Highly sensitive methods for accurate parallel quantification of nucleic acids

Abstract

To provide a next generation DNA sequencing method and use for accurate and massively parallel quantification of one or more nucleic acid targets, in large volumes of unpurified sample material.SOLUTION: Aspects of the disclosed embodiments relate to a method and a kit including probes for detecting and quantifying genetic targets in complex DNA pools, primarily used for the detection of genetic targets and variants. Aspects of the disclosed embodiments use one or more target-specific nucleic acid probes (left probe and right probe) per genetic target, and a bridge oligo or bridge oligo complex.SELECTED DRAWING: None

Description

[Technical field]

[0001] Aspects of the disclosed embodiments relate to improved next-generation DNA sequencing methods for accurate and massively parallel quantification of one or more nucleic acid targets. More specifically, aspects of the disclosed embodiments relate to methods and kits including probes for detecting and quantifying genetic targets in complex DNA pools, primarily used for detecting genetic targets and mutations. Aspects of the disclosed embodiments use one or more target-specific nucleic acid probes (left and right probes) per gene target, and a bridging oligo or bridging oligo complex. [Background technology]

[0002] With the advancement of genetic mutation research technology, the detection of genetic mutations in plants and animals is not troublesome.However, the detection and accurate quantification of genetic mutations, such as mutations, 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 the quantification methods are robust and well-established, each method is associated with specific challenges, 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 approximately $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 total amount of hands-on time scales rapidly with increasing sample numbers.

[0005] Array PCR: PCR arrays are the most reliable tools to analyze the expression of panels 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, this 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 this 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 (one target costs about $3 per sample), 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 efforts that are wasted on sequencing unrelated gene targets. For example, in cancer-associated liquid biopsies, non-targeted approaches result in wasted sequencing effort on loci that are not oncologically relevant. In fetal diagnosis, non-targeted sampling of loci significantly 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 the 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.

[0010] 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]

[0011] [Patent Document 1] International Publication No. 2018 / 109206 [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] Aspects of the disclosed embodiments provide methods using next generation sequencing for highly scalable and accurate target quantification from large sample volumes (up to tens of milliliters) and / or 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 disclosed embodiment is a method for high throughput detection of one or more target nucleotide sequences in a plurality of samples, the method comprising the steps of: (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; wherein the first probe comprises, starting from the 5' end of the molecule, a first bridging oligo-specific sequence, a first universal sequence, optionally a first sequence barcode, and a first target-specific portion at the 3' end of the first probe; and wherein the second probe, starting from the 5' end of the molecule, comprises a second target specific portion, optionally a second sequence barcode, a second universal sequence, and a second bridging oligo specific sequence at the 3' end of the second probe; and wherein the bridge oligo or bridge oligo complex capable of annealing to each other to form a bridge oligo complex has a sequence complementary to the first bridge oligo specific sequence and the second bridge oligo specific sequence in the first probe and the second probe, respectively, and optionally a third barcode; and wherein at least one of the first sequence barcode, the second sequence barcode, or the third barcode is present within the first probe, the second probe, the bridge oligo, or the bridge oligo complex, respectively; (ii) forming a hybridization complex by either: (ii-a) for each of one or more target nucleotide sequences, by contacting the first probe, the second probe, and a plurality of oligonucleotides capable of self-annealing to a bridging oligo or a bridging oligo complex capable of annealing to each other to form a bridging oligo complex, and allowing them to self-anneal into a plurality of ligation complexes, contacting nucleic acid present in each of the plurality of samples to be tested for one or more target nucleotide sequences with the ligation complexes, and allowing the first target specific portion and the second target specific portion of each of the first probe and the second probe from the ligation complexes to hybridize to essentially adjacent sections on one or more target nucleotide sequences of the plurality of samples, thereby forming one or more first hybridization complexes; Or, (ii-b) contacting nucleic acids present in each of the plurality of samples to be tested for one or more target nucleotide sequences with the first target specific portion and the second target specific portion of the first probe and the second probe, respectively, to hybridize to essentially adjacent sections on the one or more target nucleotide sequences, and contacting the hybridized one or more target nucleotide sequences and the first probe and the second probe with the bridging oligo or a bridging oligo complex capable of annealing to each other to form a bridging oligo complex, thereby forming one or more second hybridization complexes; (iii) ligating the probes in said one or more first hybridization complexes or said one or more second hybridization complexes using a ligase enzyme or a combination of an enzyme or a ligase and a DNA polymerase to obtain one or more ligated ligation complexes; (iv) amplifying nucleic acid from one or more of the ligated ligation complexes to form one or more amplified single-stranded concatemeric sequences using rolling circle amplification with a strand-displacing polymerase; and doing one of the following: (va) optionally annealing the one or more amplified single-stranded concatemeric sequences obtained in step (iv) with a specific oligonucleotide comprising a recognition sequence for an endonuclease, wherein the specific oligonucleotide anneals to the recognition sequence to form an annealed complex comprising a recognition site for the endonuclease, and cleaving the single-stranded concatemeric sequences obtained in step (iv) or cleaving the annealed complex with the endonuclease to form nucleic acid fragments; Or, (vb) contacting one or more of said single-stranded concatemer sequences with a solid support alternatively comprising a second capture moiety, such that said first capture moiety and said second capture moiety interact to link one or more of said single-stranded concatemer sequences to said solid support, and separating said solid support-linked concatemer sequences from components of the sample that are not linked to the solid support, or using a solid support capable of binding modified or unmodified DNA with high affinity, or using complementary oligonucleotides immobilized on a solid surface. (vi) subjecting the nucleic acid fragment obtained in step (iv-a) or one or more of the single-stranded concatemer sequences obtained in step (iv-b) to a high-throughput sequencing technique to determine a barcode sequence; and (vii) identifying the presence and / or number of the target nucleotide sequence in each of 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.

[0015] In another principal aspect, the disclosed embodiments relate to a kit of parts comprising a plurality of containers, the kit comprising: 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; wherein 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; wherein said 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; wherein the bridge oligo or bridge oligo complex 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; wherein 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, wherein at least one of the first probe, the second probe, the bridging oligo, or the bridging oligo complex comprises a recognition sequence for an endonuclease; wherein said kit of parts further comprises an oligonucleotide capable of annealing to said recognition sequence to provide a recognition portion for said endonuclease, and wherein the oligonucleotide anneals to the recognition sequence to provide a recognition site for the endonuclease; and wherein the bridge oligo or bridge oligo complex comprises a fourth barcode comprising a sequence capable of annealing to a target sequence of a sample. [Brief description of the drawings]

[0016] [Figure 1] 1 shows a flow diagram of a multiplex ligation assay (MLA) according to one embodiment of the present disclosure, where one embodiment of the present disclosure includes (listed from top to bottom) 10, SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, and 28. [Figure 2A] 1 shows the principle structure of a probe triplet having multiple probe entities (including SEQ ID NOs: 29, 30, and 31) according to one 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

[0017] (definition) Target nucleotide sequence: The term target nucleotide sequence may be any nucleotide sequence of interest whose detection is required. It may be understood that the given term refers to a nucleic acid molecule having a sequence of contiguous nucleotides and a complementary sequence. The target sequence in some embodiments is a nucleotide sequence that represents or is associated with a polymorphism.

[0018] 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 can be as small as one base pair.

[0019] Sample: The term sample is used herein for two or more samples having two or more target sequences. The sample provided in the method according to the disclosed embodiments may have been prepared at least to extract the target nucleic acids and make them accessible to the probes used in the disclosed embodiments. In particular, in some embodiments, the samples each contain at least two different target sequences, preferably at least 100, more preferably at least 250, more preferably at least 500, and most preferably at least 2000 or more. 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 extracts, tissue, plasma (liquid biopsy), or from the environment including 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 bodily fluids or bodily substances such as extracts from hair or skin dander.

[0020] 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 the sequence in the probe. The sections of the oligonucleotide probes that are complementary to the target sequences are designed so that for each target sequence in a 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 bridge oligos or bridge oligo complexes that are used to join the left and right probes.

[0021] 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 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.

[0022] 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.

[0023] 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.

[0024] Amplification: As used herein, the term amplification refers to the use of a DNA polymerase to increase the concentration of a particular nucleotide sequence within a mixture of nucleotide sequences. "PCR" or "Polymerase Chain Reaction" is a rapid procedure for the in vitro enzymatic amplification of specific DNA / RNA fragments. 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, DNA polymerase, can only add new nucleotides to an existing strand of DNA.

[0025] Polymerase: A polymerase is an enzyme that synthesizes long chains, or polymers, of nucleic acids. DNA polymerases and RNA polymerases are used to assemble DNA and RNA molecules, respectively, by copying a DNA or RNA template strand using base-pairing interactions.

[0026] 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 concerned with effectively screening large numbers of samples simultaneously.

[0027] Endonuclease: An endonucleases is an enzyme that cleaves double or single strands of DNA randomly or at directed locations.

[0028] As mentioned above, the present disclosure relates to methods for high throughput detection of target nucleotide sequences in a large number of samples by utilizing ligation dependent assays. The present disclosure provides methods for determining the sequence of genetic targets within a complex pool of nucleic acids using techniques enabled by next generation sequencing. The present disclosure also provides methods for profiling multiple gene targets in multiple samples, preferably very multiple samples, by utilizing ligation-dependent assays. The present disclosure further provides methods for multiplex ligation-dependent probe amplification that allows interrogation of different target nucleic acids in multiple samples. The methods of the disclosed embodiments provide multiple different probe sets for different target nucleic acids to allow sequencing of one or more target nucleotide sequences in multiple samples. In processing the sequencing data, the unique sequence identifiers are used to identify gene targets and to absolutely quantify individual samples from the sample pool.

[0029] In a first main aspect, the disclosed embodiment is a method for high throughput detection of one or more target nucleotide sequences in a plurality of samples, the method comprising the steps of: (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; wherein the first probe comprises, starting from the 5' end of the molecule, a first bridging oligo-specific sequence, a first universal sequence, optionally a first sequence barcode, and a first target-specific portion at the 3' end of the first probe; and wherein the second probe, starting from the 5' end of the molecule, comprises a second target specific portion, optionally a second sequence barcode, a second universal sequence, and a second bridging oligo specific sequence at the 3' end of the second probe; and wherein the bridge oligo or bridge oligo complex capable of annealing to each other to form a bridge oligo complex has a sequence complementary to the first bridge oligo specific sequence and the second bridge oligo specific sequence in the first probe and the second probe, respectively, and optionally a third barcode; and wherein at least one of the first sequence barcode, the second sequence barcode, or the third barcode is present within the first probe, the second probe, the bridge oligo, or the bridge oligo complex, respectively; (ii) forming a hybridization complex by either: (ii-a) for each of one or more target nucleotide sequences, by contacting the first probe, the second probe, and a plurality of oligonucleotides capable of self-annealing to a bridging oligo or a bridging oligo complex capable of annealing to each other to form a bridging oligo complex, and allowing them to self-anneal into a plurality of ligation complexes, contacting nucleic acid present in each of the plurality of samples to be tested for one or more target nucleotide sequences with the ligation complexes, and allowing the first target specific portion and the second target specific portion of each of the first probe and the second probe from the ligation complexes to hybridize to essentially adjacent sections on one or more target nucleotide sequences of the plurality of samples, thereby forming one or more first hybridization complexes; Or, (ii-b) contacting nucleic acids present in each of the plurality of samples to be tested for one or more target nucleotide sequences with the first target specific portion and the second target specific portion of the first probe and the second probe, respectively, to hybridize to essentially adjacent sections on the one or more target nucleotide sequences, and contacting the hybridized one or more target nucleotide sequences and the first probe and the second probe with the bridging oligo or a bridging oligo complex capable of annealing to each other to form a bridging oligo complex, thereby forming one or more second hybridization complexes; (iii) ligating the probes in said one or more first hybridization complexes or said one or more second hybridization complexes using a ligase enzyme or a combination of an enzyme or a ligase and a DNA polymerase to obtain one or more ligated ligation complexes; (iv) amplifying nucleic acid from one or more of the ligated ligation complexes to form one or more amplified single-stranded concatemeric sequences using rolling circle amplification with a strand-displacing polymerase; and doing one of the following: (va) optionally annealing the one or more amplified single-stranded concatemeric sequences obtained in step (iv) with a specific oligonucleotide comprising a recognition sequence for an endonuclease, wherein the specific oligonucleotide anneals to the recognition sequence to form an annealed complex comprising a recognition site for the endonuclease, and cleaving the single-stranded concatemeric sequences obtained in step (iv) or cleaving the annealed complex with the endonuclease to form nucleic acid fragments; Or, (vb) contacting one or more of said single-stranded concatemer sequences with a solid support alternatively comprising a second capture moiety, such that said first capture moiety and said second capture moiety interact to link one or more of said single-stranded concatemer sequences to said solid support, and separating said solid support-linked concatemer sequences from components of the sample that are not linked to the solid support, or using a solid support capable of binding modified or unmodified DNA with high affinity, or using complementary oligonucleotides immobilized on a solid surface. (vi) subjecting the nucleic acid fragment obtained in step (iv-a) or one or more of the single-stranded concatemer sequences obtained in step (iv-b) to a high-throughput sequencing technique to determine a barcode sequence; and (vii) identifying the presence and / or number of the target nucleotide sequence in each of 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.

[0030] In another principal aspect, the disclosed embodiments relate to a kit of parts comprising a plurality of containers, the kit comprising: 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; wherein 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; wherein said 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; wherein the bridge oligo or bridge oligo complex 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; wherein 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, wherein at least one of the first probe, the second probe, the bridging oligo, or the bridging oligo complex comprises a recognition sequence for an endonuclease; wherein said kit of parts further comprises an oligonucleotide capable of annealing to said recognition sequence to provide a recognition portion for said endonuclease, and wherein the oligonucleotide anneals to the recognition sequence to provide a recognition site for the endonuclease; and wherein the bridge oligo or bridge oligo complex comprises a fourth barcode comprising a sequence capable of annealing to a target sequence of a sample.

[0031] FIG. 1 provides a non-limiting illustration of one embodiment of the method of the present invention.

[0032] The method of the embodiment according to the present disclosure 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 ligated chemically or enzymatically with DNA ligase to form a ligated ligation complex. In aspects of the disclosed embodiment, multiple such ligated ligation complexes are generated during sample analysis in multiple samples that are analyzed.

[0033] 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 one 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.

[0034] The target sequence may include any nucleotide sequence of interest that requires detection. The target nucleotide sequences of the present disclosure may be obtained from, but are 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, for example, to assess an individual's risk for a certain type of cancer, to determine the effectiveness of a certain treatment for a certain cancer, the occurrence of drug resistance-associated 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.

[0035] As used herein, the term probe set includes a first probe, a second probe and a bridge oligo or bridge oligo complex.

[0036] In certain embodiments, the first probe starts from the 5' end of the molecule and comprises 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 starts from the 5' end of the molecule and comprises 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.

[0037] In a preferred embodiment, either the first probe or the second probe has 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 a random sequence or may have a target nucleotide sequence identifier sequence, a sample identifier sequence, and / or a molecular barcode for target enumeration.

[0038] In a preferred embodiment, the bridge oligo or bridge oligo complex may have 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 a third barcode that may be a random sequence or may have 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 for a unique definition of the complex within all ligation complexes in all samples tested.

[0039] 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. A recognition sequence is required for concatemeric sequence cleavage in step (x). In one embodiment, the recognition sequence is a recognition sequence for a restriction endonuclease such as EcoRI. In another embodiment, the recognition sequence is a recognition sequence for a homing endonuclease such as I-CeuI. In another embodiment, the recognition sequence is a recognition sequence for a cleavage system such as induced DNAaseI or CRISPR-Cas.

[0040] 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 probe, a ligation complex or a hybridization complex can be captured, i.e., bound, to a second capture moiety linked to a solid support. 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 supports.

[0041] 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 have 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 acids, peptide nucleic acids, and locked nucleic acids (e.g., as shown in Figure 3 of WO 2019 / 038372, which is incorporated herein by reference). In one embodiment, the bridging portion of the first probe or the second probe, or both, includes 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 have one or more chemically modified nucleotides independently of each other. In certain embodiments, the chemical modification allows 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.

[0042] 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.

[0043] Further, 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; (iv) a first capture moiety, such as biotin at the 5' end; Includes.

[0044] In one embodiment, 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 an embodiment 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.

[0045] Preferably, each ligation complex is unique for the combination of the first target specific sequence, the second target specific sequence and one or more barcode sequences, allowing for enumeration of target sequences after amplification and analysis of the results.

[0046] 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 ligation complex from (step ii) is contacted with a solid support comprising a second capture moiety, the first capture moiety and the second capture moiety interact with each other, resulting in a hybridization complex being linked to the solid support, and the ligation complex linked to the solid support is separated from the ligation complex not linked to the solid support (optional step (additional 1)). The ligation complex bound to the solid support is then separated from the components of the sample that are not bound to the solid support. 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.

[0047] Step (Addition 1) results in the immobilization of the ligation complex onto a solid support such as a magnetic bead or a flat surface, the latter of which can be used, for example, for fluorescent detection in certain DNA sequencing applications.

[0048] 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 consists of DNA extracted from blood, tissue, an FFPE sample, saliva, urine, or feces. In some embodiments, the sample has a volume of more than 100 microliters, e.g., more than 1 ml. In further embodiments, the sample has a nucleic acid concentration of less than 5 pmol, e.g., less than 1 pmol, e.g., less than 200 fmol. In one embodiment, the plurality of samples includes one or more blood samples, one or more saliva samples, one or more urine samples, or one or more fecal samples.

[0049] In another embodiment, one or more target nucleotide sequences in a plurality of samples are contacted with a plurality of first and second probes. The first and second target specific portions of the first and second probes, respectively, hybridize to essentially adjacent portions on the target sequence. The first and second probes hybridized to the target are then contacted with a bridging oligo, thereby forming a hybridization complex.

[0050] Then, in some embodiments, when 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, thereby linking the hybridization complex to the solid support (optional step (v)). The hybridization complexes bound to the solid support are then separated from components of the sample that are not bound to the solid support. If the solid support is a magnetic bead, the beads are immobilized using a magnet and the remaining liquid sample is removed. Optionally, a washing step is performed before continuing with the process.

[0051] In one embodiment, step (v) purifies and concentrates the nucleic acids, allowing for improved results, especially for samples of very low purity. In one embodiment, the method of the disclosed embodiments does not include a step of concentrating the nucleic acids prior to step (v). Thus, in one embodiment the method does not comprise 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 disclosed embodiments does not include a purification step after the ligation of step (vi). In other embodiments, in step (v), the ligation complex is immobilized on a solid support, such as a magnetic bead or a flat surface, the latter of which can be used for, e.g., fluorescent detection in certain DNA sequencing applications.

[0052] 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), a gap between the first and second probe, if present, can be filled by introducing a polymerase and one or more nucleotides, optionally including chemically modified nucleotides that include capture moieties, including but not limited to internal amino modifiers, internal biotin modifiers (biotin azide, biotin dT, desthiobiotin-TEG), internal thiol modifiers, alkynes (int-5-octadiynyl dU), and internal azides (NHS esters). 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, resulting in ligation of the left and right probes and including the universal sequence and / or the third barcode sequence 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 non-duplex degrading polymerase is used, 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.

[0053] In some embodiments (optional step (additional 2)), the ligated ligation complex is then subjected to DNA denaturing conditions, such as heat or alkaline conditions, to dissociate the bridging oligonucleotide from the ligated ligation complex. This dissociation exposes the universal sequence region from the ligated ligation complex, which can be used, for example, to bind to immobilized nucleotide probes for fluorescent detection applications, including certain DNA sequencing applications.

[0054] Optionally, in some embodiments (optional step (additional 3)), the ligated ligation complex is immobilized on a solid support, such as a magnetic bead or a flat surface, the latter of which can be used, for example, for fluorescent detection in certain DNA sequencing applications. The solid support may contain a second capture moiety, and the first capture moiety in the ligated ligation complex is allowed to interact with the second capture moiety, thereby linking the hybridization complex to the solid support, or the solid support may be modified to bind modified or unmodified DNA with high affinity, or the solid support may contain immobilized oligonucleotides that have affinity for the ligated ligation complex via DNA hybridization. If the solid support is a magnetic bead, the beads are immobilized using a magnet and the remaining liquid sample is removed. Optionally, a washing step is performed before continuing with the process.

[0055] Step (Additional 3) allows the immobilization of the ligation complex onto a solid support such as a magnetic bead or a flat surface, the latter of which can be used, for example, for fluorescent detection in certain DNA sequencing applications.

[0056] The ligated ligation complexes are optionally pooled from one or more target samples (step (vii)). Steps (vi) and (vii) may be performed in the order specified or in reverse order.

[0057] Nucleic acid is then amplified from one or more of the plurality of ligated ligation complexes (step (viii)). Amplification is carried out using rolling circle amplification with strand-displacing polymerases such as phi29 polymerase (UniProtKB-P03680, DPOL_BPPH2) or Bst polymerase (P52026, DPO1_GEOSE), or, optionally, chemically modified nucleotides containing capture moieties such as, but not limited to, internal amino modifiers, internal biotin modifiers (biotin azide, biotin dT, desthiobiotin-TEG), internal thiol modifiers, alkynes (int-5-octadiynyl dU), and internal azides (NHS esters). In the next step (ix), the one or more amplified single-stranded concatemer sequences obtained in step (viii) are selectively annealed with a specific oligonucleotide containing a recognition sequence for an endonuclease, where the oligonucleotide is annealed with the recognition sequence specified in step (i) so as to obtain a recognition site for the endonuclease. The specific oligonucleotide containing a recognition sequence may typically have some additional specific sequences around the recognition sequence to enable the formation of a stable double helix for cleavage.

[0058] Subsequently, the single-stranded concatemeric sequences obtained in step (viii) or the annealed complexes obtained in step (ix) are selectively cleaved with the endonuclease (step (x)), resulting in an NGS library.

[0059] 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 embodiment methods of the present disclosure, 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.

[0060] In another embodiment (optional step (additional 4)), the concatemeric sequences obtained in step (viii) are alternatively contacted with a solid support comprising a second capture moiety, so that the hybridization complex is linked to the solid support by interaction of the first capture moiety or moieties in the concatemeric sequences with the second capture moiety, and the hybridization complex linked to the solid support is separated from components of the sample that are not linked to the solid support, or a solid support capable of binding modified or unmodified DNA with high affinity is used, Alternatively, the solid support may contain immobilized oligonucleotides that have affinity to the concatemer sequences via DNA hybridization. If the solid support is a magnetic bead, the beads are immobilized using a magnet and the remaining liquid sample is removed. Optionally, a washing step is performed before continuing with the process.

[0061] Step (Addition 4) results in immobilization of the concatemeric sequences onto a solid support such as magnetic beads or flat surfaces, the latter of which can be used, for example, for fluorescent detection in certain DNA sequencing applications.

[0062] 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, wherein said primers optionally contain adapter sequences for subsequent sequencing in step (xi).

[0063] Identifying the presence and / or number of target nucleotide sequences in a plurality of samples may 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, including, but not limited to, 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 the sequence data and sequence targets quantified in silico after DNA sequencing.

[0064] 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 that are reverse complementary to the first or second universal sequence present in the ligated complex.

[0065] Advantages of the embodiments of the present disclosure include, but are not limited to, quantitative assays with low cost, high simplicity, high specificity, high sensitivity, high accuracy, high throughput, high scalability and high turnover compared to conventional nucleic acid sequencing technologies. Another aspect of the embodiments of the present disclosure is that the method 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 any prior purification or enrichment 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 embodiments of the present disclosure is that it allows for the detection and amplification of target sequences of interest using a unique probe design, i.e., a probe triplet. The probes are designed with specifically positioned modified nucleotides that improve annealing and binding efficiency. Improved binding characteristics increase the specificity, sensitivity and accuracy of the assay. Embodiments of the present disclosure 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. In a preferred embodiment, for two or more samples or two or more locus / allele combinations, the barcode sequences are used to genotype the samples for one or more sequences and / or polymorphisms, such as SNPs and / or indels.

[0066] In another aspect, an aspect of an embodiment of the present disclosure provides a kit of parts comprising a plurality of containers, wherein at least one container comprises one or more sets of first probes and second probes, and at least one container comprises one or more bridge oligos or a plurality of oligonucleotides capable of forming a bridge oligo complex; wherein 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; wherein 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; wherein the bridge oligo or bridge oligo complex comprises a sequence complementary to the first and second bridge oligo specific sequences in the first and second probes, respectively, or optionally a third barcode; wherein 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 bridging oligo, or the bridging oligo complex, respectively; wherein 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 Here, the kit of parts further comprises an oligonucleotide capable of annealing to the recognition sequence so as to provide a recognition moiety for the endonuclease.

[0067] 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.

[0068] 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.

[0069] 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.

[0070] Preferably, the first bridging moiety or the second bridging moiety, or both, contain chemically modified bases to improve binding to the bridged oligo or bridged oligo complex.

[0071] In one particular embodiment, at least one container containing the first and second sets of probes and at least one container containing the bridge oligo or multiple 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 to form a ligated complex.

[0072] One particular advantage of the embodiments of the present disclosure is that they allow the detection and amplification of target sequences of interest using unique probe designs, i.e., probe triplets. The probes are designed with improved binding properties that lead to improved assay specificity, sensitivity and accuracy. The embodiments of the present disclosure find application in fields including, but not limited to, molecular biology, evolutionary biology, metagenomics, genotyping, more specifically, cancer diagnosis or fetal chromosomal disorders, including, but not limited to, genotyping samples for one or more sequences and / or polymorphisms, such as SNPs and / or indels.

[0073] In one particular preferred embodiment, the bridge oligo or bridge oligo complex contains information to identify the sample and includes a unique barcode, where the first and second probes are universally applicable to all samples (containing only information to identify the target). Thus, in one preferred embodiment, there is provided a method or kit according to aspects of the disclosed embodiment, wherein the bridged oligo or bridged oligo complex comprises a barcode comprising a unique sequence that allows enumeration of the target sequence for each sample.

[0074] Further aspects of the present disclosure relate to the following: EMBODIMENT 1 1. A method for high throughput detection of one or more target nucleotide sequences in a plurality of samples, comprising the steps of: (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; wherein the first probe comprises, starting from the 5' end of the molecule, a first bridging oligo-specific sequence, a first universal sequence, optionally a first sequence barcode, and a first target-specific portion at the 3' end of the first probe; and wherein the second probe, starting from the 5' end of the molecule, comprises a second target specific portion, optionally a second sequence barcode, a second universal sequence, and a second bridging oligo specific sequence at the 3' end of the second probe; and wherein the bridge oligo has a sequence complementary to the first bridge oligo specific sequence and the second bridge oligo specific sequence in the first probe and the second probe, respectively, and optionally a third barcode; and wherein at least one of the first sequence barcode, the second sequence barcode, or the third barcode is present within the first probe, the second probe, the bridge oligo, or the bridge oligo complex, respectively; and wherein at least one of the first probe or the second probe or the bridging oligo comprises a recognition sequence for an endonuclease, and optionally wherein at least one of the first probe or the second probe or the bridging oligo comprises a first capture moiety. (ii) optionally, for each of one or more target nucleotide sequences, contacting in separate tubes a first probe and a second probe, a bridge oligo, or multiple oligonucleotides capable of annealing to each other to form a bridge oligo complex, and allowing them to self-anneal into multiple ligation complexes; (Additional 1) optionally contacting the ligation complex from step (ii) with a solid support comprising a second capture moiety, such that the first capture moiety and the second capture moiety interact to link the hybridization complex to the solid support, and separating the ligation complex linked to the solid support from the ligation complex not linked to the solid support; (iii) contacting nucleic acid present in each sample to be tested for the target nucleotide sequence with the ligation complex from step (ii) or (Additional 1); (iv) hybridizing the first and second target specific portions of the first and second probes from the ligation complexes from step (ii) or (Addition 1) to essentially adjacent sections on the target sequence, thereby forming a hybridization complex; (Additional 6) optionally contacting nucleic acids present in each sample to be tested for the target nucleotide sequence with the first and second target specific portions of the respective first and second probes to hybridize to essentially adjacent sections on the target sequence; (Additional 5) Optionally, contacting the first and second probes hybridized to the target from step (Additional 6) with a bridge, thereby forming a hybridization complex; (v) optionally contacting the hybridization complex from step (iv) or (additional 5) with a solid support comprising a second capture moiety such that the first capture moiety and the second capture moiety interact to link the hybridization complex to the solid support, and separating the hybridization complex linked to the solid support from components of the sample that are not linked to the solid support, or using oligonucleotides immobilized on a solid surface that have affinity for the hybridization complex through reverse complementarity to a portion of the hybridization complex; (iv) ligating the probe in the hybridization complex from step (iv) or step (additional 5) or step (v) using a ligase enzyme or a combination of an enzyme or ligase and a DNA polymerase, or, optionally, using a chemically modified nucleotide having a capture moiety (including, but not limited to, an internal amino modifier, an internal biotin modifier (biotin azide, biotin dT, desthiobiotin-TEG), an internal thiol modifier, an alkyne (int-5-octadiynyl dU), and an internal azide (NHS ester)) to provide a ligated ligation complex; (Additional 2) Optionally, subjecting the ligated ligation complex to DNA denaturing conditions such as heating or alkali treatment to dissociate the ligated complex from the hybridization complex; (Additional 3) contacting the ligated ligation complex with a solid support that alternatively contains a second capture moiety, such that the first capture moiety and the second capture moiety interact to link the hybridization complex to the solid support, and separating the hybridization complex linked to the solid support from components of the sample that are not linked to the solid support, or using a solid support that can bind modified or unmodified DNA with high affinity, or using surface-immobilized oligonucleotides; (vii) pooling the ligated ligation complexes from multiple samples; (viii) amplifying nucleic acids from one or more ligation complexes using rolling circle amplification with a strand displacing polymerase to form one or more amplified single-stranded concatemeric sequences, optionally using chemically modified nucleotides that contain capture moieties, such as, but not limited to, internal amino modifiers, internal biotin modifiers (biotin azide, biotin dT, desthiobiotin-TEG), internal thiol modifiers, alkynes (int-5-octadiynyl dU), and internal azides (NHS esters); (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, wherein said oligonucleotide anneals with said recognition sequence specified in step (i) so as to obtain a recognition site for the endonuclease; (x) cleaving the single-stranded concatemer sequence obtained in step (viii) or cleaving the annealed complex obtained in step (ix) with the endonuclease; (Additional 4) Optionally, contacting the concatemer sequence obtained in step (viii) with a solid support alternatively comprising a second capture moiety, such that the first capture moiety and the second capture moiety interact to link the concatemer sequence to the solid support, and separating the concatemer sequence linked to the solid support from components of the sample that are not linked to the solid support, or using a solid support capable of binding modified or unmodified DNA with high affinity, or using complementary oligonucleotides immobilized on a solid surface. (xi) subjecting the nucleic acid fragment obtained in step (x) to a high-throughput sequencing technique to determine a barcode sequence; and (xii) determining the presence and / or number of the target nucleotide sequence in each of a 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; Here, steps (vi) and (vii) may be performed in any order, one of steps (Addition 1), (v), (Addition 3) or (Addition 4) is performed, and one of steps (ii) or (Addition 6) is performed.

[0075] EMBODIMENT 2 2. The method of embodiment 1, wherein said plurality of samples comprises blood samples, tissue samples, FFPE samples, saliva samples, urine samples, fecal samples, or DNA extracted from any of these.

[0076] EMBODIMENT 3 The method of embodiment 1 or 2, wherein at least one of the first probe or the second probe or the bridged oligo or bridged oligo complex comprises a first capture moiety, and wherein the method comprises step (v), and wherein the method comprises step (v) and does not comprise a step of concentrating nucleic acid prior to step (v).

[0077] EMBODIMENT 4 The method of any of the above embodiments, wherein at least one of the first probe or the second probe or the bridged oligo or bridged oligo complex comprises a first capture moiety, wherein the method comprises step (v), and wherein the first capture moiety is a biotin moiety and the second capture moiety is a streptavidin moiety or an avidin moiety.

[0078] EMBODIMENT 5 The method of any one of the above embodiments 1 to 4, wherein at least one of the first probe or the second probe or the bridging oligo comprises a first capture moiety, comprising step (v), and wherein a washing step is performed between steps (v) and (vi).

[0079] EMBODIMENT 6 The bridged oligo or bridged oligo complex is (i) 1 to 5 3'-overhanging bases, and / or (ii) a 3' phosphate, and / or (iii) The method of any of embodiments 1 to 5 above, comprising one or more phosphorothioate modifications within three positions from the 3' end.

[0080] EMBODIMENT 7 The method of any of the above embodiments 1 to 6, wherein the 3' end of the first probe or the 5' end of the second probe, or both, are modified to enable the first probe to be chemically ligated to the second probe.

[0081] EMBODIMENT 8 The method of any of the above embodiments 1 to 7, wherein the bridging portion of the first probe or the second probe, or both, comprises a chemically modified base to improve binding to the bridging oligo.

[0082] EMBODIMENT 9 9. The method of any of embodiments 1 to 8 above, 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.

[0083] EMBODIMENT 10 10. The method of any one of embodiments 1 to 9 above, wherein said step (viii) is carried out using phi29 polymerase or Bst polymerase.

[0084] EMBODIMENT 11 10. The method of any of the preceding claims, wherein between steps (x) and (xi), a PCR amplification is carried out using primers that bind to universal portions of said first and second probes, wherein said primers optionally comprise an adaptor for subsequent sequencing in step (xi).

[0085] EMBODIMENT 12 12. The method of any of embodiments 1-11 above, wherein gene target enumeration is enabled by counting the number of molecular barcodes per target and per sample.

[0086] EMBODIMENT 13 13. The method of any of the above embodiments 1 to 12, wherein for two or more samples or two or more loci / allele combinations, the barcode sequences are used to determine the genotype of the samples for one or more sequences and / or polymorphisms, including SNPs and / or indels.

[0087] 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; wherein 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; wherein said 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; wherein 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; wherein 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 bridging oligo, respectively; And, wherein at least one of the first probe, the second probe, or the bridging oligo comprises a recognition sequence for an endonuclease; and wherein said kit of parts further comprises an oligonucleotide capable of annealing to said recognition sequence so as to obtain a recognition moiety for said endonuclease. EXAMPLES

[0088] (method) 1. Formation of Probe Complex The probe complex contains the genetic target, the sample index, and the sequences required for constructing an Illumina sequencing library. Three-part probe complexes can be formed (shown in Figure 2), including: (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 a sequence complementary to the first bridging oligo specific sequence and the second bridging oligo specific sequence in the first probe and the second probe, respectively.

[0089] 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).

[0090] [Table 1]

[0091] 2. Target Capture The specific genomic region containing the mutation of interest is targeted. Purified DNA (e.g., from tissue, plasma, urine or saliva) can be used as the sample, or the sample can be unpurified and only pretreated, e.g., by boiling and / or centrifugation.

[0092] 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).

[0093] [Table 2]

[0094] 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.

[0095] 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 at +42°C for 30 minutes to 2 hours. After the RCA reaction, the reaction efficiency is analyzed by measuring the concentration of single-stranded DNA (ssDNA) with a Qubit fluorometer.

[0096] 5.Enzymatic design The RCA reaction generates long concatemeric ssDNA molecules with multiple copies of the target library. Each complete target library is separated by an EcoRI restriction enzyme recognition sequence. This sequence allows sequence-specific cleavage of the long concatemers through annealing with specific oligonucleotides containing the EcoRI restriction enzyme recognition sequence, releasing the ready target libraries. These libraries are ready for further analysis after a simple purification step. The RCA products are digested with EcoRI for 1 h at +37°C.

[0097] 6. Library Purification After EcoRI digestion, library molecules are purified by electrophoresis or extraction from agarose gels using size selection beads (such as Macherey Nagel NucleoMag).

[0098] 7. Sequencing 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 fit any available sequencing platform by simple oligonucleotide modifications. Sequencing 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 probe sequences within each read, sequence the genomic regions between them, and count the number of molecular barcodes associated with each gene target.

[0099] (Experiment 1) In the first experiment, the probe mix was a collection of four different indexed 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.

[0100] 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 performed as described above. An example of a typical result is shown in Figure 3.

[0101] The ready libraries were sequenced on MiSeq and iSeq100 instruments, and the target genes were detected in the sequence data by matching the probe sequences in each read, identifying the genomic sequence regions between the probe sequences, and counting the 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 the target molecules (Figure 4).

[0102] (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 are annealed 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 complex is 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 or other strand-displacing polymerase, resulting in long concatemeric copies of the probe (116). 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 (117). In step 7, the amplified DNA is sequenced using next generation DNA sequencing. In step 8, the DNA sequencing results are converted to target counts using a bioinformatics pipeline.

[0103] 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 before sample annealing. The first 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 the 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 the 5' end that contain a segment of random nucleotides that form a molecule-specific barcode or a sample-specific barcode called barcode 1 (206). The left probe further comprises the next 15-30 bases from the 5' end and binds to the 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 the 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).

[0104] 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 comprise the portion of the right probe that binds to the gene 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 last 15-25 bases of the right probe contain a sequence for efficient bridge oligo binding, called bridge sequence 2 (220). 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.

[0105] 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 bridging oligo, called bridging sequence 2 (224), are reverse complementary to bridging sequence 2 (220) of the left probe 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.

[0106] 2B illustrates gap filling between a first probe and a second probe according to one embodiment herein, where the bridge oligo contains gap 1 (gap1) between bridging sequence 1 (228) and bridging sequence 2 (224). Gap 2 (gap2) 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 purpose 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 two gaps, i.e., gaps 1 and 2, between the first and second probes, and the subsequent action of DNA ligase ligates the bridge oligo and the left and right probes that are complementary to the target sequence, resulting in a circular complex.

[0107] 2C shows the principle structure of a probe quintet with multiple probe entities according to one embodiment of the present specification, which includes 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 bridge oligos into a circular complex.

[0108] Bases 15 to 25 of the left probe contain bridging sequence 1 (228), which may optionally contain chemically modified bases for efficient cross-linking oligo attachment, referred to as bridging sequence 1. The left probe optionally further comprises 10-20 bases following the 5' end that contain a universal sequence (204) used for library indexing. The left probe optionally further comprises 10-20 bases following from the 5' end, including a segment of random nucleotides forming a molecule-specific or sample-specific barcode called barcode 1 (206). The left probe further comprises 15-30 bases following from the 5' end, and binds to a genetic target (208). Some or all of the 228 nucleotides may contain chemical modifications that increase the affinity of the probe to its target or to the 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, called modification 1 (210).

[0109] 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, referred to 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, called bridging sequence 8 (222), are reverse complementary to bridging sequence 7 (224) of the third bridging oligo. Some or all of nucleotides 208, 216, 222 or 228 may contain chemical modifications that increase the affinity of the probe for its target or bridging oligo.

[0110] The first 15-25 bases from the 5' end of the first bridging oligo, called 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, called bridging sequence 2 (238), is reverse complementary to bridging sequence 4 (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.

[0111] 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.

[0112] The first 15-25 bases of the third bridging oligo from the 5' end, called bridging sequence 6 (242), is reverse complementary to bridging sequence 5 (240) of the second bridging oligo and contains chemically modified nucleotides to selectively increase binding. The last 15-25 bases of the first bridging oligo, called bridging sequence 7 (224), is reverse complementary to bridging sequence 8 (222) of the right probe and may contain chemically modified nucleotides to increase binding. The 3' end of the third bridging oligo may contain a phosphate (or other cleavable) moiety (234) to prevent extension during gap filling.

[0113] Incorporation by Reference The Sequence Listing (ST.26) in XML document (.xml) format is hereby incorporated by reference in its entirety.

Claims

1. 1. A method for high throughput detection of one or more target nucleotide sequences in a plurality of samples, comprising the steps of: (i) providing, for 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; wherein said first probe comprises, starting from the 5' end of the molecule, a first bridging oligo-specific sequence, a first universal sequence, optionally a first sequence barcode, and a first target-specific portion at the 3' end of the first probe; and wherein said second probe, starting from the 5' end of the molecule, comprises a second target specific portion, optionally a second sequence barcode, a second universal sequence, and a second bridging oligo specific sequence at the 3' end of the second probe; and wherein the bridge oligo or bridge oligo complex capable of annealing to each other to form a bridge oligo complex has a sequence complementary to the first bridge oligo specific sequence and the second bridge oligo specific sequence in the first probe and the second probe, respectively, and optionally a third barcode; and wherein at least one of the first sequence barcode or the second sequence barcode or the third barcode is present within the first probe or the second probe or the bridge oligo or bridge oligo complex, respectively. (ii) forming a hybridization complex by any of the following: (ii-a) for each of one or more target nucleotide sequences, by contacting the first probe, the second probe, and a plurality of oligonucleotides capable of self-annealing to the bridging oligo or a bridging oligo complex capable of annealing to each other to form a bridging oligo complex, and allowing self-annealing into a plurality of ligation complexes, contacting nucleic acid present in each of the plurality of samples to be tested for one or more target nucleotide sequences with the ligation complexes, and allowing the first target specific portion and the second target specific portion of each of the first probe and the second probe from the ligation complexes to hybridize to essentially adjacent sections on one or more target nucleotide sequences of the plurality of samples, thereby forming one or more first hybridization complexes; Or, (ii-b) contacting nucleic acids present in each of said plurality of samples to be tested for one or more target nucleotide sequences with said first target specific portion and said second target specific portion of said first probe and said second probe, respectively, to hybridize to essentially adjacent sections on one or more target nucleotide sequences, and contacting said hybridized one or more target nucleotide sequences and said first probe and said second probe with said bridging oligo or a bridging oligo complex capable of annealing to each other to form a bridging oligo complex, thereby forming one or more second hybridization complexes; (iii) ligating the probes in the one or more first hybridization complexes or the one or more second hybridization complexes to obtain one or more ligated ligation complexes; (iv) amplifying nucleic acid from one or more of the ligated ligation complexes to form one or more amplified single-stranded concatemeric sequences using rolling circle amplification with a strand-displacing polymerase; and performing one of the following: (va) optionally annealing the one or more amplified single-stranded concatemeric sequences obtained in step (iv) with a specific oligonucleotide comprising a recognition sequence for an endonuclease, wherein the specific oligonucleotide anneals with the recognition sequence to form an annealed complex comprising a recognition site for the endonuclease, and cleaving the single-stranded concatemeric sequences obtained in step (iv) or cleaving the annealed complex with the endonuclease to form nucleic acid fragments; Or, (v-b) contacting one or more of said single-stranded concatemer sequences with a solid support alternatively comprising a second capture moiety, such that said first capture moiety and said second capture moiety interact to link one or more of said single-stranded concatemer sequences to said solid support, and separating said solid support-linked concatemer sequences from components of the sample that are not linked to the solid support, or using a solid support capable of binding modified or unmodified DNA with high affinity, or using complementary oligonucleotides immobilized on a solid surface. (vi) subjecting the nucleic acid fragment obtained in step (iv-a) or one or more of the single-stranded concatemer sequences obtained in step (iv-b) to a high-throughput sequencing technique to determine a barcode sequence; and (vii) determining the presence and / or number of the target nucleotide sequence in each of 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.

2. 2. The method of claim 1, wherein the plurality of samples comprises blood samples, tissue samples, FFPE samples, saliva samples, urine samples, fecal samples, or DNA extracted from any of these.

3. The method of claim 1 or 2, wherein at least one of the first probe or the second probe or the bridged oligo or bridged oligo complex comprises a first capture moiety.

4. The method of any one of claims 1 to 3, wherein at least one of the first probe or the second probe or the bridged oligo or bridged oligo complex comprises a first capture moiety, wherein the first capture moiety is a biotin moiety and the second capture moiety is a streptavidin moiety or an avidin moiety.

5. The method according to any one of claims 1 to 4, wherein at least one of the first probe or the second probe or the bridged oligo or bridged oligo complex comprises a first capture moiety and wherein a washing step is performed between steps (ii) and (iii).

6. 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 5, comprising (iii) one or more phosphorothioate modifications within the third position from the 3' end.

7. 7. The method of any one of claims 1 to 6, 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.

8. 8. The method of any one of claims 1 to 7, 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.

9. 9. The method of any one of claims 1 to 8, 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.

10. The method according to any one of claims 1 to 9, wherein step (iv-a) is carried out using phi29 polymerase or Bst polymerase.

11. 11. The method of any one of claims 1 to 10, wherein after step (iv-a), a PCR amplification is performed using primers that bind to the first universal sequence of the first probe and the second universal sequence of the second probe, said primers optionally comprising an adapter for subsequent sequencing in step (xi).

12. The method of any one of claims 1 to 11, wherein gene target enumeration is enabled by counting the number of barcodes per target and per sample.

13. 13. The method of any one of claims 1 to 12, wherein for two or more samples or two or more loci / allele combinations, barcode sequences are used to determine the genotype of the samples for one or more sequences and / or polymorphisms including SNPs and / or indels.

14. 14. The method of any one of claims 1 to 13, further comprising contacting the ligation complex from step (ii-a) with a solid support comprising a second capture moiety and separating it from ligation complexes that are not linked to the solid support.

15. 15. The method of any one of claims 1 to 14, further comprising, after step (ii-a) or (ii-b), contacting the one or more first hybridization complexes or the one or more second hybridization complexes with a solid support comprising a second capture moiety, such that the first capture moiety and the second capture moiety interact to link the one or more first hybridization complexes or the one or more second hybridization complexes to the solid support, and separating the one or more first hybridization complexes or the one or more second hybridization complexes linked to the solid support from components of the sample that are not linked to the solid support, or using oligonucleotides immobilized on a solid surface that have affinity for the one or more first hybridization complexes or the one or more second hybridization complexes through reverse complementarity to a portion of the hybridization complex.

16. After step (iii), subjecting the ligated ligation complex to DNA denaturing conditions selected from heating or alkaline treatment to dissociate the bridging oligonucleotides from the one or more first hybridization complexes or the one or more second hybridization complexes; and / or contacting the ligated ligation complex with a solid support that alternatively comprises a second capture moiety, such that the first capture moiety and the second capture moiety interact to link one or more of the first hybridization complexes or one or more of the second hybridization complexes to the solid support; 16. The method of any one of claims 1 to 15, comprising separating the one or more first hybridization complexes or the one or more second hybridization complexes linked to the solid support from components of the sample that are not linked to a solid support, or using a solid support capable of binding modified or unmodified DNA with high affinity, or using complementary oligonucleotides immobilized on a solid surface.

17. The method of any one of claims 1 to 16, comprising pooling, after step (iii), one or more of the ligated ligation complexes from all of the multiple samples.

18. A method in which in the step (iv-a), the annealed complex is cleaved with the endonuclease instead of cleaving the single-stranded concatemer sequence.

19. 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; wherein 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; wherein said second probe starts at the 5' end of the molecule and 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; wherein said bridge oligo or bridge oligo complex comprises a sequence complementary to each of said first and second bridge oligo specific sequences in each of said first and second probes, and optionally a third barcode; wherein 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, wherein at least one of the first probe, the second probe, the bridging oligo, or the bridging oligo complex comprises a recognition sequence for an endonuclease; wherein said kit of parts further comprises an oligonucleotide capable of annealing to said recognition sequence to provide a recognition portion for said endonuclease, and wherein the oligonucleotide anneals to the recognition sequence to provide a recognition site for the endonuclease; and wherein the bridge oligo or bridge oligo complex comprises a fourth barcode comprising a sequence capable of annealing to a target sequence of a sample.

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