Methods and compositions for detecting structural rearrangements in genome

Abstract

To provide compositions, kits and methods for detecting gene fusion by sequencing.SOLUTION: Provided is a compound having Formula (I), [Olig1]-([R1]o-[R2]p)q-[L1]t-[Z]-[L2]u-[W]v-[Olig2], where o, p, q, v are 0 or 1; t and u are 0, 1 or 2; R1 is an oligonucleotide having between about 1 and about 24 nucleotides; R2 is a saturated or unsaturated, linear or cyclic aliphatic group, L1 and L2 are independently a saturated or unsaturated, linear or cyclic aliphatic group; Z is a moiety selected from a triazole, a dihydropyridazine, an amide linkage, a thioether linkage, an isoxazoline, an oxime ether etc.; W is a saturated or unsaturated, aliphatic or aromatic group; Olig1 is an oligonucleotide having between about 1 and about 30 nucleotides having a non-extendable 3' end; and Olig2 is an oligonucleotide having between about 1 and about 30 nucleotides having an extendable 3' end.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] Field of Disclosure The present disclosure relates to the field of genomics. More specifically, the present disclosure relates to the field of detecting genomic rearrangements. [Background technology]

[0002] Background of the disclosure Gene fusions are common occurrences in cancer. Some gene fusions are cancer driver mutations for which targeted therapies are being developed. The ability to detect gene fusions can be useful in cancer detection and diagnosis, tracking tumor size over time, and identifying the best personalized treatment for cancer patients. Traditional methods of detecting genomic rearrangements involve laborious multi-step procedures such as haplotype fusion PCR and ligation haplotyping. See Turner et al., (2008) Long range, high throughput haplotype determination via haplotype fusion PCR and ligation haplotyping, Nucl. Acids Res. 36:e82. More recent next-generation sequencing-based techniques can identify various gene fusions. However, this requires extensive sequencing to capture and verify a sufficient number of fusion sequences. The cost and complexity of this approach make it unsuitable for clinical use.

[0003] In some genes, the detection of gene fusions is further complicated by the presence of multiple fusion partners. For example, neurotrophic tropomyosin receptor kinase genes (NTRK 1, 2, and 3) can be fused with any number of N-terminal (5'-) partners. See Solomon et al. (2019) Identifying patients with NTRK fusion cancer, Ann. Oncol. Nov; 30 Suppl 8: viii16-viii22. Since effective therapies exist for activated NTRK, cost-effective clinical trials to identify eligible patients with NTRK gene fusions are important. Similarly, fibroblast growth factor receptor genes (FGFR2 and 3) can be fused with any number of C-terminal (3'-) partners resulting in constitutively active receptor kinase proteins. See Facchinetti et al. (2020) Facts and New Hopes on Selective FGFR Inhibitors in Solid Tumors, Clin. Cancer Res. 2020 Feb 15; 26(4): 764-774. Multiple FGFR kinase inhibitors have been developed, and pragmatic clinical trials are needed to identify eligible patients harboring FGFR gene fusions in various tumor types. Summary of the Invention

[0004] Disclosure Summary Based on the above, there is a need to identify gene fusions at lower cost and with less sequencing to increase patient access to potentially life-saving therapies.

[0005] The present disclosure relates to compositions, kits, and methods for detecting one or more gene fusions in a nucleic acid sample. In some embodiments, the disclosure provides one or more compounds, each having the formula (I): [Olig1]-([R 1 ] o -[R 2 ] p ) q -[L 1 ]t -[Z]-[L 2 ] u -[W] v -[Olig2](I),

[0006] During the ceremony,

[0007] o is 0 or 1;

[0008] p is 0 or 1;

[0009] q is 0 or 1;

[0010] t is 0, 1 or 2;

[0011] u is 0, 1 or 2;

[0012] v is 0 or 1;

[0013] R 1 is an oligonucleotide having from about 1 to about 24 nucleotides;

[0014] R 2 is a substituted or unsubstituted, saturated or unsaturated, straight chain or cyclic aliphatic group having from 2 to about 48 carbon atoms, optionally substituted with one or more heteroatoms selected from O, N or S;

[0015] L 1 and L 2 are independently a substituted or unsubstituted, saturated or unsaturated, straight chain or cyclic aliphatic group having from 1 to about 16 carbon atoms, optionally containing one or more heteroatoms selected from O, N or S, and optionally containing one or more carbonyl groups;

[0016] Z is a moiety selected from a triazole, a dihydropyridazine, a phosphate linkage, an amide linkage, a thioether linkage, an isoxazoline, a hydrozone, an oxime ether, and a chloro-s-triazine linkage;

[0017] W is a substituted or unsubstituted, saturated or unsaturated, aliphatic or aromatic group having 1 to about 12 carbon atoms, optionally substituted with one or more heteroatoms selected from O, N, S, provided that W contains at least one photocleavable, enzymatically cleavable, chemically cleavable, or pH-sensitive group;

[0018] Olig1 is an oligonucleotide comprising from about 1 to about 30 nucleotides;

[0019] Olig2 is an oligonucleotide containing about 1 to about 30 nucleotides.

[0020] In some embodiments, the compounds of formula (I) can be used to facilitate the detection of gene fusions. In that regard, the present disclosure also relates to a method for detecting gene fusions using one or more of the compounds of formula (I). In some embodiments, the compounds of formula (I) facilitate the capture of gene fusions, in which one fusion partner is utilized for the detection, amplification, and / or sequencing of one or more gene fusions in a sample (e.g., a histological sample, a cytological sample, etc.). These and other aspects of the present disclosure are further described herein.

[0021] A first aspect of the disclosure is a method for detecting a gene fusion in a nucleic acid sample, comprising: (a) treating the sample with a polymerase (e.g., a nucleic acid polymerase having polymerase activity and strand displacement activity) and a nucleic acid polymerase having formula (I): [Olig1]-([R 1 ] o -[R 2 ] p ) q -[L 1 ] t -[Z]-[L 2 ] u -[W] v -[Olig2](I),

[0022] During the ceremony,

[0023] o is 0 or 1;

[0024] p is 0 or 1;

[0025] q is 0 or 1;

[0026] t is 0, 1 or 2;

[0027] u is 0, 1 or 2;

[0028] v is 0 or 1;

[0029] R 1 is an oligonucleotide having from about 1 to about 24 nucleotides;

[0030] R 2 is a substituted or unsubstituted, saturated or unsaturated, straight chain or cyclic aliphatic group having from 2 to about 48 carbon atoms, optionally substituted with one or more heteroatoms selected from O, N or S;

[0031] L 1 and L 2 are independently a substituted or unsubstituted, saturated or unsaturated, straight chain or cyclic aliphatic group having from 1 to about 16 carbon atoms, optionally containing one or more heteroatoms selected from O, N or S, and optionally containing one or more carbonyl groups;

[0032] Z is a moiety selected from a triazole, a dihydropyridazine, a phosphate linkage, an amide linkage, a thioether linkage, an isoxazoline, a hydrozone, an oxime ether, and a chloro-s-triazine linkage;

[0033] W is a substituted or unsubstituted, saturated or unsaturated, aliphatic or aromatic group having 1 to about 12 carbon atoms, optionally substituted with one or more heteroatoms selected from O, N, S, provided that W contains at least one photocleavable, enzymatically cleavable, chemically cleavable, or pH-sensitive group;

[0034] Olig1 is an oligonucleotide comprising from about 1 to about 30 nucleotides and including an anchor sequence capable of hybridizing to a known fusion partner, wherein Olig1 has a non-extendable 3' end;

[0035] Olig2 is an oligonucleotide containing from about 1 to about 30 nucleotides and containing an extendable 3' end. contacting a compound having the formula:

[0036] (b) extending the 3' end of Olig2 of a compound having formula (I) with a polymerase, thereby generating an extension product. In some embodiments, the extension product includes a copy of a portion of the unknown fusion partner, a portion of the known fusion partner, and the fusion breakpoint, thereby forming a first strand copy of the gene fusion.

[0037] In some embodiments, Olig2 comprises a random sequence. The random sequence contains 2 to 20 nucleotides.

[0038] In some embodiments, o+p=1 and q is 1. In some embodiments, R 2 comprises a moiety having the structure of formula (IVB): [ka]

[0039] In the formula, d and e are each independently an integer ranging from 1 to 32; Q is a bond, O, S, or N(R c )(R d ) and R c and R d is independently CH or H. In some embodiments, R 2 comprises a moiety having the structure of formula (IVC): [ka]

[0040] wherein d and e are each independently an integer ranging from 1 to 32. In some embodiments, d and e range from 1 to 16. In some embodiments, d and e range from 2 to 8. In some embodiments, the method further comprises forming a second strand copy of the gene fusion by copying the first strand copy, thereby forming a double stranded copy of the gene fusion.

[0041] In some embodiments, R 1 In some embodiments, R 1 contains 4 to 8 nucleotides.

[0042] In some embodiments, v is 1. In some embodiments, the method further comprises cleaving the photocleavable, enzymatically cleavable, chemically cleavable, or pH-sensitive group of the linked primer.

[0043] In some embodiments, v is 0 and Olig2 contains a cleavage site comprising a uracil-containing nucleotide.

[0044] In some embodiments, L 1 Or L 2 At least one of comprises a substituted or unsubstituted, saturated or unsaturated, linear or cyclic aliphatic group having from 1 to about 4 carbon atoms, optionally substituted with one or more heteroatoms selected from O, N, or S, and optionally comprising one or more carbonyl groups. In some embodiments, the aliphatic group is linear. In some embodiments, the aliphatic group is linear and unsubstituted. In some embodiments, the aliphatic group is linear and unsubstituted and comprises one carbonyl group. In some embodiments, the aliphatic group is linear and substituted.

[0045] In some embodiments, the method includes sequencing copies of the gene fusion. In some embodiments, the method further comprises forming a library of double stranded copies of the gene fusion. In some embodiments, forming the library comprises attaching an adaptor to the copies of the gene fusion, the adaptor comprising a barcode and a primer binding site. In some embodiments, the method further comprises amplifying at least a portion of the formed library by universal amplification. In some embodiments, the method further comprises sequencing at least a portion of the formed library. In some embodiments, the barcode comprises a unique molecular barcode (UID), and sequencing comprises grouping sequences of the library nucleic acids into families by the UID, determining a consensus read for each family, and aligning the consensus read to a reference genome, thereby determining the sequence of the gene fusion.

[0046] In some embodiments, the method further comprises amplifying the copy strand by a method comprising: (a) dividing the sample containing the copy strand into a plurality of reaction volumes, each reaction volume comprising a forward amplification primer and a reverse amplification primer 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, the reaction comprising a step of detection with the probe; (c) determining the number of reaction volumes in which the probe is detected, thereby detecting the gene fusion. In some embodiments, the reaction volume is a droplet. In some embodiments, the detectable label comprises a combination of a fluorophore and a quencher.

[0047] In some embodiments, multiple fusions are detected in a sample by contacting the sample with two or more compounds having formula (I). In some embodiments, Olig1 of each of the two or more compounds of formula (I) is selected from the group consisting 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, ERBB5, ERBB6, ERBB7, ERBB8, ERBB9, ERBB10, ERBB11, ERBB12, ERBB13, ERBB14, ERBB15, ERBB16, ERBB17, ERBB18, ERBB19, ERBB20, ERBB21, ERBB22, ERBB23, ERBB24, ERBB25, ERBB26, ERBB27, ERBB28, ERBB29 ...9, ERBB29, ERBB29, ERBB29, ERBB29, ERBB29, ERBB29, ERBB29 G, 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, TE RT, 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, It can hybridize to genes selected from 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.

[0048] A second aspect of the disclosure is a compound having formula (I): [Olig1]-([R 1 ] o -[R2 ] p ) q -[L 1 ] t -[Z]-[L 2 ] u -[W] v -[Olig2] (I)

[0049] During the ceremony,

[0050] o is 0 or 1;

[0051] p is 0 or 1;

[0052] q is 0 or 1;

[0053] t is 0, 1 or 2;

[0054] u is 0, 1 or 2;

[0055] v is 0 or 1;

[0056] R 1 is an oligonucleotide having from about 1 to about 24 nucleotides;

[0057] R 2 is a substituted or unsubstituted, saturated or unsaturated, straight chain or cyclic aliphatic group having from 2 to about 48 carbon atoms, optionally substituted with one or more heteroatoms selected from O, N or S;

[0058] L 1 and L 2 are independently a substituted or unsubstituted, saturated or unsaturated, straight chain or cyclic aliphatic group having from 1 to about 16 carbon atoms, optionally containing one or more heteroatoms selected from O, N or S, and optionally containing one or more carbonyl groups;

[0059] Z is a moiety selected from a triazole, a dihydropyridazine, a phosphate linkage, an amide linkage, a thioether linkage, an isoxazoline, a hydrozone, an oxime ether, and a chloro-s-triazine linkage;

[0060] W is a substituted or unsubstituted, saturated or unsaturated, aliphatic or aromatic group having 1 to about 12 carbon atoms, optionally substituted with one or more heteroatoms selected from O, N, S, provided that W contains at least one photocleavable, enzymatically cleavable, chemically cleavable, or pH-sensitive group;

[0061] Olig1 is an oligonucleotide having from about 1 to about 30 nucleotides, wherein Olig1 has a non-extendable 3' end;

[0062] Olig2 is an oligonucleotide having from about 1 to about 30 nucleotides, and Olig2 has an extendable 3' end.

[0063] In some embodiments, R 2 comprises a substituted or unsubstituted, saturated or unsaturated, linear or cyclic aliphatic group having from 2 to about 32 carbon atoms, optionally substituted with one or more heteroatoms selected from O, N, or S, and optionally containing one or more carbonyl groups. 2 comprises a moiety having the structure of formula (IVA): [ka]

[0064] In the formula, d and e are each independently an integer ranging from 1 to 32; Q is a bond, O, S, N(R c )(R d ) or quaternary amine (N + H(R c )(R d )) and;R a and R bare independently H, a C1-C4 alkyl group, F, Cl or N(R c )(R d ) and R c and R d is independently CH3 or H. In some embodiments, d is 2 or 3; and e is an integer ranging from 1 to 12. In some embodiments, R 2 comprises a moiety having the structure of formula (IVB): [ka]

[0065] In the formula, d and e are each independently an integer ranging from 1 to 32; Q is a bond, O, S, or N(R c )(R d ) and R c and R d is independently CH3 or H. In some embodiments, d is 2 or 3; and e is an integer ranging from 1 to 12. In some embodiments, d is 2 or 3; and e is an integer ranging from 1 to 8.

[0066] In some embodiments, L 1 Or L 2 At least one of comprises a substituted or unsubstituted, saturated or unsaturated, straight chain or cyclic aliphatic group having from 1 to about 4 carbon atoms, optionally substituted with one or more heteroatoms selected from O, N or S, and optionally containing one or more carbonyl groups.

[0067] In some embodiments, o+p=1 and q is 1. In some embodiments, R 1 In some embodiments, R comprises from about 1 to about 16 nucleotides. 1 In some embodiments, R comprises from about 2 to about 9 nucleotides. 2 comprises a moiety having the structure of formula (IVC): [ka]

[0068] In the formula, d and e are each independently an integer ranging from 1 to 32. In some embodiments, d is 2 or 3; and e is an integer ranging from 1 to 12. In some embodiments, d is 2 or 3; and e is an integer ranging from 1 to 8. In some embodiments, d is 2 or 3; and e is an integer ranging from 1 to 8. and e is an integer ranging from 1 to 12. In some embodiments, d is 2; and e is an integer ranging from 1 to 8. In some embodiments, d is 2; and e is an integer ranging from 2 to 6.

[0069] In some embodiments, o is 0, p and q are both 1, and R 1 contains at least one PEG group, and L 1 In some embodiments, o is 0, p and q are both 1, and R 1 contains at least two PEG groups, and L 1 In some embodiments, o is 0, p and q are both 1, and R 1 contains at least three PEG groups, and L 1 In some embodiments, o is 0, p and q are both 1, and R 1 contains at least four PEG groups, and L 1 In some embodiments, o is 0, p and q are both 1, and R 1 contains at least six PEG groups, and L 1 In some embodiments, o is 0, p and q are both 1, and R 1 contains at least 8 PEG groups, and L 1 In some embodiments, o is 0, p and q are both 1, and R 1 contains at least 12 PEG groups, and L 1 contains at least one carbonyl moiety.

[0070] In some embodiments, Olig2 comprises a barcode. In some embodiments, the barcode is one or more of a unique molecular barcode (UID), a sample barcode, and an identification tag. In some embodiments, Olig2 comprises a universal primer binding site. In some embodiments, v is 0 and Olig2 comprises a cleavage site comprising a uracil-containing nucleotide. In some embodiments, Olig2 comprises a random nucleotide sequence.

[0071] In some embodiments, at least a portion of Olig1 is selected from the group consisting 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, ESR RA, ETV1, ETV4, ETV5, ETV6, EWSR1, FGR, IL2RB, INSR, JAK1, JAK2, JAK3, KIT, MAML2, MAST1, MAST2, MSMB, MUSK, MYB, MYC, NOT CH1, 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, GLIS 2, HMGA2, JAZF1, KMT2A, MALT1, MEAF6, MECOM, MKL1, MKL2, MTB, NCOA2, NUP214, NUP98, PAX5, PDGFB, PICALM, PLAG1, RBM15, RU The nucleic acid sequence comprises a nucleotide sequence capable of hybridizing to a gene selected from the group consisting of NX1, 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.

[0072] In some embodiments, Olig1 is not extendable. In some embodiments, Olig2 is extendable. In some embodiments, Olig1 comprises 1 to about 10 nucleotides. In some embodiments, Olig2 comprises 1 to about 10 nucleotides. Contains leotide.

[0073] In some embodiments, the group -([R 1 ] o -[R 2 ] p ) q The size of the -([R 1 ] o -[R 2 ] p ) q The size of the -([R 1 ] o -[R 2 ] p ) q The size of the -([R 1 ] o -[R 2 ] p ) q The size of the -([R 1 ] o -[R 2 ] p ) q The size of the -([R 1 ] o -[R 2 ] p ) q The size of the - ranges from about 20 angstroms to about 40 angstroms.

[0074] In a third aspect of the disclosure, there is provided a kit for detecting gene fusions, e.g., for detecting gene fusions between a known fusion partner and an unknown fusion partner, the kit comprising: (a) a DNA polymerase; and (b) a compound having formula (I): [Olig1]-([R 1 ] o -[R2 ] p ) q -[L 1 ] t -[Z]-[L 2 ] u -[W] v -[Olig2] (I)

[0075] During the ceremony,

[0076] o is 0 or 1;

[0077] p is 0 or 1;

[0078] q is 0 or 1;

[0079] t is 0, 1 or 2;

[0080] u is 0, 1 or 2;

[0081] v is 0 or 1;

[0082] R 1 is an oligonucleotide having from about 1 to about 24 nucleotides;

[0083] R 2 is a substituted or unsubstituted, saturated or unsaturated, straight chain or cyclic aliphatic group having from 2 to about 48 carbon atoms, optionally substituted with one or more heteroatoms selected from O, N or S;

[0084] L 1 and L 2 are independently a substituted or unsubstituted, saturated or unsaturated, straight chain or cyclic aliphatic group having from 1 to about 16 carbon atoms, optionally containing one or more heteroatoms selected from O, N or S, and optionally containing one or more carbonyl groups;

[0085] Z is a moiety selected from a triazole, a dihydropyridazine, a phosphate linkage, an amide linkage, a thioether linkage, an isoxazoline, a hydrozone, an oxime ether, and a chloro-s-triazine linkage;

[0086] W is a substituted or unsubstituted, saturated or unsaturated, aliphatic or aromatic group having from 1 to about 12 carbon atoms, optionally substituted with one or more heteroatoms selected from O, N, and S. provided that W comprises at least one photocleavable group, enzymatically cleavable group, chemically cleavable group, or pH-sensitive group;

[0087] Olig1 is an oligonucleotide having from about 1 to about 30 nucleotides, wherein Olig1 has a non-extendable 3' end;

[0088] Olig2 is an oligonucleotide having from about 1 to about 30 nucleotides, and Olig2 has an extendable 3' end.

[0089] In some embodiments, the kit further comprises a forward amplification primer and a reverse amplification primer. In some embodiments, Olig2 comprises at least one uracil-containing nucleotide and the kit further comprises uracil-N-DNA glycosylase (UNG). In some embodiments, the DNA polymerase is a reverse transcriptase and the kit further comprises a thermostable DNA-dependent DNA polymerase.

[0090] In some embodiments, at least a portion of Olig1 is selected from the group consisting 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, ESR RA, ETV1, ETV4, ETV5, ETV6, EWSR1, FGR, IL2RB, INSR, JAK1, JAK2, JAK3, KIT, MAML2, MAST1, MAST2, MSMB, MUSK, MYB, MYC, NOT CH1, 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, GLIS 2, HMGA2, JAZF1, KMT2A, MALT1, MEAF6, MECOM, MKL1, MKL2, MTB, NCOA2, NUP214, NUP98, PAX5, PDGFB, PICALM, PLAG1, RBM15, RU The nucleic acid sequence comprises a nucleotide sequence capable of hybridizing to a gene selected from the group consisting of NX1, 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.

[0091] A fourth aspect of the present disclosure is a reaction vessel containing a compound having formula (I): [Olig1]-([R 1 ] o -[R 2 ] p ) q -[L 1] t -[Z]-[L 2 ] u -[W] v -[Olig2] (I)

[0092] During the ceremony,

[0093] o is 0 or 1;

[0094] p is 0 or 1;

[0095] q is 0 or 1;

[0096] t is 0, 1 or 2;

[0097] u is 0, 1 or 2;

[0098] v is 0 or 1;

[0099] R 1 is an oligonucleotide having from about 1 to about 24 nucleotides;

[0100] R 2 is a substituted or unsubstituted, saturated or unsaturated, straight chain or cyclic aliphatic group having from 2 to about 48 carbon atoms, optionally substituted with one or more heteroatoms selected from O, N or S;

[0101] L 1 and L 2 are independently a substituted or unsubstituted, saturated or unsaturated, straight chain or cyclic aliphatic group having from 1 to about 16 carbon atoms, optionally containing one or more heteroatoms selected from O, N or S, and optionally containing one or more carbonyl groups;

[0102] Z is a moiety selected from a triazole, a dihydropyridazine, a phosphate linkage, an amide linkage, a thioether linkage, an isoxazoline, a hydrozone, an oxime ether, and a chloro-s-triazine linkage;

[0103] W is a substituted or unsubstituted, saturated or unsaturated, aliphatic or aromatic group having 1 to about 12 carbon atoms, optionally substituted with one or more heteroatoms selected from O, N, S, provided that W contains at least one photocleavable, enzymatically cleavable, chemically cleavable, or pH-sensitive group;

[0104] Olig1 is an oligonucleotide having from about 1 to about 30 nucleotides, wherein Olig1 has a non-extendable 3' end;

[0105] Olig2 is an oligonucleotide having from about 1 to about 30 nucleotides, and Olig2 has an extendable 3' end.

[0106] In some embodiments, the reaction vessel comprises at least one polymerase. In some embodiments, the at least one polymerase is a DNA polymerase. In some embodiments, the reaction vessel further comprises at least one buffer. In some embodiments, the reaction vessel further comprises at least one cofactor. In some embodiments, the reaction vessel further comprises dNTPs.

[0107] In a fifth aspect of the disclosure, there is provided (a) a compound having formula (II): [Olig1]-([R 1 ] o -[R 2 ] p ) q -[L 1 ] t -[X] (II)

[0108] During the ceremony,

[0109] o is 0 or 1;

[0110] p is 0 or 1;

[0111] q is 1 or 2;

[0112] t is 0, 1 or 2;

[0113] R 1 is an oligonucleotide having 1 to about 24 nucleotides;

[0114] R 2 is a substituted or unsubstituted, saturated or unsaturated, straight chain or cyclic aliphatic group having from 2 to about 48 carbon atoms, optionally substituted with one or more heteroatoms selected from O, N or S;

[0115] L 1 is a substituted or unsubstituted, saturated or unsaturated, linear or cyclic aliphatic group having from 1 to about 16 carbon atoms, optionally substituted with one or more heteroatoms selected from O, N or S, and optionally containing one or more carbonyl groups;

[0116] X is dibenzocyclooctyne, trans-cyclooctene, alkyne, alkene, azide, tetrazine, maleimide, N-hydroxysuccinimide, thiol, 1,3-nitrone, aldehyde, ketone, hydrazine, hydroxylamine, amino group, or phosphoramidite;

[0117] Olig1 is an oligonucleotide having about 1 to about 30 nucleotides;

[0118] (b) A compound having the formula (III): [Y]-[L 2 ] u -[W] v -[Olig2](III),

[0119] During the ceremony,

[0120] u is 0, 1 or 2;

[0121] v is 0 or 1;

[0122] Y is dibenzocyclooctyne, trans-cyclooctene, alkyne, alkene, azide, tetrazine, maleimide, N-hydroxysuccinimide, thiol, 1,3-nitrone, aldehyde, ketone, hydrazine, hydroxylamine, amino group, or phosphoramidite;

[0123] L 2 is a substituted or unsubstituted, saturated or unsaturated, linear or cyclic aliphatic group having 1 to 16 carbon atoms, optionally containing one or more heteroatoms selected from O, N or S, and optionally containing one or more carbonyl groups;

[0124] W is a substituted or unsubstituted, saturated or unsaturated aliphatic or aromatic group having 1 to 12 carbon atoms, optionally substituted with one or more heteroatoms selected from O, N, S, provided that W contains at least one photocleavable, enzymatically cleavable, chemically cleavable, or pH-sensitive group;

[0125] Olig2 is a compound that is an oligonucleotide having from about 1 to about 30 nucleotides.

[0126] In some embodiments, Olig1 comprises a non-extendable 3' end and Olig 2 comprises an extendable 3' end. In some embodiments, Olig1 comprises 1 to about 10 nucleotides. In some embodiments, Olig2 comprises 1 to about 10 nucleotides. In some embodiments, at least a portion of Olig1 is selected from the group consisting of ALK, PPARG, BRAF, EGFR, FGFR1, FGFR2, FGFR3, MET, NRG1, NTRK1, NTRK2, NTRK3, RET, ROS1, AXL, PDGFRA, P DGFB, 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, MS MB, 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, CCN B3, 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.

[0127] In some embodiments, one of X or Y comprises an alkyne moiety; the other of X or Y comprises an azide moiety. In some embodiments, the alkyne moiety is DBCO. In some embodiments, one of X or Y comprises a maleimide moiety; the other of X or Y comprises a thiol moiety. In some embodiments, one of X or Y comprises an alkene moiety; the other of X or Y comprises a tetrazine moiety. In some embodiments, the amino moiety is included and the kit further comprises s-trichlorotriazine.

[0128] In some embodiments, R 2 comprises a substituted or unsubstituted, saturated or unsaturated, linear or cyclic aliphatic group having from 2 to about 32 carbon atoms, optionally substituted with one or more heteroatoms selected from O, N, or S, and optionally containing one or more carbonyl groups. 2 comprises a moiety having the structure of formula (IVA): [ka]

[0129] In the formula, d and e are each independently an integer ranging from 1 to 32; Q is a bond, O, S, N(R c )(R d ) or quaternary amine (N + H(R c )(R d )) and;R a and R b are independently H, a C1-C4 alkyl group, F, Cl or N( Rc )(R d ) and R c and R.R. d is independently CH3 or H. In some embodiments, d is 2; and e is an integer ranging from 1 to about 12. In some embodiments, R 2 comprises a moiety having the structure of formula (IVB): [ka]

[0130] In the formula, d and e are each independently an integer ranging from 1 to 32; Q is a bond, O, S, or N(R c )(R d ) and R c and R d is independently CH3 or H. In some embodiments, is 2; and e is an integer ranging from 1 to about 12. In some embodiments, d is 2; and e is an integer ranging from 1 to about 6.

[0131] In some embodiments, o+p=1 and q is 1. In some embodiments, R 1 In some embodiments, R comprises from about 2 to about 9 nucleotides. 2 comprises a moiety having the structure of formula (IVC): [ka]

[0132] wherein d and e are each independently an integer ranging from 1 to 32. In some embodiments, d is 2; and e is an integer ranging from 1 to about 12. In some embodiments, d is 2; and e is an integer ranging from 1 to about 6. In some embodiments, o is 0, p and q are both 1, and L comprises at least one PEG group.

[0133] In some embodiments, Olig2 comprises a barcode. In some embodiments, the barcode is one or more of a unique molecular barcode (UID), a sample barcode, and an identification tag. In some embodiments, Olig2 comprises a universal primer binding site. In some embodiments, v is 0 and Olig2 comprises a cleavage site that comprises a uracil-containing nucleotide.

[0134] In some embodiments, the kit further comprises a polymerase. In some embodiments, the polymerase is a DNA polymerase. In some embodiments, the kit further comprises a nucleic acid sample comprising at least one gene fusion. In some embodiments, the kit further comprises an aliquot of a master mix. [Brief description of the drawings]

[0135] [Figure 1] FIG. 1 shows the steps of annealing and elongating a compound of formula (I). [Diagram 2] FIG. 1 shows the steps of strand displacement and strand scission to release the copy strand containing the gene fusion sequence. Detailed Description of the Invention

[0136] Detailed Description of the Disclosure overview

[0137] The present disclosure relates to compositions and kits that facilitate the detection of structural genome rearrangements in samples that contain one or more target nucleic acids. The present disclosure also relates to methods that utilize amplicon-based approaches to detect structural genome rearrangements, more specifically gene fusions. In some embodiments, the methods described herein utilize one or more compounds of formula (I) to amplify gene fusions in which one fusion partner is unknown. In some embodiments, amplification with one or more compounds of formula (I) facilitates the detection of gene fusions, with or without a sequencing step. In those embodiments in which a sequencing step is utilized, such sequencing requires a minimum sequencing depth.

[0138] definition

[0139] Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. See Sambrook et al., Molecular Cloning, A Laboratory Manual, 4th Ed. Cold Spring Harbor Lab Press (2012).

[0140] It is also to be understood that, unless expressly stated to the contrary, in any method claimed herein that includes more than one step or act, the order of the method steps or acts is not necessarily limited to the order in which the method steps or acts are described.

[0141] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly dictates otherwise. The term "comprising" is defined inclusively, such that "including A or B" means including A, B, or A and B.

[0142] As used herein and in the claims, "or" should be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" shall be interpreted as inclusive, i.e., including at least one of a number or list of elements, but including a plurality, and optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as "only one of" or "exactly one of," or, when used in the claims, "consisting of," refer to the inclusion of exactly one element of a number or list of elements. In general, the term "or" as used herein shall only be interpreted as indicating exclusive alternatives (i.e., "one or the other, but not both") when preceded by a term of exclusivity, such as "either," "one of," "only one of," or "exactly one of." "Consisting essentially of," when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0143] As used herein, terms such as "comprises," "includes," "has," and the like are used interchangeably and have the same meaning. Similarly, "comprises," "includes," "has," and the like are used interchangeably and have the same meaning. Specifically, each term is defined consistent with the general U.S. Patent Law definition of "comprising," and therefore is to be interpreted as an open term meaning "at least the following," and not to exclude additional features, limitations, aspects, and the like. Thus, for example, "a device having components a, b, and c" means that the device includes at least components a, b, and c. Similarly, the phrase "a method including steps a, b, and c" means that the method includes at least steps a, b, and c. Additionally, although steps and processes may be outlined herein in a particular order, Those skilled in the art will recognize that the sequencing steps and processes may vary.

[0144] As used herein in the specification and claims, the phrase "at least one" in connection with a list of one or more elements should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed in the list of elements, and not excluding any combination of elements in the list of elements. This definition also allows for elements other than those specifically identified in the list of elements to which the phrase "at least one" refers, may optionally be present, whether or not related to those elements specifically identified. Thus, as a non-limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B," or, equivalently, "at least one of A and / or B") can refer in one embodiment to at least one, optionally including more than one A, and no B (and optionally including elements other than B); in another embodiment to at least one, optionally including more than one B, and no A (and optionally including elements other than A); in yet another embodiment to at least one, optionally including more than one A, and at least one, optionally including more than one B (and optionally including other elements); and so forth.

[0145] As used herein, the term "adapter" refers to a nucleotide sequence that can be added to another sequence to import additional elements and properties into that sequence, including but not limited to barcodes, primer binding sites, capture moieties, labels, secondary structures.

[0146] As used herein, the term "aliphatic" refers to a linear or branched hydrocarbon chain that may be saturated or mono- or polyunsaturated. Unsaturated aliphatic groups contain one or more double and / or triple bonds. The branches of the hydrocarbon chain may include linear chains as well as non-aromatic cyclic elements. The hydrocarbon chain may be of any length and contain any number of branches, unless otherwise specified. Both the backbone and the branches may further include heteroatoms, such as, for example, B, N, O, P, S, Se, or Si.

[0147] As used herein, the term "barcode" refers to a nucleic acid sequence that can be detected and identified. Barcodes can generally be 2 or more nucleotides in length and up to about 50 nucleotides. Barcodes are designed to have at least a minimum number of differences from other barcodes in a population. Barcodes may be unique to each molecule in a sample, unique to a sample, or shared by multiple molecules in a sample. The term "multiple identifier," "MID," or "sample barcode" refers to a barcode that identifies a sample or source of a sample. Thus, all or substantially all MID barcoded polynucleotides from a single source or sample will share the same sequence MID, but all or substantially all (e.g., at least 90% or 99%) MID barcoded polynucleotides from different sources or samples will have different MID barcode sequences. Polynucleotides from different sources with different MIDs can be mixed and sequenced in parallel while maintaining the sample information encoded in the MID barcode. The term "unique molecular identifier" or "UID" refers to a barcode that identifies the polynucleotide to which it is attached. Typically, all or substantially all (e.g., at least 90% or 99%) of the UID barcodes in a mixture of UID-barcoded polynucleotides are unique. Barcodes can also be used as "identification tags" as part of a workflow. For example, a DNA molecule (e.g., cDNA) derived from RNA can be distinguished from a DNA molecule of the same sequence derived from genomic DNA by a tag that is only attached to the cDNA during cDNA synthesis. Such barcodes are sometimes referred to as "RNA identification tags" or simply "identification tags."

[0148] As used herein, the term "ctDNA" refers to free DNA released into peripheral blood from primary tumor cells, circulating tumor cells in the blood circulation, and necrotic or apoptotic tumor cells, or any combination thereof.

[0149] As used herein, the term "DNA polymerase" refers to an enzyme that performs template-directed synthesis of polynucleotides from deoxyribonucleotides. DNA polymerases include prokaryotic Pol I, Pol II, Pol III, Pol IV and Pol V, eukaryotic DNA polymerases, archaeal DNA polymerases, telomerases and reverse transcriptases. The term "thermostable polymerase" refers to an enzyme that is useful for exponential amplification of nucleic acids by polymerase chain reaction (PCR) because the enzyme is thermostable. Thermostable enzymes retain sufficient activity to effect a subsequent polynucleotide extension reaction and are not irreversibly denatured (inactivated) when subjected to high temperatures for the time required to effect denaturation of double-stranded nucleic acids.

[0150] In some embodiments, thermostable polymerases from the species Thermococcus, Pyrococcus, Sulfolobus, Methanococcus, and other archaeal B polymerases. In some cases, the nucleic acid (e.g., DNA or RNA) polymerase can be a modified naturally occurring A-type polymerase. Further embodiments of the present disclosure generally relate to methods in which the modified A-type polymerase, e.g., in a primer extension, end modification (e.g., terminal transferase, degradation or polishing) or amplification reaction, can be selected from any species of the Meiothermus, Thermotoga, or Thermomicrobium genera. Another embodiment of the disclosure generally relates to methods in which the polymerase, for example in the primer extension, end modification (e.g., terminal transferase, degrade or polish) or amplification reaction, may be isolated from either Thermus aquaticus (Taq), Thermus thermophilus, Thermus caldophilus, or Thermus filiformis. Further embodiments of the disclosure generally encompass methods in which the modified A-type polymerase, for example in the primer extension, end modification (e.g., terminal transferase, degrade or polish) or amplification reaction, may be isolated from Bacillus stearothermophilus, Sphaerobacter thermophilus, Dictoglomus thermophilum, or Escherichia coli. In another embodiment, the disclosure generally relates to methods in which the modified A-type polymerase, for example in the primer extension, end modification (e.g., terminal transferase, degrade or polish) or amplification reaction, may be a mutant Taq-E507K polymerase. Another embodiment of the disclosure generally relates to methods in which the amplification of a target nucleic acid may be performed using a thermostable polymerase.

[0151] As used herein, the term "enrichment" refers to increasing the relative amount of a target molecule in a plurality of molecules. Enrichment can increase the relative amount of target molecules to the complete or near complete elimination of non-target molecules. Examples of enrichment of target nucleic acids include linear hybridization capture, amplification, exponential amplification (PCR) and primer extension target enrichment (PETE), see, for example, U.S. Patent Application Publication Nos. 14 / 910,237, 15 / 228,806, 15 / 648,146 and International Application No. PCT / EP2018 / 085727.

[0152] As used herein, the term "gene fusion" refers to a change in a genomic sequence compared to a reference genome, including a translocation in which a portion of one gene is fused with another sequence. Some gene fusions result in a functional fusion mRNA. A subset of these gene fusions further result in a functional fusion protein. Gene fusions can be expressed in a variety of ways, including by the transcription factor 1 (TNF) that encodes the fusion protein. These fusion partners have a 5'-partner and a 3'-partner designated with reference to the mRNA they encode. The 5' fusion partner encodes the N-terminal portion of the protein, and the 3' fusion partner encodes the C-terminal portion of the protein.

[0153] As used herein, the term "heteroatom" is meant to include boron (B), oxygen (O), nitrogen (N), sulfur (S), phosphorus (P) and silicon (Si). In some embodiments, a "heterocycle" can contain one or more heteroatoms. In other embodiments, an aliphatic group can contain or be substituted with one or more heteroatoms.

[0154] As used herein, the term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as the sequence explicitly indicated.

[0155] As used herein, the term "oligonucleotide" refers to an oligomer of nucleotide or nucleoside monomeric units, where the oligomer optionally contains non-nucleotide monomeric units and / or other chemical groups attached to internal and / or external positions of the oligomer. The oligomer may be natural or synthetic and may include naturally occurring oligonucleotides or oligomers containing nucleosides with non-naturally occurring (or modified) bases, sugar moieties, phosphodiester-analog linkages, and / or alternative monomeric unit chirality and isomeric structures (e.g., 5'- to 2'-linkages, L-nucleosides, α-anomeric nucleosides, β-anomeric nucleosides, locked nucleic acids (LNA), peptide nucleic acids (PNAs)).

[0156] As used herein, the term "primer" refers to an oligonucleotide that binds to a specific region of a single-stranded template nucleic acid molecule and initiates nucleic acid synthesis via a polymerase-mediated enzymatic reaction. Typically, a primer contains less than about 100 nucleotides, and preferably less than about 30 nucleotides. A target-specific primer specifically hybridizes to a target polynucleotide under hybridization conditions. Such hybridization conditions may include, but are not limited to, hybridization in an isothermal amplification buffer (20 mM Tris-HCl, 10 mM (NH4)2SO4), 50 mM KCl, 2 mM MgSO4, 0.1% TWEEN® 20, pH 8.8, 25 °C) at a temperature of about 40 °C to about 70 °C. In addition to the target binding region, a primer may have an additional region, typically at the 5' portion. The additional region may include a universal primer binding site or a barcode. In order to perform exponential amplification, primers must be inward-facing, i.e., hybridize to opposite strands of target nucleic acid, with 3' ends facing each other. This orientation of amplification primers is sometimes called "correct orientation". Furthermore, in order to perform exponential amplification, primers hybridize to target nucleic acid within a suitable distance from each other. Under standard PCR conditions, primers hybridizing to opposite strands more than 2000 base pairs apart do not obtain sufficient amounts of product. For cfDNA samples, typical fragment sizes are 175 base pairs apart, and therefore primers hybridizing to opposite strands more than 175 base pairs apart typically do not result in amplification products.

[0157] As used herein, the terms "reference genome" and "reference genome sequence" refer to a genome sequence that is publicly available and regularly updated by the National Center for Biotechnology Information (NCBI), Refers to the entire human genome sequence (the "genome build") that is currently being constructed, GRCh38. The reference genome is searchable by chromosomal location and sequence to allow for comparison of sequences from individual samples and identification of any sequence variations in the samples.

[0158] As used herein, the term "rearranged genome" refers to a genome that contains one or more rearrangements when compared to a reference genome. It is understood that the rearranged genome also contains unrearranged sequences at other loci that are not involved in the rearrangement. In the rearranged genome, such loci have the same sequence as the corresponding reference genome loci. The term "rearranged genome sequence" refers to the rearranged sequence in the rearranged genome.

[0159] As used herein, the term "read depth" or "sequencing depth" refers to the number of times a sequence is sequenced (sequencing depth). For example, read depth can be determined by aligning multiple sequencing runs and counting the start positions of reads in non-overlapping windows of a certain size (e.g., 100 bp). Copy number polymorphism can be determined based on read depth using methods known in the art. For example, use the methods described in Yoon et al., Genome Research 2009 September; 19(9): 1586-1592; Xie et al., BMC Bioinformatics 2009 Mar. 6; 10: 80; or Medvedev et al., Nature Methods 2009 November; 6(11 Suppl): S13-20.

[0160] As used herein, the term "sample" refers to any biological sample that contains nucleic acid molecules, typically including DNA or RNA. A sample may be a tissue, cell or extract thereof, or may be a purified sample of nucleic acid molecules. The term "sample" refers to any composition that contains or is presumed to contain a target nucleic acid. The use of the term "sample" does not necessarily imply the presence of a target sequence between nucleic acid molecules present in the sample. A sample may be a sample of tissue or bodily fluid isolated from an individual, such as skin, plasma, serum, cerebrospinal fluid, lymphatic fluid, synovial fluid, urine, tears, blood cells, organ and tumor specimens, as well as in vitro cultures established from cells taken from an individual, including formalin-fixed paraffin-embedded tissue (FFPET) and nucleic acids isolated therefrom. A sample may also include acellular materials, such as cell-free blood fractions that contain cell-free DNA (cfDNA) or circulating tumor DNA (ctDNA). A sample may be collected from a non-human subject or the environment.

[0161] In some embodiments, a "sample" is a "representative sample". In some embodiments, a representative sample is a sample (or a subset of samples) that accurately reflects the overall composition, and thus the sample is an unbiased representation of the entire population. In general, this means that the different types of cells and their relative proportions or percentages in a representative sample or a portion thereof essentially accurately reflect or mimic the relative proportions or percentages of these cell types in the entire tissue specimen, typically a solid tumor or a portion thereof. Sampling is the act of reserving a portion of an object for subsequent analysis. Representative samples are generated so that a reasonably close knowledge of the studied object can be obtained. In contrast, traditional random sampling methods generally do not yield "representative samples". Selection of smaller individual sub-samples from a larger sample may be biased based on the region selected, whereas homogenization of the entire large sample, e.g., a tumor or lymph node, distributes spatially separated elements evenly throughout the sample.

[0162] As used herein, the term "sequencing" or "DNA sequencing" refers to a biochemical method for determining the order of the nucleotide bases, adenine, guanine, cytosine and thymine, in a DNA oligonucleotide. As used herein, may include, but is not limited to, parallel sequencing or any other sequencing method known to those of skill in the art, such as chain termination, rapid DNA sequencing, wandering-spot analysis, Maxam-Gilbert sequencing, dye terminator sequencing, or using any other modern automated DNA sequencing device.

[0163] As used herein, the term "target" or "target nucleic acid" refers to a nucleic acid of interest in a sample. A sample may contain multiple targets as well as multiple copies of each target.

[0164] As used herein, the term "universal primer" refers to a primer that can hybridize to a universal primer binding site. A universal primer binding site can be a natural or artificial sequence that is typically added to a target sequence in a non-target specific manner.

[0165] Composition of matter

[0166] In one aspect of the disclosure, a compound of formula (I) (also referred to herein as a "linked primer") is: [Olig1]-([R 1 ] o -[R 2 ] p ) q -[L 1 ] t -[Z]-[L 2 ] u -[W] v -[Olig2](I),

[0167] During the ceremony,

[0168] o is 0 or 1;

[0169] p is 0 or 1;

[0170] q is 0 or 1;

[0171] t is 0, 1 or 2;

[0172] u is 0, 1 or 2;

[0173] v is 0 or 1;

[0174] R 1 is an oligonucleotide having from about 1 to about 24 nucleotides;

[0175] R 2 is a substituted or unsubstituted, saturated or unsaturated, straight chain or cyclic aliphatic group having from 2 to about 48 carbon atoms, optionally substituted with one or more heteroatoms selected from O, N or S;

[0176] L 1 and L 2 are independently a substituted or unsubstituted, saturated or unsaturated, straight chain or cyclic aliphatic group having from 1 to about 16 carbon atoms, optionally containing one or more heteroatoms selected from O, N or S, and optionally containing one or more carbonyl groups;

[0177] Z is a moiety selected from a triazole, a dihydropyridazine, a phosphate linkage, an amide linkage, a thioether linkage, an isoxazoline, a hydrozone, an oxime ether, and a chloro-s-triazine linkage;

[0178] W is a substituted or unsubstituted, saturated or unsaturated, aliphatic or aromatic group having 1 to about 12 carbon atoms, optionally substituted with one or more heteroatoms selected from O, N, S, provided that W contains at least one photocleavable, enzymatically cleavable, chemically cleavable, or pH-sensitive group;

[0179] Olig1 is an oligonucleotide comprising from about 1 to about 30 nucleotides;

[0180] Olig2 is an oligonucleotide containing about 1 to about 30 nucleotides.

[0181] When a group is described as being "substituted or unsubstituted" if it is substituted, the substituents may be selected from one or more of the indicated substituents. When no substituents are indicated, the indicated "substituted" group is selected from alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heteroalicyclyl, aralkyl, heteroaralkyl, (heteroalicyclyl)alkyl, hydroxy, protected hydroxyl, alkoxy, aryloxy, acyl, mercapto, alkylthio, arylthio, cyano, cyanate, halogen, thiocarbonyl, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl. , C-amide, N-amide, S-sulfonamide, N-sulfonamide, C-carboxy, protected C-carboxy, O-carboxy, isocyanato, thiocyanato, isothiocyanato, nitro, silyl, sulfenyl, sulfinyl, sulfonyl, haloalkyl, haloalkoxy, trihalomethanesulfonyl, trihalomethanesulfonamide, ether, amino (e.g., mono- or di-substituted amino groups), and protected derivatives thereof. Any of the above groups may contain one or more heteroatoms including O, N, or S. For example, when a moiety is substituted with an alkyl group, the alkyl group may contain a heteroatom selected from O, N, or S (e.g., -(CH2-CH2-O-CH2-CH3)).

[0182] In some embodiments, Olig1 comprises about 1 to about 24 nucleotides. In other embodiments, Olig1 comprises about 1 to about 20 nucleotides. In other embodiments, Olig1 comprises about 1 to about 16 nucleotides. In still other embodiments, Olig1 comprises about 1 to about 12 nucleotides. In still other embodiments, Olig1 comprises about 2 to about 16 nucleotides. In still other embodiments, Olig1 comprises about 2 to about 12 nucleotides. In still other embodiments, Olig1 comprises about 3 to about 12 nucleotides. In still other embodiments, Olig1 comprises about 4 to about 12 nucleotides. In still other embodiments, Olig1 comprises about 3 to about 8 nucleotides. In still other embodiments, Olig1 comprises about 4 to about 8 nucleotides.

[0183] In some embodiments, Olig1 has a non-extendable 3' end. In some embodiments, the 3'-end is non-extendable due to the presence of terminator chemistry including dideoxynucleotides, 2'-phosphate nucleotides, as described, for example, in U.S. Patent No. 8,163,487, or any other 3'-O-blocked reversible terminator and 3' unblocked reversible terminator, as described, for example, in U.S. Patent Publication No. 2014 / 0242579 or Guo, J., et al., Four-color DNA sequencing with 3'-O-modified nucleotide reversible terminators and chemically cleavable fluorescent dideoxynucleotides, PNAS2008 105(27)9145-9150.

[0184] In some embodiments, Olig1 comprises an anchor sequence that can hybridize to a target sequence. In other words, at least a portion of Olig1 can hybridize to a target nucleic acid sequence. In some embodiments, the target nucleic acid sequence is a known fusion partner. Non-limiting examples of fusion partners 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, E SRRA, 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, T ERT, 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, PD These include GFB, 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.

[0185] In some embodiments, at least a portion of Olig1 is fully complementary to the target sequence. In other embodiments, Olig1 is only partially complementary to the target sequence. In either case, Olig1 is incubated in, for example, 20 mM Tris-HCl, 10 mM (NH4)2SO4, 50 mM KCl, 2 mM MgSO4, 0.1% TWEEN® 20, pH 8.8, 25° C. or 10 mM Tris-HCl, 50 mM KCl, 1.5 mM MgCl 2、 It forms a stable hybrid with a known fusion partner sequence in a buffer containing pH 8.3 at 25 °C under reaction conditions suitable for primer annealing.

[0186] In some embodiments, Olig2 comprises from about 1 to about 24 nucleotides. In other embodiments, Olig2 comprises from about 1 to about 16 nucleotides. In yet other embodiments, Olig1 comprises from about 1 to about 12 nucleotides. In yet other embodiments, Olig1 comprises from about 2 to about 9 nucleotides. In some embodiments, Olig2 comprises an extendable 3' end.

[0187] In some embodiments, Olig2 comprises a random sequence ("(N)n"). In some embodiments, the length of the random sequence can be 3, 4, 5, 6, 7, 8, or 10 or more nucleotides. To select the appropriate length of the random sequence, one skilled in the art would look for a sequence with a melting temperature (Tm) that allows a stable hybrid to form under the conditions used for hybridization of the anchor sequence. In other embodiments, Olig2 comprises a single repeating nucleotide, e.g., a poly-T oligonucleotide. In some embodiments, Olig2 is extended through the fusion breakpoint to form a copy strand that includes a portion of the upstream fusion partner, the fusion breakpoint, and a portion of the downstream fusion partner. In some embodiments, the copy strand is used for further analysis, e.g., by amplification and / or sequencing.

[0188] In some embodiments, a portion of Olig2 cannot hybridize to a target sequence. In some embodiments, the 5' portion of Olig2 may contain elements such as a universal primer binding site, a platform-specific sequencing primer binding site, a barcode (sample barcode or molecular barcode) or other tag sequence designed by the user. In some embodiments, the tag distinguishes RNA starting material from DNA starting material, as further described herein.

[0189] As noted above, in some embodiments, R 1 can be an oligonucleotide having from about 1 to about 16 nucleotides. 1 In yet another embodiment, R comprises an oligonucleotide having from about 1 to about 12 nucleotides. 1 In another embodiment, R comprises an oligonucleotide having from about 1 to about 8 nucleotides. 1 has a molecular weight in the range of about 350 g / mol to about 5200 g / mol. 1 has a molecular weight in the range of about 650 g / mol to about 300 g / mol.

[0190] As noted above, in some embodiments, R 2 may be a substituted or unsubstituted, saturated or unsaturated, linear or cyclic aliphatic group having from 2 to about 48 carbon atoms, optionally substituted with one or more heteroatoms selected from O, N, or S, and optionally containing one or more carbonyl groups. 2 comprises a substituted or unsubstituted, saturated or unsaturated, linear or cyclic aliphatic group having from 2 to about 32 carbon atoms, optionally substituted with one or more heteroatoms selected from O, N, or S, and optionally containing one or more carbonyl groups. 2comprises a substituted or unsubstituted, saturated or unsaturated, straight chain or cyclic aliphatic group having from 2 to about 28 carbon atoms, optionally substituted with one or more heteroatoms selected from O, N or S, and optionally containing one or more carbonyl groups.

[0191] In a further embodiment, R 2 may be a substituted or unsubstituted, saturated or unsaturated, linear or cyclic aliphatic group having from 2 to about 24 carbon atoms, optionally substituted with one or more heteroatoms selected from O, N or S, and optionally containing one or more carbonyl groups. 2 comprises a substituted or unsubstituted, saturated or unsaturated, linear or cyclic aliphatic group having from 2 to about 20 carbon atoms, optionally substituted with one or more heteroatoms selected from O, N or S, and optionally containing one or more carbonyl groups. 2 comprises a substituted or unsubstituted, saturated or unsaturated, straight chain or cyclic aliphatic group having between 2 and 1 to about 6 carbon atoms, optionally substituted with one or more heteroatoms selected from O, N or S, and optionally containing one or more carbonyl groups. 2 comprises a substituted or unsubstituted, saturated or unsaturated, linear or cyclic aliphatic group having from 2 to about 12 carbon atoms, optionally substituted with one or more heteroatoms selected from O, N, or S. In some embodiments, the one or more carbonyl groups can be a ketone, amide, or carboxyl. In other embodiments, R 2 does not contain a carbonyl group.

[0192] In some embodiments, R 2 comprises a moiety having the formula (IVA): [ka]

[0193] In the formula, d and e are each independently an integer ranging from 1 to 32; Q is a bond, O, S, N(R c )(R d ) or quaternary amine (N + H(R c )(R d )) and;R a and R b are independently H, a C1-C4 alkyl group, F, Cl or N( Rc )(R d ) and R c and R d is independently CH3 or H.

[0194] In some embodiments, d and e are each independently an integer in the range of 2 to 18. In some embodiments, e is in the range of 1 to 10. In other embodiments, e is in the range of 1 to 8. In still other embodiments, e is in the range of 2 to 6. In still other embodiments, e is in the range of 2 to 4. In some embodiments, d is an integer in the range of 1 to 8 and e is an integer in the range of 2 to 16. In other embodiments, d is an integer in the range of 2 to 8 and e is an integer in the range of 2 to 12. In other embodiments, d is 2 or 3 and e is an integer in the range of 2 to 12. In other embodiments, d is 2 or 3 and e is an integer in the range of 2 to 8. In other embodiments, d is 2 or 3 and e is an integer in the range of 2 to 6. In other embodiments, d is 2 or 3 and e is an integer in the range of 2 to 4. In some embodiments, R a or R b At least one of is -CH3.

[0195] In other embodiments, R 2 comprises a moiety having the formula (IVB): [ka]

[0196] In the formula, d and e are each independently an integer ranging from 1 to 32; Q is a bond, O, S, or N(R c )(R d ) and Rc and R d is independently CH3 or H.

[0197] In some embodiments, e is in the range of 1 to 10. In other embodiments, e is in the range of 1 to 8. In yet other embodiments, e is in the range of 2 to 6. In yet other embodiments, e is in the range of 2 to 4. In other embodiments, Q is O. In some embodiments, d is an integer in the range of 1 to 8 and e is an integer in the range of 2 to 16. In other embodiments, d is an integer in the range of 2 to 8 and e is an integer in the range of 2 to 12. In other embodiments, d is 2 or 3 and e is an integer in the range of 2 to 12. In other embodiments, d is 2 or 3 and e is an integer in the range of 2 to 8. In other embodiments, d is 2 or 3 and e is an integer in the range of 2 to 6. In other embodiments, d is 2 or 3 and e is an integer in the range of 2 to 4.

[0198] In yet another embodiment, R 2 comprises a moiety having the formula (IVC): [ka]

[0199] In the formula, d and e are each independently an integer ranging from 1 to 32. In one embodiment, e is in the range of 1 to 10. In another embodiment, e is in the range of 1 to 8. In yet another embodiment, e is in the range of 2 to 6. In yet another embodiment, e is in the range of 2 to 4. In some embodiments, d is in the range of 1 to 4 and e is in the range of 1 to about 8. In some embodiments, d is an integer in the range of 1 to 8 and e is an integer in the range of 2 to about 16. In other embodiments, d is an integer in the range of 2 to 8 and e is an integer in the range of 2 to about 12. In other embodiments, d is 2 or 3 and e is an integer in the range of 2 to 12. In other embodiments, d is 2 or 3 and e is an integer in the range of 2 to 8. In other embodiments, d is 2 or 3 and e is an integer in the range of 2 to 6. In other embodiments, d is 2 or 3 and e is an integer in the range of 2 to 4.

[0200] In some embodiments, R 2 comprises a solubilizing group. In some embodiments, the solubilizing group is a polyethylene glycol (PEG) group or a polypropylene glycol group. In yet other embodiments, the linker comprises from about 2 to about 8 PEG or polypropylene glycol groups. In yet other embodiments, the linker comprises about 6 PEG or polypropylene glycol groups. In yet other embodiments, the linker comprises about 4 PEG or polypropylene glycol groups. In yet other embodiments, the linker comprises 2 PEG or polypropylene glycol groups.

[0201] As noted above, in some embodiments, L 1 may be a substituted or unsubstituted, saturated or unsaturated, linear or cyclic aliphatic group having from 1 to about 16 carbon atoms, optionally substituted with one or more heteroatoms selected from O, N, or S, and optionally containing one or more carbonyl groups. 1 comprises a substituted or unsubstituted, saturated or unsaturated, linear or cyclic aliphatic group having from 1 to about 12 carbon atoms, optionally substituted with one or more heteroatoms selected from O, N or S, and optionally containing one or more carbonyl groups. 1 comprises a substituted or unsubstituted, saturated or unsaturated, straight chain or cyclic aliphatic group having from 1 to about 8 carbon atoms, optionally substituted with one or more heteroatoms selected from O, N or S, and optionally containing one or more carbonyl groups.

[0202] In a further embodiment, L 1 comprises a substituted or unsubstituted, saturated or unsaturated, straight chain or cyclic aliphatic group having from 1 to about 6 carbon atoms, optionally substituted with one or more heteroatoms selected from O, N or S, and optionally containing one or more carbonyl groups. 1comprises a substituted or unsubstituted, saturated or unsaturated, linear or cyclic aliphatic group having from 1 to about 4 carbon atoms, optionally substituted with one or more heteroatoms selected from O, N, or S, and optionally containing one or more carbonyl groups. 1 can include one or more solubilizing groups, such as PEG groups. In some embodiments, the carbonyl group is selected from ketone, amide, and carboxyl. In some embodiments, the group L 1 In some embodiments, the group L 1 includes amides.

[0203] In some embodiments, the group -([R 1 ] o -[R 2 ] p ) q - has a length ranging from about 15 angstroms to about 1000 angstroms. In other embodiments, the group -([R 1 ] o -[R 2 ] p ) q - has a length ranging from about 15 angstroms to about 500 angstroms. In yet another embodiment, the group -([R 1 ] o -[R 2 ] p ) q - has a length ranging from about 15 angstroms to about 400 angstroms. In yet another embodiment, -([R 1 ] o -[R 2 ] p ) q - has a length ranging from about 15 angstroms to about 300 angstroms. In yet other embodiments, the group -([R 1 ] o -[R 2 ] p ) q - has a length ranging from about 15 angstroms to about 250 angstroms. In yet other embodiments, the group -([R 1 ] o -[R2 ] p ) q The length ranges from about 15 angstroms to about 200 angstroms. In an embodiment, the group -([R 1 ] o -[R 2 ] p ) q - has a length ranging from about 15 angstroms to about 150 angstroms. In yet another embodiment, the group -([R 1 ] o -[R 2 ] p ) q - has a length ranging from about 15 angstroms to about 100 angstroms. In yet another embodiment, -([R 1 ] o -[R 2 ] p ) q - has a length ranging from about 15 angstroms to about 50 angstroms. In yet another embodiment, -([R 1 ] o -[R 2 ] p ) q - has a length in the range of about 20 angstroms to about 40 angstroms.

[0204] In some embodiments, o+p=1 and q is 1. In other embodiments, o is 1, p is 0, and q is 1. In yet other embodiments, o is 0, p is 1, and q is 1. In yet other embodiments, o is 0, p is 1, and q is 2.

[0205] In some embodiments, o is 1, p is 0, q is 1, and R 1 In some embodiments, o is 1, p is 0, q is 1, and R 1 comprises about 1 to about 8 nucleotides. In some embodiments, o is 1, p is 0, q is 1, and R 1 contains about 1 to about 6 nucleotides.

[0206] In some embodiments, o is 0, p and q are both 1, and R 2 In some embodiments, o is 0, p and q are both 1, and R 2 In some embodiments, o is 0, p and q are both 1, and R 2 In some embodiments, o is 0, p and q are both 1, and R 2 In some embodiments, o is 0, p and q are both 1, and R 2 In some embodiments, o is 0, p and q are both 1, and R 2 In some embodiments, o is 0, p and q are both 1, and R 2 In some embodiments, o is 0, p and q are both 1, and R 2 contains at least 16 PEG groups.

[0207] In some embodiments, o is 0, p and q are both 1, and R 2 comprises a group having the formula (IVB). In some embodiments, o is 0, p and q are both 1, and R 2 comprises a group having the formula (IVB), where e ranges from 1 to 16. In some embodiments, o is 0, p and q are both 1, and R 2 comprises a group having the formula (IVB), and e ranges from 1 to 12. In some embodiments, o is 0, p and q are both 1, and R 2 comprises a group having the formula (IVB), and e ranges from 1 to 8.

[0208] In some embodiments, o is 0, p and q are both 1, and R 2comprises a group having the formula (IVB), d is 2 or 3, and e ranges from 1 to 16. In some embodiments, o is 0, p and q are both 1, and R 2 comprises a group having the formula (IVB), d is 2 or 3, and e ranges from 1 to 12. In some embodiments, o is 0, p and q are both 1, and R 2 comprises a group having the formula (IVB), d is 2 or 3, and e ranges from 1 to 10. In some embodiments, o is 0, p and q are both 1, and R 2 comprises a group having the formula (IVB), d is 2 or 3, and e ranges from 1 to 8. In some embodiments, o is 0, p and q are both 1, and R 2 comprises a group having the formula (IVB), d is 2 or 3, and e ranges from 1 to 6. In some embodiments, o is 0, p and q are both 1, and R 2 comprises a group having the formula (IVB), d is 2 or 3, and e ranges from 1-4.

[0209] In some embodiments, o is 0, p and q are both 1, and R 2 comprises a group having the formula (IVB), d is 2, and e ranges from 1 to 12. In some embodiments, o is 0, p and q are both 1, and R 2 comprises a group having the formula (IVB), d is 2, and e ranges from 1 to 10. In some embodiments, o is 0, p and q are both 1, and R 2 comprises a group having the formula (IVB), d is 2, and e ranges from 1 to 8. In some embodiments, o is 0, p and q are both 1, and R 2 comprises a group having the formula (IVB), d is 2, and e ranges from 1 to 6. In some embodiments, o is 0, p and q are both 1, and R 2 comprises a group having the formula (IVB), d is 2 and e ranges from 1 to 4.

[0210] In some embodiments, o is 0, p and q are both 1, and R 2comprises a group having the formula (IVC). In some embodiments, o is 0, p and q are both 1, and R 2 comprises a group having the formula (IVC), and e ranges from 1 to 16. In some embodiments, o is 0, p and q are both 1, and R 2 comprises a group having the formula (IVC), and e ranges from 1 to 12. In some embodiments, o is 0, p and q are both 1, and R 2 comprises a group having the formula (IVC), and e ranges from 1 to 8. In some embodiments, o is 0, p and q are both 1, and R 2 comprises a group having the formula (IVC), and e ranges from 1 to 4. In some embodiments, o is 0, p and q are both 1, and R 2 comprises a group having the formula (IVC), d is 2 or 3, and e ranges from 1 to 16. In some embodiments, o is 0, p and q are both 1, and R 2 comprises a group having the formula (IVC), d is 2 or 3, and e ranges from 1 to 12. In some embodiments, o is 0, p and q are both 1, and R 2 comprises a group having the formula (IVC), d is 2 or 3, and e ranges from 1 to 10. In some embodiments, o is 0, p and q are both 1, and R 2 comprises a group having the formula (IVC), d is 2 or 3, and e ranges from 1 to 8. In some embodiments, o is 0, p and q are both 1, and R 2 comprises a group having the formula (IVC), d is 2 or 3, and e ranges from 1 to 6. In some embodiments, o is 0, p and q are both 1, and R 2 comprises a group having the formula (IVC), d is 2 or 3, and e ranges from 1-4.

[0211] As noted above, in some embodiments, L 2may be a substituted or unsubstituted, saturated or unsaturated, linear or cyclic aliphatic group having from 1 to about 16 carbon atoms, optionally substituted with one or more heteroatoms selected from O, N, or S, and optionally containing one or more carbonyl groups. 2 comprises a substituted or unsubstituted, saturated or unsaturated, linear or cyclic aliphatic group having from 1 to about 12 carbon atoms, optionally substituted with one or more heteroatoms selected from O, N or S, and optionally containing one or more carbonyl groups. 2 comprises a substituted or unsubstituted, saturated or unsaturated, straight chain or cyclic aliphatic group having from 1 to about 8 carbon atoms, optionally substituted with one or more heteroatoms selected from O, N or S, and optionally containing one or more carbonyl groups.

[0212] In a further embodiment, L 2 comprises a substituted or unsubstituted, saturated or unsaturated, straight chain or cyclic aliphatic group having from 1 to about 6 carbon atoms, optionally substituted with one or more heteroatoms selected from O, N or S, and optionally containing one or more carbonyl groups. 2 comprises a substituted or unsubstituted, saturated or unsaturated, linear or cyclic aliphatic group having from 1 to about 4 carbon atoms, optionally substituted with one or more heteroatoms selected from O, N, or S, and optionally containing one or more carbonyl groups. 2 can contain one or more solubilizing groups, such as PEG groups. In some embodiments, the carbonyl group is a ketone , amide, and carboxyl. In some embodiments, the group L 2 In some embodiments, the group L 2 includes amides.

[0213] In some embodiments, the compound of formula (I) comprises a cleavage site for cleaving the compound of formula (I). In some embodiments, the cleavage site is located within Olig2. In these embodiments, v is 0 and there is no W group. In these embodiments, Olig2 can comprise, for example, at least one uracil-containing nucleotide. In some embodiments, the uracil-containing nucleotide can be cleaved by adding uracil-N-DNA glycosylase (UNG) as described in US Pat., optionally in the presence of a primary amine as described in US Pat. No. 8,669,061. In some embodiments, cleavage is performed by a combination of glycosylase and endonuclease, for example, a mixture of uracil DNA glycosylase (UDG) and DNA glycosylase-lyase endonuclease VIII.

[0214] In other embodiments, the cleavage site is located outside of Olig2, such as within the group W. In some embodiments, W comprises a substituted or unsubstituted, saturated or unsaturated aliphatic or aromatic group having 1 to about 12 carbon atoms, optionally substituted with one or more heteroatoms selected from O, N, S, with the proviso that W comprises a photocleavable group, an enzymatically cleavable group, a chemically cleavable group, or a pH-sensitive group, as described above. In other embodiments, W comprises a substituted or unsubstituted, saturated or unsaturated aliphatic or aromatic group having 1 to about 8 carbon atoms, optionally substituted with one or more heteroatoms selected from O, N, S, with the proviso that W comprises a photocleavable group, an enzymatically cleavable group, a chemically cleavable group, or a pH-sensitive group.

[0215] In yet other embodiments, W comprises a substituted or unsubstituted, saturated or unsaturated aliphatic or aromatic group having 1 to about 6 carbon atoms, optionally substituted with one or more heteroatoms selected from O, N, S, with the proviso that W comprises at least one photocleavable, enzymatically cleavable, chemically cleavable, or pH-sensitive group. In further embodiments, W comprises a substituted or unsubstituted, saturated or unsaturated aliphatic or aromatic group having 1 to about 4 carbon atoms, optionally substituted with one or more heteroatoms selected from O, N, S, with the proviso that W comprises at least one photocleavable, enzymatically cleavable, chemically cleavable, or pH-sensitive group.

[0216] In some embodiments, W comprises at least one photocleavable moiety. In some embodiments, the photocleavable moiety can be cleaved upon exposure to an electromagnetic radiation source having a wavelength between about 200 nm and about 400 nm (UV) or between about 400 nm and about 800 nm (visible). Examples of suitable photocleavable moieties include arylcarbonylmethyl groups (e.g., 4-acetyl-2-nitrobenzyl, dimethylphenacyl (DMP)); 2-(alkoxymethyl)-5-methyl-α-chloroacetophenone, 2,5-dimethylbenzoyloxirane, benzoin groups (e.g., 3',5'-dimethoxybenzoin (DMB)), o-nitrobenzyl groups (e.g., 1-(2-nitrophenyl)ethyl (NPE), 1-(methoxymethyl)-2-nitrobenzene, 4,5-dimethoxy-2-nitrobenzene, 1-(2-nitrophenyl)ethyl (NPE), 1-(methoxymethyl)-2-nitrobenzene, 1-(2-nitrophenyl)ethyl (NPE), ... o-nitroanilides (e.g., acylated 5-bromo-7-nitroindoline); coumarin-4-yl-methyl groups (e.g., 7-methoxycoumarin derivatives); 9-substituted xanthenes and arylmethyl groups (e.g., o-hydroxyarylmethyl groups).

[0217] In some embodiments, at least one photocleavable moiety has a wavelength of about 700 nm and They can be cleaved upon exposure to a source of electromagnetic radiation having a wavelength between about 1000 nm. Suitable near infrared photocleavable groups include cyanine groups, including C4-dialkylamine substituted heptamethine cyanines.

[0218] In some embodiments, W comprises at least one chemically cleavable moiety. In some embodiments, the chemically cleavable moiety is a group that can be chemically cleaved by different chemical reactants, including reducing agents, or by an induced change in pH (e.g., cleavage of the group at a pH of less than 7). Non-limiting examples of chemically cleavable moieties include disulfide-based groups; diazobenzene groups (e.g., 2-(2-alkoxy-4-hydroxy-phenylazo); benzoic acid backbone; ester bond-based groups; and acid-sensitive groups (e.g., dialkoxydiphenylsilane groups or acylhydrazone groups). Electrophilically cleaved groups (e.g., p-alkoxybenzyl esters and p-alkoxybenzyl amides) are believed to be cleaved by protons and include acid-sensitive cleavage.

[0219] In some embodiments, W comprises at least one enzymatically cleavable moiety. In some embodiments, the enzymatically cleavable moiety can be cleaved by, for example, a trypsin-cleavable group and a V8 protease-cleavable group. In some embodiments, the at least one enzymatically cleavable moiety can be enzymatically cleaved by one of a USER enzyme, uracil-N-glycosylase, RNase A, β-glucuronidase, β-galactosidase, or TEV-protease.

[0220] Another aspect of the present disclosure is a compound having formula (II): [Olig1]-([R1] o -[R2] p ) q -[L1] t -[X] (II)

[0221] During the ceremony,

[0222] o is 0 or 1;

[0223] p is 0 or 1;

[0224] q is 1 or 2;

[0225] t is 0, 1 or 2;

[0226] R 1 is an oligonucleotide having from about 1 to about 24 nucleotides;

[0227] R 2 is a substituted or unsubstituted, saturated or unsaturated, straight chain or cyclic aliphatic group having from 2 to about 48 carbon atoms, optionally substituted with one or more heteroatoms selected from O, N or S;

[0228] L 1 is a substituted or unsubstituted, saturated or unsaturated, linear or cyclic aliphatic group having from 1 to about 16 carbon atoms, optionally substituted with one or more heteroatoms selected from O, N or S, and optionally containing one or more carbonyl groups;

[0229] X is dibenzocyclooctyne, trans-cyclooctene, alkyne, alkene, azide, tetrazine, maleimide, N-hydroxysuccinimide, thiol, 1,3-nitrone, aldehyde, ketone, hydrazine, hydroxylamine, amino group, or phosphoramidite;

[0230] Olig1 is an oligonucleotide containing about 1 to about 30 nucleotides.

[0231] In some embodiments, Olig1 comprises from about 1 to about 24 nucleotides. In other embodiments, Olig1 comprises from about 1 to about 16 nucleotides. In some embodiments, Olig1 has a non-extendable 3' end.

[0232] In some embodiments, Olig1 comprises an anchor sequence capable of hybridizing to a known fusion partner. Non-limiting examples of fusion partners 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, E SRRA, 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, T ERT, 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, PD These include GFB, 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.

[0233] Another aspect of the present disclosure is a compound having formula (II): [Y]-[L 2 ] u -[W] v -[Olig2] (III)

[0234] During the ceremony,

[0235] u is 0, 1 or 2;

[0236] v is 0 or 1;

[0237] Y is dibenzocyclooctyne, trans-cyclooctene, alkyne, alkene, azide, tetrazine, maleimide, N-hydroxysuccinimide, thiol, 1,3-nitrone, aldehyde, ketone, hydrazine, hydroxylamine, amino group, or phosphoramidite;

[0238] L 2 is a substituted or unsubstituted, saturated or unsaturated, linear or cyclic aliphatic group having 1 to 16 carbon atoms, optionally containing one or more heteroatoms selected from O, N or S, and optionally containing one or more carbonyl groups;

[0239] W is a substituted or unsubstituted, saturated or unsaturated aliphatic or aromatic group having 1 to 12 carbon atoms, optionally substituted with one or more heteroatoms selected from O, N, S, provided that W contains at least one photocleavable, enzymatically cleavable, chemically cleavable, or pH-sensitive group;

[0240] Olig2 is an oligonucleotide containing about 1 to about 30 nucleotides.

[0241] In some embodiments, Olig2 comprises from about 1 to about 24 nucleotides. In other embodiments, Olig2 comprises from about 1 to about 16 nucleotides. In still other embodiments, Olig2 comprises from about 1 to about 12 nucleotides. In some embodiments, Olig2 comprises an extendable 3' end. In some embodiments, Olig2 comprises a random sequence. In other embodiments, Olig2 comprises a single repeating nucleotide, such as a poly-T oligonucleotide.

[0242] Preparation of Compounds of Formulae (I), (II) and (III)

[0243] One skilled in the art will appreciate that compounds of formula (II) and formula (III) may be reacted with each other to form a compound having formula (I). In some embodiments, the group Z in formula (I) is determined by the groups X and Y in formulas (II) and (III), respectively. The table shows the groups X and Y in formulas (II) and (III) and the resulting group Z in the compound having formula (I) formed. [Table A-1] [Table A-2]

[0244] In some embodiments, the groups Olig1 and Olig2 of formula (II) and (III) are prepared according to methods known to those skilled in the art. In some embodiments, the groups Olig1 and Olig2 are synthesized using solid-phase synthesis techniques using phosphoramidite chemistry (see, for example, Protocols for Oligonucleotides and Analogs, Agrawal, S., ed., Humana Press, Totowa, NJ, 1993, the entire contents of which are incorporated herein by reference). Other methods for synthesizing Olig1, Olig2, and / or compounds of formula (II) and (III) are described in U.S. Patent Nos. 5,955,591, 6,057,431, 8,889,843 and 6,124,445; and U.S. Patent Application Publication Nos. 2008 / 0119645 and 2003 / 0153743, the disclosures of which are incorporated herein by reference in their entireties.

[0245] In some embodiments, the first step in such a process is to attach the first monomer or higher subunit containing a protected 5'-hydroxyl to a solid support, usually via a linker, using standard methods and procedures known in the art. See, e.g., Oligonucleotides and Analogues A Practical Approach, Ekstein, F. Ed., IRL Press, NY, 1991. The support-bound monomer or higher 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 compound is oxidized to produce a compound having the desired internucleotide linkage. In some embodiments, the choice of oxidizing agent determines whether the phosphite linkage is oxidized to, for example, a phosphotriester, thiophosphotriester, or dithiophosphotriester linkage.

[0246] In some embodiments, a capping step is performed before 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 the oligonucleotide strand that did not react in a given coupling cycle. In some embodiments, the cap moiety is reactive with the terminal portion of the oligonucleotide that did not participate in the coupling cycle, but not with the oligonucleotide that did participate, and is not itself reactive with the coupling reagent.

[0247] Further treatment of the oxidized oligomer with acid removes the 5'-hydroxyl protecting group, thus converting the solid support-bound oligomer into an additional compound that can then be reacted to initiate the next synthetic iteration, and this process is repeated until an oligomer of the desired length is produced.

[0248] In some embodiments, compounds of formula (II) and (III) can be reacted to form a compound of formula (I). In these embodiments, a 5'-5' bond can be formed between the compound of formula (II) and the compound of formula (II). In some embodiments, compounds of formula (I) can be reacted to form a compound of formula (II). Compounds having I) are synthesized in the 3' to 5' direction, for example, using the procedures described above. Such synthesis can be carried out using 3' amidites.

[0249] Compounds of formula (III) can be synthesized in a similar manner, except that 5'amidites are used instead of 3'amidites. Non-limiting examples of 5'amidites are shown below. In this manner, compounds of formula (III) can be synthesized in the 5' to 3' direction. In some embodiments, compounds of formula (II) and (III) can be linked via a phosphate bridge. [ka]

[0250] In some embodiments, compounds of formula (II) and formula (III) can be reacted with each other using "click chemistry". "Click chemistry" is a chemical philosophy uniquely defined by the Sharpless and Meldal groups that describes tailored chemistry to rapidly and reliably generate substances by linking small units. "Click chemistry" has been applied to a collection of reliable and autonomous organic reactions (Kolb, HC Finn, MG; Sharpless, KB Angew). Chem. Int. Ed. 2001, 40, 2004-2021). For example, the identification of copper-catalyzed azide-alkyne [3+2] cycloaddition as a reliable molecular linkage in water (Rostovtsev, VV; et al. Angew. Chem. Int. Ed. 2002, 41, 2596-2599) has been used to augment the investigation of several types of biomolecular interactions (Wang, Q.; et al. J. Am. Chem. Soc. 2003, 125, 3192-3193; Speers, AE; et al. J. Am. Chem. Soc. 2003, 125, 4686-4687; Link, AJ; Tirrell, DAJAm. Chem. Soc. 2003, 125, 11164-11165; Deiters, A.; et al. al. J. Am. Chem. Soc. 2003, 125, 11782-11783). In addition, they have been used in organic synthesis (Lee, LV; et al. J. Am. Chem. Soc. 2003, 125, 9588-9589), drug discovery (Kolb, HC; Sharpless, KBDrug Disc. Today 2003, 8, 1128-1137; Lewis, WG; et al. Angew. Chem. Int. Ed. 2002, 41, 1053-1057), and surface functionalization (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, JP; et al. Langmuir 2004, ASAP, in press; Lummerstorfer, T.; Hoffmann, HJ Phys. Chem. B 2004, in press).

[0251] In some embodiments, a precursor of a compound of formula (II) is first modified to introduce a first member of a pair of reactive functional groups capable of participating in a "click chemistry" reaction. Similarly, in some embodiments, a precursor of a compound of formula (III) is modified to introduce a second member of a 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 shown in Table 1. In some embodiments, the "click chemistry" reaction is catalyzed by an introduced reagent. In some embodiments, the introduced reagent is Cu + It is. [Table 1] Table 1: First and second members of reactive functional group pairs.

[0252] By way of example only, a precursor of the compound of formula (II) may be modified to introduce a primary halogen. Sodium azide can then be introduced, which reacts with the primary halogen such that the precursor of the compound of formula (II) is converted to an azide. In some embodiments, the precursor of the compound of formula (II) is reacted directly or indirectly through a linker with an amidite containing a primary halogen. Non-limiting examples of suitable amidites are shown below. [ka]

[0253] Also by way of example, precursors to compounds of formula (III) can be modified (such as with an amidite) to introduce moieties reactive with azides of formula (II), such as moieties that contain alkyl groups. Non-limiting examples of suitable amidites are provided below. [ka] [ka]

[0254] Another suitable reagent is a DBCO-PEG-phosphoramidite, such as DBCO-PEG4-phosphoramidite. [ka]

[0255] The resulting compounds of formula (II) and (III), each bearing a member of a reactive group capable of participating in a "click chemistry" reaction, are then reacted with each other to form a 5'-5' bond. In the above example, the azide and alkyne react to form a triazole bond.

[0256] In some embodiments, the compounds of formula (II) and (III) may each include a reactive group (X and Y, respectively) that facilitates the formation of an amide bond between the compounds. To accomplish this, in some embodiments, a precursor of each of the compounds of formula (II) and (III) may be reacted with a reagent that introduces the groups X and Y, respectively. In these embodiments, the precursor of the compound having formula (II) is modified at the 5' end with an amino moiety. For example, an amidite may be introduced into the precursor of the compound having formula (II), where the amidite includes a terminal amino moiety. Non-limiting examples of such amidite reagents include: [ka]

[0257] Similarly, the precursor of the compound having formula (III) may be modified to terminate at the 5' end with a carboxyl group. For example, an amidite may be introduced into the precursor of the compound having formula (II), the amidite containing a terminal carboxyl moiety. Non-limiting examples of such amidite reagents are as follows: [ka]

[0258] In some embodiments, the compounds of formula (II) and (III) may each include a reactive group (X and Y, respectively) that facilitates the formation of a thioether bond between the compounds. To accomplish this, in some embodiments, a precursor of each of the compounds of formula (II) and (III) may be reacted with a reagent that introduces the groups X and Y, respectively. In these embodiments, the precursor of the compound having formula (II) is modified at the 5' end with a thiol moiety. For example, an amidite may be introduced into the precursor of the compound having formula (II), the amidite including a terminal thiol moiety. Non-limiting examples of such amidite reagents include: [ka]

[0259] The compound having formula (III) may be modified so that the 5' end is terminated with a maleimide group. For example, an amidite may be introduced into the precursor of the compound having formula (III), the amidite containing a terminal maleimide moiety. Non-limiting examples of such amidite reagents are as follows: [ka]

[0260] In some embodiments, the compounds of formula (II) and (III) may each include a reactive group (X and Y, respectively) that facilitates the formation of a triazine bond between the compounds. To achieve this, in some embodiments, a precursor of each of the compounds of formula (II) and (III) may be reacted with a reagent that introduces the groups X and Y, respectively. In some embodiments, the triazine bond is a chloro-s-triazine bond. In these embodiments, the precursor of the compound having formula (II) is modified at the 5' end with an amino moiety. Similarly, the precursor of the compound having formula (II) is modified at the 5' end with an amino moiety. Non-limiting examples of suitable amidites for introducing such 5' amino groups are provided below. [ka]

[0261] After both the modification of the precursor to a compound of formula (II) and the modification of the precursor to a compound of formula (III), the formed compounds of formula (II) and (III) are reacted with a coupling reagent. In some embodiments, the coupling reagent is s-trichlorotriazine. This reaction is shown below: [ka]

[0262] In some embodiments, any precursor of the compound of formula (II) or (III) can be reacted to introduce a linker or spacer, such as a PEG-based linker or spacer. Non-limiting examples of suitable reagents for introducing a PEG-based linker or spacer are provided below. [ka]

[0263] Other reagents and methods for incorporating PEG-based linkers or spacers into precursors of compounds of formula (II) and / or (III) are described in U.S. Patent Application Publication No. 2006 / 0063147, the disclosure of which is incorporated herein by reference in its entirety.

[0264] 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 linker or spacer that includes a cleavable group. Non-limiting examples of suitable reagents are provided below. [ka]

[0265] kit

[0266] Another aspect of the disclosure is a kit, such as a kit comprising one or more compounds of formula (I). In some embodiments, the kit comprises 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 DNA polymerase. The kit may further comprise an amplification primer. In some embodiments, the kit further comprises at least one forward primer and / or reverse primer. In some embodiments, the kit comprises a forward primer capable of hybridizing to a copy of the first oligonucleotide and a reverse primer capable of hybridizing to a copy of the second oligonucleotide. In other embodiments, the kit comprises a forward primer capable of hybridizing to the first oligonucleotide and a reverse primer capable of hybridizing to a copy of the second oligonucleotide.

[0267] In other embodiments, the kit may include one or more compounds of formula (I), (II) or (III) and one or more buffers. In some embodiments, the kit includes 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.

[0268] In other embodiments, the 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).

[0269] 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 the kit further comprises s-trichlorotriazine.

[0270] In some embodiments, any of the compounds of formula (I), (II) and / or (III) may be included in a reaction vessel along 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 may occur according to the present teachings. In some embodiments, the reaction vessel comprises a well of a dPCR chip. In some embodiments, the dPCR chip may comprise, for example, a silicon substrate etched with nanoscale or smaller reaction wells. In some embodiments, the dPCR chip has low thermal mass. For example, the chip may be constructed of a thin, highly conductive material that does not store thermal energy. In some embodiments, the dPCR chip is approximately 50 mm thick.2 ~about 150mm 2 In some implementations, In terms of form, the dPCR chip is approximately 100 mm 2 The surface area of ​​the chip is 100 nm to 150 nm. Limiting the surface area can allow for greater uniformity in the heating of the chip during melt analysis, reduced run-to-run variability in melt curing analysis, reduced errors in melt curve generation, and improved discrimination of melt curves in the analysis. Other dPCR chips are described in PCT Publication No. WO / 2016 / 133783, the disclosure of which is incorporated herein by reference in its entirety.

[0271] method

[0272] Another aspect of the present disclosure is a method for detecting one or more gene fusions in which one fusion partner is unknown. In some embodiments, the method utilizes one or more compounds of formula (I). In some embodiments, the method further comprises amplifying nucleic acid and / or forming a library of amplified nucleic acid. In some embodiments, the method further comprises sequencing the library of amplified nucleic acid, thereby detecting one or more genomic rearrangements in the sample. These and other steps of the method are described herein.

[0273] Gene fusions are a common occurrence in cancer. Clinical testing of gene fusions allows for the detection and diagnosis of cancer, tracking of tumor burden over time, and the development of personalized treatment protocols for cancer patients. Particularly useful are blood-based methods to detect gene fusions. Blood-based methods access a patient's cell-free nucleic acid (cfDNA and cfRNA), including circulating tumor nucleic acid (ctDNA and ctRNA). Although blood-based tests are less invasive than biopsies, the major difficulty is detecting very small amounts of tumor-derived nucleic acid mixed with normal non-tumor-derived nucleic acid. Several commercially available tests can 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.).

[0274] In some cancer-associated gene fusions, detection of the fusion product in ctDNA is further complicated by the presence of multiple fusion partners. Tumor-associated genes with promiscuous fusions include many examples such as NTRK 1, 2 and 3 and FGFR2 and 3.

[0275] sample

[0276] The disclosed methods utilize 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 patient. In some embodiments, the sample may include a solid tissue or a fragment of a solid tumor derived from a subject or patient, for example, by biopsy. The sample may also include bodily fluids (e.g., urine, sputum, serum, plasma or lymph, saliva, sputum, sweat, tears, 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 include whole blood or blood fractions in which normal or tumor cells may be present. In some embodiments, the sample, particularly the liquid sample, may include acellular material such as cell-free tumor DNA or cell-free DNA or RNA, including cell-free fetal DNA or RNA. In some embodiments, the sample is an acellular sample, for example, a cell-free blood-derived sample in which cell-free tumor DNA or RNA or cell-free fetal DNA or RNA is present. In other embodiments, the sample is a culture sample, such as a culture or culture supernatant containing or suspected of containing nucleic acid derived from cells in the culture.

[0277] In some embodiments, the sample is a representative sample. In some embodiments, a representative sample is prepared from a tumor sample, a lymph node sample, a blood sample, and / or other tissue sample that has been homogenized (alone or together). "Homogenization" refers to a process (mechanical and / or biochemical processes) in which a biological sample is conditioned such that the composition of all fractions of the sample is equal. Representative samples (as defined herein) may be prepared by removing a portion of a homogenized sample. A homogenized sample ("homogenate") is thoroughly mixed such that removing a portion (aliquot) of the sample does not substantially change the overall composition of the remaining sample, and the components of the removed aliquot are substantially identical to the components of the remaining sample. In this disclosure, "homogenization" generally preserves the integrity of a majority of the cells in the sample, e.g., at least 50% of the cells in the sample are not ruptured or lysed as a result of the homogenization process. In other embodiments, homogenization preserves the integrity of at least 80% of the cells in the sample. In other embodiments, homogenization preserves the integrity of at least 85% of the cells in the sample. In other embodiments, homogenization preserves the integrity of at least 90% of the cells in the sample. In other embodiments, homogenization preserves the integrity of at least 95% of the cells in the sample. In other embodiments, homogenization preserves the integrity of at least 96% of the cells in the sample. In other embodiments, homogenization preserves the integrity of at least 97% of the cells in the sample. In other embodiments, homogenization preserves the integrity of at least 98% of the cells in the sample. In other embodiments, homogenization preserves the integrity of at least 99% of the cells in the sample. In other embodiments, homogenization preserves the integrity of at least 99.9% of the cells in the sample. The homogenate may be substantially dissociated into individual cells (or clusters of cells), and the resulting homogenate(s) are substantially homogenous (consisting of or composed of similar elements or uniform throughout).

[0278] In some embodiments, the input sample comprises a representative sample of cells from a tumor sample, a lymph node sample, a blood sample, or any combination thereof. In some embodiments, the input sample is from a human patient or mammalian subject that is (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 recurrence of cancer. In other embodiments, the input sample is from a healthy human patient or mammalian subject. Further 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 incorporated herein by reference in its entirety.

[0279] target nucleic acid

[0280] A target nucleic acid is a nucleic acid of interest that may be present in a sample. Each target is characterized by its nucleic acid sequence. The present disclosure allows for the detection of one or more RNA or DNA targets. In some embodiments, the DNA target nucleic acid is a gene or 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 part of a transcript of a gene or coding sequence resulting from a fusion. In some embodiments, the target nucleic acid comprises a biomarker, i.e., a gene whose variant, such as a gene fusion, is associated with a disease or condition. For example, the target nucleic acid can be selected from a panel of disease-associated markers described in U.S. Patent Application No. 14 / 774,518, filed September 10, 2015. Such a panel is available as the AVENIO ctDNA Analysis Kit (Roche Sequencing Solutions, Pleasanton, Cal.).

[0281] Of particular 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 predicted to undergo cancer-associated fusions include ALK, PPARG, BRAF, EGFR, FGFR1, FGFR2, FGFR3, MET, NRG1, NTRK1, NTRK2, NTRK3, RET, ROS1, AXL, PDGFRA, PDGFB, ABL1, ABL2, AKT1, AKT2, AKT3, ARHGAP26 ... D3, 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, NO TCH2, 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.

[0282] In some embodiments, the target nucleic acid is RNA (including mRNA). In such embodiments, the DNA polymerase that extends the compound of formula (I) is 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 that extends the compound of formula (I) is any DNA polymerase, for example, any B family DNA polymerase. The target nucleic acid can be in 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, including, for example, circulating cell-free DNA (cfDNA) or chemically degraded DNA, such as that found in chemically preserved or old 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 incorporated herein by reference in its entirety).

[0283] DNA isolation

[0284] In some embodiments, the method of the present disclosure includes a step of isolating nucleic acids. In general, any nucleic acid extraction method that results in isolated nucleic acids, including DNA, RNA, or a mixture of DNA and RNA, can be used. Genomic DNA or cellular RNA or a mixture of DNA and RNA can be extracted from tissue, cell, 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 nuclear proteins, and, optionally, removal of contaminants. Extraction of nucleic acids from preserved samples can further include a step of deparaffinization. Solution-based nucleic acid extraction methods can include salting-out methods or organic solvent or chaotropic methods. Solid-phase nucleic acid extraction methods include silica resin methods, anion exchange methods, or magnetic glass particles and paramagnetic beads (KAPA Pure). Examples of suitable nucleic acid sequences include, but are not limited to, AMPure beads (Roche Sequencing Solutions, Pleasanton, Cal.) or AMPure beads (Beckman Coulter, Brea, Cal.).

[0285] A typical extraction method involves the lysis of tissue material and cells present in the sample. The 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, and the nucleic acids can be subjected to one or more washing steps to remove contaminants from the sample, including proteins, lipids and their fragments. Finally, the bound The 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, nucleases cannot be used and care must be taken to inhibit nuclease activity during the purification process.

[0286] In some embodiments, isolation of nucleic acids utilizes epitachophoresis (ETP), as described in PCT / EP2019 / 077714, filed October 14, 2019, and PCT / EP2018 / 081049, filed November 13, 2018. ETP utilizes a device with a circular arrangement of electrodes where nucleic acids migrate and concentrate between leading and trailing electrolytes. The circular configuration allows for the concentration of nucleic acids in a very small volume collected in the center of the device. The use of ETP is particularly advantageous for plasma samples that contain large amounts of small amounts of cell-free nucleic acids.

[0287] In some embodiments, the input DNA or input RNA requires fragmentation. In such embodiments, the RNA may be fragmented by a combination of heat and a metal ion, such as magnesium. In some embodiments, the sample is heated to 85°C-94°C for 1-6 minutes in the presence of magnesium. (KAPA RNA HyperPrep Kit, KAPA Biosystems, Wilmington, Mass.) DNA may be fragmented by physical means, such as sonication, using commercially available instruments (Covaris, Woburn, Mass.) or enzymatic means (KAPA Fragmentase Kit, KAPA Biosystems).

[0288] In some embodiments, DNA repair enzymes target damaged bases in isolated nucleic acids. In some embodiments, the sample nucleic acid is partially damaged DNA from a storage sample, such as a formalin-fixed paraffin-embedded (FFPET) sample. Deamination and oxidation of bases can lead to erroneous base reads 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.

[0289] The disclosed method is applicable to several different types of nucleic acids. In some embodiments, the disclosed method utilizes isolated DNA (i.e., DNA separated from RNA by RNase digestion). In some embodiments, the disclosed method utilizes isolated RNA (i.e., RNA separated from DNA by DNase digestion). In still other embodiments, the disclosed method utilizes a mixture of DNA and RNA (i.e., isolated nucleic acid that has not been treated with nuclease).

[0290] concentrated

[0291] In some embodiments, the disclosed method 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, where the capture probes can hybridize 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 and removed from the solution of target nucleic acids and discarded. Removal can be achieved by utilizing capture probes with binding moieties that can be captured on a solid support. In other embodiments, enrichment is by retention, where the capture probes can hybridize to one or more target sequences, i.e., known sequences of 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 binding moieties that can be captured on a solid support. The captured target-probes are then subjected to a step of quantification to determine whether the target sequences are hybridized to a gene-specific capture probe and ... The hybrids are retained while the rest of the solution containing non-target sequences is discarded.

[0292] For enrichment, the capture probe may be free in solution or may be immobilized on a solid support. The probe may also contain a binding moiety (e.g., biotin) and be captured on a solid support (e.g., an avidin- or streptavidin-containing support material).

[0293] contacting the sample or the target enriched sample with a ligated primer, such as a compound of formula (I)

[0294] With reference to FIG. 1 (bottom) and FIG. 2, the disclosure provides methods for detecting gene fusions by contacting a sample with a linked primer (such as any of the primers of formula (I)). In some embodiments, the linked primer comprises a first oligonucleotide sequence (e.g., "Olig1" of formula (I)) coupled directly or indirectly via a bond (e.g., group "Z" of formula (I)) to a second oligonucleotide sequence (e.g., "Olig2" of formula (I)). In some embodiments, as shown in FIG. 1, the linked primer comprises a first oligonucleotide sequence (left side of formula (I), "Olig1") that comprises an anchor sequence that can hybridize to a known 5'-fusion partner. The linked primer comprises a "spacer" (e.g., group "-([R 1 ] o -[R 2 ] p ) q The second oligonucleotide ("Olig2" on the right side of formula (I)) contains a random sequence ("NNN") and an extendable 3' end.

[0295] As shown in Figure 1 (bottom), the sample is contacted with a nucleic acid polymerase "POL" that has polymerase activity and strand displacement activity. In some embodiments, the nucleic acid in the sample is DNA and a DNA-dependent DNA polymerase is used, such as any B-family polymerase that has strand displacement activity. In some embodiments, the nucleic acid in the sample is RNA and a reverse transcriptase is used.

[0296] In some embodiments, the nucleic acid in the sample is a mixture of DNA and RNA. Such samples can be processed into target DNA and RNA in a single tube using the methods described in U.S. Provisional Application No. 62 / 888963, "Single tube preparation of DNA and RNA for sequencing," filed August 19, 2019, and incorporated herein by reference. Briefly, the described method includes forming cDNA with a first primer having a tag that identifies the RNA starting material under conditions in which the DNA starting material is not reactive. After the cDNA is formed, the target cDNA is amplified and detected along with the target DNA by a set of common amplification primers that does not include the first primer. The end product derived from RNA is distinguished from the end product derived from DNA by the presence of an RNA-specific tag ("RNA identification tag") introduced by the first primer. In some embodiments, the 5' portion of the second oligonucleotide (e.g., "Olig2" in formula (I)) includes an RNA identification tag.

[0297] In some embodiments, a polymerase extends the 3' end of a second oligonucleotide (e.g., "Olig2" of Formula (I)) while displacing the anchor sequence of a first oligonucleotide (e.g., "Olig1" of Formula (I)) that hybridized to a known sequence of a known gene fusion partner. (Figure 1, bottom). In some embodiments, the extension product, referred to as the first copy strand, includes copies 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.

[0298] 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 the 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, In embodiments, the 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.

[0299] In some embodiments, it is desirable to remove the first oligonucleotide (e.g., "Olig1" in Formula (I)) from the first copy strand. In some embodiments, the group (e.g., group "W" in Formula (I)) between the first oligonucleotide and the second oligonucleotide (e.g., "Olig1" and "Olig2" in Formula (I)) comprises a cleavable moiety. In some embodiments, the cleavable linker is selected from a photocleavable group, an enzymatically cleavable group, a chemically cleavable group, or a pH-sensitive group. In embodiments that comprise a photocleavable moiety, the photocleavable moiety can be cleaved by introducing radiation having a particular wavelength (e.g., radiation having a wavelength in the range of about 400 nm to about 800 nm). In embodiments that comprise an enzymatically cleavable group, the enzymatically cleavable group can be cleaved by one of the USER enzymes, uracil-N-glycosylase, RNase A, β-glucuronidase, β-galactosidase, or TEV-protease. In embodiments that include chemically cleavable groups, the chemically cleavable group may be cleaved by introducing an appropriate electrophile and / or nucleophile.

[0300] In some embodiments, the compound of formula (I) does not include a group "W" (where v=0), and the cleavable moiety is included within "Olig2". In some embodiments, "Olig2" includes a cleavage site comprised of one or more uracil-containing nucleotides. In some embodiments, the strand containing 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 a primary amine, as described in U.S. Pat. No. 8,669,061. UNG recognizes uracil present in single- or double-stranded DNA and cleaves 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 incorporated herein by reference in its entirety.

[0301] In some embodiments, cleavage is performed by a combination of glycosylase and endonuclease, for example, a mixture of uracil DNA glycosylase (UDG) and DNA glycosylase-lyase endonuclease VIII. By cleaving the cleavage site, the first copy strand is separated from the first oligonucleotide (e.g., "Olig1" in formula (I)) and the linker structure (Figure 2, bottom). In some embodiments, cleavage is performed before forming the second copy strand.

[0302] In some embodiments, the first copy strand or double-stranded copy of the gene fusion is sequenced. In some embodiments, the first copy strand or double-stranded copy of the gene fusion is amplified before sequencing. As described herein, the amplification may include gene-specific primers, specific primers, or universal primers. A universal primer binding site may be introduced into the 5-part of the second oligonucleotide of the primer used to form the linking primer or the second copy strand (e.g., "Olig2" in formula (I)).

[0303] In some embodiments, the method is multiplexed, i.e., the method targets multiple genes known to be involved in gene fusion events. In such embodiments, a reaction mixture is provided that includes two or more compounds of formula (I), each of the two or more compounds of formula (I) having an anchor sequence specific to a particular gene known to be involved in gene fusion. For example, the same reaction mixture can be used to target ALK, PPARG, BRAF, EGFR, FGFR1, FGFR2, FGFR3, MET, NRG1, NTRK1, NTRK2, NTRK3, NTRK4, NTRK5, NTRK6, NTRK7, NTRK8, NTRK9, NTRK10, NTRK11, NTRK12, NTRK13, NTRK24, NTRK25, NTRK36, NTRK47, NTRK58, NTRK69, NTRK70, NTRK81, NTRK92, NTRK14, NTRK15, NTRK16, NTRK17, NTRK18, NTRK29, NTRK21, NTRK22, NTRK23, NTRK24, NTRK36, NTRK19, NTRK25, NTRK26, NTRK27, NTRK38, NTRK39, NTRK40, NTRK41, NTRK42, NTRK59, NTRK61, NTRK15, NTRK24, NTRK16, NTRK25, NTRK17, NTRK26, NTRK27, NTRK38, NTRK41, NTRK18, NTRK28, NTRK39, NTRK41, NTRK58, NTRK19, NTRK29, NTRK39, NTRK41, NTRK59, NTRK61, NTRK19, NTRK25, NTRK26, NTRK39, NTRK41, NTRK58, NTRK19, NTRK25, NTRK26, NTRK39, NTRK41, NTRK59, NTRK61, NTRK11 RK2, 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, P TK2B, RAF1, RARA, RELA, RSPO2, RSPO3, SYK, TERT, TFE3, TFEB, THADA, TMPRSS2, TSLP, TY, BCL2, BCL6, BCR, CAMTA 1, 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, STAT The compound of formula (I) may comprise two or more compounds having anchor sequences targeting one or more of: 6, 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.

[0304] In some embodiments, the linking primer is designed to accommodate short input nucleic acid.For example, cell-free DNA, including circulating tumor DNA (ctDNA), has an average length of 175 bp.In such an embodiment, the length of the linking primer should not exceed 175 bases.

[0305] amplification

[0306] In some embodiments, the disclosure includes an amplification step. The copy strands formed as shown in FIG. 2 (bottom) can be copied and amplified by linear or exponential amplification. The amplification can be isothermal or can include thermal cycling. In some embodiments, the amplification is exponential and involves PCR. In some embodiments, at least one gene-specific primer is used for the amplification, such as a primer that can hybridize to a known fusion partner. In some embodiments, the 5' portion of the linking primer contains a primer binding site for a second primer used for the amplification. In other embodiments, a universal primer binding site is added to the nucleic acid to be amplified. In some embodiments, the universal primer binding site can be added by ligating an adapter that contains a universal primer binding site. In other embodiments, the universal primer binding site is added by extending a gene-specific primer with a 5'-tail that contains a universal primer binding site. All nucleic acids with the same universal primer binding site can be conveniently amplified under the same conditions using the same primer set. The number of amplification cycles in which universal primers are used can be low, or can be high, about 10, about 20, or about 30 or more cycles, depending on the amount of product required for the subsequent steps. Because PCR using universal primers has reduced sequence bias, it is not necessary to limit the number of amplification cycles to avoid amplification bias.

[0307] Primer

[0308] In some embodiments, the present disclosure includes an amplification step that utilizes a forward primer and a reverse primer. can be target specific. A target-specific primer is composed of 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, such as a barcode or universal primer binding site, are present, they are usually located in the 5' portion of the primer.

[0309] In some embodiments, a first primer specific to a known gene sequence upstream of the fusion breakpoint can be used to amplify the copy strand formed as shown in Figure 2 (bottom). In some embodiments, a second primer is specific to a tag sequence or any other engineered sequence present in the second linking oligonucleotide.

[0310] In some embodiments, the first and second specific primers comprise a universal primer binding site in the 5' portion of the primer. After one or more rounds of specific amplification, a universal amplification is performed.

[0311] Library

[0312] In some embodiments, the 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 adapter sequences attached thereto, as described below. The nucleic acids in the library may contain elements such as barcodes and universal primer binding sites present in the adapter sequences as described herein below. In some embodiments, the additional elements are present in the adapters and are added to the library nucleic acids via adapter ligation. In other embodiments, some or all of the additional elements are present in the amplification primers and are added to the library nucleic acids prior to adapter ligation by primer extension.

[0313] In some embodiments, the library is formed from all the nucleic acids in the sample prior to the use of the fusion detection linked primers described herein. In this embodiment, an adapter molecule is added to all the nucleic acids in the sample. The method of detecting fusions using linked primers uses the library molecules as starting material. In some embodiments, universal amplification (using a universal primer that hybridizes to a primer binding site located on the adapter) is performed prior to fusion-specific amplification using linked primers. Universal amplification increases the amount of starting material for fusion-specific amplification using linked primers performed as described herein.

[0314] In some embodiments, the library molecules comprise adaptors that comprise unique molecular barcodes. Sequencing the library comprises determining sequences of the barcoded library nucleic acids, grouping the sequences into families by the unique molecular barcodes, and determining consensus reads for each family, thereby detecting gene fusions.

[0315] adapter

[0316] In some embodiments, the present disclosure utilizes an adapter nucleic acid. The adapter may be added to the nucleic acid by blunt end ligation or cohesive end ligation. In some embodiments, the adapter may be added by single strand ligation methods. In some embodiments, the adapter is added by amplification with a tiled primer having an adapter sequence at the 5' portion of the primer. Methods and compositions useful for adding adapters by ligation or amplification are described in, for example, U.S. Patent Nos. 9,476,095, 9,260,753, 8,822,150, 8,563,478, 7,741,463, 8,182,989, and 8,053,192. , the disclosures of which are incorporated herein by reference in their entireties.

[0317] In some embodiments, the adapter molecule is an in vitro synthesized artificial sequence. In other embodiments, the adapter molecule is an in vitro synthesized natural sequence. In yet other embodiments, the adapter molecule is an isolated natural molecule or an isolated non-natural molecule.

[0318] In the case of adapters added by ligation, the adapter oligonucleotides can have an overhang or blunt end at the end that is ligated to the target nucleic acid. In some embodiments, the adapters include blunt ends that can be applied for blunt-end ligation of the target nucleic acid. The target nucleic acid can be blunt-ended or blunt-ended by enzymatic treatment (e.g., "end repair"). In other embodiments, the blunt-ended DNA undergoes A-tailing, in which a single A nucleotide is added to the 3' end of one or both blunt ends. The adapters described herein are made to have a single T nucleotide extending from the blunt end to facilitate ligation between the nucleic acid and the adapter. Commercially available kits for performing adapter ligation include AVENIO ctDNA Library Prep kits or KAPA HyperPrep and HyperPlus kits (Roche Sequencing Solutions, Pleasanton, Cal.). In some embodiments, the adapter-ligated DNA can be separated from excess adapters and unligated DNA.

[0319] The adapters may further include features such as a 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 adapters include all of the above features, while in other embodiments, some of the features are added after ligation of the adapters by extending a tailed primer that contains some of the above elements.

[0320] The adaptor may further comprise a capture moiety. The capture moiety may be any moiety capable of specifically interacting with another capture molecule. Capture moiety-capture molecule pairs include avidin (streptavidin)-biotin, antigen-antibody, magnetic (paramagnetic) particle-magnet, or oligonucleotide-complementary oligonucleotide. The capture molecule may be bound to a solid support, such that any nucleic acid in which the capture moiety is present is captured on the solid support and separated from the remainder of the sample or reaction mixture. In some embodiments, the capture molecule comprises a capture moiety of a secondary capture molecule. For example, the capture moiety in the adaptor may be a nucleic acid sequence complementary to the capture oligonucleotide. The capture oligonucleotide may be biotinylated such that the adapted nucleic acid-capture oligonucleotide hybrid may be captured on a streptavidin bead.

[0321] In some embodiments, adaptor-ligated nucleic acids are enriched by capturing the capture moiety and separating the adaptor-ligated target nucleic acids from unligated nucleic acids in a sample.

[0322] In some embodiments, the stem portion of the adapter comprises modified nucleotides that increase the melting temperature of the capture oligonucleotide, such as 5-methylcytosine, 2,6-diaminopurine, 5-hydroxybutyne-2'-deoxyuridine, 8-aza-7-deazaguanosine, ribonucleotides, 2'O-methylribonucleotides, or locked nucleic acids. In another aspect, the capture oligonucleotide is modified to inhibit digestion by nucleases, such as phosphorothioate nucleotides.

[0323] In some embodiments, adapter sequences are added to the copy strands formed as shown in Figure 2 (bottom) by either adapter ligation or amplification with a tailed primer. Adapters can be added to either single-stranded or double-stranded molecules including the copy strands shown in Figure 2.

[0324] Barcode

[0325] In some embodiments, the present invention utilizes barcodes. Detection of individual molecules typically requires molecular barcodes, such as those described in U.S. Patent Nos. 7,393,665, 8,168,385, 8,481,292, 8,685,678, and 8,722,368. Unique molecular barcodes are short artificial sequences that are typically added to each molecule in a patient sample during the earliest steps of in vitro manipulation. The barcode labels the molecule and its progeny. Unique molecular barcodes (UIDs) have multiple applications. Barcodes allow tracking of individual nucleic acid molecules in a sample to assess the presence and amount of circulating tumor DNA (ctDNA) molecules, for example, in a patient's blood, 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).

[0326] The barcode may be a multiplexed sample ID (MID) used to identify the source of the samples from which the samples are mixed (multiplexed). The barcode also serves 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 a MID. In some embodiments, a single barcode is used as both a UID and a MID. In some embodiments, each barcode comprises a predetermined sequence. In other embodiments, the barcode comprises a random sequence. In some embodiments of the present disclosure, the barcode is about 4-20 bases long such that 96-384 different adapters, each with a different pair of identical barcodes, are added to the human genomic sample. One of skill in the art will recognize that the number of barcodes depends on the complexity of the sample (i.e., the expected number of unique target molecules) and can create an appropriate number of barcodes for each experiment.

[0327] Unique molecular barcodes can also be used for molecular counting and sequencing error correction. The entire progeny of a single target molecule are labeled with the same barcode, forming a barcoded family. Sequence variations that are not shared by all members of the barcoded family are discarded as artifacts, which are not true mutations. Since the entire family represents a single molecule in the original sample, barcodes can also be used for positional deduplication and target quantification (Newman, A., et al., (2016) Integrated digital error suppression for improved detection of circulating tumor DNA, Nature Biotechnology 34:547).

[0328] In some embodiments, the number of UIDs in the plurality of adaptors or barcode-containing primers can 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.

[0329] purification

[0330] In some embodiments, the disclosure includes an intermediate purification step. For example, any unused oligonucleotides, such as excess primers and excess adapters, can be purified by, for example, gel electrophoresis. The oligonucleotides are removed by a size selection method selected from affinity chromatography and size exclusion chromatography. In some embodiments, size selection can be performed using solid phase reversible immobilization (SPRI) technology by Beckman Coulter (Brea, Cal.). In some embodiments, a capture moiety is used to capture and separate the adaptor-ligated nucleic acids from the products of exponential amplification from unligated nucleic acids or excess primers. In some embodiments, excess oligonucleotides, including unused primers or adaptors, are removed using a specific capture nucleic acid that forms a closed ring structure surrounding the oligonucleotide to be removed, as described in U.S. Application No. 63 / 021875, "Remove of excess oligonucleotides from reaction mixture," filed May 8, 2020.

[0331] Sequencing

[0332] In some embodiments, the library of nucleic acids, including the copy strand, the double-stranded copy of the gene fusion sequence, and the gene fusion sequence, or their amplicons, can be subjected to nucleic acid sequencing. Sequencing can be performed according to any method known to those skilled in the art. In some embodiments, the sequencing method includes Sanger sequencing and dye terminator sequencing, as well as next-generation sequencing techniques 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, microscope-based sequencing, microfluidic Sanger sequencing, microscope-based sequencing, RNAP sequencing, tunneling current DNA sequencing, and in vitro virus sequencing. See WO2014144478, WO2015058093, WO2014106076, and WO2013068528, each of which is incorporated by reference in its entirety.

[0333] In some embodiments, sequencing can be performed by several different methods, such as using sequencing by synthesis technology. Prior art sequencing by synthesis is defined as any sequencing method that monitors the production of by-products when specific deoxynucleoside triphosphates are incorporated during the sequencing reaction (Hyman, 1988, Anal. Biochem. 174:423-436; Rhonaghi et al., 1998, Science 281:363-365). One prominent embodiment of sequencing by synthesis reaction is pyrophosphate sequencing method. In this case, the production of pyrophosphate during nucleotide incorporation is monitored by an enzyme cascade that results in the production of a chemiluminescent signal. One example of sequencing by synthesis, the 454 Genome Sequencer System (Roche Applied Science Catalog No. 04 760 085 001), is based on pyrophosphate sequencing technology. When 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.

[0334] In some embodiments, the sequencing by synthesis reaction can alternatively be based on a terminator dye type sequencing reaction. In this case, the incorporated dye deoxynucleotriphosphate (ddNTP) building block contains a detectable label, preferably a fluorescent label, that prevents further extension of the nascent DNA strand. The ddNTP building block is then coupled to the template / probe by, for example, using a DNA polymerase that contains a 3'-5' exonuclease or proofreading activity. Upon incorporation into the primer extension hybrid, the label is removed and detected.

[0335] In some embodiments, sequencing is performed using next-generation sequencing methods, such as those provided by Illumina, Inc. ("Illumina Sequencing Method"). Without wishing to be bound by any particular theory, Illumina's next-generation sequencing technology uses clonal amplification and sequencing-by-synthesis (SBS) chemistry to enable rapid and accurate sequencing. This process simultaneously identifies DNA bases as they are incorporated into the nucleic acid strand. Each base emits a unique fluorescent signal when added to the growing strand, which is used to determine the order of the DNA sequence.

[0336] In some embodiments, the sequencing method is a high-throughput single molecule sequencing method that utilizes nanopores. In some embodiments, the nucleic acids and libraries of nucleic acids formed as described herein are sequenced by a method that includes threading a biological nanopore (see US Pat. No. 10,337,060, the disclosure of which is incorporated herein by reference in its entirety) or a solid-state nanopore (see US Pat. No. 10,288,599, US Pat. No. 20180038001, US Pat. No. 10,364,507, the disclosures of which are incorporated herein by reference in their entirety). In other embodiments, the sequencing includes threading a tag through a nanopore (see US Pat. No. 8,461,854, the disclosure of which is incorporated herein by reference in its entirety) or any other currently existing or future DNA sequencing technology that utilizes nanopores.

[0337] In other embodiments, sequencing is performed by other suitable technologies for high-throughput single molecule sequencing, including the Illumina HiSeq platform (Illumina, San Diego, CA), the Ion Torrent platform (Life Technologies, Grand Island, NY), the Pacific BioSciences platform utilizing Single Molecule Real-Time (SMRT) technology (Pacific Biosciences, Menlo Park, CA), or any other currently existing or future DNA sequencing technology with or without sequencing-by-synthesis.

[0338] The sequencing step can utilize platform-specific sequencing primers. The binding sites for these primers can be introduced into the 5' portion of the amplification primers used in the amplification step. If the primer sites are not present in the library of barcoded molecules, an additional short amplification step can be performed that introduces such binding sites.

[0339] In some embodiments, the sequencing step involves sequence analysis. In some embodiments, the analysis includes a step of sequence alignment. In some embodiments, alignment is used to determine a consensus sequence from multiple sequences, for example multiple sequences with the same barcode (UID). In some embodiments, barcodes (UIDs) are used to determine a consensus from multiple sequences that all have the same barcode (UID). In other embodiments, barcodes (UIDs) are used to remove artifacts, i.e., variations that are present in some sequences but not all sequences with the same barcode (UID). Such artifacts resulting from PCR errors or sequencing errors can be removed.

[0340] In some embodiments, the number of each sequence in a sample can be quantified by quantifying the relative number of sequences with each barcode (UID) in the sample. By counting the distinct UIDs associated with each sequence variant, which represent a single molecule in a sample, the fraction of each sequence in the original sample can be determined. One of skill in the art can determine the number of sequence reads required to determine a consensus sequence. In some embodiments, the relevant number is the reads per UID ("sequence depth") required for an accurate quantitative result. In some embodiments, the desired depth is between 5 and 50 reads per UID.

[0341] In some embodiments, the sequencing step further comprises a step of error correction by consensus determination. Sequencing by synthesis of circular strands of gapped circular templates disclosed herein allows for repeated or repeated sequencing. Multiple reads at the same nucleotide position allow for correction of sequencing errors through the establishment of consensus calls for each nucleotide or for the entire sequence or a portion of the sequence. The final sequence of the nucleic acid strand is obtained from the consensus base determination at each position. In some embodiments, the consensus sequence of the nucleic acid is obtained from the 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 a step of sequence read alignment and generating a consensus sequence after the sequencing step. In some embodiments, the consensus is a simple majority consensus as described in U.S. Pat. No. 8,535,882. In other embodiments, the consensus is determined by the 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 replicate reads used to determine the consensus sequence, the sequence may be largely error-free or substantially error-free.

[0342] No Sequencing

[0343] In some embodiments, the library of nucleic acids containing the copy strand, double-stranded copy of the gene fusion sequence, and gene fusion sequence or their amplicons are detected without sequencing. Detection can be achieved by amplification, including end-point polymerase chain reaction (PCR), quantitative PCR (qPCR), or digital PCR (dPCR), including digital droplet PCR (ddPCR). In some embodiments, the detection of gene fusion is quantitative, such as the type of detection enabled by qPCR and dPCR. In other embodiments, the detection of gene fusion is qualitative, i.e., the readout is the presence or absence of a fusion-specific amplification product by gel electrophoresis, capillary electrophoresis, mass spectrometry, or another method of detecting nucleic acids of a characteristic size or molecular weight.

[0344] dPCR

[0345] In some embodiments, gene fusion-specific amplification according to the present disclosure is performed by digital PCR (dPCR), including digital droplet PCR (ddPCR).

[0346] Digital PCR is a method of quantitative amplification of nucleic acid, for example, as described in U.S. Patent No. 9,347,095, the disclosure of which is incorporated herein by reference. This process involves dividing a sample into reaction volumes, such that each volume contains no more than one copy of target nucleic acid. In some embodiments, the divided reaction volumes are aqueous droplets.

[0347] In some embodiments, the target nucleic acid in the split is a copy strand. In other embodiments, the target nucleic acid in the split is a double-stranded copy of the gene fusion sequence. Each split further comprises amplification primers, i.e., forward and reverse primers that can support exponential amplification of the target nucleic acid. In some embodiments, the forward and reverse primers can hybridize to the known fusion sequence and the 5' sequence of the second oligonucleotide (Figure 1).

[0348] Each of the digital PCR reaction volumes further comprises a detectably labeled probe capable of hybridizing to the amplicon of the forward primer and the reverse primer. In some embodiments, the probe can hybridize to a known fusion sequence. In some embodiments, the probe is designed to avoid binding to a wild-type non-fusion gene sequence.

[0349] The detectably labeled probes may be labeled with a combination of fluorophores, and the exponential amplification may be carried out using a nucleic acid polymerase that has 5'-3'-exonuclease activity.

[0350] In some embodiments, the methods of the disclosure include performing an amplification reaction using a forward primer and a reverse primer, where the reaction includes detecting the amplicon using a probe and determining the number of reaction volumes in which the probe is detected, thereby detecting the presence of the gene fusion in the sample.

Claims

1. 1. A method for detecting a gene fusion in a nucleic acid sample, comprising: (a) treating the nucleic acid sample with (i) a nucleic acid polymerase having polymerase activity and strand displacement activity, and (ii) a nucleic acid polymerase having formula (I): [Olig1]-([R １ ] ｏ -[R ２ ] ｐ ) ｑ -[L １ ] ｔ -[Z]-[L ２ ] ｕ -[W] ｖ -[Olig2] (I) (In the formula, 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; R １ is an oligonucleotide having from about 1 to about 24 nucleotides; R ２ is a substituted or unsubstituted, saturated or unsaturated, straight chain or cyclic aliphatic group having from 2 to about 48 carbon atoms, optionally substituted with one or more heteroatoms selected from O, N or S; L １ and L ２ is independently a substituted or unsubstituted, saturated or unsaturated, straight chain or cyclic aliphatic group having from 1 to about 16 carbon atoms, optionally containing one or more heteroatoms selected from O, N or S, and optionally containing 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 isoxazoline, 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 1 to about 12 carbon atoms, optionally substituted with one or more heteroatoms selected from O, N, S, provided that W contains at least one photocleavable, enzymatically cleavable, chemically cleavable, or pH-sensitive group; Olig1 is an oligonucleotide comprising from about 1 to about 30 nucleotides and including an anchor sequence capable of hybridizing to a known fusion partner, Olig1 having a non-extendable 3' end; Olig2 is an oligonucleotide comprising from about 1 to about 30 nucleotides and comprising an extendable 3' end. and (b) extending the 3' end of Olig2 with the nucleic acid polymerase, wherein the extension product comprises a copy of a portion of the unknown fusion partner, a portion of the known fusion partner, and a fusion breakpoint, thereby forming a first strand copy of the gene fusion. The method comprising:

2. 2. The method of claim 1, further comprising forming a library of double-stranded copies of the gene fusion, wherein forming the library comprises attaching adapters to the copies of the gene fusion, the adapters comprising a barcode and a primer binding site.

3. (a) dividing the sample containing the copies of the gene fusion into a plurality of reaction volumes, where each reaction volume contains forward and 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 detection step using the probe. The method includes the steps of: (c) determining the number of reaction volumes in which the probe was detected, thereby detecting the gene fusion; 2. The method of claim 1, further comprising amplifying said copies of said gene fusion by a method comprising:

4. Formula (I) [Olig1]-([R １ ] ｏ -[R ２ ] ｐ ) ｑ -[L １ ] ｔ -[Z]-[L ２ ] ｕ -[W] ｖ -[Olig2] (I) (In the formula, 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; R １ is an oligonucleotide having from about 1 to about 24 nucleotides; R ２ is a substituted or unsubstituted, saturated or unsaturated, straight chain or cyclic aliphatic group having from 2 to about 48 carbon atoms, optionally substituted with one or more heteroatoms selected from O, N or S; L １ and L ２ is independently a substituted or unsubstituted, saturated or unsaturated, straight chain or cyclic aliphatic group having from 1 to about 16 carbon atoms, optionally containing one or more heteroatoms selected from O, N or S, and optionally containing one or more carbonyl groups; Z is a moiety selected from triazole, dihydropyridazine, phosphate linkage, amide linkage, thioether linkage, isoxazoline, hydrozone, oxime ether, and chloro-s-triazine linkage; W is a substituted or unsubstituted, saturated or unsaturated aliphatic or aromatic group having 1 to about 12 carbon atoms, optionally substituted with one or more heteroatoms selected from O, N, S, provided that W contains at least one photocleavable, enzymatically cleavable, chemically cleavable, or pH-sensitive group; Olig1 is an oligonucleotide having from about 1 to about 30 nucleotides, Olig1 having a non-extendable 3' end; Olig2 is an oligonucleotide having from about 1 to about 30 nucleotides, and Olig2 has an extendable 3' end. A compound having the formula:

5. R ２ comprises a substituted or unsubstituted, saturated or unsaturated, straight chain or cyclic aliphatic group having from 2 to about 32 carbon atoms, optionally substituted with one or more heteroatoms selected from O, N or S, and optionally containing one or more carbonyl groups.

6. R ２ is represented by formula (IVA): 【Chemistry 1】 (wherein d and e are each independently an integer ranging from 1 to 32; Q is a bond, O, S, N(R ｃ ) (R ｄ ) or quaternary amine (N ＋ H (R ｃ ) (R ｄ )) and R ａ and R ｂ are independently H, C １ -C ４ Alkyl group, F, Cl or N( Ｒｃ ) (R ｄ ) and R ｃ and R ｄ is independently CH ３ or H) 5. The compound of claim 4, comprising a moiety having the structure:

7. R ２ is represented by formula (IVB): 【Chemistry 2】 (wherein d and e are each independently an integer ranging from 1 to 32; Q is a bond, O, S, or N(R ｃ ) (R ｄ ) and R ｃ and R ｄ is independently CH ３ or H) 5. The compound of claim 4, comprising a moiety having the structure:

8. L １ Or L ２ 5. The compound of claim 4, wherein at least one of comprises a substituted or unsubstituted, saturated or unsaturated, straight chain or cyclic aliphatic group having from 1 to about 4 carbon atoms, optionally substituted with one or more heteroatoms selected from O, N or S, and optionally containing one or more carbonyl groups.

9. The compound of claim 4, wherein o+p=1 and q is 1.

10. o is 0, p and q are both 1, R １ contains at least one PEG group; L １ The compound of claim 4 , wherein

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. 5. The compound of claim 4, wherein v is 0 and Olig2 comprises a cleavage site comprising at least one uracil-containing nucleotide.

14. The compound of claim 4 , wherein Olig2 comprises a random nucleotide sequence.

15. A kit for detecting a gene fusion between a known fusion partner and an unknown fusion partner, comprising a compound according to any one of claims 30 to 63 and a polymerase.

16. 13. A kit comprising: (a) Formula (II): [_]_________________ １ ] ｏ __ ２ ] ｐ ) ｑ __ １ ] ｔ _ [_] ___) (In the formula, o is 0 or 1; p is 0 or 1; q is 1 or 2; t is 0, 1 or 2; R １ is an oligonucleotide having from 1 to about 24 nucleotides; R ２ is a substituted or unsubstituted, saturated or unsaturated, straight chain or cyclic aliphatic group having from 2 to about 48 carbon atoms, optionally substituted with one or more heteroatoms selected from O, N or S; L １ is a substituted or unsubstituted, saturated or unsaturated, straight chain or cyclic aliphatic group having from 1 to about 16 carbon atoms, optionally substituted with one or more heteroatoms selected from O, N or S, and optionally containing one or more carbonyl groups; X is dibenzocyclooctyne, trans-cyclooctene, alkyne, alkene, azide, tetrazine, maleimide, N-hydroxysuccinimide, thiol, 1,3-nitrone, aldehyde, ketone, hydrazine, hydroxylamine, amino group, or phosphoramidite; Olig1 is an oligonucleotide having from about 1 to about 30 nucleotides. A first compound having the formula: (b) Formula (III): [|] ２ ] ｕ ___ ｖ ______________) (In the formula, u is 0, 1 or 2; v is 0 or 1; Y is dibenzocyclooctyne, trans-cyclooctene, alkyne, alkene, azide, tetrazine, maleimide, N-hydroxysuccinimide, thiol, 1,3-nitrone, aldehyde, ketone, hydrazine, hydroxylamine, amino group, or phosphoramidite; L ２ is a substituted or unsubstituted, saturated or unsaturated, linear or cyclic aliphatic group having 1 to 16 carbon atoms, optionally containing one or more heteroatoms selected from O, N or S, and optionally containing one or more carbonyl groups; W is a substituted or unsubstituted, saturated or unsaturated aliphatic or aromatic group having 1 to 12 carbon atoms, optionally substituted with one or more heteroatoms selected from O, N, S, provided that W comprises a photocleavable group, an enzymatically cleavable group, a chemically cleavable group, or a pH-sensitive group; Olig2 is an oligonucleotide having from about 1 to about 30 nucleotides. A second compound having the formula: Including the kit.

17. Use of a compound according to any one of claims 4 to 14 or a kit according to claims 15 to 15 in sequencing a nucleic acid molecule.

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