A highly sensitive method for accurate parallel quantification of mutant nucleic acids

Abstract

To provide a method for using next-generation sequencing for highly scalable and accurate target quantification from large volume samples (up to tens of milliliters) and / or dilute and / or non-purified sample materials.SOLUTION: The invention includes at least target-specific nucleic acid probes per genetic target (first probe, second probe and target-specific probe) and a bridge oligo or bridge oligo complex.SELECTED DRAWING: Figure 3

Description

[Technical Field]

[0001] The present disclosure relates to improved next-generation DNA sequencing methods for accurate and massively parallel quantification of one or more nucleic acid targets. More particularly, the present disclosure relates to methods and kits containing probes for detecting and quantifying gene targets in complex DNA pools, primarily used for detecting gene targets and mutations. [Background technology]

[0002] Detecting genetic variations in plants and animals has become less cumbersome due to advances in research technology. However, detecting and accurately quantifying genetic variations, such as mutations, especially in samples with weak signals, is currently still tedious, laborious, and expensive, despite the decline in sequencing costs. Various issues can be addressed more precisely, such as specificity for detecting gene signals against a consensus background, sensitivity for detecting weak gene signals, precision for accurately quantifying detected signals, throughput number of targeted gene targets per assay, cost per assay, evaluation to determine assay cost scale when assaying multiple samples in parallel, and turnover to determine the length of time from sample collection to result.

[0003] Currently, typical quantification methods for liquid biopsies and conceptually similar assays (e.g., 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. While quantification methods are robust and well-established, each method is associated with specific challenges, which are discussed in more detail below.

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

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

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

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

[0008] Next-generation sequencing-based approaches: Next-generation sequencing (NGS), also known as high-throughput sequencing, makes sequence-based gene expression analysis a "digital" alternative to analog technologies. Target counting from next-generation DNA sequencing data has become increasingly attractive as the cost of DNA sequencing continues to fall and is now used, for example, in non-invasive prenatal testing. However, current approaches suffer from high sequencing library preparation costs and sequencing effort wasted on sequencing irrelevant gene targets. For example, in cancer-related liquid biopsies, a non-targeted approach results in wasted sequencing effort on loci that are not oncologically relevant. In fetal diagnosis, non-targeted sampling of loci significantly limits statistical options for interpreting the data. Guardant Health Inc. offers a more targeted sequencing approach, in which an array of RNA capture probes enriches targets for next-generation DNA sequencing.

[0009] Akhras et al. (2007) PLoS ONE 2(2):e223 discloses a multiplex pathogen detection assay that includes barcoded target-specific probes, target circularization, and sequencing. The use of bridging oligonucleotides to ligate the target-specific probes is also disclosed. WO 2018 / 109206 describes a method for detecting an analyte in a sample using padlock probes and rolling circle amplification. The use of bridging oligos is not described. WO 2019 / 038372 describes a next-generation sequencing approach in which target sequences of interest are selectively amplified by in vitro transcription from a ligation complex containing a T7 polymerase promoter, followed by cDNA synthesis and sequencing. While this method allows for accurate and parallel detection and quantification of many target sequences in a sample, more complex, large quantities, and / or impure samples remain challenging. Therefore, in light of the above discussion, there is a need to overcome the above-mentioned shortcomings, including but not limited to specificity, sensitivity, precision, throughput, cost, validation, and turnover through accurate and massively parallel quantification of nucleic acid targets. [Prior art documents] [Patent documents]

[0010] [Patent Document 1] International Publication No. 2018 / 109206 [Patent Document 2] International Publication No. 2019 / 038372 [Non-patent literature]

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

[0012] The present invention provides methods using next-generation sequencing for highly scalable and accurate target quantification, e.g., from large sample volumes (up to tens of milliliters) and / or diluted and / or unpurified sample material. The RNA amplification step, as described in WO 2019 / 038372, is avoided, making the method simpler. Additionally, the methods of the present invention include a target sequence amplification step in which an additional target-specific probe or multiple additional target-specific probes are used to specifically amplify and thus specifically enrich for a particular sequence, such as a rare sequence. This allows for the detection of such rare sequences in samples that contain an excess of related, but non-identical, sequences. For example, this method can be used to detect rare alleles of a gene in a sample that contains an excess of other alleles. [Means for solving the problem]

[0013] In a first main aspect, the present invention provides a method for detecting one or more target nucleotide sequences in a sample, the method comprising: (i) providing, for each target nucleotide sequence in the sample, a first probe, a second probe, and a bridge oligo or oligonucleotides capable of annealing to each other to form a bridge oligo complex; the first probe comprises, starting from the 5' end of the molecule, a first bridging oligo-specific sequence, optionally a first sequence barcode, and a first target-specific portion at the 3' end of the first probe; said second probe comprising, starting at the 5' end of the molecule, a second target-specific portion, optionally a second sequence barcode, and a second bridging oligo-specific sequence at the 3' end of the second probe; the bridge oligo or bridge oligo complex comprises a sequence complementary to the first bridge oligo specific sequence and the second bridge oligo specific sequence in the first probe and the second probe, respectively, and optionally a third barcode; At least one of the first sequence barcode, the second sequence barcode, or the third barcode is present in the first probe, the second probe, the bridge oligo, or the bridge oligo complex, respectively; and Optionally, at least one of the first probe or the second probe or the bridging oligo or the bridging oligo complex comprises a recognition sequence for an endonuclease; (ii) for each of one or more target nucleotide sequences, contacting the first probe and the second probe with the bridge oligo or oligonucleotides capable of annealing to each other to form a bridge oligo complex, and allowing them to self-anneal into a plurality of ligation complexes; (iii) contacting nucleic acids present in the sample to be tested for a target nucleotide sequence with the ligation complex; (iv) hybridizing the first target-specific portion and the second target-specific portion of each of the first probe and the second probe to essentially adjacent sections on the target sequence, thereby forming a hybridization complex; (v) ligating the probes in the hybridization complex to provide a ligated ligation complex; (vi) dissociating the ligated ligation complex from the target nucleotide sequence; (vii) adding a target-specific probe comprising a sequence corresponding to the target nucleotide sequence, wherein the target-specific probe is capable of annealing to the ligated ligation complex, and allowing the target-specific probe to anneal to the ligated ligation complex, thereby forming an amplification template; (viii) amplifying nucleic acid from said amplification template using rolling circle amplification with a strand-displacing polymerase, thereby obtaining a single-stranded concatemeric sequence; (ix) optionally, providing that the recognition sequence specified in step (i) is present, performing a step of obtaining a nucleic acid fragment by: (a) cleaving the single-stranded concatemeric sequence obtained in step (vii); or (b) subjecting the amplified single-stranded concatemer sequence or sequences obtained in step (viii) to annealing with a specific oligonucleotide containing a recognition sequence for an endonuclease, wherein the oligonucleotide anneals to the recognition sequence identified in step (i) to obtain a recognition site for the endonuclease, and cleaving the annealed complex with the endonuclease; (x) subjecting the concatemer sequence obtained in step (viii) or the nucleic acid fragment obtained in step (ix) to a sequencing technique to determine the barcode sequence; and (xi) identifying the presence and / or number of the target nucleotide sequence in the sample 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 a third barcode. [Brief explanation of the drawings]

[0014] [Figure 1] FIG. 1 shows a flow diagram of a multiplex ligation assay (MLA) according to one embodiment of the present invention. [Figure 2A] FIG. 2A shows a principle combination of probes according to one embodiment of the present invention. [Figure 2B] FIG. 2B shows a principle combination of probes according to one embodiment of the present invention. [Figure 2C] FIG. 2C shows a principle combination of probes according to one embodiment of the present invention. [Figure 2D] FIG. 2D shows a principle combination of probes according to one embodiment of the present invention. [Figure 3]FIG. 3 illustrates the use of target-specific probes according to one embodiment of the present invention, here for detecting rare mutations. [Figure 4] Figure 4 shows two boosted sequencing libraries with two different target concentrations. [Figure 5] FIG. 5 shows the effect of boosting on a panel of 12 gene fusion targets. DETAILED DESCRIPTION OF THE INVENTION

[0015] (definition) Target nucleotide sequence: The term target nucleotide sequence can be any nucleotide sequence of interest whose detection is desired. It can be understood that the term, as used herein, refers to a nucleic acid molecule having a sequence of consecutive nucleotides as well as a complementary sequence. In some embodiments, the target sequence is a nucleotide sequence that represents or is associated with a polymorphism.

[0016] Polymorphism: The term polymorphism refers to the occurrence of two or more genetically determined alternative sequences or alleles in a population. A polymorphic marker or site is the locus at which sequence divergence occurs. A polymorphic locus can be as small as one base pair.

[0017] Sample: The term sample is used herein to refer to two or more samples containing two or more target sequences. Samples provided in the methods of the present invention may be prepared to extract at least the target nucleic acids and make them accessible to the probes used in the present invention. In particular, in some embodiments, the samples each contain at least two different target sequences, preferably at least 100, more preferably at least 250, more preferably at least 500, and most preferably at least 2000 or more different target sequences. The term sample can refer to two or more samples obtained from the human / animal body, including, but not limited to, urine, biopsies, saliva and other secretions, breath extracts, tissue, plasma (liquid biopsy), or two or more samples obtained from the environment, including, but not limited to, water, wastewater, soil, plants, samples containing viruses or bacteria, etc. In one embodiment, the multiple samples include a blood sample, saliva sample, urine sample, or fecal sample, a sample of other bodily fluids, or bodily material, such as extracts from hair or skin dander.

[0018] Probe: The term probe refers to a DNA or RNA fragment of variable length (usually 50-1000 bases, preferably 50-200 bases) that can be used in a DNA or RNA sample to detect the presence of a nucleotide sequence (DNA or RNA target) complementary to the sequence in the probe. The section of the oligonucleotide probe complementary to the target sequence is designed so that for each target sequence in the sample, a pair of first and second probes is provided, whereby each probe contains a complementary section at its end, resulting in each probe containing a section at its end that is complementary to a portion of the target sequence. Furthermore, the present disclosure describes bridging oligos or bridging oligo complexes used to link the first and second probes. Additionally, additional target-specific probes, or boosting probes, containing sequences corresponding to portions of the target sequence may be used.

[0019] Universal: When used to describe an amplification procedure, the term universal refers to sequences that allow a single primer or primer set to be used for multiple amplification reactions. The use of such primers greatly simplifies multiplexing in that only two primers are required to amplify multiple selected nucleic acid sequences. When the term universal is used to describe a priming site, it refers to the site to which the universal primer hybridizes. It should also be noted that a "set(s)" of universal priming sequences / primers may be used.

[0020] Hybridization: The term hybridization or hybridization refers to the process of a DNA or RNA molecule annealing to complementary DNA or RNA. Both DNA or RNA replication and transcription of DNA into RNA depend on nucleotide hybridization.

[0021] 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 can catalyze the formation of a phosphodiester bond between the ends of two polynucleotide strands that are joined to adjacent sites on complementary strands. In one embodiment, ligation can be performed chemically, particularly when both adjacent ends of the polynucleotides are modified to allow chemical ligation.

[0022] Amplification: The term amplification is used herein to refer to the use of DNA polymerase to increase the concentration of a specific nucleotide sequence within a mixture of nucleotide sequences. "PCR" or "polymerase chain reaction" is a rapid procedure for in vitro enzymatic amplification of specific DNA / RNA fragments. The DNA / RNA to be amplified can be denatured by heating the sample. The term primer is an RNA or DNA strand (generally about 18-22 bases) that serves as the starting point for DNA synthesis. It is necessary for DNA replication because the enzyme that catalyzes this process, i.e., DNA polymerase, can only add new nucleotides to an existing strand of DNA.

[0023] Polymerase: A polymerase is an enzyme that synthesizes long chains or polymers of nucleic acids. DNA polymerases and RNA polymerases are used to assemble DNA and RNA molecules, respectively, by copying DNA or RNA template strands using base-pairing interactions.

[0024] High Throughput: The term high throughput refers to the ability to process and screen many DNA samples simultaneously, as well as the ability to simultaneously screen many different loci within a single DNA sample. High throughput sequencing or screening, often abbreviated as HTS, is a method for scientific experimentation that is particularly relevant for effectively screening large numbers of samples simultaneously.

[0025] Endonuclease: An endonuclease is an enzyme that cleaves double or single strands of DNA at random or directed locations.

[0026] Barcode: The probes and oligos used in the present invention may contain one or more barcodes consisting of nucleotide sequences. The barcode sequence may include a target nucleotide sequence identifier sequence, a sample identifier sequence, and / or a molecular barcode (also called a unique molecular identifier) ​​for enumerating the target. The barcode sequence may include a random sequence.

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

[0028] In a first main aspect, the present invention provides a method for detecting one or more target nucleotide sequences in a sample, the method comprising: (i) providing, for each target nucleotide sequence in the sample, a first probe, a second probe, and a bridge oligo or oligonucleotides capable of annealing to each other to form a bridge oligo complex; the first probe comprises, starting from the 5' end of the molecule, a first bridging oligo-specific sequence, optionally a first sequence barcode, and a first target-specific portion at the 3' end of the first probe; said second probe comprising, starting at the 5' end of the molecule, a second target-specific portion, optionally a second sequence barcode, and a second bridging oligo-specific sequence at the 3' end of the second probe; the bridge oligo or bridge oligo complex comprises a sequence complementary to the first bridge oligo specific sequence and the second bridge oligo specific sequence in the first probe and the second probe, respectively, and optionally a third barcode; At least one of the first sequence barcode, the second sequence barcode, or the third barcode is present in the first probe, the second probe, the bridge oligo, or the bridge oligo complex, respectively; and Optionally, at least one of the first probe or the second probe or the bridging oligo or the bridging oligo complex comprises a recognition sequence for an endonuclease; (ii) for each of one or more target nucleotide sequences, contacting the first probe and the second probe with the bridge oligo or oligonucleotides capable of annealing to each other to form a bridge oligo complex, and allowing them to self-anneal into a plurality of ligation complexes; (iii) contacting nucleic acids present in the sample to be tested for a target nucleotide sequence with the ligation complex; (iv) hybridizing the first target-specific portion and the second target-specific portion of each of the first probe and the second probe to essentially adjacent sections on the target sequence, thereby forming a hybridization complex; (v) ligating the probes in the hybridization complex to provide a ligated ligation complex; (vi) dissociating the ligated ligation complex from the target nucleotide sequence; (vii) adding a target-specific probe comprising a sequence corresponding to the target nucleotide sequence, wherein the target-specific probe is capable of annealing to the ligated ligation complex, and allowing the target-specific probe to anneal to the ligated ligation complex, thereby forming an amplification template; (viii) amplifying nucleic acid from said amplification template using rolling circle amplification with a strand-displacing polymerase, thereby obtaining a single-stranded concatemeric sequence; (ix) optionally, providing that the recognition sequence specified in step (i) is present, performing a step of obtaining a nucleic acid fragment by: (a) cleaving the single-stranded concatemeric sequence obtained in step (vii); or (b) subjecting the amplified single-stranded concatemer sequence or sequences obtained in step (viii) to annealing with a specific oligonucleotide containing a recognition sequence for an endonuclease, wherein the oligonucleotide anneals to the recognition sequence identified in step (i) to obtain a recognition site for the endonuclease, and cleaving the annealed complex with the endonuclease; (x) subjecting the concatemer sequence obtained in step (viii) or the nucleic acid fragment obtained in step (ix) to a sequencing technique to determine the barcode sequence; and (xi) identifying the presence and / or number of the target nucleotide sequence in the sample 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 a third barcode.

[0029] In one embodiment, the method is for high-throughput detection of one or more target nucleotide sequences in a plurality of samples, wherein a plurality of samples is provided, and preferably, step (ii) is performed for each of the plurality of samples in a separate tube. In one embodiment, the plurality of samples are pooled prior to step (viii).

[0030] FIG. 1 provides a non-limiting illustration of an embodiment of the method of the present invention.

[0031] The methods of the present invention utilize four or more nucleic acid molecules, of which three target-specific nucleic acid probes (the first probe, the second probe, and the target-specific probe) are specific to a genetic target, and one or more other nucleic acid probes are typically universal (the bridge oligo or bridge oligo complex and the barcode loop oligo). The first probe and the second probe hybridize to the bridge probe or bridge oligo complex to form a ligation complex. Ligation complexes (containing one or more barcode sequences) bearing target recognition sites on the sample DNA or RNA can hybridize to complementary target sequences in the query sample. After hybridization, the first and second probes are chemically or enzymatically ligated (linked) by a DNA ligase to form a ligated ligation complex, and in the present invention, multiple such ligated ligation complexes will be formed during sample analysis in multiple samples being analyzed.

[0032] In one embodiment, "multiple samples" may refer to two or more samples obtained from the human or animal body, including, but not limited to, biopsies, saliva and other secretions, breath extracts, tissue, plasma (liquid biopsy), two or more samples obtained from the environment, including water, wastewater, soil, plants, samples containing viruses or bacteria, etc. In one embodiment, the sample is used without prior purification or enrichment of the nucleic acids, while in another embodiment, the sample may be pre-treated, for example, by lysing the cells to expose the nucleic acids.

[0033] The target sequence can include any nucleotide sequence of interest that needs to be detected. The target nucleotide sequence of the present disclosure can be obtained from, but is not limited to, the DNA fraction in the patient's blood or the DNA fraction in maternal blood. The fraction of DNA in the patient's blood can be obtained, for example, from apoptotic / necrotic cancer cells or from the fraction of DNA in maternal blood from the fetus and / or the mother. Additionally, the results of the analysis are used to assess, for example, an individual's risk for a given type of cancer, determine the effectiveness of a given treatment for a given cancer, the occurrence of drug resistance-associated mutations in tumors, or the risk of a fetus having a genetic disorder such as the common trisomy Down syndrome, Patau syndrome, and Edwards syndrome, etc. In certain embodiments, the method includes providing a plurality of different probe sets for each target nucleotide sequence.

[0034] As used herein, the term probe set(s) includes a first probe, a second probe, and one or more bridge oligos.

[0035] In certain embodiments, the first probe comprises, starting from the 5' end of the molecule, optionally a 5' phosphate, a first bridging oligo-specific sequence, optionally a first universal sequence, optionally a first sequence barcode, and a first target-specific portion at its 3' end.

[0036] In certain embodiments, the second probe comprises, starting from the 5' end of the molecule, optionally a 5' phosphate, a second target-specific portion, optionally a second sequence barcode, optionally a second universal sequence, and a second bridging oligo-specific sequence at its 3' end.

[0037] In some embodiments, either the first probe or the second probe comprises at least one of a first sequence barcode or a second sequence barcode, which 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.

[0038] The bridge oligo or bridge oligo complexes may comprise sequences complementary to the first and second bridge oligo specific sequences of the first and second probes, respectively, optionally a universal sequence, and / or a third barcode, which may comprise a random sequence or a sample or sequence identifier sequence. In this respect, the third barcode does not necessarily mean that the first and second barcodes already exist. As mentioned above, at least one barcode must be present in the ligated ligation complex so that the complex can be uniquely defined within all ligation complexes of all samples tested.

[0039] Optionally, at least one of the first probe, the second probe, one or more bridging oligos, or the target-specific probe comprises a recognition sequence for an endonuclease. The endonuclease recognition sequence allows for cleavage of the concatemeric sequence. In one embodiment, the recognition sequence is that of a restriction endonuclease, such as EcoRI. In another embodiment, the recognition sequence is that of a homing endonuclease, such as I-CeuI. In another embodiment, the recognition sequence is the recognition sequence for an induced DNAase I or CRISPR-Cas-like cleavage system. In another embodiment, the recognition sequence is a recognition sequence for a nicking endonuclease.

[0040] Optionally, at least one of the first probe, the second probe, or the one or more bridging oligos comprises a first capture moiety, which, as used herein, refers to a moiety such as a chemical group that allows a probe, ligation complex, or hybridization complex to be captured by, i.e., bound to, a second capture moiety linked to a solid support. Any suitable capture moiety known in the art can be used for this purpose, a well-known suitable example is the capture of biotinylated molecules using streptavidin-coated magnetic beads. Thus, in one embodiment, the first capture moiety is a biotin moiety, which can interact with a streptavidin or avidin moiety (second capture moiety) attached to a solid support, such as a magnetic bead. Other options include biotin derivatives such as dual biotin, desthiobiotin, or photocleavable biotin that can be used to bind to streptavidin / avidin. Further options include the use of thiol and acrydite groups for acrydite / acrylamide conjugation, alkyne and azide groups for click chemistry, and digoxigenin for anti-digoxigenin antibody conjugation. The binding partner may be provided on any solid surface, such as a bead (magnetic or otherwise) or solid support. Thus, in one embodiment of the method, at least one of the first probe, the second probe, the barcode loop oligo, or the one or more bridging oligos comprises a first capture moiety, and between steps (iv) and (v) an intermediate step (iv)(a) is carried out which comprises contacting the hybridization complex with a solid support comprising a second capture moiety, allowing the first capture moiety and the second capture moiety to interact such that the hybridization complex(es) bind to the solid support, and separating the hybridization complexes bound to the solid support from components of the sample that are not bound to the solid support.

[0041] The first target-specific portion, the second target-specific portion, the first bridging oligo-specific sequence, and / or the second bridging oligo-specific sequence preferably contain, independently of each other, at least one chemically modified nucleotide to increase probe binding. Chemical modifications that increase probe binding include, but are not limited to, ribonucleic acids, peptide nucleic acids, and locked nucleic acids (e.g., as shown in Figure 3 of WO 2019 / 038372, which is incorporated herein by reference). In one embodiment, the bridging moiety of the first probe or the second probe, or both, comprises a chemically modified base to improve binding to the bridge oligo or bridge oligo complex. In another embodiment, the first target-specific portion, the second target-specific portion, the first bridging oligo-specific sequence, and / or the second bridging oligo-specific sequence independently comprise one or more chemically modified nucleotides. In certain embodiments, the chemical modifications allow for chemical ligation of adjacent probes.

[0042] The probes bind to adjacent loci, i.e., adjacent sections of the target nucleotide sequence. However, the sections are not completely adjacent, for example, they are separated by at least 15 base pairs, e.g., at least 20 base pairs, at least 25 base pairs, or at least 30 base pairs. In preferred embodiments, the adjacent sections are separated by no more than 500 base pairs, e.g., no more than 200 base pairs, e.g., no more than 100 base pairs or no more than 50 base pairs.

[0043] In some embodiments, the first probe, the second probe, one or more bridging oligos, or the target-specific probe may include (but is not limited to) adapter sequences for DNA sequencing platforms such as Illumina MiSeq, NextSeq, or NovaSeq, which allow the resulting sequencing library to bind to the detection portion of a sequencing device such as an Illumina flow cell.

[0044] Furthermore, in some embodiments, the oligonucleotide of the bridge oligo or bridge oligonucleotides forming the bridge oligo complex comprises: (i) 1 to 5 3' overhanging bases (i.e., additional bases that do not form a duplex with the second probe), and / or (ii) a 3' phosphate, and / or (iii) one or more phosphorothioate modifications within three positions from the 3' end Includes.

[0045] In one embodiment of the method of the present invention, the method comprises the use of a plurality of oligonucleotides capable of annealing to each other to form a bridge oligo complex, said plurality of oligonucleotides comprising a barcode loop oligo, wherein the barcode loop oligo comprises, starting from the 5' end of the molecule, a third bridge oligo-specific sequence, a barcode loop sequence, and a fourth bridge oligo-specific sequence, and wherein one or more other bridge oligos comprise sequences complementary to the third bridge oligo-specific sequence and the fourth bridge oligo-specific sequence in the barcode loop oligo.

[0046] Thus, the barcode loop oligo comprises a loop section flanked by two sections that can hybridize with one or more bridge oligos to form a bridge oligo complex. The loop section does not hybridize with one or more bridge oligos or bridge oligo complexes and comprises a barcode. In one embodiment, the barcode loop sequence comprises a third barcode.

[0047] Prior to contacting the probe with a sample containing the target sequence, the first and second probes are contacted with a bridge oligo or multiple oligonucleotides capable of forming a bridge oligo complex, preferably for each sample in a separate tube, and allowed to self-anneal into a ligation complex (step (ii)). In one embodiment (shown in Figure 2B) where the bridge is not a single oligo but multiple oligonucleotides, e.g., three or five oligonucleotides, that can anneal to each other to form a bridged oligo complex, the multiple oligonucleotides may be pre-annealed before annealing with the first and second probes, or all annealing steps may be performed at once.

[0048] Preferably, each ligation complex is unique for the combination of the first target-specific sequence, the second target-specific sequence, and one or more barcode sequences, allowing for enumeration of target sequences after amplification and analysis of the results.

[0049] One or more target nucleotide sequences in the multiple samples are then contacted with the multiple ligation complexes (step (iii)). The first and second target-specific portions of the first and second probes, respectively, hybridize to essentially adjacent portions on the target sequence, thereby forming hybridization complexes (step (iv)). As mentioned above, the essentially adjacent portions on the target sequence are typically separated by between 15 and 500 base pairs.

[0050] In some embodiments, the sample has a volume of more than 100 microliters, e.g., a volume of 1 ml or more. In further embodiments, the sample has a nucleic acid concentration of less than 5 pmol, e.g., less than 1 pmol, e.g., less than 200 fmol. In one embodiment, the plurality of samples includes one or more blood samples, one or more saliva samples, one or more urine samples, or one or more fecal samples.

[0051] Subsequently, in some embodiments, if the first probe, or second probe, or bridging oligo, or nucleotide, oligonucleotide capable of forming a bridging oligo complex, comprises a first complementary moiety, the hybridization complex(es) is contacted with a solid support comprising a second capture moiety, and the first complementary moiety and the second complementary moiety are capable of interacting to bind the hybridization complex to the solid support (optional step (iv)(a)). Hybridization complexes bound to the solid support are then separated from components of the sample that are not bound to the solid support. If the solid support is magnetic beads, a magnet may be used to immobilize the beads and remove any remaining liquid sample. Optionally, a washing step is performed before the next step.

[0052] Step (iv)(a) purifies and concentrates the nucleic acids, allowing for improved results, particularly for samples of very low purity. In one embodiment, the method does not include a step of concentrating the nucleic acids prior to step (iv)(a). Thus, in one embodiment, the method does not include a step of concentrating the nucleic acids in the original sample more than 2-fold, more than 10-fold, or more than 100-fold prior to step (vi). In another embodiment, the method does not include a purification step after the ligation in step (vi).

[0053] Ligation of the probes in the formed hybridized complex is then carried out enzymatically or chemically to provide a ligated ligation complex (step (v)). Optionally, as part of step (v), any gaps between the first and second probes can be filled by introducing a polymerase and one or more nucleotides. The polymerase adds nucleotides (a) complementary to the bridging oligo sequence(s) and / or (b) complementary to the barcode sequence, thereby filling the two gaps between the first and second probes, resulting in ligation of the first and second probes and incorporation of the universal sequence and / or third barcode sequence into the bridge complementary strand. The bridge oligo or bridge oligo complex is extended from the 5' or 3' portion complementary to the ligated probe such that the target sequence identifier sequence present in the first probe or second probe is incorporated into the bridge oligo or bridge oligo complex. Preferably, a polymerase that does not degrade double-stranded DNA, such as Taq polymerase, is used so as not to interfere with ligation of the first probe to the second probe when both are annealed to the target sequence. In one embodiment, the bridge oligo, or one or more of the bridge oligonucleotides, contains multiple universal base analogs in the region not complementary to the first probe or the second probe to allow incorporation of random sequences suitable for use as molecular barcodes for target enumeration. These random sequences can thus become tertiary barcodes. In such embodiments, as part of step (v), a gap-filling step is performed using a polymerase and nucleotides to generate such random sequences. In embodiments, the plurality of universal base analogs is a plurality of 5-nitroindoles.

[0054] Before or after step (v), the ligated ligation complexes are optionally pooled from one or more target samples.

[0055] The ligated ligation complex is then dissociated from the target nucleotide sequence (step (vi)), and a target-specific probe is added and allowed to anneal to the ligated ligation complex, thereby forming an amplification template (step (vii)). The target-specific probe contains a sequence that corresponds to the target nucleotide sequence and therefore will anneal specifically to ligated ligation complexes that match that sequence. Target-specific probes optionally include capture moieties such as biotin and / or nucleotide modifications (including but not limited to phosphorothioate, LNA, and PNA modifications) that enhance binding to the target or protect the target from exonuclease activity. The inclusion of steps (vi) and (vii) allows for the selective amplification of mutant sequences, such as rare mutations, in the sample. As shown in Figure 3, if the target-specific probe sequence is selected to match the mutant sequence to be detected (e.g., a rare mutation) but not the non-mutated sequence (e.g., the bulk genome wild-type sequence), it will promote selective amplification of the mutant sequence in the next step and facilitate its subsequent detection. Typically, in step (vii), a large excess of target-specific probe is added relative to the amount of target sequence, so that the formation of amplification templates is favored over reannealing of ligated ligation complexes with the target sequence.

[0056] Nucleic acid is then amplified from the amplification template (step (viii)). As shown in Figure 3, amplification can be initiated from a bridging oligo and / or a target-specific probe. Amplification from the annealed target-specific probe results in selective amplification of mutant target sequences containing the target-specific probe sequence ("match") over non-mutant target sequences that do not contain the target-specific probe sequence ("mismatch"). Amplification is carried out using rolling circle amplification with a strand-displacing polymerase such as phi29 polymerase (UniProtKB-P03680; DPOL_BPPH2) or Bst polymerase (P52026; DPO1_GEOSE).

[0057] A single-stranded concatemeric sequence is obtained as a result of step (viii).

[0058] Optionally, if the recognition sequence specified in step (i) is present, step (ix) is carried out to obtain the nucleic acid fragment by: (a) cleaving the single-stranded concatemeric sequence obtained in step (viii); or (b) annealing the amplified single-stranded concatemer sequence or sequences obtained in step (viii) with a specific oligonucleotide containing a recognition sequence for an endonuclease, wherein the oligonucleotide anneals to the recognition sequence identified in step (i) to provide a recognition site for the endonuclease, and cleaving the annealed complex with the endonuclease.

[0059] Optionally, after amplification, the solid support, if present, is removed and the supernatant is used for further processing, for example, if the solid support is magnetic particles, these can be removed using a magnet. In some other embodiments of the methods of the present invention, the interaction between the first capture moiety and the second capture moiety is interrupted immediately after step (v), after step (vi), or after step (vii). For example, if the first capture moiety is biotin and the second capture moiety is streptavidin, the interaction can be disrupted by adding excess soluble biotin. If the streptavidin is bound to magnetic particles, it can be removed using a magnet.

[0060] Next, in step (x), the concatemer sequence obtained in step (viii), or, if step (ix) is performed, the nucleic acid fragment obtained in step (ix), is subjected to high-throughput sequencing technology to determine the barcode sequence.

[0061] Optionally, PCR amplification is carried out immediately prior to step (x) using primers that bind to the universal portions of the first and second probes, wherein said primers optionally contain adapter sequences for subsequent sequencing in step (x).

[0062] In another embodiment, the sequencing in step (x) is performed using nanopore sequencing, and optionally the concatemeric sequences obtained in step (viii) are fragmented using translocation complexes. Techniques suitable for nanopore sequencing have been reviewed in Wang et al., 2021 Nat Biotechnol 39(11):1348.

[0063] Identifying the presence and / or number of target nucleotide sequences in the plurality of samples may be carried out by determining at least a portion of the first and / or second target specificity portions, and / or at least a portion of the first and / or second barcodes, and / or at least a portion of the third barcode using high-throughput sequencing technology (steps (x) and (xi)), for example, using a next-generation sequencing platform, including but not limited to Illumina iSeq, MiSeq, HiSeq, NextSeq, or NovaSeq. Preferably, gene target enumeration is enabled by counting the number of molecular barcodes per target and per sample, which are deconvoluted from the sequence data and quantified in silico after DNA sequencing.

[0064] Advantages of both aspects of the present invention include, but are not limited to, quantitative assays with low cost, high simplicity, high specificity, high sensitivity, high accuracy, high throughput, high scalability and high turnover compared to conventional nucleic acid sequencing technologies. Another aspect of the present invention is that the methods of the present invention enable accurate and massively parallel quantification of multiple nucleic acid targets in multiple samples, including human and animal populations, and including large amounts of unpurified sample material. As noted above, in preferred embodiments, samples such as urine samples are used without prior purification or concentration of nucleic acids, hi other embodiments, the sample may be pre-treated, for example, by lysing cells to expose nucleic acids. One particular advantage of the present invention is that it uses a unique probe design, i.e., a probe triplet, to enable the detection and amplification of target sequences of interest. The probes are designed with specially positioned modified nucleotides that improve annealing and binding efficiency. Improved binding characteristics increase the specificity, sensitivity, and accuracy of the assay. The methods of the present invention are also applicable to the study of genetic variants and find diagnostic and prognostic applications, including, but not limited to, genotyping a sample for one or more sequences and / or polymorphisms, such as SNPs and / or indels, cancer diagnosis, or fetal chromosomal disorders from maternal blood. In a preferred embodiment, for two or more samples or two or more locus / allele combinations, the barcode sequences are used to genotype the samples for one or more sequences and / or polymorphisms, such as SNPs and / or indels.

[0065] In another aspect, the present invention provides a kit of parts comprising a plurality of containers, wherein at least one container comprises a set of one or more first probes and second probes, and at least one container comprises one or more bridge oligos or a plurality of oligonucleotides capable of forming a bridge oligo complex with a barcode loop oligo; wherein the first probe comprises, starting from the 5' end of the molecule, a first bridging oligo-specific sequence, optionally a first sequence barcode, and a first target-specific portion at the 3' end of the first probe; wherein the second probe comprises, starting at the 5' end of the molecule, a second target-specific portion, optionally a second sequence barcode, and a second bridging oligo-specific sequence at the 3' end of the second probe; wherein the bridge oligo or bridge oligo complex comprises sequences complementary to the first and second bridge oligo specific sequences in the first and second probes, respectively, and optionally a third barcode; wherein at least one of the first sequence barcode, the second sequence barcode, or the third barcode is present in the first probe, the second probe, the bridging oligo, or the bridging oligo complex, respectively; wherein the kit further comprises a target-specific probe comprising a sequence corresponding to the target nucleotide sequence, wherein the target-specific probe is capable of annealing to the ligated ligation complex; and wherein, optionally, at least one of the first probe, the second probe, the bridging oligo, or the bridging oligo complex comprises a recognition sequence for an endonuclease; and wherein, optionally, said kit of parts further comprises an oligonucleotide capable of annealing to said recognition sequence so as to obtain a recognition site for said endonuclease.

[0066] 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.

[0067] Preferably, the bridge oligo or one of the bridge oligonucleotides comprises one or more chemically modified nucleotides in the sequence complementary to the sequence of the first probe, the sequence complementary to the sequence of the second probe, or both.

[0068] 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.

[0069] Preferably, the bridging moiety of the first probe or the second probe or both or the bridging oligo or oligonucleotides of the plurality of bridging oligonucleotides comprises a chemically modified base that allows for improved binding to the bridging oligo or bridging oligo complex.

[0070] In a specific embodiment, at least one container containing the first and second sets of probes and at least one container containing a bridge oligo or oligonucleotides that can anneal to each other to form a bridge oligo complex are the same container, in which case the probes may have been pre-annealed to form a ligation complex.

[0071] One particular advantage of the present invention is that it allows for the detection and amplification of target sequences of interest using unique probe designs that are designed with improved binding properties leading to increased assay specificity, sensitivity, and accuracy. The present invention finds application in the fields of molecular biology, evolutionary biology, metagenomics, genotyping, more particularly, but not limited to, cancer diagnosis or fetal chromosomal disorders, including, but not limited to, genotyping a sample for one or more sequences and / or polymorphisms such as SNPs and / or indels.

[0072] In one particularly preferred embodiment, the bridge oligo or bridge oligo complex comprises a unique barcode with information for identifying the sample. In such a case, the first and second probes are generally applicable to all samples (only comprising information for identifying the target). Thus, in one preferred embodiment, a method or kit according to the present invention is provided, in which the bridge oligo or bridge oligo complex comprises a barcode with a unique sequence that allows enumeration of the target sequence for each sample. [Example]

[0073] (method) 1. Probe Complex Formation The probe complexes contain sequences necessary for genome targeting, sample indexing, and construction of an Illumina sequencing library.

[0074] A three-part probe complex is formed (see Figure 2) that contains: (a) a first probe starting at the 5' end of the molecule and having a first bridging oligo-specific sequence and a first target-specific portion at the 3' end of the first probe; (b) a second probe starting at the 5' end of the molecule and having a second target-specific portion, a second sequence barcode, and a second bridging oligo-specific sequence at the 3' end of the second probe; and (c) a bridge oligo having a sequence complementary to the first bridge oligo-specific sequence and the second bridge oligo-specific sequence in the first probe and the second probe, respectively;

[0075] The probe complex is constructed by combining equimolar amounts of all three parts (bridge, right arm, left arm) in an annealing reaction, which is carried out in a thermocycler (annealing program in Table 1).

[0076] [Table 1]

[0077] 2.Target capture The specific genomic region containing the mutation of interest is targeted. The sample can be purified DNA (e.g., from tissue, plasma, urine, or saliva), or it can be unpurified and only requires pretreatment such as boiling and / or centrifugation. The probe complex hybridizes to the target region through complementary base sequence interactions. To initiate target capture, the reaction probe and target DNA are mixed and incubated in a thermal cycler (Target Capture and Gap Fill Program in Table 2).

[0078] [Table 2]

[0079] 3. Gap-filling Reaction After target capture, the probe complexes from the separate targeting reactions are pooled, followed by extension and ligation by adding a combination of Phusion DNA polymerase, nucleotides, and Ampligase DNA ligase and incubating at +45°C for 45 minutes.

[0080] 4. Exonuclease Treatment After gap filling, linear molecules are removed by adding 1 μl of thermolabile exonuclease 1 (NEB, #M0568L) and 1 μl of RecJF exonuclease (NEB, #M0264L) and incubating at +37°C for 30 minutes. The exonucleases are inactivated by incubating at +92°C for 12 minutes.

[0081] 5. Rolling Circle Amplification After extension and ligation, the circular probe molecules are aligned with target-specific probes and subjected to rolling circle amplification (RCA). For the RCA reaction, the target capture reaction is mixed with the boost oligo and briefly denatured. The reaction is then mixed with an RCA reaction mix containing EquipPhi29 (Thermo Scientific) polymerase. The reaction is incubated at +42°C for 30 minutes to 2 hours. After the RCA reaction, the reaction efficiency is analyzed by measuring the concentration of single-stranded DNA (ssDNA) using a Qubit fluorometer.

[0082] 6. Enzyme digestion The RCA reaction generates long concatemeric ssDNA molecules containing multiple copies of the target library. Each complete target library is separated by an EcoRI restriction enzyme recognition sequence. This sequence allows for sequence-specific cleavage of the long concatemers by annealing with specific oligonucleotides containing the EcoRI restriction enzyme recognition sequence, liberating the ready-to-use target library. These libraries are ready for further analysis after a simple purification step. The RCA product is digested with EcoRI at +37°C for 1 hour.

[0083] 7. Library PCR The digested RCA products are extended into a sequencing library in a PCR reaction, and the truncated sequence adapters present in the appropriate probes are extended to flow-cell compatible full-length sequence adapters.

[0084] 8. Library Purification After library PCR, the library molecules are purified by electrophoresis or by extracting them from an agarose gel using size selection beads (such as Macherey Nagel NucleoMag).

[0085] 9. Sequencing The purified MiSeq or iSeq100-compatible libraries are sequenced using state-of-the-art sequencing instruments. Importantly, the libraries can be converted to fit any available sequencing platform through simple oligonucleotide modifications. Sequence data are processed using a combination of Unix command-line tools and the Python and R programming languages. In summary, the rationale for sequence processing is to identify probe sequences within each read, sequence the genomic region between them, and count the number of molecular barcodes associated with each gene target.

[0086] (Test 1) In the first experiment, the probe mix was a collection of four different indexed probe complexes, resulting in four replicate reactions, targeting the 12 gene fusions listed in Figure 5. The target oligonucleotides had unique recognition sequences that allowed for the identification of each target. For the samples, two types of synthetic target oligonucleotides were mixed at equal concentrations for each of the 12 gene fusions. Target capture, extension and ligation reactions, rolling circle amplification, and subsequent EcoRI digestion were performed as described above. An example of the resulting sequencing library is shown in Figure 4. The prepared libraries were sequenced on an iSeq100 instrument, and the target regions within the sequence data were detected by matching the probe sequences within each read, identifying the genomic sequence regions between the probe sequences, and counting the molecular barcodes. The counting data accurately reflected the boosting status of each gene fusion target (Figure 5). In Figure 5, data related to non-boosting are marked with the letter "N" in the graph. Unmarked bars are related to boosting.

[0087] (Detailed description of Figures 1, 2 and 3) Figure 1 shows the workflow of one embodiment of the invention described in the detailed description. In step 1, nucleic acids (DNA or RNA) in a sample (102) are contacted with a set of ligation complexes (104). The ligation complexes anneal on the target nucleic acid (106). In step 2, optionally, the target-bound ligation complexes are captured from the sample material, leaving behind sample impurities (103). In step 3, the annealed ligation complexes are ligated to obtain a ligated ligation complex. In step 4, the ligated complexes from multiple samples were pooled together. In step 5, the ligated ligation complex is dissociated from the target nucleotide sequence, a target-specific probe containing a sequence corresponding to the target nucleotide sequence is added, and the target-specific probe is allowed to anneal to the ligated ligation complex. Target-specific probes anneal specifically to selected rare mutations (114) and optionally contain modifications (including, but not limited to, phosphorothioate modifications) that enhance binding to the target or protect against exonuclease activity. In step 6, the probe sequence is amplified by rolling circle amplification using phi29 polymerase or other strand-displacing polymerases to generate long concatemeric copies of the probe, further boosting the amplification of rare mutations (116). In step 7, the concatemeric probe copies are optionally cleaved into monomeric units using a restriction endonuclease such as EcoRI or a homing nuclease such as I-CeuI, and optionally further amplified using PCR or emulsion PCR (117). In step 8, the amplified DNA is sequenced using next generation DNA sequencing. In step 9, the DNA sequencing results are converted into target numbers using a bioinformatic pipeline.

[0088] 2A illustrates gap filling between a first probe and a second probe according to one embodiment of the present invention, where the bridging oligo contains Gap 1 between bridging sequence 1 (228) and bridging 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. This process can use a mixture of Stoffel fragment, Taq polymerase, or Phusion polymerase and a DNA ligase such as Ampligase. The polymerase adds (a) nucleotides complementary to the universal bridge oligo sequence and (b) nucleotides complementary to the target sequence, thereby filling two gaps, Gap 1 and Gap 2, between the first and second probes. The subsequent action of DNA ligase results in ligation of the bridge oligo and the first and second probes complementary to the target sequence to form a circular complex.

[0089] Figure 2B shows the principle structure of a probe quintet with multiple probe entities according to one embodiment of the present invention, where the multiple probe entities include a first probe, a second probe, and a bridge consisting of three oligos. Here, the probe complex contains gaps between the first probe and the second bridge (228 and 236), between the second bridge and the second probe (240 and 222), between the first and third bridge oligos (238 and 242), and between the first and second probe (208 and 216). These gaps are filled by introducing a polymerase and one or more nucleotides. This process can use a mixture of Stoffel fragment, Taq polymerase, or Phusion polymerase with a DNA ligase such as Ampligase. The polymerase fills these gaps, and the subsequent action of the DNA ligase ligates the probe and bridge oligo to form a circular complex.

[0090] Bases 15-25 of the first probe comprise bridging sequence 1 (228), which optionally contains chemically modified bases for efficient cross-linking oligo binding, referred to as bridging sequence 1. The first probe also optionally contains 10-20 bases from the 5' end that contain a universal sequence used for library indexing (204). The first probe further optionally comprises 10-20 bases from the 5' end that include a segment of random nucleotides that form a molecule-specific barcode or a sample-specific barcode, referred to as barcode 1 (206). The first probe further comprises 15-30 bases from the 5' end that bind to the genetic target (208). Some or all of the 228 nucleotides may contain chemical modifications that increase the affinity of the probe for the target or for the crosslink (226). The last base of the first probe optionally contains a phosphate moiety for enzymatic ligation or a modification that allows chemical ligation to the 5' end of an adjacent probe, called modification 1 (210).

[0091] The first base of the second probe optionally contains a phosphate moiety for enzymatic ligation, or a modification that allows chemical ligation to the 5' end of an adjacent probe, referred to as modification 2 (214). The second probe includes a portion of the second probe that binds to the gene target (216) within 15 to 30 bases from the 5' end. The next 10-20 bases from the 5' end of the second probe optionally contain a segment of random nucleotides that forms a molecule-specific or sample-specific barcode called barcode 2 (218). The next 10-20 bases from the 5' end of the second probe may also contain a universal sequence (220). The last 15-25 bases of the second probe, called bridging sequence 8 (222), are reverse complementary to bridging sequence 7 of the third bridging oligo (224). Some or all of nucleotides 208, 216, 222, or 228 may contain chemical modifications that increase the affinity of the probe for the target or bridging oligo.

[0092] The first 15-25 bases from the 5' end of the first bridge oligo, called bridge sequence 3 (226), are reverse complementary to bridge sequence 1 of the second probe (228) and optionally contain chemically modified nucleotides to enhance binding. The last 15-25 bases of the first bridge oligo, referred to as bridge sequence 2 (238), are reverse complementary to bridge sequence 4 of the second bridge oligo (236) and optionally contain chemically modified nucleotides to enhance binding. The 5' end of the first bridge oligo optionally contains a capture moiety (230) that is used to capture the ligation complex.

[0093] The first 15-25 bases from the 5' end of the second bridge oligo, referred to as bridge sequence 5 (240), are reverse complementary to bridge sequence 6 (242) of the third bridge oligo and optionally contain chemically modified nucleotides to enhance binding. The last 15-25 bases of the second bridge oligo, referred to as bridge sequence 4 (236), are reverse complementary to the sequence of bridge sequence 2 of the first bridge oligo (238) and optionally contain chemically modified nucleotides to enhance binding.

[0094] The first 15-25 bases from the 5' end of the third bridge oligo, referred to as bridge sequence 6 (242), are reverse complementary to the sequence of bridge sequence 5 of the second bridge oligo (240) and optionally contain chemically modified nucleotides to enhance binding. The last 15-25 bases of the first bridging oligo, called bridging sequence 7 (224), are reverse complementary to the sequence of bridging sequence 8 of the second probe (222) and optionally contain chemically modified nucleotides to enhance binding. The 3' end of the third bridge oligo optionally contains a phosphate (or other cleavable) moiety (234) to prevent extension during gap filling.

[0095] Figure 2C shows an embodiment involving the use of a barcode loop oligo, where the multiple probe entities include a first probe (202), a second probe (201), a bridge oligo (200), and a barcode loop oligo (217). Here, the probe complex contains gaps between the first probe and the barcode loop oligo (210 and 213), between the second probe and the barcode loop oligo (207 and 215), and between the first and second probe (203 and 204). These gaps are filled by introducing a polymerase and one or more nucleotides, a process that can use a mixture of Stoffel fragment, Taq polymerase, or Phusion polymerase and a DNA ligase such as Ampligase. Polymerase fills in these gaps, and the subsequent action of DNA ligase ligates the probe, bridge, and barcode loop oligos into a circular complex.

[0096] The first probe comprises a 15-25 base cross-linking sequence (210) that optionally contains chemically modified bases for efficient cross-linking oligonucleotide binding, and the first probe further comprises 15-30 bases from the 5' end that bind to the gene target (203). Some or all of the 210 nucleotides may contain chemical modifications that increase the affinity of the probe for the target or crosslink (209). The last base of the first probe optionally contains a phosphate moiety for enzymatic ligation or a modification that allows chemical ligation to the 5' end of an adjacent probe (205).

[0097] The first base of the second probe optionally contains a phosphate moiety for enzymatic ligation or a modification that allows chemical ligation to the 5' end of an adjacent probe (206). The 15-30 bases from the 5' end of the second probe comprise the portion of the second probe that binds to the genetic target (204). The last 15-25 bases of the second probe (207) are reverse complementary to the bridge oligo (208). Some or all of nucleotides 203, 204, 207, or 210 may contain chemical modifications that increase the affinity of the probe for the target or bridge oligo.

[0098] The first 15-25 bases from the 5' end of the bridge oligo (209) are reverse complementary to the bridge oligo-specific sequence of the first probe (210) and optionally contain chemically modified nucleotides to enhance binding. The last 15-25 bases of the bridge oligo (208) are reverse complementary to the sequence of the second probe (207) and optionally contain chemically modified nucleotides to enhance binding. The 5' end of the bridge oligo optionally includes a capture moiety (211) that is used to capture the ligation complex. Additionally, the bridge oligo contains sequences 214 and 216 that are complementary to sequences 213 and 215 of the barcode loop oligo. The 3' end of the bridge oligo optionally contains a phosphate (or other cleavable) moiety (212) to prevent extension during gap filling.

[0099] The first 15-25 bases from the 5' end of the barcode loop oligo (215) are reverse complementary to the bridging oligo sequence 216. The barcode loop oligo includes a loop region that contains the barcode (218). The last 15-25 bases of the barcode loop oligo (213) are reverse complementary to the bridging oligo sequence 214.

[0100] 2D shows a quattroplet principle structure of a probe quadruplet according to one embodiment of the present invention. The multiple probe entities include a first probe (202), a second probe (201), a bridge oligo (200), and a barcode loop oligo (217). Here, the probe complex includes gaps between the first probe and the barcode loop oligo (210 and 213), between the second probe and the barcode loop oligo (207 and 215), and between the first and second probes (203 and 204). These gaps are filled by introducing a polymerase and one or more nucleotides. This process can use a mixture of Stoffel fragment, Taq polymerase, or Phusion polymerase and a DNA ligase such as Ampligase. The polymerase fills these gaps, and the subsequent action of the DNA ligase ligates the probe, bridge, and barcode loop oligos into a circular complex.

[0101] The first probe comprises a cross-linking sequence (210) that optionally contains chemically modified bases for efficient cross-linking oligo binding. The first probe further comprises binding sites for an amplification primer (221) and a barcode sequence (222), and a sequence (203) that binds to the genetic target 15-30 bases from the 5' end. Some or all of the 210 nucleotides may contain chemical modifications that increase the affinity of the probe for the target or crosslink (209). The last base of the first probe optionally contains a phosphate moiety for enzymatic ligation or a modification that allows chemical ligation to the 5' end of an adjacent probe (205).

[0102] The first base of the second probe optionally contains a phosphate moiety for enzymatic ligation or a modification that allows chemical ligation to the 5' end of an adjacent probe (206). The 15-30 bases from the 5' end of the second probe contain the portion of the second probe that binds to the genetic target (204). The second probe further comprises a binding site for an amplification primer (223), a sequencing adapter sequence (224), a recognition site for a restriction endonuclease such as EcoRI (225) and another sequencing adapter sequence (207). The last 15-25 bases of the second probe (207) are reverse complementary to the bridge oligo (208). Some or all of nucleotides 203, 204, 207, or 210 may contain chemical modifications that increase the affinity of the probe for the target or bridge oligo.

[0103] The first 15-25 bases from the 5' end of the bridge oligo (209) are reverse complementary to the bridge oligo-specific sequence of the first probe (210) and optionally contain chemically modified nucleotides to enhance binding. The last 15-25 bases of the bridge oligo (208) are reverse complementary to the sequence of the second probe (207) and optionally contain chemically modified nucleotides to enhance binding. The portion of the bridge oligo (220) that is not reverse complementary to either end of the barcode loop oligo optionally contains a recognition site for a restriction endonuclease. The 5' end of the bridge oligo optionally includes a capture moiety (211) that is used to capture the ligation complex. Additionally, the bridge oligo includes sequences 214 and 216 that are complementary to sequences 213 and 215 of the barcode loop oligo. The 3' end of the bridge oligo optionally includes a phosphate (or other cleavable) moiety (212) to prevent extension during gap filling.

[0104] The first 15-25 bases from the 5' end of the barcode loop oligo (215) are reverse complementary to the bridging oligo sequence 216. The barcode loop oligo includes a loop region that contains the barcode (218). The last 15-25 bases of the barcode loop oligo (213) are reverse complementary to the bridging oligo sequence 214.

[0105] FIG. 3 shows how the workflow of one embodiment of the described invention enhances the detection of rare mutations in samples that also contain bulk genomes that do not contain mutations. In step 1, the target-bound ligation complex undergoes gap filling and ligation. In step 2, the ligated ligation complex is dissociated from the target nucleotide sequence, a target-specific probe containing a sequence corresponding to the target nucleotide sequence is added, and the target-specific probe is allowed to anneal to the ligated ligation complex. In steps 3 and 4, the probe sequence is amplified by rolling circle amplification using phi29 polymerase or other strand-displacing polymerases, resulting in long concatemeric copies of the probe. Amplification initiated from the target-specific probe occurs only if the target-specific probe matches the target sequence. If there is a mismatch, amplification only occurs from the bridging oligo.

Claims

1. 1. A method for detecting one or more target nucleotide sequences in a sample, the method comprising: (i) providing, for each target nucleotide sequence in the sample, a first probe, a second probe, and a bridge oligo or oligonucleotides capable of annealing to each other to form a bridge oligo complex; the first probe comprises, starting at the 5' end of the molecule, a first bridging oligo-specific sequence, a first sequence barcode, and a first target-specific portion at the 3' end of the first probe; the second probe comprises, starting at the 5' end of the molecule, a second target-specific portion, a second sequence barcode, and a second bridging oligo-specific sequence at the 3' end of the second probe; the bridge oligo or bridge oligo complex comprises a sequence complementary to the first bridge oligo-specific sequence and the second bridge oligo-specific sequence in the first probe and the second probe, respectively, and a third barcode; At least one of the first sequence barcode, the second sequence barcode, or the third barcode is present in the first probe, the second probe, the bridge oligo, or the bridge oligo complex, respectively; and At least one of the first probe, the second probe, the bridging oligo, or the bridging oligo complex contains a recognition sequence for an endonuclease; (ii) for each of one or more target nucleotide sequences, contacting the first probe and the second probe with the bridge oligo or oligonucleotides capable of annealing to each other to form a bridge oligo complex, and annealing the first probe and the second probe with the bridge oligo or oligonucleotides to form a plurality of ligation complexes; (iii) contacting nucleic acids present in the sample to be tested for a target nucleotide sequence with the ligation complex; (iv) hybridizing the first target-specific portion and the second target-specific portion of each of the first probe and the second probe to adjacent sections of the target nucleotide sequence that are between 15 and 500 base pairs apart, thereby forming a hybridization complex; (v) ligating the probes in the hybridization complex to provide a ligated ligation complex; (vi) dissociating the ligated ligation complex from the target nucleotide sequence; (vii) adding a target-specific probe comprising a sequence corresponding to the target nucleotide sequence, wherein the target-specific probe is capable of annealing to the ligated ligation complex, and allowing the target-specific probe to anneal to the ligated ligation complex, thereby forming an amplification template; (viii) amplifying nucleic acid from said amplification template using rolling circle amplification with a strand-displacing polymerase, thereby obtaining a single-stranded concatemeric sequence; (ix) performing a step of obtaining a nucleic acid fragment, provided that the recognition sequence specified in step (i) is present, by: (a) cleaving the single-stranded concatemeric sequence obtained in step (viii); or (b) subjecting the amplified single-stranded concatemer sequence or sequences obtained in step (viii) to annealing with a specific oligonucleotide containing a recognition sequence for an endonuclease, wherein the oligonucleotide anneals to the recognition sequence identified in step (i) to obtain a recognition site for the endonuclease, and cleaving the annealed complex with the endonuclease; (x) subjecting the concatemer sequences obtained in step (viii) or the nucleic acid fragments obtained in step (ix) to a sequencing technique to determine the barcode sequence; and (xi) identifying the presence and / or number of the target nucleotide sequence in the sample by determining the sequence of 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 sequence barcode and / or the second sequence barcode, and / or at least a portion of a third barcode; one or more oligonucleotides of the bridge oligo or plurality of bridge oligonucleotides comprise a plurality of universal base analogs or random nucleotides in a region that is not complementary to the first probe or the second probe, which allows for the incorporation of random sequences suitable for use as molecular barcodes for target enumeration, and prior to step (v), a gap-filling step is carried out using a polymerase and nucleotides to generate such random sequences; The method, wherein the plurality of universal base analogs is a plurality of 5-nitroindoles.

2. 10. The method of claim 1 for high-throughput detection of one or more target nucleotide sequences in multiple samples, wherein multiple samples are provided and step (ii) is performed for each of the samples in separate tubes.

3. 3. The method of claim 2, wherein the plurality of samples are pooled prior to step (viii).

4. at least one of the first probe, the second probe, the bridging oligo, or the oligonucleotides among the plurality of bridging oligonucleotides comprises a first capture moiety; and between steps (iv) and (v), contacting the hybridization complex with a solid support containing a second capture moiety; allowing the first capture moiety and the second capture moiety to interact such that the hybridization complex binds to the solid support; and 4. The method according to any one of claims 1 to 3, further comprising an intermediate step (iv)(a) of separating hybridization complexes bound to the solid support from components of the sample that are not bound to the solid support.

5. 5. The method of claim 4, which does not include a step of concentrating the nucleic acid prior to step (iv)(a).

6. 5. The method of claim 4, wherein the first capture moiety is a biotin moiety and the second capture moiety is a streptavidin moiety or an avidin moiety.

7. The method of claim 5 , wherein the first capture moiety is a biotin moiety and the second capture moiety is a streptavidin moiety or an avidin moiety.

8. 10. The method of claim 1, wherein the sample or samples comprise a blood sample, a saliva sample, a urine sample, or a fecal sample.

9. It involves the use of multiple oligonucleotides that can anneal to each other to form a bridged oligo complex, the plurality of oligonucleotides comprises a barcode loop oligo; the barcode loop oligo comprises, starting at the 5' end of the molecule, a third bridge oligo-specific sequence, a barcoded loop sequence that may comprise the third barcode, and a fourth bridge oligo-specific sequence; and 2. The method of claim 1, wherein one or more other bridge oligos comprise sequences complementary to the third bridge oligo-specific sequence and the fourth bridge oligo-specific sequence in the barcode loop oligo.

10. The bridged oligo or bridged oligo complex is (i) 1 to 5 3' overhanging bases, and / or (ii) a 3' phosphate, and / or (iii) comprising one or more phosphorothioate modifications within three positions from the 3' end.

11. 2. The method of claim 1, 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.

12. 2. The method of claim 1, wherein the bridging portion of the first probe or the second probe, or both, or the bridge oligo or one of the plurality of bridge oligonucleotides, comprises a chemically modified base that improves binding to the bridge oligo or bridge oligo complex.

13. 2. The method of claim 1, wherein the first target-specific portion, the second target-specific portion, the first bridging oligo-specific sequence, and / or the second bridging oligo-specific sequence independently comprise one or more chemically modified nucleotides.

14. 2. The method of claim 1, wherein the bridge oligo or an oligonucleotide of the plurality of bridge oligonucleotides comprises one or more chemically modified nucleotides.

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

16. PCR amplification is carried out immediately prior to step (x) using primers that bind to the universal portions of the first and second probes; 2. The method of claim 1, wherein the primer comprises an adapter for subsequent sequencing in step (x).

17. the sequencing of step (x) is carried out using nanopore sequencing; The method of claim 1, wherein the concatemeric sequences obtained in step (viii) are fragmented using a translocation complex.

18. 10. The method of claim 1, wherein gene target enumeration is enabled by counting the number of molecular barcodes per target and per sample.

19. 10. The method of claim 1, wherein for two or more samples, or for two or more locus / allele combinations, barcode sequences are used to genotype the samples for one or more sequences and / or polymorphisms of SNPs and / or indels.

20. 1. A kit of parts comprising a plurality of containers for use in a method for detecting one or more target nucleotide sequences in a sample, comprising: At least one container contains one or more sets of first probes and second probes, and at least one container contains one or more bridge oligos or a plurality of oligonucleotides capable of forming a bridge oligo complex; the first probe begins at the 5' end of the molecule and comprises a first bridging oligo-specific sequence, a first sequence barcode, and a first target-specific portion at the 3' end of the first probe; the second probe begins at the 5' end of the molecule and comprises a second target-specific portion, a second sequence barcode, and a second bridging oligo-specific sequence at the 3' end of the second probe; the bridge oligo or bridge oligo complex comprises a sequence complementary to the first and second bridge oligo specific sequences in the first and second probes, respectively, and a third barcode; at least one of the first sequence barcode, the second sequence barcode, or the third barcode is present in the first probe, the second probe, a bridge oligo, or a bridge oligo complex, respectively; The kit further comprises a target-specific probe comprising a sequence corresponding to the target nucleotide sequence, the target-specific probe being capable of annealing to the ligated ligation complex; and At least one of the first probe, the second probe, the bridge oligo, or the bridge oligo complex comprises a recognition sequence for an endonuclease; and The kit of parts further comprises an oligonucleotide capable of annealing to said recognition sequence to provide a recognition site for said endonuclease.

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