Patent Application
20230053146
This patent application claims priority from Italian patent application no. 102019000024159 filed on Dec. 16, 2019, the entire disclosure of which is incorporated herein by reference.
The present invention relates to a method and a kit for whole genome amplification and analysis of target molecules, in particular quantification of proteins, in a biological sample, in particular in a single cell sample.
Methods for single-cell analysis allow to obtain information on the status of a cell without the complication resulting from heterogeneity in a bulk sample. Analysis of proteome and genome in the same single cell provides correlations between the cell's phenotype and its genotype, thus enabling unique insights into diverse biological and pathological processes. This is particularly true for tumors, in which somatically acquired genetic heterogeneity and its effect on transcription and protein translation are key components of the initiation and evolution of cancer.
Whole Genome Amplification (WGA) is useful to analyze the genome from single cells in order to obtain more DNA and simplify and/or allow different types of genetic analyses, including sequencing, SNP detection etc. WGA with a LM-PCR based on a Deterministic Restriction Site (DRS-WGA) is known from EP1109938.
WO 2017/178655 and WO 2019/016401 teach a simplified method to prepare massively parallel sequencing libraries from DRS-WGA (e.g. Ampli1™ WGA) or MALBAC for low-pass whole genome sequencing and copy number profiling.
Recently, methods for the simultaneous analysis of genome and transcriptome in single cells have been developed. The 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 by which messenger RNAs from single cells isolated by hand are first transcribed to single stranded cDNA and then amplified together with genomic DNA through a quasilinear whole-genome amplification. Two different libraries, one from cDNA and one from genomic DNA are then prepared and sequenced. In another approach, Macaulay, I. C., 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 the polyadenylated mRNA molecules from a fully lysed single cell. The mRNA is then amplified using a modified Smart-seq2 protocol (Picelli, S. et al., 2013, Smart-seg2 for sensitive full-length transcriptome profiling in single cells. Nature Methods, 10(11), 1096-1100. http://doi.org/10.1038/nmeth.2639), while the gDNA can be amplified with available whole genome amplification methods and sequenced. These methods, while useful to link genotype to messenger transcription, do not allow to get a direct detection of proteins, the translation and turn-over/degradation of which are actively regulated in the cell.
Currently, the most widely applied single-cell protein detection approaches rely on targeting specific proteins using tagged antibodies. Fluorescence-based detection and quantitation of proteins by fluorescence-activated cell sorting (FACS) or fluorescence microscopy allow protein detection in single cells with a low level of multiplexing by means of fluorescently labeled antibodies recognizing specific cell proteins. However this approach is generally limited to 10-15 simultaneous measurements as fluorophore-based highly multiplexed assays are challenged by spectral overlap between the emission spectra of multiple dyes. Moreover, complex algorithms are needed to deconvolute the overlapping spectra.
Fluidigm mass cytometer (CyTOF™) employs metal-containing polymer tagged (MAXPAR™) antibodies to detect proteins. The instrument is based on a non-optical physical principle of detection and a different chemical nature of labels. The fluorescent labels are replaced by specially designed multi-atom elemental tags and detection takes advantage of the high resolution, sensitivity, and speed of analysis of Time-of-Flight Mass Spectrometry (TOF-MS). Since many available stable isotopes can be used as tags, many proteins can potentially be detected simultaneously 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]. The work 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 RNAs and proteins in single cells based on Proximity Ligation Assay for RNA (PLAYR). PLAYR enables highly multiplexed quantification of transcripts in single cells by mass-cytometry enabling simultaneous quantification of more than 40 different mRNAs and proteins. Finally, mass cytometry allowed to investigate multiple cellular processes and phenotypic characteristics, along with proteins and messenger RNAs transcription, such as protein phosphorylation (Bendall, S. C. 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 cellular proliferation (Behbehani, G. K. 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).
The limitations of these approaches are that:
Methods for the detection of proteins through oligonucleotide-labeled antibodies have been 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, which teaches a technique (Proximity Ligation Assay; PLA) in which the coordinated and proximal binding of a target protein by two DNA aptamers promotes ligation of oligonucleotides linked to each aptamer affinity probe. The ligation of two such proximity probes gives rise to an amplifiable DNA sequence that reflects the identity and amount of the target protein. The method 3PLA (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 the proximity ligation method by using three recognition events and allows to detect as little as a hundred target molecules. In 3PLA, sets of three oligonucleotide-modified antibody reagents bind individual target proteins to give rise to a detectable signal by proximity ligation. The 3′ and 5′ ends of oligonucleotides on two proximity probes are capable of hybridizing to an oligonucleotide present on a third proximity probe forming a complex involving the three probes and the target protein. This allows the two oligonucleotides to be connected via the intermediary fragment by two ligation reactions, templated by the third proximity probe, forming a specific, amplifiable DNA strand that can be detected by qPCR. Proximity Extension Assay (PEA) is a variation of PLA in which 2 oligonucleotide-labeled antibodies bind an individual protein, the oligonucleotides partially anneal at their 3′ end and an extension by a polymerase produces an amplifiable DNA sequence which 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). While the above disclosed methods were not designed specifically for single-cell protein detection, the Fluidigm C1™ single cell auto prep system was employed to automate the preparation of amplifiable targets of 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™ 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 the analysis of transcriptome or genomic DNA sequence by whole exome sequencing and targeted DNA sequencing, however the methods cannot be easily combined to obtain information on genotype and phenotype from the same single cells.
Thus, PLA and PEA assays, in which detection is based on qPCR, are sensitive and highly specific, but are limited in their throughput and can only detect proteins.
U.S. Pat. No. 9,714,937 by NanoString Technologies, Inc. teaches methods for the detection of proteins through the use of a capture antibody conjugated to a moiety, such as biotin, specific for a first region of a target protein and a detection antibody, for a second region of the target protein, with a nanoreporter comprising multiple detachable labels linked to the detection antibody through hybridization to a linker oligonucleotide. The two antibodies form a complex with the target protein which can form a bond with a matrix or a bead with high affinity for the moiety. The target is detected and quantified by counting the number of nanoreporter molecules. Commercially available assays from Nanostring, Inc. based on nCounter® digital molecular barcoding technology, detect proteins using uniquely oligonucleotide-labeled antibodies targeting specific protein epitopes. The unique single-stranded DNA tags are detected using a combination of a biotinylated capture probe and a reporter probe made by a single-stranded DNA molecule annealed to a series of fluorescently labeled RNA segments. The linear order of these labels creates a unique barcode for each target of interest. Complexes are then immobilized to an imaging surface through a non-covalent bond between biotin and immobilized streptavidin molecules and fluorescent barcodes are imaged and counted. The number of counts per protein-specific barcode is a digital measure directly related to the number of molecules present in the sample. Protein detection can be combined to messenger RNAs detection by using capture probe-reporter probe couples designed on specific target RNAs. About 30 protein targets and 770 mRNA targets can be analyzed in a single analysis.
The disadvantages of this method is that it requires large quantities of cells to profile RNAs (equivalent to 2,500 cells) and/or to profile proteins (equivalent to 100,000 cells) and that it is not suitable, as is, to profile single cells. Potentially the method can be used to detect other analytes, such as genomic DNA, however it cannot provide a direct readout of the genomic sequence but only a signal of presence/absence of a known sequence. Being based on hybridization it is also partially tolerant to sequence variants and may not be able to distinguish different sequence variants.
An NGS-based method for integrated analysis of multiple proteins and RNA transcripts in single cells, named cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq), was first described in Stoeckius et al., 2017, Simultaneous epitope and transcriptome measurement in single cells, Nature Methods volume 14, pages 865-868, and US 2018/0251825. The method relies on oligonucleotide-labeled antibodies which are used to integrate cellular protein and transcriptome measurements into a single-cell readout through a 3′-poly adenosine tail present on antibody tags analogous to that present on messenger RNAs. The method is compatible with droplet-based approaches for sample partitioning in single cells and single-cell library preparation, such as that provided by 10× Genomics. In more detail, in the CITE-seq method, cells stained with oligonucleotide-labeled antibodies for cell surface epitopes are partitioned in oil droplets containing lysing enzymes and barcoded beads by microfluidics means. Barcoded antibodies and mRNAs from each single cell/droplet are captured by beads with a unique cell barcode. mRNAs are then retro-transcribed and amplified along with the oligo from barcoded antibodies, generating NGS libraries ready for sequencing. Finally, sequence counts are used for the quantification of 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, RNA expression and protein quantification assay (REAP-seq), based on DNA-labeled antibodies and droplet microfluidics, by which proteins can be quantified using 82 barcoded antibodies and the transcription of >20,000 can be profiled in single cells. Both the above mentioned methods exploit the DNA polymerase activity of the reverse transcriptase to simultaneously extend the primed oligo-labeled antibodies with a poly(dT) cell barcode and synthesize complementary DNA from mRNA in the same reaction. On the other hand, other methods, also based on droplet approaches, are available for the analysis of genome-wide copy number profiles or for the analysis of the genome sequence in single cells. For example, the commercial solution Chromium Single cell CNV Solution by 10× Genomics allows 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 panels of genes.
The drawbacks of these methods are that:
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 that simultaneously allows analysis of genome-wide copy-number profiles/genome sequence and analysis of protein expression on the same single cells, overcoming in particular one or more of the following drawbacks of the state of the art:
This object is achieved by the method as defined in claim 1.
A further object of the present invention is to provide a kit as defined in claim 17.
Unless defined otherwise, 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 pertains. Although many methods and materials similar or equivalent to those described herein may be used in the practice or testing of the present invention, preferred methods and materials are described below. Unless mentioned otherwise, the techniques described herein for use with the invention are standard methodologies well known to persons of ordinary skill in the art.
By “ab-oligo mix” there is intended a solution containing all ab-oligos that target a cell's internal (namely “internal ab-oligo mix”) and/or external (namely “external ab-oligo mix”) epitopes and/or isotypic control ab-oligos.
By “antibody-oligonucleotide conjugate” or “ab-oligo conjugate” or “ab-oligo” there are intended synthetic molecules derived by the chemical conjugation of an antibody molecule with ssDNA oligonucleotide molecules. Chemical conjugation is usually performed using specific chemical reactions that enable the linking of the two molecules in a covalent manner. The antibody:oligonucleotide stoichiometry can be controlled in order to have a specified ratio. During WGA initial steps, the antibody moiety is usually digested and only the oligonucleotide part remains. For the sake of simplicity, in the description these molecules will still be referred to as ab-oligo molecules or ab-oligo amplicons.
By the acronym “APC” there is intended the fluorophore allophycocyanin.
By “binding agent barcode sequence (BAB)” there is intended a unique DNA oligonucleotide sequence that identifies the binding agent.
By “balanced PCR amplification” there is intended the feature of a PCR to perform an amplification of multiple targets whereby, in each PCR cycle, substantially every target molecule is amplified.
By “binding agent” there is intended a molecule (such as, byway of a non-limiting example, antibodies, affibodies, ligands, aptamers, synthetic binding proteins, small molecules) able to specifically bind designated target molecules, for example proteins or glycosylated proteins or phosphorylated proteins.
By “CITE-Seq” or “Cellular Indexing of Transcriptomes and Epitopes by Sequencing” there is intended the method developed by Stoeckius et al. for simultaneous protein quantification and mRNA sequencing in single-cells.
By “CyTOF” or “cytometry by time of flight” there is intended an equipment carrying out mass-cytometry technique, enabling the quantification of proteins in single cells using mass spectrometry in combination with cytometry. Cells are stained with binding agents conjugated with heavy metal isotopes.
By the term “conjugate” there is intended a molecule obtained from the covalent conjugation of a binding agent and a tagged oligonucleotide.
By “copy number alteration (CNA)” there is intended a somatic change in copy-numbers of a genomic region, defined in general with respect to the same individual genome.
By “DNA library purification” there is intended a process whereby the DNA library material is separated from unwanted reaction components such as enzymes, dNTPs, salts and/or other molecules which are not part of the desired DNA library. Examples of DNA library purification processes are purification with Agencourt AMPure, or Merck Millipore Amicon spin-columns or solid-phase reversible immobilization (SPRI)-beads such as from Beckman Coulter.
By “DNA library quantification” there is intended a process whereby the DNA library material is quantified. Example of DNA library quantification processes are quantification using QuBit technology, electrophoretic assays (Agilent Bioanalyzer 2100, Perkin Elmer LabChip technologies) or RT-PCR PicoGreen system (Kapa Biosystems).
By “dynamic range” there is intended the ratio between the largest and smallest values that a certain quantity can assume.
By “library primers” there is intended ssDNA molecules that act as primers in order to generate massively-parallel sequenceable libraries from tagged oligonucleotides.
By “low-pass whole genome sequencing” or “lowpass sequencing” there is intended a whole genome sequencing at mean sequencing depth lower than 1.
By “massive-parallel sequencing” or “next generation sequencing (NGS)” there is intended a method of sequencing DNA comprising the creation of a library of DNA molecules spatially and/or time separated, clonally sequenced (with or without prior clonal amplification). Examples include Illumina platform (Illumina Inc), IonTorrent platform (ThermoFisher Scientific Inc), Pacific Biosciences platform, MinIon (Oxford Nanopore Technologies Ltd).
By “Multiple Annealing and Looping Based Amplification Cycles (MALBAC)” there is intended a quasilinear whole genome amplification method (Zong et al., 2012, Genome-wide detection of single-nucleotide and copy-number variations of a single human cell, Science. December 21; 338(6114):1622-6. doi: 10.1126/science.1229164.). MALBAC primers have an 8 nucleotide 3′ random sequence to hybridize to the template, and a 27 nucleotides 5′ common sequence (GTG AGT GAT GGT TGA GGT AGT GTG GAG). After first extension, semiamplicons are used as templates for another extension yielding a full amplicon which has complementary 5′ and 3′ ends. Following few cycles of quasi-linear amplification, full amplicon can be exponentially amplified with subsequent PCR cycles.
By the term “oligonucleotide” or “oligo” there is intended an oligomeric molecule comprising a sequence of nucleotides such as, by way of non-limiting example, deoxyribonucleic acid (DNA), ribonucleic acid (RNA), locked nucleic acid (LNA), peptide nucleic acid (PNA).
By “tagged oligonucleotide” or “tagged oligo” there is intended an oligonucleotide molecule (e.g. ssDNA molecule) that is directly conjugated to a binding agent (e.g. a primary antibody). The tagged oligo is used for indirect quantification of the target-molecule (e.g. a protein) which is a ligand for the binding agent.
By the acronym “PE” there is intended the fluorophore phycoerythrin.
By “PFA 2%” there is intended a solution at 2% w/v of paraformaldehyde in phosphate buffer saline.
By “primary WGA DNA library (pWGAlib)” there is intended a DNA library obtained from a WGA reaction.
By the term “re-amplification” or “re-amp” there is intended a PCR reaction where all or a substantial portion of the primary WGA DNA library is further amplified.
By “residues” there are intended the amino acid residues present in polypeptide chains in proteins.
By “sequencing barcode” there is intended a polynucleotide sequence which, when sequenced within one sequencer read, allows to assign that read to a specific sample associated with that barcode.
By “UMI” or “Unique Molecular Identifier sequence” there is intended degenerate or partially-degenerate (i.e. random or semi-random) oligonucleotide sequences which are virtually unique for each ssDNA or dsDNA molecule.
By “universal WGA-primer” or “WGA PCR Primer” there is intended the additional oligonucleotide ligated to each fragment generated by the action of the restriction enzyme. Universal WGA-primer are used in DRS-WGA such as Ampli1™ WGA
The method for whole genome amplification and analysis of multiple target molecules in a biological sample including genomic DNA and target molecules according to the present invention comprises the following steps.
In step a), the biological sample is provided. The biological sample is preferably a single cell, but can also be a sample comprising several cells.
In step b), the biological sample is contacted with at least one binding agent, which is directed to at least one of the target molecules, conjugated with a tagged oligonucleotide, so that—when 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.
The binding agent 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 binding agent is an antibody. Binding agents bind target molecules with specific stoichiometry such as monoclonal antibodies or enzyme substrates, or with unspecified stoichiometry such as polyclonal antibodies or small molecules. The former enable a better quantification of the target with respect to the latter. Binding agents are chemically conjugated to tagged oligos via covalent bond interactions or non-covalent interactions. In the former case, both oligos and binding agents possess reactive moieties which enable reciprocal binding. Binding agent:oligo stoichiometry can be controlled during conjugation procedures.
A non limiting list of examples of binding-agent/target molecules is reported in the following Table 1.
Oligonucleotides used as tagged oligonucleotides are preferably ssDNA or dsDNA molecules with a chemical modification on the 5′ or 3′ end. This modification is used for the covalent conjugation with the relative binding agent.
Conjugates formed by the tagged oligos conjugated with the binding agents may target both extra-cellular and intracellular epitopes. “External” and “internal” conjugates can be applied to the biological sample as two separate mixes containing the conjugates at their final staining concentration. Firstly, an external mix is applied to label external epitopes. Secondly, cells are permeabilized using detergents or similar means and an internal mix is applied to the sample to label internal epitopes. Alternatively, external and internal conjugates can be mixed together as well to perform a one-step staining. The final staining concentration varies for each binding agent and must be determined experimentally.
The tagged oligo sequence is preferably shorter than 300 bases, more preferably shorter than 120 bases to facilitate conjugation with the binding agent and to reduce cost. In a preferred embodiment, the tagged oligo sequence is between 60-80 nucleotides.
With reference to
The payload sequence contains the necessary information for target counting.
The unique molecular identifier sequence (UMI) is preferably a degenerate or semi-degenerate sequence in the range from 10 to 30 nucleotides. Preferably the UMI has a length of at least 10 bases, corresponding to a theoretical 4{circumflex over ( )}10=1,048,576 different combinations, which is enough for most target molecules. For highly abundant target molecules, a longer UMI may be used to increase the possible combinations, such as 12 bases. Using semi-degenerate bases, the possible combination decrease and the UMI length is preferably increased, for example up to 20 or 30 bases. Semi-degenerate UMIs may be advantageously used for introducing reference points which can be used in the read-out to re-align the sequence preventing an overestimation of the distinct UMIs present. The UMI sequence may be located at either the 5′ or the 3′ of the BAB. The UMI sequence is preferentially located just after the Read 1 sequencing primer annealing site to increase the complexity of the first sequenced bases. This is advantageous for Illumina sequencing platforms as initial sequencing steps require high complexity for cluster discrimination. BAB is a fixed sequence for each conjugate molecule. BAB are designed in order to avoid features which may interfere with primary PCR amplification and sequencing steps, such as homopolymers, hairpins, and/or heteroduplex formation [Frank, D. N., 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 the pool of all possible BAB sequences of a defined length in order to maximize their relative Hamming distance, thus minimizing the possibility that any PCR or sequencing error might lead to an incorrect assignment of the sequenced read. BAB length must be selected based on the number of target molecules to be detected. Preferably the BAB has a length of at least 10 nucleotides, corresponding to theoretical 4{circumflex over ( )}10=1,048,576 different combinations which are reduced to about 2000 possible BAB sequences after applying filters on GC content (for example: [30% . . . 70%]), absence of homopolymers, absence of hairpins and minimum hamming distance (preferably ≥3 nt).
The first tagged oligonucleotide amplification sequence (5-TOS) is located at the 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 while processing samples which may hamper proper UMI counting.
With reference to
In a preferred embodiment 5-TOS and 3-TOS sequences are designed to avoid the formation of hairpins and other intramolecular stable secondary structures within the amplification temperature range, as they may hamper tagged oligo amplification.
Tagged oligos and their amplification primers are optimized to achieve high sensitivity, extensive dynamic range, balanced PCR amplification and reproducibility.
In preferred embodiments of the present invention the UMI sequence length was selected (n=10) to quantify targets in the range of 0 to ˜106 molecules.
Dynamic range was characterized by amplification of tagged oligos with a concentration spanning four orders of magnitude (from 102 to 106 molecules).
Balanced PCR amplification was characterized by performing different cycles of amplification on the same starting sample. As shown in
Sensitivity was characterized by amplification of a pool of tagged oligos in different quantities (down to 40 molecules). As shown in
In step c) of the method according to the invention, a separating step is carried out to selectively remove unbound binding agent, thus obtaining a labeled biological sample. The separating step is typically carried out by washing in a suitable buffer solution and collecting the labelled biological sample by centrifugation.
In step d), a whole genome amplification of said genomic DNA and an amplification of the tagged oligonucleotide conjugated with the at least one binding agent are carried out simultaneously on the labeled biological sample. The whole genome amplification of the genomic DNA is carried out by either deterministic restriction-site whole genome amplification (DRS-WGA), or by Multiple Annealing and Looping Based Amplification Cycles whole genome amplification (MALBAC).
In step e) a massively parallel sequencing library is prepared from the amplified tagged oligonucleotide. In step f), the massively parallel sequencing library is sequenced.
In step g), the sequences of the binding-agent barcode sequence (BAB) and unique molecular identifier sequence (UMI) are retrieved from each sequencing read.
In step h), the number of distinct unique molecular identifier sequences (UMI) is counted for each binding agent.
Steps e), f), g) and h) will be disclosed in greater detail with reference to specific embodiments later on in the description.
The above-disclosed method preferably further comprises a step of isolating a single cell from the biological sample. Isolation may be carried out by sorting cells, in particular e.g. using a cell sorter such as DEPArray® NxT (Menarini Silicon Biosystems S.p.A.), or—as an alternative—by partitioning cells into droplets. The step of isolating is preferably performed after step c) and before step d).
The above-disclosed method preferably comprises a step of purifying the massively parallel sequencing library before step f).
In more specific terms but with no intent to limit the scope of the present description, the above-disclosed method, also designated as Ampli1 Protein (A1-P), allows the quantification of proteins and whole genome genetic characterization of single cells. Single or multiple proteins quantification in single-cells is achieved using a panel of binding agents (in particular, antibodies (Ab)) conjugated with tagged oligonucleotides. These oligonucleotides are designed to unambiguously identify the conjugated antibody by means of a DNA barcode sequence and to quantify the abundance of the epitope of interest by means of a random or partially degenerate sequence, namely Unique Molecular Identifiers (UMI), which is used for epitope quantification. Biological samples are 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 DEPArraym N×T system) and their genomic content can be amplified by whole genome amplification (i.e Ampli1™ Whole-Genome-Amplification kit). During the latter step o right after it, tagged oligonucleotides are pre-amplified to avoid any downsampling during NGS library preparation procedure. Specific primers, namely “library primers” are used to generate NGS (Illumina) libraries ready to be sequenced. Tagged Oligonucleotides are designed to be compatible with the Ampli1™ WGA (A1-WGA) workflow, enabling single-cell genetic analyses (for example Ampli1™ LowPass) in parallel with protein quantification using A1-P.
In the following, three specific embodiments of the present invention are disclosed, which respectively employ a different number of primers for tagged oligo amplification and whole genome amplification.
In a first preferred embodiment and with reference to
a) the first tagged oligonucleotide amplification sequence of nucleic acid (5-TOS), comprising in turn a 5′ whole genome amplification handle sequence (5-WGAH) and a first amplification handle sequence (1AH);
b) the payload sequence (PL);
c) the second tagged oligonucleotide amplification sequence of nucleic acid (3-TOS), comprising in turn a second amplification handle sequence of nucleic acid (2AH) and a 3′ whole genome amplification handle sequence (3-WGAH).
The 3-WGAH is the reverse complementary sequence of 5-WGAH enabling simultaneous amplification of gDNA and tagged oligonucleotides during whole genome amplification. The 1AH and the 2AH are located at the 5′ and 3′ ends of the payload sequence, respectively, and are used for subsequent library generation. The 1AH and 2AH are preferably designed to avoid stable intramolecular secondary structures, such as hairpins, which may inhibit tagged oligo amplification. Between each aforementioned sequence, fixed sequences may be present.
The whole genome amplification and the amplification of the tagged oligo are preferably carried out using a single primer.
In a second preferred embodiment and with reference to
a) the first tagged oligonucleotide amplification sequence of nucleic acid (5-TOS), comprising in turn a 5′ whole genome amplification handle sequence (5-WGAH) and a first amplification handle sequence (1AH);
b) the 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 (E-p) is used
for the extension of the tagged oligonucleotide, said at least one oligonucleotide (E-p) comprising from 5′ to 3′ at least:
d) the 5′ whole genome amplification handle sequence (5-WGAH);
e) a 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 binding-agent barcode sequence (BAB-RC).
In other words, amplification of the tagged oligo occurs by annealing of E-p to AS located at the 3′ of the tagged oligo (
After the extension of the tagged oligo and E-p, the WGA primer will perform the amplification of tagged oligos, within the resulting larger molecule. The spacer sequence (SS) increases the length of the amplicons generated by the tagged oligos. The increased fragment length destabilizes intramolecular secondary structures, such as hairpins induced by the fragment's complementary ends, thus decreasing their melting temperature (
In a third preferred embodiment and with reference to
At least one first primer is used for whole genome amplification and at least one second and one third primer are used for the amplification of the tagged oligonucleotides, the at least one second primer having a sequence identical to the first amplification handle sequence (1AH) and the at least one third primer having a sequence reverse complementary to the second amplification handle sequence (2AH-RC).
Tagged oligo amplification primers are designed to have a melting temperature that is compatible with at least the first 10-15 cycles of the WGA PCR thermal profile.
Preferably, the at least one second primer and at least third primer are added in step d).
Step e) of preparing a massively parallel sequencing library from the amplified tagged oligonucleotide is preferably performed 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 sequence reverse complementary to the second amplification handle sequence (2AH-RC).
Library primers are therefore advantageously used to generate NGS libraries for binding agent quantification analysis with a single PCR step. The specific examples of library primers reported in the present description are used to generate libraries compatible with the Illumina sequencing platform, without this intending to limit the scope of invention of the present invention.
In a preferred embodiment forward and reverse primers are designed on the basis of Illumina adapters (Illumina), comprising from 5′ to 3′:
1) an Illumina adapter sequence (IA): required for Illumina sequencing;
2) A first amplification handle sequence (1AH) or a sequence reverse complementary to the second amplification handle sequence (2AH-RC): these sequences anneal with the reverse complement of the first amplification handle sequence and with the second amplification handle sequence, respectively, on the tagged oligo, which are double stranded following tagged oligo amplification.
After the generation of the library, at least one purification step is preferably performed, followed by library quantification and pooling necessary for the subsequent sequencing procedure. Sequencing is preferentially performed as paired-end sequencing and two reads, each deriving from a strand of the library DNA molecule, are generated.
The same approach can be used to produce NGS libraries for other sequencing platforms, such as for example Ion Torrent.
Analysis of the paired-end read sequences generated from the NGS libraries are analysed according to the following steps:
1. sub-sequences extraction. Sub-sequences corresponding to UMI, BAB and amplification handle sequence/s (1AH and/or 2AH) are extracted from both sequencing reads for each tagged oligo molecule.
2. Read re-alignment. In case sub-sequences of BAB and/or amplification handle sequence/s do not match reference sequences (with a tolerance of 0.5-2 mismatches every 5 bases), sub-sequences position is offset by a variable amount, ranging from −n to +n, where n is the maximum offset allowed (for example n=8) and sub-sequences are re-extracted. For each iteration the Hamming distance from reference sequences is calculated and the offset returning the lowest distance is selected and all sub-sequences (UMI, BAB, amplification handle sequences) are extracted.
3. Reads filtering. Reads the BAB and/or handle sub-sequences of which differ from reference sequences more than a defined amount of bases are discarded as low quality reads.
4. UMI determination. UMI sequences from read pairs are expected to be perfectly complementary in the absence of sequencing errors. In the presence of any differences between the first strand UMI and second strand complementary UMI sequences:
a. the read pair may be discarded as low confidence or
b. a consensus between the two sequences can be calculated by selecting, for each position of the UMI, the base, among the sequences from the two sequencing reads, which have the highest base calling score as reported by the sequencer base caller.
It should be noted that the first method (a) is less prone to lead to an overestimation of target molecules due to sequencing biases but may lose true binding events between the binding agent and the target molecule, which are instead recovered with the second method (b).
5. Target molecules quantification. Counting of target molecules is performed by determining the number of distinct UMI sequences for each BAB sequence, representing a specific binding agent, in the analyzed sample.
A kit according to the present invention comprises:
a) at least one binding agent directed to at least one target molecule in a biological sample conjugated with a tagged oligonucleotide, the tagged oligonucleotide comprising:
b) at least one primer for carrying out a whole genome amplification and at least one primer for carrying out an amplification of the tagged oligonucleotide, the at least one primer for carrying out a whole genome amplification and at least one primer for carrying out an amplification of the tagged oligonucleotide either having the same sequence or having different sequence.
In a preferred embodiment, the kit comprises an oligonucleotide for extending the tagged oligonucleotide.
In a preferred embodiment, the at least one tagged oligonucleotide has a sequence corresponding to SEQ ID NO:1, the primers for carrying out the amplification of the tagged oligonucleotide are two and have respectively sequence SEQ ID NO:2 and SEQ ID NO:3. The kit further preferably comprises one or more first library primer/s and one or more second library primer/s. More preferably, said first library primer/s has/have a sequence selected from the group consisting of SEQ ID NO:8 to SEQ ID NO:15 and said second library primer/s has/have a sequence selected from the group consisting of SEQ ID NO:16 to SEQ ID NO:27.
In another preferred embodiment, the at least one tagged oligonucleotide has a sequence corresponding to SEQ ID NO:28, and the oligonucleotide for carrying out the extension of the tagged oligonucleotide is one and has a sequence corresponding to SEQ ID NO:29. The kit preferably further comprises one or more first library primer/s and one or more second library primer/s. More preferably, said first library primer/s has/have a sequence selected from the group consisting of SEQ ID NO:30 to SEQ ID NO:37 and said second library primer/s has/have a sequence selected from the group consisting of SEQ ID NO:38 to SEQ ID NO:49.
In this example tagged oligos were designed to perform amplification with the set-up according to
As shown in
Oligo P5-Synth (SEQ ID NO:1): the white box indicates the internal domain with the UMI and Binding agent barcode. Grey boxes with black border indicate the first and second amplification handle sequences.
P5 library primer (SEQ ID NO:8): the forward primer used for NGS library generation. In the grey box the annealing site with Oligo P5-Synth. In the short dashed box the sequencing primer site; in dashed-dotted box the i5 index used for multiplexing sequencing reactions; in the long dashed box the index sequencing primer/flow cell adapter sequence.
Synth library primer (SEQ ID NO:16): the reverse primer used for NGS library generation. In the grey box the annealing site with Oligo P5-Synth. In the short dashed box the sequencing primer site; in dashed-dotted box the i5 index used for multiplexing sequencing reactions; in the long dashed box the index sequencing primer/flow cell adapter sequence.
As can be seen in
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 hindrance inhibition effects on subsequent PCR reactions. Antibodies were covalently bound to tagged oligos using amines normally present in the antibody from lysine, glutamine, arginine and asparagine residues, through an amino-reactive reagent. Four Ab-oligos (Table 2) have been generated with the tagged oligos and antibody oligo conjugation was performed by Expedeon Ltd (25 Norman Way, Over, Cambridge CB24 5QE, United Kingdom) with an antibody:tagged oligo stoichiometry of 1:2. Epitope localization: indicates the position respect to the cell membrane.
Ab-oligos were used to stain two different types of cell lines. The first cell type was SK-BR-3 cells which are a breast tumor derived cell line which overexpresses cytokeratin and the Her2 protein. The second cell type was Peripheral Blood Mononuclear Cells (PBMCs) which are white blood cells extracted from whole blood which express CD45 and negligible levels of cytokeratin and Her2.
SK-BR-3 cells (ATCC® HTB-30m, ATCC) were grown in culture according to the manufacturer's procedure. PBMCs were extracted from human blood samples. Both cell types were fixed using PFA 2% according to a customised protocol.
Cell staining with Ab-oligos was performed on 100,000−50,000 previously fixed and permeabilised cells. Cells were collected by centrifugation at 1000×g, for 5 minutes at RT. Cells were washed with at least 1 mL of Running Buffer (autoMACS Running Buffer, ref. 130-091-221, Miltenyi Biotec) and collected by centrifugation. This last step was repeated two times. External Ab-oligos and their isotype Ab-oligo controls are diluted in 100 μl of Running Buffer down to their working concentration. External Ab-oligo mix (Ab-oligo tag3) was added to cells and incubated 15 minutes at RT. Sample was subsequently washed twice with 1 mL of Running Buffer and collected by centrifugation. Goat anti Mouse IgG2a—PE antibody in 500 μl of Running Buffer was added and incubated for 30 minutes at +4° C. This step enabled the staining of PBMC cells in PE. The sample was washed twice with Running Buffer. Internal Ab-oligos and their isotype Ab-oligo controls are diluted in 200 μl of Inside Perm Buffer (Inside Stain Kit, Ref. 130-090-477, Miltenyi Biotec) down to their working concentrations. Internal Ab-oligo mix (Ab-oligo tag1, 2 and 4) was added to cells and incubated for 10 minutes at RT. Sample was washed twice with 1 mL of Inside Perm Buffer and collected by centrifugation. A mix of Hoechst and Goat anti Mouse IgG1—APC antibody in 500 μl of Inside Perm Buffer was added and incubated for 30 minutes at +4° C. This step enabled the staining of SK-BR-3 cells in PE and all cell nuclei. The sample was washed twice with Running Buffer.
The addition of secondary antibodies conjugated to fluorophores enabled the identification of SK-BR-3 cells (APC channel) and PBMCs (PE channel) by fluorescence. Moreover the fluorescence levels reflect the relative abundance of Ab-oligo. Single-cells were purified using DEPArray™ N×T system (Menarini Silicon Biosystems) based on their immunofluorescent labeling (
Specifically,
Alternatively, to perform tagged oligo amplification after WGA a customized reaction mix of forward/reverse primers and Ampli1™ PCR kit reagents were prepared according to Table 3, left inset. Added 15 μl of the reaction mix to each tube containing WGA products. Each sample was incubated according to thermal profile indicated in Table 3, right inset.
Left inset: reaction mixture composition of tagged oligo amplification reaction. Right inset: thermal cycling program for the tagged oligo amplification.
Library preparation was performed by taking an aliquot of 1 μl of WGA containing Ab-oligo amplicons amplified using Ampli1™ PCR kit using P5 and Lib1 library primers at a final concentration of 0.5 μm. PCR thermal cycling profile is indicated in Table 5. Each sample had a different combination of NGS library primers for dual indexing in order to demultiplex data during bioinformatic analysis. The list of the library primers used is reported in Table 4. P5 library primers are forward primers that can be used with tagged oligo P5-Synth.
Thermal cycling profile for NGS library generation. The number of cycles during step 3 vary depending on the number of cells that have been recovered and the effective quantity of total Ab-oligos in cells. Usually 27 amplification cycles at stage 3 resulted in sufficient amplicon quantity from a single cell.
Library samples were purified using Agencourt AmPure XP beads (Beckman-Coulter). NGS DNA quantification was performed using KARA SYBR® FAST qPCR kit (Kapa Biosystems). Each NGS library was checked using Agilent Bioanalyzer 2100 (Agilent) showing a library electropherogram that was typically composed of a single peak at 185 bp (
Samples were pooled together and sequencing was performed on MiSeq system (Illumina) using MiSeq Reagent Kit v3 150-cycle (Ref. MS-102-3001, Illumina). Data analysis was performed using a custom software developed in Python. Quantification of protein targets according to UMIs counting is reported in
In this example tagged oligos were designed to perform amplification with the set-up according to
Quantification of protein targets according to UMIs counting is reported in
In this example, tagged oligos were added directly in single cells. The tagged oligos, namely “P5-Lib1” (SEQ ID NO: 28), were designed to be amplifiable by Ampli1™ WGA kit (Menarini Silicon Biosystems). The tagged oligo amplification primer has sequence SEQ ID NO: 29 (forward and reverse primers are identical, sharing the sequence of 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 sequences are identical to those described in Example 1. The second amplification handle sequence consists of a 3′-WGA handle sequence and an additional 5 bp sequence at the 5′ end of it (
More in detail,
Oligo P5-Lib1: The white filled box with thick borders indicates the internal domain with the UMI and Binding agent barcode. Grey filled boxes with thick borders indicate the annealing sites for the two library primers. The grey boxes with thin borders are the WGA handle sequences (Lib1).
P5 library primer: the forward primer used for NGS library generation. In the grey filled box the annealing site with tagged oligo P5-Lib1. In the short dashed box the sequencing primer site; in dashed-dotted box the i5 index used for multiplexing sequencing reactions; in the long dashed box the index sequencing primer/flow cell adapter sequence.
Lib1 library primer: the reverse primer used for NGS library generation. In the grey filled box the annealing site with tagged oligo P5-Lib1: this sequence is composed of a part of Lib1 reverse complement sequence and a small tail (ACCAC) enabling annealing only to the 3′ end of the Oligo P5-Lib1. In the short dashed box the sequencing primer site; in dashed-dotted box the i5 index used for multiplexing sequencing reactions; in the long dashed box the index sequencing primer/flow cell adapter sequence.
The forward library primer was identical to Index 2 (i5) Adapters (Illumina), whereas the reverse library primer was identical to Index 1 (i7) Adapters with the addition of a reverse complementary sequence of the second amplification handle sequence (
SK-BR-3 cells (ATCC® HTB-30™, ATCC) were grown in culture according to the manufacturer's procedure and were fixed using PFA 2% according to a customized protocol. Single-cells were purified using DEPArray™ N×T system (Menarini Silicon Biosystems) based on their morphology. P5-Lib1 and P5-Synth tagged oligos were added directly inside tubes containing single cells. Different quantities of each oligo were added within each single cell and performed Ampli1™ WGA. Samples containing P5-Synth tagged oligos were amplified as in Example 1.
Tagged oligos library generation was performed by taking an aliquot of 1 μl of WGA containing Ab-oligo amplicons was amplified using Ampli1™ PCR kit using P5 and Lib1 library primers at a final concentration of 0.5 μm. PCR thermal cycling profile is indicated in Table 5. Each sample had a different combination of NGS library primers for dual indexing in order to demultiplex data during bioinformatic analysis. The list of the library primers used is reported in Table 6.
An aliquot of 10 μl WGA samples were purified with SPRI beads (Beckmann Coulter) and subsequently processed with Ampli1™ LowPass kit to generate NGS libraries for CNA analysis. Spiking of tagged oligos in single cell before WGA procedure did not affect downstream genetic analyses (
Advantages
The method for whole genome amplification and analysis of multiple target molecules in a biological sample according to the present invention allows to obtain simultaneously analysis of genome-wide copy-number profiles/genome sequence and protein expression on the same single cells.
The method of the present invention enables to perform whole genome amplification of genomic DNA, useful to enable further analysis such as genome-wide copy number profiling by low-pass sequencing or targeted sequencing of panels of genes of interest, and to detect and perform a digital quantification of a panel of multiple proteins down to single-cell resolution, using very few samples, as it may be the case where only few (down to a single one) circulating tumour cells (CTCs) are available. This is particularly advantageous over measuring each molecular type in different cells in a genetically heterogeneous sample where differences in genotype, phenotype and environment may confound and completely prevent the correlation of genotype (copy number of sequence alterations) with phenotype (protein expression).
The method according to the invention surprisingly advances the state of the art with performances previously thought unachievable by the skilled in the art, in one or more of the following dimensions, given by way of non-limiting example:
The primary field of application of the method is oncology, but the method can be applied to other fields such as mosaic disorders such as dermatological or overgrowth phenotypes.
| Number | Date | Country | Kind |
|---|---|---|---|
| 102019000024159 | Dec 2019 | IT | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/IB2020/062053 | 12/16/2020 | WO |
