High sensitivity method for accurately quantifying nucleic acids in parallel

Abstract

Aspects of the disclosed embodiments relate to next generation DNA sequencing methods and uses for accurate and massively parallel quantification of one or more nucleic acid targets, e.g., in large quantities of unpurified sample material. The invention also relates to a method and a kit, wherein the kit comprises a probe for detecting and quantifying a gene target in a complex sample. Comprising one or more target-specific nucleic acid probes (left and right probes) for each gene target, and bridge oligonucleotides or bridge oligonucleotide complexes.

Description

[0001] Incorporation by Reference

[0002] The Sequence Listing in the form of an XML file (.XML) (ST.26) is incorporated herein by reference in its entirety. Technical Field

[0003] 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 gene targets in complex DNA pools, primarily for gene target and variant detection. Aspects of the disclosed embodiments use one or more target-specific nucleic acid probes (left probe and right probe) and bridge oligonucleotides or bridge oligonucleotide complexes for each gene target. Background Art

[0004] With the advancement of technology for studying gene variation, detecting gene variation in plants and animals is not cumbersome. However, despite the decline in sequencing costs, detecting and accurately quantifying gene variation (such as mutations), especially in samples with weaker signals, is still cumbersome, laborious and expensive. Various issues can be more accurately stated as follows: such as specificity to detect gene signals in a common background, sensitivity to detect weak gene signals, accuracy to accurately quantify detected signals, the number of fluxes of targeted gene targets detected each time, the cost of each assay, the scale of the assay cost when multiple samples are assayed in parallel, and the turn-over to determine the length of time from sampling to results.

[0005] Currently, typical quantitative 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 quantitative methods are robust and well-established approaches, each approach is associated with specific issues discussed in more detail below:

[0006] Quantitative PCR: Quantitative PCR (qPCR) is a technique that involves the amplification of targeted DNA molecules during the PCR process (i.e. in real time). Real-time PCR is divided into quantitative use (quantitative real-time PCR), and can also be used semi-quantitatively, i.e. above / below a certain amount of DNA molecules (semi-quantitative real-time PCR). Quantitative PCR (qPCR) is the gold standard for quantification of gene targets. Currently, the laboratory cost of a qPCR reaction is about 2$. However, considering the considerable hands-on time (labor costs) required to set up the reaction, the need for standard curves, and the replicates for each quantified target, the actual cost is actually much higher. Since each gene target requires a separate quantification experiment, the hands-on time increases dramatically with the number of samples.

[0007] Array PCR: PCR arrays are the most reliable tool for analyzing the expression of a panel of genes for a pathway- or disease-focused group. Each 96-well plate, 384-well plate, or 100-well disk PCR array includes SYBR Green optimized primer assays for a thorough investigation of a panel of focused genes. A new iteration of qPCR technology is array qPCR, which miniaturizes individual qPCR reactions. Array PCR reduces the cost of individual qPCR reactions and increases the scalability of the approach to multiple targets and samples. However, the approach is currently limited to profiling 384 targets from 12 samples (or conversely, 12 targets from 384 samples) at a cost of thousands of dollars per chip plus the huge capital cost of the readout infrastructure. Therefore, profiling thousands of samples using the above setup remains prohibitively expensive.

[0008] 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. This method is relatively cost-effective (one target per sample costs about $3), but the actual time to prepare, set up and run a separate experiment for each target in each sample makes it difficult to scale to thousands of samples.

[0009] Multiplex ligation-dependent probe amplification (MLPA) provides a method to simplify the detection of multiple gene targets in a single sample. However, MLPA only provides relative quantification of targets and requires a separate detection experiment for each sample. Recently, a variation of MLPA has introduced the concept of DNA barcoding. This concept allows for better quantitative resolution and sample multiplexing compared to the traditional MLPA workflow.

[0010] Next-generation sequencing-based methods: Next-generation sequencing (NGS), also known as high-throughput sequencing, makes sequence-based gene expression analysis a "digital" alternative to analog technologies. As the cost of DNA sequencing continues to decrease, target counting from next-generation DNA sequencing data is becoming increasingly attractive and is currently being used, for example, for NIPT screening. However, current methods suffer from high sequencing library preparation costs and sequencing effort wasted on sequencing non-relevant gene targets. For example, in cancer-related liquid biopsies, non-targeted approaches result in waste of sequencing effort for non-relevant loci in oncology. In fetal diagnostics, non-targeted sampling of loci greatly limits the statistical options for interpreting the data. GuardantHealth Inc. offers a more targeted sequencing approach in which a series of RNA capture probes enrich for targets for next-generation DNA sequencing.

[0011] Akhras et al. (2007) PLoS ONE 2(2): e223 discloses a multiplex pathogen detection assay involving barcoded target-specific probes, target circularization and sequencing. Also disclosed is the use of bridge oligonucleotides to link target-specific probes.

[0012] WO2018109206 describes a method for detecting an analyte in a sample using padlock probes and rolling circle amplification. The use of bridge oligonucleotides is not described.

[0013] WO2019038372 describes a next-generation sequencing method in which a target sequence of interest is selectively amplified from a ligation complex containing a T7 polymerase promoter by in vitro transcription, followed by cDNA synthesis and sequencing. Although this method allows accurate and parallel detection and quantification of many target sequences in a sample, it is still challenging for more complex, large-volume, diluted and / or impure samples.

[0014] Therefore, in light of the foregoing discussion, there is a need to overcome the aforementioned shortcomings, such as, but not limited to, specificity, sensitivity, accuracy, throughput, cost, scaling, and turnaround, through accurate and massively parallel quantification of nucleic acid targets. Summary of the invention

[0015] Aspects of the disclosed embodiments provide methods for highly sensitive, scalable and accurate target quantification using next generation sequencing in large sample volumes (up to tens of milliliters) and / or diluted and / or unpurified sample materials. Moreover, RNA amplification steps such as those described in WO2019038372 are avoided, making the method simpler.

[0016] In a first main aspect, aspects of the disclosed embodiments relate to a method for high throughput detection of one or more target nucleotide sequences in a plurality of samples, the method comprising the steps of:

[0017] (i) providing, for each target nucleotide sequence in each sample, a first probe, a second probe, and a bridge oligonucleotide or a plurality of oligonucleotides that can anneal to each other to form a bridge oligonucleotide complex;

[0018] wherein the first probe comprises a first bridge oligonucleotide specific sequence, a first universal sequence, an optional first sequence barcode, and a first target specific portion located at the 3' end of the first probe, starting at the 5' end of the molecule;

[0019] and wherein the second probe comprises, from the 5' end of the molecule, a second target-specific portion, an optional second sequence barcode, a second universal sequence, and a second bridge oligonucleotide-specific sequence at the 3' end of the second probe;

[0020] and wherein the bridge oligonucleotide or the plurality of oligonucleotides capable of annealing to each other to form a bridge oligonucleotide complex comprises sequences complementary to a first bridge oligonucleotide-specific sequence in the first probe and a second bridge oligonucleotide-specific sequence in the second probe, respectively, and an optional third barcode;

[0021] and wherein at least one of the first sequence barcode or the second sequence barcode or the third barcode is present in the first probe or the second probe or the bridge oligonucleotide or the bridge oligonucleotide complex, respectively;

[0022] and wherein at least one of the first probe or the second probe or the bridge oligonucleotide or the bridge oligonucleotide complex comprises a recognition sequence for an endonuclease;

[0023] and wherein, optionally, at least one of the first probe or the second probe or the bridge oligonucleotide or the plurality of oligonucleotides capable of annealing to each other to form a bridge oligonucleotide complex comprises a first capture portion;

[0024] (ii) forming a hybridization complex by:

[0025] (ii-a) for each of the one or more target nucleotide sequences, contacting a first probe, a second probe, and a bridge oligonucleotide or a plurality of oligonucleotides capable of annealing to each other to form a bridged oligonucleotide complex and allowing self-annealing to form a plurality of ligation complexes;

[0026] contacting the nucleic acid present in each of the plurality of samples of one or more target nucleotide sequences to be detected with the ligation complex; and

[0027] allowing the first target-specific portion of the first probe and the second target-specific portion of the second probe from the ligation complex to hybridize to substantially adjacent segments of one or more target nucleotide sequences in each of the plurality of samples, thereby forming one or more first hybridization complexes;

[0028] or

[0029] (ii-b) contacting the nucleic acid present in each of the multiple samples of the one or more target nucleotide sequences to be detected with the first target-specific portion of the first probe and the second target-specific portion of the second probe to hybridize with substantially adjacent segments 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 a bridge oligonucleotide or a plurality of oligonucleotides that can anneal to each other to form a bridge oligonucleotide complex, thereby forming one or more second hybridization complexes;

[0030] (iii) ligating the probes in the one or more first hybridization complexes or the one or more second hybridization complexes using a ligase or a combination of a ligase and a DNA polymerase to provide one or more ligated ligation complexes;

[0031] (iv) amplifying the nucleic acid from the one or more ligated ligation complexes by rolling circle amplification using a strand displacement polymerase to form an amplified one or more single-stranded concatemer sequences;

[0032] And do one of the following:

[0033] (va) optionally, annealing the amplified one or more single-stranded concatemer sequences obtained in step (iv) with a specific oligonucleotide containing a recognition sequence for an endonuclease, wherein the specific oligonucleotide anneals to the recognition sequence to form an annealed complex containing a recognition site for the endonuclease; and

[0034] Cutting the single-stranded concatemer sequence obtained in step (iv) or cutting the annealed complex with the endonuclease to form nucleic acid fragments;

[0035] or

[0036] (vb) contacting one or more single-stranded concatemer sequences with a solid support, wherein the solid support may alternatively include a second capture portion, allowing the first capture portion and the second capture portion to interact, thereby allowing the one or more single-stranded concatemer sequences to be attached to the solid support, and separating the concatemer sequences attached to the solid support from components of the sample that are not attached to the solid support; or using a solid support that can bind to modified or unmodified DNA with high affinity, or using complementary oligonucleotides immobilized on a solid surface;

[0037] (vi) subjecting the nucleic acid fragments obtained in step (va) or the one or more single-stranded concatemer sequences obtained in step (vb) to a high-throughput sequencing technique to determine the barcode sequence; and

[0038] (vii) identifying the presence and / or quantity of a 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.

[0039] In another main aspect, aspects of the disclosed embodiments relate to a kit of parts comprising a plurality of containers, wherein at least one container holds one or more sets of first probes and second probes, and at least one container holds one or more bridge oligonucleotides or a plurality of oligonucleotides capable of forming a bridge oligonucleotide complex;

[0040] wherein the first probe comprises, from the 5' end of the molecule, a first bridge oligonucleotide specific sequence, an optional first sequence barcode, and a first target specific portion located at the 3' end of the first probe;

[0041] wherein the second probe comprises, starting from the 5' end of the molecule, a second target-specific portion, an optional second sequence barcode, and a second bridge oligonucleotide-specific sequence located at the 3' end of the second probe;

[0042] wherein the bridge oligonucleotide or bridge oligonucleotide complex comprises sequences complementary to the first bridge oligonucleotide specific sequence in the first probe and the second bridge oligonucleotide specific sequence in the second probe, respectively, and an optional third barcode;

[0043] 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 oligonucleotide, or the bridge oligonucleotide complex, respectively;

[0044] and wherein at least one of the first probe or the second probe or the bridge oligonucleotide or the bridge oligonucleotide complex comprises a recognition sequence for an endonuclease;

[0045] Wherein, the kit further comprises an oligonucleotide capable of annealing with the recognition sequence to obtain a recognition site for the endonuclease; and wherein the bridge oligonucleotide or the bridge oligonucleotide complex comprises a fourth barcode, and the fourth barcode comprises a sequence capable of annealing with a target sequence of the sample. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1A flow chart of a multiple ligation assay (MLA) according to an embodiment of the present invention is shown, including (listed from top to bottom) 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.

[0047] Figure 2A The schematic structure of a probe triplet (including SEQ ID Nos. 29, 30, and 31) having multiple probe entities according to an embodiment of the present invention is shown.

[0048] Figure 2B Gap filling between a first probe and a second probe according to embodiments herein is shown.

[0049] Figure 2C Gap filling between the first and second probes and the bridge complex according to embodiments herein is shown.

[0050] Figure 3 The RCA products in the workflow are shown before (lane 2) and after (lane 1) digestion with restriction endonucleases.

[0051] Figure 4 The linear response of the experimental flow to logarithmically decreasing amounts of gene target in four replicate reactions is shown, inferred from next-generation DNA sequencing data by enumerating molecular barcodes. Each row represents three concentrations of target sequence. The response is linear over three orders of magnitude. DETAILED DESCRIPTION

[0052] definition:

[0053] Target nucleotide sequence: The term "target nucleotide sequence" can be any nucleotide sequence of interest that needs to be detected. It should be understood that the given term refers to a continuous nucleotide sequence as well as a nucleic acid molecule with a complementary sequence. In some embodiments, the target sequence is a nucleotide sequence that represents a polymorphism or is associated with a polymorphism.

[0054] 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 a genetic locus where sequence differences occur. A polymorphic locus can be as small as one base pair.

[0055] Sample: The term "sample" is used herein for two or more samples containing two or more target sequences. The samples provided according to the methods of the disclosed embodiments may have been prepared so as to extract at least target nucleic acids and make those nucleic acids accessible to the probes used in the disclosed embodiments. In particular, in some embodiments, each sample includes 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" may refer to, but is not limited to, two or more samples obtained from a human / animal body, including urine, biopsy, saliva and other secretions, exhaled water extracts, tissues, plasma (liquid biopsy samples); or two or more samples obtained from the environment, including water, wastewater, soil, plants; or two or more samples containing viruses or bacteria, etc. In one embodiment, the plurality of samples includes a blood sample, a saliva sample, a urine sample or a stool sample, a sample of another body fluid, or an extract of a body material, such as hair or dandruff.

[0056] Probe: The term "probe" is a DNA or RNA fragment of variable length (usually 50 to 1000 bases long, preferably 50 to 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 segment of the oligonucleotide probe complementary to the target sequence is designed so that for each target sequence in the sample, a pair of left and right probes is provided, whereby each probe contains a segment complementary to a portion of the target sequence at its end. In addition, the present disclosure describes a bridge oligonucleotide or a bridge oligonucleotide complex for joining the left probe and the right probe.

[0057] Universal: When used to describe an amplification procedure, the term "universal" refers to a sequence that enables multiple amplification reactions to be performed using a single primer or set of primers. The use of such primers greatly simplifies multiplexing, as only two primers are required to amplify multiple selected nucleic acid sequences. When used to describe a primer site, the term "universal" refers to the site to which a universal primer will hybridize. It should also be noted that a "set" of universal priming sequences / primers can be used.

[0058] Hybridization: The term "hybridization" describes the process by which deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) molecules anneal to complementary DNA or RNA. DNA or RNA replication and transcription of DNA into RNA both rely on nucleotide hybridization.

[0059] Ligation: The term "ligation" refers to the joining of two nucleic acid fragments by the action of an enzyme. DNA ligase is an enzyme that catalyzes the formation of a phosphodiester bond between (the ends of) two polynucleotide chains that bind at adjacent sites on complementary strands. In one embodiment, ligation can also be performed chemically, particularly if both adjacent ends of the polynucleotide are modified to enable chemical ligation.

[0060] Amplification: As used herein, the term "amplification" refers to the use of a DNA polymerase to increase the concentration of a specific nucleotide sequence in a mixture of nucleotide sequences. "PCR" or "polymerase chain reaction" is a rapid procedure for 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 (usually about 18 to 22 bases) that serves as a starting point for DNA synthesis. This is necessary for DNA replication because the enzyme that catalyzes this process, DNA polymerase, can only add new nucleotides to existing DNA strands.

[0061] Polymerase: A polymerase is an enzyme that synthesizes long chains of nucleic acids or nucleic acid polymers. DNA polymerase and RNA polymerase are used to assemble DNA and RNA molecules respectively by copying strands of DNA or RNA templates through base pairing interactions.

[0062] High throughput: The term "high throughput" refers to the ability to process and screen large numbers of DNA samples simultaneously; and the ability to screen a large number of different loci simultaneously in a single DNA sample. High-throughput sequencing or screening, often abbreviated as HTS, is a scientific experimental method that is particularly suitable for efficiently screening large numbers of samples simultaneously.

[0063] Endonuclease: Endonuclease is an enzyme that cleaves double-stranded or single-stranded DNA at random or specific locations.

[0064] As described above, the present disclosure relates to methods for high-throughput detection of target nucleotide sequences in a very large number of samples using a ligation-dependent assay. The present disclosure provides methods for determining the sequence of a gene target in a complex nucleic acid pool using technology allowed by next-generation sequencing. The present disclosure also provides methods for profiling a variety of gene targets in multiple samples (preferably in a very large number of samples) by utilizing a ligation-dependent assay. The present disclosure provides methods for multiple ligation-dependent probe amplification, which enables querying different target nucleic acids in multiple samples. The methods of the embodiments of the present disclosure enable sequencing of one or more target nucleotide sequences in multiple samples, providing multiple different probe sets for different target nucleic acids. When processing sequencing data, unique sequence identifiers are used to identify gene target nuclei and to absolutely quantify individual samples in a sample pool.

[0065] In a first major aspect, aspects of the disclosed embodiments relate to a method for high-throughput detection of one or more target nucleotide sequences in a plurality of samples, the method comprising the steps of:

[0066] (i) providing, for each target nucleotide sequence in each sample, a first probe, a second probe, and a bridge oligonucleotide or a plurality of oligonucleotides that can anneal to each other to form a bridge oligonucleotide complex;

[0067] wherein the first probe comprises, from the 5' end of the molecule, a first bridge oligonucleotide specific sequence, a first universal sequence, an optional first sequence barcode, and a first target specific portion located at the 3' end of the first probe;

[0068] and wherein the second probe comprises, from the 5' end of the molecule, a second target-specific portion, an optional second sequence barcode, a second universal sequence, and a second bridge oligonucleotide-specific sequence at the 3' end of the second probe;

[0069] and wherein the bridge oligonucleotide or the plurality of oligonucleotides capable of annealing to each other to form a bridge oligonucleotide complex comprises sequences complementary to a first bridge oligonucleotide-specific sequence in the first probe and a second bridge oligonucleotide-specific sequence in the second probe, respectively, and an optional third barcode;

[0070] and wherein at least one of the first sequence barcode or the second sequence barcode or the third barcode is present in the first probe or the second probe or the bridge oligonucleotide or the bridge oligonucleotide complex, respectively;

[0071] and wherein at least one of the first probe or the second probe or the bridge oligonucleotide or the bridge oligonucleotide complex comprises a recognition sequence for an endonuclease;

[0072] and wherein, optionally, at least one of the first probe or the second probe or the bridge oligonucleotide or the plurality of oligonucleotides capable of annealing to each other to form a bridge oligonucleotide complex comprises a first capture portion;

[0073] (ii) forming a hybridization complex by:

[0074] (ii-a) for each of the one or more target nucleotide sequences, contacting the first probe, the second probe, and a bridge oligonucleotide or a plurality of oligonucleotides capable of annealing to each other to form a bridge oligonucleotide complex and allowing self-annealing into a plurality of ligation complexes;

[0075] contacting the nucleic acid present in each of the plurality of samples of one or more target nucleotide sequences to be detected with the ligation complex; and

[0076] allowing the first target-specific portion of the first probe and the second target-specific portion of the second probe from the ligation complex to hybridize to substantially adjacent segments of one or more target nucleotide sequences in each of the plurality of samples, thereby forming one or more first hybridization complexes;

[0077] or

[0078] (ii-b) contacting the nucleic acid present in each of the multiple samples of the one or more target nucleotide sequences to be detected with the first target-specific portion of the first probe and the second target-specific portion of the second probe to hybridize with substantially adjacent segments 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 a bridge oligonucleotide or a plurality of oligonucleotides that can anneal to each other to form a bridge oligonucleotide complex, thereby forming one or more second hybridization complexes;

[0079] (iii) ligating the probes in the one or more first hybridization complexes or the one or more second hybridization complexes using a ligase or a combination of a ligase and a DNA polymerase to provide one or more ligated ligation complexes;

[0080] (iv) amplifying nucleic acid from the one or more ligated ligation complexes by rolling circle amplification using a strand displacement polymerase to form an amplified one or more single-stranded concatemer sequences;

[0081] And do one of the following:

[0082] (va) optionally, annealing the amplified one or more single-stranded concatemer sequences obtained in step (iv) with a specific oligonucleotide containing a recognition sequence for an endonuclease, wherein the specific oligonucleotide anneals to the recognition sequence to form an annealed complex containing a recognition site for the endonuclease; and

[0083] Cutting the single-stranded concatemer sequence obtained in (iv) or cutting the annealed complex with the endonuclease to form nucleic acid fragments;

[0084] or

[0085] (vb) contacting one or more single-stranded concatemer sequences with a solid support, which may alternatively include a second capture portion, allowing the first capture portion and the second capture portion to interact, thereby allowing the one or more single-stranded concatemer sequences to be attached to the solid support, and separating the concatemer sequences attached to the solid support from components of the sample that are not attached to the solid support; or using a solid support that can bind to modified or unmodified DNA with high affinity, or using complementary oligonucleotides immobilized on a solid surface;

[0086] (vi) subjecting the nucleic acid fragments obtained in step (va) or the one or more single-stranded concatemer sequences obtained in step (vb) to a high-throughput sequencing technique to determine the barcode sequence; and

[0087] (vii) identifying the presence and / or quantity of a 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.

[0088] In another main aspect, aspects of the disclosed embodiments relate to a kit of parts comprising a plurality of containers, wherein at least one container holds one or more sets of first probes and second probes, and at least one container holds one or more bridge oligonucleotides or a plurality of oligonucleotides capable of forming a bridge oligonucleotide complex;

[0089] wherein the first probe comprises, from the 5' end of the molecule, a first bridge oligonucleotide specific sequence, an optional first sequence barcode, and a first target specific portion located at the 3' end of the first probe;

[0090] wherein the second probe comprises, from the 5' end of the molecule, a second target-specific portion, an optional second sequence barcode, and a second bridge oligonucleotide-specific sequence located at the 3' end of the second probe;

[0091] wherein the bridge oligonucleotide or bridge oligonucleotide complex comprises sequences complementary to the first bridge oligonucleotide specific sequence in the first probe and the second bridge oligonucleotide specific sequence in the second probe, respectively, and an optional third barcode;

[0092] 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 oligonucleotide, or the bridge oligonucleotide complex, respectively;

[0093] and wherein at least one of the first probe or the second probe or the bridge oligonucleotide or the bridge oligonucleotide complex comprises a recognition sequence for an endonuclease;

[0094] Wherein, the kit further comprises an oligonucleotide capable of annealing with the recognition sequence to obtain a recognition site for the endonuclease; and wherein the bridge oligonucleotide or the bridge oligonucleotide complex comprises a fourth barcode, and the fourth barcode comprises a sequence capable of annealing with a target sequence of the sample.

[0095] Figure 1 Non-limiting illustrations of embodiments of the methods of the disclosed embodiments are provided.

[0096] The method of the disclosed embodiment utilizes three nucleic acid probes, two target-specific nucleic acid probes (left probe and right probe) are specific to the gene target, and one nucleic acid probe is generally universal (bridge oligonucleotide or bridge oligonucleotide complex). The left probe and the right probe hybridize with the bridge probe or the bridge oligonucleotide complex to form a connection complex. The connection complex (including one or more barcode sequences) with the target identification site on the sample DNA or RNA is hybridized with the complementary target sequence of the query sample. After hybridization, the left probe and the right probe are chemically connected or enzymatically connected by DNA ligase to form a connected connection complex. In the aspects of the disclosed embodiment, during the sample analysis process of multiple samples to be analyzed, multiple such connected connection complexes will be formed.

[0097] In an embodiment, "multiple samples" may refer to, but are not limited to, two or more samples obtained from a human or animal body, including biopsy samples, saliva and other secretions, exhaled water extracts, tissues, plasma (liquid biopsy samples); two or more samples obtained from the environment, including water, wastewater, soil, plants; or two or more samples containing viruses or bacteria, etc. In one embodiment, the samples used do not require prior purification or concentration of nucleic acids. In another embodiment, the samples may be pre-treated, such as lysing cells to expose nucleic acids.

[0098] The target sequence may include any nucleotide sequence of interest that needs to be detected. The target nucleotide sequence disclosed herein can be obtained from a portion of DNA in the patient's blood or a portion of DNA in the maternal blood, but is not limited thereto. A portion of the DNA in the patient's blood can be obtained from apoptotic / necrotic cancer cells, or a portion of the DNA in the maternal blood can be of fetal and / or maternal origin. Further, the analysis results are used, for example, to assess the risk of an individual developing a given type of cancer, to determine the efficacy of a given treatment for a given cancer, the development of resistance-related mutations in tumors, or the risk of a fetus carrying a genetic disease (such as common trisomy Down syndrome, Patou syndrome, and Edwards syndrome). In certain embodiments, the method includes providing a plurality of different probe sets for each target nucleotide sequence.

[0099] As used herein, the term "probe set" includes a first probe, a second probe, and a bridge oligonucleotide or a bridge oligonucleotide complex.

[0100] In certain embodiments, the first probe comprises, from the 5' end of the molecule, an optional 5' phosphate, a first bridge oligonucleotide specific sequence, an optional first universal sequence, an optional first sequence barcode, and a first target-specific portion at its 3' end. In certain embodiments, the second probe comprises, from the 5' end of the molecule, an optional 5' phosphate, a second target-specific portion, an optional second sequence barcode, an optional second universal sequence, and a second bridge oligonucleotide specific sequence at its 3' end.

[0101] In a preferred embodiment, the first probe or the second probe comprises at least one of a first sequence barcode or a second sequence barcode. The first sequence barcode or the second sequence barcode or both may be a random sequence, or may comprise a target nucleotide sequence identifier sequence, a sample identifier sequence and / or a molecular barcode for target enumeration.

[0102] In a preferred embodiment, the bridge oligonucleotide or bridge oligonucleotide complex comprises a sequence complementary to the first bridge oligonucleotide specific sequence in the first probe and the second bridge oligonucleotide specific sequence in the second probe, respectively, an optional universal sequence, and / or, or may comprise a third barcode, which may be a random sequence or may comprise a sample or sequence identifier sequence. In this regard, the third barcode does not necessarily mean that the first barcode and the second barcode are already present. As previously described, at least one barcode should be present in the connected ligation complex, which enables the complex to be uniquely defined in all ligation complexes in all test samples.

[0103] In addition, at least one of the first probe or the second probe or the bridge oligonucleotide or the bridge oligonucleotide complex includes a recognition sequence for an endonuclease. The recognition sequence is required for cutting the concatemer sequence 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 guide DNAaseI or CRISPR-Cas class cutting system.

[0104] Alternatively, at least one of the first probe or the second probe or the bridge oligonucleotide or the bridge oligonucleotide complex includes a first capture portion. When used herein, "first capture portion" refers to a portion, such as a chemical group, that allows the probe, the connection complex or the hybridization complex to be connected to the second capture portion of the solid support to capture the captured (i.e., combined with it). Any suitable capture portion known in the art can be used for this purpose. Well-known suitable examples are to capture biotinylated molecules using magnetic beads coated with streptavidin. Therefore, in one embodiment, the first capture portion is a biotin portion, which can interact with the streptavidin or avidin portion (second capture portion) connected to a solid support (such as a magnetic bead). Other selections include biotin derivatives, such as bibiotin, desthiobiotin or photolyzable biotin, which can be used to conjugate with streptavidin / avidin. Further options include the use of thiol and acrydite groups for acrylamide / acrylamide conjugation, the use of alkyne and azide groups for click chemistry, and the use of digoxigenin for conjugation with anti-digoxigenin antibodies. The conjugation partner can be disposed on any solid surface, such as beads (magnetic or other) or a solid support.

[0105] Preferably, the first target-specific portion, the second target-specific portion, the first bridge oligonucleotide-specific sequence and / or the second bridge oligonucleotide-specific sequence independently of each other comprise at least one chemically modified nucleotide 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 in WO2019038372 Figure 3 In one embodiment, the first probe or the second probe or the bridging portion of the two includes chemically modified bases to allow improved binding to the bridge oligonucleotide or bridge oligonucleotide complex. In another embodiment, the first target-specific portion, the second target-specific portion, the first bridge oligonucleotide specific sequence and / or the second bridge oligonucleotide specific sequence independently include one or more chemically modified nucleotides. In certain embodiments, chemical modification allows chemical connection of adjacent probes. In some embodiments, the probes described above are combined with completely adjacent genetic loci or with a distance of at most 500 base pairs, for example, at most 200 base pairs, such as at most 50 base pairs, preferably at most 40 base pairs, more preferably at most 30 base pairs, more preferably at most 20 base pairs, more preferably at most 10 base pairs, and most preferably at most 5 base pairs.

[0106] In some embodiments, the first probe or the second probe or the bridge oligonucleotide or the bridge oligonucleotide complex may include an adapter sequence for a DNA sequencing platform such as (but not limited to) Illumina. These adapter sequences allow the resulting sequencing library to be combined with a detection component of a sequencing device (such as an Illumina flow cell).

[0107] Furthermore, in some embodiments, the bridge oligonucleotide or bridge oligonucleotide complex comprises:

[0108] (i) one to five 3' overhanging bases (ie, additional bases that do not form a duplex with the second probe), and / or

[0109] (ii) 3′ phosphate, and / or

[0110] (iii) one or more phosphorothioate modifications within three positions from the 3' end,

[0111] (iv) A first capture moiety, such as biotin at the 5' end.

[0112] In one embodiment, preferably for each sample in a separate tube, the first probe and the second probe are contacted with a bridge oligonucleotide or a plurality of oligonucleotides capable of forming a bridge oligonucleotide complex and allowed to self-anneal to form a ligation complex (step (ii)) before the probes are contacted with the sample containing the target sequence. In an embodiment (herein in Figure 2C ), the plurality of oligonucleotides may be pre-annealed prior to annealing with the first probe and the second probe, or all annealing steps may be performed at once.

[0113] 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. This enables enumeration of target sequences after amplification and analysis of the results.

[0114] In some embodiments, if at least one of the first probe or the second probe or the bridge oligonucleotide or the bridge oligonucleotide complex comprises a first capture portion, the ligated complex from (step ii) is contacted with a solid support comprising a second capture portion, and the first capture portion and the second capture portion are allowed to interact, so that the hybridization complex is attached to the solid support, and the ligated complex attached to the solid support is separated from the ligated complex not attached to the solid support (optional step (additional step 1)). Thereafter, the ligated complex attached to the solid support is separated from the components of the sample that are not attached to the solid support. If the solid support is a magnetic bead, the magnetic beads can be fixed using a magnet, and the remaining liquid sample can be removed. Optionally, a washing step is performed before continuing;

[0115] The step (additional step 1) allows the ligation complex to be immobilized on a solid support, such as magnetic beads or a flat surface, the latter of which can be used, for example, for fluorescence detection in certain DNA sequencing applications.

[0116] Thereafter, one or more target nucleotide sequences in the plurality of samples are contacted with the plurality of ligation complexes (step (iii)). The first target-specific portion of the first probe and the second target-specific portion of the second probe hybridize to substantially adjacent segments on the target sequence to form a hybridization complex (step (iv)). In some embodiments, the sample is composed of DNA extracted from blood, tissue, FFPE samples, saliva, urine, or feces. In some embodiments, the volume of the sample exceeds 100 microliters, for example, exceeds 1 ml. In another embodiment, the nucleic acid concentration of the sample is less than 5 pmol, for example, less than 1 pmol, for example, less than 200 fmol. In one embodiment, the plurality of samples include one or more blood samples, one or more saliva samples, one or more urine samples, or one or more fecal samples.

[0117] In another embodiment, one or more target nucleotide sequences in multiple samples are contacted with multiple first probes and second probes. The first target-specific portion of the first probe and the second target-specific portion of the second probe hybridize to substantially adjacent segments on the target sequence. Subsequently, the first probe and the second probe that have been targeted for hybridization are contacted with a bridge oligonucleotide to form a hybridization complex.

[0118] Subsequently, in some embodiments, if at least one of the first probe or the second probe or the bridge oligonucleotide or the bridge oligonucleotide complex includes a first capture portion, the hybrid complex is contacted with a solid support containing a second capture portion, and the first capture portion and the second capture portion are allowed to interact so that the hybrid complex is connected to the solid support (optional step (v)). Thereafter, the hybrid complex connected to the solid support is separated from the components of the sample that are not connected to the solid support. If the solid support is a magnetic bead, the magnetic beads can be fixed using a magnet, and the remaining liquid sample can be removed. Optionally, a washing step is performed before continuing.

[0119] In one embodiment, step (v) results in purification and enrichment of nucleic acids, resulting in improved results, particularly for highly impure samples. In one embodiment, the method of the disclosed embodiment does not include a step of enriching nucleic acids before step (v). Therefore, in one embodiment, the method does not include a step of concentrating the nucleic acids in the original sample more than 2 times, more than 10 times, or more than 100 times before step (vi). In another embodiment, the method of the disclosed embodiment does not include a purification step after the connection in step (vi). In another embodiment, step (v) allows the connection complex to be fixed on a solid support, such as magnetic beads or a flat surface, which can be used, for example, for fluorescence detection in certain DNA sequencing applications.

[0120] Subsequently, the probes in the formed hybridization complex are ligated enzymatically or chemically to provide a ligated ligated complex (step (vi)). Optionally, as part of step (vi), the gap between the first probe and the second probe (if present) can be filled by introducing a polymerase and one or more nucleotides, wherein the one or more nucleotides optionally include a chemically modified nucleotide containing a capture moiety, wherein the capture moiety includes, but is 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). The polymerase adds nucleotides that are (a) complementary to the universal bridge oligonucleotide sequence and / or (b) complementary to the barcode sequence, thereby filling the two gaps between the first probe and the second probe, ligating the left probe and the right probe, and incorporating the universal sequence and / or the third barcode sequence into the bridge complementary strand. The bridge oligonucleotide or bridge oligonucleotide complex extends from the 5' site or the 3' site to be complementary to the connected probe, so that the target sequence identifier sequence present in the first probe or the second probe is integrated into the bridge oligonucleotide or the bridge oligonucleotide complex. Preferably, a polymerase that does not destroy double-stranded DNA, such as Taq polymerase, is used so that the connection between the first probe and the second probe is not disturbed when the first probe and the second probe anneal to the target sequence.

[0121] Subsequently, in some embodiments (optional step (additional step 2)), the ligated ligation complexes are subjected to DNA denaturing conditions, such as heat or alkaline conditions, to dissociate the bridging oligonucleotide from the ligated ligation complexes. This dissociation will expose the universal sequence regions of the ligated ligation complexes, which can be used, for example, to bind these ligated ligation complexes to immobilized nucleotide probes for fluorescent detection applications including certain DNA sequencing applications.

[0122] Optionally, in some embodiments (optional step (additional step 3)), the ligated ligation complex is contacted with a solid support (such as magnetic beads or a flat surface), which can be used, for example, for fluorescence detection in certain DNA sequencing applications. The solid support may include a second capture portion that allows the first capture portion and the second capture portion in the ligated ligation complex to interact so that the ligation complex is connected to the solid support, or the solid support may be modified to be able to bind to unmodified or modified DNA with high affinity, or the solid support may include an immobilized oligonucleotide that has affinity for the ligated ligation complex through DNA hybridization. If the solid support is a magnetic bead, the magnetic beads can be immobilized using a magnet and the remaining liquid sample can be removed. Optionally, a washing step is performed before continuing.

[0123] The step (additional step 3) results in the immobilization of the ligated ligation complex on a solid support (eg magnetic beads or a flat surface), which in the case of a flat surface can be used, for example, for fluorescence detection in certain DNA sequencing applications.

[0124] Then, optionally, the ligated ligation complexes are pooled from one or more target samples (step (vii)). Steps (vi) and (vii) may be performed in the order specified or alternatively in reverse order.

[0125] Next, nucleic acid is amplified from one or more ligated ligation complexes (step (viii)). Amplification is performed by rolling circle amplification using a strand displacement polymerase, such as phi29 polymerase (UniProtKB-P03680; DPOL_BPPH2) or Bst polymerase (P52026; DPO1_GEOSE), wherein the strand displacement polymerase optionally comprises a chemically modified nucleotide containing a capture portion, the capture portion 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). In the following step (ix), the amplified one or more single-stranded concatemer sequences obtained in step (viii) are optionally annealed with a specific oligonucleotide containing a recognition sequence for a nuclease, wherein the oligonucleotide anneals to the recognition sequence in step (i), thereby obtaining a recognition site for the nuclease. Specific oligonucleotides containing a recognition sequence will usually contain some additional specific sequences around the recognition sequence to enable formation of a stable duplex for cleavage.

[0126] The single-stranded concatemer sequence obtained in step (viii) or the annealed complex obtained in step (ix) is then optionally cleaved with the endonuclease (step (x)), thereby generating an NGS library.

[0127] Optionally, after amplification, the solid support (if present) is removed and the supernatant is used for subsequent processing. For example, if the solid support is a magnetic particle, it can be removed using a magnet. In some other embodiments of the method of the disclosed embodiment, the interaction between the first capture part and the second capture part is immediately destroyed after step (vi), after step (vii), or after step (viii). For example, if the first capture part is biotin and the second capture part is streptavidin, the interaction can be destroyed by adding an excess of soluble biotin. If the streptavidin is bound to the magnetic particles, it can then be removed using a magnet.

[0128] In another embodiment (optional step (additional step 4)), the concatemer sequence obtained in step (viii) is contacted with a solid support, which may alternatively include a second capture portion, allowing the first capture portion or multiple first capture portions in the concatemer sequence to interact with the second capture portion, so that the hybrid complex is attached to the solid support, and the hybrid complex attached to the solid support is separated from the components in the sample that are not attached to the solid support, or a solid support that can bind to modified or unmodified DNA with high affinity is used, or the solid support may include fixed oligonucleotides that have affinity for the concatemer sequence through DNA hybridization. If the solid support is a magnetic bead, the magnetic beads can be fixed using a magnet, and the remaining liquid sample can be removed. Optionally, a washing step is performed before continuing.

[0129] The step (additional step 4) results in the immobilization of the concatemer sequences on a solid support (eg magnetic beads or a flat surface), which in the case of a flat surface can be used, for example, for fluorescence detection in certain DNA sequencing applications.

[0130] In addition, optionally, between step (x) and step (xi), PCR amplification is optionally performed using primers that bind to the common portion of the first probe and the second probe, wherein the primers optionally include an adapter sequence for sequencing in the subsequent step (xi).

[0131] The presence and / or quantity of target nucleotide sequences in a plurality of samples can be identified by determining at least a portion of the first and / or second target-specific portion, at least a portion of the first and / or second barcode, and / or at least a portion of the third barcode by high-throughput sequencing technology (step (xi) and step (xii)), for example, using a next-generation sequencing platform, including but not limited to Illumina iSeq, MiSeq, HiSeq, NextSeq or NovaSeq. Preferably, enumeration of gene targets is allowed by counting the number of molecular barcodes per target and per sample. Samples are separated from the sequence data (deconvolution) and sequence targets are quantified in silico following DNA sequencing.

[0132] In a preferred embodiment, the molecule is further amplified using a first primer and a second primer to provide an amplification product. Preferably a universal first primer and a universal second primer are used which are reverse complementary to the first or second universal sequence present in the ligation complex.

[0133] Compared with traditional nucleic acid sequencing technology, the advantages of the disclosed embodiments include, but are not limited to, low cost, high simplicity, high specificity, high sensitivity, high accuracy, high throughput, high scalability and high turnover quantitative determination. Another aspect of the disclosed embodiments is that the methods of the disclosed embodiments enable accurate and large-scale parallel quantification of multiple nucleic acid targets in multiple samples including human and animal populations and including large volumes of unpurified sample materials. As mentioned, in a preferred embodiment, the samples used, such as urine samples, do not require prior purification or concentration of nucleic acids. In another embodiment, the sample can be pretreated, such as lysing cells to expose nucleic acids. A particular advantage of the disclosed embodiments is the ability to use a unique probe design (i.e., a probe triplet) to detect and amplify target sequences of interest. The probe design has specially positioned modified nucleotides that can improve annealing and binding efficiency. Improved binding properties result in higher detection specificity, sensitivity and accuracy. The methods of the disclosed embodiments are also applicable to studying genetic variants and find applications in diagnosis and prognosis, including but not limited to genotyping samples of one or more sequences and / or polymorphisms (such as SNPs and / or insertions and deletions (indels)), cancer diagnosis, or fetal chromosomal diseases from maternal blood. In a preferred embodiment, for two or more samples or for two or more loci / allele combinations, one or more sequences and / or polymorphisms (e.g., SNPs and / or insertions and deletions) in the sample are genotyped using a barcode sequence.

[0134] In another aspect, aspects of the disclosed embodiments provide a kit comprising a plurality of containers, wherein at least one container holds one or more sets of first probes and second probes, and at least one container holds one or more bridge oligonucleotides or a plurality of oligonucleotides capable of annealing to each other to form a bridge oligonucleotide complex;

[0135] wherein the first probe comprises, from the 5' end of the molecule, a first bridge oligonucleotide specific sequence, an optional first sequence barcode, and a first target specific portion located at the 3' end of the first probe;

[0136] wherein the second probe comprises, starting from the 5' end of the molecule, a second target-specific portion, an optional second sequence barcode, and a second bridge oligonucleotide-specific sequence located at the 3' end of the second probe;

[0137] wherein the bridge oligonucleotide or bridge oligonucleotide complex comprises a sequence complementary to a first bridge oligonucleotide specific sequence in the first probe and a second bridge oligonucleotide specific sequence in the second probe, and an optional third barcode;

[0138] 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 oligonucleotide, or the bridge oligonucleotide complex, respectively;

[0139] wherein at least one of the first probe or the second probe or the bridge oligonucleotide or the bridge oligonucleotide complex comprises a recognition sequence for an endonuclease;

[0140] And wherein, the complete kit further comprises an oligonucleotide capable of annealing with the recognition sequence, thereby obtaining the recognition site of the nuclease.

[0141] 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.

[0142] Preferably, the bridge oligonucleotide or bridge oligonucleotide complex includes one or more chemically modified nucleotides in the sequence complementary to the first probe sequence, or in the sequence complementary to the second probe sequence, or in both.

[0143] 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.

[0144] Preferably, the bridging portion of the first probe or the second probe, or both, comprises a chemically modified base to allow for improved binding to the bridge oligonucleotide or bridge oligonucleotide complex.

[0145] In a specific embodiment, at least one container containing the first and second probe groups and at least one container containing a plurality of oligonucleotides that can anneal to each other to form a bridge oligonucleotide complex are the same container. In this case, the three probes can be pre-annealed and form a connected complex.

[0146] A particular advantage of the disclosed embodiments is the ability to use unique probe designs (i.e., probe triplets) to detect and amplify target sequences of interest. The probes are designed to have improved binding properties, resulting in higher assay specificity, sensitivity, and accuracy. Aspects of the disclosed embodiments are applied to the fields of molecular biology, evolutionary biology, metagenomics, genotyping, more specifically, but not limited to cancer diagnosis or fetal chromosomal diseases, including but not limited to genotyping one or more sequences and / or polymorphisms (such as SNPs and / or indels) of a sample.

[0147] In a particularly preferred embodiment, the bridging oligonucleotide or bridging oligonucleotide complex includes information for identifying the sample and includes a unique barcode. In this case, the first and second probes are universally applicable to all samples (and only include information for identifying the target). Therefore, in a preferred embodiment, a method or kit according to aspects of the disclosed embodiments is provided, wherein the bridging oligonucleotide or bridging oligonucleotide complex includes a barcode containing a unique sequence that enables the target sequence of each sample to be enumerated.

[0148] Additionally, aspects of the disclosed embodiments relate to:

[0149] Embodiment 1: A method for high-throughput detection of one or more target nucleotide sequences in multiple samples, the method comprising the following steps:

[0150] (i) providing, for each target nucleotide sequence in each sample: a first probe, a second probe, and a bridge oligonucleotide;

[0151] wherein the first probe comprises, from the 5' end of the molecule, a first bridge oligonucleotide specific sequence, an optional first sequence barcode, and a first target specific portion located at the 3' end of the first probe;

[0152] and wherein the second probe comprises, from the 5' end of the molecule, a second target-specific portion, an optional second sequence barcode, and a second bridge oligonucleotide-specific sequence at the 3' end of the second probe;

[0153] and wherein the bridge oligonucleotide comprises a sequence complementary to a first bridge oligonucleotide-specific sequence in the first probe and a second bridge oligonucleotide-specific sequence in the second probe, respectively, and an optional third barcode;

[0154] and wherein at least one of the first sequence barcode or the second sequence barcode or the third barcode is present in the first probe or the second probe or the bridge oligonucleotide, respectively;

[0155] and wherein at least one of the first probe or the second probe or the bridge oligonucleotide comprises a recognition sequence for an endonuclease;

[0156] and wherein, optionally, at least one of the first probe or the second probe or the bridge oligonucleotide comprises a first capture moiety;

[0157] (ii) optionally, for each of the one or more target nucleotide sequences, in separate tubes, contacting the first probe and the second probe, a bridge oligonucleotide or a plurality of oligonucleotides capable of annealing to each other to form a bridge oligonucleotide complex and allowing self-annealing to form a plurality of ligation complexes;

[0158] (Additional step 1) Optionally, contacting the ligated complex from step (ii) with a solid support comprising a second capture moiety, allowing the first capture moiety and the second capture moiety to interact, such that the hybridization complex is attached to the solid support, and the ligated complex attached to the solid support is separated from the ligated complex not attached to the solid support;

[0159] (iii) contacting the nucleic acid present in each of the samples of the target nucleotide sequence to be detected with the ligation complex from step (ii) or (additional step 1);

[0160] (iv) allowing the first target-specific portion of the first probe and the second target-specific portion of the second probe from the ligation complex of step (ii) or (additional step 1) to hybridize to substantially adjacent segments on the target sequence, thereby forming a hybridization complex;

[0161] (Additional Step 6) Optionally, contacting the nucleic acid present in each of the samples of the target nucleotide sequence to be detected with the first target-specific portion of the first probe and the second target-specific portion of the second probe to hybridize to substantially adjacent segments on the target sequence;

[0162] (Additional step 5) Optionally, contacting the target-hybridized first and second probes from step (Additional step 6) with the bridge, thereby forming a hybridization complex;

[0163] (v) Optionally, contacting the hybridization complex from step (iv) or step (additional step 5) with a solid support containing a second capture portion, allowing the first capture portion and the second capture portion to interact, so that the hybridization complex is attached to the solid support, and the hybridization complex attached to the solid support and the components in the sample that are not attached to the solid support are separated; or using oligonucleotides immobilized on a solid surface, which have affinity for the hybridization complex by being reverse complementary to a portion of the hybridization complex;

[0164] (vi) ligating the probes in the hybridization complex from step (iv) or step (additional step 5) or step (v) using a ligase or a combination of ligases or a DNA polymerase to provide a ligated ligation complex, optionally using a modified nucleotide containing 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));

[0165] (Additional step 2) Optionally, subjecting the ligated ligation complex to DNA denaturing conditions, such as heating or alkaline treatment, to dissociate the bridge oligonucleotide from the hybridization complex;

[0166] (Additional step 3) Optionally, contacting the linked ligation complex with a solid support, which may alternatively include a second capture portion, allowing the first capture portion and the second capture portion to interact, so that the hybridization complex is linked to the solid support, and the hybridization complex linked to the solid support is separated from the components of the sample that are not linked to the solid support; or using a solid support that can bind to the modified or unmodified DNA with high affinity, or using complementary oligonucleotides immobilized on a solid surface;

[0167] (vii) pooling the ligated ligation complexes from multiple samples;

[0168] (viii) amplifying nucleic acid from the one or more ligated ligation complexes by rolling circle amplification with a strand displacement polymerase to form an amplified one or more single-stranded concatemer sequence, optionally using modified nucleotides containing a capture moiety (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));

[0169] (ix) optionally, annealing the amplified one or more single-stranded concatemer sequences obtained in step (viii) with a specific oligonucleotide containing a recognition sequence for an endonuclease, wherein the oligonucleotide anneals to the specific recognition sequence in step (i), thereby obtaining a recognition site for the endonuclease;

[0170] (x) cleaving the single-stranded concatemer sequence obtained in step (viii) or the annealed complex obtained in step (ix) with the endonuclease;

[0171] (Additional step 4) Optionally, contacting the concatemer sequence obtained in step (viii) with a solid support, which may alternatively include a second capture portion, allowing the first capture portion and the second capture portion to interact, so that the concatemer sequence is attached to the solid support, and separating the concatemer sequence attached to the solid support and the component of the sample not attached to the solid support; or using a solid support capable of binding to the modified or unmodified DNA with high affinity, or using a complementary oligonucleotide immobilized on a solid surface;

[0172] (xi) subjecting the nucleic acid fragments obtained in step (x) to high-throughput sequencing technology to determine the barcode sequence; and

[0173] (xii) identifying the presence and / or quantity of a target nucleotide sequence in 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;

[0174] wherein steps (vi) and (vii) may be performed in any order;

[0175] wherein one of step (additional step 1), step (v), (additional step 3) or (additional step 4) is completed,

[0176] Wherein one of step (ii) or (additional step 6) is completed.

[0177] Embodiment 2: The method according to embodiment 1, wherein the plurality of samples comprises a blood sample, a tissue sample, a FFPE sample, a saliva sample, a urine sample or a stool sample or DNA extracted from any of these.

[0178] Embodiment 3: The method according to embodiment 1 or 2, wherein at least one of the first probe or the second probe or the bridge oligonucleotide comprises a first capture portion, wherein the method comprises step (v), and the method does not comprise a step of enriching nucleic acid before step (v).

[0179] Embodiment 4: According to the method of any one of embodiments 1 to 3, wherein at least one of the first probe or the second probe or the bridge oligonucleotide includes a first capture portion, wherein the method includes step (v), and wherein the first capture portion is a biotin portion and the second capture portion is a streptavidin portion or an avidin portion.

[0180] Embodiment 5: The method according to any one of embodiments 1 to 4, wherein at least one of the first probe or the second probe or the bridge oligonucleotide comprises a first capture portion, wherein the method comprises step (v), and wherein a washing step is performed between steps (v) and (vi).

[0181] Embodiment 6: The method according to any one of embodiments 1 to 5, wherein the bridge oligonucleotide comprises:

[0182] (i) one to five bases of 3' overhang, and / or

[0183] (ii) 3' phosphate, and / or

[0184] (iii) one or more phosphorothioate modifications within three positions from the 3′ end,

[0185] Embodiment 7: The method according to any one of embodiments 1 to 6, wherein the 3' end of the first probe or the 5' end of the second probe, or both, is modified to allow chemical ligation of the first probe to the second probe.

[0186] Embodiment 8: The method according to any one of 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 bridge oligonucleotide.

[0187] Embodiment 9: A method according to any one of embodiments 1 to 8, wherein the first target-specific portion, the second target-specific portion, the first bridge oligonucleotide-specific sequence and / or the second bridge oligonucleotide-specific sequence independently comprise one or more chemically modified nucleotides.

[0188] Embodiment 10: The method according to any one of embodiments 1 to 9, wherein step (viii) is performed using phi29 polymerase or Bst polymerase.

[0189] Embodiment 11: According to any one of claims 1 to 9, a method wherein between step (x) and step (xi), PCR amplification is performed using primers that bind to the common portion of the first and second probes, wherein the primers optionally include a linker for subsequent sequencing in step (xi).

[0190] Embodiment 12: The method according to any one of embodiments 1 to 11, wherein enumeration of gene targets is enabled by counting the number of molecular barcodes per target and per sample.

[0191] Embodiment 13: A method according to any one of embodiments 1 to 12, wherein for two or more samples or for two or more loci / allele combinations, one or more sequences and / or polymorphisms (such as SNPs and / or insertions and deletions (indels)) of the samples are genotyped using barcode sequences.

[0192] Embodiment 14: A kit comprising a plurality of containers, wherein 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 oligonucleotides;

[0193] wherein the first probe comprises, from the 5' end of the molecule, a first bridge oligonucleotide specific sequence, an optional first sequence barcode, and a first target specific portion located at the 3' end of the first probe;

[0194] wherein the second probe comprises, starting from the 5' end of the molecule, a second target-specific portion, an optional second sequence barcode, and a second bridge oligonucleotide-specific sequence located at the 3' end of the second probe;

[0195] wherein the bridge oligonucleotide comprises a sequence complementary to a first bridge oligonucleotide specific sequence in the first probe and a second bridge oligonucleotide specific sequence in the second probe, respectively, and an optional third barcode;

[0196] 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 bridge oligonucleotide, respectively;

[0197] and wherein at least one of the first probe or the second probe or the bridge oligonucleotide comprises a recognition sequence for an endonuclease;

[0198] And wherein, the complete kit further comprises an oligonucleotide capable of annealing with the recognition sequence to obtain the recognition site of the nuclease.

[0199] Example

[0200] method

[0201] 1. Probe Complex Formation

[0202] The probe complex contains the sequences required for genome targeting, sample indexing, and construction of Illumina sequencing libraries.

[0203] A probe complex can be formed comprising the following three parts (as shown in Figure 2):

[0204] (a) a first probe having a first bridge oligonucleotide-specific sequence from the 5' end of the molecule, and a first target-specific portion located at the 3' end of the first probe;

[0205] (b) a second probe having a second target-specific portion from the 5' end of the molecule, a second sequence barcode, and a second bridge oligonucleotide-specific sequence located at the 3' end of the second probe; and

[0206] (c) a bridge oligonucleotide having a sequence complementary to a first bridge oligonucleotide-specific sequence in the first probe and a second bridge oligonucleotide-specific sequence in the second probe, respectively;

[0207] The probe complex was constructed by combining all three parts (bridge, right arm and left arm) in equimolar amounts in an annealing reaction. The reaction was performed in a thermal cycler (annealing program see Table 1).

[0208] Table 1

[0209]

[0210] 2. Target capture

[0211] Targeting specific genomic regions containing mutations of interest. Purified DNA (e.g. from tissue, plasma, urine or saliva) can be used as a sample, or the sample can be unpurified and simply pre-treated, such as boiling and / or centrifugation.

[0212] The probe complex is hybridized to the target region through base sequence complementary interaction. To initiate target capture, the reaction probe and target DNA are mixed and incubated in a thermal cycler (target capture and gap filling procedures are shown in Table 2).

[0213] Table 2

[0214]

[0215] 3. GapFill reaction

[0216] After target capture, the probe complexes are extended and ligated by adding a combination of Phusion DNA polymerase, nucleotides, and Ampligase DNA ligase and incubating at +45°C for 45 minutes.

[0217] 4. Rolling Circle Amplification

[0218] After extension and ligation, the circular probe molecules were subjected to rolling circle amplification (RCA). For the RCA reaction, the target capture reaction was mixed with an RCA reaction mixture containing EquipPhi29 (Thermo Scientific) polymerase. The reaction was incubated at +42°C for 30 minutes to 2 hours. After the RCA reaction, the reaction efficiency was analyzed by measuring the concentration of single-stranded DNA (ssDNA) with a Qubit fluorometer.

[0219] 5. Enzyme Digestion

[0220] The RCA reaction generates long concatemer ssDNA molecules with multiple copies of the target library. Each complete target library is separated by an EcoRI restriction enzyme recognition sequence. This sequence enables sequence-specific cleavage of the long concatemers and releases the prepared target libraries by annealing with a specific oligonucleotide containing the EcoRI restriction enzyme recognition sequence. After a simple purification step, these libraries can be used for further analysis. The RCA products are digested with EcoRI for 1 hour at +37°C.

[0221] 6. Library Purification

[0222] After EcoRI digestion, library molecules are purified by extraction from agarose gels after electrophoresis or using size selective beads (such as Macherey Nagel NucleoMag).

[0223] 7. Sequencing

[0224] Purified MiSeq or iSeq100 compatible libraries are sequenced using state-of-the-art sequencing instruments. Importantly, these libraries can be converted to fit any existing sequencing platform through simple oligonucleotide modifications. Sequencing data is processed using a combination of Unix command-line tools and Python and R programming languages. In short, sequence processing works by identifying the probe sequences in each read, sequencing the genomic region between them, and counting the number of molecular barcodes associated with each gene target.

[0225] Experiment 1

[0226] In the first experiment, the probe mix was a collection of four different index probes that resulted in four replicate reactions. They targeted the EML-ALK fusion. The target oligonucleotides had a unique recognition sequence that allowed each target to be identified.

[0227] As a sample, three synthetic target oligonucleotides were mixed in logarithmic increasing concentrations. Target capture, extension and ligation reactions, rolling circle amplification and subsequent EcoRI digestion were performed as described above. Typical results are shown in Figure 2. Figure 3 shown.

[0228] Prepared libraries were sequenced using MiSeq and iSeq100 instruments to detect target regions in sequence data by matching probe sequences in each read, identifying genomic sequence regions between probe sequences, and counting molecular barcodes. The count data accurately reflects the number of inserted template molecules, and the detected signal is highly specific to the presence of target molecules ( Figure 4 ).

[0229] Figure 1 and detailed description of Figure 2

[0230] Figure 1The workflow of one embodiment of the present disclosure is shown. In step 1, the nucleic acid (DNA or RNA) in the sample (102) is contacted with a set of ligation complexes (104). The ligation complexes are annealed on the target nucleic acid (106). In step 2, the ligation complexes that bind to the target are optionally captured from the sample material, leaving behind sample impurities (103). In step 3, the annealed ligation complexes are ligated to obtain ligated ligation complexes. In step 4, the ligated ligation complexes from multiple samples (110) are brought together (112). In step 5, the probe sequence is amplified by rolling circle amplification using phi29 polymerase or other chain displacement polymerases to obtain long concatemer copies of the probe (116). In step 6, the concatemer probe copies are optionally cut into monomer units using a restriction endonuclease (e.g., EcoRI) or a homing nuclease (e.g., I-CeuI), and are 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 into target counts using a bioinformatics pipeline.

[0231] Figure 2A The principle structure of the probe triplet with multiple probe entities according to the embodiments of the present invention is shown. Multiple probe entities include left probes, right probes and bridge oligonucleotides assembled before sample annealing. The first base (202) of the left probe optionally includes a phosphate portion for enzyme connection or allows chemical connection to the 5' end of the adjacent probe, which is called modification 1. The 15-25 bases (204) of the left probe include bridge binding sequence 1, which may further include chemically modified bases for effectively combining bridge oligonucleotides, which are called bridge site 1. The left probe further optionally includes subsequent 10-20 bases (206) from the 5' end, which include fragments of random nucleotides, which form a molecule-specific barcode or sample-specific barcode called barcode 1. The left probe further includes subsequent 15-30 bases (208) from the 5' end, which are combined with the gene target. Some or all of the nucleotides of 204 or 208 may include chemical modifications to increase the affinity of the probe to the target or bridge oligonucleotide (226). The last base (210) of the left probe optionally includes a phosphate moiety for enzymatic ligation or a modification that allows chemical ligation to the 5' end of the adjacent probe, referred to as modification 1.

[0232] The first base (214) of the right probe optionally includes a phosphate moiety for enzyme ligation or a modification that allows chemical ligation to the 5' end of an adjacent probe, referred to as modification 2. The 15-30 bases (216) of the right probe from the 5' end include the portion of the right probe that binds to the gene target. The subsequent 10-20 bases (218) of the right probe from the 5' end optionally include a fragment of random nucleotides that form a molecule-specific barcode or a sample-specific barcode, referred to as barcode 2. The last 15-25 bases (220) of the right probe include a sequence that effectively binds to a bridge oligonucleotide, referred to as bridge sequence 2. Some or all of the nucleotides 204, 208, 216, or 220 may include chemical modifications to increase the affinity of the probe for the target or bridge oligonucleotide.

[0233] The first 15-25 bases (228) at the 5' end of the bridge oligonucleotide, called bridge sequence 3, are reverse complementary to the bridge sequence 1 (204) of the right probe, and optionally include chemically modified nucleotides to increase binding. The last 15-25 bases (224) of the bridge, called bridge sequence 2, are reverse complementary to the bridge sequence 2 sequence (220) of the left probe, and optionally include chemically modified nucleotides to increase binding. The bridge sequence (226) here includes a recognition site for a restriction endonuclease (such as EcoRI) or a homing endonuclease (such as I-CeuI). The 5' end of the bridge oligonucleotide optionally includes a capture portion (230) for capturing the ligation complex.

[0234] Figure 2B Gap filling between the first probe and the second probe according to an embodiment of the present invention is shown. Here, the bridge oligonucleotide comprises gap 1 between bridge sequence 1 (228) and bridge sequence 2 (224). Gap 2 is formed between the target binding portions of probes 1 and 2 (208 and 216). These gaps are filled by introducing a polymerase and one or more nucleotides. In this process, a mixture of Stoffel fragments, Taq polymerase or Phusion polymerase, and a DNA ligase (e.g., Ampligase) can be used. The polymerase adds nucleotides that are (a) complementary to the universal bridge oligonucleotide sequence and (b) complementary to the target sequence, thereby filling the two gaps between the first probe and the second probe, namely gap 1 and gap 2, and the subsequent action of the DNA ligase connects the left probe and the right probe that are complementary to the bridge oligonucleotide and the target sequence into a circular complex.

[0235] Figure 2CThe principle structure of the probe five-piece set with multiple probe entities according to the embodiment of this paper is shown.Multiple probe entities include left probe, right probe and the bridge composed of three oligonucleotides.Here, the probe complex includes the gap 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 the third bridge oligonucleotide (238 and 242) and between the left and right probes (208 and 216).Fill these gaps by introducing polymerase and one or more nucleotides.In this process, a mixture of Stoffel fragment, Taq polymerase or Phusion polymerase and DNA ligase (such as Ampligase) can be used.Polymerase fills these gaps, and the subsequent action of DNA ligase makes probe and bridge oligonucleotide connect into annular complex.

[0236] The 15-25 bases (228) of the left probe include a bridge binding sequence 1, which optionally includes a base for chemical modification for effectively binding to a bridge oligonucleotide, referred to as bridge sequence 1. The left probe further optionally includes a subsequent 10-20 bases (204) from the 5' end, which includes a universal sequence for library indexing. The left probe further optionally includes a subsequent 10-20 bases (206) from the 5' end, which includes a fragment of random nucleotides, which forms a molecule-specific barcode or sample-specific barcode referred to as barcode 1. The left probe further includes a subsequent 15-30 bases (208) from the 5' end, which bind to the gene target. Some or all of the nucleotides of 228 may include chemical modifications to increase the affinity of the probe for the target or bridge (226). The last base (210) of the left probe optionally includes a phosphate portion for enzyme connection or a modification that allows chemical connection to the 5' end of the adjacent probe, referred to as modification 1.

[0237] The first base (214) of the right probe optionally includes a phosphate portion for enzyme connection or a modification that allows chemical connection to the 5' end of the adjacent probe, which is referred to as modification 2. The 15-30 bases (216) of the right probe from the 5' end include the portion where the right probe binds to the gene target. The subsequent 10-20 bases (218) of the right probe from the 5' end optionally include a fragment of random nucleotides, which form a molecule-specific barcode or a sample-specific barcode (referred to as barcode 2). The subsequent 10-20 bases (220) of the right probe from the 5' end optionally include a universal sequence. The last 15-25 bases (222) of the right probe are referred to as bridge sequence 8, which is reversely complementary to the bridge sequence 7 of the third bridge oligonucleotide (224). Some or all of the nucleotides of 208, 216, 222 or 228 may include chemical modifications to increase the affinity of the probe to the target or bridge oligonucleotide.

[0238] The first 15-25 bases (226) of the first bridge oligonucleotide from the 5' end are called bridge sequence 3, which is reverse complementary to the bridge sequence 1 (228) of the right probe, and optionally includes chemically modified nucleotides to increase binding. The last 15-25 bases (238) of the first bridge oligonucleotide are called bridge sequence 2, which is reverse complementary to the bridge sequence 4 (236) sequence of the second bridge oligonucleotide, and optionally includes chemically modified nucleotides to increase binding. The 5' end of the first bridge oligonucleotide optionally includes a capture portion (230) for capturing the ligation complex.

[0239] The first 15-25 bases (240) of the second bridge oligonucleotide from the 5' end are called bridge sequence 5, which is reverse complementary to the bridge sequence 6 (242) of the third bridge oligonucleotide, and optionally includes chemically modified nucleotides to increase binding. The last 15-25 bases (236) of the second bridge oligonucleotide are called bridge sequence 4, which is reverse complementary to the bridge sequence 2 (238) sequence of the first bridge oligonucleotide, and optionally includes chemically modified nucleotides to increase binding.

[0240] The first 15-25 bases (242) of the third bridge oligonucleotide from the 5' end are called bridge sequence 6, which is reverse complementary to the bridge sequence 5 (240) sequence of the second bridge oligonucleotide, and optionally includes chemically modified nucleotides to increase binding. The last 15-25 bases (224) of the first bridge oligonucleotide are called bridge sequence 7, which is reverse complementary to the bridge sequence 8 (222) sequence of the right probe, and optionally includes chemically modified nucleotides to increase binding. The 3' end of the third bridge oligonucleotide optionally includes a phosphate (or other cleavable) portion (234) to prevent extension during gap filling.

Claims

1. A method for high-throughput detection of one or more target nucleotide sequences in multiple samples, the method comprising the following steps: (i) providing, for each target nucleotide sequence in each sample, a first probe, a second probe, and a bridge oligonucleotide or a plurality of oligonucleotides that can anneal to each other to form a bridge oligonucleotide complex; wherein the first probe comprises, from the 5' end of the molecule, a first bridge oligonucleotide specific sequence, a first universal sequence, an optional first sequence barcode, and a first target specific portion located at the 3' end of the first probe; and wherein the second probe comprises, from the 5' end of the molecule, a second target-specific portion, an optional second sequence barcode, a second universal sequence, and a second bridge oligonucleotide-specific sequence at the 3' end of the second probe; and wherein the bridge oligonucleotide or the plurality of oligonucleotides capable of annealing to each other to form a bridge oligonucleotide complex comprises sequences complementary to a first bridge oligonucleotide-specific sequence in the first probe and a second bridge oligonucleotide-specific sequence in the second probe, respectively, and an optional third barcode; and wherein at least one of the first sequence barcode or the second sequence barcode or the third barcode is present in the first probe or the second probe or the bridge oligonucleotide or the bridge oligonucleotide complex, respectively; and wherein at least one of the first probe or the second probe or the bridge oligonucleotide or the bridge oligonucleotide complex comprises a recognition sequence for an endonuclease; and wherein, optionally, the first probe or the second probe or the bridge oligonucleotide or at least one of the plurality of oligonucleotides capable of annealing to each other to form a bridge oligonucleotide complex comprises a first capture portion; (ii) forming a hybridization complex by: (ii-a) for each of the one or more target nucleotide sequences, contacting the first probe, the second probe, and the bridge oligonucleotide or a plurality of oligonucleotides capable of annealing to each other to form a bridge oligonucleotide complex and allowing self-annealing to form a plurality of ligation complexes; contacting the nucleic acid present in each of the plurality of samples to be tested for the one or more target nucleotide sequences with the ligation complex; and allowing the first target-specific portion of the first probe and the second target-specific portion of the second probe from the ligation complex to hybridize to substantially adjacent segments of the one or more target nucleotide sequences in each of the plurality of samples, thereby forming one or more first hybridization complexes; or (ii-b) contacting the nucleic acid present in each of the multiple samples of the one or more target nucleotide sequences to be detected with the first target-specific portion of the first probe and the second target-specific portion of the second probe to hybridize with substantially adjacent segments on the one or more target nucleotide sequences, and contacting the hybridized one or more target nucleotide sequences and the first and second probes with the bridge oligonucleotide or a plurality of oligonucleotides capable of annealing to each other to form a bridge oligonucleotide 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 using a ligase or a combination of a ligase and a DNA polymerase to provide one or more ligated ligation complexes; (iv) amplifying nucleic acid from the one or more ligated ligation complexes by rolling circle amplification using a strand displacement polymerase to form an amplified one or more single-stranded concatemer sequences; And do one of the following: (va) optionally, annealing the amplified one or more single-stranded concatemer sequences obtained in step (iv) with a specific oligonucleotide containing a recognition sequence for an endonuclease, wherein the specific oligonucleotide anneals to the recognition sequence to form an annealed complex containing a recognition site for the endonuclease; and Cutting the single-stranded concatemer sequence obtained in step (iv) or cutting the annealed complex with the endonuclease to form nucleic acid fragments; or (vb) contacting the one or more single-stranded concatemer sequences with a solid support, wherein the solid support may alternatively include a second capture portion, allowing the first capture portion and the second capture portion to interact, thereby attaching the one or more single-stranded concatemer sequences to the solid support, and separating the concatemer sequences attached to the solid support from components of the sample that are not attached to the solid support; or using a solid support that can bind to 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 (va) or the one or more single-stranded concatemer sequences obtained in step (vb) to high-throughput sequencing technology to determine the barcode sequence; and (vii) identifying the presence and / or quantity of a target nucleic acid 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. The method according to claim 1, wherein: The plurality of samples includes a blood sample, a tissue sample, a FFPE sample, a saliva sample, a urine sample or a stool sample or DNA extracted from any of these.

3. A method according to any one of the preceding claims, wherein: At least one of the first probe or the second probe or the bridge oligonucleotide or the bridge oligonucleotide complex comprises a first capture portion.

4. A method according to any one of the preceding claims, wherein: At least one of the first probe or the second probe or the bridge oligonucleotide or the bridge oligonucleotide complex comprises a first capture moiety, and wherein the first capture moiety is a biotin moiety and the second capture moiety is a streptavidin moiety or an avidin moiety.

5. A method according to any one of the preceding claims, wherein: At least one of the first probe or the second probe or the bridge oligonucleotide or the bridge oligonucleotide complex comprises a first capture portion, and wherein a washing step is performed between step (ii) and step (iii).

6. A method according to any one of the preceding claims, wherein: The bridge oligonucleotide or bridge oligonucleotide complex comprises: (i) one to five bases of 3' overhang, and / or (ii) 3' phosphate, and / or (iii) one or more phosphorothioate modifications within three positions from the 3' end.

7. A method according to any one of the preceding claims, wherein: The 3' end of the first probe or the 5' end of the second probe, or both, are modified to allow chemical ligation of the first probe to the second probe.

8. A method according to any one of the preceding claims, wherein: The bridging portion of the first probe or the second probe, or both, includes a chemically modified base to improve binding to the bridge oligonucleotide or bridge oligonucleotide complex.

9. A method according to any one of the preceding claims, wherein: The first target-specific portion, the second target-specific portion, the first bridge oligonucleotide-specific sequence and / or the second bridge oligonucleotide-specific sequence independently of each other comprise one or more chemically modified nucleotides.

10. A method according to any one of the preceding claims, wherein: Step (va) was performed using either phi29 polymerase or Bst polymerase.

11. A method according to any one of the preceding claims, wherein: After step (va), 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, wherein the primers optionally include adapters for subsequent sequencing in step (xi).

12. A method according to any one of the preceding claims, wherein: Enumeration of gene targets is allowed by counting the number of barcodes per target and per sample.

13. A method according to any one of the preceding claims, wherein: For two or more samples or for two or more loci / allele combinations, the barcode sequences are used to genotype one or more sequences and / or polymorphisms, including SNPs and / or indels, of the samples.

14. The method according to any one of the preceding claims, further comprising: The ligated complex from step (ii-a) is contacted with a solid support containing a second capture portion, the first capture portion and the second capture portion are allowed to interact, so that the ligated complex is attached to the solid support, and the ligated complex attached to the solid support is separated from the ligated complex not attached to the solid support.

15. A method according to any one of the preceding claims, wherein: The method further includes: after step (ii-a) or step (ii-b), by contacting the one or more first hybridization complexes or the one or more second hybridization complexes with a solid support containing a second capture portion, allowing the first capture portion and the second capture portion to interact, thereby connecting 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 connected to the solid support from components in the sample that are not connected to the solid support; or using oligonucleotides immobilized on the solid surface, which have affinity for the one or more first hybridization complexes or the one or more second hybridization complexes by being reverse complementary to a portion of the hybridization complex.

16. A method according to any one of the preceding claims, wherein: After step (iii), subjecting the ligated ligation complexes to a DNA denaturing condition selected from heating or alkaline treatment to dissociate the bridge oligonucleotide from the one or more first hybridization complexes or the one or more second hybridization complexes; and / or The linked ligated complex is contacted with a solid support, wherein the solid support may alternatively include a second capture portion, allowing the first capture portion and the second capture portion to interact with each other, so that the one or more first hybridization complexes or the one or more second hybridization complexes are linked to the solid support, and the one or more first hybridization complexes or the one or more second hybridization complexes linked to the solid support and the components of the sample not linked to the solid support are separated; or a solid support capable of binding to modified or unmodified DNA with high affinity is used, or complementary oligonucleotides are immobilized on a solid surface.

17. A method according to any one of the preceding claims, wherein: After step (iii), the one or more ligated ligation complexes are pooled from all of the plurality of samples.

18. A method according to any one of the preceding claims, wherein: In step (va), the annealed complex is cleaved by the endonuclease rather than the single-stranded concatemer sequence.

19. A kit comprising a plurality of containers, wherein: at least one container holds one or more sets of first probes and second probes, and at least one container holds one or more bridge oligonucleotides or a plurality of oligonucleotides capable of forming a bridge oligonucleotide complex; wherein the first probe comprises, starting from the 5' end of the molecule, a first bridge oligonucleotide specific sequence, an optional first sequence barcode, and a first target specific portion located at the 3' end of the first probe; wherein the second probe comprises, starting from the 5' end of the molecule, a second target-specific portion, an optional second sequence barcode, and a second bridging oligonucleotide-specific sequence located at the 3' end of the second probe; wherein the bridge oligonucleotide or bridge oligonucleotide complex comprises sequences complementary to the first bridge oligonucleotide specific sequence in the first probe and the second bridge oligonucleotide specific sequence in the second probe, respectively, and an optional 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 oligonucleotide, or the bridge oligonucleotide complex, respectively; and wherein at least one of the first probe or the second probe or the bridge oligonucleotide or the bridge oligonucleotide complex comprises a recognition sequence for an endonuclease; Wherein, the kit further comprises an oligonucleotide capable of annealing with the recognition sequence to obtain a recognition site for the endonuclease; and wherein the bridge oligonucleotide or bridge oligonucleotide complex comprises a fourth barcode, and the fourth barcode comprises a sequence capable of annealing with a target sequence of a sample.

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