Methods for accurate parallel quantification of nucleic acid in diluted or non-purified samples

Abstract

To provide methods for using next generation sequencing for highly scalable and accurate target quantification from large volume samples (up to tens of milliliters) and / or diluted and / or non-purified sample material.SOLUTION: The disclosure relates to a method and a kit comprising probes for detecting and quantifying genetic targets in a complex DNA pool. The invention finds particular application in the field of detection of disease-causing genetic alterations in highly impure samples, such as samples obtained from the human or animal body, including without limitation, urine, biopsies, saliva and other secretions, exhaled moisture extracts, tissue, blood plasma (liquid biopsies) or the like. The invention uses one or more target-specific nucleic acid probes per genetic target (left probe and right probe) and a bridge oligo or a bridge oligo complex.SELECTED DRAWING: Figure 2B

Description

[Technical field]

[0001] The present disclosure relates to an improved next-generation DNA sequencing method for accurate and massively parallel quantification of one or more nucleic acid targets. More specifically, the disclosure relates to a method and kit including probes for detecting and quantifying gene targets in complex DNA pools, primarily used for detecting gene targets and variants. The present invention finds particular application in the field of detecting disease-causing gene mutations in highly impure samples, such as samples obtained from the human or animal body, including but not limited to urine, biopsies, saliva and other secretions, breath water extracts, tissues, plasma (liquid biopsies), etc. The present invention uses one or more target-specific nucleic acid probes (left and right probes) for each gene target, and bridge oligos or bridge oligo complexes. [Background technology]

[0002] The detection of genetic variation in some plants and animals is not troublesome due to the advancement of research technology.However, the detection and accurate quantification of genetic variation, such as mutation, especially in samples with weak signals, is still currently troublesome, laborious and expensive, despite the reduction in sequencing costs.Various issues can be more precisely indicated, such as the specificity for detecting genetic signals against consensus background, the sensitivity for detecting weak genetic signals, the accuracy for accurately quantifying detected signals, the throughput number of targeted genetic targets per assay, the cost per assay, the evaluation for determining the assay cost scale when multiple samples are assayed in parallel, and the turnover for determining the length of time from sample collection to result.

[0003] Currently, typical quantification methods for liquid biopsies and conceptually similar assays (such as antibiotic resistance gene detection) include quantitative PCR (qPCR), array qPCR, digital PCR, multiplex ligation-dependent probe amplification (MLPA), or quantification from next-generation DNA sequencing data. Although quantification methods are robust and well-established, each method is associated with specific problems, which are described in more detail below.

[0004] Quantitative PCR: Quantitative PCR (qPCR) is a technique that involves the amplification of target DNA molecules during PCR, i.e. in real time. Real-time PCR can be used quantitatively (quantitative real-time PCR) and semi-quantitatively, i.e. for more / less than a certain amount of DNA molecules (semi-quantitative real-time PCR). Quantitative PCR (qPCR) is the gold standard for gene target quantification. Currently, the laboratory cost of a qPCR reaction is about $2. However, taking into account the considerable hands-on time (labor cost) to set up the reaction, the need for standard curves, and replicates for each quantification target, in fact the actual cost is much higher. Because a separate quantification experiment is required for each gene target, the amount of hands-on time changes rapidly with increasing numbers of samples.

[0005] Array PCR: PCR arrays are the most reliable tools for analyzing the expression of a panel of genes focused on relevant pathways or diseases. PCR arrays in 96-well plates, 384-well plates, or 100-well disks each contain SYBR Green optimized primer assays for a thoroughly studied panel of focused gene panels. A newer iteration of qPCR technology is array qPCR, which miniaturizes individual qPCR reactions. Array PCR lowers the cost of individual qPCR reactions and improves the scalability of the method to multiple targets and samples. However, the method is currently limited to 384 targets from 12 samples (or conversely, 12 targets from 384 samples), at a cost of several thousand dollars per chip, plus the large capital costs of readout infrastructure. Thus, profiling thousands of samples using the mechanism remains prohibitively expensive.

[0006] Digital PCR: Digital polymerase chain reaction (digital PCR, DigitalPCR, dPCR, or dePCR) is a method that provides absolute quantification of targets through droplet microfluidics and fluorescence detection. Although this methodology is relatively cost-effective (cost of one target per sample is about $3), the hands-on time to prepare, set up, and run experiments for each target in each sample is insufficient at the scale of thousands of samples.

[0007] Multiplex ligation-dependent probe amplification (MLPA) offers an approach to simplify the detection of multiple gene targets in an individual sample. However, MLPA only provides relative quantification of targets and requires separate detection experiments for each sample. More recently, variants of MLPA introduce concepts from DNA barcoding. This concept allows for better quantitative separation and sample multiplexing than traditional MLPA workflows.

[0008] Next-generation sequencing-based approaches: Next-generation sequencing (NGS), also known as high-throughput sequencing, makes sequence-based gene expression analysis a "digital" alternative to analog techniques. Target counting from next-generation DNA sequencing data is becoming increasingly attractive as the cost of DNA sequencing continues to fall, and is currently used, for example, in NIPT screening. However, current approaches suffer from high sequencing library preparation costs, and sequencing effort wasted on sequencing irrelevant gene targets. For example, in cancer-associated liquid biopsies, non-targeted approaches result in wasted sequencing effort on oncologically irrelevant loci. In fetal diagnosis, non-targeted sampling of loci considerably limits statistical options for interpreting the data. Guardant Health Inc offers a more targeted sequencing approach, in which an array of RNA capture probes enriches targets for next-generation DNA sequencing.

[0009] Akhras et al. (2007) PLoS ONE 2(2):e223 discloses a multiplex pathogen detection assay that includes barcoded target-specific probes, target circularization and sequencing. The use of bridging oligonucleotides to ligate target-specific probes is also disclosed. WO 2019 / 038372 describes a next-generation sequencing approach to selectively amplify and sequence target sequences of interest. This method allows for accurate and parallel detection and quantification of many target sequences in a sample, but more complex, large quantities and / or impure samples remain challenging. Thus, in light of the above discussion, there is a need to overcome the above-mentioned shortcomings, including but not limited to specificity, sensitivity, precision, throughput, cost, validation and turnover through accurate and massively parallel quantification of nucleic acid targets. [Prior art documents] [Patent documents]

[0010] [Patent Document 1] International Publication No. 2019 / 038372 [Non-patent literature]

[0011] [Non-Patent Document 1] Akhras et al. (2007) PLoS ONE 2(2):e223 Summary of the Invention [Problem to be solved by the invention]

[0012] The present invention provides methods using next generation sequencing for highly scalable and accurate target quantification from, for example, large sample volumes (up to tens of milliliters) and / or diluted and / or unpurified sample material. [Means for solving the problem]

[0013] In a first main aspect, the present invention provides a method for high throughput detection of one or more target nucleotide sequences in a plurality of samples, comprising: (i) for each target nucleotide sequence in each of said samples, providing a first probe, a second probe and a bridge oligo or a plurality of oligonucleotides capable of annealing to each other to form a bridge oligo complex; said first probe starting from the 5' end of the molecule and comprising a first bridging oligo-specific sequence, optionally a first sequence barcode, and a first target specific portion at the 3' end of the first probe; said second probe starting at the 5' end of the molecule and comprising a second target specific portion, optionally a second sequence barcode, and a second bridging oligo specific sequence at the 3' end of the second probe; the bridge oligo or bridge oligo complex has a sequence complementary to the first bridge oligo specific sequence and the second bridge oligo specific sequence of the first probe and the second probe, respectively, and optionally a third barcode; at least one of the first sequence barcode or the second sequence barcode or the third barcode is present in the first probe or the second probe or the bridge oligo or bridge oligo complex, respectively; At least one of the first probe or the second probe or the bridge oligo or bridge oligo complex comprises a first capture moiety; (ii) for each of said one or more target nucleotide sequences, contacting said first probe and said second probe with said bridge oligo or a plurality of oligonucleotides capable of forming a bridge oligo complex, preferably for each of said samples in separate tubes, and allowing self-annealing into a plurality of ligation complexes; (iii) contacting nucleic acid present in each of the samples to be tested for the target nucleotide sequence with the ligation complex; (iv) hybridizing the first and second target-specific portions of the first and second probes to essentially adjacent portions on the target sequence, thereby forming a hybridization complex; (v) contacting the hybridization complex with a solid support comprising a second capture moiety and allowing the first capture moiety to interact with the second capture moiety such that the hybridization complex is linked to the solid support; (vi) separating hybridization complexes bound to the solid support from components of the sample that are not bound to the solid support; (vii) ligating the probes in the hybridization complex to provide a ligated ligation complex; (viii) optionally pooling the ligated ligation complexes from the multiple samples; (ix) amplifying nucleic acid from one or more of the ligated ligation complexes; (x) subjecting the nucleic acid obtained in step (ix) to high throughput sequencing techniques to determine the barcode sequence; and (xi) identifying the presence and / or number of said target nucleotide sequences in said plurality of samples by determining at least a portion of said first target specific portion and / or said second target specific portion, and / or at least a portion of said first barcode and / or said second barcode, and / or at least a portion of said third barcode; The method wherein steps (vii) and (viii) may be performed in any order.

[0014] In a second main aspect, the present invention provides a method for high throughput detection of one or more target nucleotide sequences in a plurality of samples, comprising: (i) providing a respective target nucleotide sequence for each of said samples; The single probe starts at the 5' end of the molecule and comprises a first target specific portion, a spacer portion comprising a barcode, and a second target specific portion at the 3' end of the second probe; a bridge oligo having a sequence complementary to the spacer portion or a portion of the spacer portion of the single probe, the single probe or the bridge oligo comprises a first capture moiety; (ii) contacting and self-annealing said single probe for each of said one or more target nucleotide sequences with said bridge oligo for each of said samples, preferably in separate tubes; (iii) contacting nucleic acids present in the plurality of samples to be tested for the target nucleotide sequence with the single probe annealed to the bridge oligo; (iv) hybridizing the first target-specific portion and the second target-specific portion of the single probe to essentially adjacent portions on the target sequence, thereby forming a hybridization complex; (v) contacting the hybridization complex with a solid support comprising a second capture moiety and allowing the first capture moiety to interact with the second capture moiety such that the hybridization complex is linked to the solid support; (vi) separating hybridization complexes bound to the solid support from components of the sample that are not bound to the solid support; (vii) ligating the 5' and 3' ends of the single probe to provide a ligated ligation complex; (viii) optionally pooling the ligated ligation complexes from the multiple samples; and (ix) amplifying nucleic acid from the one or more ligated ligation complexes; (x) subjecting the nucleic acid obtained in step (ix) to high throughput sequencing techniques to determine the barcode sequence; and (xi) identifying the presence and / or number of the target nucleotide sequences in the plurality of samples by determining at least a portion of the first target specific portion and / or the second target specific portion, and / or at least a portion of the first barcode and / or the second barcode, and / or at least a portion of the third barcode; The method, wherein steps (vii) and (viii) may be performed in any order. [Brief description of the drawings]

[0015] [Figure 1] FIG. 1 shows a flow diagram of a multiplex ligation assay (MLA) according to one embodiment of the present specification. [Figure 2A] 1 illustrates the principle structure of a probe triplet having multiple probe entities according to an embodiment of the present specification; [Figure 2B] 1 illustrates gap filling between a first probe and a second probe according to an embodiment herein. [Figure 2C]1 illustrates gap filling between a first probe, a second probe, and a cross-linked complex according to an embodiment of the present disclosure. [Diagram 3] Gel electrophoresis of two DNA sequencing libraries prepared using the workflow described in the Examples (lanes 6-7), as well as two negative control experiments in which incorrect oligonucleotide targets were provided to the probes (lanes 8-9). [Figure 4] The number of molecules detected accurately reflects the amount of spike-in synthetic oligonucleotide target. Each row represents three spike-in concentrations of a specified mutation. The response is linear over four orders of magnitude. [Diagram 5] The signals detected are highly specific to the presence or absence of the probes and their oligonucleotide targets. Two different oncogenic mutants of PIK3CA were tested. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] (definition) Target nucleotide sequence: The term target nucleotide sequence can be any nucleotide sequence of interest whose detection is required. It can be understood that the given term refers to a nucleic acid molecule having a sequence of consecutive nucleotides as well as a complementary sequence. The target sequence in some embodiments is a nucleotide sequence that represents or is associated with a polymorphism.

[0017] Polymorphism: The term polymorphism refers to the occurrence of two or more genetically determined alternative sequences or alleles in a population. A polymorphic marker or site is the locus at which sequence divergence occurs. A polymorphic locus may be as small as one base pair.

[0018] Sample: The term sample is used herein for two or more samples comprising two or more target sequences. The samples provided in the method according to the invention may have been prepared to extract at least the target nucleic acids and make them accessible to the probes used in the invention. In particular, in some embodiments, the samples each comprise at least two different target sequences, preferably at least 100, more preferably at least 250, more preferably at least 500, most preferably at least 2000 or more different target sequences. The term sample can refer to two or more samples obtained from the human / animal body, including but not limited to urine, biopsy, saliva and other secretions, breath water extracts, tissue, plasma (liquid biopsy), or two or more samples obtained from the environment, including but not limited to water, wastewater, soil, plants, samples containing viruses or bacteria, etc. In one embodiment, the multiple samples include blood samples, saliva samples, urine samples or fecal samples, samples of other body fluids or bodily substances such as extracts from hair or skin dander.

[0019] Probe: The term probe refers to a fragment of DNA or RNA of variable length (usually 50-1000 bases long, preferably 50-200 bases long) that can be used in DNA or RNA samples to detect the presence of a nucleotide sequence (DNA or RNA target) that is complementary to a sequence in the probe. The section of the oligonucleotide probe that is complementary to the target sequence is designed such that for each target sequence in the sample, a pair of left and right probes are provided, whereby the probes each contain a section at their end that is complementary to a portion of the target sequence. Alternatively, a single probe is provided that contains two sections that are complementary to portions of the target sequence separated by a spacer section. Additionally, the disclosure describes bridged oligos or bridged oligo complexes that are used to join the left and right probes or hybridize with the spacer section of a single probe.

[0020] Universal: When used to describe an amplification procedure, the term universal refers to a sequence that allows a single primer or set of primers to be used for multiple amplification reactions. The use of such primers greatly simplifies multiplexing in that only two primers are required to amplify multiple selected nucleic acid sequences. When the term universal is used to describe a priming site, it is the site to which the universal primer hybridizes. It should also be noted that a "set" of universal priming sequences / primers may be used.

[0021] Hybridization: The term hybridization or hybridisation refers to the process of annealing a deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) molecule to a complementary DNA or RNA. Both DNA or RNA replication and transcription of DNA into RNA depend on nucleotide hybridisation.

[0022] Ligation: The term ligation is the joining of two nucleic acid fragments by the action of an enzyme. DNA ligase is an enzyme that can catalyze the formation of a phosphodiester bond between the (ends of) two polynucleotide strands that are joined to adjacent sites on complementary strands. In one embodiment, ligation can be performed chemically, especially if both adjacent ends of the polynucleotides are modified to allow chemical ligation.

[0023] Amplification: The term amplification as used herein refers to the use of a polymerase-based reaction to increase the concentration of a specific nucleotide sequence within a mixture of nucleotide sequences. "PCR" or "polymerase chain reaction" is a rapid procedure for in vitro enzymatic amplification of specific DNA / RNA. The DNA / RNA to be amplified can be denatured by heating the sample. The term primer is a short strand of RNA or DNA (generally about 18-22 bases) that serves as the starting point for DNA synthesis. It is necessary for DNA replication because the enzyme that catalyzes this process, i.e. DNA polymerase, can only add new nucleotides to an existing strand of DNA. T7 RNA polymerase can both transcribe and amplify a single DNA molecule into multiple copies of RNA that can be converted back into cDNA.

[0024] Polymerase: A polymerase is an enzyme that synthesizes long chains or polymers of nucleic acid. DNA polymerase and RNA polymerase are used to assemble DNA and RNA molecules, respectively, by copying DNA or RNA template strands using base pairing interactions. The specific polymerase used herein, T7 RNA polymerase, is an RNA polymerase from the T7 bacteriophage that catalyzes the formation of RNA in the 5'→3' direction. T7 RNA polymerase requires a partially double-stranded DNA template and Mg as a cofactor. 2+ ion is required for the synthesis of RNA. T7 RNA polymerase can both transcribe and amplify a single DNA molecule into multiple copies of RNA. Another specific polymerase used herein, phi29 polymerase, is a strand-displacing DNA polymerase derived from bacteriophage phi29. Phi29 polymerase is highly evolved and therefore is an ideal polymerase for rolling circle amplification of circular DNA templates to generate long concatemeric sequences.

[0025] High throughput: The term high throughput refers to the ability to process and screen many DNA samples simultaneously, as well as the ability to simultaneously screen many different loci within a single DNA sample. High throughput sequencing or screening, often abbreviated as HTS, is a method for scientific experimentation that is particularly relevant for effectively screening large numbers of samples simultaneously.

[0026] Uracil-specific excision reagent (USER): A reagent that allows linearization of a circular DNA molecule by cleavage in the presence of deoxyuridine nucleotides.

[0027] As mentioned above, the present disclosure relates to a method for high-throughput detection of target nucleotide sequences in a large number of samples by utilizing ligation-dependent assays. The present disclosure provides a method for determining the sequence of gene targets in a complex nucleic acid pool using technology that is permitted by next-generation sequencing. The present disclosure also provides a method for profiling multiple gene targets in a large number of samples, preferably a large number of samples, by utilizing ligation-dependent assays. The present disclosure further provides a method for multiplex ligation-dependent probe amplification that allows interrogation of different target nucleic acids in multiple samples. The method of the present invention provides multiple different probe sets for different target nucleic acids to allow sequencing of one or more target nucleotide sequences in multiple samples. When processing the sequencing data, unique sequence identifiers are used to identify gene targets from the sample pool and to absolutely quantify individual samples.

[0028] In a first main aspect, the present invention relates to a method for high throughput detection of one or more target nucleotide sequences in a plurality of samples, the method comprising: 1. A method for high throughput detection of one or more target nucleotide sequences in a plurality of samples, comprising: (i) for each target nucleotide sequence in each of said samples, providing a first probe, a second probe and a bridge oligo or a plurality of oligonucleotides capable of annealing to each other to form a bridge oligo complex; said first probe starting from the 5' end of the molecule and comprising a first bridging oligo-specific sequence, optionally a first sequence barcode, and a first target specific portion at the 3' end of the first probe; said second probe starting at the 5' end of the molecule and comprising a second target specific portion, optionally a second sequence barcode, and a second bridging oligo specific sequence at the 3' end of the second probe; the bridge oligo or bridge oligo complex has a sequence complementary to the first bridge oligo specific sequence and the second bridge oligo specific sequence of the first probe and the second probe, respectively, and optionally a third barcode; at least one of the first sequence barcode or the second sequence barcode or the third barcode is present in the first probe or the second probe or the bridge oligo or bridge oligo complex, respectively; At least one of the first probe or the second probe or the bridge oligo or bridge oligo complex comprises a first capture moiety; (ii) for each of said one or more target nucleotide sequences, contacting said first probe and said second probe with said bridge oligo or a plurality of oligonucleotides capable of forming a bridge oligo complex, preferably for each of said samples in separate tubes, and allowing self-annealing into a plurality of ligation complexes; (iii) contacting nucleic acid present in each of the samples to be tested for the target nucleotide sequence with the ligation complex; (iv) hybridizing the first and second target-specific portions of the first and second probes to essentially adjacent portions on the target sequence, thereby forming a hybridization complex; (v) contacting the hybridization complex with a solid support comprising a second capture moiety and allowing the first capture moiety to interact with the second capture moiety such that the hybridization complex is linked to the solid support; (vi) separating hybridization complexes bound to the solid support from components of the sample that are not bound to the solid support; (vii) ligating the probes in the hybridization complex to provide a ligated ligation complex; (viii) pooling the ligated ligation complexes from the multiple samples; and (ix) amplifying nucleic acid from one or more of the ligated ligation complexes; (x) subjecting the nucleic acid obtained in step (ix) to high throughput sequencing techniques to determine the barcode sequence; and (xi) identifying the presence and / or number of said target nucleotide sequences in said plurality of samples by determining at least a portion of said first target specific portion and / or said second target specific portion, and / or at least a portion of said first barcode and / or said second barcode, and / or at least a portion of said third barcode; The method wherein steps (vii) and (viii) may be performed in any order.

[0029] FIG. 1 provides a non-limiting illustration of one embodiment of the method of the present invention.

[0030] The method of the present invention utilizes three or more nucleic acid probes, of which two target-specific nucleic acid probes (left and right probes) are specific to a genetic target, and one nucleic acid probe or complex is typically universal (bridge oligo or bridge oligo complex). The left and right probes hybridize to the bridge probe or bridge oligo complex to form a ligation complex. The ligation complex (containing one or more barcode sequences) with a target identification site on the sample DNA or RNA can hybridize to a complementary target sequence of the interrogation sample. After hybridization, the left and right probes are chemically or enzymatically ligated by DNA ligase to form a ligated ligation complex. In the present invention, a plurality of such ligated ligation complexes will be formed during sample analysis in a plurality of samples to be analyzed.

[0031] In one embodiment, "multiple samples" may refer to two or more samples obtained from the human or animal body, including, but not limited to, biopsies, saliva and other secretions, breath extracts, tissue, plasma (liquid biopsy), two or more samples obtained from the environment, including water, wastewater, soil, plants, samples containing viruses or bacteria, etc. In a preferred embodiment, the sample is used without prior purification or concentration of nucleic acids. In another embodiment, the sample may be pre-treated, for example, by lysing cells to expose nucleic acids.

[0032] The target sequence may include any nucleotide sequence of interest that requires detection. The target nucleotide sequence of the present disclosure may be obtained from, but is not limited to, a fraction of DNA in the patient's blood or a fraction of DNA in maternal blood. The fraction of DNA in the patient's blood may be obtained from apoptotic / necrotic cancer cells or from a fraction of DNA in maternal blood from fetus and / or mother. Furthermore, the results of the analysis may be used to assess, for example, an individual's risk for a certain type of cancer, determine the effectiveness of a certain treatment for a certain cancer, the occurrence of drug resistance-related mutations in tumors, or the risk of a fetus having a genetic disorder, such as common trisomy Down's syndrome, Patau's syndrome, and Edwards' syndrome, etc. In certain embodiments, the method includes providing a plurality of different probe sets for each target nucleotide sequence.

[0033] As used herein, the term probe set includes a first probe, a second probe and a bridge oligo (or a bridge oligo complex), or a single probe and a bridge oligo (or a bridge oligo complex).

[0034] In certain embodiments, the first probe comprises, starting from the 5' end of the molecule, optionally a 5' phosphate, a first bridging oligo-specific sequence, optionally a first universal sequence, optionally a first sequence barcode, and a first target specific portion at its 3' end. In certain embodiments, the second probe comprises, starting from the 5' end of the molecule, optionally a 5' phosphate, a second target specific portion, optionally a second sequence barcode, optionally a second universal sequence, and a second bridging oligo-specific sequence at its 3' end.

[0035] In a preferred embodiment, either the first probe or the second probe comprises at least one of a first sequence barcode or a second sequence barcode. The first sequence barcode or the second sequence barcode, or both, may be random sequences or may comprise a target nucleotide sequence identifier sequence, a sample identifier sequence, and / or a molecular barcode for target enumeration.

[0036] In a preferred embodiment, the bridge oligo or bridge oligo complex may contain a third barcode that contains a sequence complementary to the first and second bridge oligo specific sequences in the first and second probes, respectively, optionally a universal sequence, and / or may be a random sequence, or may contain a sample or sequence identifier sequence. In this respect, the third barcode does not necessarily mean that the first and second barcodes are already present. As mentioned above, at least one barcode should be present in the ligated ligation complex, which allows the complex to be uniquely defined within all ligation complexes in all samples tested.

[0037] Furthermore, at least one of the first probe or the second probe or the bridged oligo or the bridged oligo complex comprises a first capture moiety. A first capture moiety, as used herein, refers to a moiety such as a chemical group, by which the second capture moiety linked to the solid support can capture, i.e. bind, to the probe, the ligation complex or the hybridization complex. Any suitable capture moiety known in the art can be used for this purpose. A well-known suitable example is the capture of biotinylated molecules using streptavidin-coated magnetic beads. Thus, in one embodiment, the first capture moiety is a biotin moiety, which can interact with a streptavidin or avidin moiety (second capture moiety) linked to a solid support such as a magnetic bead. Other options include biotin derivatives such as dual biotin, desthiobiotin or photocleavable biotin, which can be used for conjugation with streptavidin / avidin. Further options include thiol and acryldite groups for acryldite / acrylamide conjugation, alkyne and azide groups for click chemistry, and the use of digoxigenin for anti-digoxigenin antibody conjugation. The conjugation partners can be provided on any solid surface, such as beads (magnetic or otherwise), or solid support.

[0038] In some embodiments, the first, second or bridging probe or bridging oligo complex contains a promoter sequence of T7 RNA polymerase. The promoter sequence uses T7 RNA polymerase to amplify the ligated ligation complex in step (ix). The promoter sequence of T7 RNA polymerase is preferably present in the bridging oligo or bridging oligo complex, but the promoter can also be present in the first or second probe instead of being present in the bridging oligo or bridging oligo complex. However, in such a case, the design of the probe and oligo must be such that T7 RNA polymerase can transcribe all the sequences required for sample and target identification and target sequence enumeration.

[0039] The first target specific portion, the second target specific portion, the first bridging oligo specific sequence, and / or the second bridging oligo specific sequence preferably contain at least one chemically modified nucleotide independently of each other to increase probe binding. Chemical modifications to increase probe binding include, but are not limited to, ribonucleic acid, peptide nucleic acid, and locked nucleic acid (e.g., as shown in Figure 3 of WO2019 / 038372, which is incorporated herein by reference). In one embodiment, the bridging portion of the first probe or the second probe, or both, contains a chemically modified base to improve binding to the bridging oligo or bridging oligo complex. In another embodiment, the first target specific portion, the second target specific portion, the first bridging oligo specific sequence, and / or the second bridging oligo specific sequence contain one or more chemically modified nucleotides independently of each other. In certain embodiments, the chemical modifications allow adjacent probes to be chemically ligated. In some embodiments, the probes bind to completely adjacent loci or up to 500 base pairs apart, such as up to 200 base pairs apart, for example up to 50 base pairs apart, preferably up to 40 base pairs apart, more preferably up to 30 base pairs apart, more preferably up to 20 base pairs apart, more preferably up to 10 base pairs apart, most preferably up to 5 base pairs apart.

[0040] Prior to contacting the probe with a sample containing the target sequence, the first and second probes are contacted with a bridge oligo or multiple oligonucleotides capable of forming a bridge oligo complex, preferably for each sample in separate tubes, to allow self-annealing to a ligation complex (step (ii)). In embodiments where the bridge is not one oligo but multiple oligonucleotides, e.g., three or five oligonucleotides, that can anneal to each other to form a bridge oligo complex (as shown in FIG. 2C herein), the multiple oligonucleotides may be pre-annealed before annealing with the first and second probes, or all annealing steps may be performed once.

[0041] Preferably, each ligation complex is unique to the combination of the first target specific sequence, the second target specific sequence and one or more barcode sequences, allowing for enumeration of the target sequences after amplification and analysis of the results.

[0042] Then, one or more target nucleotide sequences in the multiple samples are contacted with the multiple ligation complexes (step (iii)). The first and second target specific portions of the first and second probes, respectively, hybridize to essentially adjacent portions on the target sequence, thereby forming hybridization complexes (step (iv)). In some embodiments, the sample has a volume of more than 100 microliters, such as more than 1 ml. In further embodiments, the sample has a nucleic acid concentration of less than 5 pmol, such as less than 1 pmol, such as less than 200 fmol.

[0043] The hybridization complex is then contacted with a solid support comprising a second capture moiety, allowing the first capture moiety to interact with the second capture moiety such that the hybridization complex is linked to the solid support (step (v)). The hybridization complex linked to the solid support is then separated from components of the sample that are not linked to the solid support (step (vi)). If the solid support is a magnetic bead, a magnet may be used to immobilize the beads and remove the remaining liquid sample. Optionally, a washing step is performed before proceeding to step (vii).

[0044] Steps (v) and (vi) purify and concentrate the nucleic acid, allowing for improved results, especially for samples with very low purity. In one embodiment, the method of the present invention does not include a step of concentrating the nucleic acid before step (vi). Thus, in one embodiment, the method does not include a step of concentrating the nucleic acid in the original sample more than 2-fold, more than 10-fold, or more than 100-fold before step (vi). In another embodiment, the method of the present invention does not include a purification step after the ligation of step (vii).

[0045] Ligation of the probes in the formed hybridized complex is then carried out enzymatically or chemically to provide a ligated ligation complex (step (vii)). Optionally as part of step (vii), any gaps between the first and second probes can be filled by introducing a polymerase and one or more nucleotides. The polymerase adds nucleotides that are (a) complementary to the universal bridge oligo sequence or bridge oligo complex and / or (b) complementary to the barcode sequence, thereby filling the two gaps between the first and second probes, such that the left and right probes are ligated and the universal sequence and / or the third barcode sequence is included in the bridge complementary strand. The bridge oligo is extended from the 5' or 3' site complementary to the ligated probe such that the target sequence identifier sequence present in the first or second probe is incorporated into the bridge oligo. Preferably, a polymerase is used that does not degrade double-stranded DNA, such as Taq polymerase, so as not to interfere with the ligation of the first probe to the second probe when both are annealed to the target sequence.

[0046] The ligated ligation complexes are then pooled from one or more target samples (step (viii)). Steps (vii) and (viii) may be performed in the order specified or in reverse order.

[0047] Nucleic acid from one or more ligated ligation complexes is then amplified (step (ix)).

[0048] In one embodiment, the amplification in step (ix) is carried out by PCR using primers that bind to the universal portions of the first and second probes.

[0049] In another embodiment, at least one of the first probe or the second probe or the bridging oligo or the bridging oligo complex comprises a recognition sequence for an endonuclease, and step (ix) comprises: (a) amplifying nucleic acid from one or more ligated ligation complexes using rolling circle amplification with a strand-displacing polymerase; (b) optionally subjecting the resulting amplified one or more single-stranded concatemer sequences to annealing with a specific oligonucleotide containing said recognition sequence, said specific oligonucleotide annealing to said recognition sequence in such a way as to provide a recognition site for said endonuclease; (c) optionally cleaving the resulting single-stranded concatemeric sequences or the resulting annealed complexes with said endonuclease. Suitable strand-displacing polymerases include phi29 polymerase or Bst polymerase. A specific oligonucleotide containing a recognition sequence will typically contain some additional specific sequence around the recognition sequence to allow the formation of a stable double helix for cleavage.

[0050] In some embodiments, no cleavage is performed and a subsequent high-throughput sequencing step (x) is performed on the concatemeric sequences.

[0051] In another embodiment, the amplification in step (ix) is performed using T7 RNA polymerase, which binds to the double-stranded T7 RNA polymerase promoter site on the oligo sequence of the ligated ligation complex and transcribes the RNA downstream. Amplification of the synthesized RNA from one or more ligated ligation complexes may be performed, for example, by using emulsion reverse transcriptase (RT)-PCR or T7 RNA polymerase. Removal of free sample and probe nucleic acids can be performed by adding DNA-specific exonucleases and endonucleases to the pooled amplification reaction after RNA synthesis by T7 RNA polymerase but before cDNA synthesis or RT-PCR. In one embodiment, the probe or bridging oligo or bridging oligo complex contains a deoxyuridine moiety that allows linearization by cleavage with a uracil-specific excision reagent (USER). This allows RNA transcription from the linearized ligation complex using T7 RNA polymerase, which initiates RNA synthesis from the T7 RNA polymerase promoter incorporated in the bridging oligo or bridging oligo complex or the first or second probe. The T7 RNA polymerase promoter sequence and the deoxyuridine moiety should be in such a position that the T7 RNA polymerase can transcribe all the information required for enumeration of different targets in different samples. Between the T7 RNA polymerase and the deoxyuridine moiety there should be the following sequence: At least one sequence that allows identification of the target; At least one sequence allowing identification of the sample, and At least one unique barcode sequence that allows for the determination of the copy number of the target sequence in the sample. Alternatively, cDNA is prepared from the RNA molecule using a DNA-oligonucleotide molecule that is reverse-complementary to the universal site. The RNA molecule is optionally converted to cDNA and amplified by PCR or emulsion PCR using a primer that binds to the universal portion of the probe. Alternatively, RT-PCR or emulsion RT-PCR may be used.

[0052] Optionally, after amplification, the solid support is removed and the supernatant is used for further processing, for example, if the solid support is a magnetic particle, these can be removed using a magnet.

[0053] In another embodiment of the method of the present invention, the interaction between the first capture moiety and the second capture moiety is interrupted immediately after step (vi), after step (vii) or after step (viii).For example, if the first capture moiety is biotin and the second capture moiety is streptavidin, the interaction can be interrupted by adding an excess of soluble biotin.If the streptavidin is bound to a magnetic particle, it can then be removed using a magnet.

[0054] Regardless of the amplification method in step (ix), in some embodiments, the nucleotide molecule (RNA molecule, DNA molecule or cDNA molecule) is (further) amplified with a first primer and a second primer to obtain an amplification product. Preferably, a universal first primer and a universal second primer are used that are reverse complementary to the first or second universal sequence present in the ligated complex.

[0055] Identifying the presence and / or number of target nucleotide sequences in a plurality of samples can be performed by determining at least a portion of the first and / or second target specificity portion, at least a portion of the first and / or second barcode, and / or at least a portion of the third barcode by high-throughput next-generation sequencing technology, for example, using a next-generation sequencing platform including, but not limited to, Illumina iSeq, MiSeq, HiSeq, NextSeq or NovaSeq (steps (x) and (xi)). Preferably, gene target enumeration is enabled by counting the number of molecular barcodes per target and per sample. Samples are separated (deconvoluted) from sequence data and sequence targets are quantified in silico after DNA sequencing.

[0056] As mentioned above, in a second main aspect, the present invention relates to a method for high throughput detection of one or more target nucleotide sequences in a plurality of samples, the method comprising: 1. A method for high throughput detection of one or more target nucleotide sequences in a plurality of samples, comprising: (i) providing a respective target nucleotide sequence for each of said samples; The single probe starts at the 5' end of the molecule and comprises a first target specific portion, a spacer portion comprising a barcode, and a second target specific portion at the 3' end of the second probe; a bridge oligo having a sequence complementary to the spacer portion or a portion of the spacer portion of the single probe, the single probe or the bridge oligo comprises a first capture moiety; (ii) contacting and self-annealing said single probe for each of said one or more target nucleotide sequences with said bridge oligo for each of said samples, preferably in separate tubes; (iii) contacting nucleic acids present in the plurality of samples to be tested for the target nucleotide sequence with the single probe annealed to the bridge oligo; (iv) hybridizing the first target-specific portion and the second target-specific portion of the single probe to essentially adjacent portions on the target sequence, thereby forming a hybridization complex; (v) contacting the hybridization complex with a solid support comprising a second capture moiety and allowing the first capture moiety to interact with the second capture moiety such that the hybridization complex is linked to the solid support; (vi) separating hybridization complexes bound to the solid support from components of the sample that are not bound to the solid support; (vii) ligating the 5' and 3' ends of the single probe to provide a ligated ligation complex; (viii) optionally pooling the ligated ligation complexes from the multiple samples; and (ix) amplifying nucleic acid from the one or more ligated ligation complexes; (x) subjecting the nucleic acid obtained in step (ix) to high throughput sequencing techniques to determine the barcode sequence; and (xi) identifying the presence and / or number of the target nucleotide sequences in the plurality of samples by determining at least a portion of the first target specific portion and / or the second target specific portion, and / or at least a portion of the first barcode and / or the second barcode, and / or at least a portion of the third barcode; The steps (vii) and (viii) may be carried out in any order.

[0057] The embodiments described above for the first aspect of the invention apply mutatis mutandis to this second aspect of the invention.

[0058] In one embodiment, a single probe comprises two or more oligonucleotides that are chemically linked.

[0059] In one embodiment the spacer portion comprises more than 25 bases, such as more than 50 bases, such as more than 75 or more than 100 bases, such as between 20 and 1000 bases, such as between 50 and 1000 bases.

[0060] Advantages of both aspects of the invention include, but are not limited to, quantitative assays at low cost, high simplicity, high specificity, high sensitivity, high accuracy, high throughput, high scalability and high turnover compared to conventional nucleic acid sequencing techniques. Another aspect of the invention is that the method of the invention allows for accurate and massively parallel quantification of multiple nucleic acid targets in multiple samples, including human and animal populations, and including large amounts of raw sample material. As mentioned above, in a preferred embodiment, a sample, such as a urine sample, is used without prior purification or concentration of the nucleic acid. In another embodiment, the sample may be pretreated, for example, by lysing cells to expose the nucleic acid. One particular advantage of the invention is that it allows for the detection and amplification of target sequences of interest using a unique probe design, i.e., probe triplets. The probes are designed with specially positioned modified nucleotides that improve annealing and binding efficiency. The improved binding characteristics improve the specificity, sensitivity and accuracy of the assay. The methods of the invention are also applicable to the study of genetic variants and find diagnostic and prognostic applications including, but not limited to, genotyping a sample for one or more sequences and / or polymorphisms, such as SNPs and / or indels, cancer diagnosis, or fetal chromosomal disorders from maternal blood, etc. In a preferred embodiment, for two or more samples or two or more locus / allele combinations, the barcode sequence is used to genotype the sample for one or more sequences and / or polymorphisms, such as SNPs and / or indels.

[0061] In another aspect, the invention provides a kit for use in the method of the invention. In one embodiment, the kit of parts comprises a plurality of containers, At least one container contains one or more sets of first probes and second probes, and at least one container contains one or more bridge oligos or a plurality of oligonucleotides capable of forming a bridge oligo complex; said first probe starting at the 5' end of the molecule and comprising a first bridging oligo-specific sequence and, optionally, a first sequence barcode and a first target-specific portion at the 3' end of the first probe; said second probe starting at the 5' end of the molecule and comprising a second target specific portion and, optionally, a second sequence barcode and a second bridging oligo specific sequence at the 3' end of the second probe; said bridge oligo or bridge oligo complex comprising a sequence complementary to said first and second bridge oligo specific sequences in each of said first and second probes, respectively, and optionally a third barcode; At least one of the first sequence barcode, the second sequence barcode, or the third barcode is present in the first probe, the second probe, the bridge oligo, or the bridge oligo complex, respectively; and At least one of the first probe or the second probe or the bridged oligo or bridged oligo complex comprises a first capture moiety capable of linking to a second capture moiety linked to a solid support.

[0062] Preferably, the 3' end of the first probe or the 5' end of the second probe, or both, are modified to allow chemical ligation of the first probe to the second probe.

[0063] Preferably, the bridged oligo or bridged oligo complex contains one or more chemically modified nucleotides in the sequence complementary to the sequence of the first probe or the sequence complementary to the sequence of the second probe or both.

[0064] Optionally, the first probe, the second probe, or the bridge oligo or bridge oligo complex contains a deoxyuridine moiety that allows linearization by cleavage with a uracil-specific cleavage reagent.

[0065] Preferably, the 3' end of the first probe or the 5' end of the second probe, or both, are modified to allow chemical ligation of the first probe to the second probe.

[0066] Preferably, the bridging moiety of the first probe or the second probe, or both, contains chemically modified bases to improve binding to the bridge oligo or bridge oligo complex.

[0067] In one particular embodiment, at least one container that contains the first and second probe sets and at least one container that contains a plurality of oligonucleotides that can form bridge oligo or bridge oligo complex is one and the same container.In such a case, three or more probes can be pre-annealed and ligated to form a complex.

[0068] One particular advantage of the present invention is that it uses a unique probe design, i.e., a probe triplet, to allow the detection and amplification of target sequences of interest.The probe is designed with improved binding properties, which leads to improved assay specificity, sensitivity and accuracy.The present invention finds application in the fields of molecular biology, evolutionary biology, metagenomics, genotyping, more specifically, including but not limited to the genotyping of samples for one or more sequences and / or polymorphisms, such as SNPs and / or indels, including but not limited to cancer diagnosis or fetal chromosomal disorders.

[0069] In one particular preferred embodiment, the bridge oligo or bridge oligo complex comprises information for identifying the sample and comprises a unique identifier.In such a case, the first and second probes are generally applicable to all samples (only comprise information for identifying the target).Therefore, in one preferred embodiment, the method or kit according to the present invention is provided, in which the bridge oligo or bridge oligo complex comprises a barcode that comprises a unique sequence that allows the enumeration of the target sequence of each sample.

[0070] In a further aspect, the present invention relates to a kit of parts comprising a plurality of containers, at least one container containing one or more single probes and at least one container containing one or more bridge oligos; The single probe comprises, starting from the 5' end of the molecule, a first target specific portion, a spacer portion comprising a barcode, and a second target specific portion at the 3' end of the second probe; the bridge oligo has a sequence complementary to the spacer portion or a portion of the spacer portion of the single probe; and The single probe or the bridging oligo comprises a first capture moiety that can be linked to a second capture moiety that is linked to a solid support.

[0071] Furthermore, the present invention relates to: Embodiment 1: A method for high throughput detection of one or more target nucleotide sequences in a plurality of samples, comprising: (i) for each target nucleotide sequence in each of said samples, providing a first probe, a second probe and a bridge oligo or a plurality of oligonucleotides capable of annealing to each other to form a bridge oligo complex; said first probe starting from the 5' end of the molecule and comprising a first bridging oligo-specific sequence, optionally a first sequence barcode, and a first target specific portion at the 3' end of the first probe; said second probe starting at the 5' end of the molecule and comprising a second target specific portion, optionally a second sequence barcode, and a second bridging oligo specific sequence at the 3' end of the second probe; the bridge oligo or bridge oligo complex has a sequence complementary to the first bridge oligo specific sequence and the second bridge oligo specific sequence of the first probe and the second probe, respectively, and optionally a third barcode; at least one of the first sequence barcode or the second sequence barcode or the third barcode is present in the first probe or the second probe or the bridge oligo or bridge oligo complex, respectively; At least one of the first probe or the second probe or the bridged oligo or bridged oligo complex comprises a first capture moiety; (ii) for each of said one or more target nucleotide sequences, contacting said first probe and said second probe with said bridge oligo or a plurality of oligonucleotides capable of forming a bridge oligo complex, preferably for each of said samples in separate tubes, and allowing self-annealing into a plurality of ligation complexes; (iii) contacting nucleic acid present in each of the samples to be tested for the target nucleotide sequence with the ligation complex; (iv) hybridizing the first and second target-specific portions of the first and second probes to essentially adjacent portions on the target sequence, thereby forming a hybridization complex; (v) contacting the hybridization complex with a solid support comprising a second capture moiety and allowing the first capture moiety to interact with the second capture moiety such that the hybridization complex is linked to the solid support; (vi) separating hybridization complexes bound to the solid support from components of the sample that are not bound to the solid support; (vii) ligating the probes in the hybridization complex to provide a ligated ligation complex; (viii) pooling the ligated ligation complexes from the multiple samples; and (ix) amplifying nucleic acid from one or more of the ligated ligation complexes; (x) subjecting the nucleic acid obtained in step (ix) to high throughput sequencing techniques to determine the barcode sequence; and (xi) identifying the presence and / or number of said target nucleotide sequences in said plurality of samples by determining at least a portion of said first target specific portion and / or said second target specific portion, and / or at least a portion of said first barcode and / or said second barcode, and / or at least a portion of said third barcode; The method wherein steps (vii) and (viii) may be performed in any order.

[0072] Embodiment 2: The method of embodiment 1, wherein the plurality of samples comprises a blood sample, a saliva sample, a urine sample, or a fecal sample.

[0073] Embodiment 3: The method of embodiment 1 or 2, wherein the first capture moiety is a biotin moiety and the second capture moiety is a streptavidin moiety or an avidin moiety.

[0074] Embodiment 4: The method of any one of embodiments 1 to 3, wherein a washing step is performed between steps (vi) and (vii).

[0075] Embodiment 5: The method according to any one of embodiments 1 to 4, wherein the sequencing is performed by next generation DNA or RNA sequencing.

[0076] Embodiment 6: The method according to any one of embodiments 1 to 5, wherein the 3' end of the first probe or the 5' end of the second probe, or both, are modified to allow the first probe to be chemically ligated to the second probe.

[0077] Embodiment 7: The method of any one of embodiments 1 to 6, wherein the bridging moiety of the first probe or the second probe, or both, comprises a chemically modified base for improved binding to the bridging oligo.

[0078] Embodiment 8: The method of any one of embodiments 1 to 7, wherein the first target-specific portion, the second target-specific portion, the first bridging oligospecific sequence, and / or the second bridging oligospecific sequence independently of each other contain one or more chemically modified nucleotides.

[0079] Embodiment 9: A promoter sequence of T7 RNA polymerase is present in the first probe, the second probe or the bridge oligo or bridge oligo complex; The method according to any one of embodiments 1 to 8, wherein said amplification in step (ix) comprises RNA synthesis from said one or more ligated ligation complexes using T7 RNA polymerase that initiates RNA synthesis from said T7 RNA polymerase promoter incorporated in said ligated ligation complexes.

[0080] Embodiment 10: The method of embodiment 9, wherein the first probe, the second probe, or the bridging oligo comprises a deoxyuridine moiety that allows for linearization by cleavage with a uracil-specific cleavage reagent.

[0081] Embodiment 11: The method of embodiment 9, wherein prior to step (x), cDNA is prepared from the RNA molecule using a DNA-oligonucleotide molecule that is reverse complementary to a universal site present in the first probe or the second probe or the bridging oligo or bridging oligo complex.

[0082] Embodiment 12: At least one of the first probe or the second probe or the bridging oligo or the bridging oligo complex comprises a recognition sequence for an endonuclease; (a) the amplification of nucleic acids from the one or more ligated ligation complexes in step (ix) is carried out using rolling circle amplification with a strand displacing polymerase, followed by (b) optionally subjecting the resulting amplified one or more single-stranded concatemer sequences to annealing with a specific oligonucleotide containing the recognition sequence, the specific oligonucleotide annealing to the recognition sequence in such a way as to provide a recognition site for the endonuclease; and (c) optionally cleaving the resulting amplified one or more single-stranded concatemer sequences or the resulting annealed complexes with an endonuclease;

[0083] Embodiment 13: The method according to any one of embodiments 1 to 8, wherein the amplification in step (ix) is carried out by PCR using primers that bind to the universal portions of the first and second probes.

[0084] Embodiment 14: The method of any one of embodiments 1 to 13, wherein gene target enumeration is enabled by counting the number of molecular barcodes per target and per sample.

[0085] Embodiment 15: The method according to any one of embodiments 9 to 14, wherein the nucleotide molecule is amplified with a first primer and a second primer to obtain an amplification product.

[0086] Embodiment 16: A kit of parts comprising a plurality of containers, At least one container contains one or more sets of first probes and second probes, and at least one container contains one or more bridge oligos or a plurality of oligonucleotides capable of forming a bridge oligo complex; said first probe starting at the 5' end of the molecule and comprising a first bridging oligo-specific sequence and, optionally, a first sequence barcode and a first target-specific portion at the 3' end of the first probe; said second probe starting at the 5' end of the molecule and comprising a second target specific portion and, optionally, a second sequence barcode and a second bridging oligo specific sequence at the 3' end of the second probe; said bridge oligo or bridge oligo complex comprising a sequence complementary to said first and second bridge oligo specific sequences in each of said first and second probes, respectively, and optionally a third barcode; At least one of the first sequence barcode, the second sequence barcode, or the third barcode is present in the first probe, the second probe, the bridge oligo, or the bridge oligo complex, respectively; and At least one of the first probe or the second probe or the bridged oligo or bridged oligo complex comprises a first capture moiety capable of linking to a second capture moiety linked to a solid support. EXAMPLES

[0087] (method) In one example, the starting material was 5 ml of urine. The process began by denaturing the proteins in the sample material by heating the sample to +95° C. for 15 minutes. The sample material was then centrifuged at 18000×g for 10 minutes to remove precipitated proteins and other debris, and the supernatant was collected for subsequent steps.

[0088] A three-part probe complex was formed (shown in FIG. 2) that contained: (a) a first probe having, starting at the 5' end of the molecule, a first bridging oligo-specific sequence and a first target-specific portion at the 3' end of the first probe; (b) a second probe having, starting at the 5' end of the molecule, a second target specific portion, a second sequence barcode, and a second bridging oligo specific sequence at the 3' end of the second probe; (c) a bridging oligo having a sequence complementary to the first and second bridging oligo specific sequences in the first and second probes, respectively, and a 5' biotin.

[0089] The three-part probe complex was added to the sample supernatant at femtomolar concentrations and allowed to anneal (shown in FIG. 1, step 1).

[0090] Dynabeads MyOne Streptavidin C1 magnetic particles were then added to the reaction and incubated at room temperature for 1 hour to allow biotin and streptavidin to interact and bind the hybridization complex to the particles, after which the particles were collected using a magnet and washed with Tween 20 containing TE buffer to remove sample impurities.

[0091] After a washing step, the bead-bound probe complex was extended (gap-filling) (Figure 2B) and ligated by adding a combination of DreamTaq DNA polymerase and Ampligase DNA ligase and incubating for 1 h at +45° C. The resulting extended and ligated product was cleaved by adding a uracil-specific enzyme mix, targeting the uracil moiety in the left-hand probe (New England Biolabs, used according to the manufacturer's instructions).

[0092] After extension, ligation and cleavage, the bead-bound molecules were washed with T7 reaction buffer and subjected to RNA synthesis by adding a combination of T7 RNA polymerase and RiboLock RNase inhibitor and incubating for 1 h at +37°C. The magnetic particles were then removed using a magnet and the supernatant was retained for further processing.

[0093] The RNA in the supernatant was prepared for cDNA synthesis by adding complementary oligonucleotides and incubating for 5 min at +75°C. RiboLock RNase inhibitor and M-MLV reverse transcriptase were then added to the reaction and incubated for 1 h at +37°C.

[0094] The cDNA was subsequently prepared into an Illumina-compatible DNA sequencing library using indexed PCR primers and PCR amplification with Phusion Hot Start II DNA polymerase.

[0095] Finally, the libraries were sequenced using an Illumina MiSeq or iSeq, and the sequencing data were processed using a combination of Unix command line tools and the Python and R programming languages. Briefly, the rationale for the sequencing process is to identify the probe sequences within each read, sequence the genomic regions between them, and count the number of molecular barcodes associated with each gene target.

[0096] (experiment) In the first experiment, urine samples were spiked with femtomolar concentrations of synthetic oligonucleotides that resemble typical oncogenic mutations in genes such as AKT1, CD74-ROS1, CHEK2, EGFR, EML4-ALK, KRAS, PIK3CA and TP53. Biotin-containing tripartite probes were added to target these genes. The first, second and crosslinking probes were 61, 85 and 50 nt in length, respectively.

[0097] The spiked samples were treated according to the method described above. The results were analyzed by gel electrophoresis. An example of a typical result is shown in Figure 3.

[0098] Target genes were detected within the sequence data by matching probe sequences within each read, identifying genomic sequence regions between the probe sequences, and counting molecular barcodes. The counting data accurately reflected the number of spike-in template molecules, and the response was linear over four orders of magnitude (Figure 4). The detected signal was highly specific to the presence or absence of the target molecule (Figure 5).

[0099] In another example, the starting material was 1 ml of saliva. The samples were treated similarly, except that they were boiled and centrifuged before the assay was performed. For these samples, the detected signal was also highly specific for the presence or absence of the target molecule.

[0100] (Detailed description of Figures 1 and 2) Figure 1 shows the workflow of one embodiment of the present invention described, where T7 RNA polymerase is used for amplification. As mentioned above, other amplification methods are possible. In step 1, the nucleic acid (DNA or RNA) in the sample (102) is contacted with a set of ligation complexes (104). The ligation complexes anneal onto the target nucleic acid (106). In step 2, the target-bound ligation complexes are captured from the sample material leaving behind sample impurities (103). In step 3, the annealed ligation complexes are ligated to obtain ligated ligation complexes. In step 4, the ligated ligation complexes from multiple samples (110) are pooled together (112). In step 5, T7 RNA polymerase is used to synthesize RNA from the ligated ligation complexes. Probe and sample DNA may be removed using a mixture of endonucleases and exonucleases. The amplified RNA is converted to cDNA (116) and may be amplified using PCR or emulsion PCR. In step 6, the amplified DNA is sequenced using next generation DNA sequencing. In step 7, the DNA sequencing results are converted to target counts using a bioinformatics pipeline.

[0101] FIG. 2A shows the principle structure of a probe triplet with multiple probe entities according to one embodiment of the present specification. The multiple probe entities include a left probe, a right probe and a bridge oligo assembled prior to sample annealing. The first base of the left probe optionally includes a phosphate moiety for enzymatic ligation or a modification that allows chemical ligation to the 5' end of the adjacent probe, called modification 1 (202). Bases 15-25 of the left probe include bridge sequence 1 (204), which may further include a chemically modified base for efficient bridge oligo binding, called bridge site 1. The aforementioned segment also optionally includes a reverse complement sequence of a T7 RNA polymerase promoter. As mentioned above, this promoter sequence may be present in the first probe or the second probe, but not in the bridge oligo, provided that the oligos and probes are designed to allow the T7 RNA polymerase to transcribe all the information required for enumeration of target sequences in different samples. The following 15-30 bases of the left probe optionally include a universal binding site for a PCR primer, referred to herein as universal site 1 (206). The left probe further includes 10-20 bases following from the 5' end, optionally including a segment of random nucleotides forming a molecule-specific or sample-specific barcode, referred to as barcode 1 (208). The left probe further includes 15-30 bases following from the 5' end, which bind to a genetic target (210). Some or all of the nucleotides of 204 or 210 may include chemical modifications that increase the affinity of the probe to the target or bridging oligo (226). The last base of the left probe optionally includes a phosphate moiety for enzymatic ligation or a modification that allows chemical ligation to the 5' end of an adjacent probe, referred to as modification 1 (210).

[0102] The first base of the right probe optionally contains a phosphate moiety for enzymatic ligation or a modification that allows for chemical ligation to the 5' end of an adjacent probe, referred to as modification 2 (214). The 15-30 bases from the 5' end of the right probe contain the portion of the right probe that binds to the genetic target (216). The next 10-20 bases from the 5' end of the right probe optionally contain a segment of random nucleotides that form a molecule-specific or sample-specific barcode, referred to as barcode 2 (218). The next 15-30 bases from the 5' end of the right probe optionally contain a universal binding site for a PCR primer, referred to as universal site 2 (220). The last 15-25 bases of the right probe contain a sequence for efficient bridging oligo binding, referred to as bridging sequence 2 (222). The aforementioned segment also optionally contains the reverse complement of a T7 RNA polymerase promoter. As mentioned above, this promoter sequence can be present in the first probe or the second probe, but not in the bridging oligo, provided that the oligos and probes are designed to allow T7 RNA polymerase to transcribe all the information required for enumeration of the target sequences in different samples. Some or all of nucleotides 216 or 222 can contain chemical modifications that increase the affinity of the probe to the target or bridging oligo (224). The last base of the right probe optionally contains a phosphate moiety for enzymatic ligation, or a modification that allows chemical ligation to the 5' end of the adjacent probe, called modification 2.

[0103] The first 15-25 bases from the 5' end of the bridge oligo, referred to as bridge sequence 3 (226), are reverse complementary to bridge sequence 1 (204) of the right probe and may contain chemically modified nucleotides to increase binding. The 15-25 bases following the bridge, referred to as bridge sequence 4 (224), are reverse complementary to bridge sequence 2 sequence (222) of the left probe and may contain chemically modified nucleotides to increase binding. Bridge sequence 3 (226) or bridge sequence 4 (224) optionally contain a sequence for a T7 RNA polymerase promoter. The 5' end of the bridge oligo contains a first capture moiety (228) that is used to capture the ligation complex.

[0104] FIG. 2B shows gap filling between the first and second probes according to one embodiment of the present specification, where the bridge oligo includes gap sequence 1 (230) between bridge sequence 3 (226) and bridge sequence 4 (224). Gap sequence (230) may optionally include a sequence of a T7 RNA polymerase promoter. The gap between the left and right probes is filled by introducing a polymerase and one or more nucleotides. For this process, Stoffel fragment, Taq polymerase or Phusion polymerase can be used. The polymerase adds nucleotides that are (a) complementary to the universal bridge oligo sequence and (b) complementary to the target sequence, thereby filling the two gaps, gap 1 and gap 2 between the first and second probes, resulting in ligation of the left and right probes that are complementary to the bridge oligo. In this way, the barcode, if present in the bridge oligo at position 230, is incorporated into the complementary sequence. Optionally, the bridge oligo probe 224 extends from the 5' or 3' site complementary to the ligated probe such that the barcode (if present in probe 1 or probe 2) and / or the target sequences 210 and 216 are incorporated into the bridge oligo, thereby forming a ligated ligation complex. However, care should be taken to ensure that the action of the polymerase does not interfere with the ligation of the probe at the site of the target sequence. For example, it is possible to use a polymerase that stops its action when it reaches a double-stranded DNA portion, e.g., when present in the portion of the first and second probes that are hybridized to the target sequence. A T7 RNA polymerase promoter sequence can be embedded in the ligation complex within positions 226, 230 or 224 of the bridge oligo. A deoxyuridine portion can be embedded between positions 204 and 206 or within 206 of the left probe.

[0105] FIG. 2C shows the principle structure of a probe quintet with multiple probe entities according to one embodiment of the present specification. The multiple probe entities include a left probe, a right probe, and a bridge consisting of three oligos. Here, the probe complex includes gaps between the left probe and the second bridge (228 and 236), between the second bridge and the right probe (240 and 222), between the first bridge oligo and the third bridge oligo (238 and 242), and between the left probe and the right probe (208 and 216). These gaps are filled by introducing a polymerase and one or more nucleotides. For this process, a mixture of Stoffel fragment, Taq polymerase or Phusion polymerase, and a DNA ligase such as Ampligase can be used. The polymerase fills these gaps, and the subsequent action of the DNA ligase ligates the probe and the bridge oligo into a circular complex.

[0106] The 15-25 bases of the left probe include bridging sequence 1 (228), which may include chemically modified bases for efficient bridge oligo attachment, referred to as bridging sequence 1. The left probe optionally further includes 10-20 bases continuing from the 5' end, including a universal sequence (204) used for library indexing. The left probe optionally further includes 10-20 bases continuing from the 5' end, including a segment of random nucleotides forming a molecule-specific or sample-specific barcode, referred to as barcode 1 (206). The left probe further includes 15-30 bases continuing from the 5' end, which bind to the genetic target (208). Some or all of the 228 nucleotides may include chemical modifications (226) that increase the affinity of the probe to its target or bridge oligo. The last base of the left probe optionally includes a phosphate moiety for enzymatic ligation, or a modification that allows chemical ligation to the 5' end of an adjacent probe, referred to as modification 1 (210).

[0107] The first base of the right probe optionally contains a phosphate moiety for enzymatic ligation, or a modification that allows chemical ligation to the 5' end of an adjacent probe, as modification 2 (214). The 15-30 bases from the 5' end of the right probe contain the portion of the right probe that binds to the genetic target (216). The next 10-20 bases from the 5' end of the right probe optionally contain a segment of random nucleotides that form a molecule-specific barcode or a sample-specific barcode, as barcode 2 (218). The next 10-20 bases from the 5' end of the right probe optionally contain a universal sequence (220). The last 15-25 bases of the right probe, referred to as bridging sequence 8 (222), are reverse complementary to bridging sequence 7 of the third bridging oligo (224). Some or all of nucleotides 208, 216, 222, or 228 may contain chemical modifications that increase the affinity of the probe to the target or bridging oligo.

[0108] The first 15-25 bases from the 5' end of the first bridging oligo, referred to as bridging sequence 3 (226), are reverse complementary to bridging sequence 1 (228) of the right probe and may contain chemically modified nucleotides to increase binding. The last 15-25 bases of the first bridging oligo, referred to as bridging sequence 2 (238), are reverse complementary to bridging sequence 4 sequence (236) of the second bridging oligo and may contain chemically modified nucleotides to increase binding. The 5' end of the first bridging oligo optionally contains a capture moiety (230) that is used to capture the ligation complex.

[0109] The first 15-25 bases from the 5' end of the second bridging oligo, referred to as bridging sequence 5 (240), are reverse complementary to bridging sequence 6 (242) of the third bridging oligo and may contain chemically modified nucleotides to selectively increase binding. The last 15-25 bases of the second bridging oligo, referred to as bridging sequence 4 (236), are reverse complementary to bridging sequence 2 (238) of the first bridging oligo and may contain chemically modified nucleotides to selectively increase binding.

[0110] The first 15-25 bases of the third bridging oligo from the 5' end, referred to as bridging sequence 6 (242), are reverse complementary to the bridging sequence 5 sequence (240) of the second bridging oligo and may optionally contain chemically modified nucleotides to increase binding. The last 15-25 bases of the first bridging oligo, referred to as bridging sequence 7 (224), are reverse complementary to the bridging sequence 8 sequence (222) of the right probe and may optionally contain chemically modified nucleotides to increase binding. The 3' end of the third bridging oligo may optionally contain a phosphate (or other cleavable) moiety (234) to prevent extension during gap filling.

[0111] A T7 RNA polymerase promoter sequence can be embedded in the ligation complex within positions 226, 238 or 242 or 224 of the bridge oligo. A deoxyuridine moiety can be embedded between positions 204 and 228, or within 206 of the left probe.

Claims

1. 1. A method for high throughput detection of one or more target nucleotide sequences in a plurality of samples, comprising: (i) detecting each target nucleotide sequence in each of said samples by: providing a first probe, a second probe and a bridge oligo or a plurality of oligonucleotides capable of annealing to each other to form a bridge oligo complex; said first probe starting from the 5' end of the molecule comprises a first bridging oligo-specific sequence, optionally a first sequence barcode, and a first target-specific portion at the 3' end of the first probe; said second probe starting at the 5' end of the molecule and comprising a second target specific portion, optionally a second sequence barcode, and a second bridging oligo specific sequence at the 3' end of the second probe; the bridge oligo or bridge oligo complex has a sequence complementary to the first bridge oligo specific sequence and the second bridge oligo specific sequence of the first probe and the second probe, respectively, and optionally a third barcode; at least one of the first sequence barcode or the second sequence barcode or the third barcode is present in the first probe or the second probe or the bridge oligo or bridge oligo complex, respectively; At least one of the first probe or the second probe or the bridged oligo or bridged oligo complex comprises a first capture moiety; (ii) for each of said one or more target nucleotide sequences, contacting said first probe and said second probe with said bridge oligo or a plurality of oligonucleotides capable of forming a bridge oligo complex, preferably for each of said samples in separate tubes, and allowing self-annealing into a plurality of ligation complexes; (iii) contacting nucleic acid present in each of the samples to be tested for the target nucleotide sequence with the ligation complex; (iv) hybridizing the first and second target-specific portions of the first and second probes to essentially adjacent portions on the target sequence, thereby forming a hybridization complex; (v) contacting the hybridization complex with a solid support comprising a second capture moiety and allowing the first capture moiety to interact with the second capture moiety such that the hybridization complex is linked to the solid support; (vi) separating hybridization complexes bound to the solid support from components of the sample that are not bound to the solid support; (vii) ligating the probes in the hybridization complex to provide a ligated ligation complex; (viii) optionally pooling the ligated ligation complexes from the multiple samples; (ix) amplifying nucleic acid from one or more of the ligated ligation complexes; (x) subjecting the nucleic acid obtained in step (ix) to high throughput sequencing techniques to determine the barcode sequence; and (xi) identifying the presence and / or number of said target nucleotide sequences in said plurality of samples by determining at least a portion of said first target specific portion and / or said second target specific portion, and / or at least a portion of said first barcode and / or said second barcode, and / or at least a portion of said third barcode; The method wherein steps (vii) and (viii) may be performed in any order.

2. 2. The method of claim 1, wherein the plurality of samples comprises blood samples, saliva samples, urine samples or fecal samples, samples of other bodily fluids or extracts from bodily substances.

3. The method of claim 1 or 2, wherein the first capture moiety is a biotin moiety and the second capture moiety is a streptavidin moiety or an avidin moiety.

4. The method according to any one of claims 1 to 3, wherein a washing step is carried out between steps (vi) and (vii).

5. The method according to any one of claims 1 to 4, wherein the sequencing is carried out by next generation DNA or RNA sequencing.

6. 6. The method of any one of claims 1 to 5, wherein the 3' end of the first probe or the 5' end of the second probe, or both, are modified to enable chemical ligation of the first probe to the second probe.

7. 7. The method of any one of claims 1 to 6, wherein the bridging moiety of the first probe or the second probe, or both, comprises a chemically modified base for improved binding to the bridged oligo or bridged oligo complex.

8. 8. The method of any one of claims 1 to 7, wherein the first target-specific portion, the second target-specific portion, the first bridging oligospecific sequence, and / or the second bridging oligospecific sequence independently comprise one or more chemically modified nucleotides.

9. a promoter sequence for T7 RNA polymerase is present in the first probe, the second probe, or the bridge oligo or bridge oligo complex; 9. The method of any one of claims 1 to 8, wherein the amplification in step (ix) comprises RNA synthesis from the one or more ligated ligation complexes using T7 RNA polymerase that initiates RNA synthesis from the T7 RNA polymerase promoter incorporated in the ligated ligation complexes.

10. 10. The method of claim 9, wherein the first probe, the second probe, or the bridge oligo or bridge oligo complex comprises a deoxyuridine moiety that allows linearization by cleavage with a uracil-specific cleavage reagent.

11. 10. The method of claim 9, wherein prior to step (x), a cDNA is prepared from the RNA molecule using a DNA-oligonucleotide molecule that is reverse-complementary to a universal site present in the first probe or the second probe or the bridging oligo or bridging oligo complex.

12. At least one of the first probe or the second probe or the bridging oligo or the bridging oligo complex comprises a recognition sequence for an endonuclease; (a) the amplification of nucleic acids from the one or more ligated ligation complexes in step (ix) is carried out using rolling circle amplification with a strand displacing polymerase, followed by (b) optionally subjecting the resulting amplified one or more single-stranded concatemer sequences to annealing with a specific oligonucleotide containing the recognition sequence, the specific oligonucleotide annealing to the recognition sequence in such a way as to provide a recognition site for the endonuclease; and The method of any one of claims 1 to 8, further comprising (c) selectively cleaving the resulting amplified one or more single-stranded concatemeric sequences or the resulting annealed complexes with an endonuclease.

13. The method according to any one of claims 1 to 8, wherein the amplification in step (ix) is carried out by PCR using primers that bind to universal portions of the first and second probes.

14. The method of any one of claims 1 to 13, wherein gene target enumeration is enabled by counting the number of molecular barcodes per target and per sample.

15. The method according to any one of claims 9 to 14, wherein the nucleotide molecule is amplified with the first and second primers to provide an amplification product.

16. A kit of parts including a plurality of containers, At least one container contains one or more sets of first probes and second probes, and at least one container contains one or more bridge oligos or a plurality of oligonucleotides capable of forming a bridge oligo complex; said first probe starting from the 5' end of the molecule comprises a first bridging oligo-specific sequence and, optionally, a first sequence barcode and a first target-specific portion at the 3' end of the first probe; said second probe starting at the 5' end of the molecule and comprising a second target specific portion and, optionally, a second sequence barcode and a second bridging oligo specific sequence at the 3' end of the second probe; said bridge oligo or bridge oligo complex comprising a sequence complementary to said first and second bridge oligo specific sequences in each of said first and second probes, respectively, and optionally a third barcode; At least one of the first sequence barcode or the second sequence barcode or the third barcode is present in the first probe or the second probe or the bridge oligo or bridge oligo complex, respectively; and At least one of the first probe or the second probe or the bridged oligo or bridged oligo complex comprises a first capture moiety capable of linking to a second capture moiety linked to a solid support.

17. 1. A method for high throughput detection of one or more target nucleotide sequences in a plurality of samples, comprising: (i) providing a respective target nucleotide sequence for each of said samples; The single probe starts at the 5' end of the molecule and comprises a first target specific portion, a spacer portion comprising a barcode, and a second target specific portion at the 3' end of the second probe; a bridge oligo having a sequence complementary to the spacer portion or a portion of the spacer portion of the single probe, the single probe or the bridge oligo comprises a first capture moiety; (ii) contacting and self-annealing said single probe for each of said one or more target nucleotide sequences with said bridge oligo for each of said samples, preferably in separate tubes; (iii) contacting nucleic acids present in the plurality of samples to be tested for the target nucleotide sequence with the single probe annealed to the bridge oligo; (iv) hybridizing the first target-specific portion and the second target-specific portion of the single probe to essentially adjacent portions on the target sequence, thereby forming a hybridization complex; (v) contacting the hybridization complex with a solid support comprising a second capture moiety and allowing the first capture moiety to interact with the second capture moiety such that the hybridization complex is linked to the solid support; (vi) separating hybridization complexes bound to the solid support from components of the sample that are not bound to the solid support; (vii) ligating the 5' and 3' ends of the single probe to provide a ligated ligation complex; (viii) optionally pooling the ligated ligation complexes from the multiple samples; (ix) amplifying nucleic acid from the one or more ligated ligation complexes; (x) subjecting the nucleic acid obtained in step (ix) to high throughput sequencing techniques to determine the barcode sequence; and (xi) identifying the presence and / or number of said target nucleotide sequences in said plurality of samples by determining at least a portion of said first target specific portion and / or said second target specific portion, and / or at least a portion of said first barcode and / or said second barcode, and / or at least a portion of said third barcode; The method, wherein steps (vii) and (viii) may be performed in any order.

18. The method of claim 17, wherein the single probe comprises two or more oligonucleotides chemically linked together.

19. Method according to claim 17 or 18, further comprising the features according to any one of claims 2 to 15.

20. A kit of parts including a plurality of containers, At least one container contains one or more single probes, and at least one container contains one or more bridge oligos; The single probe comprises, starting from the 5' end of the molecule, a first target-specific portion, a spacer portion comprising a barcode, and a second target-specific portion at the 3' end of the second probe; the bridge oligo has a sequence complementary to the spacer portion or a portion of the spacer portion of the single probe; and The single probe or the bridging oligo comprises a first capture moiety capable of being linked to a second capture moiety linked to a solid support.

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