LOYZER MELISSA (US)
ROCHE DIAGNOSTICS GMBH (DE)
ROCHE SEQUENCING SOLUTIONS INC (US)
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PATENT CLAIMS 1. A method of detecting a gene fusion in a nucleic acid sample, the method comprising (a) contacting the nucleic acid sample with (i) a nucleic acid polymerase having a polymerase activity and a strand displacement activity, and (ii) a compound having Formula (I): [Olig1]–([R1]o–[R2]p)q–[L1]t–[Z]–[L2]u–[W]v–[Olig2] (I), wherein o is 0 or 1; p is 0 or 1; q is 0 or 1; t is 0, 1 or 2; u is 0, 1 or 2; v is 0 or 1; R1 is an oligonucleotide having between about 1 and about 24 nucleotides; R2 is a substituted or unsubstituted, saturated or unsaturated, linear or cyclic aliphatic group having between 2 and about 48 carbon atoms, optionally substituted with one or more heteroatoms selected from O, N, or S; L1 and L2 are independently a substituted or unsubstituted, saturated or unsaturated, linear or cyclic aliphatic group having between 1 and about 16 carbon atoms, optionally including with one or more heteroatoms selected from O, N, or S, and optionally including one or more carbonyl groups; Z is a moiety selected from a triazole, a dihydropyridazine, a phosphate linkage, an amide linkage, a thioether linkage, an isooxazoline, a hydrozone, an oxime ether, and a chloro-s-triazine linkage; W is a substituted or unsubstituted, saturated or unsaturated, aliphatic or aromatic group having between 1 and about 12 carbon atoms, optionally substituted with one or more heteroatoms selected from O, N, S, provided that W includes at least one photocleavable, enzymatically cleavable, chemically cleavable, or pH sensitive group; Olig1 is an oligonucleotide comprising between about 1 and about 30 nucleotides and comprising an anchor sequence capable of hybridizing to a known fusion partner, and wherein Olig1 has a non-extendable 3' end; and Olig2 is an oligonucleotide comprising between about 1 and about 30 nucleotides and comprising an extendable 3' end; and (b) extending the 3’-end of Olig2 with the nucleic acid polymerase, wherein an extension product comprises a copy of a portion of an unknown fusion partner, a portion of the known fusion partner, and a fusion breakpoint, thereby forming a first strand copy of the gene fusion. 2. The method of claim 1, further comprising forming a library of double-stranded copies of the gene fusions; wherein the forming of the library comprises: attaching adaptors to copies of gene fusions wherein adaptors comprise barcodes and primer binding sites. 3. The method of claim 1, further comprising amplifying the copy of the gene fusion by a method comprising: (a) partitioning the sample comprising the copy of the gene fusion into a plurality of reaction volumes; wherein each reaction volume comprises a forward and a reverse amplification primers capable of hybridizing to the copy strand and the complement of the copy strand, and a first detectably-labeled probe; (b) performing an amplification reaction, wherein the reaction comprises a step of detection with the probe; (c) determining a number of reaction volumes where the probe has been detected thereby detecting the gene fusion. 4. A compound having Formula (I), [Olig1]–([R1]o–[R2]p)q–[L1]t–[Z]–[L2]u–[W]v–[Olig2] (I), wherein o is 0 or 1; p is 0 or 1; q is 0 or 1; t is 0, 1 or 2; u is 0, 1 or 2; v is 0 or 1; R1 is an oligonucleotide having between about 1 and about 24 nucleotides; R2 is a substituted or unsubstituted, saturated or unsaturated, linear or cyclic aliphatic group having between 2 and about 48 carbon atoms, optionally substituted with one or more heteroatoms selected from O, N, or S; L1 and L2 are independently a substituted or unsubstituted, saturated or unsaturated, linear or cyclic aliphatic group having between 1 and about 16 carbon atoms, optionally including with one or more heteroatoms selected from O, N, or S, and optionally including one or more carbonyl groups; Z is a moiety selected from a triazole, a dihydropyridazine, a phosphate linkage, an amide linkage, a thioether linkage, an isooxazoline, a hydrozone, an oxime ether, and a chloro-s-triazine linkage; W is a substituted or unsubstituted, saturated or unsaturated, aliphatic or aromatic group having between 1 and about 12 carbon atoms, optionally substituted with one or more heteroatoms selected from O, N, S, provided that W includes at least one photocleavable, enzymatically cleavable, chemically cleavable, or pH sensitive group; Olig1 is an oligonucleotide having between about 1 and about 30 nucleotides, and wherein Olig1 has a non-extendable 3' end; and Olig2 is an oligonucleotide having between about 1 and about 30 nucleotides, and wherein Olig 2 has an extendable 3' end. 5. The compound of claim 4, wherein R2 comprises a substituted or unsubstituted, saturated or unsaturated, linear or cyclic aliphatic group having between 2 and about 32 carbon atoms, optionally substituted with one or more heteroatoms selected from O, N, or S, and optionally including one or more carbonyl groups. 6. The compound of claim 4, wherein R2 comprises a moiety having the structure of Formula (IVA): wherein d and e are integers each independently ranging from 1 to 32; Q is a bond, O, S, N(Rc)(Rd) or a quaternary amine (N+H(Rc)(Rd)); Ra and Rb are independently H, a C1-C4 alkyl group, F, Cl, or N(Rc)(Rd); and Rc and Rd are independently CH3 or H. 7. The compound of claim 4, wherein R2 comprises a moiety having the structure of Formula (IVB): wherein d and e are integers each independently ranging from 1 to 32; Q is a bond, O, S, or N(Rc)(Rd); and Rc and Rd are independently CH3 or H. 8. The compound of claim 4, wherein at least one of L1 or L2 comprises a substituted or unsubstituted, saturated or unsaturated, linear or cyclic aliphatic group having between 1 and about 4 carbon atoms, optionally substituted with one or more heteroatoms selected from O, N, or S, and optionally including one or more carbonyl groups. 9. The compound of claim 4, wherein o + p = 1, and q is 1. 10. The compound of claim 4, wherein o is 0 and p and q are both 1, R1 comprises at least one PEG group, and L1 comprises at least one carbonyl moiety. 11. The compound of claim 4, wherein Olig2 comprises a barcode. 12. The compound of claim 4, wherein Olig2 comprises a universal primer binding site. 13. The compound of claim 4, wherein v is 0, and Olig2 includes a cleavage site including at least one uracil-containing nucleotide. 14. The compound of claim 4, wherein Olig2 comprises a random nucleotide sequence. 15. A kit or detecting gene fusions between a known fusion partner and an unknown fusion partner, the kit comprising the compound of any one of claims 30 – 63, and a polymerase. 16. A kit comprising: (a) a first compound having Formula (II): [Olig1]–([R1]o–[R2]p)q–[L1]t–[X] (II), wherein o is 0 or 1; p is 0 or 1; q is 1 or 2; t is 0, 1 or 2; R1 is an oligonucleotide having between 1 and about 24 nucleotides; R2 is a substituted or unsubstituted, saturated or unsaturated, linear or cyclic aliphatic group having between 2 and about 48 carbon atoms, optionally substituted with one or more heteroatoms selected from O, N, or S; L1 is a substituted or unsubstituted, saturated or unsaturated, linear or cyclic aliphatic group having between 1 and about 16 carbon atoms, optionally substituted with one or more heteroatoms selected from O, N, or S, and optionally including one or more carbonyl groups; X is a dibenzocyclooctyne, a trans-cyclooctene, an alkyne, an alkene, an azide, a tetrazine, a maleimide, a N-hydroxysuccinimide, a thiol, a 1,3-nitrone, an aldehyde, a ketone, a hydrazine, a hydroxylamine, an amino group, or a phosphoramidite; and Olig1 is an oligonucleotide having between about 1 to about 30 nucleotides; (b) a second compound having Formula (III): [Y]–[L2]u–[W]v–[Olig2] (III), wherein u is 0, 1 or 2; v is 0 or 1; Y is a dibenzocyclooctyne, a trans-cyclooctene, an alkyne, an alkene, an azide, a tetrazine, a maleimide, a N-hydroxysuccinimide, a thiol, a 1,3-nitrone, an aldehyde, a ketone, a hydrazine, a hydroxylamine, an amino group, or a phosphoramidite; L2 is a substituted or unsubstituted, saturated or unsaturated, linear or cyclic aliphatic group having between 1 and 16 carbon atoms, optionally including with one or more heteroatoms selected from O, N, or S, and optionally including one or more carbonyl groups; W is a substituted or unsubstituted, saturated or unsaturated, aliphatic or aromatic group having between 1 and 12 carbon atoms, optionally substituted with one or more heteroatoms selected from O, N, S, provided that W includes a photocleavable, enzymatically cleavable, chemically cleavable, or pH sensitive group; and Olig2 is an oligonucleotide having between about 1 and about 30 nucleotides. 17. Use of the compound of any of one of claims 4 - 14 or a kit of claim 15 – 15 in sequencing a nucleic acid molecule. |
[0245] In some embodiments, the groups Olig1 and Olig2 of Formulas (II) and (III) are prepared according to methods known to those of ordinary skill in the art. In some embodiments, the groups Olig1 and Olig2 are synthesized using solid-phase synthesis techniques employing phosphoramidite chemistry, (see, e.g., Protocols for Oligonucleotides and Analogs, Agrawal, S., ed., Humana Press, Totowa, N.J., 1993, hereby incorporated by reference in its entirety). Other methods of synthesizing Olig1, Olig2, and/or the compounds of Formulas (II) and (III) are described in U.S. Patent Nos.5,955,591, 6,057,431, 8,889,843, and 6,124,445; and in U.S. Patent Publication Nos. 2008/0119645 and 2003/0153743, the disclosures of which are hereby incorporated by reference herein in their entireties. [0246] In some embodiments, the first step in such a process is the attachment of a first monomer or higher order subunit containing a protected 5′-hydroxyl to a solid support, usually through a linker, using standard methods and procedures known in the art. See for example, Oligonucleotides and Analogues A Practical Approach, Ekstein, F. Ed., IRL Press, N.Y, 1991. The support-bound monomer or higher order first synthon is then treated to remove the 5′-protecting group. In some embodiments, this is accomplished by treatment with acid. In some embodiments, the solid support bound monomer is then reacted with a phosphoramidite to form a phosphite linkage. In some embodiments, the phosphite-containing compounds are oxidized to produce compounds having a desired internucleotide linkage. In some embodiments, the choice of oxidizing agent will determine whether the phosphite linkage will be oxidized to, for example, a phosphotriester, thiophosphotriester, or a dithiophosphotriester linkage. [0247] In some embodiments, a capping step is performed either prior to or after oxidation of the phosphite triester, thiophosphite triester, or dithiophosphite triester. In some embodiments, the capping step involves attachment of a "cap" moiety to oligonucleotide chains that have not reacted in a given coupling cycle. The cap moiety, in some embodiments, is reactive with the terminal portion of oligonucleotides that did not participate in the coupling cycle but is not reactive with oligonucleotides that did participate and, moreover, is not itself reactive with the coupling reagents. [0248] Further treatment of the oxidized oligomer with an acid removes the 5′-hydroxyl protecting group, and thus transforms the solid support bound oligomer into a further compound which may be subsequently reacted to begin the next synthetic iteration. This process is repeated until an oligomer of desired length is produced. [0249] In some embodiments, the compounds of Formula (II) and (III) may be reacted to form a compound of Formula (I). In these embodiments, a 5' to 5' linkage may be formed between the compounds of Formula (II) and those of Formula (II). In some embodiments, compounds having Formula (II) are synthesized, such as using the procedures described above, in the 3' to 5' direction. Such a synthesis may be carried out using 3' amidites. [0250] The compounds of Formula (III) may also be synthesized in a similar manner but using 5' amidites instead of 3' amidites. Non-limiting examples of 5' amidites are set forth below. In this manner, the compounds of Formula (III) may be synthesized in the 5' to 3' direction. In some embodiments, the compounds of Formulas (II) and (III) may be linked through a phosphate linkage. [0251] In some embodiments, the compounds of Formula (II) and Formula (III) may be reacted with each other using "click chemistry." "Click chemistry" is a chemical philosophy, independently defined by the groups of Sharpless and Meldal, that describes chemistry tailored to generate substances quickly and reliably by joining small units together. "Click chemistry" has been applied to a collection of reliable and self-directed organic reactions (Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Angew). Chem. Int. Ed. 2001, 40, 2004-2021). For example, the identification of the copper catalyzed azide-alkyne [3+2] cycloaddition as a highly reliable molecular connection in water (Rostovtsev, V. V.; et al. Angew. Chem. Int. Ed.2002, 41, 2596- 2599) has been used to augment several types of investigations of biomolecular interactions (Wang, Q.; et al. J. Am. Chem. Soc.2003, 125, 3192-3193; Speers, A. E.; et al. J. Am. Chem. Soc. 2003, 125, 4686-4687; Link, A. J.; Tirrell, D. A. J. Am. Chem. Soc. 2003, 125, 11164-11165; Deiters, A.; et al. J. Am. Chem. Soc.2003, 125, 11782-11783). In addition, applications to organic synthesis (Lee, L. V.; et al. J. Am. Chem. Soc.2003, 125, 9588-9589), drug discovery (Kolb, H. C.; Sharpless, K. B. Drug Disc. Today 2003, 8, 1128-1137; Lewis, W. G.; et al. Angew. Chem. Int. Ed. 2002, 41, 1053-1057), and the functionalization of surfaces (Meng, J.-C.; et al. Angew. Chem. Int. Ed.2004, 43, 1255-1260; Fazio, F.; et al. J. Am. Chem. Soc.2002, 124, 14397-14402; Collman, J. P.; et al. Langmuir 2004, ASAP, in press; Lummerstorfer, T.; Hoffmann, H. J. Phys. Chem. B 2004, in press) have also appeared. [0252] In some embodiments, precursors of the compounds of Formula (II) are first modified to introduce a first member of a pair of reactive functional groups capable of participating in a "click chemistry" reaction. Likewise, in some embodiments, precursors of the compounds of Formula (III) are modified to introduce a second member of the pair of reactive functional groups capable of participating in a "click chemistry" reaction. In some embodiments, the first and second members of the pair of reactive functional groups capable of participating in a "click chemistry" reaction are identified in Table 1. In some embodiments, the "click chemistry" reaction is catalyzed with an introduced reagent. In some embodiments, the introduced reagent is Cu + . [0253] By way of example only, a precursor to a compound of Formula (II) may be modified to introduce a primary halogen. Subsequently, sodium azide may be introduced, which reacts with the primary halogen such that the precursor to the compound of Formula (II) is converted to the azide. In some embodiments, the precursor to the compound of Formula (II) is reacted with an amidite including the primary halogen either directly or indirectly through a linker. A non-limiting example of a suitable amidite is illustrated below: [0254] Again by way of example, a precursor to a compound of Formula (III) may be modified (such as with an amidite) to introduce a moiety which is reactive with the azide of Formula (II), such as a moiety including an alkyl group. Non-limiting examples of suitable amidites are provided below:
[0255] Another suitable reagent is DBCO-PEG-Phosphoramidite, such as DBCO-PEG4- Phosphoramidite: [0256] The resulting compounds of Formula (II) and (III), each bearing a member of the reactive groups capable of participating in a "click chemistry" reaction, are then allowed to react with each other to form the 5' to 5' linkage. In the example provided above, the azide and alkyne will reactive to form a triazole linkage. [0257] In some embodiments, the compounds of Formulas (II) and (III) may each include reactive groups (X and Y, respectively) that facilitate the formation of an amide linkage between the compounds. To achieve this, in some embodiments precursors to the compounds of each of Formulas (II) and (III) may be reacted with a reagent which introduces the groups X and Y, respectively. In these embodiments, a precursor to a compound having Formula (II) is modified with an amino moiety at a 5' end. For instance, an amidite may be introduced to a precursor of a compound having Formula (II), where the amidite includes a terminal amino moiety. Non-limiting examples of such amidite reagents include the following: [0258] Similarly, a precursor to a compound having Formula (III) may also be modified at a 5' end to terminate in a carboxyl group. For instance, an amidite may be introduced to a precursor of a compound having Formula (III), where the amidite includes a terminal carboxyl moiety. A non-limiting example of such an amidite reagent is: [0259] In some embodiments, the compounds of Formulas (II) and (III) may each include reactive groups (X and Y, respectively) that facilitate the formation of a thioether linkage between the compounds. To achieve this, in some embodiments precursors to the compounds of each of Formulas (II) and (III) may be reacted with a reagent which introduces the groups X and Y, respectively. In these embodiments, a precursor to a compound having Formula (II) is modified with a thiol moiety at a 5' end. For instance, an amidite may be introduced to a precursor to a compound having Formula (II), where the amidite includes a terminal thiol moiety. Non-limiting examples of such amidite reagents include the following: [0260] A compound having Formula (III) may also be modified at a 5' end to terminate in a maleimide group. For instance, an amidite may be introduced to a precursor to a compound having Formula (III), where the amidite includes a terminal maleimide moiety. A non-limiting example of such an amidite reagent is: [0261] In some embodiments, the compounds of Formulas (II) and (III) may each include reactive groups (X and Y, respectively) that facilitate the formation of a triazine linkage between the compounds. To achieve this, in some embodiments precursors to the compounds of each of Formulas (II) and (III) may be reacted with a reagent which introduces the groups X and Y, respectively. In some embodiments, the triazine linkage is a chloro-s-triazine linkage. In these embodiments, a precursor to a compound having Formula (II) is modified with an amino moiety at a 5' end. Likewise, a precursor to a compound having Formula (III) is modified with an amino moiety at a 5' end. Non-limiting examples of suitable amidites for introducing such a 5' amino group are set forth below: [0262] Following the modification of both the precursor to the compound of Formula (II) and the precursor to the compound of Formula (III), the formed compounds of Formula (II) and (III) are then reacted with a coupling reagent. In some embodiments, the coupling reagent is s- trichlorotriazine. This reaction is illustrated below: [0263] In some embodiments, any precursor of a compound of Formula (II) or (III) may be reacted to introduce a linker or spacer, such as a PEG-based linker or spacer. A non-limiting example of a suitable reagent to introduce a PEG-based linker or spacer is set forth below: [0264] Other reagents and methods for incorporating a PEG-based linker or spacer into the precursors of the compounds of Formulas (II) and/or (III) are described in U.S. Patent Publication No. 2006/0063147, the disclosure of which is hereby incorporated by reference herein in its entirety. [0265] In some embodiments, any precursor to a compound of Formula (II) or (III) may be reacted to introduce a linker or spacer, such as a linker or spacer including a cleavable group. Non-limiting examples of a suitable reagents are set forth below: [0266] KITS [0267] Another aspect of the present disclosure are kits, such as kits including one or more of the compounds of Formula (I). In some embodiments, the kit includes one or more compounds of Formula (I) and a polymerase. In some embodiments, the polymerase is a DNA polymerase. In some embodiments, the DNA polymerase is a thermostable DNA-dependent DAN polymerase. The kit may further include amplification primers. In some embodiments, the kit further comprises at least one of a forward primer and/or a reverse primer. In some embodiments, the kit includes a forward primer capable of hybridizing to a copy of the first oligonucleotide and a reverse primer capable of hybridizing to the second oligonucleotide. In other embodiments, the kit includes a forward primer capable of hybridizing to the first oligonucleotide and a reverse primer capable of hybridizing to a copy of the second oligonucleotide. [0268] In other embodiments, a kit may include one or more of the compounds of Formulas (I), (II), or (III) and one or more buffers. In some embodiments, a kit comprises one or more compounds of Formula (I) and a master mix. In some embodiments, the master mix includes two or more of an enzyme, a buffer, a cofactor (e.g. MgCl2 or MgSO4), water, and dNTPs. In some embodiments, the master mix further includes template DNA. [0269] In other embodiments, a kit may include a compound of Formula (II) and a compound of Formula (III). In some embodiments, the compound of Formula (II) includes a first reactive group capable of reacting with a second reactive group of the compound of Formula (III). [0270] In some embodiments, the first reactive group comprises an alkyne moiety; and the second reactive group comprises an azide moiety. In some embodiments, the alkyne moiety is DBCO. In some embodiments, the first reactive group comprises a maleimide moiety; and the second reactive group comprises a thiol moiety. In some embodiments, the first reactive group comprises an alkene moiety and the second reactive group comprises a tetrazine moiety. In some embodiments, both the first and second reactive groups are amino moieties, and wherein the kit further comprises s-trichlorotriazine. [0271] In some embodiments, any of the compounds of Formulas (I), (II), and/or (III) may be included in a reaction vessel, together with one or more additional components. As used herein, the term "reaction vessel" generally refers to any container, chamber, device, or assembly, in which a reaction can occur in accordance with the present teachings. In some embodiments, the reaction vessel includes a well of a dPCR chip. In some embodiments, dPCR chips may include, for example, a silicon substrate etched with nano-scale or smaller reaction wells. In some embodiments, a dPCR chip has a low thermal mass. For example, the chip may be constructed of thin, highly conductive materials that do not store heat energy. In some embodiments, a dPCR chip has a surface area of from about 50 mm 2 to about 150 mm 2 . In some embodiments a dPCR chip has a surface area of about 100 mm 2 . Limiting the surface area may allow for greater uniformity of heating of the chip during melt analysis and a reduction in run-to-run variation in the melt cure analysis, a reduction in errors in melt curve generation, and increased discrimination of melt curves in the analysis. Other dPCR chips are describes in PCT Publication No. WO/2016/133783, the disclosure of which is hereby incorporated by reference herein in its entirety. [0272] METHODS [0273] Another aspect of the present disclosure is a method of detecting one or more gene fusions where one fusion partner is unknown. In some embodiments, the methods utilize one or more of the compounds of Formula (I). In some embodiments, the method further comprises, amplifying nucleic acids and/or forming a library of amplified nucleic acids. In some embodiments, the method further comprises sequencing a library of amplified nucleic acids thereby detecting one or more genomic rearrangements in the sample. These and other steps of the method are described herein. [0274] Gene fusions are a common occurrence in cancer. Clinical tests for gene fusions enable detection and diagnosis of cancer, tracking tumor burden over time, and developing an individualized treatment protocol for a cancer patient. Of special utility are blood-based methods of detecting gene fusions. Blood based methods access patient's cell-free nucleic acids (cfDNA and cfRNA), which includes circulating tumor nucleic acids (ctDNA and ctRNA). While blood- based tests are less invasive than a biopsy, the major difficultly is detecting very small amounts of tumor-derived nucleic acid mixed with normal, non-tumor derived nucleic acid. Several commercially available tests are able to detect mutations in ctDNA including single nucleotide variations (SNVs), copy number variations (CNVs) and gene fusions (e.g., AVENIO ctDNA Test Kit, Roche Sequencing Solutions, Pleasanton, Cal.) [0275] For some cancer-related gene fusions, detecting fusion products in ctDNA is further complicated by occurrence of multiple fusion partners. Tumor related genes with promiscuous fusions include many examples such as NTRK 1, 2 and 3, and FGFR 2 and 3. [0276] Sample [0277] The methods of the present disclosures utilizes a sample containing one or more nucleic acids, including one or more target nucleic acids. In some embodiments, the sample is derived from a subject or a patient. In some embodiments the sample may comprise a fragment of a solid tissue or a solid tumor derived from the subject or the patient, e.g., by biopsy. The sample may also comprise body fluids (e.g., urine, sputum, serum, plasma or lymph, saliva, sputum, sweat, tear, cerebrospinal fluid, amniotic fluid, synovial fluid, pericardial fluid, peritoneal fluid, pleural fluid, cystic fluid, bile, gastric fluid, intestinal fluid, or fecal samples). The sample may comprise whole blood or blood fractions where normal or tumor cells may be present. In some embodiments, the sample, especially a liquid sample may comprise cell-free material such as cell-free DNA or RNA including cell-free tumor DNA or tumor RNA of cell-free fetal DNA or fetal RNA. In some embodiments, the sample is a cell-free sample, e.g., cell-free blood-derived sample where cell-free tumor DNA or tumor RNA or cell-free fetal DNA or fetal RNA are present. In other embodiments, the sample is a cultured sample, e.g., a culture or culture supernatant containing or suspected to contain nucleic acids derived from the cells in the culture. [0278] In some embodiments, the sample is a representative sample. In some embodiments, the representative sample is prepared from a tumor sample, a lymph node sample, a blood sample, and/or other tissue samples which are homogenized (alone or together). "Homogenization" refers to a process (such as a mechanical process and/or a biochemical process) whereby a biological sample is brought to a state such that all fractions of the sample are equal in composition. Representative samples (as defined herein) may be prepared by removal of a portion of a sample that has been homogenized. A homogenized sample (a "homogenate") is mixed well such that removing a portion of the sample (an aliquot) does not substantially alter the overall make-up of the sample remaining and the components of the aliquot removed is substantially identical to the components of the sample remaining. In the present disclosure the "homogenization" will in general preserve the integrity of the majority of the cells within the sample, e.g., at least 50% of the cells in the sample will not be ruptured or lysed as a result of the homogenization process. In other embodiments, homogenization will preserve the integrity of at least 80% of the cells in the sample. In other embodiments, homogenization will preserve the integrity of at least 85% of the cells in the sample. In other embodiments, homogenization will preserve the integrity of at least 90% of the cells in the sample. In other embodiments, homogenization will preserve the integrity of at least 95% of the cells in the sample. In other embodiments, homogenization will preserve the integrity of at least 96 of the cells in the sample. In other embodiments, homogenization will preserve the integrity of at least 97% of the cells in the sample. In other embodiments, homogenization will preserve the integrity of at least 98% of the cells in the sample. In other embodiments, homogenization will preserve the integrity of at least 99% of the cells in the sample. In other embodiments, homogenization will preserve the integrity of at least 99.9% of cells in the same. The homogenates may be substantially dissociated into individual cells (or clusters of cells) and the resultant homogenate or homogenates are substantially homogeneous (consisting of or composed of similar elements or uniform throughout). [0279] In some embodiments, the input sample comprises a representative sample of cells derived from a tumor sample, lymph node sample, blood sample, or any combination thereof. In some embodiments, the input sample is derived from a human patient or mammalian subject (i) diagnosed with cancer, (ii) suspected of having cancer, (iii) at risk of developing cancer; (iv) at risk of relapse or recurrence of cancer; and/or (v) suspected of having cancer recurrence. In other embodiments, the input sample is derived from a healthy human patient or mammalian subject. Additional methods of generating representative samples and/or preparing representative samples for downstream processing are described in PCT Application No. PCT/US19/62857, the disclosure of which is hereby incorporated by reference herein in its entirety. [0280] Target Nucleic Acids [0281] Target nucleic acids are the nucleic acid of interest that may be present in the sample. Each target is characterized by its nucleic acid sequence. The present disclosure enables detection of one or more RNA or DNA targets. In some embodiments, the DNA target nucleic acid is a gene or a gene fragment (including exons and introns) involved in a fusion event or an intergenic region where a fusion breakpoint is located. The RNA target nucleic acid is a transcript or a portion of the transcript of a gene or coding sequence resulting from fusion. In some embodiments, the target nucleic acid comprises a biomarker, i.e., a gene whose variants such as gene fusion are associated with a disease or condition. For example, the target nucleic acids can be selected from panels of disease-relevant markers described in U.S. Patent Application Ser. No. 14/774,518 filed on September 10, 2015. Such panels are available as AVENIO ctDNA Analysis kits (Roche Sequencing Solutions, Pleasanton, Cal.) [0282] Of special interest are target genes known to undergo gene fusions in tumors. For example, ALK, RET, ROS, FGFR2, FGFR3 and NTRK1 are known to undergo fusions resulting in an abnormally active kinase phenotype. Other genes known or expected to undergo fusions relevant for cancer include ALK, PPARG, BRAF, EGFR, FGFR1, FGFR2, FGFR3, MET, NRG1, NTRK1, NTRK2, NTRK3, RET, ROS1, AXL, PDGFRA, PDGFB , ABL1, ABL2, AKT1, AKT2, AKT3, ARHGAP26, BRD3, BRD4, CRLF2, CSF1R, EPOR, ERBB2, ERBB4, ERG, ESR1, ESRRA, ETV1, ETV4, ETV5, ETV6, EWSR1, FGR, IL2RB, INSR, JAK1, JAK2, JAK3, KIT, MAML2, MAST1, MAST2, MSMB, MUSK, MYB, MYC, NOTCH1, NOTCH2, NUMBL, NUT, PDGFRB, PIK3CA, PKN1, PRKCA, PRKCB, PTK2B, RAF1, RARA, RELA, RSPO2, RSPO3, SYK, TERT, TFE3, TFEB, THADA, TMPRSS2, TSLP, TY, BCL2, BCL6, BCR, CAMTA1, CBFB, CCNB3, CCND1, CIC, CRFL2, DUSP22, EPC1, FOXO1, FUS, GLI1, GLIS2, HMGA2, JAZF1, KMT2A, MALT1, MEAF6, MECOM, MKL1, MKL2, MTB, NCOA2, NUP214, NUP98, PAX5, PDGFB, PICALM, PLAG1, RBM15, RUNX1, RUNX1T1, SS18, STAT6, TAF15, TAL1, TCF12, TCF3, TFG, TYK2, USP6, YWHAE, AR, BRCA1, BRCA2, CDKN2A, ERB84, FLT3, KRAS, MDM4, MYBL1, NF1, NOTCH4, NUTM1, PRKACA, PRKACB, PTEN, RAD51B, and RB1. [0283] In some embodiments, the target nucleic acid is RNA (including mRNA). In such embodiments, the DNA polymerase extending the compound of Formula (I) is a reverse transcriptase. In other embodiments, the target nucleic acid is DNA, including cellular DNA or cell-free DNA (cfDNA) including circulating tumor DNA (ctDNA) and cell-free fetal DNA. In such embodiments, the DNA polymerase extending the compound of Formula (I) is any DNA polymerase, e.g. any B family DNA polymerase. The target nucleic acid may be present in a short or long form. In some embodiments, longer target nucleic acids are fragmented by enzymatic or physical treatment as described below. In some embodiments, the target nucleic acid is naturally fragmented, e.g., includes circulating cell-free DNA (cfDNA) or chemically degraded DNA such as the one found in chemically preserved or ancient samples. In some embodiments, the ctDNA or cfDNA is derived from a representative sample (see PCT Application No. PCT/US19/62857, the disclosure of which is hereby incorporated by reference herein in its entirety). [0284] DNA Isolation [0285] In some embodiments, the method of the present disclosure comprises a step of isolating nucleic acids. Generally, any method of nucleic acid extraction that yields isolated nucleic acids comprising DNA, RNA or a mixture of DNA and RNA may be used. Genomic DNA or cellular RNA or a mixture of DNA and RNA may be extracted from tissues, cells, or liquid biopsy samples (including blood or plasma samples) using solution-based or solid-phase based nucleic acid extraction techniques. Nucleic acid extraction can include detergent-based cell lysis, denaturation of nucleoproteins, and optionally removal of contaminants. Extraction of nucleic acids from preserved samples may further include a step of deparaffinization. Solution based nucleic acid extraction methods may comprise salting out methods or organic solvent or chaotropic methods. Solid-phase nucleic extraction methods can include but are not limited to silica resin methods, anion exchange methods or magnetic glass particles and paramagnetic beads (KAPA Pure Beads, Roche Sequencing Solutions, Pleasanton, Cal.) or AMPure beads (Beckman Coulter, Brea, Cal.) [0286] A typical extraction method involves lysis of tissue material and cells present in the sample. Nucleic acids released from the lysed cells can be bound to a solid support (beads or particles) present in solution or in a column, or membrane where the nucleic acids may undergo one or more washing steps to remove contaminants including proteins, lipids and fragments thereof from the sample. Finally, the bound nucleic acids can be released from the solid support, column or membrane and stored in an appropriate buffer until ready for further processing. Because both DNA and RNA must be isolated, no nucleases may be used, and care should be taken to inhibit any nuclease activity during the purification process. [0287] In some embodiments, nucleic acid isolation utilizes epitachophoresis (ETP) as described in PCT/EP2019/077714 filed on October 14, 2019 and PCT/EP2018/081049 filed on November 13, 2018. ETP utilizes a device with a circular arrangement of electrodes where the nucleic acid migrates and concentrates between a leading electrolyte and a trailing electrolyte. The circular configuration allows concentrating nucleic acids in a very small volume collected in the center of the device. The use of ETP is especially advantageous for blood plasma samples containing small amounts of cell-free nucleic acid in a large volume. [0288] In some embodiments, the input DNA or input RNA require fragmentation. In such embodiments, RNA may be fragmented by a combination of heat and metal ions, e.g., magnesium. In some embodiments, the sample is heated to 85°-94°C for 1-6 minutes in the presence of magnesium. (KAPA RNA HyperPrep Kit, KAPA Biosystems, Wilmington, Mass). DNA can be fragmented by physical means, e.g., sonication, using commercially available instruments (Covaris, Woburn. Mass.) or enzymatic means (KAPA Fragmentase Kit, KAPA Biosystems). [0289] In some embodiments, the DNA repair enzymes target damaged bases in the isolated nucleic acids. In some embodiments, sample nucleic acid is partially damaged DNA from preserved samples, e.g., formalin-fixed paraffin embedded (FFPET) samples. Deamination and oxidation of bases can result in an erroneous base read during the sequencing process. In some embodiments, the damaged DNA is treated with uracil N-DNA glycosylase (UNG/UDG) and/or 8-oxoguanine DNA glycosylase. [0290] The methods of the present disclosure are applicable to multiple different types of nucleic acids. In some embodiments, the methods of the present disclosure utilizes isolated DNA (i.e., DNA separated from RNA by RNase digestion). In some embodiments, the methods of the present disclosure utilizes isolated RNA (i.e., RNA separated from DNA by DNase digestion). In yet other embodiments, the methods of the present disclosure utilizes a mixture of DNA and RNA (i.e., isolated nucleic acids not treated with a nuclease). [0291] Enrichment [0292] In some embodiments, the methods of the present disclosure further comprises a step of target enrichment. In some embodiments, the method utilizes a pool of oligonucleotide probes (e.g., capture probes). In some embodiments, enrichment is by subtraction in which case capture probes are capable of hybridizing to abundant undesired sequences including ribosomal RNA (rRNA) or abundantly expressed genes (e.g., globin). In the case of subtraction, the undesired sequences are captured by the capture probes, removed from the solution of target nucleic acids and discarded. Removal may be accomplished by utilizing capture probes with a binding moiety that can be captured on solid support. In other embodiments, enrichment is by retention in which case, capture probes are capable of hybridizing to one or more target sequences, i.e., known sequences of the fusion partner genes. In some embodiments, the target sequences are hybridized to gene-specific capture probes and removed from the solution, e.g., utilizing capture probes with a binding moiety that can be captured on solid support. The captured target-probe hybrids are retained while the remainder of the solution containing non-target sequences is discarded. [0293] For enrichment, the capture probes may be free in solution or fixed to solid support. The probes may also comprise a binding moiety (e.g., biotin) and be capable of being captured on solid support (e.g., avidin or streptavidin containing support material). [0294] Contacting the Sample or Target Enriched Sample with a Linked Primer, Such as with a Compound of Formula (I) [0295] Referring to Figure 1 (bottom) and Figure 2, the present disclosure provides a method of detecting a gene fusion by contacting a sample with a linked primer (such as any of those of Formula (I). In some embodiments, the linked primer comprise a first oligonucleotide sequence (e.g. "Olig1" of Formula (I)) coupled directly or indirectly through a linkage (e.g. group "Z" of Formula (I)) to a second oligonucleotide sequence (e.g. "Olig2" of Formula (I)). In some embodiments, and as depicted in Figure 1, the linked primer comprises a first oligonucleotide sequence (left side, "Olig1" of Formula (I)) which includes an anchor sequence capable of hybridizing to a known 5'-fusion partner. The linked primer also includes a "Spacer" (e.g. the group "–([R 1 ]o–[R 2 ]p)q–" of Formula (I)). The second oligonucleotide (right side, "Olig2" of Formula (I)) comprises a random sequence ("NNN") and an extendable 3'-end. [0296] As shown in Figure 1 (bottom), the sample is contacted with a nucleic acid polymerase having a polymerase activity and a strand displacement activity ("POL"). In some embodiments, the nucleic acid in the sample is DNA and a DNA-dependent DNA polymerase is used, e.g., any B-family polymerase with a strand displacement activity. In some embodiments, the nucleic acid in the sample is RNA and a reverse transcriptase is used. [0297] In some embodiments, the nucleic acid in the sample is a mixture of DNA and RNA. Such a sample can be processed to target DNA and RNA in a single tube using the method described in U.S. Provisional Application Ser. No. 62/888963 “Single tube preparation of DNA and RNA for sequencing,” filed on August 19, 2019 and incorporated herein by reference. Briefly, the method described comprises forming cDNA with a first primer having a tag identifying the RNA starting material under conditions where DNA starting material is not reactive. After cDNA is formed, target cDNA is amplified and detected along with the target DNA by a common set of amplification primers not including the first primer. Final products originating from RNA are distinguished from final products originating from DNA by the presence of the RNA–specific tag (“RNA-identifying tag”) introduced by the first primer. In some embodiments, the 5'-portion of the second oligonucleotide (e.g. "Olig2" of Formula (I)) includes an RNA-identifying tag. [0298] In some embodiments, the polymerase extends the 3'-end of the second oligonucleotide (e.g. "Olig2" of Formula (I)) while displacing the anchor sequence of the first oligonucleotide (e.g. "Olig1" of Formula (I)) hybridized to the known sequence of the known gene fusion partner. (Figure 1, bottom). In some embodiments, the extension product, referred to as a first copy strand, contains a copy of a portion of the 3'-fusion partner and a portion of the 5'-fusion partner, thereby forming a first strand copy of the gene fusion. [0299] In some embodiments, the first copy strand is copied to form a second copy thereby forming a double-stranded copy of the gene fusion. In some embodiments, a primer complementary to a sequence in a known fusion partner can be used to form the second copy strand. In some embodiments, this primer is also an amplification primer. In some embodiments, this primer comprises one or more additional features in the 5'-portion selected from a sample barcode, a molecular barcode, a universal primer binding site, and a sequencing platform-specific primer binding site. [0300] In some embodiments, it is desirable to remove the first oligonucleotide (e.g. "Olig1"of Formula (I)) from the first copy strand. In some embodiments, a group (e.g. the group "W" of Formula (I)) between the first and second oligonucleotides (e.g. "Olig1" and "Olig2" of Formula (I) includes a cleavable moiety. In some embodiments, the cleavable linker is selected from a photocleavable, enzymatically cleavable, chemically cleavable, or pH-sensitive group. In those embodiments including a photocleavable moiety, the photocleavable moiety may be cleaved by introducing radiation having a specific wavelength (e.g. radiation having a wavelength ranging from between about 400nm to about 800nm). In those embodiments including a enzymatically cleavable group, the enzymatically cleavable group may be cleaved by one of a USER enzyme, uracil-N-glycosylase, an RNase A, a beta-glucuronidase, a beta-galactosidase, or a TEV-protease. In those embodiments including a chemically cleavable group, the chemically cleavable group may be cleaved by introducing an appropriate electrophile and/or nucleophile. [0301] In some embodiments, the compound of Formula (I) does not include a group "W" (where v = 0) and a cleavable moiety is included within "Olig2." In some embodiments, "Olig2" comprises a cleavage site comprised of one or more uracil-containing nucleotides. In some embodiments, the strand comprising the uracil-containing nucleotide (e.g., the first copy strand) is cleaved by contacting the reaction mixture with Uracil-N-DNA glycosylase (UNG), optionally in the presence of primary amines as described in U. S. Pat. No.8,669,061. UNGs recognize uracils present in single-stranded or double-stranded DNA and cleave the N-glycosidic bond between the uracil base and the deoxyribose, leaving an abasic site. See e.g. U.S. Pat. No. 6,713,294, the disclosure of which is hereby incorporated by reference herein in its entirety. [0302] In some embodiments, cleavage is performed by a combination of a glycosylase and an endonuclease, e.g., a mixture of Uracil DNA glycosylase (UDG) and a DNA glycosylase- lyase Endonuclease VIII. Cleaving the cleavage sites separates the first copy strand from the first oligonucleotide (e.g. "Olig1" of Formula (I)) and from the linker structure (Figure 2, bottom). In some embodiments, the cleavage takes place prior to forming the second copy strand. [0303] In some embodiments, the first copy strand or the double-stranded copy of the gene fusion are sequenced. In some embodiments, prior to sequencing, the first copy strand or the double-stranded copy of the gene fusion are amplified prior to sequencing. As described herein, amplification can include gene specific primers, specific primers or universal primers. Universal primer binding sites may be introduced in the 5-portions of the second oligonucleotide (e.g. "Olig2" of Formula (I)) of the linked primer or the primer used to form the second copy strand. [0304] In some embodiments, the method is multiplexed, meaning that the method is targeting multiple genes known to be involved in gene fusion events. In such embodiments, a reaction mixture is provided which comprises two or more of the compounds of Formula (I), where each of the two or more compounds of Formula (I) have an anchor sequence specific to a particular gene known to be a involved in gene fusion. For example, the same reaction mixture may contain two or more compounds of Formula (I) with anchor sequences targeting one or more of ALK, PPARG, BRAF, EGFR, FGFR1, FGFR2, FGFR3, MET, NRG1, NTRK1, NTRK2, NTRK3, RET, ROS1, AXL, PDGFRA, PDGFB , ABL1, ABL2, AKT1, AKT2, AKT3, ARHGAP26, BRD3, BRD4, CRLF2, CSF1R, EPOR, ERBB2, ERBB4, ERG, ESR1, ESRRA, ETV1, ETV4, ETV5, ETV6, EWSR1, FGR, IL2RB, INSR, JAK1, JAK2, JAK3, KIT, MAML2, MAST1, MAST2, MSMB, MUSK, MYB, MYC, NOTCH1, NOTCH2, NUMBL, NUT, PDGFRB, PIK3CA, PKN1, PRKCA, PRKCB, PTK2B, RAF1, RARA, RELA, RSPO2, RSPO3, SYK, TERT, TFE3, TFEB, THADA, TMPRSS2, TSLP, TY, BCL2, BCL6, BCR, CAMTA1, CBFB, CCNB3, CCND1, CIC, CRFL2, DUSP22, EPC1, FOXO1, FUS, GLI1, GLIS2, HMGA2, JAZF1, KMT2A, MALT1, MEAF6, MECOM, MKL1, MKL2, MTB, NCOA2, NUP214, NUP98, PAX5, PDGFB, PICALM, PLAG1, RBM15, RUNX1, RUNX1T1, SS18, STAT6, TAF15, TAL1, TCF12, TCF3, TFG, TYK2, USP6, YWHAE, AR, BRCA1, BRCA2, CDKN2A, ERB84, FLT3, KRAS, MDM4, MYBL1, NF1, NOTCH4, NUTM1, PRKACA, PRKACB, PTEN, RAD51B, and RB1. [0305] In some embodiments, the linked primers are designed to accommodate short input nucleic acids. For example, cell-free DNA, including circulating tumor DNA (ctDNA) averages 175 bp in length. In such embodiments, the length of the linked primer may not exceed 175 bases. [0306] Amplification [0307] In some embodiments, the present disclosure comprises an amplification step. The copy strand formed as illustrated in Figure 2 (bottom), can be copied and amplified by linear or exponential amplification. Amplification may be isothermal or involve thermocycling. In some embodiments, the amplification is exponential and involves PCR. In some embodiments, at least one gene-specific primer, e.g., a primer capable of hybridizing the known fusion partner is used for amplification. In some embodiments, the 5'-portion of the linked primer comprises a primer binding site for a second primer used in amplification. In other embodiments, universal primer binding sites are added to the nucleic acid to be amplified. In some embodiments, the universal primer binding sites may be added by ligating an adaptor comprising the universal primer binding sites. In other embodiments, the universal primer binding sites are added by extending a gene specific primer having a 5'-tail comprising the universal primer binding site. All nucleic acids having the same universal primer binding sites can be conveniently amplified with the same set of primers and under the same conditions. The number of amplification cycles where universal primers are used can be low but also can be about 10, about 20 or as high as about 30 or more cycles, depending on the amount of product needed for the subsequent steps. Because PCR with universal primers has reduced sequence bias, the number of amplification cycles need not be limited to avoid amplification bias. [0308] Primers [0309] In some embodiments, the present disclosure involves an amplification step utilizing a forward and a reverse primer. One or both of the forward and reverse primers may be target-specific. A target specific primer comprises at least a 3'-portion that is specific for (i.e., at least partially complementary to and forms a stable hybrid with) the target nucleic acid. If additional sequences are present, such as a barcode or a universal primer binding site, they are typically located in the 5'-portion of the primer. [0310] In some embodiments, to amplify the copy strand formed as shown in Figure 2 (bottom), a first primer specific for a known gene sequence upstream of the fusion breakpoint may be used. In some embodiments, a second primer is specific for the tag sequence or any other engineered sequence present in the second linked oligonucleotide. [0311] In some embodiments, the first and the second specific primers comprise a universal primer binding site in the 5'-portion of the primer. After one or more rounds of specific amplification, universal amplification is performed. [0312] Library [0313] In some embodiments, the present disclosure is a library of nucleic acids enriched for fusion-specific nucleic acids as described herein. The library comprises double-stranded nucleic acid molecules flanked by adaptor sequences attached thereto as described below. The nucleic acids in the library may comprise elements such as barcodes and universal primer binding sites present in adaptor sequences as described herein below. In some embodiments, the additional elements are present in adaptors and are added to the library nucleic acids via adaptor ligation. In other embodiments, some or all of the additional elements are present in amplification primers and are added to the library nucleic acids prior to adaptor ligation by extension of the primers. [0314] In some embodiments, the library is formed from all nucleic acids in the sample prior to the use of fusion detection linked primers described herein. In this embodiment, adaptor molecules are added to all nucleic acids in the sample. The method of detecting fusions with linked primers uses the library molecules as starting material. In some embodiments, universal amplification (with universal primers hybridizing to primer binding sites located in adaptors) takes place prior to fusion-specific amplification with linked primers. The universal amplification increases the amount of starting material for fusion-specific amplification with linked primers performed as described herein. [0315] In some embodiments, library molecules include adaptors comprising unique molecular barcodes. Sequencing the library comprises determining the sequence of barcoded library nucleic acids, grouping the sequences into families by unique molecular barcodes, and determining a consensus read for each family thereby detecting the gene fusion. [0316] Adaptor [0317] In some embodiments, the present disclosure utilizes an adaptor nucleic acid. The adaptor may be added to the nucleic acid by a blunt-end ligation or a cohesive end ligation. In some embodiments, the adaptor may be added by single-strand ligation method. In some embodiments, the adaptor is added by amplification with tiled primers having the adaptor sequence in the 5'-portion of the primer. The methods and compositions useful for adding adaptors by ligation or amplification are described e.g., in U.S. Patent Nos. 9476095, 9260753, 8822150, 8563478, 7741463, 8182989 and 8053192, the disclosures of which are hereby incorporated by reference herein in their entireties. [0318] In some embodiments, adaptor molecules are in vitro synthesized artificial sequences. In other embodiments, adaptor molecules are in vitro synthesized naturally occurring sequences. In yet other embodiments, adaptor molecules are isolated naturally occurring molecules or isolated non-naturally occurring molecules. [0319] In the case of adaptor added by ligation, the adaptor oligonucleotide can have overhangs or blunt ends on the terminus to be ligated to the target nucleic acid. In some embodiments, the adaptor comprises blunt ends to which a blunt-end ligation of the target nucleic acid can be applied. The target nucleic acids may be blunt-ended or may be rendered blunt-ended by enzymatic treatment (e.g., "end repair"). In other embodiments, the blunt-ended DNA undergoes A-tailing where a single A nucleotide is added to the 3'-end of one or both blunt ends. The adaptors described herein are made to have a single T nucleotide extending from the blunt end to facilitate ligation between the nucleic acid and the adaptor. Commercially available kits for performing adaptor ligation include AVENIO ctDNA Library Prep Kit or KAPA HyperPrep and HyperPlus kits (Roche Sequencing Solutions, Pleasanton, Cal.). In some embodiments, the adaptor ligated DNA may be separated from excess adaptors and unligated DNA. [0320] The adaptor may further comprise features such as universal primer binding site (including a sequencing primer-binding site) a barcode sequence (including a sample barcode (SID) or a unique molecular barcode or identifier (UID or UMI). In some embodiments, the adaptors comprise all of the above features while in other embodiments, some of the features are added after adaptor ligation by extending tailed primers that contain some of the elements described above. [0321] The adaptor may further comprise a capture moiety. The capture moiety may be any moiety capable of specifically interacting with another capture molecule. Capture moieties – capture molecule pairs include avidin (streptavidin) – biotin, antigen – antibody, magnetic (paramagnetic) particle – magnet, or oligonucleotide – complementary oligonucleotide. The capture molecule can be bound to a solid support so that any nucleic acid on which the capture moiety is present is captured on solid support and separated from the rest of the sample or reaction mixture. In some embodiments, the capture molecule comprises a capture moiety for a secondary capture molecule. For example, a capture moiety in the adaptor may be a nucleic acid sequence complementary to a capture oligonucleotide. The capture oligonucleotide may be biotinylated so that adapted nucleic acid-capture oligonucleotide hybrid can be captured on a streptavidin bead. [0322] In some embodiments, the adaptor-ligated nucleic acid is enriched via capturing the capture moiety and separating the adaptor-ligated target nucleic acids from unligated nucleic acids in the sample. [0323] In some embodiments, the stem portion of the adaptor includes a modified nucleotide increasing the melting temperature of the capture oligonucleotide, e.g., 5-methyl cytosine, 2,6-diaminopurine, 5-hydroxybutynl-2'-deoxyuridine, 8-aza-7-deazaguanosine, a ribonucleotide, a 2'O-methyl ribonucleotide or a locked nucleic acid. In another aspect, the capture oligonucleotide is modified to inhibit digestion by a nuclease, e.g., by a phosphorothioate nucleotide. [0324] In some embodiments, adaptor sequences are added to the copy strand formed as shown in Figure 2 (bottom) either by ligation of adaptors or by amplification with tailed primers. The adaptors may be added to either a single strand or a double-stranded molecule comprising the copy strand shown in Figure 2. [0325] Barcodes [0326] In some embodiments, the present disclosure utilizes a barcode. Detecting individual molecules typically requires molecular barcodes such as described in U.S. Patent Nos. 7,393,665, 8,168,385, 8,481,292, 8,685,678, and 8,722,368. A unique molecular barcode is a short artificial sequence added to each molecule in the patient's sample typically during the earliest steps of in vitro manipulations. The barcode marks the molecule and its progeny. The unique molecular barcode (UID) has multiple uses. Barcodes allow tracking each individual nucleic acid molecule in the sample to assess, e.g., the presence and amount of circulating tumor DNA (ctDNA) molecules in a patient's blood in order to detect and monitor cancer without a biopsy (Newman, A., et al., (2014) An ultrasensitive method for quantitating circulating tumor DNA with broad patient coverage, Nature Medicine doi:10.1038/nm.3519). [0327] A barcode can be a multiplex sample ID (MID) used to identify the source of the sample where samples are mixed (multiplexed). The barcode may also serve as a unique molecular ID (UID) used to identify each original molecule and its progeny. The barcode may also be a combination of a UID and an MID. In some embodiments, a single barcode is used as both UID and MID. In some embodiments, each barcode comprises a predefined sequence. In other embodiments, the barcode comprises a random sequence. In some embodiments of the present disclosure, the barcodes are between about 4-20 bases long so that between 96 and 384 different adaptors, each with a different pair of identical barcodes are added to a human genomic sample. A person of ordinary skill would recognize that the number of barcodes depends on the complexity of the sample (i.e., expected number of unique target molecules) and would be able to create a suitable number of barcodes for each experiment. [0328] Unique molecular barcodes can also be used for molecular counting and sequencing error correction. The entire progeny of a single target molecule is marked with the same barcode and forms a barcoded family. A variation in the sequence not shared by all members of the barcoded family is discarded as an artifact and not a true mutation. Barcodes can also be used for positional deduplication and target quantification, as the entire family represents a single molecule in the original sample (Newman, A., et al., (2016) Integrated digital error suppression for improved detection of circulating tumor DNA, Nature Biotechnology 34:547). [0329] In some embodiments, the number of UIDs in the plurality of adaptors or barcode- containing primers may exceed the number of nucleic acids in the plurality of nucleic acids. In some embodiments, the number of nucleic acids in the plurality of nucleic acids exceeds the number of UIDs in the plurality of adaptors. [0330] Purification [0331] In some embodiments, the present disclosure comprises intermediate purification steps. For example, any unused oligonucleotides such as excess primers and excess adaptors are removed, e.g., by a size selection method selected from gel electrophoresis, affinity chromatography and size exclusion chromatography. In some embodiments, size selection can be performed using Solid Phase Reversible Immobilization (SPRI) technology from Beckman Coulter (Brea, Cal.). In some embodiments, a capture moiety is used to capture and separate adaptor-ligated nucleic acids from unligated nucleic acids or excess primers from the products of exponential amplification. In some embodiments, the excess oligonucleotides including unused primers or adaptors are removed using a specific capture nucleic acid that forms a closed circular structure that encloses the oligonucleotide to be removed as described in the U.S. Application Ser. No. 63/021875 “Removal of excess oligonucleotides from a reaction mixture,” filed on May 8, 2020. [0332] Sequencing [0333] In some embodiments, the copy strands, double stranded copies of gene fusion sequences and libraries of nucleic acids including gene fusion sequences, or amplicons thereof can be subjected to nucleic acid sequencing. Sequencing may be performed according to any method known to those of ordinary skill in the art. In some embodiments, sequencing methods include Sanger sequencing and dye-terminator sequencing, as well as next-generation sequencing technologies such as pyrosequencing, nanopore sequencing, micropore-based sequencing, nanoball sequencing, MPSS, SOLiD, Illumina, Ion Torrent, Starlite, SMRT, tSMS, sequencing by synthesis, sequencing by ligation, mass spectrometry sequencing, polymerase sequencing, RNA polymerase (RNAP) sequencing, microscopy-based sequencing, microfluidic Sanger sequencing, microscopy-based sequencing, RNAP sequencing, tunneling currents DNA sequencing, and in vitro virus sequencing. See WO2014144478, WO2015058093, WO2014106076 and WO2013068528, each of which is hereby incorporated by reference in its entirety. [0334] In some embodiments, sequencing can be performed by a number of different methods, such as by employing sequencing by synthesis technology. Sequencing by synthesis according to the prior art is defined as any sequencing method which monitors the generation of side products upon incorporation of a specific deoxynucleoside-triphosphate during the sequencing reaction (Hyman, 1988, Anal. Biochem.174:423-436; Rhonaghi et al., 1998, Science 281:363-365). One prominent embodiment of the sequencing by synthesis reaction is the pyrophosphate sequencing method. In this case, generation of pyrophosphate during nucleotide incorporation is monitored by an enzymatic cascade which results in the generation of a chemo- luminescent signal. The 454 Genome Sequencer System (Roche Applied Science cat. No.04760 085 001), an example of sequence by synthesis, is based on the pyrophosphate sequencing technology. For sequencing on a 454 GS20 or 454 FLX instrument, the average genomic DNA fragment size is in the range of 200 or 600 bp, respectively, as described in the product literature. [0335] In some embodiments, a sequencing by synthesis reaction can alternatively be based on a terminator dye type of sequencing reaction. In this case, the incorporated dye deoxynucleotriphosphates (ddNTPs) building blocks comprise a detectable label, which is preferably a fluorescent label that prevents further extension of the nascent DNA strand. The label is then removed and detected upon incorporation of the ddNTP building block into the template/primer extension hybrid for example by using a DNA polymerase comprising a 3′-5′ exonuclease or proofreading activity. [0336] In some embodiments, sequencing is performed using a next-generation sequencing method such as that provided by Illumina, Inc. (the "Illumina Sequencing Method"). Without wishing to be bound by any particular theory, the Illumina next-generation sequencing technology uses clonal amplification and sequencing by synthesis (SBS) chemistry to enable rapid, accurate sequencing. The process simultaneously identifies DNA bases while incorporating them into a nucleic acid chain. Each base emits a unique fluorescent signal as it is added to the growing strand, which is used to determine the order of the DNA sequence. [0337] In some embodiments, the sequencing method is a high-throughput single molecule sequencing method utilizing nanopores. In some embodiments, the nucleic acids and libraries of nucleic acids formed as described herein are sequenced by a method involving threading through a biological nanopore (see US10337060, the disclosure of which is hereby incorporated by reference herein in its entirety) or a solid-state nanopore (see US10288599, US20180038001, US10364507, the disclosures of which are hereby incorporated by reference herein in their entireties). In other embodiments, sequencing involves threading tags through a nanopore. (see US8461854, the disclosure of which is hereby incorporated by reference herein in its entirety) or any other presently existing or future DNA sequencing technology utilizing nanopores. [0338] In other embodiments, sequencing is performed by other suitable technologies of high-throughput single molecule sequencing. include the Illumina HiSeq platform (Illumina, San Diego, Cal.), Ion Torrent platform (Life Technologies, Grand Island, NY), Pacific BioSciences platform utilizing the Single Molecule Real-Time (SMRT) technology (Pacific Biosciences, Menlo Park, Cal.) or any other presently existing or future DNA sequencing technology that does or does not involve sequencing by synthesis. [0339] The sequencing step may utilize platform-specific sequencing primers. Binding sites for these primers may be introduced in 5'-portions of the amplification primers used in the amplification step. If no primer sites are present in the library of barcoded molecules, an additional short amplification step introducing such binding sites may be performed. [0340] In some embodiments, the sequencing step involves sequence analysis. In some embodiments, the analysis includes a step of sequence aligning. In some embodiments, aligning is used to determine a consensus sequence from a plurality of sequences, e.g., a plurality having the same barcodes (UID). In some embodiments barcodes (UIDs) are used to determine a consensus from a plurality of sequences all having an identical barcode (UID). In other embodiments, barcodes (UIDs) are used to eliminate artifacts, i.e., variations existing in some but not all sequences having an identical barcode (UID). Such artifacts resulting from PCR errors or sequencing errors can be eliminated. [0341] In some embodiments, the number of each sequence in the sample can be quantified by quantifying relative numbers of sequences with each barcode (UID) in the sample. Each UID represents a single molecule in the original sample and counting different UIDs associated with each sequence variant can determine the fraction of each sequence in the original sample. A person skilled in the art will be able to determine the number of sequence reads necessary to determine a consensus sequence. In some embodiments, the relevant number is reads per UID ("sequence depth") necessary for an accurate quantitative result. In some embodiments, the desired depth is 5-50 reads per UID. [0342] In some embodiments, the step of sequencing further includes a step of error correction by consensus determination. Sequencing by synthesis of the circular strand of the gapped circular template disclosed herein enables iterative or repeated sequencing. Multiple reads of the same nucleotide position enable sequencing error correction through establishment of a consensus call for each nucleotide or for the entire sequence or for a part of the sequence. The final sequence of a nucleic acid strand is obtained from the consensus base determinations at each position. In some embodiments, a consensus sequence of a nucleic acid is obtained from a consensus obtained by comparing the sequences of complementary strands or by comparing the consensus sequences of complementary strands. In some embodiments, the present disclosure comprises after the sequencing step, a step of sequence read alignment and a step of generating a consensus sequence. In some embodiments, consensus is a simple majority consensus described in U.S. Patent 8535882. In other embodiments, consensus is determined by Partial Order Alignment (POA) method described in Lee et al. (2002) "Multiple sequence alignment using partial order graphs," Bioinformatics, 18(3):452-464 and Parker and Lee (2003) "Pairwise partial order alignment as a supergraph problem – aligning alignments revealed," J. Bioinformatics Computational Biol., 11:1-18. Based on the number of iterative reads used to determine a consensus sequence, the sequence may be largely free or substantially free of errors. [0343] No sequencing [0344] In some embodiments, the copy strands, double stranded copies of gene fusion sequences and libraries of nucleic acids including gene fusion sequences, or amplicons thereof are detected without sequencing. The detection may be accomplished by amplification, including by end-point polymerase chain reaction (PCR), quantitative PCR (qPCR) or digital PCR (dPCR), including digital droplet PCR (ddPCR). In some embodiments, detection of gene fusions is quantitative, such as the type of detection enabled by qPCR and dPCR. In other embodiments, detection of gene fusion is qualitative, i.e., the read-out is the presence or absence of the fusion- specific amplification product by gel electrophoresis, capillary electrophoresis, mass- spectrometry, or another method of detecting a nucleic acid of a characteristic size or characteristic molecular weight. [0345] dPCR [0346] In some embodiments, gene fusion-specific amplification according to the present disclosure is conducted by digital PCR (dPCR) including digital droplet PCR (ddPCR). [0347] Digital PCR is a method of quantitative amplification of nucleic acids described e.g., in U.S. Patent No. 9,347,095, the disclosure of which is hereby incorporated by reference herein. The process involves partitioning a sample into reaction volumes so that each volume comprises one or fewer copies of the target nucleic acid. In some embodiments, the partitioned reaction volume is an aqueous droplet. [0348] In some embodiments, the target nucleic acid in partitions is the copy strand. In other embodiments, the target nucleic acid in partitions is the double stranded copy of the gene fusion sequence. Each partition further comprises amplification primers, i.e., a forward and a reverse primer capable of supporting exponential amplification of the target nucleic acid. In some embodiments, the forward and a reverse primer are capable of hybridizing to the known fusion sequence and to the 5'-sequence of the second oligonucleotide (Figure 1). [0349] Each of the digital PCR reaction volumes further comprises a detectably-labeled probe capable of hybridizing to an amplicon of the forward and reverse primers. In some embodiments, the probe is capable of hybridizing to the known fusion sequence. In some embodiments, the probe is designed to avoid binding to the wild-type non-fusion gene sequence. [0350] The detectably labeled probe may be labeled with a combination of a fluorophore and the exponential amplification may be performed with a nucleic acid polymerase having a 5'- 3'-exonuclease activity. [0351] In some embodiments, the method of the present disclosure comprises performing an amplification reaction with the forward and reverse primers, wherein the reaction comprises a step of detecting the amplicon with the probe and determining a number of reaction volumes where the probe has been detected thereby detecting the presence of a gene fusion in the sample.