Method for accurate parallel detection and quantification of nucleic acids - Patent Application 20070122997

Abstract

To provide a next generation DNA sequencing method for accurate and massively parallel quantification of one or more nucleic acid targets, for example, large volumes of unpurified sample materials, the method and a kit comprising probes for detecting and quantifying genetic targets in complex samples.SOLUTION: The invention includes two target-specific nucleic acid probes per genetic target, a barcode loop oligo and a bridge oligo or bridge oligo complex.SELECTED DRAWING: Figure 2A

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 for use in detecting gene targets and mutations. The present invention uses one or more target-specific nucleic acid probes, a barcode loop oligo, and one or more bridge oligos for each gene target. [Background technology]

[0002] The detection of genetic variation in plants and animals has become less tedious due to the advancement of research technology.However, the detection and accurate quantification of genetic variation, such as mutation, especially in samples with weak signals, is still currently laborious, laborious, and expensive, despite the reduction in sequencing costs.Various issues can be more precisely posed, such as the specificity of detecting genetic signals against consensus background, the sensitivity of detecting weak genetic signals, the accuracy of accurately quantifying detected signals, the throughput number of targeted genetic targets per assay, the cost per assay, the evaluation of the scale of assay cost when multiple samples are assayed in parallel, and the turnover time of determining the length of time from sample collection to results.

[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 considering the substantial hands-on time (labor costs) to set up the reaction, the need for standard curves, and replicates for each quantification target, the actual cost is in fact much higher. Because a separate quantification experiment is required for each gene target, the amount of hands-on time scales rapidly with increasing 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 the use of separate barcode loop oligonucleotides for sample identification. The use of barcode loop oligonucleotides allows for simultaneous target detection and sample indexing, permitting efficient sample pooling, resulting in more cost-effective and flexible quantitative assays, and provides an alternative method for introducing unique molecular identifier (UMI) sequences. The resulting protocol eliminates the requirement for separate sample indexing, thereby enabling shorter read lengths, offering advantages for both short-read and nanopore sequencing applications. [Means for solving the problem]

[0013] In a first principal aspect, the present invention provides a method for high-throughput detection of one or more target nucleotide sequences in a plurality of samples, said method comprising: (i) providing, to each target nucleotide sequence in each of said samples, a first probe, a second probe, a barcode loop oligo, and a bridge oligo, or a plurality of bridge oligonucleotides capable of annealing to the barcode loop oligo to form a bridge oligo complex; the first probe comprises a first bridging oligo-specific sequence at the 5' end of the first probe and a first target-specific portion at the 3' end of the first probe; the second probe comprises a second target-specific portion at the 5' end of the second probe and a second bridging oligo-specific sequence at the 3' end of the second probe; the barcode loop oligo, starting at the 5' end of the molecule, comprises a third bridge oligo-specific sequence, a barcode loop sequence, and a fourth bridge oligo-specific sequence; the bridge oligo or the plurality of bridge oligonucleotides comprise sequences 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 sequences complementary to the third bridge oligo-specific sequence and the fourth bridge oligo-specific sequence in the barcode loop oligo; and optionally, at least one of the oligonucleotides of the first probe, the second probe, the barcode loop oligo, the bridge oligo, or the plurality of bridge oligonucleotides comprises a recognition sequence for an endonuclease; (ii) for each of said one or more target nucleotide sequences, contacting said first probe and said second probe with said barcode loop oligo and said bridge oligo or oligonucleotides for each of said samples, preferably in separate tubes, and allowing self-annealing into a ligation complex; (iii) contacting nucleic acids present in each of the samples to be tested for the target nucleotide sequence with the ligation complex; (iv) hybridizing the first target-specific portion and the second target-specific portion of the first probe and the second probe, respectively, to essentially adjacent portions on the target sequence, thereby forming a plurality of hybridization complexes; (v) optionally pooling said hybridization complexes from said plurality of samples; (vi) ligating the probes in the hybridization complex to form a ligated ligation complex; (vii) amplifying nucleic acids from one or more ligated ligation complexes using rolling circle amplification with a displacing polymerase, thereby obtaining single-stranded concatemeric sequences; (viii) optionally, if the designated recognition sequence is present in step (i), performing a step of obtaining a nucleic acid fragment by the following method; (a) cleaving the single-stranded concatemeric sequence obtained in step (vii); or (b) subjecting the single-stranded concatemeric sequence obtained in step (vii) to annealing with a specific oligonucleotide containing a recognition sequence for an endonuclease, wherein said oligonucleotide anneals with the recognition sequence identified in step (i), thereby providing a recognition site for the endonuclease, and cleaving the annealed complex with said endonuclease; (ix) subjecting the concatemer sequence obtained in step (vii) or the nucleic acid fragment obtained in step (viii) to a high-throughput sequencing technique to determine the barcode sequence; and (x) identifying the presence and / or number of target nucleotide sequences 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 a barcode sequence corresponding to a barcode in a barcode loop oligo; The method wherein steps (v) and (vi) may be performed in any order. [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] Figure 3 shows the RCA products from the workflow before (lane 2) and after (lane 1) digestion with restriction endonucleases. [Figure 4] Figure 4 shows the linear response of the experimental workflow to a logarithmically decreasing number of gene targets in four replicate reactions, 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 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 a locus at which sequence differences occur. 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 fragment of DNA or RNA of variable length (usually 50-1000 bases long, preferably 50-200 bases long) that can be used in a DNA or RNA sample to detect the presence of a nucleotide sequence (DNA or RNA target) that is complementary to the sequence in the probe. The sections of the oligonucleotide probes that are complementary to the target sequence are 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, such that the probes each contain a section at their end that is complementary to a portion of the target sequence. Additionally, the present disclosure describes bridge oligos or bridge oligo complexes that are used to link a first probe and a second probe. Additionally, the present disclosure describes barcode loop oligos that include a loop section flanked by two sections that can hybridize to one or more bridge oligos, where the loop section does not hybridize to one or more bridge oligos and includes a barcode.

[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 cuts or nicks 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 relates to a method for high throughput detection of one or more target nucleotide sequences in a plurality of samples, said method comprising: (i) providing, to each target nucleotide sequence in each of said samples, a first probe, a second probe, a barcode loop oligo, and a bridge oligo, or a plurality of bridge oligonucleotides capable of annealing to the barcode loop oligo to form a bridge oligo complex; the first probe comprises a first bridging oligo-specific sequence at the 5' end of the first probe and a first target-specific portion at the 3' end of the first probe; the second probe comprises a second target-specific portion at the 5' end of the second probe and a second bridging oligo-specific sequence at the 3' end of the second probe; the barcode loop oligo, starting at the 5' end of the molecule, comprises a third bridge oligo-specific sequence, a barcode loop sequence, and a fourth bridge oligo-specific sequence; the bridge oligo or the plurality of bridge oligonucleotides comprise sequences 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 sequences complementary to the third bridge oligo-specific sequence and the fourth bridge oligo-specific sequence in the barcode loop oligo; and optionally, at least one of the oligonucleotides of the first probe, the second probe, the barcode loop oligo, the bridge oligo, or the plurality of bridge oligonucleotides comprises a recognition sequence for an endonuclease; (ii) for each of said one or more target nucleotide sequences, contacting said first probe and said second probe with said barcode loop oligo and said bridge oligo or oligonucleotides for each of said samples, preferably in separate tubes, and allowing them to self-anneal into a ligation complex; (iii) contacting nucleic acids present in each of the samples to be tested for the target nucleotide sequence with the ligation complex; (iv) hybridizing the first target-specific portion and the second target-specific portion of the first probe and the second probe, respectively, to essentially adjacent portions on the target sequence, thereby forming a hybridization complex (or optionally, a single hybridization complex); (v) optionally pooling said hybridization complexes from said plurality of samples; (vi) ligating the probes in the hybridization complex to form a ligated ligation complex; (vii) amplifying nucleic acids from one or more ligated ligation complexes using rolling circle amplification with a displacing polymerase, thereby obtaining single-stranded concatemeric sequences; (viii) optionally, if the designated recognition sequence is present in step (i), performing a step of obtaining a nucleic acid fragment by the following method; (a) cleaving the single-stranded concatemeric sequence obtained in step (vii); or (b) subjecting the single-stranded concatemeric sequence obtained in step (vii) to annealing with a specific oligonucleotide containing a recognition sequence for an endonuclease, wherein said oligonucleotide anneals with the recognition sequence identified in step (i), thereby providing a recognition site for the endonuclease, and cleaving the annealed complex with said endonuclease; (ix) subjecting the concatemer sequence obtained in step (vii) or the nucleic acid fragment obtained in step (viii) to a high-throughput sequencing technique to determine the barcode sequence; and (x) identifying the presence and / or number of target nucleotide sequences 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 a barcode sequence corresponding to a barcode in a barcode loop oligo; The method wherein steps (v) and (vi) may be performed in any order.

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

[0030] The methods of the present invention utilize four or more nucleic acid molecules, of which two target-specific nucleic acid probes (first probe and second probe) are specific to a genetic target, and two or more other nucleic acid probes or complexes are typically universal (bridge oligo or bridge oligo complex and barcode loop oligo). The first probe, second probe and barcode loop oligo hybridize to one or more bridge probes 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 with a DNA ligase to form a ligated ligation complex. In the present invention, a plurality of such ligated ligation complexes will be formed during sample analysis in the plurality of samples being analyzed.

[0031] In one embodiment, "multiple samples" may refer to two or more samples obtained from the human or animal body, including, but not limited to, biopsies, saliva and other secretions, breath extracts, tissue, plasma (liquid biopsy), two or more samples obtained from the environment, including water, wastewater, soil, plants, samples containing viruses or bacteria, etc. In 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.

[0032] 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.

[0033] As used herein, the term probe set includes a first probe, a second probe, a barcode loop oligo, and one or more bridge oligos.

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

[0035] 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.

[0036] The bridge oligo or bridge oligo complexes each comprise a sequence complementary to the first and second bridge oligo-specific sequences of the first and second probes, respectively, and optionally a sequence complementary to the universal sequence and the third and fourth bridge oligo-specific sequences in the barcode loop oligo.

[0037] The barcode loop oligo starts at the 5' end of the molecule and comprises a third bridge oligo-specific sequence, a barcode loop sequence, and a fourth bridge oligo-specific sequence. The barcode can be used to uniquely define the complex within every ligation complex in every sample tested. The barcode loop oligo may have any suitable length, for example, between 30 and 100 bp in length, e.g., between 40 and 60 bp in length.

[0038] Optionally, at least one of the first probe, the second probe, the barcode loop oligo, or the one or more bridge oligos comprises a recognition sequence for an endonuclease that allows 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.

[0039] In one embodiment, the barcode loop oligo comprises one or more recognition sequences for an endonuclease, such as a nicking endonuclease. In a further embodiment, the barcode loop oligo comprises two recognition sequences that can anneal to each other to yield a double-stranded endonuclease recognition site. In a further embodiment, the method does not include the above-identified step (viii), but instead includes, between steps (vii) and (ix), the step of annealing two recognition sequences within the barcode loop oligo and cleaving the resulting double-stranded endonuclease recognition site with an endonuclease, such as a nicking endonuclease, that has specificity for said recognition site.

[0040] Optionally, at least one of the first probe, second probe, barcode loop oligo, or one or more bridge 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, and 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, the second bridging oligo-specific sequence, the third bridging oligo-specific sequence and / or the fourth bridging oligo-specific sequence optionally 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, the second probe, the barcode loop oligo, or all three of these comprises a chemically modified base to improve binding to one or more bridging oligos. In another embodiment, the first target-specific portion, the second target-specific portion, the first bridging oligospecific sequence, the second bridging oligospecific sequence, the third bridging oligospecific sequence, and / or the fourth bridging oligospecific sequence independently contain one or more chemically modified nucleotides. In certain embodiments, the chemical modifications allow for chemical ligation of adjacent probes.

[0042] In some embodiments, the probes bind to perfectly adjacent loci, i.e., adjacent sections of the target nucleotide sequence, or up to 500 base pairs apart, e.g., up to 200 base pairs apart, up to 50 base pairs apart, up to 40 base pairs apart, up to 30 base pairs apart, up to 20 base pairs apart, up to 10 base pairs apart, up to 5 base pairs apart, etc.

[0043] In some embodiments, the first probe, the second probe, the barcode loop oligo, or one or more bridge oligos may include, but are not limited to, adapter sequences for DNA sequencing platforms such as Illumina, which allow the resulting sequencing library to bind to the detection portion of a sequencing device such as an Illumina flow cell.

[0044] Additionally, in some embodiments, the oligonucleotide of the bridge oligonucleotide or bridge oligonucleotides 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] Prior to contacting the probe with a sample containing the target sequence, the first probe, second probe and barcode loop oligo 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 anneal into a ligation complex (step (ii)). The bridge oligo and barcode loop may be pre-annealed before annealing with the first and second probes, or all annealing steps may be performed at once. In embodiments where the bridge is not one oligo but multiple oligonucleotides, such as two oligonucleotides that can anneal to the barcode loop oligo to form a bridge oligo complex (shown herein as 2B): The multiple oligonucleotides may be pre-annealed with the barcode loop oligo before annealing with the first and second probes, or all annealing steps may be performed at once, or the barcode loop oligo may be annealed to the pre-annealed probe complex during target capture.

[0046] 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.

[0047] 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 may be directly adjacent or may be separated by a gap of up to 500 base pairs.

[0048] 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.

[0049] Subsequently, in some embodiments, when at least one of the first probe, the second probe, the barcode loop oligo, or one or more bridging oligos comprises a first capture moiety, the hybridization complex(es) is contacted with a solid support comprising a second capture moiety, and the first and second capture moieties are allowed to interact such that the hybridization complex binds 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.

[0050] 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).

[0051] Ligation of the probes in the formed hybridized complex is then carried out enzymatically or chemically to provide a ligated ligation complex (step (vi)). Optionally, as part of step (vi), gaps, if any, between the first probe and the second probe, between the first probe and the barcode loop oligo, and / or between the second probe and the barcode loop oligo can be filled by introducing a polymerase and one or more nucleotides. The polymerase adds a nucleotide (a) complementary to the bridge oligo sequence(s), thereby filling the gap between the first probe, the second probe and the barcode loop oligo, resulting in ligation of the probes and incorporation of the barcode loop oligo 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 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, comprises multiple universal base analogs in the region not complementary to the first probe, the second probe, or the barcode loop oligo to allow incorporation of random sequences suitable for use as molecular barcodes for target enumeration. In such embodiments, as part of step (vi), 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.

[0052] The ligated ligation complexes are then optionally pooled from one or more target samples (step (vi)). Steps (v) and (vi) may be performed in the order specified or in reverse order.

[0053] The nucleic acid from one or more ligated ligation complexes is then amplified, thereby obtaining a single-stranded concatemeric sequence (step (vii)). Amplification is carried out using rolling circle amplification using a strand-displacing polymerase such as phi29 polymerase (UniProtKB-P03680; DPOL_BPPH2) or Bst polymerase (P52026; DPO1_GEOSE).

[0054] In one embodiment, steps (a) and (b) are performed after step (Vi) and before step (Vii), wherein step (a) comprises dissociating the ligated ligation complex from the target nucleotide sequence, and step (b) comprises 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, allowing the target-specific probe to anneal to the ligated ligation complex, thereby forming an amplification template. In such embodiments, the amplification template is amplified in step (vii) by rolling circle amplification using a strand-displacing polymerase.

[0055] Optionally, if the recognition sequence identified in step (i) is present, step (viii) is carried out to obtain the nucleic acid fragment by: (a) cleaving the single-stranded concatemeric sequence obtained in step (vii); or (b) annealing the single-stranded concatemeric sequence obtained in step (vii) with a specific oligonucleotide containing a recognition sequence for an endonuclease, wherein the oligonucleotide anneals to the recognition sequence identified in step (i) so as to obtain a recognition site for the endonuclease, and cleaving the annealed complex with the endonuclease.

[0056] Subsequently, in step (ix), the concatemer sequences obtained in step (vii), or, if step (viii) is performed, the nucleic acid fragments obtained in step (viii), are subjected to high-throughput sequencing techniques to determine the barcode sequences.

[0057] 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 method 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 interrupted by adding excess soluble biotin. If the streptavidin is bound to magnetic particles, it can be removed using a magnet.

[0058] Further, optionally, PCR amplification is carried out immediately prior to step (ix) 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 (ix).

[0059] In another embodiment, the sequencing in step (ix) is performed using nanopore sequencing, and optionally the concatemeric sequences obtained in step (vii) are fragmented using a translocation complex. Techniques suitable for nanopore sequencing have been reviewed in Wang et al., 2021 Nat Biotechnol 39(11):1348.

[0060] 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 barcodes using high-throughput sequencing technology (steps (ix) and (x)), 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.

[0061] 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 allow for pooling of multiple samples by indexing the samples early in the workflow, improving the cost and speed of the assay. Another aspect of the present invention is that the methods of the present invention allow for 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 allows for the detection and amplification of target sequences of interest using a unique probe design, i.e., a probe triplet. The probe is designed with specially positioned modified nucleotides that improve annealing and binding efficiency. The 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, etc. In a preferred embodiment, for two or more samples or two or more locus / allele combinations, barcode sequences are used to genotype the samples for one or more sequences and / or polymorphisms, such as SNPs and / or indels.

[0062] In another aspect, the present invention provides a kit of parts comprising a plurality of containers, wherein at least one container comprises one or more sets of first probes and second probes, at least one container comprises barcode loop oligos, and at least one container comprises one or more bridge oligos or a plurality of bridge oligonucleotides capable of forming a bridge oligo complex with the barcode loop oligos; the first probe comprises a first bridging oligo-specific sequence at the 5' end of the first probe and a first target-specific portion at the 3' end of the first probe; the second probe comprises a second target-specific sequence at the 5' end of the second probe and a second bridging oligo target-specific portion at the 3' end of the second probe; the barcode loop oligo, starting from the 5' end of the molecule, comprises a third bridge oligo-specific sequence, a barcode loop sequence, and a fourth bridge oligo-specific sequence; the bridge oligo or bridge oligonucleotides comprise sequences complementary to the first and second bridge oligo-specific sequences in the first and second probes, respectively, and sequences complementary to the third and fourth bridge oligo-specific sequences in the barcode loop oligo; Optionally, at least one of the oligonucleotides of the first probe, the second probe, the barcode loop oligo, the bridge oligo, or the plurality of bridge oligonucleotides comprises a recognition sequence for an endonuclease; and Optionally, the kit of parts further comprises an oligonucleotide capable of annealing to said recognition sequence so as to provide a recognition site for said endonuclease. Preferably, the 3' end of the first probe or the 5' end of the second probe, or both, are modified to allow chemical ligation of the first probe to the second probe.

[0063] Preferably, the 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.

[0064] 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 and second probes.

[0065] Preferably, the bridging portion of the first probe or the second probe or both, or the oligonucleotide of the barcode loop oligo, the bridge oligo, or the plurality of bridge oligonucleotides, comprises a chemically modified base to improve binding to the bridge oligo or bridge oligo complex.

[0066] In one particular embodiment, at least one container containing the first and second sets of probes, at least one container containing the barcode loop oligo, and at least one container containing the bridge oligo or oligonucleotides that can anneal to each other to form a bridge oligo complex are one and the same container. In such cases, the four or more probes may be pre-annealed to form a ligated complex.

[0067] 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.

[0068] In one particularly preferred embodiment, the barcode loop oligo 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 barcode loop oligo comprises a barcode with a unique sequence that allows enumeration of the target sequence for each sample. [Example]

[0069] (method) 1. Probe Complex Formation The probe complexes contain sequences necessary for genome targeting, sample indexing, and construction of an Illumina sequencing library.

[0070] A four-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; (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; and (d) A loop oligo with a sequence complementary to the bridge oligo.

[0071] 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).

[0072] [Table 1]

[0073] 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, 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). A pre-annealed barcode loop (containing the specific index sequence for each sample) is added to the targeting reaction.

[0074] [Table 2]

[0075] 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.

[0076] 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.

[0077] 5. Rolling Circle Amplification After extension and ligation, the circular probe molecules are subjected to rolling circle amplification (RCA). In the RCA reaction, the target capture reaction is mixed with an RCA reaction mix containing EquipPhi29 (Thermo Scientific) polymerase. The reaction is incubated at +42°C for 30 minutes to 2 hours. After the RCA reaction, the reaction efficiency is analyzed by measuring the concentration of single-stranded DNA (ssDNA) using a Qubit fluorometer.

[0078] 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 primed target library. The RCA products are digested with EcoRI at +37°C for 1 hour.

[0079] 7. Library PCR The digested RCA products are extended into a sequencing library in a PCR reaction, where the truncated sequence adapters present in the appropriate probes are extended to flow-cell compatible full-length sequence adapters.

[0080] 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).

[0081] 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.

[0082] (Test 1) In the first experiment, the probe mix was a collection of four loop-indexed probe complexes contained in five separate samples: the first four samples had a single loop index, and the fifth sample was a pooled sample of loops 1 through 4. They targeted four gene fusions. The target oligonucleotides had unique recognition sequences that allowed for the identification of each target. Three synthetic target oligonucleotides were mixed at logarithmically increasing concentrations as samples. Target capture, extension and ligation reactions, rolling circle amplification, subsequent EcoRI digestion, and library preparation PCR were performed as described above. An example of the resulting sequencing library is shown in Figure 3. The prepared libraries were sequenced on MiSeq and iSeq100 instruments. Target regions were detected within the sequence data by matching probe sequences within each read, identifying genomic sequence regions between the probe sequences, and counting molecular barcodes. The counting data accurately reflected the proportion of pooled loop oligo molecules (Figure 4).

[0083] Detailed Description of Figures 1 and 2 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 ligated ligation complexes from multiple samples (110) are pooled together (112). In step 4, the annealed and pooled ligation complexes are ligated to obtain a ligated ligation complex. In step 5, the probe sequence is amplified by rolling circle amplification using phi29 polymerase or other strand-displacing polymerase to generate long concatemeric copies of the probe (116). In step 6, 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 further amplified using PCR or emulsion PCR (117) as needed. In step 7, the amplified DNA is sequenced using next generation DNA sequencing. In step 8, the DNA sequencing results are converted into target numbers using a bioinformatic pipeline.

[0084] 2A shows the principle structure of a probe quadruplet according to one embodiment of the present invention, in which 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 or nicks 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. 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.

[0085] Between 15 and 25 bases of the first probe comprise a cross-linking sequence (210) that optionally contains chemically modified bases for efficient cross-linking oligo binding. The first probe further comprises 15 to 30 bases from the 5' end that bind to the genetic target (203). Some or all of the 210 nucleotides may contain chemical modifications that increase the affinity of the probe for the target or the cross-link (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).

[0086] 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.

[0087] 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 contains a capture moiety (211) used to capture the ligation complex. Additionally, the bridge oligo contains sequences 214 and 216 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.

[0088] 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.

[0089] Figure 2B shows the principle structure of a probe set having multiple probe entities according to one embodiment of the present invention. The probes correspond to those in Figure 2A, except that this embodiment includes two bridge oligos (200 and 220), where bridge oligos 200 include 209 and 214, and bridge oligos 220 include 208 and 216.

[0090] Figure 2C shows the principle structure of a probe set having multiple probe entities according to one embodiment of the present invention. The probes correspond to those of Figure 2A, except that the bridge oligo 200 includes a sequence (219) containing multiple universal base analogs that allows for the incorporation of random sequences suitable for use as molecular barcodes for target enumeration. Either or both of the sequences 219 shown may be present.

[0091] 2D shows the 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 has 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.

[0092] Between 15 and 25 bases of the first probe contain a bridge-binding sequence (210) that optionally contains chemically modified bases for efficient bridge oligo binding. The first probe further contains binding sites for an amplification primer (221) and a barcode sequence (222), and, 15 to 30 bases from the 5' end, a sequence (203) that binds to the genetic target. Some or all of the 210 nucleotides may contain chemical modifications that increase the affinity of the probe for the target or the bridge (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).

[0093] 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 contains a binding site for an amplification primer (223), a sequencing adapter sequence (224), a recognition site for a restriction endonuclease, such as EcoRI or a homing endonuclease (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.

[0094] 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 to enable enzymatic degradation of the non-loop-containing structure. The 5' end of the bridge oligo optionally contains a capture portion (211) 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.

[0095] 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.

Claims

1. 1. A method for high throughput detection of one or more target nucleotide sequences in a plurality of samples, the method comprising: (i) providing, to each target nucleotide sequence in each of said samples, a first probe, a second probe, a barcode loop oligo, and a bridge oligo, or a plurality of bridge oligonucleotides capable of annealing to the barcode loop oligo to form a bridge oligo complex; the first probe comprises a first bridging oligo-specific sequence at the 5' end of the first probe and a first target-specific portion at the 3' end of the first probe; the second probe comprises a second target-specific portion at the 5' end of the second probe and a second bridging oligo-specific sequence at the 3' end of the second probe; the barcode loop oligo, starting from the 5' end of the molecule, comprises a third bridge oligo-specific sequence, a barcode loop sequence, and a fourth bridge oligo-specific sequence; the bridge oligo or the plurality of bridge oligonucleotides comprise sequences 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 sequences complementary to the third bridge oligo-specific sequence and the fourth bridge oligo-specific sequence in the barcode loop oligo; and at least one of the oligonucleotides of the first probe, the second probe, the barcode loop oligo, the bridge oligo, or the plurality of bridge oligonucleotides comprises a recognition sequence for an endonuclease; (ii) for each of the one or more target nucleotide sequences, contacting the first probe and the second probe with the barcode loop oligo and the bridge oligo or oligonucleotides for each of the samples in separate tubes, and annealing the first probe, the second probe, the barcode loop oligo, and the bridge oligo or oligonucleotides to form a ligation complex; (iii) contacting nucleic acids present in each of the samples to be tested for the target nucleotide sequence with the ligation complex; (iv) hybridizing the first target-specific portion and the second target-specific portion of the first probe and the second probe, respectively, to immediately adjacent portions or adjacent portions separated by up to 500 bases on the target nucleotide sequence, thereby forming a plurality of hybridization complexes; (v) pooling the hybridization complexes from the plurality of samples; (vi) ligating the probes in the hybridization complex to form a ligated ligation complex; (vii) amplifying nucleic acids from one or more ligated ligation complexes using rolling circle amplification with a strand-displacing polymerase, thereby obtaining single-stranded concatemeric sequences; (viii) optionally, when the designated recognition sequence is present in step (i), carrying out a step of obtaining a nucleic acid fragment by the following method; (a) cleaving the single-stranded concatemeric sequence obtained in step (vii); or (b) subjecting the single-stranded concatemeric sequence obtained in step (vii) to annealing with a specific oligonucleotide containing a recognition sequence for an endonuclease, wherein said oligonucleotide anneals with the recognition sequence identified in step (i), thereby providing a recognition site for the endonuclease, and cleaving the annealed complex with said endonuclease; (ix) subjecting the concatemer sequence obtained in step (vii) or the nucleic acid fragment obtained in step (viii) to a high-throughput sequencing technique to determine a barcode sequence; and (x) identifying the presence and / or number of target nucleotide sequences 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 a barcode sequence corresponding to a barcode in a barcode loop oligo; If the above (viii) is not optionally included, the barcode loop oligo comprises one or more recognition sequences for an endonuclease, the barcode loop oligo comprising two recognition sequences that can anneal to each other to provide a double-stranded endonuclease recognition site; between steps (vii) and (ix), a step of annealing the two recognition sequences in the barcode loop oligo and cleaving the resulting double-stranded endonuclease recognition site with an endonuclease having specificity for the recognition site; The method wherein steps (v) and (vi) may be performed in any order.

2. the bridge oligo or one or more of the plurality of bridge oligonucleotides comprises a plurality of universal base analogs that allow incorporation of a random sequence suitable for use as a molecular barcode for target enumeration in a region that is not complementary to the first probe, the second probe, or the barcode loop oligo, wherein the plurality of universal base analogs are a plurality of 5-nitroindoles; 2. The method of claim 1, wherein as part of step (vi), a gap-filling step is carried out using a polymerase and nucleotides to generate such random sequences.

3. 10. The method of claim 1, wherein the first probe, the second probe, or the bridge oligo or one of the plurality of bridge oligonucleotides further comprises a sequence barcode.

4. After step (v) and before step (vii), steps (a) and (b) are carried out; step (a) comprises dissociating the ligated ligation complex from the target nucleotide sequence; and step (b) comprises adding a target-specific probe comprising a sequence corresponding to the target nucleotide sequence; the target-specific probe is capable of annealing to the ligated ligation complex, allowing the target-specific probe to anneal to the ligated ligation complex, thereby forming an amplification template; and 2. The method of claim 1, wherein in step (vii), the amplification template is amplified by rolling circle amplification using a strand-displacing polymerase.

5. at least one of the first probe, the second probe, the barcode loop oligo, the bridge oligo, or the oligonucleotides of the plurality of bridge oligonucleotides comprises a first capture moiety; Between steps (iv) and (v), contacting the hybridization complex with a solid support comprising a second capture moiety; the first capture moiety and the second capture moiety interact such that the hybridization complex binds to a solid support; 2. The method of claim 1, wherein an intermediate step (iv)(a) is performed, comprising separating the hybridization complexes bound to the solid support from components of the sample that are not bound to the solid support.

6. 10. The method of claim 1, wherein the plurality of samples comprises blood samples, saliva samples, urine samples, or fecal samples.

7. the bridge oligo or oligonucleotides of the plurality of bridge oligonucleotides (i) 1 to 5 3' overhanging bases, and / or (ii) a 3' phosphate, and / or (iii) one or more phosphorothioate modifications within three positions from the 3' end 10. The method of claim 1, comprising:

8. 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.

9. 2. The method of claim 1, wherein the bridging portion of the first probe or the second probe, or both, or the barcode loop oligo, 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.

10. 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.

11. 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.

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

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

14. the sequencing of step (ix) is carried out using nanopore sequencing; The method of claim 1, wherein the concatemeric sequences obtained in step (vii) are fragmented using a transposition complex.

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

16. 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.

17. 1. A kit of parts comprising a plurality of containers for use in a method for high throughput detection of one or more target nucleotide sequences in a plurality of samples, comprising: at least one container contains one or more sets of first probes and second probes, at least one container contains barcode loop oligos, and at least one container contains one or more bridge oligos or a plurality of bridge oligonucleotides capable of forming a bridge oligo complex with the barcode loop oligos; the first probe comprises a first bridging oligo-specific sequence at the 5' end of the first probe and a first target-specific portion at the 3' end of the first probe; the second probe comprises a second target-specific portion at the 5' end of the second probe and a second bridging oligo-specific sequence at the 3' end of the second probe; the barcode loop oligo, starting from the 5' end of the molecule, comprises a third bridge oligo-specific sequence, a barcode loop sequence, and a fourth bridge oligo-specific sequence; the bridge oligo or the plurality of bridge oligonucleotides comprise sequences complementary to the first and second bridge oligo-specific sequences in the first and second probes, respectively, and sequences complementary to the third and fourth bridge oligo-specific sequences in the barcode loop oligo; at least one of the first probe, the second probe, the barcode loop oligo, the bridge oligo, or the oligonucleotides among the plurality of bridge oligonucleotides 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.

Images