Highly sensitive methods for accurate parallel quantification of nucleic acids

Abstract

To provide a next generation DNA sequencing method for accurate and massively parallel quantification of one or more nucleic acid targets, in large volumes of unpurified sample material.SOLUTION: The present invention provides a method and a kit for detecting genetic targets and mutations, specifically, a method for the high-throughput detection of one or more target nucleotide sequence in a plurality of samples, comprising one or more target-specific nucleic acid probes for detecting and quantifying genetic targets in complex DNA pools, and a bridge oligo or a bridge oligo complex.SELECTED DRAWING: Figure 2A

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 present disclosure relates to a method and kit that includes probes for detecting and quantifying genetic targets in complex DNA pools, which are primarily used for detecting genetic targets and variants. The present invention uses one or more target-specific nucleic acid probes (left and right probes) for each genetic target, and bridging oligos or bridging oligo complexes. [Background technology]

[0002] The detection of genetic variation in some plants and animals is not troublesome due to the advancement of genetic variation research technology.However, the detection and accurate quantification of genetic variation, such as mutation, especially in samples with weak signals, is currently still 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., 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 substantial hands-on time (labor cost) to set up the reaction, the need for standard curves, and replicates for each quantification target, the actual cost is in fact much higher. Because a separate quantification experiment is required for each gene target, the amount of hands-on time scales 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, where 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 2018 / 109206 shows a method for detecting an analyte in a sample using padlock probes and rolling circle amplification. The use of bridging oligos is not described. WO 2019 / 038372 describes a next-generation sequencing approach in which target sequences of interest are selectively amplified by in vitro transcription from a ligation complex containing a promoter for T7 polymerase, followed by cDNA synthesis and sequencing. 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. 2018 / 109206

[0011] [Patent Document 2] International Publication No. 2019 / 038372 [Non-patent literature]

[0012] [Non-Patent Document 1] Akhras et al. (2007) PLoS ONE 2(2):e223 Summary of the Invention [Problem to be solved by the invention]

[0013] The present invention provides methods using next generation sequencing for highly scalable and accurate target quantification from large sample volumes (up to tens of milliliters) and / or from diluted and / or unpurified sample material. Furthermore, an RNA amplification step as described in WO2019 / 038372 is avoided, making the method simpler. [Means for solving the problem]

[0014] 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) providing, to each target nucleotide sequence in each of said samples, 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; The second probe, starting from the 5' end of the molecule, comprises 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; and The bridge oligo or bridge oligo complex comprises 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; and 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 bridging oligo or the bridging oligo complex comprises a recognition sequence for an endonuclease; and Optionally, at least one of the first probe or the second probe or the bridging oligo or a plurality of oligonucleotides capable of annealing to each other to form a bridging oligo complex comprises a first capture moiety; The process and (ii) for each of said one or more target nucleotide sequences, contacting said first probe and said second probe, preferably for each of said samples in separate tubes, with a plurality of nucleotides capable of annealing to each other to form said bridge oligo or said bridge oligo complex, 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) optionally contacting the hybridization complex 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, and separating the hybridization complex bound to the solid support from components of the sample that are not bound to the solid support; (vi) ligating the probes in the hybridization complex to provide a ligated ligation complex; (vii) optionally pooling the ligated ligation complexes from the plurality of samples; (viii) amplifying nucleic acid from one or more of the ligated ligation complexes using rolling circle amplification with a strand-displacing polymerase; (ix) optionally subjecting the amplified one or more single-stranded concatemer sequences obtained in step (viii) to annealing with a specific oligonucleotide containing a recognition sequence for an endonuclease, said oligonucleotide annealing with the recognition sequence specified in step (i) so as to obtain a recognition site for the endonuclease; (x) optionally cleaving the single-stranded concatemeric sequence obtained in step (viii) or the annealed complex obtained in step (ix) with an endonuclease; (xi) subjecting the nucleic acid fragment obtained in step (x) or the concatemer sequence obtained in step (viii) to a high-throughput sequencing technique to determine the barcode sequence; and (xii) 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 is such that steps (vi) and (vii) may be carried out 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]The RCA products from the workflow are shown before (lane 2) and after (lane 1) digestion with restriction endonuclease. [Figure 4] Figure 1 shows the linear response of the experimental workflow to the number of gene targets, which decreases logarithmically during four replicate reactions, as inferred from next-generation DNA sequencing data by enumerating molecular barcodes. Each row shows three concentrations of target sequences. The response is linear over three orders of magnitude. 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: A probe is a fragment of DNA or RNA of variable length (usually 50-1000 bases long, preferably 50-200 bases long) that can be used in a DNA or RNA sample 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 is provided, whereby the probes each contain a section at their end that is complementary to a portion of the target sequence. Additionally, the disclosure describes a bridge oligo or bridge oligo complex that is used to join the left and right probes.

[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 the in vitro enzymatic amplification of a specific DNA / RNA. The DNA / RNA to be amplified can be denatured by heating the sample. The term primer is a short strand (generally about 18-22 bases) of RNA or DNA 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.

[0024] Polymerase: A polymerase is an enzyme that synthesizes long chains, or polymers, of nucleic acids. DNA polymerase and RNA polymerase are used to assemble DNA and RNA molecules, respectively, by copying a DNA or RNA template strand using base-pairing interactions.

[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] Endonuclease: Endonucleases are enzymes that cleave double or single strands of DNA randomly or at directed locations.

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

[0028] 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) providing, to each target nucleotide sequence in each of said samples, 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; The second probe, starting from the 5' end of the molecule, comprises 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; and The bridge oligo or bridge oligo complex comprises 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; and 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 bridging oligo or the bridging oligo complex comprises a recognition sequence for an endonuclease; and Optionally, at least one of the first probe or the second probe or the bridging oligo or a plurality of oligonucleotides capable of annealing to each other to form a bridging oligo complex comprises a first capture moiety; The process and (ii) for each of said one or more target nucleotide sequences, contacting said first probe and said second probe, preferably for each of said samples in separate tubes, with a plurality of nucleotides capable of annealing to each other to form said bridge oligo or said bridge oligo complex, 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) optionally contacting the hybridization complex 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, and separating the hybridization complex bound to the solid support from components of the sample that are not bound to the solid support; (vi) ligating the probes in the hybridization complex to provide a ligated ligation complex; (vii) pooling the ligated ligation complexes from the multiple samples; (viii) amplifying nucleic acid from one or more of the ligated ligation complexes using rolling circle amplification with a strand-displacing polymerase; (ix) optionally subjecting the amplified one or more single-stranded concatemer sequences obtained in step (viii) to annealing with a specific oligonucleotide containing a recognition sequence for an endonuclease, said oligonucleotide annealing with the recognition sequence specified in step (i) so as to obtain a recognition site for the endonuclease; (x) optionally cleaving the single-stranded concatemeric sequence obtained in step (vii) or the annealed complex obtained in step (ix) with an endonuclease; (xi) subjecting the nucleic acid fragment obtained in step (x) or the concatemer sequence obtained in step (viii) to a high-throughput sequencing technique to determine the barcode sequence; and (xii) 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 is such that steps (vi) and (vii) may be carried out 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 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 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 enrichment of the nucleic acids, hi another embodiment, the sample may be pretreated, for example by lysing cells to expose the nucleic acids. In one embodiment, the sample is used without prior purification or enrichment of the nucleic acids, hi another embodiment, the sample can be pretreated, for example, cells can be lysed to expose the 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 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] Additionally, 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. The recognition sequence is required for the concatemer sequence cleavage in step (x). In one embodiment, it is a recognition sequence for a restriction endonuclease such as EcoRI. In another embodiment, it is a recognition sequence for a homing endonuclease such as I-CeuI. In another embodiment, it is a recognition sequence for a cleavage system such as induced DNAaseI or CRISPR-Cas.

[0038] Optionally, 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 a second capture moiety linked to a solid support can capture, i.e. bind, a probe, a ligation complex or a 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.

[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 modification chemically ligates adjacent probes.In some embodiments, the probes bind to completely adjacent loci or at a distance of up to 500 base pairs, for example at a distance of up to 200 base pairs, for example at a distance of up to 50 base pairs, preferably at a distance of up to 40 base pairs, more preferably at a distance of up to 30 base pairs, more preferably at a distance of up to 20 base pairs, more preferably at a distance of up to 10 base pairs, and most preferably at a distance of up to 5 base pairs.

[0040] In some embodiments, the first probe or the second probe or the cross-linking probe or the cross-linking oligo complex may include an adapter sequence for a DNA sequencing platform, such as, but not limited to, Illumina, which allows the resulting sequencing library to be coupled to the detection portion of a sequencing device, such as an Illumina flow cell.

[0041] Additionally, in some embodiments, the bridged oligo or bridged oligo complex comprises: (i) 1 to 5 3' overhanging bases (i.e., additional bases that do not form a duplex with the second probe), and / or (ii) a 3' phosphate, and / or (iii) one or more phosphorothioate modifications within three positions from the 3' end; Includes.

[0042] Before 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 a separate tube, to allow self-annealing to the 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 (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 at once.

[0043] 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.

[0044] One or more target nucleotide sequences in the multiple samples are then 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. In one embodiment, the multiple samples include one or more blood samples, one or more saliva samples, one or more urine samples, or one or more fecal samples.

[0045] Then, in some embodiments, if at least one of the first probe or the second probe or the bridging oligo or the bridging oligo complex comprises a first capture moiety, the hybridization complex is contacted with a solid support comprising a second capture moiety, and the first capture moiety and the second capture moiety are allowed to interact such that the hybridization complex is linked to the solid support (optional 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.

[0046] Step (v) purifies and concentrates the nucleic acids, allowing improved results, especially for samples of very low purity. In one embodiment, the method of the invention does not include a step of concentrating the nucleic acids prior to step (v). Thus, in one embodiment the method does not include a step of concentrating the nucleic acids in the original sample by more than 2-fold, more than 10-fold, or more than 100-fold prior to step (vi). In another embodiment, the method of the invention does not include a purification step after the ligation in step (vi).

[0047] Ligation of the probes in the formed hybridized complex is then carried out enzymatically or chemically to provide a ligated ligation complex (step (vi)). Optionally as part of step (vi), 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 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 or bridge oligo complex 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 or bridge oligo complex. Preferably, a polymerase is used that does not degrade double-stranded DNA, such as Taq polymerase, so as not to interfere with ligation of the first probe to the second probe when both are annealed to the target sequence.

[0048] The ligated ligation complexes are then pooled from one or more target samples (step (vii)). Steps (vi) and (vii) may be performed in the order specified or in reverse order.

[0049] Nucleic acid is then amplified from one or more of the plurality of ligated ligation complexes (step (viii)). The amplification is carried out using rolling circle amplification with a strand-displacing polymerase such as phi29 polymerase (UniProtKB-P03680, DPOL_BPPH2) or Bst polymerase (P52026, DPO1_GEOSE). In the next step (ix), the amplified one or more single-stranded concatemer sequences obtained in step (viii) are subjected to selective annealing with a specific oligonucleotide containing a recognition sequence of an endonuclease, where said oligonucleotide anneals to said recognition sequence specified in step (i) so as to obtain a recognition site of the endonuclease. The specific oligonucleotide containing a recognition sequence may typically contain some additional specific sequences around the recognition sequence to allow the formation of a stable double helix for cleavage.

[0050] Subsequently, the single-stranded concatemeric sequences obtained in step (viii) or the annealed complexes obtained in step (ix) are selectively cleaved with said endonuclease (step (x)), resulting in an NGS library.

[0051] Optionally, after amplification, if a solid support is present, it is removed and the supernatant is used for further processing. For example, if the solid support is a magnetic particle, it can be removed using a magnet. In some other embodiments 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 (vi), 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 streptavidin is bound to a magnetic particle, it can then be removed using a magnet.

[0052] Further, optionally, a PCR amplification is performed between step (x) and step (xi) using primers that bind to the universal portions of the first and second probes, said primers optionally comprising an adaptor for subsequent sequencing in step (xi).

[0053] 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 using high throughput next generation sequencing technology, such as, but not limited to, next generation sequencing platforms including Illumina iSeq, MiSeq, HiSeq, NextSeq or NovaSeq (steps (xi) and (xii)). 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.

[0054] In a preferred embodiment, the molecule is further amplified with a first primer and a second primer to provide an amplification product.Preferably, a universal first primer and a universal second primer are used, which are complementary to the first or second universal sequence present in the ligated complex.

[0055] Advantages of the present 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 present invention is that the method of the present 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 present 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.

[0056] In another aspect, the invention provides a kit of parts as follows: The kit of parts includes a plurality of containers, at least one container including one or more sets of first and second probes, and at least one container including one or more bridge oligos or a plurality of oligonucleotides capable of forming a bridge oligo complex; the first probe, starting at 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; the second probe starts at the 5' end of the molecule and comprises 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; the bridge oligo or bridge oligo complex comprises sequences complementary to the first and second bridge oligo specific sequences of the first and second probes, respectively, and optionally a third barcode; said bridge oligo or bridge oligo complex comprises a sequence complementary to each of the first and second bridge oligo specific sequences in each of said first and second probes, 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 bridging oligo or the bridging oligo complex, respectively; 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 the bridge oligo complex, respectively; and 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 The kit of parts further comprises an oligonucleotide capable of annealing to the recognition sequence to provide a recognition moiety for the endonuclease.

[0057] Preferably, the 3' ends of the plurality of first probes or the 5' ends of the plurality of second probes, or both, are modified to allow chemical ligation of the plurality of first probes to the plurality of second probes.

[0058] 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.

[0059] 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.

[0060] 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.

[0061] In one particular embodiment, at least one container that contains the first and second set of probes and at least one container that contains the bridge oligo or a plurality of oligonucleotides that can be annealed to each other to form a bridge oligo complex are one and the same container.In such a case, the three probes may be pre-annealed and ligated to form a complex.

[0062] 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.

[0063] Furthermore, the present invention relates to: EMBODIMENT 1 1. A method for high throughput detection of one or more target nucleotide sequences in a plurality of samples, comprising: (i) providing, for each target nucleotide sequence in each of said samples, a first probe, a second probe and a bridge oligo; The 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; and The second probe, starting from the 5' end of the molecule, comprises 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; and The bridge oligo or bridge oligo complex comprises 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; and 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 bridging oligo or the bridging oligo complex comprises a recognition sequence for an endonuclease; and Optionally, at least one of the first probe or the second probe or the bridging oligo comprises a first capture moiety; The process and (ii) for each of said one or more target nucleotide sequences, contacting said first probe and said second probe with said bridged oligo complex, preferably for each of said samples in separate tubes, and allowing self-annealing into multiple 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) optionally contacting the hybridization complex 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, and separating the hybridization complex bound to the solid support from components of the sample that are not bound to the solid support; (vi) ligating the probes in the hybridization complex to provide a ligated ligation complex; (vii) pooling the ligated ligation complexes from the multiple samples; (viii) amplifying nucleic acid from one or more of the ligated ligation complexes using rolling circle amplification with a strand-displacing polymerase; (ix) subjecting the amplified one or more single-stranded concatemer sequences obtained in step (viii) to annealing with a specific oligonucleotide containing a recognition sequence for an endonuclease, said oligonucleotide annealing with the recognition sequence specified in step (i) so as to obtain a recognition site for the endonuclease; (x) cleaving the single-stranded concatemeric sequence obtained in step (viii) or the annealed complex obtained in step (ix) with an endonuclease; (xi) subjecting the nucleic acid fragments obtained in step (x) to a high-throughput sequencing technique to determine the barcode sequence; and (xii) 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 (vi) and (vii) may be performed in any order.

[0064] EMBODIMENT 2 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.

[0065] EMBODIMENT 3 At least one of the first probe or the second probe or the bridging oligo comprises a first capture moiety; 3. The method of embodiment 1 or 2, comprising step (v) and not comprising a step of concentrating the nucleic acid prior to step (v).

[0066] EMBODIMENT 4 At least one of the first probe or the second probe or the bridging oligo comprises a first capture moiety; 4. The method according to any one of the preceding embodiments, comprising step (v), wherein the first capture moiety is a biotin moiety and the second capture moiety is a streptavidin moiety or an avidin moiety.

[0067] EMBODIMENT 5 At least one of the first probe or the second probe or the bridging oligo comprises a first capture moiety; 5. The method according to any one of the preceding embodiments, comprising step (v), wherein a washing step is carried out between steps (v) and (vi).

[0068] EMBODIMENT 6 The bridge oligo is (i) 1 to 5 3' overhanging bases, and / or (ii) a 3' phosphate, and / or (iii) one or more phosphorothioate modifications within three positions from the 3' end The method according to any one of embodiments 1 to 5, comprising:

[0069] EMBODIMENT 7 7. The method according to any one of the preceding claims, 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.

[0070] EMBODIMENT 8 8. The method of any one of the preceding claims, wherein the bridging moiety of the first probe or the second probe, or both, comprises a chemically modified base to improve binding to the bridged oligo or bridged oligo complex.

[0071] EMBODIMENT 9 9. The method of any one of the preceding claims, wherein the first target-specific portion, the second target-specific portion, the first bridging oligospecific sequence, and / or the second bridging oligospecific sequence independently contain one or more chemically modified nucleotides.

[0072] EMBODIMENT 10 10. The method according to any one of the preceding claims, wherein step (viii) is carried out using phi29 polymerase or Bst polymerase.

[0073] EMBODIMENT 11 11. The method of any of the preceding claims, wherein PCR amplification is carried out between step (x) and step (xi) using primers that bind to universal portions of the first and second probes, said primers optionally comprising an adaptor for subsequent sequencing in step (xi).

[0074] EMBODIMENT 12 12. The method of any of the preceding claims, wherein gene target enumeration is enabled by counting the number of molecular barcodes per target and per sample.

[0075] EMBODIMENT 13 13. The method of any of the preceding claims, wherein for two or more samples or two or more loci / allele combinations, barcode sequences are used to genotype the samples for one or more sequences and / or polymorphisms, such as SNPs and / or indels.

[0076] EMBODIMENT 14 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; the 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 from 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; the bridge oligo comprises a sequence complementary to each of the first and second bridge oligo specific sequences in each of the first and second probes, 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, or the bridge oligo, respectively; and At least one of the first probe or the second probe or the bridging oligo comprises a recognition sequence for an endonuclease; and The kit of parts further comprises an oligonucleotide capable of annealing to said recognition sequence so as to provide a recognition moiety for said endonuclease. EXAMPLES

[0077] (method) 1. Probe Complex Formation The probe complex contains the genetic target, the sample index, and the sequences required for constructing an Illumina sequencing library. A three-part probe complex was formed (shown in Figure 2), which contains: (a) a first probe having, starting from 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 from 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; and (c) a bridging oligo having sequences complementary to the first bridging oligo specific sequence and the second bridging oligo specific sequence in the first probe and the second probe, respectively.

[0078] The probe complex is constructed by combining equimolar amounts of all three parts (bridge, right arm, left arm) in an annealing reaction. The reaction is carried out in a thermocycler (annealing program shown in Table 1).

[0079] [Table 1]

[0080] 2. Target Capture The specific genomic region containing the mutation of interest is targeted. Purified DNA (e.g., from tissue, plasma, urine, saliva) can be used as the sample, or the sample can be unpurified and only pretreated, e.g., by boiling and / or centrifugation.

[0081] The probe complex hybridizes to the target region through complementary interactions of base sequences. To initiate target capture, the reaction probe and target DNA are mixed and incubated in a thermal cycler (target capture and gap-filling program shown in Table 2).

[0082] [Table 2]

[0083] 3.Gap filling After target capture, the probe complex is extended and ligated by adding a combination of Phusion DNA polymerase, nucleotides, and Ampligase DNA ligase and incubating at +45°C for 45 minutes.

[0084] 4. Rolling Circle Amplification After extension and ligation, the circular probe molecules are subjected to rolling circle amplification (RCA). For the RCA reaction, the target capture reaction is mixed with an RCA reaction mix containing EquipPhi29 (Thermo Scientific) polymerase. The reaction is incubated for 30 min to 2 h at +42° C. After the RCA reaction, the reaction efficiency is analyzed by measuring the concentration of single-stranded DNA (ssDNA) with a Qubit fluorometer.

[0085] 5.Enzymatic design The RCA reaction generates long concatemeric ssDNA molecules with multiple copies of the target library, with each complete target library separated by an EcoRI restriction enzyme recognition sequence. This sequence allows for sequence-specific cleavage of long concatemers via annealing with specific oligonucleotides containing the EcoRI restriction enzyme recognition sequence and release of a primed target library. These libraries are ready for further analysis after a simple purification step: the RCA products are digested with EcoRI for 1 hour at +37°C.

[0086] 6. Library Purification After EcoRI digestion, library molecules are purified by extraction from agarose gels following electrophoresis or using size selection beads (such as Macherey Nagel NucleoMag).

[0087] 7. Screening The purified MiSeq or iSeq100 compatible libraries are subjected to sequence analysis using state-of-the-art sequencing instruments. Importantly, the libraries can be converted to be compatible with any available sequencing platform by simple oligonucleotide modifications.Sequence data are 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 in each read, sequence the genomic regions between them, and count the number of molecular barcodes associated with each gene target.

[0088] (Experiment 1) In the first experiment, the probe mix was a collection of four different index probes resulting in four replicate reactions, each of which targeted the EML-ALK fusion. The targeting oligonucleotides had a unique recognition sequence that allowed for the identification of each target.

[0089] As samples, three synthetic target oligonucleotides were mixed at logarithmically increasing concentrations. Target capture, extension and ligation reactions, rolling circle amplification and subsequent digestion with EcoRI were carried out as described above. An example of a typical result is shown in Figure 3.

[0090] The ready libraries were sequenced on MiSeq and iSeq100 instruments, and target genes were detected in the sequence data by matching probe sequences in 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 detected signals were highly specific for the presence or absence of target molecules (Figure 4).

[0091] (Detailed description of Figures 1 and 2) Figure 1 shows the workflow of one embodiment of the present invention described. In step 1, nucleic acid (DNA or RNA) in a sample (102) is contacted with a set of ligation complexes (104). The ligation complexes anneal onto a target nucleic acid (106). In step 2, target-bound ligation complexes were selectively captured from the sample material leaving behind sample impurities (103). In step 3, the annealed ligation complexes are ligated to obtain a ligated ligation complex. In step 4, the ligated ligation complexes from multiple samples (110) are pooled together (112). In step 5, the probe sequence is amplified by rolling circle amplification using phi29 polymerase (UniProtKB-P03680, DPOL_BPPH2) or other strand-displacing polymerase, resulting in long concatemeric copies of the probe (1116). In step 6, optionally the concatemeric probe copies are cleaved into monomeric units using a restriction endonuclease such as EcoRI or a homing nuclease such as I-CeuI and optionally further amplified using PCR or emulsion PCR. In step 7, the amplified DNA is sequenced using next generation DNA sequencing. In step 8, the DNA sequencing results are converted into target counts using a bioinformatics pipeline.

[0092] 2A shows the principle structure of a probe triplet with multiple probe entities according to one embodiment of the present specification, which includes a left probe, a right probe and a bridge oligo assembled before sample annealing. The first base of the left probe optionally contains a phosphate moiety for enzymatic ligation or a modification allowing chemical ligation to the 5' end of an adjacent probe, called modification 1 (202). Bases 15 to 25 of the left probe contain cross-linking sequence 1 (204), which may further contain chemically modified bases for efficient cross-linking oligo binding, referred to as cross-linking site 1. The left probe optionally further comprises 10-20 bases following from the 5' end, including a segment of random nucleotides forming a molecular specific barcode or sample specific barcode, referred to as barcode 1 (206). The left probe further comprises 15-30 bases following from the 5' end, which bind to a genetic target (208). Some or all of the nucleotides of 204 or 208 may contain chemical modifications that increase the affinity of the probe to its target or bridging oligo (226). The last base of the left probe optionally contains 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).

[0093] 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, called 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, called barcode 2 (218). The last 15-25 bases of the right probe contain a sequence for efficient bridge oligo binding, called bridge sequence 2 (222). Some or all of nucleotides 204, 208, 216 or 220 may contain chemical modifications that increase the affinity of the probe for the target or bridge oligo.

[0094] The first 15-25 bases from the 5' end of the bridging oligo, called bridging sequence 3 (228), are reverse complementary to bridging sequence 1 (204) of the right probe and may optionally contain chemically modified nucleotides to increase binding. The last 15-25 bases of the bridge, called bridging sequence 4 (224), are reverse complementary to bridging sequence 2 of the left probe (220) and may optionally contain chemically modified nucleotides to increase binding. Here, the bridging sequence contains a recognition site for a restriction endonuclease such as EcoRI or a homing endonuclease such as I-CeuI (226). The 5' end of the bridging oligo optionally contains a capture moiety (230) that is used to capture the ligation complex.

[0095] 2B illustrates gap filling between a first probe and a second probe according to one embodiment herein, where the bridge oligo contains gap 1 between bridge sequence 1 (228) and bridge sequence 2 (224). Gap 2 is formed between the target binding sites of probes 1 and 2 (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 adds nucleotides that are (a) complementary to the universal bridge oligo sequence and (b) complementary to the target sequence, thereby filling the two gaps, i.e., between the first and second probes, and the subsequent action of the DNA ligase results in the ligation of the bridge oligo and the left and right probes complementary to the target sequence into a circular complex.

[0096] 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 contains 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 and third bridge oligos (238 and 242), and between the left and right probes (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 results in the ligation of the probe and the bridge oligo into a circular complex.

[0097] The 15-25 bases of the left probe include bridging sequence 1 (228), which may optionally include chemically modified bases for efficient bridging oligo attachment, referred to as bridging sequence 1. The left probe further optionally includes 10-20 bases continuing from the 5' end that include a universal sequence (204) used for library indexing. The left probe further optionally includes 10-20 bases continuing from the 5' end that include 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 that 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 bridging 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).

[0098] 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, called 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, called 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, called 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 its target or bridging oligo.

[0099] 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 optionally 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 optionally 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.

[0100] 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 optionally contain chemically modified nucleotides to 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 optionally contain chemically modified nucleotides to increase binding.

[0101] 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 bridging sequence 5 (240) of the second bridging oligo and may contain chemically modified nucleotides to selectively increase binding. The last 15-25 bases of the first bridging oligo, called bridging sequence 7 (224), are reverse complementary to the bridging sequence 8 sequence (222) of the right probe and may contain chemically modified nucleotides to increase binding. The 3' end of the third bridge oligo may contain a phosphate (or other cleavable) moiety (234) to prevent extension during gap filling.

Claims

1. 1. A method for high throughput detection of one or more target nucleotide sequences in a plurality of samples, comprising: (i) detecting a target nucleotide sequence in each of the plurality of 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 comprises a first bridging oligo-specific sequence, and / or optionally a first sequence barcode, and a first target-specific portion at the 3' end of the first probe; The second probe, starting from the 5' end of the molecule, comprises a second target specific portion, and / or optionally a second sequence barcode, and a second bridging oligo specific sequence at the 3' end of the second probe; and The bridge oligo or bridge oligo complex comprises 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 / or optionally a third barcode; and 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 bridging oligo or the bridging oligo complex comprises a recognition sequence for an endonuclease; and At least one of the first probe or the second probe or the bridge oligo or a plurality of oligonucleotides capable of annealing to each other to form a bridge oligo complex comprises a first capture moiety; (ii) for each of the one or more target nucleotide sequences, contacting the first probe and the second probe with the bridge oligo or a plurality of nucleotides capable of annealing to each other to form the bridge oligo complex, to form a plurality of ligation complexes by self-annealing, the plurality of ligation complexes are constructed by combining in equimolar amounts the first probe, the second probe and the bridge oligo or the bridge oligo complexes by annealing to each other; (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 nucleotide sequence, thereby forming a hybridization complex; (vi) ligating the first and second probes in the hybridization complex to bridge the gap between the first and second probes to provide a ligated ligation complex; (viii) amplifying a target nucleotide sequence from one or more of the ligated ligation complexes using rolling circle amplification with a strand-displacing polymerase to obtain one or more single-stranded concatemeric sequences; (ix) subjecting the amplified one or more single-stranded concatemer sequences obtained in step (viii) to annealing with a specific oligonucleotide containing a recognition sequence for an endonuclease, said specific oligonucleotide annealing with the recognition sequence specified in step (i) so as to obtain an annealed complex having a recognition site for said endonuclease; (x) cleaving the single-stranded concatemeric sequence obtained in step (viii) or the annealed complex obtained in step (ix) with the endonuclease; (xi) subjecting the fragments cleaved in step (x) or the single-stranded concatemer sequences obtained in step (viii) to a high-throughput sequencing technique to determine the barcode sequence of the first sequence barcode, the second sequence barcode, or the third barcode; and (xii) identifying the presence and / or number of said target nucleotide sequences in said plurality of samples by determining at least a portion of the first target specificity portion and / or the second target specificity portion, and / or at least a portion of the first sequence barcode and / or the second sequence barcode, and / or at least a portion of the third barcode.

2. At least one of the first probe, the second probe, and the bridge oligo or a plurality of oligonucleotides capable of annealing to each other to form a bridge oligo complex comprises a first capture moiety; 2. The method of claim 1, further comprising, after step (iv), step (v) of contacting the hybridization complex with a solid support comprising a second capture moiety, allowing the first capture moiety and the second capture moiety to interact such that the hybridization complex is linked to the solid support, and separating the hybridization complex bound to the solid support from components of the sample that are not bound to the solid support.

3. The method of claim 1 or 2, further comprising, after step (vi), a step (vii) of pooling the ligated ligation complexes from the multiple samples.

4. The method of any one of claims 1 to 3, wherein the multiple samples include blood samples, saliva samples, urine samples or fecal samples.

5. At least one of the first probe or the second probe or the bridge oligo or the bridge oligo complex comprises a first capture moiety; The method of claim 2, comprising step (v) and not comprising a step of concentrating nucleic acids contained in the sample prior to step (v).

6. At least one of the first probe or the second probe or the bridge oligo or the bridge oligo complex comprises a first capture moiety; 6. The method of claim 2 or 5, comprising step (v), wherein the first capture moiety is a biotin moiety and the second capture moiety is a streptavidin moiety or an avidin moiety.

7. At least one of the first probe or the second probe or the bridge oligo or the bridge oligo complex comprises a first capture moiety; 7. The method according to claim 2, 5 or 6, comprising step (v), wherein a washing step is carried out between steps (v) and (vi).

8. The bridged oligo or bridged oligo complex is (i) 1 to 5 3' overhanging bases, and / or (ii) a 3′ phosphate, and / or The method of any one of claims 1 to 7, comprising (iii) one or more phosphorothioate modifications within the third position from the 3' end.

9. The method of claim 1, wherein the first target-specific portion, the second target-specific portion, the first bridging oligospecific sequence, and / or the second bridging oligospecific sequence independently contain one or more chemically modified nucleotides.

10. The method of claim 1, wherein step (viii) is carried out using phi29 polymerase or Bst polymerase.

11. The method of any one of claims 1 to 10, wherein the first probe comprises a first universal sequence and the second probe comprises a second universal sequence, PCR amplification is carried out between step (x) and step (xi) using primers that bind to the first and second universal sequences, and the primers comprise an adapter for subsequent sequencing in step (xi).

12. The method of claim 1, wherein the first sequence barcode or the second sequence barcode, or both, comprise a molecular barcode; 12. The method of any one of claims 1 to 11, wherein in step (xii), identifying the presence and / or number of target nucleotide sequences is performed by counting the number of molecular barcodes per target and per sample.

13. A method according to any one of claims 1 to 12, wherein the first sequence barcode, the second sequence barcode or a third barcode are used for two or more samples or two or more loci / allele combinations to determine the genotype of the sample for one or more sequences and / or polymorphisms.

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