Methods and compositions for background reduction in nucleic acid detection

Abstract

Methods and compositions relating to multiplex target detection, comprising use of at least one pool of one or more single-stranded termini complementary to a target nucleic acid and comprising a double-stranded region formed by intermolecular hybridization between the padlock probe and an accessory or blocker oligonucleotide.

Description

ENMRA-44283.601METHODS AND COMPOSITIONS FOR BACKGROUND REDUCTION IN NUCLEIC ACID DETECTIONThe present application claims priority to U.S. Provisional Application Serial No.63 / 747,813, filed January 21, 2025, which is incorporated herein by reference.SEQUENCE LISTINGThe text of the computer readable sequence listing filed herewith, titled “44283-601_SEQUENCE_LISTING”, created January 21, 2026, having a file size of 23,432 bytes, is hereby incorporated by reference in its entirety.FIELD OF THE INVENTIONThe technology herein relates to compositions, methods, systems, and kits for using circularizable probes, e.g., in detecting and quantifying molecules, particularly nucleic acid molecules, and for determining relative amounts of molecules, e.g., fetal, and maternal DNA, gene dosage, etc., and for reducing background in assays using circularization of probes for detection, especially in multiplexed reactions.BACKGROUND OF THE INVENTIONDetection of the presence of, or variations in the numbers of molecules in a sample is a useful way of characterizing the sample and the source of the sample. For example, variations in gene dosage are clinically significant indicators of disease states, e.g., in a subject from whom a sample is collected. Variations in gene dosage arise due to errors in DNA replication and can occur in germ line cells, leading to congenital defects and even embryonic demise, or in somatic cells, often resulting in cancer. These replication anomalies can cause deletion or duplication of parts of genes, full-length genes and their surrounding regulatory regions, megabase-long portions of chromosomes, or entire chromosomes. Analysis of other biomolecules is also clinically important. For example, variations in amounts of RNA or protein may indicate changes in expression of a gene associated with a disease state.Quantifying variations in numbers of molecules finds application broadly in analyzing nucleic acids for any purpose, e.g., for characterizing nucleic acids and nucleic acid mixtures,ENMRA-44283.601such as nucleic acids indicative of cancer or other disease in a subject, nucleic acids indicative of microbes, e.g., viral and bacterial microbes and mixtures of microbes in a sample, etc.SUMMARY OF THE INVENTIONThe technology provided herein relates to compositions comprising a plurality of different target-specific padlock probes that share a common linker sequence, wherein the common linkers are hybridized to blocker oligonucleotides that share a sequence complementary to at least a portion of the common linker sequence, wherein a complex comprising a padlock probe hybridized to a blocker oligonucleotide is referred to as a blocked padlock probe. The technology provides methods and compositions for forming a population of circularized molecules from a plurality of different target-specific blocked padlock probes. The technology finds use in numerous different assays and methods using circularized oligonucleotides, e.g., in assay detection, signal generation, etc. Embodiments of the technology include but are not limited to:1. A composition comprising a first set of blocked padlock probes, each blocked padlock probe comprising in sequence a first targeting arm, a linker, and a second targeting arm, wherein the first set of blocked padlock probes comprises a population of padlock probe strands each comprising a different pair of first and second targeting arms, wherein each padlock probe strand in the population of padlock probes comprises a first common linker sequence hybridized to a first common blocker oligonucleotide.2. The composition of embodiment 1, wherein in the first set of blocked padlock probes, 25% to 75%, preferably 30% to 70%, preferably 35% to 65%, preferably 40% to 60%, preferably 45% to 55%, preferably about 50% of the first common linker sequence is hybridized to a first blocker oligonucleotide in a duplex.3. The composition of embodiment 1 or embodiment 2, further comprising a second set of blocked padlock probes, , wherein the second set of blocked padlock probes comprises a second population of padlock probe strands each comprising a different pair of first and second targeting arms, wherein each padlock probe strand in the second set of padlock probes comprises a second common linker sequence hybridized to a second common blocker oligonucleotide,ENMRA-44283.601wherein the second common linker sequence is not the same as the first common linker sequence.4. The composition of embodiment 3, wherein in the second set of blocked padlock probes, 25% to 75%, preferably 30% to 70%, preferably 35% to 65%, preferably 40% to 60%, preferably 45% to 55%, preferably about 50% of the second common linker sequence is hybridized to a second blocker oligonucleotide in a duplex.5. The composition of any one of embodiments 1 to 4, further comprising a sample comprising nucleic acid molecules.6. The composition of any one of embodiments 1 to 5, further comprising a ligase.7. The composition of any one of embodiments 1 to 6, wherein the blocked padlock probes comprise circularized probes.8. A method of making circularized padlock probes, comprising:a) providing a set of blocked padlock probes according to any one of embodiments 1 to 4 and a sample comprising nucleic acid molecules;b) hybridizing padlock probe strands of the set of blocked padlock probes to the nucleic acid molecules to generate hybridized padlocked probes; andc) in a reaction mixture, ligating hybridized padlock probe strands to form a plurality of circularized padlock probes.9. The method of embodiment 8, further comprising treating the plurality of circularized padlock probes with at least one nuclease, preferably an exonuclease, wherein circularized padlock probes are not substrate for the at least one nuclease.10. The method of embodiment 8 or embodiment 9, further comprisinga) forming at least one complex comprising an oligonucleotide primer hybridized to a circularized padlock probe,ENMRA-44283.601b) detecting formation of the at least one complex in a process comprising extending the primer in the complex in a rolling circle amplification (RCA) reaction to form RCA product.11. The method of embodiment 10, wherein the oligonucleotide primer is attached to a solid support.12. The method of embodiment 11, wherein the oligonucleotide primer is covalently attached to the solid support.13. The method of embodiment 11 or embodiment 12, wherein the solid support comprises one or more of metal, glass, quartz, plastic, silicon, carbon, graphite, diamond, and ceramic.14. The method of embodiment 13, wherein the solid support comprises glass.15. The method of any one of embodiments 11 to 14, wherein the solid support comprises a polymeric coating, wherein the oligonucleotide primer is attached to the polymeric coating.DEFINITIONSTo facilitate an understanding of the present invention, a number of terms and phrases are defined below:Throughout the specification and claims, the following terms take the meanings explicitly associated herein, unless the context clearly dictates otherwise. The phrase “in one embodiment” as used herein does not necessarily refer to the same embodiment, though it may. Furthermore, the phrase “in another embodiment” as used herein does not necessarily refer to a different embodiment, although it may. Thus, as described below, various embodiments of the invention may be readily combined, without departing from the scope or spirit of the invention.ENMRA-44283.601In addition, as used herein, the term “or” is an inclusive “or” operator and is equivalent to the term “and / or” unless the context clearly dictates otherwise. The term “based on” is not exclusive and allows for being based on additional factors not described, unless the context clearly dictates otherwise. In addition, throughout the specification, the meaning of “a,” “an,” and “the” include plural references. The meaning of “in” includes “in” and “on.”The transitional phrase “consisting essentially of’ as used in claims in the present application limits the scope of a claim to the specified materials or steps “and those that do not materially affect the basic and novel characteristic(s)” of the claimed invention, as discussed in In re Herz, 537 F.2d 549, 551-52, 190 USPQ 461, 463 (CCPA 1976). For example, a composition “consisting essentially of’ recited elements may contain an unrecited contaminant at a level such that, though present, the contaminant does not alter the function of the recited composition as compared to a pure composition, i.e., a composition “consisting of’ the recited components.The term “sample” in the present specification and claims is used in its broadest sense. On the one hand it is meant to include a specimen or culture (e.g., microbiological cultures). On the other hand, it is meant to include both biological and environmental samples. A sample may include a specimen of synthetic origin. Biological samples may be animal, including human, fluid, solid (e. ., stool) or tissue, as well as liquid and solid food and feed products and ingredients such as dairy items, vegetables, meat and meat by-products, and waste. Biological samples may be obtained from all of the various families of domestic animals, as well as feral or wild animals, including, but not limited to, such animals as canines, felines, ungulates, bear, fish, lagomorphs, rodents, marsupials, etc.Environmental samples include environmental material such as surface matter, soil, water, and industrial samples, as well as samples obtained from food and dairy processing instruments, apparatus, equipment, utensils, disposable, and non-disposable items. These examples are not to be construed as limiting the sample types applicable to the present invention.The term “target” as used herein refers to a molecule sought to be sorted out from other molecules for assessment, measurement, or other characterization. For example, a target nucleic acid may be sorted from other nucleic acids in a sample, e.g., by probe binding, amplification, isolation, capture, etc. When used in reference to a hybridization-based detection, e.g.,ENMRA-44283.601polymerase chain reaction, “target” refers to the region of nucleic acid bounded by the primers used for polymerase chain reaction, while when used in an assay in which target DNA is not amplified, e.g., in capture by molecular inversion probes (MIPs), also referred to herein as padlock probes (PLPs), a target comprises the site bounded by the hybridization of the targetspecific arms of the PLP, such that the PLP can be ligated and the presence of the target nucleic acid can be detected.The term “source of target nucleic acid” refers to any sample that contains nucleic acids (RNA or DNA). Particularly preferred sources of target nucleic acids are biological samples including, but not limited to blood, plasma, serum, saliva, urine, feces, gastrointestinal fluid, cerebral spinal fluid, pleural fluid, milk, lymph, sputum, and semen.As used herein, the “sensitivity” of a given assay (or set of assays used together) refers to the percentage of samples that report a particular form or variant, e.g., a mutation, gene duplication, chromosome duplication, above a threshold value that distinguishes between samples exhibiting a variant phenotype (e.g., cancerous cells, aneuploidy) and samples exhibiting a normal or wild-type phenotype e.g., non-cancerous cells, euploidy). In some embodiments, a “positive” is defined as a clinically-confirmed variant that reports an assay result associated with the presence of the disease or condition to be detected, and a false negative is defined as a clinically-confirmed variant that reports an assay result associated with the absence of the disease or condition. The value of sensitivity, therefore, reflects the probability that a given diagnostic assay performed on a known variant or diseased sample will produce a result indicative of the presence of the variation or disease. As defined here, the clinical relevance of a calculated sensitivity value represents an estimation of the probability that a given assay would detect the presence of a clinical condition when applied to a subject with that condition. Using the technology described herein, it may be possible to achieve a certain level of accuracy without the need for generating sequence reads. The accuracy may refer to sensitivity, it may refer to specificity, or it may refer to some combination thereof. The desired level of accuracy may be between 90% and 95%; it may be between 95% and 98%; it may be between 98% and 99%; it may be between 99% and 99.5%; it may be between 99.5% and 99.9%; it may be between 99.9% and 99.99%; it may be between 99.99% and 99.999%, it may be between 99.999% and 100%. Levels of accuracy above 95% may be referred to as high accuracy.ENMRA-44283.601As used herein, the “specificity” of a given assay (or set of assays used together) refers to the percentage of normal samples that report an assay result associated with the presence of the disease or condition to be detected, and a false positive is defined as a clinically-confirmed normal sample that reports an assay result associated with the presence of the disease or condition. The value of specificity, therefore, reflects the probability that a given diagnostic assay performed on a known normal sample will produce a result indicative of the presence of the variation or disease. As defined here, the clinical relevance of the calculated specificity value represents an estimation of the probability that a given marker would detect the absence of a clinical condition when applied to a subject without that condition.The term “gene” refers to a DNA sequence that comprises control and coding sequences necessary for the production of an RNA having a non-coding function (e.g., a ribosomal or transfer RNA), a polypeptide or a precursor. The RNA or polypeptide can be encoded by a full-length coding sequence or by any portion of the coding sequence so long as the desired activity or function is retained.The term “chromosome-specific” as used herein refers to a sequence that is found only in that particular type of chromosome.As used herein, the term “hybridization” is used in reference to the pairing of complementary nucleic acids. Hybridization and the strength of hybridization (i.e., the strength of the association between the nucleic acids) is influenced by such factors as the degree of complementary between the nucleic acids, stringency of the conditions involved, and the Tmof the formed hybrid. “Hybridization” methods involve the annealing of one nucleic acid to another, complementary nucleic acid, i.e., a nucleic acid having a complementary nucleotide sequence. The ability of two polymers of nucleic acid containing complementary sequences to find each other and anneal through base pairing interaction is a well-recognized phenomenon. The initial observations of the “hybridization” process by Marmur and Lane, Proc. Natl. Acad. Sci. USA 46:453 (1960) and Doty et al., Proc. Natl. Acad. Sci. USA 46:461 (1960) have been followed by the refinement of this process into an essential tool of modem biology.The term “oligonucleotide” as used herein is defined as a molecule comprising two or more deoxyribonucleotides or ribonucleotides, preferably at least 5 nucleotides, more preferably at least about 10-15 nucleotides and more preferably at least about 15 to 30 nucleotides. TheENMRA-44283.601exact size will depend on many factors, which in turn depend on the ultimate function or use of the oligonucleotide. The oligonucleotide may be generated in any manner, including chemical synthesis, DNA replication, reverse transcription, PCR, or a combination thereof.Because mononucleotides are reacted to make oligonucleotides in a manner such that the 5' phosphate of one mononucleotide pentose ring is attached to the 3' oxygen of its neighbor in one direction via a phosphodiester linkage, an end of an oligonucleotide is referred to as the “5' end” if its 5' phosphate is not linked to the 3' oxygen of a mononucleotide pentose ring and as the “3' end” if its 3' oxygen is not linked to a 5' phosphate of a subsequent mononucleotide pentose ring. As used herein, a nucleic acid sequence, even if internal to a larger oligonucleotide, also may be said to have 5' and 3' ends. A first region along a nucleic acid strand is said to be upstream of another region if the 3' end of the first region is before the 5' end of the second region when moving along a strand of nucleic acid in a 5' to 3' direction.When two different, non-overlapping oligonucleotides anneal to different regions of the same linear complementary nucleic acid sequence, and the 3' end of one oligonucleotide points towards the 5' end of the other, the former may be called the “upstream” oligonucleotide and the latter the “downstream” oligonucleotide. Similarly, when two overlapping oligonucleotides are hybridized to the same linear complementary nucleic acid sequence, with the first oligonucleotide positioned such that its 5' end is upstream of the 5' end of the second oligonucleotide, and the 3' end of the first oligonucleotide is upstream of the 3' end of the second oligonucleotide, the first oligonucleotide may be called the “upstream” oligonucleotide and the second oligonucleotide may be called the “downstream” oligonucleotide.The term “primer” refers to an oligonucleotide that is capable of acting as a point of initiation of synthesis when placed under conditions in which primer extension is initiated, e.g.. in the presence of nucleotides and a suitable nucleic acid polymerase. An oligonucleotide “primer” may occur naturally, may be made using molecular biological methods, e.g., purification of a restriction digest, or may be produced synthetically. In preferred embodiments, a primer is composed of or comprises DNA.A primer is selected to be “substantially” complementary to a strand of specific sequence of the template. A primer must be sufficiently complementary to hybridize with a template strand for primer elongation to occur. A primer sequence need not reflect the exact sequence of the template. For example, a non-complementary nucleotide fragment may be attached to the 5' endENMRA-44283.601of the primer, with the remainder of the primer sequence being substantially complementary to the strand. Non-complementary bases or longer sequences can be interspersed into the primer, provided that the primer sequence has sufficient complementarity with the sequence of the template to hybridize and thereby form a template primer complex for synthesis of the extension product of the primer.The term “sequence variation” as used herein refers to differences in nucleic acid sequence between two nucleic acids. For example, a wild-type structural gene and a mutant form of this wild-type structural gene may vary in sequence by the presence of single base substitutions and / or deletions or insertions of one or more nucleotides. These two forms of the structural gene are said to vary in sequence from one another. A second mutant form of the structural gene may exist. This second mutant form is said to vary in sequence from both the wild-type gene and the first mutant form of the gene.The term “nucleotide analog” as used herein refers to modified or non-naturally occurring nucleotides including but not limited to analogs that have altered stacking interactions such as 7-deaza purines (i.e., 7-deaza-dATP and 7-deaza-dGTP); base analogs with alternative hydrogen bonding configurations (e.g., such as Iso-C and Iso-G and other non-standard base pairs described in U.S. Patent No. 6,001,983 to S. Benner); non-hydrogen bonding analogs (e.g., nonpolar, aromatic nucleoside analogs such as 2,4-difluorotoluene, described by B.A. Schweitzer and E.T. Kool, J. Org. Chem., 1994, 59, 7238-7242, B.A. Schweitzer and E.T. Kool, J. Am. Chem. Soc., 1995, 117, 1863-1872); “universal” bases such as 5-nitroindole and 3 -nitropyrrole; and universal purines and pyrimidines (such as “K” and “P” nucleotides, respectively; P. Kong, etal., Nucleic Acids Res., 1989, 17, 10373-10383, P. Kong etal., Nucleic Acids Res., 1992, 20, 5149-5152). Nucleotide analogs include base analogs, and comprise modified forms of deoxyribonucleotides as well as ribonucleotides, and include but are not limited to modified bases and nucleotides described in U.S. Pat. Nos. 5,432,272; 6,001,983; 6,037,120; 6,140,496; 5,912,340; 6,127,121 and 6,143,877, each of which is incorporated herein by reference in their entireties; heterocyclic base analogs based on the purine or pyrimidine ring systems, and other heterocyclic bases.The term “duplex” refers to the state of nucleic acids in which the base portions of the nucleotides on one strand are bound through hydrogen bonding their complementary bases arrayed on a second strand. The condition of being in a duplex form reflects on the state of theENMRA-44283.601bases of a nucleic acid. By virtue of base pairing, the strands of nucleic acid also generally assume the tertiary structure of a double helix, having a major and a minor groove. The assumption of the helical form is implicit in the act of becoming duplexed.The term “template” refers to a strand of nucleic acid on which a complementary copy is built from nucleoside triphosphates through the activity of a template-dependent nucleic acid polymerase. Within a duplex the template strand is, by convention, depicted and described as the “bottom” strand. Similarly, the non-template strand is often depicted and described as the “top” strand.As applied to polynucleotides, the term “substantial identity” denotes a characteristic of a polynucleotide sequence, wherein the polynucleotide comprises a sequence that has at least 85 percent sequence identity, preferably at least 90 to 95 percent sequence identity, more usually at least 99 percent sequence identity as compared to a reference sequence over a comparison window of at least 20 nucleotide positions, frequently over a window of at least 25-50 nucleotides, wherein the percentage of sequence identity is calculated by comparing the reference sequence to the polynucleotide sequence, which may include deletions or additions which total 20 percent or less of the reference sequence over the window of comparison. The reference sequence may be a subset of a larger sequence, for example, as a splice variant of the full-length sequences.The term “label” as used herein refers to any atom or molecule that can be used to provide a detectable (preferably quantifiable) effect, and that can be attached to a nucleic acid or protein. Labels include but are not limited to dyes; radiolabels such as32P; binding moieties such as biotin; haptens such as digoxigenin; luminogenic, phosphorescent or fluorogenic moieties; mass tags; and fluorescent dyes alone or in combination with moieties that can suppress (“quench”) or shift emission spectra by fluorescence resonance energy transfer (FRET). FRET is a distance-dependent interaction between the electronic excited states of two molecules (e.g., two dye molecules, or a dye molecule and a non-fluorescing quencher molecule) in which excitation is transferred from a donor molecule to an acceptor molecule without emission of a photon. (Stryer et al. , 1978, Ann. Rev. Biochem., 47:819; Selvin, 1995, Methods Enzymol., 246:300, each incorporated herein by reference). As used herein, the term “donor” refers to a fluorophore that absorbs at a first wavelength and emits at a second, longer wavelength. The term “acceptor” refers to a moiety such as a fluorophore, chromophore, or quencher that has an absorptionENMRA-44283.601spectrum that overlaps the donor’s emission spectrum, and that is able to absorb some or most of the emitted energy from the donor when it is near the donor group (typically between 1-100 nm). If the acceptor is a fluorophore, it generally then re-emits at a third, still longer wavelength; if it is a chromophore or quencher, it then releases the energy absorbed from the donor without emitting a photon. In some embodiments, changes in detectable emission from a donor dye (e.g., when an acceptor moiety is near or distant) are detected. In some embodiments, changes in detectable emission from an acceptor dye are detected. In preferred embodiments, the emission spectrum of the acceptor dye is distinct from the emission spectrum of the donor dye such that emissions from the dyes can be differentiated (e.g., spectrally resolved) from each other.In some embodiments, a donor dye is used in combination with multiple acceptor moieties. In a preferred embodiment, a donor dye is used in combination with a non-fluorescing quencher and with an acceptor dye, such that when the donor dye is close to the quencher, its excitation is transferred to the quencher rather than the acceptor dye, and when the quencher is removed e.g., by cleavage of a probe), donor dye excitation is transferred to an acceptor dye. In particularly preferred embodiments, emission from the acceptor dye is detected. See, e.g., Tyagi, el al., Nature Biotechnology 18:1191 (2000), which is incorporated herein by reference.Labels may provide signals detectable by fluorescence (e.g., simple fluorescence, FRET, time-resolved fluorescence, fluorescence polarization, / c.), radioactivity, colorimetry, gravimetry, X-ray diffraction or absorption, magnetism, enzymatic activity, characteristics of mass or behavior affected by mass (e.g., MALDI time-of-flight mass spectrometry), and the like. A label may be a charged moiety (positive or negative charge) or alternatively, may be charge neutral. Labels can include or consist of nucleic acid or protein sequence, so long as the sequence comprising the label is detectable.In some embodiment a label comprises a particle for detection. In preferred embodiments, the particle is a phosphor particle. In particularly preferred embodiments, the phosphor particle is an up-converting phosphor particle (see, e.g., Ostermayer, F.W. Preparation and properties of infrared-to-visible conversion phosphors. Metall. Trans. 752, 747-755

[1971] ). In some embodiments, rare earth-doped ceramic particles are used as phosphor particles.Phosphor particles may be detected by any suitable method, including but not limited to up-converting phosphor technology (UPT), in which up-converting phosphors transfer low energy infrared (IR) radiation to high-energy visible light. While the present invention is not limited toENMRA-44283.601any particular mechanism, in some embodiments the UPT up-converts infrared light to visible light by multi-photon absorption and subsequent emission of dopant-dependent phosphorescence. See, e.g., U.S. Patent No. 6,399,397, Issued June 4, 2002 to Zarling, et al. van De Rijke, et al., Nature Biotechnol. 19(3):273-6

[2001] ; Corstjens, et al., IEE Proc.Nanobiotechnol. 152(2):64

[2005] , each incorporated by reference herein in its entirety.As used herein, the terms “solid support” or “support” refer to any material that provides a substrate structure to which another material can be attached. A support or substrate may be, but need not be, solid. Support materials include smooth solid supports (e.g., smooth metal, glass, quartz, plastic, silicon, wafers, carbon (e.g., diamond), and ceramic surfaces, etc.), as well as textured and porous materials. Solid supports need not be flat. Supports include any type of shape, including spherical shapes (e.g., beads). Support materials also include, but are not limited to, gels, hydrogels, aerogels, rubbers, polymers, and other porous and / or non-rigid materials.As used herein, the terms “bead” and “particle” are used interchangeably, and refer to a small support, typically a solid support, that is capable of moving about when in a solution (e.g., it has dimensions smaller than those of the enclosure or container in which the solution resides). In some embodiments, beads may settle out of a solution when the solution is not mixed (e.g., by shaking, thermal mixing, vortexing), while in other embodiments, beads may be suspended in solution in a colloidal fashion. In some embodiments, beads are completely or partially spherical or cylindrical. However, beads are not limited to any particular three-dimensional shape. In some embodiments, beads or particles may be paramagnetic. For example, in some embodiments, beads and particles comprise a magnetic material, e.g., ferrous oxide.A bead or particle is not limited to any particular size, and in a preparation comprising a plurality of particles, the particles may be essentially uniform in size (e.g., in diameter) or may be a mixture of different sizes. In some embodiments, beads comprise or consist of nanoparticles, e.g., particles of less than about 1000 nm, 900 nm, 800 nm, 700 nm, 600 nm, 500 nm, 400 nm, 300 nm, 200 nm, 100 nm, 90 nm, 80 nm, 70 nm, 60 nm, 50 nm, 40 nm, 30 nm, 20 nm, 10 nm, 5 nm, or 1 nm in diameter. In some embodiments, the nanoparticle beads between 5 and 20 nm average diameter.Materials attached to a solid support may be attached to any portion of the solid support (e.g., may be attached to an interior portion of a porous solid support material, or to an exterior portion, or to a flat portion on an otherwise non-flat support, or vice versa). In preferredENMRA-44283.601embodiments of the technology, biological molecules such as nucleic acid or protein molecules are attached to solid supports. A biological material is “attached” to a solid support when it is affixed to the solid support through chemical or physical interaction. In some embodiments, attachment is through a covalent bond. However, attachments need not be covalent and need not be permanent. In some embodiments, an attachment may be undone or disassociated by a change in condition, e.g, by temperature, ionic change, addition or removal of a chelating agent, or other changes in the solution conditions to which the surface and bound molecule are exposed.In some embodiments, materials are attached to a first support and are localized to the surface of a second support. For example, in some embodiments, materials that comprise a ferrous or magnetic particle may be magnetically localized to a surface or a region of a surface, such as a planar surface of a slide or well.As used herein in reference to a support or substrate, e.g., for a coating or for attachment of a molecule, the term “surface” broadly refers to a portion of a support or substrate that is accessible for a purpose. For example, a portion of a bead or vessel or plate that is accessible to be coated, functionalized, attached to a moiety, e.g., an oligonucleotide or other macromolecule, or otherwise treated, may be considered a “surface” of the bead or plate, even if the surface is on an interior portion of the bead or vessel (e.g, within a pore, within a sintered matrix, inside a well, etc.) Similarly, a portion of a matrix that is flexible and / or porous (e.g., a hydrogel, aerogel, mesh, and that is accessible for a purpose, e.g, to be coated, functionalized, attached to a moiety, derivatized, etc., may be considered a surface of the matrix. In certain embodiments, a support may comprise a support surface, sometimes termed a first surface, which is the surface of the structural support material, e.g, in the absence of a coating or modifying layer, and may further comprise substrate surface, sometimes termed a second surface, which is the surface that is accessible for a purpose after the support surface is modified, e.g., by coating with a polymer or other coating. In some embodiments, the substrate surface comprises functional groups capable of complexing covalently or non-covalently with the one or more analytes, such as oligonucleotides or polypeptides that comprise reactive or binding groups suitable for complexing with the substrate surface functional groups.In some embodiments, a target molecule, e.g., a biological material, is attached to a solid support through a “spacer molecule” or “linker group.” Such spacer molecules are molecules that have a first portion that attaches to the biological material and a second portion that attachesENMRA-44283.601to the solid support. Spacer molecules typically comprise a chain of atoms, e.g., carbon atoms, that provide additional distance between the first portion and the second portion. Thus, when attached to the solid support, the spacer molecule permits separation between the solid support and the biological material, but is attached to both. Examples of linkers and spacers include but are not limited to carbon chains, e.g., C3 and C6 (hexanediol), r,2'-dideoxyribose (dSpacer); photocleavable (PC) spacers; triethylene glycol (TEG); and hexa-ethylene glycol spacers (Integrated DNA Technologies, Inc.).As used herein, the terms “array” and “microarray” refer a surface or vessel comprising a plurality of pre-defined loci that are addressable for analysis of the locus, e.g., to determine a result of an assay. Analysis at a locus in an array is not limited to any particular type of analysis and includes, e.g., analysis for detection of an atom, molecule, chemical reaction, light or fluorescence emission, suppression, or alteration (e.g., in intensity or wavelength) indicative of a result at that locus. Examples of pre-defined loci include a grid or any other pattern, wherein the locus to be analyzed is determined by its known position in the array pattern. Microarrays, for example, are described generally in Schena, “Microarray Biochip Technology,” Eaton Publishing, Natick, MA, 2000. Examples of arrays include but are not limited to supports with a plurality of molecules non-randomly bound to the surface (e.g., in a grid or other regular pattern) and vessels comprising a plurality of defined reaction loci (e.g., wells) in which molecules or signal-generating reactions may be detected. In some embodiments, an array comprises a patterned distribution of wells that receive beads, e.g., as described above for the SIMOA technology. See also U.S. Patent Nos. 9,057,730; 9,556,429; 9,481,883; and 9,376,677, each of which is incorporated herein by reference in its entirety, for all purposes.As used herein, the terms “dispersed” and “dispersal” as used in reference to loci or sites, e.g., on a support or surface, refers to a collection of loci or sites that are distributed or scattered on or about the surface, wherein at least some of the loci are sufficiently separated from other loci that they are individually detectable or resolvable, one from another, e.g., by a detector such as a microscope. Dispersed loci may be in an ordered array, or they may be in an irregular distribution or dispersal, as described below.As used herein, the term “irregular” as used in reference to a dispersal or distribution of loci or sites, e.g., on a solid support or surface, refers to distribution of loci on or in a surface in a non- arrayed manner. For example, molecules may be irregularly dispersed on a surface byENMRA-44283.601application of a solution of a particular concentration that provides a desired approximate average distance between the molecules on the surface, but at sites that are not pre-defined by or addressable any pattern on the surface or by the means of applying the solution (e.g., inkjet printing). In such embodiments, analysis of the surface may comprise finding the locus of a molecule by detection of a signal wherever it may appear (e.g., scanning a whole surface to detect fluorescence anywhere on the surface). This contrasts to locating a signal by analysis of a surface or vessel only at predetermined loci (e.g., points in a grid array), to determine how much (or what type of) signal appears at each locus in the grid.As used herein, the term “distinct” in reference to signals refers to signals that can be differentiated one from another, e.g., by spectral properties such as fluorescence emission wavelength, color, absorbance, mass, size, fluorescence polarization properties, charge, etc., or by capability of interaction with another moiety, such as with a chemical reagent, an enzyme, an antibody, etc.As used herein, the term “nucleic acid detection assay” refers to any method of determining the nucleotide composition of a nucleic acid of interest. Nucleic acid detection assay include but are not limited to, DNA sequencing methods, probe hybridization methods, structure specific cleavage assays (e.g., the INVADER assay, (Hologic, Inc.) and are described, e.g., in U.S. Patent Nos. 5,846,717; 5,985,557; 5,994,069; 6,001,567; 6,090,543; and 6,872,816;Lyamichev etal., Nat. Biotech., 17:292 (1999), Hall etal., PNAS, USA, 97:8272 (2000), and US Pat. No. 9,096,893, each of which is herein incorporated by reference in its entirety for all purposes); enzyme mismatch cleavage methods (e.g., Variagenics, U.S. Pat. Nos. 6,110,684, 5,958,692, 5,851,770, herein incorporated by reference in their entireties); polymerase chain reaction (PCR), described above; branched hybridization methods (e.g., Chiron, U.S. Pat. Nos.5,849,481, 5,710,264, 5,124,246, and 5,624,802, herein incorporated by reference in their entireties); rolling circle amplification (e.g., U.S. Pat. Nos. 6,210,884, 6,183,960 and 6,235,502, herein incorporated by reference in their entireties); the variation of rolling circle amplification called “RAM amplification” (see, e.g., US 5,942,391, incorporated herein by reference in its entirety; NASBA (e.g., U.S. Pat. No. 5,409,818, herein incorporated by reference in its entirety); molecular beacon technology (e.g., U.S. Pat. No. 6,150,097, herein incorporated by reference in its entirety); E-sensor technology (Motorola, U.S. Pat. Nos. 6,248,229, 6,221,583, 6,013,170, and 6,063,573, herein incorporated by reference in their entireties); cycling probe technology (e.g.,ENMRA-44283.601U.S. Pat. Nos. 5,403,711, 5,011,769, and 5,660,988, herein incorporated by reference in their entireties); Dade Behring signal amplification methods (e.g., U.S. Pat. Nos. 6,121,001, 6,110,677, 5,914,230, 5,882,867, and 5,792,614, herein incorporated by reference in their entireties); ligase chain reaction (e.g., Barany Proc. Natl. Acad. Sci USA 88, 189-93 (1991)); and sandwich hybridization methods (e.g., U.S. Pat. No. 5,288,609, herein incorporated by reference in its entirety).In some embodiments, target nucleic acid is amplified (e.g., by PCR) and amplified nucleic acid is detected simultaneously using an invasive cleavage assay. Assays configured for performing a detection assay (e.g., invasive cleavage assay) in combination with an amplification assay are described in U.S. Pat. No. 9,096,893, incorporated herein by reference in its entirety for all purposes. Additional amplification plus invasive cleavage detection configurations, termed the QuARTS method, are described in, e.g., in U.S. Pat. Nos. 8,361,720; 8,715,937; 8,916,344; and 9,212,392, each of which is incorporated herein by reference for all purposes. The term “invasive cleavage structure” as used herein refers to a cleavage structure comprising i) a target nucleic acid, ii) an upstream nucleic acid (e.g., an invasive or “INVADER” oligonucleotide), and iii) a downstream nucleic acid (e.g., a probe), where the upstream and downstream nucleic acids anneal to contiguous regions of the target nucleic acid, and where an overlap forms between the a 3' portion of the upstream nucleic acid and duplex formed between the downstream nucleic acid and the target nucleic acid. An overlap occurs where one or more bases from the upstream and downstream nucleic acids occupy the same position with respect to a target nucleic acid base, whether or not the overlapping base(s) of the upstream nucleic acid are complementary with the target nucleic acid, and whether or not those bases are natural bases or non-natural bases. In some embodiments, the 3' portion of the upstream nucleic acid that overlaps with the downstream duplex is a non-base chemical moiety such as an aromatic ring structure, e.g., as disclosed, for example, in U.S. Pat. No. 6,090,543, incorporated herein by reference in its entirety. In some embodiments, one or more of the nucleic acids may be attached to each other, e.g., through a covalent linkage such as nucleic acid stem-loop, or through a non-nucleic acid chemical linkage (e.g., a multi-carbon chain). As used herein, the term “flap endonuclease assay” includes “INVADER” invasive cleavage assays and QuARTS assays, as described above.ENMRA-44283.601As used herein, the terms “digital PCR,” “single molecule PCR” and “single molecule amplification” refer to PCR and other nucleic acid amplification methods that are configured to provide amplification product or signal from a single starting molecule. Typically, samples are divided, e.g., by serial dilution or by partition into small enough portions (e.g., in microchambers or in emulsions) such that each portion or dilution has, on average as assessed according to Poisson distribution, no more than a single copy of the target nucleic acid. Methods of single molecule PCR are described, e.g., in US 6,143,496, which relates to a method comprising dividing a sample into multiple chambers such that at least one chamber has at least one target, and amplifying the target to determine how many chambers had a target molecule; US 6,391,559; which relates to an assembly for containing and portioning fluid; and US 7,459,315, which relates to a method of dividing a sample into an assembly with sample chambers where the samples are partitioned by surface affinity to the chambers, then sealing the chambers with a curable “displacing fluid.” See also US 6,440,706 and US 6,753,147, and Vogelstein, et al., Proc. Natl. Acad. Sci. USA Vol. 96, pp. 9236-9241, August 1999. See also US 20080254474, describing a combination of digital PCR combined with methylation detection.The term “sequencing”, as used herein, is used in a broad sense and may refer to any technique known in the art that allows the order of at least some consecutive nucleotides in at least part of a nucleic acid to be identified, including without limitation at least part of an extension product or a vector insert. In some embodiments, sequencing allows the distinguishing of sequence differences between different target sequences. Exemplary sequencing techniques include targeted sequencing, single molecule real-time sequencing, electron microscopy-based sequencing, transistor-mediated sequencing, direct sequencing, random shotgun sequencing, Sanger dideoxy termination sequencing, targeted sequencing, exon sequencing, whole-genome sequencing, sequencing by hybridization, pyrosequencing, capillary electrophoresis, gel electrophoresis, duplex sequencing, cycle sequencing, single-base extension sequencing, solidphase sequencing, high-throughput sequencing, massively parallel signature sequencing, emulsion PCR, co-amplification at lower denaturation temperature-PCR (COLD-PCR), multiplex PCR, sequencing by reversible dye terminator, paired-end sequencing, near-term sequencing, exonuclease sequencing, sequencing by ligation, short-read sequencing, singlemolecule sequencing, sequencing-by-synthesis, real-time sequencing, reverse-terminatorENMRA-44283.601sequencing, ion semiconductor sequencing, nanoball sequencing, nanopore sequencing, 454 sequencing, Solexa Genome Analyzer sequencing, miSeq (Illumina), HiSeq 2000 (Illumina), HiSeq 2500 (Illumina), Illumina Genome Analyzer (Illumina), Ion Torrent PGM™ (Life Technologies), MinlON™ (Oxford Nanopore Technologies), real-time SMRT™ technology (Pacific Biosciences), the Probe-Anchor Ligation (cP AL™) (Complete Genomics / BGI), SOLiD® sequencing, MS-PET sequencing, mass spectrometry, and a combination thereof. In some embodiments, sequencing comprises detecting the sequencing product using an instrument, for example but not limited to an ABI PRISM® 377 DNA Sequencer, an ABI PRISM® 310, 3100, 3100-Avant, 3730, or 3730x1 Genetic Analyzer, an ABI PRISM® 3700 DNA Analyzer, or an Applied Biosystems SOLiD™ System (all from Applied Biosystems), a Genome Sequencer 20 System (Roche Applied Science), or a mass spectrometer. In certain embodiments, sequencing comprises emulsion PCR. In certain embodiments, sequencing comprises a high throughput sequencing technique, for example but not limited to, massively parallel signature sequencing (MPSS).As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably in reference to a chain of two or more amino acids linked together by peptide bonds. Polypeptides may be synthetic or naturally occurring, and may be short, e.g, between two about 30 amino acid residues, or may be hundreds or thousands of amino acid residues in length. Polypeptides may be composed of the 20 main naturally-occurring amino acids, or may comprise one or more non-natural amino acids, e.g., peptide nucleic acid residues, which comprise pyrimidine or purine bases on a peptide chain backbone, or modified versions of natural amino acids (e.g., modified in the structure of the side groups).As used herein, the terms “digital sequencing,” “single-molecule sequencing,” and “next generation sequencing (NGS)” are used interchangeably and refer to determining the nucleotide sequence of individual nucleic acid molecules. Systems for individual molecule sequencing include but are not limited to the 454 FLX™ or 454 TITANIUM™ (Roche), the SOLEXA™ / Illumina Genome Analyzer (Illumina), the HELISCOPE™ Single Molecule Sequencer (Helicos Biosciences), and the SOLID™ DNA Sequencer (Life Technologies / Applied Biosystems) instruments), as well as other platforms still under development by companies such as Intelligent Biosystems and Pacific Biosystems. See also U.S. Patent No. 7,888,017, entitled “Non-invasiveENMRA-44283.601fetal genetic screening by digital analysis,” relating to digital analysis of maternal and fetal DNA, e.g, cfDNA.As used herein, the term “probe” or “hybridization probe” refers to an oligonucleotide (i.e., a sequence of nucleotides), whether occurring naturally as in a purified restriction digest or produced synthetically, recombinantly or by PCR amplification, that is capable of hybridizing, at least in part, to another oligonucleotide of interest. A probe may be single-stranded or doublestranded. Probes are useful in the detection, identification, and isolation of particular sequences. In some preferred embodiments, probes used in the present invention will be labeled with a “reporter molecule,” so that is detectable in any detection system, including, but not limited to enzyme (e.g., ELISA, as well as enzyme-based histochemical assays), fluorescent, radioactive, and luminescent systems. It is not intended that the present invention be limited to any particular detection system or label.The terms “padlock probe” (PLP) and “molecular inversion probe” (MIP) are used interchangeably herein and refer to a circularizable oligonucleotide molecules that comprise a pair of unique polynucleotide arms (“targeting arms” that hybridize to a target nucleic acid to form a nick or gap and a polynucleotide linker (e.g., a universal linker). In some embodiments, the unique polynucleotide arms hybridize to a target strand immediately adjacent to each other to form a ligatable nick while in some embodiments, one the hybridized PLP must be further modified (e.g., by polymerase extension, base excision, and / or flap cleavage) to form a ligatable nick. Ligation of a PLP probe to form a circular nucleic acid is typically indicative of the presence of the complementary target strand. In some embodiments, PLPs may comprise one or more unique molecular tags (or unique molecular identifiers).In some embodiments, a PLP may comprise more than one unique molecular tags, such as, two unique molecular tags, three unique molecular tags, or more. In some embodiments, the unique polynucleotide arms in each PLP are located at the 5' and 3' ends of the , while the unique molecular tag(s) and the polynucleotide linker are located internal to the 5' and 3' ends of the PLP. For example, the PLPs that are used in some embodiments of this disclosure comprise in sequence the following components: first unique polynucleotide arm - first unique molecular tag - polynucleotide linker - second unique molecular tag - second unique polynucleotide arm. In some embodiments, the PLP is a 5' phosphorylated single- stranded nucleic acid (e.g., DNA)ENMRA-44283.601molecule. See, for example, WO 2017 / 020023, filed July 29, 2016, and WO 2017 / 020024, filed July 29, 2016, each of which is incorporated by reference herein for all purposes.As used herein, the terms “blocker oligonucleotide” and linear structure modifier (LSM) are used interchangeably, and refer to an oligonucleotide that is complementary to a region of a padlock probe, and that hybridizes to a portion of the padlock probe to create a double-stranded region within the padlock probe. Preferably, a blocker oligonucleotide hybridizes within at least a portion of the polynucleotide linker, and more preferably, a blocker oligonucleotide does not hybridize within any portion either targeting arm of the padlock probe.The term “blocked padlock probe” as used herein refers to a padlock probe hybridized to a blocker oligonucleotide, preferably comprising in order: a first targeting arm - a duplex region comprising a hybridized blocker oligonucleotide - a second targeting arm, wherein the first and second targeting arms are preferably single stranded.As used herein, the terms “circular nucleic acid” and “circularized nucleic acid” as used, for example, in reference to probe nucleic acids (e.g., a circularized probe), refers to nucleic acid strands, e.g., padlock probes, that are joined at the ends, e.g., by ligation, to form a continuous circular strand of nucleic acid.The unique molecular tag may be any tag that is detectable and can be incorporated into or attached to a nucleic acid (e.g., a polynucleotide) and allows detection and / or identification of nucleic acids that comprise the tag. In some embodiments the tag is incorporated into or attached to a nucleic acid during sequencing (e.g., by a polymerase). Non-limiting examples of tags include nucleic acid tags, nucleic acid indexes or barcodes, radiolabels (e.g., isotopes), metallic labels, fluorescent labels, chemiluminescent labels, phosphorescent labels, fluorophore quenchers, dyes, proteins (e.g., enzymes, antibodies or parts thereof, linkers, members of a binding pair), the like or combinations thereof. In some embodiments, particularly sequencing embodiments, the tag (e.g., a molecular tag) is a unique, known and / or identifiable sequence of nucleotides or nucleotide analogues (e.g., nucleotides comprising a nucleic acid analogue, a sugar and one to three phosphate groups). In some embodiments, tags are six or more contiguous nucleotides. A multitude of fluorophore-based tags are available with a variety of different excitation and emission spectra. Any suitable type and / or number of fluorophores can be used as a tag. In some embodiments 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 20 or more, 30 or more, 50 or more, 100 or more, 500ENMRA-44283.601or more, 1000 or more, 10,000 or more, 100,000 or more different tags are utilized in a method described herein (e.g., a nucleic acid detection and / or sequencing method). In some embodiments, one or two types of tags (e.g., different fluorescent labels) are linked to each nucleic acid in a library. In some embodiments, chromosome-specific tags are used to make chromosomal counting faster or more efficient. Detection and / or quantification of a tag can be performed by a suitable method, machine or apparatus, non-limiting examples of which include flow cytometry, quantitative polymerase chain reaction (qPCR), gel electrophoresis, a luminometer, a fluorometer, a spectrophotometer, a suitable gene- chip or microarray analysis, Western blot, mass spectrometry, chromatography, cytofluorimetric analysis, fluorescence microscopy, a suitable fluorescence or digital imaging method, confocal laser scanning microscopy, laser scanning cytometry, affinity chromatography, manual batch mode separation, electric field suspension, a suitable nucleic acid sequencing method and / or nucleic acid sequencing apparatus, the like and combinations thereof.In the PLPs, the unique polynucleotide arms are designed to hybridize immediately upstream and downstream of a specific target sequence (or site) in a nucleic acid target, e.g., in an RNA, cfDNA, or genomic nucleic acid sample. In some embodiments, hybridization of a PLP to a target sequence produces a ligatable nick without a gap, i.e., the two arms of the PLP hybridize to contiguous sequences in the target strand such that no overlap or gap is formed upon hybridization. See, e.g., M. Nilsson, etal. “Padlock probes: circularizing oligonucleotides for localized DNA detection.” Science. 265 (5181): 2085-2088 (1994); I. Baner, et al., Nucleic Acids Res., 26 (22):5073-5078 (1998). In other embodiments the hybridized PLP / target nucleic acid complex requires modification to produce a ligatable nick. For example, in some embodiments, hybridization leaves a gap that is filled, e.g., by polymerase extending a 3' end of the PLP, prior to ligation, while in other embodiments, hybridization forms an overlapping flap structure that must be modified, e.g., by a flap endonuclease or a 3' exonuclease, to produce a ligatable nick. In some embodiments, PLPS comprise unique molecular tags are short nucleotide sequences that are randomly generated. In some embodiments, the unique molecular tags do not hybridize to any sequence or site located on a genomic nucleic acid fragment or in a genomic nucleic acid sample. In some embodiments, the polynucleotide linker in the PLPs are universal in all the PLPs used in embodiments of this disclosure.ENMRA-44283.601In some embodiments, the PLPs are introduced to nucleic acid fragments derived from a test subject (or a reference subject) to perform capture of target sequences or sites (or control sequences or sites) located on a nucleic acid sample (e. ., a genomic DNA). In some embodiments, fragmenting aids in capture of target nucleic acid by molecular inversion probes. In some embodiments, for example, when the nucleic acid sample is comprised of cell free nucleic acid, fragmenting may not be necessary to improve capture of target nucleic acid by molecular inversion probes. For example, in some types of samples, cell free nucleic acid is fragmented in the sample such that further fragmentation is not necessary and may even be detrimental capture of the target nucleic acids. As described in greater detail herein, after capture of the target sequence (e.g., locus) of interest, the captured target may be subjected to enzymatic gap-fdling and ligation steps, such that a copy of the target sequence is incorporated into a circlelike structure. In some embodiments, nucleic acid analogs, e.g., containing labels, haptens, etc., may be incorporated in the filled section, for use, e.g., in downstream detection, purification, or other processing steps. Capture efficiency of the PLP to the target sequence on the nucleic acid fragment can, in some embodiments, be improved by lengthening the hybridization and gapfilling incubation periods. (See, e.g., Turner E H, el al., Nat Methods. 2009 Apr. 6:1-2.).In some embodiments, the PLPs that are used according to the disclosure to capture a target site or target sequence comprise in sequence the following components:first targeting polynucleotide arm - first unique targeting molecular tag - polynucleotide linker -second unique targeting molecular tag - second targeting polynucleotide arm.In some embodiments, the PLPs that are used in the disclosure to capture a control site or control sequence comprise in sequence the following components:first control polynucleotide arm - first unique control molecular tag - polynucleotide linker -second unique control molecular tag - second control polynucleotide arm.PLP technology may be used to detect or amplify particular nucleic acid sequences in complex mixtures. One of the advantages of using the PLP technology is in its capacity for a high degree of multiplexing, which allows thousands of target sequences to be captured in a single reaction containing thousands of PLPs. Various aspects of PLP technology are described in, for example, Hardenbol et al., “Multiplexed genotyping with sequence-tagged molecular inversion probes,” Nature Biotechnology, 21(6): 673-678 (2003); Hardenbol et al., “Highly multiplexed molecular inversion probe genotyping: Over 10,000 targeted SNPs genotyped in aENMRA-44283.601single tube assay,” Genome Research, 15: 269-275 (2005); Burmester et al., “DMET microarray technology for pharmacogenomics-based personalized medicine,” Methods in Molecular Biology, 632: 99-124 (2010); Sissung etal., “Clinical pharmacology and pharmacogenetics in a genomics era: the DMET platform,” Pharmacogenomics, 11(1): 89-103 (2010); Deeken, “The Affymetrix DMET platform and pharmacogenetics in drug development,” Current Opinion in Molecular Therapeutics, 11(3): 260-268 (2009); Wang et al., “High quality copy number and genotype data from FFPE samples using Molecular Inversion Probe (PLP) microarrays,” BMC Medical Genomics, 2:8 (2009); Wang et al., “Analysis of molecular inversion probe performance for allele copy number determination,” Genome Biology, 8(11): R246 (2007); Ji et al., “Molecular inversion probe analysis of gene copy alternations reveals distinct categories of colorectal carcinoma,” Cancer Research, 66(16): 7910-7919 (2006); and Wang etal., “Allele quantification using molecular inversion probes (PLP),” Nucleic Acids Research, 33(21): el83 (2005), each of which is hereby incorporated by reference in its entirety for all purposes. See also in U.S. Pat. Nos. 6,858,412; 5,817,921; 6,558,928; 7,320,860; 7,351,528; 5,866,337; 6,027,889 and 6,852,487, each of which is hereby incorporated by reference in its entirety for all purposes.PLP technology has previously been successfully applied to other areas of research, including the novel identification and subclassification of biomarkers in cancers. See, e.g., Brewster et al., “Copy number imbalances between screen- and symptom-detected breast cancers and impact on disease-free survival,” Cancer Prevention Research, 4(10): 1609-1616 (2011); Geiersbach etal., “Unknown partner for USP6 and unusual SSI 8 rearrangement detected by fluorescence in situ hybridization in a solid aneurysmal bone cyst,” Cancer Genetics, 204(4): 195-202 (2011); Schiffman et al., “Oncogenic BRAF mutation with CDKN2A inactivation is characteristic of a subset of pediatric malignant astrocytomas,” Cancer Research, 70(2): 512-519 (2010); Schiffman et al., “Molecular inversion probes reveal patterns of 9p21 deletion and copy number aberrations in childhood leukemia,” Cancer Genetics and Cytogenetics, 193(1): 9-18 (2009); Press et al., “Ovarian carcinomas with genetic and epigenetic BRCA1 loss have distinct molecular abnormalities,” BMC Cancer, 8:17 (2008); and Deeken et al., “A pharmacogenetic study of docetaxel and thalidomide in patients with castration-resistant prostate cancer using the DMET genotyping platform,” Pharmacogenomics, 10(3): 191-199 (2009), each of which is hereby incorporated by reference in its entirety for all purposes.PLP technology has also been applied to the identification of new drug-relatedENMRA-44283.601biomarkers. See, e.g., Caldwell et al., “CYP4F2 genetic variant alters required warfarin dose,” Blood, 111(8): 4106-4112 (2008); and McDonald et al., “CYP4F2 Is a Vitamin KI Oxidase: An Explanation for Altered Warfarin Dose in Carriers of the V433M Variant,” Molecular Pharmacology, 75: 1337-1346 (2009), each of which is hereby incorporated by reference in its entirety for all purposes. Other PLP applications include drug development and safety research. See, e.g., Mega et al., “Cytochrome P-450 Polymorphisms and Response to Clopidogrel,” New England Journal of Medicine, 360(4): 354-362 (2009); Dumaual et al., “Comprehensive assessment of metabolic enzyme and transporter genes using the Affymetrix Targeted Genotyping System,” Pharmacogenomics, 8(3): 293-305 (2007); and Daly et al., “Multiplex assay for comprehensive genotyping of genes involved in drug metabolism, excretion, and transport,” Clinical Chemistry, 53(7): 1222-1230 (2007), each of which is hereby incorporated by reference in its entirety for all purposes. Further applications of PLP technology include genotype and phenotype databasing. See, e.g., Man et al., “Genetic Variation in Metabolizing Enzyme and Transporter Genes: Comprehensive Assessment in 3 Major East Asian Subpopulations with Comparison to Caucasians and Africans,” Journal of Clinical Pharmacology, 50(8): 929-940 (2010), which is hereby incorporated by reference in its entirety for all purposes.The term “capture” or “capturing,” as used herein, refers to the binding or hybridization reaction between a padlock probe and its corresponding targeting site. In some embodiments, upon capturing, a circular replicon or a PLP replicon is produced or formed. In some embodiments, the targeting site is a deletion (e.g., partial, or full deletion of one or more exons). In some embodiments, a target PLP is designed to bind to or hybridize with a naturally occurring (e.g., wild-type) genomic region of interest where a target deletion is expected to be located. The target PLP is designed not to bind to a genomic region exhibiting the deletion. In these embodiments, binding or hybridization between a target PLP and the target site of deletion is expected to not occur. The absence of such binding or hybridization indicates the presence of the target deletion. In these embodiments, the phrase “capturing a target site” or the phrase “capturing a target sequence” refers to detection of a target deletion by detecting the absence of such binding or hybridization. As used in reference to other oligonucleotides, e.g., “capture oligonucleotide” the term refers to a binding or hybridization reaction between the captureENMRA-44283.601oligonucleotide and a nucleic acid to be captured, e.g., to be immobilized, removed from solution, or otherwise be manipulated by hybridization to the capture oligonucleotide.The term “PLP replicon” or “circular replicon,” as used herein, refers to a circular nucleic acid molecule generated via a capturing reaction (e.g., a binding or hybridization reaction between a PLP and its targeted sequence). In some embodiments, the PLP replicon is a singlestranded circular nucleic acid molecule. In some embodiments, a targeting PLP captures or hybridizes to a target sequence or site. After the capturing reaction or hybridization, in some embodiments, a ligation reaction mixture is introduced to ligate the nick formed by hybridization of the two targeting polynucleotide arms to form single-stranded circular nucleotide molecules, i.e., a targeting PLP replicon, while in some embodiments, hybridization of the PLP leaves a gap, and a ligation / extension mixture is introduced to extend and ligate the gap region between the two targeting polynucleotide arms to form a targeting PLP replicon. In some embodiments, a control PLP captures or hybridizes to a control sequence or site. After the capturing reaction or hybridization, a ligation reaction mixture is introduced to ligate the nick formed by hybridization of the two control polynucleotide arms, or a ligation / extension mixture is introduced to extend and ligate the gap region between the two control polynucleotide arms to form single-stranded circular nucleotide molecules, i.e., a control PLP replicon. PLP replicons may be amplified through a polymerase chain reaction (PCR) to produce a plurality of targeting PLP amplicons, which are double-stranded nucleic acid molecules. PLP replicons find particular application in rolling circle amplification, or RCA. RCA is an isothermal nucleic acid amplification technique where a DNA polymerase continuously adds single nucleotides to a primer annealed to a circular template, which results in a long concatemer of single stranded DNA that contains tens to hundreds to thousands of tandem repeats (complementary to the circular template). See, e.g., M. Ali, et al. “Rolling circle amplification: a versatile tool for chemical biology, materials science and medicine.” Chemical Society Reviews. 43 (10): 3324-3341, which is incorporated herein by reference in its entirety, for all purposes. See also WO 2015 / 083002, which is incorporated herein by reference in its entirety, for all purposes.Polymerases typically used in RCA for DNA amplification are Phi29, Bst, and Vent exo-DNA polymerases, with Phi29 DNA polymerase being preferred in view of its superior processivity and strand displacement abilityENMRA-44283.601The term “amplicon,” as used herein, refers to a nucleic acid generated via amplification reaction (e.g., a PCR reaction). In some embodiments, the amplicon is a single-stranded nucleic acid molecule. In some embodiments, the amplicon is a double-stranded nucleic acid molecule. In some embodiments, a targeting PLP replicon is amplified using conventional techniques to produce a plurality of targeting PLP amplicons, which are double-stranded nucleotide molecules. In some embodiments, a control PLP replicon is amplified using conventional techniques to produce a plurality of control PLP amplicons, which are double-stranded nucleotide molecules.As used herein, the phrase “not substantially complementary” as used in reference to a probe flap or arm means that the flap portion is sufficiently non-complementary not to hybridize selectively to a nucleic acid sequence, e.g., a target nucleic acid or amplified DNA, under the designated annealing conditions or stringent conditions, encompassing the terms “substantially non-complementary” and “perfectly non-complementary.”The term “signal” as used herein refers to any detectable effect, such as would be caused or provided by a label or by action or accumulation of a component or product in an assay reaction.As used herein, the term “detector” refers to a system or component of a system, e.g., an instrument (e.g. a camera, fluorimeter, charge-coupled device, scintillation counter, solid state nanopore device, etc..) or a reactive medium (X-ray or camera film, pH indicator, etc.), that can convey to a user or to another component of a system (e.g., a computer or controller) the presence of a signal or effect. A detector is not limited to a particular type of signal detected, and can be a photometric or spectrophotometric system, which can detect ultraviolet, visible or infrared light, including fluorescence or chemiluminescence; a radiation detection system; a charge detection system; a system for detection of an electronic signal, e.g., a current or charge perturbation; a spectroscopic system such as nuclear magnetic resonance spectroscopy, mass spectrometry or surface enhanced Raman spectrometry; a system such as gel or capillary electrophoresis or gel exclusion chromatography; or other detection system known in the art, or combinations thereof.The term “detection” as used herein refers to quantitatively or qualitatively identifying an analyte (e.g., DNA, RNA, or a protein), e.g., within a sample. The term “detection assay” as used herein refers to a kit, test, or procedure performed for the purpose of detecting an analyte within a sample. Detection assays produce a detectable signal or effect when performed in the presenceENMRA-44283.601of the target analyte, and include but are not limited to assays incorporating the processes of hybridization, nucleic acid cleavage (e.g., exo- or endonuclease), nucleic acid amplification, nucleotide sequencing, primer extension, nucleic acid ligation, antigen- antibody binding, interaction of a primary antibody with a secondary antibody, and / or conformational change in a nucleic acid (e.g., an oligonucleotide) or polypeptide (e.g., a protein or small peptide).As used herein, the term “prenatal or pregnancy-related disease or condition” refers to any disease, disorder, or condition affecting a pregnant woman, embryo, or fetus. Prenatal or pregnancy-related conditions can also refer to any disease, disorder, or condition that is associated with or arises, either directly or indirectly, as a result of pregnancy. These diseases or conditions can include any and all birth defects, congenital conditions, or hereditary diseases or conditions. Examples of prenatal or pregnancy -related diseases include, but are not limited to, Rhesus disease, hemolytic disease of the newborn, beta-thalassemia, sex determination, determination of pregnancy, a hereditary Mendelian genetic disorder, chromosomal aberrations, a fetal chromosomal aneuploidy, fetal chromosomal trisomy, fetal chromosomal monosomy, trisomy 8, trisomy 13 (Patau Syndrome), trisomy 16, trisomy 18 (Edwards syndrome), trisomy 21 (Down syndrome), X-chromosome linked disorders, trisomy X (XXX syndrome), monosomy X (Turner syndrome), XXY syndrome, XYY syndrome, XYY syndrome, XXXY syndrome, XXYY syndrome, XYYY syndrome, XXXXX syndrome, XXXXY syndrome, XXXYY syndrome, XXYYY syndrome, Fragile X Syndrome, fetal growth restriction, cystic fibrosis, a hemoglobinopathy, fetal death, fetal alcohol syndrome, sickle cell anemia, hemophilia, Klinefelter syndrome, dup(17)(pl 1.2pl.2) syndrome, endometriosis, Pelizaeus-Merzbacher disease, dup(22)(qll.2qll.2) syndrome, cat eye syndrome, cri-du-chat syndrome, Wolf-Hirschhorn syndrome, Williams-Beuren syndrome, Charcot-Marie-Tooth disease, neuropathy with liability to pressure palsies, Smith-Magenis syndrome, neurofibromatosis, Alagille syndrome, Velocardiofacial syndrome, DiGeorge syndrome, steroid sulfatase deficiency, Prader-Willi syndrome, Kallmann syndrome, microphthalmia with linear skin defects, adrenal hypoplasia, glycerol kinase deficiency, Pelizaeus-Merzbacher disease, testis-determining factor on Y, azospermia (factor a), azospermia (factor b), azospermia (factor c), 1 p36 deletion, phenylketonuria, Tay-Sachs disease, adrenal hyperplasia, Fanconi anemia, spinal muscular atrophy, Duchenne’s muscular dystrophy, Huntington’s disease, myotonic dystrophy, Robertsonian translocation, Angelman syndrome, tuberous sclerosis, ataxia telangieltasia, openENMRA-44283.601spina bifida, neural tube defects, ventral wall defects, small-for-gestational-age, congenital cytomegalovirus, achondroplasia, Marfan’s syndrome, congenital hypothyroidism, congenital toxoplasmosis, biotinidase deficiency, galactosemia, maple syrup urine disease, homocystinuria, medium-chain acyl Co-A dehydrogenase deficiency, structural birth defects, heart defects, abnormal limbs, club foot, anencephaly, arhinencephaly / holoprosencephaly, hydrocephaly, anophthalmos / microphthalmos, anotia / microtia, transposition of great vessels, tetralogy of Fallot, hypoplastic left heart syndrome, coarctation of aorta, cleft palate without cleft lip, cleft lip with or without cleft palate, oesophageal atresia / stenosis with or without fistula, small intestine atresia / stenosis, anorectal atresia / stenosis, hypospadias, indeterminate sex, renal agenesis, cystic kidney, preaxial polydactyly, limb reduction defects, diaphragmatic hernia, blindness, cataracts, visual problems, hearing loss, deafness, X-linked adrenoleukodystrophy, Rett syndrome, lysosomal disorders, cerebral palsy, autism, aglossia, albinism, ocular albinism, oculocutaneous albinism, gestational diabetes, Arnold-Chiari malformation, CHARGE syndrome, congenital diaphragmatic hernia, brachydactlia, aniridia, cleft foot and hand, heterochromia, Dwamian ear, Ehlers Danlos syndrome, epidermolysis bullosa, Gorham’s disease, Hashimoto’s syndrome, hydrops fetalis, hypotonia, Klippel-Feil syndrome, muscular dystrophy, osteogenesis imperfecta, progeria, Smith Lemli Opitz symdrom, chromatelopsia, X-linked lymphoproliferative disease, omphalocele, gastroschisis, pre-eclamp si a, eclampsia, pre-term labor, premature birth, miscarriage, delayed intrauterine growth, ectopic pregnancy, hyperemesis gravidarum, morning sickness, or likelihood for successful induction of labor.In some NIPT embodiments, the technology described herein further includes estimating a fetal fraction for a sample, wherein the fetal fraction is used to aid in the determination of whether the genetic data from the test subject is indicative of an aneuploidy. Methods for determining or calculating fetal fraction are known in the art.As used herein, the term “valid detection assay” refers to a detection assay that has been shown to accurately predict an association between the detection of a target and a phenotype (e.g., medical condition). Examples of valid detection assays include, but are not limited to, detection assays that, when a target is detected, accurately predict the phenotype medical 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, or 99.9% of the time. Other examples of valid detection assays include, but are not limited to, detection assays that qualify as and / or are marketed asENMRA-44283.601Analyte-Specific Reagents (i.e., as defined by FDA regulations) or In-Vitro Diagnostics (i.e., approved by the FDA).As used herein, the term “kit” refers to any delivery system for delivering materials. In the context of reaction assays, such delivery systems include systems that allow for the storage, transport, or delivery of reaction reagents (e.g, oligonucleotides, enzymes, etc. in the appropriate containers) and / or supporting materials (e.g, buffers, written instructions for performing the assay etc.) from one location to another. For example, kits include one or more enclosures (e.g., boxes) containing the relevant reaction reagents and / or supporting materials. As used herein, the term “fragmented kit” refers to a delivery system comprising two or more separate containers that each contain a subportion of the total kit components. The containers may be delivered to the intended recipient together or separately. For example, a first container may contain an enzyme for use in an assay, while a second container contains oligonucleotides. The term “fragmented kit” is intended to encompass kits containing Analyte specific reagents (ASR’s) regulated under section 520(e) of the Federal Food, Drug, and Cosmetic Act, but are not limited thereto. Indeed, any delivery system comprising two or more separate containers that each contains a subportion of the total kit components are included in the term “fragmented kit.” In contrast, a “combined kit” refers to a delivery system containing all of the components of a reaction assay in a single container (e.g., in a single box housing each of the desired components). The term “kit” includes both fragmented and combined kits.As used herein, the term “system” refers to two or more items, components, or articles, e.g., samples, reagents, instruments, containers, instruction information, used together for a particular purpose. In some embodiments, some, or all of the components of a system are provided together, e.g., in a kit, while in some embodiments, at least some of the components of the system are collected for use individually, e.g, by a user, for use together for a particular purpose.As used herein, the term “information” refers to any collection of facts or data. In reference to information stored or processed using a computer system(s), including but not limited to internets, the term refers to any data stored in any format (e.g, analog, digital, optical, < / < .). As used herein, the term “information related to a subject” refers to facts or data pertaining to a subject (c.g, a human, plant, or animal). The term “genomic information” refers to information pertaining to a genome including, but not limited to, nucleic acid sequences, genes,ENMRA-44283.601allele frequencies, RNA expression levels, protein expression, phenotypes correlating to genotypes, etc. “Allele frequency information” refers to facts or data pertaining allele frequencies, including, but not limited to, allele identities, statistical correlations between the presence of an allele and a characteristic of a subject (e.g., a human subject), the presence or absence of an allele in an individual or population, the percentage likelihood of an allele being present in an individual having one or more particular characteristics, etc.BRIEF DESCRIPTION OF THE DRAWINGSThe patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee.Fig. 1 provides a schematic diagram of single-stranded, unligated or “open circle” padlock probe (SEQ ID NO: 4) aligned with three different complementary blocker oligonucleotides (LSMs 1 (SEQ ID NO: 3), 2 (SEQ ID NO: 2) , and 3 (SEQ ID NO: 1)). The padlock probe comprises targeting arms (3). The linker between the targeting arms comprises a primer binding site (e.g., a complement of a “grafting primer” (indicated as cGP), a surfacebound primer that primes, for example, rolling circle replication) and a set-specific sequence common to all padlock probes within a set (shown here as “AF647,” in this instance referring to the wavelength of an ALEXA fluorophore associated with a set of padlock probes directed to a particular chromosome) and a universal insert sequence (e.g, a sequence shared by all padlock probes across some or all different probe sets).Fig. 2 provides a schematic diagram showing a padlock probe aligned (SEQ ID NO: 4) with 24 different variations of blocker oligonucleotides (LSMs (SEQ ID NOs: 3, 2, 1 and 5-25). For multiplexing, a multiplex PLP pool (e.g, >20k padlock probes) with numerous unique pairs of arm sequences that specifically hybridize to different target nucleic acids may share a common linker sequence between the Arml and Arm2. As shown, blocker oligonucleotides can be of different lengths, e.g, to hybridize to more or less of the linker and / or to have higher or lower melting temperatures with respect to the padlock probe, to, for example, allow different levels of structural control between the PLPs in the pool that share the same linker sequence.Fig. 3 provides a graph showing results of a negative control sample composed of a highly multiplexed pool of un-circularized padlock probes incubated at 60°C in a capture andENMRA-44283.601ligation reaction buffer (CL), in the absence of target DNA template and ligase, which results in increasing amounts of unwanted off-target detected objects that correlate with the incubation time.Fig. 4 provides a graph showing results from use of three different padlock probe pools, each producing signal one of three separate fluorophore channels (channel 1, 2, or 3) and targeting three different chromosomes. The different padlock probe pools were used together in a single capture and ligation reaction mixture with no DNA template (no-template ligation, or NTL). NTL reactions including either no blocker oligonucleotide (0 LSM), or 10 pM LSM1, LSM2, or LSM3 blocker oligonucleotides (see Fig. 2) were run in parallel. LSM1, LSM2, and LSM3 each only target the linker of the channel 3 PLP probe pool. These data show significant percentage drop in the number of detected objects (counts) in each well (53-62.5% drop) in channel 3, with only 10.7 to 28.6% drops in channels 1 and 2.Fig. 5 provides a table of the top performing blocker oligonucleotides from the variants diagrammed in Fig. 2. All LSMs reduced background signal (no-target ligations (NTLs) in negative controls), but some also significantly reduce total counts in samples that included target DNA (not shown). Acceptable LSMs significantly reduce NTL counts while retaining ability to detect specific targets, as shown for all LSMs in the table. Some LSMs (e.g., LSM23) decreased NTL counts while also increasing specific template detection in positive samples.Fig. 6 provides a graph examining the effects of the 24 variants of blocker oligonucleotides (Fig. 2), comparing LSM length to the percent reduction in counts in assays containing target and in NTL reactions. These data show that longer LSMs correlate with decreased assay performance.Fig. 7 provides graphs of data showing results when the primer binding site (shown as cGP in Figs. 1 and 2) of the linker sequence is used to bind circularized PLPs onto a surface. If blocking oligonucleotides are not successfully digested and removed from circularized probes, then they may reduce or prevent hybridization of the circles to oligonucleotides on the surface to the surface. It was determined that using a blocking oligonucleotide covering up to about 50% of the GP site still results in successful assay performance.Fig. 8 provides graphs showing results when blocker oligonucleotides are designed uniquely against the four different linker sequences of four sets of padlock probes (each set of padlock probes sharing the same linker sequence). The blocked padlock probe sets wereENMRA-44283.601combined and included in a capture and ligation reaction. Blue data points represent the result of adding LSMs to “no template” control samples, where a significant reduction in background signal is observed. In contrast, adding the same four-LSM combination mix to genomic DNA samples in order to target different chromosomal features showed no reduction in target-specific capture, and target DNA was detected by each of the four sets of blocked padlock probes.DETAILED DESCRIPTION OF THE INVENTIONAlthough the technology is discussed in reference to particular DNA capture and detection reactions, the technology is not limited to any particular type of, or any particular type of assay to be performed using circularizable probes.A goal in molecular diagnostics has been to achieve accurate, sensitive detection of analytes in as little time as possible with the least amount of labor and steps as possible. One manner in which this is achieved is the multiplex detection of analytes in samples, allowing multiple detection events in a single reaction vessel or solution. However, many of the existing diagnostic methods, including multiplex reactions,In development of the technology, it has been determined that padlock probes that are partially double-stranded, e.g, that have oligonucleotide hybridized to the linker portion of the linear padlock probe, exhibit decreased background in no-target control reactions. While not being limited to any particular mechanism of action, it is noted that duplexed DNA is much less flexible than single stranded DNA, and the presence of a duplexed region in a padlock probe decreases its flexibility, and the degree to which single-stranded portions of the padlock probe , e.g., the pair of targeting arms, may fold or otherwise interact or be reacted to produce background signal in the absence of a target nucleic acid.In technologies that produce circularized nucleic acid molecules, c'.g, for rolling circle amplification, an exemplary method for assaying the ligation of padlock probes that have been circularized by ligation may comprise the following steps:a) providing a ligation mixture comprising circularized nucleic acid probes and linear nucleic acids;b) treating the ligation mixture with at least one exonuclease, wherein circularized nucleic acid probes are not substrate for the at least one exonuclease;ENMRA-44283.601c) forming a plurality of complexes, each complex comprising an oligonucleotide primer hybridized to a circularized nucleic acid probe from the treated ligation mixture; andd) detecting formation of the plurality of complexes in a process comprising extending primers in the complexes in a rolling circle amplification (RCA) reaction to form RCA products.An exemplary assay for detecting RCA products may comprise;ii) hybridizing labeled probes to the RCA products, preferably wherein RCA products with hybridized labeled probes are localized to a support, e. ., at dispersed loci; andiii) counting RCA products at loci on the support, e.g., by microscopy.See, e.g., US Patent Nos. 11,230,731 and 11,186,863, each of which is incorporated herein by reference in its entirety, for all purposes.The technology relates to the field of nucleic acid detection methods in general and more particularly relates to the specific detection and counting of nucleic acids in a highly multiplexed detection assay, e.g., using greater than 10,000; 20,000; 30,000; 40,000; 50,000; 60,000; 70,000; 80,000; 90,000; preferably greater than 100,000 unique padlock probe sequences together. An aspect of using such a large numbers of different probes in a single reaction (a “highly multiplexed” reaction) is that intra- and intermolecular interactions between the different probes may generate detectable events or background signal, even when no target nucleic acids are present in the reaction, e.g., as shown in Fig. 3. Unwanted reactions among padlock probes or any other type of DNA probe reduces sensitivity.The technology provides compositions and methods that use auxiliary nucleic acids to modify and block portions of the padlock probes, decreasing background signal and increasing the accuracy of specific target detection and quantification. This technology comprises using oligonucleotides, termed linear structure modifier or “blocker” oligonucleotides, that partially complement and hybridize to single-stranded padlock probes, to, e.g., minimize formation of higher order structures (intramolecular folding, intermolecular hybridization) in the multiplexENMRA-44283.601probe mixture, thereby reducing background signal from the reaction. As used herein, a complex comprising a blocker oligonucleotide hybridized to a padlock probe, e.g., to a linker region of the padlock probe, is referred to as a blocked padlock probe.The sensitivity and specificity of multiplexed assays using padlock probes are derived in part through the judicious choice of oligonucleotide probes used together as a “probe set.” Despite careful design, a negative side effect of highly multiplexed probe assays (e.g., >100k unique padlock probes) is background noise attributed to non-specific hybridization of the padlock probe sequences to non-target molecules. For example, non-specific hybridization of any of the following may result in an increase in background noise: cross-hybridization of one or more PLPs with other PLPs; self-complementarity of PLPs; non-specific hybridization of PLPs to non-target sample DNA rather than to the target of interest. Non-specific hybridization of highly multiplexed PLP assays has the effect of reducing the sensitivity of the assay.In addition, long oligonucleotides are more prone to forming complex secondary structures and other higher order structures, and a greater the number of probes in a reaction mixture results in greater numbers of nonspecific interactions. To minimize higher order structure formation during probing reactions, linear structural modifiers (LSM), e.g., oligonucleotides that hybridize to the padlock probes, were designed to limit the number of structural configurations that cross-reactive single-stranded padlock probes might form. The technology reduces probe cross-reactivity and non-specific products.In embodiments of the technology, a single-stranded oligonucleotide, preferably shorter in length than a padlock probe, is hybridized to its complement within a padlock probe, e.g., in a padlock probe pool. See, e.g., Figs. 1 and 2, showing exemplary blocker oligonucleotides, (LSMs) aligned with an exemplary padlock probe sequence. Preferably, the blocker oligonucleotide hybridizes within the linker region of padlock probes in the PLP mixture, and is not complementary to the target-specific arms of the padlock probe. Preferably the blocker oligonucleotides are added to the padlock probes to create blocked padlock probes before the padlock probes are used in a target capture reaction.In certain embodiments, a population of different padlock probes containing different target-specific arms share a common region, e.g., in the linker portion, such that a single blocker probe can block hundreds or thousands (e.g., >20,000) of different padlock probes, resulting in the ability to reduce non-specific interactions. Designing multiple different blocker oligos, oneENMRA-44283.601for each unique linker used in a plurality of sets of padlock probes in which each padlock probe in a set of probes shares a common linker, and each different set of padlock probes uses a different linker, allows very high levels of multiplex assay design.In preferred embodiments, the technology provides blocked padlock probes, which are altered in structure to reduce or eliminate non-specific and other undesired hybridizations with other padlock probes in the reaction, effectively “inactivating” padlock probes for interactions other than the intended hybridization with the specific target for which they have been designed. Such inactivation refers to the reduced ability of the probe to hybridize to sequences other than their intended target sequence. As used herein, “inactivation” of probes does not require complete loss of non-specific hybridization, and reduction in non-specific hybridization is sufficient.In the presence of a complementary target nucleic acid, the blocked padlock probe can be circularized to form a PLP replicon suitable for detection. In some embodiments, the PLP is simply ligated using a nick repair enzyme, e.g., T4 DNA ligase, AMPLIGASE thermostable DNA ligase, etc., while in some embodiments closing of the probe to form a circle comprises additional modification of the probe to create a ligatable nick, e.g., cleavage of an overlap between the termini, filling of a gap between the termini using a nucleic acid polymerase, etc.A target site or sequence, as used herein, refers to a portion or region of a nucleic acid sequence that is sought to be sorted out from other nucleic acids in the sample that have other sequences, which is informative for determining the presence or absence of a genetic disorder or condition (e.g., the presence or absence of mutations, polymorphisms, deletions, insertions, aneuploidy etc.). A control site or sequence, as used herein, refers to a site that has known or normal copy numbers of a particular control gene. In some embodiments, the targeting PLPs comprise in sequence the following components: first targeting polynucleotide arm - first unique targeting molecular tag - polynucleotide linker - second unique targeting molecular tag - second targeting polynucleotide arm. In some embodiments, a target population of the targeting PLPs are used in the methods of the disclosure. In the target population, the pairs of the first and second targeting polynucleotide arms in each of the targeting PLPs are identical and are substantially complementary to first and second regions in the nucleic acid that, respectively, flank the target site. See, e.g., WO 2017 / 020023 and WO 2017 / 020024, each of which is incorporated herein by reference in its entirety.ENMRA-44283.601In some embodiments, the length of each of the targeting polynucleotide arms is between 18 and 35 base pairs. In some embodiments, the length of each of the targeting polynucleotide arms is 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 base pairs, or any size range between 18 and 35 base pairs. In some embodiments, the length of each of the control polynucleotide arms is between 18 and 35 base pairs. In some embodiments, the length of each of the control polynucleotide arms is 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 base pairs, or any size ranges between 18 and 35 base pairs. In some embodiments, each of the targeting polynucleotide arms has a melting temperature between 55°C and 70°C. In some embodiments, each of the targeting polynucleotide arms has a melting temperature at 56 °C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, or any temperature between 55°C and 70°C. In some embodiments, each of the control polynucleotide arms has a melting temperature between 55°C and 70°C. In some embodiments, each of the control polynucleotide arms has a melting temperature at 56°C, 57°C, 58°C, 59°C, 60°C, 61 °C, 62°C, 63 °C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, or any temperature between 55°C and 70°C.In some embodiments, each of the targeting polynucleotide arms has a GC content between 20% and 80%. In some embodiments, each of the targeting polynucleotide arms has a GC content of 20-30%, 30-40%, or 30-50%, or 30-60%, or 40-50%, or 40-60%, or 40-70%, or 50-60%, or 50-70%, or 50-80%, or any range of GC content between 20% and 80%, or any specific percentage between 20% and 80%. In some embodiments, each of the control polynucleotide arms has a GC content between 20% and 80%. In some embodiments, each of the control polynucleotide arms has a GC content of 20-30%, 30-40%, or 30-50%, or 30-60%, or 40-50%, or 40-60%, or 40-70%, or 50-60%, or 50-70%, or 50-80%, or any range of GC content between 20% and 80%, or any specific percentage between 20% and 80%.In some embodiments, the polynucleotide linker is not substantially complementary to any genomic region of the sample or the subject. In some embodiments, the polynucleotide linker has a length of 30 to 40 nucleotides. In some embodiments, the polynucleotide linker has a length of 31, 32, 33, 34, 35, 36, 37, 38, or 39 nucleotides, or any interval between 30 and 40 nucleotides, and including 30 or 40 nucleotides. In some embodiments, the polynucleotide linker has a melting temperature with respect to a complementary strand of between 60°C and 80°C. In some embodiments, the polynucleotide linker has a melting temperature of 60°C, 65°C, 70°C,ENMRA-44283.60175°C, or 80°C, or any interval between 60°C and 80°C, or any specific temperature between 60°C and 80°C. In some embodiments, the polynucleotide linker has a GC content between 40% and 60%. In some embodiments, the polynucleotide linker has a GC content of 40%, 45%, 50%, 55%, or 60%, or any interval between 40% and 60%, or any specific percentage between 40% and 60%.In certain embodiments, the methods described herein are used to detect exonic deletions or insertions or duplication. In some embodiments, the target site (or sequence) is a deletion or insertion or duplication in a gene of interest or a genomic region of interest. In some embodiments, the target site is a deletion or insertion or duplication in one or more exons of a gene of interest. In some embodiments, the target multiple exons are consecutive. In some embodiments, the target multiple exons are non-consecutive. In some embodiments, the first and second targeting polynucleotide arms of PLPs are designed to hybridize upstream and downstream of the deletion (or insertion, or duplication) or deleted (or inserted, or duplicated) genomic region (e.g, one or more exons) in a gene or a genomic region of interest. In some embodiments, the first or second targeting polynucleotide arm of PLPs comprises a sequence that is substantially complementary to the genomic region of a gene of interest that encompasses the target deletion or duplication site (e.g., exons or partial exons).Circular DNA molecules such as ligated PLPs are suitable substrates for amplification using rolling circle amplification (RCA). In certain embodiments of RCA, a rolling circle replication primer hybridizes to a circular nucleic acid molecule, e.g, a ligated PLP, or circularized cfDNA. Extension of the primer using a strand-displacing DNA polymerase (e.g., (p29 (Phi29), Bst Large Fragment, and Klenow fragment of E. coli Pol I DNA polymerases) results in long single-stranded DNA molecules containing repeats of a nucleic acid sequence complementary to the PLP circular molecule. In preferred embodiments, primers are immobilized, e.g, to a surface.A number of different “front-end” methods for recognizing target nucleic acid and producing a new product are described, for example, in WO 2019 / 195346 and WO 2020 / 206170, which are both incorporated herein by reference in their entireties for all purposes. In some embodiments, such methods comprise use of a “back end” technology for detecting and / or measuring production of the new product from the front-end method. In certain embodiments, back-end technologies are based on immobilizing nucleic acid on a surface, preferably a surfaceENMRA-44283.601coated with organic polymer. In preferred embodiments, technologies that comprise binding PLPs on a surface by hybridization to surface-immobilized oligonucleotide primers, and detecting the bound PLPs, e.g., using rolling circle amplification.The technology may be carried out in a single, “addition only” workflow reaction, allowing for rapid production of highly multiplexed targeted capture of chromosomal targets, blocker oligonucleotides are added to the master mix of the existing capture reaction (see Fig 4). One universal blocker oligonucleotide is designed and added for each set of PLPs that share a common linker. The data show that blocker oligonucleotides are specific for each linker (as shown by linker-specific channels of detection) and reduce non-templated ligations (NTLs) significantly (up to 70%; see Fig. 5). Furthermore, once a particular pool of PLPs is ‘inactivated’ from cross-reactivity, the pool is also unable to take part in cross-reactivity with padlock probes from another set having a different unique linker. As a result, NTLs across all channels is reduced, even if the blocker oligonucleotide targeted the linkers of probes for only one channel (see Fig. 4).During development of the technology, it was also determined that longer blocker oligonucleotide duplexes correlate with decreased assay performance (Fig. 6). While any length of blocker oligonucleotide may be used, including blocker oligonucleotides that may comprise 5' or 3' tails that are not complementary to the blocker-binding site in a linker in a pool of padlock probes, it was found that there is an optimal length of duplex and coverage aspect of the best performing LSMs in these data. While all LSMs decreased background noise, LSMs that formed longer duplexed regions in the blocked padlock probes also tended to reduce assay performance. It was found that LSMs work optimally when 50% or less of the primer binding site in the linker (cGP in Figs. 1 and 2) was double stranded (see Fig 7). Nonetheless, up to about 75% of the padlock probe linker sequence (e.g., as shown in Fig.2) can be duplexed to a blocker oligonucleotide and still achieve optimal performance (see Fig. 5).In some embodiments, reducing background noise generated from the whole diagnostic system comprises designing a different blocker oligonucleotide for each PLP pool, each of which has its own linker that dictates in which channel it is detected. The technology provided herein allows adding the different variants of blocker oligonucleotides only once, in a step at the beginning of the workflow, as a component of the target capture ligation reaction. For example, to mitigate high counts in no-DNA controls across four different PLP pools (e.g. a total of 80kENMRA-44283.601PLPs), four different blocker oligonucleotides can be designed, one for each pool, and the blocker oligonucleotides can be mixed together before use, or added separately before or after the different PLP pools are combined. A reduction in off-target object detection between 50-80% can be observed across all channels (see Fig. 8).In some embodiments, some LSMs can be designed to partially cover a junction between the linker and an arm, although the arms within a pool are highly heterogeneous. As such, mixed bases can be used selectively in the portion of such a blocking oligonucleotide on the arm side of the junction (see, e.g., LSM23 and LSM24 in Fig. 2).In some embodiments, the double-stranded region of a blocked padlock probe is preferably more stable (having a higher melting temperature, e.g.) than duplexes between the padlock probe and any mismatched target sequences. In this way, the double-stranded section of the blocked padlock probe will be thermodynamically favored over undesired 5' or 3' arm partial duplexes formed with non-target molecules.As discussed above, after ligation to circularize padlock probe, the ligation mixture may be treated with an exonuclease to remove linear nucleic acids, e.g., individual blocking oligonucleotides, unligated padlock probes, and unused blocked padlock probe complexes. Preferably a ligation reaction comprising blocked padlock probes is treated with cocktail of single-strand and double-strand specific exonucleases to which the circularized molecules are resistant.ENMRA-44283.601Additional materials incorporated herein by reference1. M. Nilsson, et al., Real-time monitoring of rolling-circle amplification using a modified molecular beacon design Nucleic Acids Research, 30(14):e66 (2002)2. M. Nilsson, et al. “Padlock probes: circularizing oligonucleotides for localized DNA detection.” Science. 265 (5181): 2085-2088 (1994)3. J. Baner, et al., Signal amplification of padlock probes by rolling circle replication Nucleic Acids Research 26 (22): 5073-5078 (1998)4. Paluzzi, VE et al., Near-Quantitative Preparation of Short Single-Stranded DNA Circles, Angew. Chem. Int. Ed. 2023, 62, e202218443;5. US Patent No. 5,854,033 toLizardi6. US Patent No. 6,221,603 toMahtani7. US Patent No. 11,230,731 to Sekedat, et al.8. US Patent No. 11,186,863 to Sekedat, et al.9. WO 2015 / 083002 “Multiplex Detection of Nucleic Acids”10. Zhang, DY., and Eiu, B., Detection of target nucleic acids and proteins by amplification of circularizable probes, Expert Rev. Mol. Diagn. 3(2):237-248 (2003)All literature and similar materials cited in this application, including the publications described in the Bibliography above, and including but not limited to patents, patent applications, articles, books, treatises, and internet web pages, are expressly incorporated by reference in their entireties for any purpose. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which the various embodiments described herein belong. When definitions of terms in incorporated references appear to differ from the definitions provided in the present teachings, the definition provided in the present teachings shall control.ENMRA-44283.601Various modifications and variations of the described compositions, methods, and uses of the technology will be apparent to those skilled in the art without departing from the scope and spirit of the technology as described. Although the technology has been described in connection with specific exemplary embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in molecular biology, molecular diagnostics, nucleic acid’s structure, biochemistry, medical science, or related fields are intended to be within the scope of the claims.

Claims

ENMRA-44283.601CLAIMSWe claim:

1. A composition comprising a first set of blocked padlock probes, each blocked padlock probe comprising in sequence a first targeting arm, a linker, and a second targeting arm, wherein the first set of blocked padlock probes comprises a population of padlock probe strands each comprising a different pair of first and second targeting arms, wherein each padlock probe strand in the population of padlock probes comprises a first common linker sequence hybridized to a first common blocker oligonucleotide.

2. The composition of claim 1, wherein in the first set of blocked padlock probes, 25% to 75%, preferably 30% to 70%, preferably 35% to 65%, preferably 40% to 60%, preferably 45% to 55%, preferably about 50% of the first common linker sequence is hybridized to a first blocker oligonucleotide in a duplex.

3. The composition of claim 1 or claim 2, further comprising a second set of blocked padlock probes, , wherein the second set of blocked padlock probes comprises a second population of padlock probe strands each comprising a different pair of first and second targeting arms, wherein each padlock probe strand in the second set of padlock probes comprises a second common linker sequence hybridized to a second common blocker oligonucleotide, wherein the second common linker sequence is not the same as the first common linker sequence.

4. The composition of claim 3, wherein in the second set of blocked padlock probes, 25% to 75%, preferably 30% to 70%, preferably 35% to 65%, preferably 40% to 60%, preferably 45% to 55%, preferably about 50% of the second common linker sequence is hybridized to a second blocker oligonucleotide in a duplex.

5. The composition of any one of claims 1 to 4, further comprising a sample comprising nucleic acid molecules.

6. The composition of any one of claims 1 to 5, further comprising a ligase.ENMRA-44283.6017. The composition of any one of claims 1 to 6, wherein the blocked padlock probes comprise circularized probes.

8. A method of making circularized padlock probes, comprising:a) providing a set of blocked padlock probes according to any one of claims 1 to 4 and a sample comprising nucleic acid molecules;b) hybridizing padlock probe strands of the set of blocked padlock probes to the nucleic acid molecules to generate hybridized padlocked probes; andc) in a reaction mixture, ligating hybridized padlock probe strands to form a plurality of circularized padlock probes.

9. The method of claim 8, further comprising treating the plurality of circularized padlock probes with at least one nuclease, preferably an exonuclease, wherein circularized padlock probes are not substrate for the at least one nuclease.

10. The method of claim 8 or claim 9, further comprisinga) forming at least one complex comprising an oligonucleotide primer hybridized to a circularized padlock probe,b) detecting formation of the at least one complex in a process comprising extending the primer in the complex in a rolling circle amplification (RCA) reaction to form RCA product.

11. The method of claim 10, wherein the oligonucleotide primer is attached to a solid support.

12. The method of claim 11, wherein the oligonucleotide primer is covalently attached to the solid support.

13. The method of claim 11 or claim 12, wherein the solid support comprises one or more of metal, glass, quartz, plastic, silicon, carbon, graphite, diamond, and ceramic.ENMRA-44283.60114. The method of claim 13, wherein the solid support comprises glass.

15. The method of any one of claims 11 to 14, wherein the solid support comprises a polymeric coating, wherein the oligonucleotide primer is attached to the polymeric coating.

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