Methods and kits for whole genome amplification and analysis of target molecules in biological samples - Patents.com

Abstract

Disclosed is a method for whole genome amplification and analysis of multiple target molecules in a biological sample containing genomic DNA and target molecules, the method comprising the steps of: contacting the biological sample with at least one binder for at least one of the target molecules, the binder being conjugated to a tagged oligonucleotide comprising a binder barcode sequence (BAB) and a unique molecular identifier sequence (UMI); obtaining a labeled biological sample by performing a separation step to selectively remove unbound binders; simultaneously performing whole genome amplification and amplification of the tagged oligonucleotides on the labeled biological sample; creating a massively parallel sequencing library from the amplified tagged oligonucleotides; sequencing the massively parallel sequencing library; searching for the sequences of the BAB and UMI from each sequencing read; and counting the number of different UMIs for each binder.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This patent application claims priority from Italian Patent Application No. 102019000024159, filed December 16, 2019, the entire disclosure of which is incorporated herein by reference.

[0002] The present invention relates to methods and kits for whole genome amplification and analysis of target molecules, in particular protein quantification, in biological samples, in particular single cell samples. [Background technology]

[0003] Methods for single-cell analysis make it possible to obtain information about the cellular context without the complexities arising from the heterogeneity of bulk samples. Analysis of the proteome and genome in the same single cell provides a correlation between the phenotype of a cell and its genotype, thus enabling unique insights into diverse biological and pathological processes. This is particularly true for tumors, where the genetic heterogeneity acquired by somatic cells and its effects on transcription and protein translation are key components of cancer initiation and development.

[0004] Whole genome amplification (WGA) is useful for analyzing genomes from single cells in order to obtain more DNA and to simplify and / or enable various types of genetic analysis, including sequencing, SNP detection, etc. WGA using deterministic restriction site-based LM-PCR (DRS-WGA) is known from EP1109938.

[0005] WO 2017 / 178655 and WO 2019 / 016401 teach simplified methods for generating massively parallel sequencing libraries from DRS-WGA (e.g., Ampli1™ WGA) or MALBAC for low-pass whole genome sequencing and copy number profiling.

[0006] Recently, a method for simultaneous analysis of the genome and transcriptome in single cells has been developed. A paper by Dey S. et al., 2015, Integrated genome and transcriptome sequencing of the same cell. Nature Biotechnology, 33(3), 285-289. http: / / doi.org / 10.1038 / nbt.3129 teaches a method in which messenger RNA from manually isolated single cells is first transcribed into single-stranded cDNA and then amplified along with genomic DNA through quasi-linear whole genome amplification. Two different libraries, one derived from cDNA and the other from genomic DNA, are then generated and sequenced. In another approach, Macaulay, IC, et al., 2015, G&T-seq: Parallel sequencing of single-cell genomes and transcriptomes. Nature Methods, 12(6), 519-522. http: / / doi.org / 10.1038 / nmeth.3370, mRNA is physically separated from gDNA using oligo-dT-coated beads to capture and isolate polyadenylated mRNA molecules from fully lysed single cells. Subsequently, mRNA is amplified using a modified Smart-seq2 protocol (Picelli, S. et al., 2013, Smart-seq2 for sensitive full-length transcriptome profiling in single cells. Nature Methods, 10(11), 1096-1100. http: / / doi.org / 10.1038 / nmeth.2639), while gDNA can be amplified and sequenced using available whole genome amplification methods. Although these methods are useful for relating genotype to messenger transcription, they do not allow for direct detection of proteins whose transcription and turnover / degradation are actively regulated in cells.

[0007] Currently, the most widely applied single-cell protein detection approach relies on targeting specific proteins using tagged antibodies. Fluorescence-based detection and quantification of proteins by fluorescence-activated cell sorting (FACS) or fluorescence microscopy allows protein detection in single cells with low multiplexing levels, using fluorescently labeled antibodies that recognize specific cellular proteins. However, this approach is generally limited to simultaneous measurements of 10–15 species, as highly multiplexed fluorophore-based assays are challenged by spectral overlap between the emission spectra of multiple dyes. Furthermore, complex algorithms are required to deconvolute overlapping spectra.

[0008] The Fluidigm Mass Cytometer (CyTOF™) utilizes metal-containing polymer-tagged (MAXPAR™) antibodies to detect proteins. The instrument is based on a non-optical physical detection principle and different chemical properties of the labels. Fluorescent labels are replaced by specially designed multi-atom element tags, and detection utilizes the high-resolution, high-sensitivity, and high-speed analysis of time-of-flight mass spectrometry (TOF-MS). Because many available stable isotopes can be used as tags, it is possible to simultaneously detect many proteins in individual cells [Ornatsky, O. et al, 2010, Highly multiparametric analysis by mass cytometry. Journal of Immunological Methods, 361(1-2), 1-20. http: / / doi.org / 10.1016 / j.jim.2010.07.002]. A study by Frei et al., 2016, Highly multiplexed simultaneous detection of RNAs and proteins in single cells. Nature Methods, 13(3), 269-275. http: / / doi.org / 10.1038 / nmeth.3742 teaches a method for simultaneous detection of RNA and proteins in single cells based on the Proximity Ligation Assay for RNA (PLAYR). PLAYR enables highly multiplexed quantification of transcripts in single cells by mass cytometry, which allows simultaneous quantification of over 40 different mRNAs and proteins.Finally, mass cytometry has made it possible to investigate multiple intracellular processes and phenotypic features, such as protein phosphorylation (Bendall, SC et. Al., 2011, Single-Cell Mass Cytometry of Differential Immune and Drug Responses Across a Human Hematopoietic Continuum. Science, 332(6030), 687-696. http: / / doi.org / 10.1126 / science.1198704) and cell proliferation (Behbehani, GK et al., 2012, Single-cell mass cytometry adapted to measurements of the cell cycle. Cytometry Part A, 81A(7), 552-566. http: / / doi.org / 10.1002 / cyto.a.22075), along with protein and messenger RNA transcription.

[0009] The limitations of these approaches are: - Due to the dynamics of ion flight in the mass spectrometer, the throughput of mass cytometry lags behind that of fluorescence-based instruments. In addition, the sensitivity of mass reporters falls short of even a few relatively quantum-efficient fluorophores (e.g., phycoerythrin), making it more difficult to measure molecular features expressed at very low levels using mass cytometry (Spitzer, MH et al., 2016, Mass Cytometry: Single Cells, Many Features. Cell, 165(4), 780-791. http: / / doi.org / 10.1016 / j.cell.2016.04.019). - Importantly, since the cells are nebulized and ionized, they cannot be recovered after analysis and therefore the genomic DNA cannot be analyzed.

[0010] A method for detecting proteins through oligonucleotide-labeled antibodies is described in a paper by Fredriksson et al., 2002, Protein detection using proximity-dependent DNA ligation assays. Nature Biotechnology, 20(5), 473-477. http: / / doi.org / 10.1038 / nbt0502-473. This paper teaches a technique (proximity ligation assay; PLA) in which cooperative and close binding of a target protein by two DNA aptamers promotes ligation of oligonucleotides linked to aptamer affinity probes. Ligation of two such proximity probes generates an amplifiable DNA sequence that reflects the identity and quantity of the target protein. The 3PLA method (Schallmeiner, E. et al., 2007, Sensitive protein detection via triple-binder proximity ligation assays. Nature Methods, 4(2), 135-137. http: / / doi.org / 10.1038 / nmeth974) extends the sensitivity and specificity of proximity ligation assays by using three recognition events, enabling the detection of as few as 100 target molecules. In 3PLA, a set of three oligonucleotide-modified antibody reagents binds to individual target proteins, generating a detectable signal via proximity ligation. The 3' and 5' ends of the oligonucleotides on two proximity probes can hybridize to oligonucleotides present on a third proximity probe, forming a complex containing the three probes and the target protein. This allows the two oligonucleotides to be linked via an intermediate fragment by two ligation reactions and templated by the third proximity probe to form a specific, amplifiable DNA strand that can be detected by qPCR.The proximity extension assay (PEA) is a variation of PLA, in which two oligonucleotide-labeled antibodies bind to separate proteins, the oligonucleotides partially anneal at their 3' ends, and polymerase extension generates an amplifiable DNA sequence that can be detected by qPCR (Lundberg, M. et al., 2011, Homogeneous antibody-based proximity extension assays provide sensitive and specific detection of low-abundant proteins in human blood. Nucleic Acids Research, 39(15). http: / / doi.org / 10.1093 / nar / gkr424). Although the methods disclosed above were not specifically designed for single-cell protein detection, the Fluidigm C1™ single-cell automated preparation system was utilized to automate the preparation of amplifiable targets for a panel of 92 proteins in up to 96 single cells per run using the PEA assay (Egidio C. et al., 2014, A Method for Detecting Protein Expression in Single Cells Using the C1). TM Single-Cell Auto Prep System (TECH2P.874), J Immunol, 192 (1 Supplement) 135.5). The Fluidigm C1 microfluidic system supports a range of single-cell biology methods for analysis of transcriptome or genomic DNA sequences by whole-exome sequencing and targeted DNA sequencing; however, these methods cannot be easily combined to obtain genotypic and phenotypic information from the same single cell.

[0011] Thus, although PLA and PEA assays, whose detection is based on qPCR, are sensitive and highly specific, their throughput is limited and they can only detect protein.

[0012] US Patent No. 9,714,937 to NanoString Technologies teaches a method for protein detection by using a capture antibody conjugated to a moiety (e.g., biotin) specific for a first region of the target protein and a detection antibody specific for a second region of the target protein, together with a nanoreporter containing multiple detachable labels linked to the detection antibody via hybridization to a linker oligonucleotide. The two antibodies form a complex with the target protein, which can be bound to a matrix or bead with high affinity for the moiety. The target is detected and quantified by counting the number of nanoreporter molecules. Nanostring's commercially available assay, based on its nCounter® digital molecular barcoding technology, detects proteins using unique oligonucleotide-labeled antibodies targeting specific protein epitopes. Unique single-stranded DNA tags are detected using a combination of a biotinylated capture probe and a reporter probe made of a single-stranded DNA molecule annealed to a series of fluorescently labeled RNA segments. The linear order of these labels generates a unique barcode for each target of interest. The complex is then immobilized on an imaging surface via a non-covalent bond between biotin and immobilized streptavidin molecules, and the fluorescent barcodes are imaged and counted. The number of counts per protein-specific barcode is a digital measurement directly related to the number of molecules present in the sample. Protein detection can be combined with messenger RNA detection using capture probe-reporter probe pairs designed against specific target RNAs. Approximately 30 protein targets and 770 mRNA targets can be analyzed in a single run.

[0013] The drawbacks of this method are that it requires a large number of cells for RNA profiling (equivalent to 2500 cells) and / or protein profiling (equivalent to 100,000 cells), and is not suitable for profiling single cells as such. This method could potentially be used to detect other analytes, such as genomic DNA; however, it cannot provide a direct readout of the genomic sequence, but can only provide a signal of the presence / absence of a known sequence. Because it is based on hybridization, it is also partially tolerant of sequence variants and may not be able to distinguish between different sequence variants.

[0014] An NGS-based method for integrated analysis of multiple protein and RNA transcripts in single cells, termed cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq), was first described by Stoeckius et al., 2017, Simultaneous epitope and transcriptome measurement in single cells, Nature Methods volume 14, pages 865-868, and US 2018 / 0251825. This method relies on oligonucleotide-labeled antibodies used to integrate cellular protein and transcriptome measurements into a single-cell readout through a 3'-polyadenosine tail present on the antibody tag, similar to that present on messenger RNA. This method is compatible with droplet-based approaches for sample partitioning and single-cell library generation in single cells, such as those offered by 10X Genomics. More specifically, in the CITE-seq method, cells stained with oligonucleotide-labeled antibodies against cell surface epitopes are partitioned by microfluidics into oil droplets containing lytic enzymes and barcoded beads. The barcoded antibodies and mRNA from each single cell / droplet are captured by beads bearing a unique cell barcode. The mRNA is then retrotranscribed and amplified with barcoded antibody-derived oligos to generate a sequencing-ready NGS library. Finally, sequence counts are used to quantify the barcoded antibodies.Similarly, Peterson et al., 2017, Multiplexed quantification of proteins and transcripts in single cells, Nature Biotech., (35) 10:936-939, teaches a method called RNA Expression and Protein Quantification Assay (REAP-seq) based on DNA-labeled antibodies and droplet microfluidics, which can quantify proteins using 82 barcoded antibodies and profile over 20,000 transcripts in single cells. Both of the above-mentioned methods utilize the DNA polymerase activity of reverse transcriptase to extend oligo-labeled antibodies primed with poly(dT) cellular barcodes and simultaneously synthesize complementary DNA from mRNA in the same reaction. On the other hand, other methods, also based on droplet approaches, are available for analyzing genome-wide copy number profiles or for analyzing genome sequences in single cells. For example, Chromium Single Cell CNV Solution, a solution commercially available from 10X Genomics, enables copy number profiling of hundreds to thousands of single cells, and Mission Bio's Tapestri® platform provides single-cell targeted DNA sequencing for sequence and CNV analysis of a panel of genes.

[0015] The disadvantages of these methods are: - Both methods for simultaneous transcriptome / proteome profiling mentioned above do not allow the simultaneous analysis of genomic sequences together with proteins or transcripts, since genomic DNA does not possess the polyadenosine tail necessary to amplify it. Dropseq-based methods for copy number and / or targeted sequencing are only suitable for the analysis of genomic DNA, but do not provide any phenotypic information such as transcriptional profiles or quantification of surface markers or other proteins of single cells. - In droplet-based single-cell segmentation approaches, single cells and all their information content are essentially "destroyed" during the process, and it is not possible to recover single cells for further analysis after the procedure is complete. [Prior art documents] [Patent documents]

[0016] [Patent Document 1] EP1109938 [Patent Document 2] WO 2017 / 178655 [Patent Document 3] WO 2019 / 016401 [Patent Document 4] US 9,714,937 [Patent Document 5] US 2018 / 0251825 [Non-patent literature]

[0017] [Non-Patent Document 1] Dey S. et al., 2015, Integrated genome and transcriptome sequencing of the same cell. Nature Biotechnology, 33(3), 285-289. http: / / doi.org / 10.1038 / nbt.3129 [Non-patent document 2] Macaulay, IC, et al., 2015, G&T-seq: Parallel sequencing of single-cell genomes and transcriptomes. Nature Methods, 12(6), 519-522. http: / / doi.org / 10.1038 / nmeth.3370 [Non-patent document 3] Picelli, S. et al., 2013, Smart-seq2 for sensitive full-length transcriptome profiling in single cells. Nature Methods, 10(11), 1096-1100. http: / / doi.org / 10.1038 / nmeth.2639 [Non-patent document 4] Ornatsky, O. et al, 2010, Highly multiparametric analysis by mass cytometry. Journal of Immunological Methods, 361(1-2), 1-20. http: / / doi.org / 10.1016 / j.jim.2010.07.002 [Non-patent document 5] Frei et al., 2016, Highly multiplexed simultaneous detection of RNAs and proteins in single cells. Nature Methods, 13(3), 269-275. http: / / doi.org / 10.1038 / nmeth.3742 [Non-patent document 6] Bendall, SC et. Al., 2011, Single-Cell Mass Cytometry of Differential Immune and Drug Responses Across a Human Hematopoietic Continuum. Science, 332(6030), 687-696. http: / / doi.org / 10.1126 / science.1198704 [Non-Patent Document 7] Behbehani, GK et al., 2012, Single-cell mass cytometry adapted to measurements of the cell cycle. Cytometry Part A, 81A(7), 552-566. http: / / doi.org / 10.1002 / cyto.a.22075 [Non-patent document 8] Spitzer, MH et al., 2016, Mass Cytometry: Single Cells, Many Features. Cell, 165(4), 780-791. http: / / doi.org / 10.1016 / j.cell.2016.04.019 [Non-Patent Document 9] Fredriksson et al., 2002, Protein detection using proximity-dependent DNA ligation assays. Nature Biotechnology, 20(5), 473-477. http: / / doi.org / 10.1038 / nbt0502-473 [Non-Patent Document 10] Schallmeiner, E. et al., 2007, Sensitive protein detection via triple-binder proximity ligation assays. Nature Methods, 4(2), 135-137. http: / / doi.org / 10.1038 / nmeth974 [Non-Patent Document 11] Lundberg, M. et al., 2011, Homogeneous antibody-based proximity extension assays provide sensitive and specific detection of low-abundant proteins in human blood. Nucleic Acids Research, 39(15). http: / / doi.org / 10.1093 / nar / gkr424 [Non-Patent Document 12] Egidio C. et al., 2014, A Method for Detecting Protein Expression in Single Cells Using the C1 TM Single-Cell Auto Prep System (TECH2P.874), J Immunol, 192 (1 Supplement) 135.5 [Non-Patent Document 13] Stoeckius et al., 2017, Simultaneous epitope and transcriptome measurement in single cells, Nature Methods volume 14, pages 865-868 [Non-Patent Document 14] Peterson et al., 2017, Multiplexed quantification of proteins and transcripts in single cells, Nature biotech., (35) 10:936-939 Summary of the Invention [Problem to be solved by the invention]

[0018] It is therefore an object of the present invention to provide a method for whole genome amplification and analysis of multiple target molecules in a biological sample, which allows the simultaneous analysis of the genome-wide copy number profile / genomic sequence and the analysis of protein expression for the same single cell, thereby overcoming in particular one or more of the following disadvantages of the state of the art: - the inability to detect and quantify proteins and analyze genomes in the same sample down to single cell resolution; - The inability to reanalyze single cells for additional targeted genomic information. [Means for solving the problem]

[0019] This object is achieved by the method defined in claim 1.

[0020] A further object of the present invention is to provide a kit as defined in claim 17. [Brief explanation of the drawings]

[0021] [Figure 1] 1A and 1B show the overall structure of two possible embodiments of tagged oligonucleotides without (FIG. 1A) or with (FIG. 1B) a 3' tagged oligonucleotide sequence according to the invention. PL = payload sequence; 5-TOS = first tagged oligonucleotide amplification sequence; 3-TOS = second tagged oligonucleotide amplification sequence; UMI = unique molecular identifier sequence; BAB = binder barcode sequence. [Figure 2] One shows a graph representing a tagged oligonucleotide library from two different tagged oligos that are prone to intramolecular hairpin formation between 5-TOS and 3-TOS at different temperatures ("with hairpin": Tm=67°C; "without hairpin": Tm=45°C). Expected UMI counts are shown on the x-axis, and UMIs counted after sequencing are shown on the y-axis. [Figure 3]Figure 1 shows a graph depicting amplification of an oligo mixture at different amounts using 27 PCR cycles. Each dilution was performed from three independent dilution replicates. The number of distinct UMIs predicted to be observed is shown on the x-axis, and the experimentally observed UMIs are shown on the y-axis. [Figure 4] Figure 4 shows three graphs of amplification of four oligo mixtures with different BABs at different amounts. Each dilution has four data points, one for each oligo. Figure 4A: 23 PCR cycles were performed. Figure 4B: 27 PCR cycles were performed. Figure 4C: 35 PCR cycles were performed. The number of distinct UMIs predicted to be observed is shown on the x-axis, and the experimentally observed UMIs are shown on the y-axis. [Figure 5] Figure 1 shows the structure of an embodiment in accordance with the invention of a tagged oligo with single primer amplification. Additional footnotes: 5-WGAH = 5' WGA handle sequence; 3-WGAH = 3' WGA handle sequence; 1AH = first amplification handle sequence; 2AH = second amplification handle sequence. [Figure 6] 6A-6B show the structure of another embodiment of a tagged oligo with at least a second primer amplification according to the present invention. Figure 6A: Structure of a tagged oligo and a relative extension oligonucleotide with an annealing site corresponding to a BAB. Figure 6B: Structure of a tagged oligo and a relative extension oligonucleotide with an annealing site not corresponding to a BAB. Additional footnotes: Ep = 5' extension oligonucleotide sequence; SS = spacer sequence; AS = annealing sequence; AS-RC = annealing sequence reverse complement. [Figure 7] FIG. 1 shows a graph of in silico prediction of the melting temperature ([Na+]=150 mM; [Mg++]=4 mM) of hairpins induced by 15 nt-long complementary sequences located at the ends of ssDNA molecules as a function of molecular length. [Figure 8] FIG. 1 shows the structure of another embodiment of a tagged oligo with at least three rounds of primer amplification according to the present invention. [Figure 9]

[0033] Figure 9A shows a general scheme for library generation. Figure 9A: Generation of a library from tagged oligos according to the embodiment of Figure 5. Figure 9B: Generation of a library from tagged oligos according to the embodiment of Figure 6A. Figure 9C: Generation of a library from tagged oligos according to the embodiment of Figure 8. Additional footnotes: 2AH-RC = second amplification handle sequence reverse complement. [Figure 10] FIG. 1 shows the design of P5-Synth oligos and the corresponding library primers disclosed in Example 1. [Figure 11] FIG. 1 shows a scheme for NGS library generation of oligo P5-Synth using library primers according to Example 1. [Figure 12] Figure 1 shows a scatter plot of PBMC cells and SK-BR-3 cells stained with Ab-oligos and secondary fluorescent antibodies. Fluorescence levels in the APC channel are shown on the x-axis, which are proportional to the amount of Ab-oligo tag1, tag2, and tag4. Fluorescence levels in the PE channel are shown on the y-axis, which are proportional to the amount of Ab-oligo tag3. [Figure 13] FIG. 1 shows an electropherogram from a library made with P5-synth tagged oligos from a single cell according to Example 1. [Figure 14-1] 14A-14D show protein quantification results from single cells processed according to the embodiment of FIG. 8 using tagged oligo amplification after WGA. UMI counts for cytokeratin (FIG. 14A), Her2 (FIG. 14B), CD45 (FIG. 14C), and IgG1 isotype control (FIG. 14D) quantification, respectively. Number of UMIs is shown on the y-axis, and cell types isolated according to FIG. 9 are shown on the x-axis. [Figure 14-2] 14A-14D show protein quantification results from single cells processed according to the embodiment of FIG. 8 using tagged oligo amplification after WGA. UMI counts for cytokeratin (FIG. 14A), Her2 (FIG. 14B), CD45 (FIG. 14C), and IgG1 isotype control (FIG. 14D) quantification, respectively. Number of UMIs is shown on the y-axis, and cell types isolated according to FIG. 9 are shown on the x-axis. [Figure 15-1]15A-15D show protein quantification results from single cells processed according to the embodiment of FIG. 8 using tagged oligo amplification during WGA. UMI counts for cytokeratin (FIG. 15A), Her2 (FIG. 15B), CD45 (FIG. 15C), and IgG1 isotype control (FIG. 15D) quantification, respectively. Number of UMIs is shown on the y-axis, and cell types isolated according to FIG. 9 are shown on the x-axis. [Figure 15-2] 15A-15D show protein quantification results from single cells processed according to the embodiment of FIG. 8 using tagged oligo amplification during WGA. UMI counts for cytokeratin (FIG. 15A), Her2 (FIG. 15B), CD45 (FIG. 15C), and IgG1 isotype control (FIG. 15D) quantification, respectively. Number of UMIs is shown on the y-axis, and cell types isolated according to FIG. 9 are shown on the x-axis. [Figure 16] FIG. 1 shows the design of P5-Lib1 oligos and the corresponding library primers disclosed in Example 3. [Figure 17] FIG. 1 shows a scheme for NGS library generation of P5-Lib1 oligos using library primers. [Figure 18]

[0033] Figures 18A-18C show examples of low-pass profiles for CNA analysis obtained from single cells. Figure 18A: Single-cell CNA profile spiked with P5-Lib1 oligo and processed according to the embodiment of Figure 5. Figure 18B: Single-cell CNA profile spiked with P5-Synth oligo and processed according to the embodiment of Figure 8. Figure 18C: Single-cell CNA profile without tagged oligo spiking. All profiles correspond to SK-BR-3 cells, with typical gains and losses. Small variations are due to single-cell genomic heterogeneity. [Figure 19] Figure 1 shows a graph representing tagged oligo libraries obtained from single-cell spiking with P5-Synth and P5-Libl, with predicted UMI counts on the x-axis and UMIs counted after sequencing on the y-axis. DETAILED DESCRIPTION OF THE INVENTION

[0022] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although a number of methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described below. Unless otherwise noted, the techniques described herein for use in connection with the present invention are standard methodology well known to those skilled in the art.

[0023] By "ab-oligo mix" is intended a solution containing all ab-oligos targeting epitopes and / or isotype control ab-oligos inside (i.e., "internal ab-oligo mix") and / or outside (i.e., "external ab-oligo mix") cells.

[0024] By "antibody-oligonucleotide conjugate" or "ab-oligo conjugate" or "ab-oligo" is intended a synthetic molecule derived by chemical conjugation of an antibody molecule with an ssDNA oligonucleotide molecule. Chemical conjugation is usually performed using specific chemical reactions that allow for covalent linkage of the two molecules. The antibody:oligonucleotide stoichiometry can be controlled to have a specified ratio. During the initial stages of WGA, the antibody portion is usually digested, leaving only the oligonucleotide portion. For simplicity, these molecules will still be referred to as ab-oligo molecules or ab-oligo amplicons in the description.

[0025] The acronym "APC" refers to the fluorophore allophycocyanin.

[0026] By "binder barcode sequence" (BAB) is intended a unique DNA oligonucleotide sequence that identifies a binder.

[0027] By "balanced PCR amplification" is intended a feature of PCR for multiple target amplification whereby substantially all target molecules are amplified in each PCR cycle.

[0028] By "binding agent" is intended a molecule (including but not limited to antibodies, affibodies, ligands, aptamers, synthetic binding proteins, small molecules, etc.) that can specifically bind to a designated target molecule (e.g., a protein or a glycosylated protein or a phosphorylated protein).

[0029] By "CITE-Seq" or "Cellular Indexing of Transcriptomes and Epitopes by Sequencing" is intended the method developed by Stoeckius et al. for simultaneous protein quantification and mRNA sequencing in single cells.

[0030] "CyTOF" or "Time of Flight Cytometry" refers to an instrument that performs a mass cytometry technique that allows for the quantification of proteins in single cells using mass spectrometry combined with cytometry. Cells are stained with binding agents conjugated with heavy metal isotopes.

[0031] The term "conjugate" intends a molecule resulting from the covalent conjugation of a binding agent with a tagged oligonucleotide.

[0032] By "copy number alteration" (CNA) is intended a somatic change in the copy number of a genomic region, generally defined relative to the genome of the same individual.

[0033] By "DNA library purification" is intended a process by which DNA library material is separated from unwanted reaction components, such as enzymes, dNTPs, salts, and / or other molecules that are not part of the desired DNA library. Examples of DNA library purification processes are purification using Agencourt AMPure, or Merck Millipore Amicon spin columns or solid phase reversible immobilization (SPRI) beads, such as those from Beckman Coulter.

[0034] By "DNA library quantification" is intended a process by which DNA library material is quantified. Examples of DNA library quantification processes are quantification using QuBit technology, electrophoretic assays (Agilent Bioanalyzer 2100, Perkin Elmer LabChip technologies), or the RT-PCR PicoGreen system (Kapa Biosystems).

[0035] By "dynamic range" is intended the ratio between the maximum and minimum values ​​that a quantity can assume.

[0036] By "library primer" is intended a ssDNA molecule that functions as a primer to generate a massively parallel sequenceable library from tagged oligonucleotides.

[0037] By "low-pass whole genome sequencing" or "low-pass sequencing" is intended whole genome sequencing at an average sequencing depth of less than 1.

[0038] "Massively parallel sequencing" or "next generation sequencing" (NGS) refers to a method of sequencing DNA that involves generating libraries of spatially and / or temporally separated DNA molecules that are clonally sequenced (with or without prior clonal amplification). Examples include the Illumina platform (Illumina), the IonTorrent platform (ThermoFisher Scientific), the Pacific Biosciences platform, and MinIon (Oxford Nanopore Technologies).

[0039] "Multiple annealing and looping-based amplification cycles" (MALBAC) refers to a quasi-linear whole genome amplification method (Zong et al., 2012, Genome-wide detection of single-nucleotide and copy-number variations of a single human cell, Science. Dec 21;338(6114):1622-6. doi: 10.1126 / science.1229164.). MALBAC primers have an 8-nucleotide 3' random sequence for hybridizing to the template and a 27-nucleotide 5' consensus sequence (GTG AGT GAT GGT TGA GGT AGT GTG GAG). After the first extension, the semi-amplicon is used as a template for another extension to generate a complete amplicon with complementary 5' and 3' ends. Following several cycles of quasi-linear amplification, the complete amplicon can be exponentially amplified using subsequent PCR cycles.

[0040] The term "oligonucleotide" or "oligo" refers to an oligomeric molecule comprising a sequence of nucleotides, such as, but not limited to, deoxyribonucleic acid (DNA), ribonucleic acid (RNA), locked nucleic acid (LNA), peptide nucleic acid (PNA), and the like.

[0041] By "tagged oligonucleotide" or "tagged oligo" is intended an oligonucleotide molecule (e.g., an ssDNA molecule) that is directly conjugated to a binding agent (e.g., a primary antibody). The tagged oligo is used for indirect quantification of a target molecule (e.g., a protein) that is a ligand for the binding agent.

[0042] The acronym "PE" refers to the fluorophore phycoerythrin.

[0043] By "PFA 2%" is intended a 2% w / v solution of paraformaldehyde in phosphate buffered saline.

[0044] By "primary WGA DNA library" (pWGAlib) is intended the DNA library obtained from a WGA reaction.

[0045] The term "reamplification" or "reamp" refers to a PCR reaction in which all or a substantial portion of the primary WGA DNA library is further amplified.

[0046] By "residue" is intended an amino acid residue occurring in a polypeptide chain of a protein.

[0047] By "sequencing barcode" is intended a polynucleotide sequence that, when sequenced within a single sequencer read, allows the read to be assigned to the specific sample associated with the barcode.

[0048] By "UMI" or "unique molecular identifier sequence" is intended a degenerate or partially degenerate (i.e., random or semi-random) oligonucleotide sequence that is substantially unique for each ssDNA or dsDNA molecule.

[0049] By "universal WGA primer" or "WGA PCR primer" is intended an additional oligonucleotide ligated to each fragment generated by the action of a restriction enzyme. Universal WGA primers are used in DRS-WGA, such as Ampli1™ WGA.

[0050] Detailed Description of the Invention The method for whole genome amplification and analysis of multiple target molecules in a biological sample containing genomic DNA and target molecules according to the present invention comprises the following steps.

[0051] In step a), a biological sample is provided, which is preferably a single cell, although samples containing several cells can also be used.

[0052] In step b), the biological sample is contacted with one binding agent for at least one target molecule conjugated to a tagged oligonucleotide, whereby, if the at least one target molecule is present in the biological sample, the at least one binding agent binds to the at least one target molecule.

[0053] The binder is preferably selected from the group consisting of an antibody or fragment thereof, an aptamer, a small molecule, a peptide, and a protein. The target molecule is preferably selected from the group consisting of a protein, a peptide, a glycoprotein, a carbohydrate, a lipid, and a combination thereof. More preferably, the binder is an antibody. The binder binds to a target molecule with a specific stoichiometry, such as a monoclonal antibody or an enzyme substrate, or with an unspecified stoichiometry, such as a polyclonal antibody or a small molecule. The former allows for relatively better quantification of the target compared to the latter. The binder is chemically conjugated to the tagged oligo via covalent or non-covalent interactions. In the former case, both the oligo and the binder possess reactive moieties that allow them to bind to each other. The binder:oligo stoichiometry can be controlled during the conjugation procedure.

[0054] A non-limiting list of examples of binding agents / target molecules is reported in Table 1 below.

[0055] [Table 1]

[0056] The oligonucleotides used as tagged oligonucleotides are preferably ssDNA or dsDNA molecules with chemical modifications at the 5' or 3' ends that are used for covalent conjugation with the relevant binding agent.

[0057] The conjugate formed by the tagged oligo conjugated with the binding agent may target both extracellular and intracellular epitopes. The "external" and "internal" conjugates can be added to a biological sample as two separate mixes containing the conjugates at their final staining concentrations. First, the external mix is ​​added to label the external epitopes. Second, the cells are permeabilized using a detergent or similar means, and the internal mix is ​​added to the sample to label the internal epitopes. Alternatively, the external and internal conjugates can be mixed together to perform one-step staining. The final staining concentration will vary for each binding agent and must be determined experimentally.

[0058] The tagged oligo sequence is preferably shorter than 300 bases, more preferably shorter than 120 bases, to facilitate conjugation with a binding agent and reduce costs. In a preferred embodiment, the tagged oligo sequence is 60 to 80 nucleotides.

[0059] Referring to FIG. 1A, the tagged oligonucleotides are: i) a nucleic acid payload sequence (PL) comprising a binder barcode sequence (BAB) and a unique molecular identifier sequence (UMI), and ii) at least one first tagged oligonucleotide amplification sequence (5-TOS) of a nucleic acid; Includes:

[0060] The payload sequence contains the necessary information for target counting.

[0061] The unique molecular identifier sequence (UMI) is preferably a degenerate or semi-degenerate sequence ranging from 10 to 30 nucleotides. Preferably, the UMI has a length of at least 10 bases, which is sufficient for most target molecules and theoretically corresponds to 4^10 = 1,048,576 different combinations. For highly abundant target molecules, a longer UMI, such as 12 bases, can be used to increase the number of possible combinations. Using semi-degenerate bases reduces the number of possible combinations, and the UMI length is preferably increased, for example, to a maximum of 20 or 30 bases. Semi-degenerate UMIs can be advantageously used to introduce a reference point that can be used in the readout to realign sequences and prevent overestimation of the different UMIs present. The UMI sequence can be located either 5' or 3' of the BAB. The UMI sequence is preferentially located immediately after the Read1 sequencing primer annealing site to increase the complexity of the bases sequenced initially. This is advantageous for the Illumina sequencing platform, since the initial sequencing step requires high complexity for cluster identification. BABs are fixed sequences for each conjugate molecule. BABs are designed to avoid features that may interfere with the primary PCR amplification and sequencing steps (such as homomultimers, hairpins, and / or heteroduplex formation) [Frank, DN, 2009, BARCRAWL and BARTAB: software tools for the design and implementation of barcoded primers for highly multiplexed DNA sequencing. BMC Bioinformatics, 10, 362. http: / / doi.org / 10.1186 / 1471-2105-10-362], and are selected from a pool of all possible BAB sequences of a defined length to maximize their relative Hamming distance, thereby minimizing the possibility that any PCR or sequencing error may result in incorrect assignment of the sequenced read. BAB length must be selected based on the number of target molecules to be detected.Preferably, the BABs have a length of at least 10 nucleotides, which, after applying filters for GC content (e.g., [30%..70%]), absence of homomultimers, absence of hairpins, and minimum Hamming distance (preferably 3 nt or greater), reduces to approximately 2000 possible BAB sequences, theoretically corresponding to 4^10 = 1,048,576 different combinations.

[0062] The first tagged oligonucleotide amplification sequence (5-TOS) is located 5' of the tagged oligo. This sequence is required for tagged oligo amplification and subsequent library generation. Tagged oligo amplification is necessary to avoid any bias due to loss of molecules during sample processing, which may prevent proper UMI counting.

[0063] Referring to Figure 1B, the target oligonucleotide preferably further comprises at least one second tagged oligonucleotide amplification sequence (3-TOS). This sequence is located 3' of the tagged oligo. This sequence is necessary for tagged oligo amplification and subsequent library generation. Tagged oligo amplification is necessary to avoid any bias due to loss of molecules during sample processing, which may prevent proper UMI counting.

[0064] In a preferred embodiment, the 5-TOS and 3-TOS sequences are designed to avoid the formation of hairpins and other intramolecularly stable secondary structures within the amplification temperature range, which may interfere with tagged oligo amplification. Figure 2 shows a graph representing a tagged oligonucleotide library from two different tagged oligos, one of which is prone to intramolecular hairpin formation between 5-TOS and 3-TOS at different temperatures ("with hairpin": Tm = 67°C, SEQ ID NO: 50, ΔG = -11.15 kcal / mol; "without hairpin": Tm = 45°C, SEQ ID NO: 51, ΔG = -1.52 kcal / mol). The x-axis shows predicted UMI counts, and the y-axis shows UMIs counted after sequencing.

[0065] The tagged oligos and their amplification primers are optimized to achieve maximum sensitivity, wide dynamic range, balanced PCR amplification, and reproducibility.

[0066] In a preferred embodiment of the present invention, the UMI sequence length is from 0 to about 10 6 A range of molecules were selected to quantify targets (n=10).

[0067] The dynamic range spans four orders of magnitude (10 2 ~10 6 The amplification of tagged oligos with different concentrations of UMIs was characterized. Figure 3 shows a graph representing the amplification of the oligo mixture at different amounts using 27 PCR cycles. Each dilution was performed from three independent dilution replicates. The x-axis shows the number of different UMIs predicted to be observed, and the y-axis shows the experimentally observed UMIs. The 10-fold difference between the observed and predicted UMIs before amplification was 100%. 2 ~10 6 A highly linear correlation can be observed over the range of molecules.

[0068] Equilibrated PCR amplification was characterized by performing different cycles of amplification on the same starting sample. As shown in Figures 4A and 4B, amplification of the same pool of tagged oligos using different numbers of total PCR cycles (23 and 27 PCR cycles, respectively) did not result in differences in the observed UMIs, indicating that the number of PCR cycles does not affect UMI counts.

[0069] Sensitivity was characterized by amplifying a pool of tagged oligos at different amounts (down to 40 molecules). As shown in Figure 4C, 2 It is possible to quantify down to 100 tagged oligo molecules. It should be noted that serially diluted solution experiments are prone to sampling bias due to the highly uneven distribution of molecules within the volume, which is particularly relevant for highly dilute solutions. Thus, the observed quantification limits may be an underestimation related to the experimental setup rather than limitations of the assay.

[0070] In step c) of the method according to the invention, a separation step is carried out to selectively remove unbound binding agent, thereby obtaining the labeled biological sample, which is typically carried out by washing in a suitable buffer solution and recovering the labeled biological sample by centrifugation.

[0071] In step d), the labeled biological sample is subjected to simultaneous whole genome amplification of the genomic DNA and amplification of tagged oligonucleotides conjugated with at least one binding agent, either by deterministic restriction site whole genome amplification (DRS-WGA) or by multiplex annealing and looping-based amplification cycle whole genome amplification (MALBAC).

[0072] In step e), a massively parallel sequencing library is generated from the amplified tagged oligonucleotides.

[0073] In step f) the massively parallel sequencing library is sequenced.

[0074] In step g), the sequences of the binder barcode sequence (BAB) and the unique molecular identifier sequence (UMI) are retrieved from each sequencing read.

[0075] In step h), for each binder, the number of different unique molecular identifier sequences (UMI) is counted.

[0076] Steps e), f), g) and h) will be disclosed in more detail below in the description with reference to specific embodiments.

[0077] The method disclosed above preferably further comprises a step of isolating single cells from the biological sample. Isolation can be carried out by sorting the cells, in particular using a cell sorter such as a DEPArray® NxT (Menarini Silicon Biosystems), or alternatively by dividing the cells into droplets. The isolation step is preferably carried out after step c) and before step d).

[0078] The method disclosed above preferably comprises a step of purifying the massively parallel sequencing library prior to step f).

[0079] The method disclosed above, also referred to as Ampli1 protein (A1-P) in more specific terms but not intended to limit the scope of this description, enables protein quantification and whole-genome genetic characterization of single cells. Single- or multiple-protein quantification in single cells is achieved using a panel of binding agents (particularly antibodies (Ab)) conjugated with tagged oligonucleotides. These oligonucleotides are designed to unambiguously identify the conjugated antibodies using DNA barcode sequences and to quantify the abundance of epitopes of interest using random or partially degenerate sequences (i.e., unique molecular identifiers (UMIs)) used for epitope quantification. A biological sample is labeled with one or more Ab-oligo conjugates, each with a unique DNA barcode sequence. Subsequently, single cells or pools of cells can be isolated by different means (i.e., DEPArray™ NxT system), and their genomic content can be amplified by whole-genome amplification (i.e., Ampli1™ Whole Genome Amplification Kit). During or immediately after the latter step, the tagged oligonucleotides are pre-amplified to avoid any downsampling during the NGS library preparation procedure. Specific primers, i.e., "library primers," are used to generate NGS (Illumina) libraries ready for sequencing. The tagged oligonucleotides are designed to be compatible with the Ampli1™ WGA (A1-WGA) workflow, allowing single-cell genetic analysis (e.g., Ampli1™ LowPass) in parallel with protein quantification using A1-P.

[0080] Below, three specific embodiments of the invention are disclosed, each utilizing a different number of primers for tagged oligo amplification and whole genome amplification.

[0081] In a first preferred embodiment, referring to FIG. 5, the tagged oligonucleotide comprises, from 5′ to 3′, at least: a) a nucleic acid first tagged oligonucleotide amplification sequence (5-TOS) further comprising a 5' whole genome amplification handle sequence (5-WGAH) and a first amplification handle sequence (1AH); b) payload sequence (PL); c) a second tagged oligonucleotide amplification sequence of nucleic acid (3-TOS), further comprising a second amplification handle sequence of nucleic acid (2AH) and a 3' whole genome amplification handle sequence (3-WGAH); Includes:

[0082] 3-WGAH is the reverse complementary sequence of 5-WGAH, enabling simultaneous amplification of gDNA and tagged oligonucleotides during whole genome amplification. 1AH and 2AH are located at the 5' and 3' ends of the payload sequence, respectively, and are used for subsequent library generation. 1AH and 2AH are preferably designed to avoid stable intramolecular secondary structures, such as hairpins, that may inhibit tagged oligo amplification. A fixed sequence may exist between each of the above sequences.

[0083] Whole genome amplification and amplification of tagged oligos are preferably performed using a single primer.

[0084] In a second preferred embodiment, referring to Figures 6A and 6B, the tagged oligonucleotide comprises, from 5' to 3', at least: a) a nucleic acid first tagged oligonucleotide amplification sequence (5-TOS) further comprising a 5' whole genome amplification handle sequence (5-WGAH) and a first amplification handle sequence (1AH); b) payload sequence (PL); c) Optionally, an annealing sequence (AS) Includes:

[0085] At least one primer is used for whole genome amplification and amplification of the tagged oligonucleotide, and at least one oligonucleotide (Ep) is used for extension of the tagged oligonucleotide, and the at least one oligonucleotide (Ep) comprises, in a 5' to 3' direction, at least: d) 5′ whole genome amplification handle sequence (5-WGAH); e) spacer sequence (SS); f) a second amplification handle sequence (2AH); and g) a sequence reverse complementary to the annealing sequence (AS-RC) or a sequence reverse complementary to the binder barcode sequence (BAB-RC) Includes:

[0086] In other words, amplification of the tagged oligo occurs through annealing of the Ep to the AS located 3' of the tagged oligo via the annealing sequence reverse complement (AS-RC) located at the 3' end of the Ep (Figure 6A), thus triggering 3' extension of both the tagged oligo and the Ep within the reaction, which then generates a WGA primer-amplifiable molecule. Alternatively, the AS can match the BAB sequence, and the Ep anneals to the BAB sequence via the BAB reverse complement sequence (BAB-RC), as shown in Figure 6B. The first option (annealing to the AS) has the advantage that a single Ep can be used with any BAB, thus reducing production costs and protocol complexity. The second option (annealing to the BAB) can be advantageously used to normalize signals from targets with large differences in abundance. This can be achieved, as a non-limiting example, by using a limiting amount of primer for potentially highly abundant targets or by using different BAB annealing temperatures to reduce amplification of highly abundant tagged oligos. The annealing temperature can be adjusted depending on the BAB length and / or composition.

[0087] After extension of the tagged oligo and Ep, the WGA primer will amplify the tagged oligo within the resulting relatively large molecule. The spacer sequence (SS) increases the length of the amplicon generated by the tagged oligo. The increased fragment length destabilizes intramolecular secondary structures, such as hairpins, induced by the complementary ends of the fragments, thus lowering their melting temperatures (Figure 7), which favors tagged oligo amplification together with other WGA fragments (M. Zuker. Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res. 31 (13), 3406-15, (2003)). The sequence length of the extension oligonucleotide (Ep) is preferably within the range of 60 to 300 bases. More preferably, the sequence length of the extension oligonucleotide (Ep) is within the range of 120 to 200 bases.

[0088] In a third preferred embodiment, referring to FIG. 8, the tagged oligonucleotide comprises, from 5′ to 3′, at least: a) a first tagged oligonucleotide amplification sequence (5-TOS) of a nucleic acid corresponding to a first amplification handle sequence (1AH); b) payload sequence (PL); c) a second tagged oligonucleotide amplification sequence (3-TOS) of a nucleic acid corresponding to the second amplification handle sequence (2AH); Includes:

[0089] At least one first primer is used for whole genome amplification, and at least one second primer and one third primer are used for amplification of the tagged oligonucleotide, wherein at least one second primer has a sequence identical to the first amplification handle sequence (1AH), and at least one third primer has a sequence reverse complementary to the second amplification handle sequence (2AH-RC).

[0090] The tagged oligo amplification primers are designed to have melting temperatures that are compatible with at least the first 10-15 cycles of the WGA PCR temperature profile.

[0091] Preferably, at least one second primer and at least a third primer are added in step d).

[0092] Step e) of generating a massively parallel sequencing library from the amplified tagged oligonucleotides is preferably carried out by a PCR reaction using at least one first library primer comprising a 3' sequence corresponding to the first amplification handle sequence (1AH) and at least one second library primer comprising a 3' sequence corresponding to the reverse complementary sequence to the second amplification handle sequence (2AH-RC).

[0093] Therefore, the library primers are advantageously used to generate NGS libraries for binding agent quantification analysis using a single PCR step. The specific examples of library primers reported in this description are used to generate libraries that are compatible with the Illumina sequencing platform, without intending to limit the scope of the invention.

[0094] In a preferred embodiment, the forward and reverse primers are designed based on Illumina adapters (Illumina) and comprise, from 5' to 3': 1) Illumina adapter sequences (IA) required for Illumina sequencing; - index sequencing primer / flow cell binding sequence (this region is required for flow cell binding) as well as the annealing sequence for the i5 / i7 index sequencing primer; - i5 / i7 index: index used for NGS multiplex reactions; - Read1 / read2 sequencing primers: annealing sequences to Illumina sequencing primers as well as for amplifying libraries from tagged oligos. 2) A sequence reverse complementary to the first amplification handle sequence (1AH) or the second amplification handle sequence (2AH-RC): These sequences anneal to the reverse complement of the first amplification handle sequence and the second amplification handle sequence, respectively, on the tagged oligo, which is double-stranded after tagged oligo amplification.

[0095] Figure 9 shows the structure of library primers used for generating massively parallel sequencing libraries from amplified tagged oligonucleotides in embodiments according to Figure 5 (Figure 9A), Figure 6A (Figure 9B), and Figure 8 (Figure 9C), respectively.

[0096] Library generation is preferably followed by at least one purification step, followed by the necessary pooling for library quantification and subsequent sequencing procedures, which is preferentially performed as paired-end sequencing, generating two reads, each derived from a strand of a library DNA molecule.

[0097] The same approach can be used to generate NGS libraries for other sequencing platforms, such as Ion Torrent.

[0098] Analysis of paired-end read sequences generated from NGS libraries is performed according to the following steps: 1. Subsequence Extraction: Subsequences corresponding to the UMI, BAB, and amplification handle sequences (1AH and / or 2AH) are extracted from both sequencing reads for each tagged oligo molecule.

[0099] 2. Read realignment. If a subsequence of the BAB and / or amplification handle sequence does not match the reference sequence (allowing 0.5-2 mismatches per 5 bases), the subsequence position is offset by a variable amount ranging from -n to +n, where n is the maximum offset allowed (e.g., n=8), and the subsequence is re-extracted. For each iteration, the Hamming distance from the reference sequence is calculated, the offset that returns the minimum distance is selected, and all subsequences (UMI, BAB, amplification handle sequences) are extracted.

[0100] 3. Read filtering. Reads whose BAB and / or handle subsequences differ from the reference sequence by more than a specified amount of bases are discarded as low-quality reads.

[0101] 4. UMI Determination. The UMI sequences from a read pair are predicted to be perfectly complementary in the absence of sequencing errors. In the presence of any differences between the first-strand UMI and the second-strand complementary UMI sequence, a. The read pair may be discarded as low confidence, or b. The consensus between two sequences can be calculated by selecting, for each position of the UMI, the base from the sequences from the two sequencing reads that have the highest base-calling scores reported by the sequencer's base caller.

[0102] It should be noted that the first method (a) is less likely to cause overestimation of target molecules due to sequencing bias, but may miss true binding events between binders and target molecules that would instead be recovered using the second method (b).

[0103] 5. Target Molecule Quantification: Target molecule counting is performed by determining the number of distinct UMI sequences for each BAB sequence that represent specific binders in the analyzed sample.

[0104] The kit according to the present invention comprises: a) at least one binding agent for at least one target molecule in a biological sample conjugated to a tagged oligonucleotide, wherein the tagged oligonucleotide comprises: i) a nucleic acid payload sequence (PL) comprising a binder barcode sequence (BAB) and a unique molecular identifier sequence (UMI); ii) at least one first tagged oligonucleotide amplification sequence (5-TOS); a binder; b) at least one primer for performing whole genome amplification and at least one primer for performing tagged oligonucleotide amplification, the primers having the same sequence; All at least one primer for performing genome amplification and at least one primer for performing amplification of the tagged oligonucleotide Includes:

[0105] In a preferred embodiment, the kit includes an oligonucleotide for extending the tagged oligonucleotide.

[0106] In a preferred embodiment, at least one tagged oligonucleotide has a sequence corresponding to SEQ ID NO: 1, and there are two types of primers for amplifying the tagged oligonucleotide, each having the sequences of SEQ ID NO: 2 and SEQ ID NO: 3. The kit preferably further comprises one or more first library primers and one or more second library primers. More preferably, the first library primer has a sequence selected from the group consisting of SEQ ID NO: 8 to SEQ ID NO: 15, and the second library primer has a sequence selected from the group consisting of SEQ ID NO: 16 to SEQ ID NO: 27.

[0107] In another preferred embodiment, at least one tagged oligonucleotide has a sequence corresponding to SEQ ID NO: 28, and there is one oligonucleotide for extending the tagged oligonucleotide, which has a sequence corresponding to SEQ ID NO: 29. The kit preferably further comprises one or more first library primers and one or more second library primers. More preferably, the first library primer has a sequence selected from the group consisting of SEQ ID NOs: 30 to 37, and the second library primer has a sequence selected from the group consisting of SEQ ID NOs: 38 to 49. [Example]

[0108] Example 1 In this example, tagged oligos were designed for WGA followed by amplification using the setup according to Figure 8. The tagged oligo, designated "P5-Synth" (SEQ ID NO: 1, Figure 10, NNNNNNNNNN:UMI sequence), was designed to be compatible with the Ampli1™ WGA kit (Menarini Silicon Biosystems). The first amplification handle sequence (1AH) was identical to the last 19 bases of the Index2 (i5) adapter from Illumina TruSeq DNA and RNA CD Indexes. The second amplification handle sequence (2AH) was generated in silico to avoid any intramolecular secondary structure and possible matches to the human genome. The melting temperatures of both amplification handle sequences were designed to be similar to those of the WGA primers. Tagged oligo amplification primers (SEQ ID NO: 2 and SEQ ID NO: 3) were designed according to the first and second amplification handle sequences.

[0109] As shown in Figure 10, the forward library primer was identical to the Index2 (i5) adapter (Illumina), while the reverse library primer was identical to the Index1 (i7) adapter with the reverse complement of the second amplification handle sequence added. More specifically, Figure 10 shows the design of the P5-Synth oligos and corresponding library primers.

[0110] Oligo P5-Synth (SEQ ID NO: 1): The white box indicates the internal domain containing the UMI and binder barcode. The grey box with black border indicates the first and second amplification handle sequences.

[0111] P5 library primer (SEQ ID NO: 8): Forward primer used for NGS library generation. The annealing site with oligo P5-Synth is shown in the gray box. The sequencing primer site is shown in the short-dashed box; the i5 index used to multiplex the sequencing reaction is shown in the dash-dotted box; and the index sequencing primer / flow cell adapter sequence is shown in the long-dashed box.

[0112] Synth library primer (SEQ ID NO: 16): Reverse primer used for NGS library generation. The annealing site with oligo P5-Synth is shown in the gray box. The sequencing primer site is shown in the short-dashed box; the i5 index used to multiplex the sequencing reaction is shown in the dash-dotted box; and the index sequencing primer / flow cell adapter sequence is shown in the long-dashed box.

[0113] As can be seen in Figure 11, during library generation, Illumina Index 1 and 2 adapters are added to the 5' and 3' ends of the tagged oligos, respectively.

[0114] In this example, tagged oligos were conjugated via a 5' amino modifier. A C6 or C12 spacer was present between the amine moiety and the 5' of the oligo to avoid any steric inhibitory effects on the subsequent PCR reaction. Antibodies were covalently attached to tagged oligos via amino-reactive reagents, using amines commonly present in antibodies from lysine, glutamine, arginine, and asparagine residues. Four Ab-oligos (Table 2) were generated using tagged oligos, and antibody oligoconjugation was performed by Expedeon (25 Norman Way, Over, Cambridge CB24 5QE, United Kingdom) with an antibody:tagged oligo stoichiometry of 1:2. Epitope localization: Indicates location relative to the cell membrane.

[0115] [Table 2]

[0116] The Ab-oligos were used to stain two different types of cell lines. The first cell type was SK-BR-3 cells, a breast cancer-derived cell line that overexpresses cytokeratin and Her2 proteins. The second cell type was peripheral blood mononuclear cells (PBMCs), white blood cells extracted from whole blood that express CD45 and negligible levels of cytokeratin and Her2.

[0117] SK-BR-3 cells (ATCC® HTB-30™, ATCC) were grown in culture according to the manufacturer's protocol. PBMCs were extracted from human blood samples. Both cell types were fixed with PFA 2% according to a customized protocol.

[0118] Cell staining with Ab-oligos was performed on 100,000–50,000 pre-fixed and permeabilized cells. Cells were harvested by centrifugation at 1,000 × g for 5 min at room temperature. Cells were washed with at least 1 mL of running buffer (autoMACS running buffer, ref. 130-091-221, Miltenyi Biotec) and harvested by centrifugation. This last step was repeated twice. External Ab-oligos and their isotype Ab-oligo controls were diluted to their working concentrations in 100 μL of running buffer. The external Ab-oligo mix (Ab-oligo tag3) was added to the cells and incubated for 15 min at room temperature. Subsequently, the samples were washed twice with 1 mL of running buffer and harvested by centrifugation. Goat anti-mouse IgG2a-PE antibody in 500 μL of running buffer was added and incubated for 30 min at +4°C. This step allowed for staining of PBMC cells with PE. The samples were washed twice with running buffer. The internal Ab-oligos and their isotype Ab-oligo controls were diluted to their working concentrations in 200 μL of Inside Perm buffer (Inside Staining Kit, Ref. 130-090-477, Miltenyi Biotec). The internal Ab-oligo mix (Ab-oligo tags 1, 2, and 4) was added to the cells and incubated for 10 min at RT. The samples were washed twice with 1 mL of Inside Perm buffer and collected by centrifugation. A mix of Hoechst and goat anti-mouse IgG1-APC antibodies in 500 μL of Inside Perm buffer was added and incubated for 30 min at +4°C. This step allowed for the staining of SK-BR-3 cells and all cell nuclei with PE. The samples were washed twice with running buffer.

[0119] Addition of a secondary antibody conjugated to a fluorophore allowed identification of SK-BR-3 cells (APC channel) and PBMCs (PE channel) by fluorescence. Furthermore, the fluorescence level reflected the relative abundance of the Ab-oligo. Single cells were purified based on their immunofluorescent labeling using the DEPArray™ NxT system (Menarini Silicon Biosystems) (Figure 12).

[0120] Specifically, Figure 12 shows scatter plots of PBMCs and SK-BR-3 cells stained with Ab-oligos and secondary fluorescent antibodies. The x-axis shows the fluorescence level in the APC channel, which is proportional to the amount of Ab-oligo tag1, tag2, and tag4. The y-axis shows the fluorescence level in the PE channel, which is proportional to the amount of Ab-oligo tag3. The scatter plots are divided into four quadrants, each containing a specific cell type based on their immunofluorescence levels: 1) PBMCs (high levels of CD45 and low levels of CK and Her2); 2) double-positive cells (high levels of CD45, CK, and Her2); 3) double-negative cells (low levels of CD45, CK, and Her2); and 4) SK-BR-3 cells (low levels of CD45 and high levels of CK and Her2). Single cells, highlighted by hollow / solid squares / circles, were isolated and used for library generation.

[0121] Alternatively, for tagged oligo amplification after WGA, a customized reaction mix of forward / reverse primers and Ampli1™ PCR kit reagents was prepared according to the left insert of Table 3. 15 μL of the reaction mix was added to each tube containing the WGA product. Each sample was incubated according to the temperature profile shown in the right insert of Table 3.

[0122] [Table 3]

[0123] Left insert: Reaction mixture composition for tagged oligo amplification reaction. Right insert: Thermal cycling program for tagged oligo amplification.

[0124] Library generation was performed by taking 1 μL aliquots of the WGA-containing Ab-oligo amplicons amplified using the Ampli1™ PCR kit with P5 and Lib1 library primers at a final concentration of 0.5 μM. The PCR thermal cycling profile is shown in Table 5. Each sample had a different combination of NGS library primers for dual indexing to demultiplex the data during bioinformatics analysis. The list of library primers used is reported in Table 4. The P5 library primer is a forward primer that can be used with the tagged oligo P5-Synth.

[0125] [Table 4A]

[0126] [Table 4B]

[0127] [Table 5]

[0128] Thermal cycling profile for NGS library generation. The number of cycles in step 3 varies depending on the number of cells harvested and the effective amount of total Ab-oligos in the cells. Typically, 27 amplification cycles in stage 3 produced sufficient amplicon amounts from a single cell.

[0129] Library samples were purified using Agencourt AmPure XP beads (Beckman-Coulter). NGS DNA quantification was performed using the KAPA SYBR® FAST qPCR kit (Kapa Biosystems). Each NGS library was examined using an Agilent Bioanalyzer 2100 (Agilent), which showed that the library electropherogram typically consisted of a single peak at 185 bp (Figure 13).

[0130] Samples were pooled together and sequenced on a MiSeq system (Illumina) using the MiSeq Reagent Kit v3 150 cycles (Ref. MS-102-3001, Illumina). Data analysis was performed using custom software developed by Python. Quantification of protein targets according to UMI counts is reported in Figure 14. As expected, SK-BR-3 cells showed high expression of cytokeratin and Her2 and relatively low levels of CD45, while PBMCs exhibited the opposite behavior. Protein expression levels were higher in double-positive cells, especially for the isotype control, indicating that such cells were more susceptible to nonspecific staining. Conversely, double-negative cells had relatively low levels of all four targets.

[0131] Example 2 In this example, tagged oligos were designed for amplification during WGA using the setup according to Figure 8. The experimental procedure was identical to Example 1 with the following exceptions: tagged oligo amplification primers were added directly to the primary PCR reaction mix at a final concentration of 0.02 μM. Library generation and data analysis were performed as described in Example 1.

[0132] Quantification of protein targets according to UMI counts is reported in Figure 15. As expected, SK-BR-3 cells showed high expression of cytokeratin and Her2 and very low levels of CD45, while PBMCs exhibited the opposite behavior. Protein expression levels were higher in double-positive cells, especially for the isotype control, indicating that such cells were relatively susceptible to nonspecific staining. Conversely, double-negative cells had relatively low levels for all four targets. According to the results from Example 1, it can be inferred that tagged oligo amplification is feasible both during and after WGA. However, it should be noted that absolute UMI counts differ significantly between the two procedures. The difference between the two cell types is more in line with what is expected for the CD45 target, which has lower expression compared to CK when tagged oligo amplification is performed during WGA.

[0133] Example 3 In this example, tagged oligos were directly added to single cells. A tagged oligo designated "P5-Lib1" (SEQ ID NO: 28) was designed to be amplifiable by the Ampli1™ WGA kit (Menarini Silicon Biosystems). The tagged oligo amplification primer had the sequence of SEQ ID NO: 29 (the forward and reverse primers were identical and shared the sequence of the Ampli1 WGA Lib1 primer). Specifically, the 5'-WGA handle sequence was identical to the Lib1 WGA primer, while the 3'-WGA handle sequence was the reverse complement sequence of the Lib1 WGA primer. The first amplification handle sequence was identical to that described in Example 1. The second amplification handle sequence consisted of the 3'-WGA handle sequence and an additional 5 bp sequence at its 5' end (Figure 16).

[0134] More specifically, Figure 16 shows the design of the P5-Synth oligos and corresponding library primers.

[0135] Oligo P5-Lib1: The white solid box with a thick border indicates the internal domain containing the UMI and binder barcode. The gray solid box with a thick border indicates the annealing sites for the two library primers. The gray box with a thin border is the WGA handle sequence (Lib1).

[0136] P5 library primer: forward primer used for NGS library generation. The annealing site with tagged oligo P5-Lib1 is shown in the gray solid box. The sequencing primer site is shown in the short-dashed box; the i5 index used to multiplex the sequencing reaction is shown in the dash-dotted box; and the index sequencing primer / flow cell adapter sequence is shown in the long-dashed box.

[0137] Lib1 library primer: reverse primer used for NGS library generation. The annealing site with tagged oligo P5-Lib1 is shown in the gray solid box: this sequence consists of a portion of the Lib1 reverse complement sequence and a small tail (ACCAC) that allows annealing only to the 3' end of oligo P5-Lib1. The sequencing primer site is shown in the short-dashed box; the i5 index used to multiplex the sequencing reaction is shown in the dash-dotted box; and the index sequencing primer / flow cell adapter sequence is shown in the long-dashed box.

[0138] The forward library primer was identical to the Index2 (i5) adapter (Illumina), while the reverse library primer was identical to the Index1 (i7) adapter with the reverse complement of the second amplification handle sequence added (Figure 16). Thus, during library construction, Illumina Index1 and 2 adapters are added to the 5' and 3' ends of the tagged oligos, respectively (Figure 17).

[0139] SK-BR-3 cells (ATCC® HTB-30™, ATCC) were grown in culture according to the manufacturer's protocol and fixed with 2% PFA according to a customized protocol. Single cells were purified based on their morphology using the DEPArray™ NxT system (Menarini Silicon Biosystems). P5-Lib1 and P5-Synth tagged oligos were added directly to the inside of a tube containing single cells. Different amounts of each oligo were added to each single cell, and Ampli1™ WGA was performed. Samples containing P5-Synth tagged oligos were amplified as in Example 1.

[0140] Tagged oligo library generation was performed by taking 1 μL aliquots of the WGA-containing Ab-oligo amplicons amplified using the Ampli1™ PCR kit with P5 and Lib1 library primers at a final concentration of 0.5 μM. The PCR thermal cycling profile is shown in Table 5. Each sample had a different combination of NGS library primers for dual indexing to demultiplex the data during bioinformatics analysis. A list of the library primers used is reported in Table 6.

[0141] [Table 6A]

[0142] [Table 6B]

[0143] Aliquots of 10 μL WGA samples were purified using SPRI beads (Beckmann Coulter) and subsequently processed using the Ampli1™ LowPass kit to generate NGS libraries for CNA analysis. Spiking tagged oligos into single cells prior to the WGA procedure did not affect downstream genetic analysis ( FIG. 18 ). Tagged oligos designed to fit the workflows shown in FIGS. 5 and 8 did not affect or interfere with the WGA procedure. Furthermore, it was still possible to obtain NGS libraries from tagged oligonucleotides under both conditions. Both tagged oligonucleotides could be accurately quantified, demonstrating the robustness of both methodologies as well as the tagged oligo design ( FIG. 19 ).

[0144] advantage The method for whole genome amplification and analysis of multiple target molecules in biological samples according to the present invention allows for simultaneous obtaining of genome-wide copy number profile / genomic sequence and analysis of protein expression for the same single cell.

[0145] The method of the present invention allows for further analysis, such as genome-wide copy number profiling by low-pass sequencing or targeted sequencing of a panel of genes of interest, and allows for whole genome amplification of genomic DNA, which is useful for detecting and performing digital quantification of a panel of multiple proteins down to single-cell resolution, using very small samples, as few (down to one) circulating tumor cells (CTCs) may be available. This is particularly advantageous for measuring each molecular type in different cells in genetically heterogeneous samples, where differences in genotype, phenotype, and environment can confound and completely prevent the correlation between genotype (copy number of sequence variations) and phenotype (protein expression).

[0146] The method according to the present invention surprisingly advances the state of the art, with achievements previously considered unattainable by those skilled in the art in one or more of the following aspects, given as non-limiting examples: Digital quantification of proteins in single cells down to a few hundred copies per cell. The use of WGA inherent in the process achieves the above with the added possibility of obtaining additional genetic material for investigation of other characteristics of the single cell, as well as the possibility of reliably reanalyzing the single cell for confirmation, which is not possible using droplet-based approaches such as those proposed by 10x Genomics.

[0147] The main field of application of this method is oncology, but the method can be applied in other fields such as mosaic disorders or neoproliferative phenotypes, such as dermatology.

Claims

1. 1. A method for whole genome amplification and analysis of multiple target molecules in a biological sample containing genomic DNA and target molecules, comprising: a) contacting the biological sample with at least one binding agent for at least one of the target molecules conjugated to a tagged oligonucleotide, wherein the tagged oligonucleotide: i) a nucleic acid payload sequence (PL) comprising a binder barcode sequence (BAB) and a unique molecular identifier sequence (UMI), and ii) at least one first tagged oligonucleotide amplification sequence (5-TOS) of the nucleic acid; whereby, if at least one target molecule is present in the biological sample, the at least one binding agent binds to the at least one target molecule; b) obtaining the labeled biological sample by performing a separation step to selectively remove unbound binding agent; c) administering to the labeled biological sample: - i) deterministic restriction site whole genome amplification (DRS-WGA), or ii) Multiple Annealing and Looping-Based Amplification Cycle Whole Genome Amplification (MALBAC) whole genome amplification of the genomic DNA by - amplifying the tagged oligonucleotide conjugated to said at least one binding agent; wherein whole genome amplification and amplification of the tagged oligonucleotides are performed simultaneously; d) generating a massively parallel sequencing library from the amplified tagged oligonucleotides; e) sequencing the massively parallel sequencing library; f) searching for the sequences of the binder barcode sequence (BAB) and unique molecular identifier sequence (UMI) from each sequencing read; g) counting the number of distinct unique molecular identifier sequences (UMIs) for each binding agent A method comprising:

2. 10. The method of claim 1, wherein the tagged oligonucleotide further comprises at least one second tagged oligonucleotide amplification sequence (3-TOS).

3. 3. The method of claim 1 or 2, wherein the unique molecular identifier sequence (UMI) is a degenerate or semi-degenerate sequence ranging from 10 to 30 nucleotides.

4. 2. The method of claim 1, further comprising the step of isolating a single cell from the biological sample, wherein the isolating step is performed after step b) and before step c).

5. The method of claim 4, wherein the isolating step is carried out by sorting the cells.

6. 5. The method of claim 4, wherein the isolating step is performed by dividing the cells into droplets.

7. 2. The method of claim 1, further comprising purifying the massively parallel sequencing library prior to step e).

8. The at least one binder is a) an antibody or a fragment thereof; b) aptamers, c) small molecules; d) peptides, and e) protein 2. The method of claim 1, selected from the group consisting of:

9. The target molecule is a) proteins, b) peptides, c) glycoproteins, d) carbohydrates, e) lipids, and f) any combination thereof 2. The method of claim 1, selected from the group consisting of:

10. The tagged oligonucleotide comprises, in a 5' to 3' direction, at least a) a nucleic acid first tagged oligonucleotide amplification sequence (5-TOS) further comprising a 5' whole genome amplification handle sequence (5-WGAH) and a first amplification handle sequence (1AH); b) payload sequence (PL); c) a second tagged oligonucleotide amplification sequence of nucleic acid (3-TOS), further comprising a second amplification handle sequence of nucleic acid (2AH) and a 3' whole genome amplification handle sequence (3-WGAH); 3. The method of claim 2, comprising:

11. 2. The method of claim 1, wherein the whole genome amplification and the amplification of the tagged oligos are performed using a single primer.

12. The tagged oligonucleotide comprises, in a 5' to 3' direction, at least a) a nucleic acid first tagged oligonucleotide amplification sequence (5-TOS) further comprising a 5' whole genome amplification handle sequence (5-WGAH) and a first amplification handle sequence (1AH); b) payload sequence (PL); c) Optionally, an annealing sequence (AS) At least one primer is used for whole genome amplification and amplification of the tagged oligonucleotide, and at least one oligonucleotide (Ep) is used for extension of the tagged oligonucleotide, and the at least one oligonucleotide (Ep) comprises, from 5' to 3', at least: d) 5′ whole genome amplification handle sequence (5-WGAH); e) spacer sequence (SS); f) a second amplification handle sequence (2AH); and g) a sequence reverse complementary to the annealing sequence (AS-RC) or a sequence reverse complementary to the binder barcode sequence (BAB-RC) 2. The method of claim 1, comprising:

13. The tagged oligonucleotide comprises, in a 5' to 3' direction, at least a) a first tagged oligonucleotide amplification sequence (5-TOS) of a nucleic acid corresponding to a first amplification handle sequence (1AH); b) payload sequence (PL); c) a second tagged oligonucleotide amplification sequence (3-TOS) of a nucleic acid corresponding to the second amplification handle sequence (2AH); wherein at least one first primer is used for whole genome amplification, and at least one second primer and one third primer are used for amplification of the tagged oligonucleotide; 2. The method of claim 1, wherein the at least one second primer has a sequence identical to the first amplification handle sequence (1AH) and the at least one third primer has a sequence that is reverse complementary to the second amplification handle sequence (2AH-RC).

14. 14. The method of claim 13, wherein the at least one second primer and at least one third primer are added in step c).

15. 11. The method of claim 10, wherein step d) of generating a massively parallel sequencing library from the amplified tagged oligonucleotides is performed by a PCR reaction using at least one first library primer comprising a 3' sequence corresponding to a first amplification handle sequence (1AH) and at least one second library primer comprising a 3' sequence corresponding to a reverse complementary sequence to a second amplification handle sequence (2AH-RC).

16. a) at least one binding agent for at least one target molecule in a biological sample conjugated to a tagged oligonucleotide, wherein the tagged oligonucleotide comprises: i) a nucleic acid payload sequence (PL) comprising a binder barcode sequence (BAB) and a unique molecular identifier sequence (UMI); ii) at least one first tagged oligonucleotide amplification sequence (5-TOS); a binder; b) at least one primer for performing whole genome amplification and at least one primer for performing amplification of the tagged oligonucleotide, the at least one primer for performing whole genome amplification and at least one primer for performing amplification of the tagged oligonucleotide having the same sequence; 10. A kit for carrying out the method of claim 1, comprising:

17. 17. The kit of claim 16, further comprising an oligonucleotide for extending the tagged oligonucleotide.

18. 17. The kit of claim 16, wherein the at least one tagged oligonucleotide has a sequence corresponding to SEQ ID NO: 1, and there are two types of primers for amplifying the tagged oligonucleotide, the primers having the sequences of SEQ ID NO: 2 and SEQ ID NO: 3, respectively.

19. 19. The kit of any one of claims 16 to 18, further comprising one or more first library primers and one or more second library primers.

20. 20. The kit of claim 19, wherein the first library primer has a sequence selected from the group consisting of SEQ ID NO:8 to SEQ ID NO:15, and the second library primer has a sequence selected from the group consisting of SEQ ID NO:16 to SEQ ID NO:

27.

21. 17. The kit of claim 16, wherein the at least one tagged oligonucleotide has a sequence corresponding to SEQ ID NO: 28, and the primer for amplifying the tagged oligonucleotide is one type and has a sequence corresponding to SEQ ID NO:

29.

22. 22. The kit of claim 21, further comprising one or more first library primers and one or more second library primers.

23. 23. The kit of claim 22, wherein the first library primer has a sequence selected from the group consisting of SEQ ID NO:30 to SEQ ID NO:37, and the second library primer has a sequence selected from the group consisting of SEQ ID NO:38 to SEQ ID NO:49.

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