some sequencing evidence is quantified as shown in FIG. 4 and sequencing reads 406. This number is used to directly infer the number of diploid yeast cells formed during the aggregation assay, without taking into account the variation introduced during the assay period.

[0059] Quantifying the number of original diploid formation events as a surrogate property of diploid formation, based on quantification of unique barcode-barcode sequences, can be used as a basis to improve and more precisely quantify the uncertainty in estimating PPI affinities from sequencing data.

[0060] Constructs and barcodes As used herein, the term "nucleic acid construct" refers to a contiguous polynucleotide or DNA molecule that can be integrated into a yeast strain. In some implementations, the nucleic acid construct comprises (a) a homology arm located at the 5' end of the nucleic acid construct, (b) a first expression cassette comprising a gene encoding a synthetic adhesion protein (SAP) that binds to a cell wall glycosylphosphatidylinositol (GPI)-anchored protein, (c) a second expression cassette comprising a first marker, (d) a unique primer binding site, (e) an oligonucleotide molecular barcode, (f) a recombination site, and (g) a homology arm located at the 3' end of the nucleic acid construct. In some introduction forms, components (a) to (g) of the nucleic acid construct are arranged in a 5' to 3' direction on the nucleic acid construct, in which case component (a) is on the 5' side of component (b), which is on the 5' side of component (c), which is on the 5' side of component (d), which is on the 5' side of component (e), which is on the 5' side of component (f), which is on the 5' side of component (g), and component (g) is located at the 3' end of the nucleic acid construct.

[0061] In some implementations, a nucleic acid construct comprising a first expression cassette encoding a synthetic adhesion protein (SAP) may be integrated into the genome of a yeast cell at a user-defined genomic target site. In other implementations, the nucleic acid construct comprising a first expression cassette encoding a SAP may be, for example, a 2 micron plasmid or a centromeric plasmid that does not integrate into the yeast genome.

[0062] As used herein, the term "expression cassette" refers to a DNA sequence comprising a promoter, an open reading frame, and a terminator. In certain embodiments, a nucleic acid construct comprises one or more expression cassettes. For example, a nucleic acid construct may comprise one, two, three, or more expression cassettes. In certain embodiments, a nucleic acid construct comprises a first expression cassette comprising a fusion gene encoding a first SAP bound to a first cell wall GPI-anchored protein, and a second expression cassette comprising a first marker. In some embodiments, the SAP of the first expression cassette of the first nucleic acid construct is fused to a sexual aggregation protein Aga2, and the SAP of the first expression cassette of the second nucleic acid construct is fused to a sexual aggregation protein Aga2, as shown in Figures 1 and 2A-2C.

[0063] In some modes of introduction, the nucleic acid construct contains recombination sites that allow for certain site-specific recombination events when the nucleic acid construct integrates into a genomic target region and mating occurs. In other modes of introduction, the nucleic acid construct does not integrate into the yeast genome and site-specific recombination events occur between extrachromosomal nucleic acid constructs, such as 2 micron plasmids or centromeric plasmids.

[0064] In some implementations, the recombination sites are located near the barcoded SAP expression cassette and are configured such that recombination causes a chromosomal translocation that places two barcodes, derived from the first and second nucleic acid constructs, respectively, previously integrated on the same chromosome of the first and second yeast strains, on the same chromosome of the diploid yeast cell. In some implementations, the recombination sites of the first and second nucleic acid constructs are designed such that recombination does not disrupt the chromosome or cause cell killing. The site-specific recombination events at the recombination sites are controlled by a site-specific recombinase that catalyzes and mediates the site-specific recombination events between the two DNA recombination sites.

[0065] In some introduction forms, one or both of the yeast strains contain an exogenous recombinase. The recombinase is expressed only in the diploid cells after mating. For example, the second recombinant yeast strain can express a transcription factor and the first recombinant yeast strain contains an exogenous recombinase, or the first recombinant yeast strain can express a transcription factor and the second recombinant yeast strain contains an exogenous recombinase. It is also possible for both strains to contain an exogenous recombinase and a transcription factor. When expressed in the diploid cells, the recombinase mediates recombination between site-specific Cre recombination sites.

[0066] In some embodiments, only one of the multiple strains contains an inducible promoter that controls the expression of the exogenous recombinase. To express the recombinase only in the diploid cells after mating, an inducible transcription factor (e.g., Zev4) is controllably induced (i.e., Zev4 is activated with β-estradiol, which allows Zev4 to enter the nucleus and then activate promoter pZ4 there), and activates transcription from its promoter located upstream of the recombinase. Thus, adding an inducer (i.e., beta-estradiol) to the cells after mating activates the expression of the exogenous recombinase and causes a chromosomal translocation in the diploids that pair together the two barcodes.

[0067] In some embodiments, each nucleic acid construct comprises a unique primer binding site. The unique primer binding site is designed to allow amplification using a set of primers that amplifies only the target nucleic acid fragments that contain two unique barcodes from properly recombined diploid cells. The pool of target nucleic acid fragments is then sequenced, for example, using next-generation sequencing.

[0068] As used herein, a primer or pair of primers refers to a pair of natural or synthetic oligonucleotides (i.e., forward and reverse primers) that, when double-stranded with a polynucleotide template, act as an initiation point for nucleic acid synthesis and have the ability to extend from their 3' ends along the template so that a target nucleic acid fragment is formed. In another mode of introduction, the unique primer binding site of the first nucleic acid construct and the unique primer binding site of the second nucleic acid construct are integrated into the same chromosome, and after mating and chromosomal translocation, the primer binding sites are usable to amplify a target nucleotide sequence that includes both the unique barcode of the first nucleic acid construct and the unique barcode of the second nucleic acid construct, or both a portion of the unique barcode of the first nucleic acid construct and a portion of the unique barcode of the second nucleic acid construct.

[0069] In one embodiment, the unique barcode of the first nucleic acid construct and the unique barcode of the second nucleic acid construct are integrated into the same chromosomal locus, and after mating and chromosomal translocation, they are within about 5,000, 4,000, 3,000, 2,000, 1,000, 900, 800, 700, 600, 500, 400, 300, 200, or 100 base pairs. In some embodiments, using next generation sequencing, paired-end reading is used to read the barcodes at either end of the target nucleic acid fragment.

[0070] In another mode of introduction, recombination occurs in the diploid cell after mating between a first extrachromosomal nucleic acid construct encoding a first SAP coupled to a first oligonucleotide molecular barcode and a second extrachromosomal nucleic acid construct encoding a second SAP coupled to a second oligonucleotide molecular barcode, such that the unique primer binding site of the first nucleic acid construct and the unique primer binding site of the second nucleic acid construct are present on the same molecule, and the primer binding sites can be used to amplify a target nucleotide sequence that includes both the unique barcode of the first nucleic acid construct and the unique barcode of the second nucleic acid construct, or both a portion of the unique barcode of the first nucleic acid construct and a portion of the unique barcode of the second nucleic acid construct.

[0071] In the new method, each nucleic acid construct comprises an oligonucleotide molecular barcode. In some implementations, each barcode is specific to a particular SAP that comprises a particular POI. In other implementations, multiple unique barcode sequences are associated with a particular SAP that comprises a particular POI. Within multiple nucleic acid constructs encoding a single POI, each construct may comprise a unique oligonucleotide molecular barcode, such that a single POI is associated with multiple unique oligonucleotide molecular barcodes.

[0072] Oligonucleotide molecular barcodes used in the compositions and methods disclosed herein can be, for example, about 5 to 40 nucleotides in length; about 10 to 35 nucleotides in length; about 15 to 30 nucleotides in length; about 20 to 25 nucleotides in length. In some implementations, the oligonucleotide molecular barcodes are 10, 15, 20, 25, or 30 nucleotides in length.

[0073] In some embodiments, the barcode is not specifically selected. Rather, it is added with a degenerate primer that contains a region containing random base pairs (e.g., in SAP library sequential screening). In some implementations, the oligonucleotide molecular barcodes are synthesized as a degenerate library by nucleic acid synthesis methods well known in the art, and combined with a library of constructs encoding a library of POIs by nucleic acid assembly methods, such as isothermal in vitro recombination methods.

[0074] FIG. 5A is a schematic diagram of several portions of a nucleic acid construct in which an ORF encoding a POI is synthesized with multiple oligonucleotide molecular barcode sequences, with each ORF linked to a different unique oligonucleotide molecular barcode sequence. A 5A depicts a plurality of nucleic acid constructs including an ORF 500 encoding a unique oligonucleotide molecular barcode sequence 504, each construct including a unique oligonucleotide molecular barcode sequence 504. The sequence diversity of the oligonucleotide molecular barcode sequences 504 in the different nucleic acid constructs is represented by the various patterns shown in the schematic diagram of FIG. 5A. Primer binding sites 502 are used to amplify the combined unique barcode-barcode sequences following a cell fusion event as described above. In some implementations, the ORF 500, primer binding sites 502, and oligonucleotide molecular barcode sequences 504 may be synthesized by one of several DNA synthesis methods known in the art.

[0075] FIG. 5B is a schematic diagram of several portions of a nucleic acid construct, in which a library of oligonucleotide molecular barcode sequences are individually synthesized and assembled with an ORF encoding a POI by an isothermal in vitro assembly method, resulting in multiple nucleic acid constructs, each of which contains an ORF encoding a POI, with each ORF linked to a different unique oligonucleotide molecular barcode sequence. A5B represents a plurality of nucleic acid constructs including ORF 506 encoding a POIA, and a primer binding site 508 that is used to amplify the combined unique barcode-barcode sequence following a cell fusion event, as described above. The library of oligonucleotide molecular barcode sequences 510 can be individually synthesized by one of several DNA synthesis methods known in the art. The sequence diversity of the oligonucleotide molecular barcode sequences 510 is represented by the various patterns shown in the schematic diagram of FIG. 5B. The resulting library of diverse oligonucleotide molecular barcode sequences can be combined with ORF 506 and primer binding site 508 by an isothermal in vitro assembly method, such that a single POIA encoded by ORF 506 links to a diverse plurality of oligonucleotide molecular barcode sequences 510.

[0076] In the new method, for a given POI A -For a pair of POIα, A The number of observed unique barcode-barcode combinations detected by downstream sequencing compared to the total number of possible barcode-barcode combinations for the POIα pair is A and is used to estimate the number of diploid formation events mediated by SAP involving POIα.

[0077] Protein of interest In some implementations, the compositions and methods include a first protein of interest (POI) and a library of a second POI. The library of the second POI may include a plurality of user-specified or randomly added mutants of the POI and a wild-type protein. The library of the second POI may include a plurality of protein species encoded by a plurality of genes, for example human genes. In other implementations, the methods include a library of a first POI and a library of a second POI. The plurality of user-specified or randomly added mutants of the first POI or the second POI may include variants of the POI with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid substitutions. The amino acid substitutions may be selected to introduce charge changes and / or conformational changes to the POI, and wild-type amino acids may be replaced with natural or unnatural amino acids.

[0078] In some implementations, amino acid substitutions can be generated by performing site saturation mutagenesis (SSM) to generate an SSM library of POI variants. In some implementations, the library of the first POI or the second POI can be generated by alanine scanning. In some implementations, the library of the first POI or the second POI can be generated by random mutagenesis, such as using error-prone PCR or another method of introducing changes into the amino acid sequence of the expressed protein. The library of the first POI and the library of the second POI or the library of the first POI and the library of the second POI are assayed for binding affinity according to the methods disclosed herein, such that affinity is measured individually in a pairwise parallelized high-throughput manner for interactions between the first POI and each of the multiple second POIs, or between each of the multiple first POIs and each of the multiple second POIs.

[0079] The first POI library or the second POI library may include a plurality of user-specified or randomly added mutants of the POI and a wild-type POI. The plurality of user-specified or randomly added mutants of the POI may include variants of the target protein having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid substitutions. The amino acid substitutions may be selected to introduce a charge change in the POI or a conformational change in the POI, and the wild-type amino acid may be replaced with a natural or unnatural amino acid.

[0080] In some implementations where a library of a first POI is assayed for binding affinity relative to a library of a second POI, the assay can be a yeast two-hybrid system, synthetic yeast agglutination in liquid culture, or another parallel high-throughput sequential library screening method. The binding affinity of the interaction between mutant POIs compared to the binding affinity between wild-type POIs can be measured by any number of methods for quantifying protein binding affinity, including yeast two-hybrid screening, biolayer interferometry, ELISA, quantitative ELISA, surface plasmon resonance, FACS-based enrichment, synthetic yeast agglutination in liquid culture, or any other measurement of protein interaction strength. For example, synthetic yeast agglutination in liquid culture is described in US Patent Application Publication No. 2017 / 0205421.

[0081] In some introduction forms, the first POI and the second POI are full-length proteins. In other introduction forms, the first POI and the second POI are truncated proteins. In other introduction forms, the first POI and the second POI are fusion proteins. In other introduction forms, the first POI and the second POI are tagged proteins. Tagged proteins include epitope-tagged proteins, such as FLAG-tagged, HA-tagged, His-tagged, Myc-tagged proteins, among others known in the art. In some introduction forms, the first POI is a full-length protein and the second POI is a truncated protein. The first POI and the second POI can each be a full-length protein, a truncated protein, a fusion protein, a tagged protein, or a combination thereof.

[0082] In some implementations, the first POI is an antibody or a truncated portion of an antibody polypeptide. In other implementations, the library of first POIs is an antibody, a library of truncated antibody polypeptides, or a library of antibody mutants generated by site-saturation mutagenesis, alanine scanning, or other methods of introducing multiple amino acid variants known in the art. Antibodies, also known as immunoglobulins, are relatively large multi-unit protein structures that specifically recognize and bind to a unique molecule or molecules. In most antibodies, two heavy chain polypeptides of approximately 50 kDA and two light chain polypeptides of approximately 25 kDA are linked by disulfide bonds to form a larger Y-shaped multi-unit structure. The variable and hypervariable regions, which display the variability of amino acid sequences at the tips of the Y-shaped structure, confer specificity to a given antibody for recognizing its target.

[0083] In some implementations, the first POI is a single chain variable fragment (scFv), a fusion protein consisting of the variable regions of the heavy (VH) and light (VL) chains of an immunoglobulin linked by a short linker peptide. In some implementations, the library of the first POI is a library of scFvs or a library of scFv mutants generated by site-saturation mutagenesis, alanine scanning, or other methods of introducing multiple amino acid variants known in the art.

[0084] In some implementations, the first POI is an antigen-binding fragment (Fab), which is the region of an antibody that binds to an antigen. The Fab may contain one constant domain and one variable domain of each of the heavy and light chains, and also contains the paratope region of the antibody. In some implementations, the library of the first POI is a library of Fabs, or a library of Fab mutants generated by site-saturation mutagenesis, alanine scanning, or other methods of introducing multiple amino acid variants known in the art.

[0085] In some implementation forms, the first POI can be a portion of a single domain antibody, which is an antigen-binding fragment of an antibody only in the heavy chain, or a VHH. A VHH comprises one variable domain of a heavy chain antibody. In some implementation forms, the library of first POIs is a library of VHHs, or a library of VHH mutants generated by site-saturation mutagenesis, alanine scanning, or other methods of introducing multiple amino acid variants known in the art.

[0086] In some implementations, the first POI is an E3 ubiquitin ligase. In other implementations, the library of first POIs is a library of E3 ubiquitin ligases or a library of E3 ubiquitin ligase mutants generated by site-saturation mutagenesis, among other methods. E3 ubiquitin ligases include MDM2, CRL4, CRBN , SCFβ -TrCPE3 ubiquitin ligases include E2 ubiquitin-conjugating enzymes, which are loaded with ubiquitin, recognize their target protein substrates, and catalyze the transfer of the ubiquitin molecule from the E2 to the protein substrate for subsequent degradation by the proteasome complex.

[0087] In some implementations, the second POI is a target protein that contains a degron. In other implementations, the library of second POIs is a library of polypeptides that contain degrons or a library of polypeptides that contain degron mutants generated by site-saturation mutagenesis, among other methods. Degrons are portions of polypeptides that mediate protein degradation, in some cases regulated by the ubiquitin proteasome system. Degrons can contain short amino acid motifs, post-translational modifications, such as phosphorylation, structural motifs, and / or sugar modifications.

[0088] In some transfection forms in which the second POI is a degron, the degron can be fluorescently tagged, i.e., by expressing the degron as a fusion protein containing a genetically encoded fluorescent tag, such as green fluorescent protein (GFP), red fluorescent protein (RFP), mCherry, M Scarlet, tdTomato, among others.

[0089] In some implementations, the second POI is an E3 ubiquitin ligase. The library of second POIs may include, for example, polypeptide substrates known in the art for associating with E3 ubiquitin ligases. The library of second POIs may further include, for example, previously known full-length mapped E3 ubiquitin ligase substrate domains; high-throughput oligonucleotide-encodeable truncated E3 ubiquitin ligase substrates; E3 ubiquitin ligase substrate species modified by site-saturation mutagenesis; previously defined degron motifs; or computationally predicted degron motifs. The library of second POIs may include a plurality of user-specified mutants of the polypeptide substrate and a wild-type polypeptide substrate. The plurality of user-specified mutants of the POI may include variants of the POI with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid substitutions. The amino acid substitutions may be generated by site-saturation mutagenesis. A library of a first POI and a second POI can be assayed for binding affinity, where affinity is measured individually in a pairwise parallel high-throughput manner for interactions between the first POI and each of multiple user-specified mutants of the second POI.

[0090] Yeast strains and culture conditions In sequential library screening of PPIs based on the methods disclosed herein, yeast sexual aggregation is re-engineered. For example, the native protein-protein interaction between native sexual aggregation proteins in S. cerevisiae (whose binding is essential for mating in liquid culture) is replaced by an interaction between two proteins of interest expressed as multiply barcoded synthetic adhesion proteins on the surface of recombinant haploid yeast cells. To construct recombinant yeast strains for use in the methods disclosed herein, isogenic fragments for yeast transformation or plasmid assembly can be PCR amplified from existing plasmids, yeast genomic DNA, animal or human cDNA, animal or human genomic DNA, cDNA gel extracted from plasmid digests, or commercially synthesized by conventional DNA synthesis. Plasmids can be constructed by isothermal assembly methods and verified using Sanger sequencing (which can also be used to identify the diverse multiple oligonucleotide molecular barcode sequences linked to each ORF encoding SAP or SAP variants).

[0091] In some implementations, a MATa haploid strain optimized for surface display, such as EBY100, can be used as the parent strain. A parent MAT alpha haploid surface display strain can be constructed using mating, sporulation, tetrad separation, and screening with a selectable marker. Isogenic chromosomal integration can be performed by digestion of the plasmid with Pme1, followed by standard lithium acetate yeast transformation protocols. SSM libraries of SAP can be transformed into yeast using nuclease-assisted chromosomal integration. A parent strain containing a landing pad, such as a Scel landing pad, can be grown in galactose medium for 6 hours before transformation. Recycling of the URA3 gene can be achieved by growing the strain to saturation without URA selection and plating on 5-FOA.

[0092] In some introductions, isogenic strains can be constructed individually and multiple oligonucleotide molecular barcodes associated with each SAP can be determined using Sanger or next generation sequencing. A library of yeast strains can be produced that are all of the same mating type and display a unique barcoded SAP, with each ORF encoding a SAP linked to multiple oligonucleotide molecular barcode sequences. Each haploid strain in the library can be grown individually to saturation, assessed for surface expression intensity as previously described, and mixed in equal amounts. After growth to saturation, cells can be harvested by centrifugation and lysed by heating to 70°C for 5 minutes in 200 mM LiOAc and 1% SDS. Cell debris can be removed and incubated with 0.05 mg / mL RNase A at 37°C for 4 hours. Ethanol precipitation can be performed to purify and concentrate the genomic DNA. A primary qPCR can be performed to amplify the barcode region using standard adapters, and the PCR product is used as a template for a secondary qPCR to ligate the index barcodes and standard Illumina adapters for next generation sequencing. The fragments can be gel extracted, quantified using Qubit, and analyzed on a commercial next generation sequencing platform.

[0093] In some implementations, for large-scale library matings constructed using nuclease-assisted chromosomal integration, mating libraries can be grown individually to saturation in 3 mL YPD medium. 1 mL of MATa culture and 2 mL of MATalpha culture can be mixed and genome prepared based on standard conditions. This genomic DNA can be used as a template for two separate qPCR reactions, one to amplify the MATa expression cassette and barcode, and the other to amplify the MATalpha expression cassette and barcode. A secondary PCR can be used to add different sequencing index barcodes and Illumina adapters. These fragments can be sequenced using a commercially available next-generation sequencing platform, such as Illumina MiSeq. 2.5 μL of MATa culture and 5 μL of MATalpha culture can be combined in 3 mL YPD medium and processed similarly to the small-scale batch mating.

[0094] In some implementations, for small-scale libraries, after the library of synthesized barcodes is assembled with a nucleic acid construct containing an SAP expression cassette, the oligonucleotide molecular barcode sequences for each SAP are determined using Sanger sequencing (see, for example, FIG. 5B). For large-scale libraries, next-generation sequencing may be required to map each SAP to its associated oligonucleotide molecular barcode sequences and to determine the starting concentration of each SAP-expressing strain. Next-generation sequencing of fragments amplified from diploid genomic DNA after SAP-mediated fusion events provides identification information for unique combined barcode-barcode pairs occurring within the same fragment (see, for example, FIG. 4), with each unique combined sequence representing an individual mating event.

[0095] In some implementations, multiplexed SAP barcoding and recombination schemes can be used to analyze entire protein interaction networks in a single assay. Single MATa and MATa parental strains, such as yNGYSDa and yNGYSDa, can be constructed and the multiplexed-barcoded SAP cassettes or plasmids carrying multiplexed-barcoded SAP cassettes can be transformed into strains following conventional yeast transformation protocols. In addition to knocking out Sag1 and complementary lysine and leucine markers in the parental strains, yNGYSDa contains a CRE recombinase expression cassette with the inducible promoter pZ4 and constitutively expresses ZEV4 (an activator of the pZ4 promoter with an estradiol-binding domain for nuclear localization).

[0096] SAP cassettes can be assembled in standardized vectors, such as pNGYSDa or pNGYSDα, for integration into the corresponding yeast parent strain. In addition to the surface expression cassette, each vector backbone can contain one or more of the following: a mating type-specific fluorescent reporter cassette, one of several oligonucleotide molecular barcode sequences, a mating type-specific primer binding site, and a lox recombination site. Upon cell-cell fusion and mating, β-estradiol (βE) can be added to induce CRE recombinase expression in the fused diploid cells, and the barcodes from each haploid chromosome are integrated, allowing next-generation sequencing to be used to identify unique barcode-barcode combinations, each representing an individual unique cell-cell fusion event mediated by a pair of interacting SAPs (see Figures 1-4).

[0097] According to methods described in more detail below, the number of unique combined barcode sequences detected by sequencing estimates the number of unique cell-cell fusion events for each SAP interaction in the network, providing a relative interaction strength for each PPI in the network. AUsing the estimated number of diploid yeast cells formed per -POIα pairing and adjusting for the proportion of the input library constructed with each POI, PPI affinity can be estimated by reference to a set of PPI standards with known affinity, i.e., positive and negative controls.

[0098] Yeast strains for use in the methods disclosed herein are described, many of which can undergo multiple transformations. For example, a displayer strain compatible with CRE recombinase assays may require the integration of Aga1 under the control of a constitutive promoter, knockout of the native sexual agglutinin protein, integration of a fluorescent reporter, integration of CRE recombinase and GAVN or HygMX and ZEV4, and integration of multiple barcoded surface expression cassettes with lox sites. For each integration, a plasmid can be constructed that contains the required yeast cassette, E. coli resistance marker, and replication origin, as well as 5' and 3' regions with homology to the yeast genome for integration.

[0099] statistical analysis After next generation sequencing and quantifying the number of unique barcode-barcode combinations detected in the sequencing reads, the number of diploid formation events can be estimated. The estimation is essentially equivalent to the classic "balls-into-bins" problem in probability theory: if you throw an unknown number of balls into n bins, and then quantify the number of bins that contain the balls, how many balls were thrown? In a scenario where the number of bins greatly exceeds the number of balls, and the balls are thrown randomly, the estimated number of balls is simply equal to the number of bins that contain the balls.

[0100] Applying that here, a "bin" is the number of possible unique barcode-barcode combinations (i.e., POIs). AThe number of "balls in the bin" is equal to the number of unique barcode-barcode combinations detected after sequencing. When the number of possible unique barcode-barcode combinations significantly exceeds the number of diploid formation events expected to occur (expected to be the case for the library sequential PPI assay described herein), the number of diploid formation events can be estimated to be equal to the number of unique barcode-barcode combinations detected after sequencing. To offset the possibility that some barcode pairs may have paired more than once, which is unlikely here, the total number of diploid formation events can be estimated by solving the Poisson rate parameter that matches the observed proportion of barcode pairs: Estimate of diploids formed = number of possible barcode pairs * - ln(proportion of unobserved barcode pairs) POI A Using the estimated number of diploid yeast cells formed per -POIα pairing and adjusting for the proportion of the input library constructed with each POI, PPI affinity can be estimated by reference to a set of PPI standards with known affinity, i.e., positive and negative controls. EXAMPLES

[0101] The compositions and methods disclosed herein are further described in the following examples, which do not limit the scope of the compositions and methods described in the claims. Several embodiments have been described for the present invention. Nevertheless, it will be understood that various modifications can be made without departing from the spirit and scope of the present invention.

[0102] Example 1 - Synthetic yeast aggregation using multiplexed barcoding This example demonstrates a synthetic yeast aggregation assay in liquid culture for library-on-library characterization of protein-protein interactions (PPIs) that utilizes the multiplexed barcoding approach described herein to combine yeast surface display and sexual aggregation to link protein binding to S. cerevisiae mating, where each SAP of a library of SAPs is associated with multiple unique oligonucleotide molecular barcodes. A given pair of interacting SAPs mediated mating between a-type recombinant haploid yeast cells and α-type recombinant haploid yeast cells, after which CRE recombinase expression was induced in the diploids, and a βEa recombination event at the lox sites integrated both SAP-Aga2 fusion expression cassettes on the same chromosome, resulting in a barcode linked to the first SAP and a barcode linked to the second SAP adjacent to each other.

[0103] In this example, each SAP in the SAP library was linked to many unique barcodes, so that each diploid fusion event and subsequent recombination event produced a unique barcode-barcode combination. A single fragment containing both barcodes was then amplified by PCR using primers annealing to Pf and Pr (primers specific to the primers derived from the first and second nucleic acid constructs integrated into the genomic target site) and sequenced to identify interacting SAP pairs.

[0104] A multiplexed barcoding and recombination scheme was developed to analyze the entire protein interaction network within a single liquid culture. A library of 36,000 POIs (mutation variants of antibodies) was analyzed against a library of 500 POIs (antibody targets of interest to assess cross-reactivity). Single MATa and MATα parental strains, yNGYSDa and yNGYSDα, were constructed. Multiplexed barcoded SAP expression cassettes were assembled by combining the SAP library with a library of unique oligonucleotide molecular barcodes by an isothermal assembly method.

[0105] The multiplexed barcoded SAP cassettes were transformed into yeast strains with seven unique oligonucleotide molecular barcodes linked to each SAP. In addition to knockout of Sag1 and complementary lysine and leucine markers in the parental strains, yNGYSDa contained a CRE recombinase expression cassette with the inducible promoter pZ4. yNGYSDa constitutively expressed ZEV4, an activator of the pZ4 promoter with an estradiol binding domain for nuclear localization. The SAP cassettes were assembled in one of two standardized vectors, pNGYSDa or pNGYSDa, for integration into the corresponding yeast parent strains. In addition to the surface expression cassette, each vector backbone contained a mating type-specific fluorescent reporter cassette, a unique randomized 10-nucleotide barcode with seven unique barcodes linked to each SAP, a mating type-specific primer binding site, and a lox recombination site.

[0106] Upon mating, the addition of β-estradiol (βE) induces CRE recombinase expression in diploid cells, integrating the barcodes derived from each haploid chromosome, allowing next-generation sequencing to determine the number of unique diploid formation events that occurred during the assay. The number of unique barcode-barcode combinations for each SAP pair was quantified from the unique NGS sequencing reads, providing a highly accurate estimate of the number of original diploid formation events via digital quantification.

[0107] FIG. 6 is a plot of the data obtained from this example, and shows a histogram of potential and observed barcode pairs for each POI pair with some observed sequencing data. A For -POIα, there were approximately 48 potential unique barcode-barcode combinations with 7 unique barcodes linked to each POI. Very few barcode pairs were observed for the vast majority of POI pairs, and the POIs A An average of 0.5 barcodes was observed across -POIα pairs. However, the distribution of potential barcode pairs shifts substantially toward higher values. This example illustrates a situation in which a multiplexed barcoding scheme is particularly useful for improving quantitative accuracy.

[0108] Figure 7 shows the distribution of sequencing reads observed for POI pairs, where it was estimated with high confidence that 10 diploid yeast were formed during the synthetic yeast aggregation assay in this example, for which exactly 10 unique barcode-barcode combinations were observed in the sequencing data, with at least 200 possible barcode-barcode combinations predicted for each POI pair. As expected, the plot reveals a wide distribution of sequencing read numbers for unique POI pairs, demonstrating that the process of yeast growth, PCR amplification, and next generation sequencing introduces considerable variation in the number of final sequencing reads generated per POI pair.

[0109] Digital quantification, enabled by multiplexed barcoding of POIs, allows us to infer with high confidence that 10 diploids were formed per POI pair. Without multiplexed barcoding, this inference would be skewed to suggest that POI pairs with a higher number of sequencing reads had higher affinity, which is not necessarily the case.

[0110] Figure 8 confirms that the multiplexed barcoding method can accurately estimate the number of diploid yeast cells formed during mating for each pair of binding POIs. If the number of diploid yeast cells formed during the assay is correctly estimated, the variability observed during repeated experiments should approximately follow a Poisson distribution (μ=the estimated number of diploids formed during the assay for a pair of POIs). Figure 8 shows the distribution of estimated diploids for the pair of POIs (10 estimated diploids were formed during this experiment). The assay was performed two more times as biological replicates. As shown in Figure 8, there is a close agreement between the experimental observations and the statistical predictions, suggesting that the initial estimate of 10 diploid events in the first replicate was quite accurate.

[0111] To further demonstrate the improvement of SAP multiplex barcoding over SAP simple barcoding in estimating PPI affinities, we calculated confidence intervals for PPI networks of increasing size. Figure 9 shows the impact of multiplex barcoding on the estimation of uncertainty in PPI affinities. A Four networks of SARS-CoV2-POIα interactions were measured using a synthetic yeast agglutination assay. All networks shared a core set of 14 standard positive and negative controls, 42 additional variants of positive controls, 33 anti-SARS-CoV2 antibodies, 60 RBD domains from multiple SARS-CoV2 strains and variants, 47 anti-flaA antibodies, and 5 flaA variants. This core set of interactions was expanded into a much larger network by adding additional variants of antibodies and associated targets.

[0112] For each network, the correct behavior for uncertainty quantification accounts for 95% of the measurements from the smallest network (high confidence estimate, represented by the horizontal line located at the top of the plot shown in Figure 9 ) and falls within the nominal 95% confidence interval calculated for the larger network.

[0113] The confidence intervals calculated using multiplexed barcoding capture 70-90% of the data. These results suggest that the confidence intervals are slightly narrower, but still close to the nominal target of 95%. In contrast, the previously utilized method without multiplexed barcoding (using ±2 experimental standard deviations in biological replicates to construct the confidence intervals) produces very narrow confidence intervals, with only 20-40% of measurements with high confidence falling within the nominal 95% confidence window. This example illustrates how multiplexed barcodes can be assigned to each POI to substantially improve the ability to quantify the uncertainty in PPI affinity measurements derived from synthetic yeast agglutination assays.

[0114] Other embodiments Although several embodiments of the invention have been described, it will be understood that various modifications can be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.

Claims

1. 1. A method for quantifying a unique cell fusion event, comprising: providing a first quantity of cells, each cell of the first quantity of cells comprising an exogenous nucleic acid vector of a first library of exogenous nucleic acid vectors, each of the exogenous nucleic acid vectors in the first library comprising a first open reading frame (ORF) linked to an oligonucleotide molecular barcode sequence selected from a first plurality of oligonucleotide molecular barcode sequences; providing a second quantity of cells, each cell of the second quantity of cells comprising an exogenous nucleic acid vector of a second library of exogenous nucleic acid vectors, each of the exogenous nucleic acid vectors in the second library comprising a second ORF linked to an oligonucleotide molecular barcode sequence selected from a second plurality of oligonucleotide molecular barcode sequences; combining the first amount of cells and the second amount of cells in a liquid medium to produce a culture; growing the culture for a time and under conditions sufficient to allow a fusion event to occur between cells of the first amount of cells and cells of the second amount of cells to produce a plurality of fused cells, wherein a recombination event occurs between a first exogenous nucleic acid vector and a second exogenous nucleic acid vector within the fused cells to produce a combined oligonucleotide molecular barcode sequence; and Sequencing the combined oligonucleotide molecular barcode sequences from the culture; and determining, for each pair of first and second ORFs, a first number of unique pairs of first and second oligonucleotide molecular barcode sequences present in combined oligonucleotide molecular barcodes observed in said culture; determining a second number of possible combined oligonucleotide molecular barcode sequences for each pair of the first ORF and the second ORF; calculating an estimated number of unique fusion events in the culture based on the first number and the second number; A method comprising:

2. 2. The method of claim 1, wherein the first amount of cells and the second amount of cells are yeast cells.

3. 3. The method of claim 1 or claim 2, wherein the first quantity of cells are a-type haploid yeast cells and the second quantity of cells are alpha-type haploid yeast cells.

4. The first ORF encodes the protein of interest "a" (POI ａ 4. The method of claim 2 or claim 3, wherein the first ORF encodes a first ORF, and the second ORF encodes a protein of interest "α" (POIα).

5. POI ａ 5. The method of claim 4, wherein each ORF encoding a POIα is operably linked to an oligonucleotide molecular barcode sequence selected from the first plurality of oligonucleotide molecular barcode sequences, and each ORF encoding a POIα is operably linked to an oligonucleotide molecular barcode sequence selected from the second plurality of oligonucleotide molecular barcode sequences.

6. Each POI ａ The method of claim 4 or claim 5, wherein each POIα is expressed on the cell surface of the first amount of cells and each POIα is expressed on the cell surface of the second amount of cells.

7. 7. The method of any one of claims 3 to 6, wherein at least one of the first amount of cells or the second amount of cells is rendered incapable of mating according to any naturally occurring sexual aggregation process, such that the first amount of recombinant haploid yeast cells and the second amount of recombinant haploid yeast cells are incapable of mating according to any naturally occurring sexual aggregation process.

8. Each POI ａ and each POIα is a synthetic adhesion protein (SAP).

9. Each POI ａ and each POIα is i) the fusion protein bound to a cell wall glycosylphosphatidylinositol (GPI)-anchored protein present on the surface of the first amount of recombinant haploid yeast cells or a portion of a second amount of haploid yeast cells; or ii) a glycosylphosphatidylinositol (GPI)-anchored fusion protein present on the surface of a first amount of haploid yeast cells or a portion of said second amount of haploid yeast cells; The method according to any one of claims 4 to 8.

10. 13. The method of any one of the preceding claims, wherein the first plurality of oligonucleotide molecular barcode sequences comprises three or more unique oligonucleotide molecular barcode sequences, the second plurality of oligonucleotide molecular barcode sequences comprises three or more unique oligonucleotide molecular barcode sequences, or both the first plurality of oligonucleotide molecular barcode sequences and the second plurality of oligonucleotide molecular barcode sequences each comprise three or more unique oligonucleotide molecular barcode sequences.

11. 13. The method of any one of the preceding claims, wherein the first plurality of oligonucleotide molecular barcode sequences comprises 10 or more unique oligonucleotide molecular barcode sequences, the second plurality of oligonucleotide molecular barcode sequences comprises 10 or more unique oligonucleotide molecular barcode sequences, or both the first plurality of oligonucleotide molecular barcode sequences and the second plurality of oligonucleotide molecular barcode sequences each comprise 10 or more unique oligonucleotide molecular barcode sequences.

12. 13. The method of any one of the preceding claims, wherein the first plurality of oligonucleotide molecular barcode sequences comprises 100 or more unique oligonucleotide molecular barcode sequences, the second plurality of oligonucleotide molecular barcode sequences comprises 100 or more unique oligonucleotide molecular barcode sequences, or both the first plurality of oligonucleotide molecular barcode sequences and the second plurality of oligonucleotide molecular barcode sequences each comprise 100 or more unique oligonucleotide molecular barcode sequences.

13. 13. The method of any one of the preceding claims, wherein the first plurality of oligonucleotide molecular barcode sequences comprises 1000 or more unique oligonucleotide molecular barcode sequences, the second plurality of oligonucleotide molecular barcode sequences comprises 1000 or more unique oligonucleotide molecular barcode sequences, or both the first plurality of oligonucleotide molecular barcode sequences and the second plurality of oligonucleotide molecular barcode sequences each comprise 1000 or more unique oligonucleotide molecular barcode sequences.

14. 13. The method of any one of the preceding claims, wherein the second number of possible oligonucleotide molecular barcode pairs is nine or more.

15. 2. The method of any one of the preceding claims, wherein the second number of possible oligonucleotide molecular barcode pairs is 100 or more.

16. 2. The method of any one of the preceding claims, wherein the second number of possible oligonucleotide molecular barcode pairs is 10,000 or more.

17. POI ａ Library of 10 or more POIs ａ and / or the library of POIα comprises 10 or more POIα.

18. POI ａ Library of over 100 POIs ａ and / or the library of POIα comprises 100 or more POIα.

19. POI ａ Library of over 1000 POIs ａ and / or the library of POIα comprises 1000 or more POIα.

20. POI ａ Library of over 10,000 POIs ａ and / or the library of POIα comprises 10,000 or more POIα.

21. 2. The method of any one of the preceding claims, wherein the first exogenous nucleic acid vector and the second exogenous nucleic acid vector each further comprise a unique primer binding site, a recombination site, and a selectable marker.

22. 2. The method of any one of the preceding claims, wherein each cell of the first amount of cells and each cell of the second amount of cells further comprises an exogenous recombinase.

23. 23. The method of claim 22, wherein the exogenous recombinase mediates the recombination event.

24. 13. The method of any one of the preceding claims, wherein a portion of a first oligonucleotide molecular barcode sequence and a portion of a second oligonucleotide molecular barcode sequence are sequenced to obtain a plurality of sequencing reads, each sequencing read comprising a portion of the first oligonucleotide molecular barcode sequence and a portion of the second oligonucleotide molecular barcode sequence.

25. i) each cell of said first amount of cells lacks either a functional Aga1 or a functional Aga2 protein; or ii) each cell of the second quantity of cells lacks a functional Sag1 protein; or iii) each cell of the first quantity of cells lacks either a functional Aga1 or a functional Aga2 protein, and each cell of the second quantity of cells lacks a functional Sag1 protein; The method according to any one of claims 7 to 24.

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