High-resolution spectral chromosome banding method for detecting chromosomal abnormalities

Abstract

A method for detecting structural variations in chromosomes by labeling single-stranded chromatids with different colored probes is disclosed. The hybridization patterns of the labeled probes generate spectral profiles that enable high-resolution detection of structural variations, facilitating the distinction between benign and deleterious structural variations. Furthermore, the spectral profiles provide information about complex structural variations that may result from more than one rearrangement of chromosomal segments. The spectral profiles can be used to generate data tables, which can then be subjected to node analysis to identify structural features of interest.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This patent application claims the benefit of co-pending U.S. Provisional Patent Application No. 62 / 945,850, filed December 9, 2019, which is incorporated herein by reference in its entirety.

[0002] The present disclosure relates generally to the detection of chromosomal structural variations, and more particularly to chromosome-specific combinatorial labeling for the detection of potentially deleterious structural variations, including, but not limited to, translocations, amplifications, deletions, and inversions. [Background technology]

[0003] Directed genomic hybridization (dGH) is a single-cell method for mapping the structure of the genome of single-stranded metaphase chromosomes. dGH techniques can facilitate the detection of a broader range of genomic structural variants than previously possible.

[0004] One way chromosomes are prepared for dGH is through the CO-FISH technique. Developed in the 1990s, CO-FISH allows fluorescent probes to specifically target sites on either chromatid, but not both. In S.M. Bailey et al., "Strand-Specific Fluorescence in situ Hybridization: The CO-FISH Family" (Cytogenet. Genome Res. 107:11-14 (2004)), chromosome organization is studied using strand-specific FISH (fluorescence or fluorescence in situ hybridization) [CO-FISH: chromosome orientation-FISH], which involves removing the newly replicated strand from the DNA of metaphase (mitosis) chromosomes, so that one single-stranded target DNA is present in each mitotic chromatid, where the base sequence of each chromatid is the complement of the base sequence of the other. This is achievable because each newly replicated double helix present in the new chromatid contains one parental DNA strand and one newly synthesized strand, which is removed because it has been made photosensitive during replication.

[0005] Structural variants are broadly defined as changes in the sequence or order of a segment of a genome compared to a "normal" genome. Simple variants include single occurrences of unbalanced translocations, balanced translocations, homologous translocations, inversions, duplications, insertions, and deletions. Complex variants include multiple simple variants in a single cell, losses or gains of genomic material, losses or gains of entire chromosomes, and simple variants combined with the more common DNA damage known as chromosomal thrombosis. Variant heterogeneity, defined as different structural variants appearing in a genome, individual cells of the same organism, cell cultures, or cell batches, can involve simple or complex structural variants. Mosaicism of structural variants arises when dividing cells spontaneously generate structural variants, resulting in the proliferation of both variant-free offspring and variant-containing daughter offspring.

[0006] Structural variants are distinct from base-level changes such as single nucleotide polymorphisms (SNiPs) or short insertions and deletions (INDELs). Structural variants arise when the ends of multiple double-strand breaks are incorrectly rejoined or incorrectly repaired. Depending on the subsequent reproductive capacity of the rearrangement-carrying cell, the effects of the resulting structural variant may be limited to a single cell, affect a subset of an organism's tissues, or, if it arises in an embryonic cell, may be inherited and affect the organism's lineage.

[0007] Whenever a DNA double-strand break (DSB) occurs, there is a possibility of DNA misrepair, resulting in chromosomal structural variants. DSBs can arise endogenously during normal cellular metabolic processes, such as replication and transcription. DSBs are estimated to occur naturally in actively metabolizing cells at a rate of approximately 50 per cell per cell cycle, and repair occurs both during replication and by replication-independent pathways. Double-strand breaks are of particular concern when induced by exogenous factors that exceed spontaneous rates, either through radiation exposure, medical interventions such as chemotherapy with certain drugs, or during gene editing processes. Most DSBs are repaired by non-homologous end joining (NHEJ), which operates throughout the cell cycle. In this process, the broken ends are detected, processed, and ligated back together. This is an error-prone process because previously existing base pair sequences are not necessarily repaired with high fidelity. Nevertheless, this rejoining process (restoration) restores the linear continuity of chromosomes and does not result in structural abnormalities. However, when two or more DSBs occur in sufficiently close spatial and temporal proximity, the broken ends of one break pair, along with the two loose ends of the other break pair, may incorrectly rejoin with the ends of another break pair, resulting in structural abnormalities. Examples include balanced and unbalanced translocations, inversions, or deletions. There is also a DSB repair process involving homologous recombination (HR), sometimes referred to as homology-directed repair (HDR). Homology-directed repair (HDR) occurs after replication when identical homologous sequences become available and are in close proximity. The HDR pathway does not operate in G1 or G0 cells, where the levels of the Rad51 protein required for HDR are very low or absent. However, as part of a gene editing process (such as the CRISPR system), the sequence to be edited is targeted and one or more DSBs are introduced to insert the desired sequence using HDR. Therefore, whenever a DSB is introduced, there is always the possibility of spontaneous or other DSBs incorrectly rejoining, resulting in structural abnormalities. Structural variants are highly associated with many human diseases because they result in copy number variations and can significantly affect gene function.The contribution of structural variants to genetic variation is estimated to be 10-30 times higher than that of SNiPs or INDELs. Therefore, methods for detecting structural variants are needed to detect chromosomal abnormalities and distinguish benign from harmful genetic abnormalities.

[0008] These structural variants, regardless of how they are formed, may be harmless, non-genotoxic, or may adversely affect cellular function, cause genomic instability, kill cells, or form genotoxic products. Non-harmless structural variants can adversely affect cells and contribute to disease through the formation of oncogenes, gene inactivation or knockout, disruption of regulatory elements, loss of heterozygosity, gene or promoter duplication, and other mechanisms that disrupt essential metabolic pathways or activate inactive metabolic pathways. When structural variants are congenital, even if they do not result in any overt pathology, meiotic crossover errors caused by misalignment can result in genetic abnormalities in the offspring of affected individuals. In a typical Mendelian pattern, recessive structural variants inherited from both parents can cause disease in offspring that is not active in either parent. X-linked structural variants preferentially affect male offspring because the Y chromosome of an XY pair lacks a compensatory normal gene.

[0009] The detection and identification of nonrecurrent SVs in individual cells resulting from DSB misrepair, as well as SVs present in individual genomes and their expression in individual cells (heterogeneity / mosaicism), are clinically relevant and important across a wide range of human diseases and conditions. Misrepairs and resulting structural variants must be measured due to the potential for both cell death and risk to the patient's DNA. Next-generation sequencing and Sanger sequencing attempt to provide this data through short- and long-read whole-genome sequencing and analysis, but are insufficient as single methods. Two types of approaches are commonly used to detect structural variants: array-based detection / comparative genomic hybridization (array cGH) and sequence-based computational analysis. Each of these can measure several misrepair products through SV detection algorithms and may be more effective when used in conjunction with cross-validation findings. Because these techniques measure the sequence of DNA bases and not the relationships or structure of genes, promoters, or large DNA segments in single cells, they can only be used to generate hypotheses about genome structure through bioinformatic reconstruction. While sequence-based methods may be sufficient for targeted measurement of known structural variants, de novo measurement of structural variation using sequence-based methods has been shown to yield numerous false-positive and false-negative results, making the technique largely impractical. Summary of the Invention

[0010] It is therefore an object of the present disclosure to provide a highly sensitive method for high-resolution detection of chromosomal structural variants. Additional objects, advantages, and novel features of the present disclosure will be set forth in part in the description which follows, and in part will become apparent to those skilled in the art upon reading the following description, or may be learned by practice of the disclosed method. The objects and advantages of the disclosed method may be realized and attained by means of the instrumentalities and combinations particularly pointed out in the appended claims.

[0011] The following numbered paragraphs

[0012] ~

[0147] includes a description of a wide range of combinations of the technical features of the present invention disclosed in this specification.

[0012] 1. A method for detecting at least one structural variation in a chromosome, comprising: a) generating a pair of single-stranded sister chromatids from a chromosome, each sister chromatid comprising one or more target DNA sequences; b) contacting one or both single-stranded sister chromatids with two or more oligonucleotide probes, each of the probes being single-stranded, unique, and complementary to at least a portion of the target DNA sequence, each of the probes comprising at least one label, and at least two of the probes complementary to the target DNA sequence comprising labels of different colors, such that a spectral profile of one or both single-stranded sister chromatids is generated by the hybridization pattern of the at least two probes to one or both single-stranded sister chromatids; c) detecting the spectral profile of one or both single-stranded sister chromatids; d) comparing the spectral profile of step (c) with a reference spectral profile representing a control; e) determining the presence of at least one structural mutation based on at least one spectral difference between the spectral profile of either or both of step (c) and a reference spectral profile.

[0013] 2. The method of embodiment 1, wherein the spectral profile of step (c) is of one single-stranded sister chromatid and the reference spectral profile is of the other single-stranded sister chromatid.

[0014] 3. The method of embodiment 1 or 2, wherein the structural mutation is selected from the group consisting of a chromosomal segment copy number change, a chromosomal copy number change, an inversion, a translocation, a sister chromatid recombination, a micronucleus formation, a chromosomal thrombus recombination event, and any combination thereof.

[0015] 4. The method of any one of aspects 1 to 3, wherein the structural variation is a change in the copy number of a segment of a chromosome, and the change is selected from the group consisting of amplification, deletion, and any combination thereof.

[0016] 5. The method of any one of aspects 1 to 4, wherein the probe is 25 to 75 nucleotides in length.

[0017] 6. The method of any one of aspects 1 to 5, wherein the probe is 30 to 50 nucleotides in length.

[0018] 7. The method of any one of aspects 1 to 6, wherein the probe is 37 to 43 nucleotides in length.

[0019] 8. The method of any one of aspects 1 to 7, wherein the label on the probe is a fluorescent dye conjugated at the 5' end of the probe.

[0020] 9. The method of any one of embodiments 1 to 8, wherein the probes complementary to the target DNA sequence on each single-stranded sister chromatid comprise labels of at least two different colors.

[0021] 10. The method of any one of embodiments 1-9, wherein the probes complementary to the target DNA sequence on each single-stranded sister chromatid comprise labels of at least three different colors.

[0022] 11. The method of any one of embodiments 1-10, wherein the probes complementary to the target DNA sequence on each single-stranded sister chromatid comprise labels of at least four different colors.

[0023] 12. The method of any one of embodiments 1-11, wherein the probes complementary to the target DNA sequence on each single-stranded sister chromatid comprise labels of at least five different colors.

[0024] 13. The method of any one of embodiments 1-12, wherein the probes complementary to the target DNA sequence on each single-stranded sister chromatid comprise labels of at least six different colors.

[0025] 14. The method of any one of embodiments 1-13, wherein the probes complementary to the target DNA sequence on each single-stranded sister chromatid comprise labels of at least seven different colors.

[0026] 15. The method of any one of embodiments 1-14, wherein the probes complementary to the target DNA sequence on each single-stranded sister chromatid comprise labels of at least eight different colors.

[0027] 16. The method of any one of embodiments 1-15, wherein the probes complementary to the target DNA sequence on each single-stranded sister chromatid comprise labels of at least nine different colors.

[0028] 17. The method of any one of embodiments 1-16, wherein the probes complementary to the target DNA sequence on each single-stranded sister chromatid comprise at least 10 different color labels.

[0029] 18. The method of any one of aspects 1-17, wherein at least one label is selected from the group consisting of a label detectable in the visible light spectrum, a label detectable in the infrared light spectrum, a label detectable in the ultraviolet light spectrum, and any combination thereof.

[0030] 19. The method of any one of embodiments 1-18, wherein the at least one label is selected from the group consisting of a label on the end of the probe, a label on the side of the probe, one or more labels on the body of the probe, and any combination thereof.

[0031] 20. The method of any one of aspects 1-19, wherein at least one label is a base label on a sugar or amidite functional group of the probe.

[0032] 21. The method of any one of aspects 1-20, wherein detecting the spectral profile comprises using a narrow band filter and processing the spectral information by software.

[0033] 22. The method of any one of aspects 1-21, wherein detecting the spectral profile specifically excludes one or more spectral regions of the spectral profile.

[0034] 23. The method of any one of aspects 1-22, wherein step (e) is performed with the aid of artificial intelligence.

[0035] 24. A method for detecting at least one structural variation in a chromosome, comprising: a) generating a pair of single-stranded sister chromatids from a chromosome, each sister chromatid comprising one or more target DNA sequences; b) after step a), contacting one or both single-stranded sister chromatids with a stain; c) after step a), contacting one or both single-stranded sister chromatids with two or more oligonucleotide probes, each of the probes being single-stranded, unique, and complementary to at least a portion of the target DNA sequence, each of the probes comprising at least one label, and at least two of the probes complementary to the target DNA sequence comprising labels of different colors, such that a spectral profile of one or both single-stranded sister chromatids is generated by the hybridization pattern of the at least two probes to one or both single-stranded sister chromatids; d) detecting the spectral profile of one or both single-stranded sister chromatids; e) detecting the staining pattern of one or both single-stranded sister chromatids; f) comparing the spectral profile of either or both of step (d) with a reference spectral profile representative of a control, and further comparing the staining pattern of either or both of step (e) with the reference staining pattern representative of a control; g) determining the presence of at least one structural mutation based on at least one spectral difference between either or both of the spectral profiles of step (d) and a reference spectral profile, and further based on at least one staining difference between either or both of the staining patterns of step (e) and the reference staining pattern.

[0036] 25. The method of embodiment 24, wherein the spectral profile of step (d) is of one single-stranded sister chromatid and the reference spectral profile is of the other single-stranded sister chromatid.

[0037] 26. The method of aspect 24 or 25, wherein the staining pattern of step (e) is of one single-stranded sister chromatid and the reference staining pattern is of the other single-stranded sister chromatid.

[0038] 27. The method of any one of aspects 24-26, wherein the structural mutation is selected from the group consisting of a change in the copy number of a segment of a chromosome, a change in the copy number of a chromosome, an insertion, a deletion, an inversion, a balanced translocation, an unbalanced translocation, a sister chromatid recombination, a micronucleus formation, a chromosomal thrombus thrombus event, a loss or gain of genetic material, a loss or gain of one or more entire chromosomes, and any combination thereof.

[0039] 28. The method of embodiment 27, wherein the structural variation is a change in the copy number of a segment of a chromosome, and the change is selected from the group consisting of amplification, deletion, and any combination thereof.

[0040] 29. The method of any one of aspects 24 to 28, wherein the probe is 25 to 75 nucleotides in length.

[0041] 30. The method of any one of aspects 24 to 29, wherein the probe is 30 to 50 nucleotides in length.

[0042] 31. The method of any one of aspects 24 to 30, wherein the probe is 37 to 43 nucleotides in length.

[0043] 32. The method of any one of aspects 24 to 31, wherein the label on the probe is a fluorescent dye conjugated at the 5' end of the probe.

[0044] 33. The method of any one of embodiments 24 to 32, wherein the probes complementary to the target DNA sequence on each single-stranded sister chromatid comprise labels of at least two different colors.

[0045] 34. The method of any one of embodiments 24 to 33, wherein the probes complementary to the target DNA sequence on each single-stranded sister chromatid comprise labels of at least three different colors.

[0046] 35. The method of any one of embodiments 24-34, wherein the probes complementary to the target DNA sequence on each single-stranded sister chromatid comprise labels of at least four different colors.

[0047] 36. The method of any one of embodiments 24 to 35, wherein the probes complementary to the target DNA sequence on each single-stranded sister chromatid comprise labels of at least five different colors.

[0048] 37. The method of any one of embodiments 24 to 36, wherein the probes complementary to the target DNA sequence on each single-stranded sister chromatid comprise labels of at least six different colors.

[0049] 38. The method of any one of embodiments 24 to 37, wherein the probes complementary to the target DNA sequence on each single-stranded sister chromatid comprise labels of at least seven different colors.

[0050] 39. The method of any one of embodiments 24 to 38, wherein the probes complementary to the target DNA sequence on each single-stranded sister chromatid comprise labels of at least eight different colors.

[0051] 40. The method of any one of embodiments 24 to 39, wherein the probes complementary to the target DNA sequence on each single-stranded sister chromatid comprise labels of at least nine different colors.

[0052] 41. The method of any one of embodiments 24 to 40, wherein the probes complementary to the target DNA sequences on each single-stranded sister chromatid comprise labels of at least 10 different colors.

[0053] 42. The method according to any one of 24 to 41, wherein the staining agent is selected from the group consisting of DAPI, Hoechst 33258, and actinomycin D.

[0054] 43. The method of any one of aspects 24-42, wherein at least one label is selected from the group consisting of a label detectable in the visible light spectrum, a label detectable in the infrared light spectrum, a label detectable in the ultraviolet light spectrum, and any combination thereof.

[0055] 44. The method of any one of aspects 24 to 43, wherein at least one label is selected from the group consisting of a label on the end of the probe, a label on the side of the probe, a label in the body of the probe, and any combination thereof.

[0056] 45. The method of embodiment 44, wherein at least one label is a body label on a sugar or amidite functional group of the probe.

[0057] 46. ​​The method of any one of aspects 24-45, wherein detecting the spectral profile comprises using a narrow band filter and processing the spectral information by software.

[0058] 47. The method of any one of aspects 24-46, wherein detecting the spectral profile specifically excludes one or more spectral regions of the spectral profile.

[0059] 48. The method of any one of aspects 24-47, wherein step (e) is performed with the aid of artificial intelligence.

[0060] 49. A method for detecting at least one structural variation in a chromosome, comprising: a) generating a pair of single-stranded sister chromatids from a chromosome, each sister chromatid comprising one or more target DNA sequences; b) after step a), contacting the single-stranded sister chromatids with oligonucleotide markers complementary to a repeated sequence on the single-stranded sister chromatid that is not the target DNA sequence, each of the markers comprising at least one label; c) after step a), contacting one or both single-stranded sister chromatids with two or more oligonucleotide probes, each of the probes being single-stranded, unique, and complementary to at least a portion of the target DNA sequence, each of the probes comprising at least one label, and at least two of the probes complementary to the target DNA sequence comprising labels of different colors, such that a spectral profile of one or both single-stranded sister chromatids is generated by the hybridization pattern of the at least two probes to one or both single-stranded sister chromatids; d) detecting the spectral profile of one or both single-stranded sister chromatids; e) detecting the marker hybridization pattern of one or both single-stranded sister chromatids; f) comparing the spectral profile of step (d) with a reference spectral profile representative of a control, and further comparing the marker hybridization pattern of step (e) with a reference marker hybridization pattern representative of a control; g) determining the presence of at least one structural mutation based on at least one spectral difference between either or both of the spectral profiles of step (d) and a reference spectral profile, and further based on at least one marker hybridization pattern difference between either or both of the marker hybridization patterns of step (e) and the reference marker hybridization pattern.

[0061] 50. The method of embodiment 49, wherein the spectral profile of step (d) is of one single-stranded sister chromatid and the reference spectral profile is of the other single-stranded sister chromatid.

[0062] 51. The method of embodiment 49 or 50, wherein the marker hybridization pattern in step (e) is for one single-stranded sister chromatid and the reference marker hybridization pattern is for the other single-stranded sister chromatid.

[0063] 52. The method of any one of aspects 49 to 51, wherein the structural mutation is selected from the group consisting of a chromosomal segment copy number change, a chromosomal copy number change, an inversion, a translocation, a sister chromatid recombination, a micronucleus formation, a chromosomal thrombus thrombus event, and any combination thereof.

[0064] 53. The method of any one of aspects 49 to 52, wherein the structural variation is a change in the copy number of a segment of a chromosome, and the change is selected from the group consisting of an amplification, a deletion, and any combination thereof.

[0065] 54. The method of any one of aspects 49 to 53, wherein the probe is 25 to 75 nucleotides in length.

[0066] 55. The method of any one of aspects 49 to 54, wherein the probe is 30 to 50 nucleotides in length.

[0067] 56. The method of any one of aspects 49 to 55, wherein the probe is 37 to 43 nucleotides in length.

[0068] 57. The method of any one of aspects 49 to 56, wherein the label on the probe is a fluorescent dye conjugated at the 5' end of the probe.

[0069] 58. The method of any one of embodiments 49 to 57, wherein the probes complementary to the target DNA sequence on each single-stranded sister chromatid comprise labels of at least two different colors.

[0070] 59. The method of any one of embodiments 49-58, wherein the probes complementary to the target DNA sequence on each single-stranded sister chromatid comprise labels of at least three different colors.

[0071] 60. The method of any one of embodiments 49-59, wherein the probes complementary to the target DNA sequence on each single-stranded sister chromatid comprise labels of at least four different colors.

[0072] 61. The method of any one of aspects 49-60, wherein the probes complementary to the target DNA sequence on each single-stranded sister chromatid comprise labels of at least five different colors.

[0073] 62. The method of any one of embodiments 49-61, wherein the probes complementary to the target DNA sequences on each single-stranded sister chromatid comprise labels of at least six different colors.

[0074] 63. The method of any one of embodiments 49 to 62, wherein the probes complementary to the target DNA sequence on each single-stranded sister chromatid comprise labels of at least seven different colors.

[0075] 64. The method of any one of embodiments 49 to 63, wherein the probes complementary to the target DNA sequence on each single-stranded sister chromatid comprise labels of at least eight different colors.

[0076] 65. The method of any one of embodiments 49 to 64, wherein the probes complementary to the target DNA sequence on each single-stranded sister chromatid comprise labels of at least nine different colors.

[0077] 66. The method of any one of embodiments 49 to 65, wherein the probes complementary to the target DNA sequences on each single-stranded sister chromatid comprise labels of at least 10 different colors.

[0078] 67. The method of any one of embodiments 49-66, wherein at least one label is selected from the group consisting of a label detectable in the visible light spectrum, a label detectable in the infrared light spectrum, a label detectable in the ultraviolet light spectrum, and any combination thereof.

[0079] 68. The method of any one of embodiments 49 to 67, wherein at least one label is selected from the group consisting of a label on the end of the probe, a label on the side of the probe, a label in the body of the probe, and any combination thereof.

[0080] 69. The method of embodiment 68, wherein at least one label is a backbone label on a sugar or amidite functional group of the probe.

[0081] 70. The method of any one of aspects 49-69, wherein detecting the spectral profile comprises using a narrow band filter and processing the spectral information by software.

[0082] 71. The method of any one of aspects 49-70, wherein detecting the spectral profile specifically excludes one or more spectral regions of the spectral profile.

[0083] 72. The method of any one of aspects 49-71, wherein step (e) is performed with the aid of artificial intelligence.

[0084] 73. A computer-implemented method for detecting at least one structural variation in a chromosome, comprising: a) generating a pair of single-stranded sister chromatids from a chromosome, each sister chromatid comprising one or more target DNA sequences; b) contacting one or both single-stranded sister chromatids with two or more oligonucleotide probes, each of the probes being single-stranded, unique, and complementary to at least a portion of the target DNA sequence, each of the probes comprising at least one label, and at least two of the probes complementary to the target DNA sequence comprising labels of different colors, such that a spectral profile of one or both single-stranded sister chromatids is generated by the hybridization pattern of the at least two probes to one or both single-stranded sister chromatids; c) detecting the spectral profile of one or both single-stranded sister chromatids; d) comparing the spectral profile of step (c) with a reference spectral profile representing a control; and e) determining the presence of at least one structural mutation based on at least one spectral difference between either or both of the spectral profiles of step (c) and a reference spectral profile, wherein steps (d) and (e) are calculated on a computer system.

[0085] 74. A computer-readable program storage device comprising: a) generating a pair of single-stranded sister chromatids from a chromosome, each sister chromatid comprising one or more target DNA sequences; b) contacting one or both single-stranded sister chromatids with two or more oligonucleotide probes, each of the probes being single-stranded, unique, and complementary to at least a portion of the target DNA sequence, each of the probes comprising at least one label, and at least two of the probes complementary to the target DNA sequence comprising labels of different colors, such that a spectral profile of one or both single-stranded sister chromatids is generated by the hybridization pattern of the at least two probes to one or both single-stranded sister chromatids; c) detecting the spectral profile of one or both single-stranded sister chromatids; d) comparing the spectral profile of step (c) with a reference spectral profile representing a control; and e) determining the presence of at least one structural variation based on at least one spectral difference between the spectral profile of either or both of step (c) and a reference spectral profile.

[0086] 75. A method for detecting at least one structural variation in a chromosome, comprising: a) generating a pair of single-stranded sister chromatids from a chromosome, the sister chromatids comprising one or more target DNA sequences; b) contacting the single-stranded sister chromatid with two or more oligonucleotide probes, each of the probes being single-stranded, unique, and complementary to at least a portion of the target DNA sequence, each of the probes comprising at least one label, and at least two of the probes comprising labels of different colors, such that a spectral profile of the single-stranded sister chromatid is generated by the hybridization pattern of the at least two probes to the single-stranded sister chromatid; c) detecting the spectral profile of single-stranded sister chromatids; d) comparing the spectral profile of step (c) with a reference spectral profile representing a control; e) determining the presence of at least one structural variant based on at least one spectral difference between the spectral profile of step (c) and a reference spectral profile.

[0087] 76. The method of embodiment 75, wherein the reference spectral profile is that of the other single-stranded sister chromatid.

[0088] 77. The method of embodiment 75 or 76, wherein the structural mutation is selected from the group consisting of a change in the copy number of a chromosomal segment, a change in the copy number of a chromosome, an inversion, a translocation, a sister chromatid recombination, a micronucleus formation, a chromosomal thrombus thrombus event, and any combination thereof.

[0089] 78. The method of any one of embodiments 75 to 77, wherein the structural variation is a change in the copy number of a segment of a chromosome, and the change is selected from the group consisting of an amplification, a deletion, and any combination thereof.

[0090] 79. The method of any one of embodiments 75 to 78, wherein the probe is 25 to 75 nucleotides in length.

[0091] 80. The method of any one of aspects 75-79, wherein the probe is 30-50 nucleotides in length.

[0092] 81. The method of any one of aspects 75-80, wherein the probe is 37-43 nucleotides in length.

[0093] 82. The method of any one of aspects 75-81, wherein the label on the probe is a fluorescent dye conjugated at the 5' end of the probe.

[0094] 83. The method of any one of embodiments 75-82, wherein the probes complementary to the target DNA sequence on each single-stranded sister chromatid comprise labels of at least two different colors.

[0095] 84. The method of any one of embodiments 76-83, wherein the probes complementary to the target DNA sequence on each single-stranded sister chromatid comprise labels of at least three different colors.

[0096] 85. The method of any one of embodiments 76 to 84, wherein the probes complementary to the target DNA sequence on each single-stranded sister chromatid comprise labels of at least four different colors.

[0097] 86. The method of any one of embodiments 76 to 85, wherein the probes complementary to the target DNA sequence on each single-stranded sister chromatid comprise labels of at least five different colors.

[0098] 87. The method of any one of embodiments 76-86, wherein the probes complementary to the target DNA sequences on each single-stranded sister chromatid comprise labels of at least six different colors.

[0099] 88. The method of any one of embodiments 76 to 87, wherein the probes complementary to the target DNA sequences on each single-stranded sister chromatid comprise labels of at least seven different colors.

[0100] 89. The method of any one of embodiments 76 to 88, wherein the probes complementary to the target DNA sequences on each single-stranded sister chromatid comprise labels of at least eight different colors.

[0101] 90. The method of any one of embodiments 76-89, wherein the probes complementary to the target DNA sequence on each single-stranded sister chromatid comprise labels of at least nine different colors.

[0102] 91. The method of any one of embodiments 76 to 90, wherein the probes complementary to the target DNA sequences on each single-stranded sister chromatid comprise labels of at least 10 different colors.

[0103] 92. The method of any one of aspects 76-91, wherein step (d) and step (e) are calculated on a computer system.

[0104] 93. The method of any one of embodiments 76-92, wherein at least one label is selected from the group consisting of a label detectable in the visible light spectrum, a label detectable in the infrared light spectrum, a label detectable in the ultraviolet light spectrum, and any combination thereof.

[0105] 94. The method of any one of embodiments 76 to 93, wherein at least one label is selected from the group consisting of a label on the end of the probe, a label on the side of the probe, a label in the body of the probe, and any combination thereof.

[0106] 95. The method of embodiment 94, wherein at least one label is a body label on a sugar or amidite functional group of the probe.

[0107] 96. The method of any one of aspects 76-95, wherein detecting the spectral profile comprises using a narrow band filter and processing the spectral information by software.

[0108] 97. The method of any one of aspects 76-96, wherein detecting the spectral profile specifically excludes one or more spectral regions of the spectral profile.

[0109] 98. The method of any one of aspects 76-97, wherein step (e) is performed with the aid of artificial intelligence.

[0110] 99. The method of any one of aspects 76 to 98, further comprising, after step a), contacting the single-stranded sister chromatids with a staining agent, detecting a staining pattern of the sister chromatids, comparing the staining pattern to a reference staining pattern representing a control, and determining the presence of at least one structural variant based in part on at least one staining difference between the staining pattern of the sister chromatids and the reference staining pattern.

[0111] 100. The method of any one of aspects 76 to 99, further comprising, after step a), contacting the single-stranded sister chromatid with oligonucleotide markers complementary to a repeated sequence on the single-stranded sister chromatid that is not the target DNA sequence, wherein each of the markers comprises at least one label; detecting a marker hybridization pattern of the sister chromatid; comparing the marker hybridization pattern to a reference marker hybridization pattern representing a control; and determining the presence of at least one structural mutation based in part on at least one marker hybridization pattern difference between the marker hybridization pattern of the sister chromatid and the reference marker hybridization pattern.

[0112] 101. The method of any one of embodiments 1, 24, 49, 73, 74, 75, 99, and 100, wherein the reference spectral profile lacks at least one structural variation.

[0113] 102. The method of any one of embodiments 1, 24, 49, 73, 74, 75, 99, and 100, wherein the reference spectral profile comprises at least one structural variant.

[0114] 103. The method of any one of embodiments 1, 24, 49, 73, 74, 75, 99, and 100, wherein the reference spectral profile comprises an intentional distribution of labeled probes.

[0115] 104. The method of embodiment 24 or 99, wherein the reference staining pattern lacks at least one structural mutation.

[0116] 105. The method of embodiment 24 or 99, wherein the reference staining pattern comprises at least one structural variation.

[0117] 106. The method of embodiment 49 or 100, wherein the reference marker hybridization pattern lacks at least one structural variation.

[0118] 107. The method of embodiment 49 or 100, wherein the reference marker hybridization pattern comprises at least one structural variation.

[0119] 108. The method of embodiment 49 or 100, wherein the reference marker hybridization pattern comprises a deliberate distribution of labeled probes.

[0120] 109. A method for identifying one or more structural features of a target DNA strand, the method being processor-implemented, comprising: a) receiving a spectral profile representing at least one sequence of base pairs of a target DNA strand, the spectral profile including frequency data corresponding to the base sequence of the target DNA strand, the frequency data including at least two color channels; b) converting the spectral profile into a data table of the target DNA strand, the data table including position data and intensity data of at least two color channels of the base sequence; c) comparing the data table of the subject DNA strand with a reference feature lookup table including one or more feature nodes representing normal and / or abnormal features of a corresponding control DNA strand to identify one or more normal and / or abnormal features of the subject DNA strand, wherein each of the one or more feature nodes is defined by a color band representing a base subsequence of the control DNA strand starting from a starting base and ending at a terminal base.

[0121] 110. Receiving a spectral profile: a) generating a pair of single-stranded sister chromatids from a chromosome, wherein the target DNA strand is comprised of at least a portion of the single-stranded sister chromatid, and the target DNA strand comprises one or more target DNA sequences; b) contacting one or both single-stranded sister chromatids with two or more oligonucleotide probes, each of the probes being single-stranded, unique, and complementary to at least a portion of the target DNA sequence, each of the probes comprising at least one label, and at least two of the probes complementary to the target DNA sequence comprising labels of different colors corresponding to at least two color channels, such that a spectral profile of one or both single-stranded sister chromatids is generated by the hybridization pattern of the at least two probes to one or both single-stranded sister chromatids, thereby generating a spectral profile of base sequences on the target DNA strand; c) detecting a spectral profile of the base sequence of the target DNA strand.

[0122] 111. The method of aspect 109 or 110, wherein transforming the spectral profile includes segmenting the spectral profile into a plurality of regions, each of the regions having a color corresponding to one of the at least two color channels.

[0123] 112. The method of embodiment 111, wherein each of the regions is defined by a position parameter and a size parameter.

[0124] 113. The method of any one of aspects 109-112, wherein the conversion is performed by a machine learning / AI algorithm.

[0125] 114. The method of any one of aspects 109 to 113, wherein each feature node represents at least a portion of a genetic element, a structural variation, or a combination thereof.

[0126] 115. The method of embodiment 114, wherein the genetic element is selected from the group consisting of a protein coding region, a region affecting transcription, a region affecting translation, a region affecting post-translational modification, and any combination thereof.

[0127] 116. The method of embodiment 114, wherein the genetic element is selected from the group consisting of an exon, an intron, a 5' untranslated region, a 3' untranslated region, a promoter, an enhancer, a silencer, an operator, a terminator, a polyA tail, an inverted terminal repeat, an mRNA stability element, and any combination thereof.

[0128] 117. The method of embodiment 114, wherein the variation is selected from the group consisting of a change in the copy number of a chromosomal segment, a change in the copy number of a chromosome, an inversion, a translocation, a sister chromatid recombination, a micronucleus formation, a chromosomal thrombus thrombus event, and any combination thereof.

[0129] 118. The method of any one of aspects 109-117, wherein the comparison is performed for each of a plurality of feature lookup tables.

[0130] 119. The method of embodiment 118, wherein each of the plurality of feature lookup tables corresponds to a different genetic element of interest.

[0131] 120. The method of any one of aspects 109-119, wherein the comparison is performed by a machine learning / AI algorithm.

[0132] 121. A method of processing data representing a target DNA strand, the method being implemented on a processor, comprising: a) receiving a spectral profile representing at least one sequence of bases of a target DNA strand, the spectral profile including frequency data corresponding to the base sequence of the target DNA strand, the frequency data including at least two color channels; b) converting the spectral profile into a data table of the target DNA strand, the data table including position data and intensity data of at least two color channels of the base sequence; c) storing the data table in a memory.

[0133] 122. The method of embodiment 121, further comprising determining one or more normal and / or abnormal features of the target DNA strand by comparing a data table representing the target DNA strand with a reference feature lookup table including one or more feature nodes representing normal and / or abnormal features of a corresponding control DNA strand, wherein each of the one or more feature nodes is defined by a color band and a base subsequence starting from a starting base and ending at a terminal base.

[0134] 123. The method of embodiment 121 or 122, further comprising merging base-level data of the subject DNA strand into a data table.

[0135] 124. The method of any one of aspects 121 to 123, further comprising defining one or more feature nodes representing normal and / or abnormal features of the control DNA strand, each of the one or more feature nodes being defined by a color band and a base subsequence starting from a starting base and ending at a terminal base.

[0136] 125. The method of aspect 124, wherein one or more feature nodes are defined by a trained machine learning algorithm.

[0137] 126. The method of embodiment 124, further comprising receiving and converting each of a plurality of control DNA strands, and storing a plurality of resulting data tables in memory.

[0138] 127. The method of embodiment 126, wherein the multiple control DNA strands originate from the same genomic region and are obtained from different patient samples.

[0139] 128. The method of aspect 126, further comprising: receiving a query for a particular feature node of the one or more defined feature nodes; and processing the query using the plurality of resulting data tables.

[0140] 129. A method for identifying the chromosomal origin of extrachromosomal DNA (ECDNA), comprising: a) contacting ECDNA from a cell with two or more oligonucleotide probes, each of the probes being single-stranded, unique, and complementary to at least a portion of the ECDNA, and each of the probes comprising at least one label; b) contacting at least one chromosome or at least one single-stranded sister chromatid of a chromosome from the same cell with the same probe of step (a); c) detecting the spectral profile of the ECDNA and detecting the spectral profile of at least one chromosome or at least one single-stranded sister chromatid of the chromosome; d) comparing the spectral profiles of step (c); e) identifying the at least one chromosome or at least one single-stranded sister chromatid of the chromosome as the DNA source of the ECDNA based on at least one similarity between the spectral profile of the ECDNA and the spectral profile of the at least one chromosome or at least one single-stranded sister chromatid of the chromosome.

[0141] 130. The method of embodiment 129, further comprising identifying the location on at least one chromosome or at least one single-stranded sister chromatid of a chromosome from which the DNA of the ECDNA originated based on the comparison of step d).

[0142] 131. The method of embodiment 130, wherein the origin of the ECDNA from at least one chromosome or at least one single-stranded sister chromatid of a chromosome is due to amplification of DNA at the position.

[0143] 132. The method of embodiment 130, wherein at least one cancer gene is identified on ECDNA.

[0144] 133. The method of embodiment 129, wherein the ECDNA is selected from the group consisting of episomal DNA and vector-integrated DNA.

[0145] 134. The method of any one of embodiments 1-72, 75-108, and 129-133, wherein at least one target region on at least one chromosome or at least one single-stranded sister chromatid of the chromosome is identified for target enrichment, and the at least one chromosome or at least one single-stranded sister chromatid of the chromosome is contacted with a target enrichment probe.

[0146] 135. The method of any one of aspects 1-72, 75-108, and 129-134, wherein the comparison of spectral profiles comprises spectral analysis of breathing on at least one other band on the same chromosome or the same single-stranded sister chromatid.

[0147] 136. The method of any one of aspects 1-72, 75-108, and 129-135, wherein contacting at least one chromosome or at least one single-stranded sister chromatid of a chromosome with two or more oligonucleotide probes comprises embedding a sample comprising at least one chromosome or at least one single-stranded sister chromatid of a chromosome in a swellable hydrogel and chemically binding the sample to the hydrogel, further wherein the hydrogel swells to increase spatial resolution across the x-axis, y-axis, and z-axis. [Brief explanation of the drawings]

[0148] [Figure 1-1] An example of an intrachromosomal rearrangement is illustrated comparing banded and monochrome dGH paints. 1a: Normal chromosome 2 prepared for dGH and hybridized with chromosome 2 dGH paint with multicolored bands. 1b: Chromosome 2 with a deletion, where the missing bands are identified. [Figure 1-2] 2a: Normal chromosome 2 prepared for dGH and hybridized with monochrome chromosome 2 dGH paint. 2b: Chromosome 2 with a deletion (region unknown). [Figure 1-3] 1c: Chromosome 2 with amplification, where a region with extra bands was identified. 1d: Chromosome 2 with sister chromatid recombination event (only visible for one replication cycle complete repair event), where it was identified as SCR because the bands were in the correct order (not inversion). 1e: Chromosome 2 with inversion event, where it was identified by the reversed order of the bands. [Figure 1-4] 2c: Chromosome 2 with amplification (amplified region unknown). 2d: Chromosome 2 with either an SCR or an inversion event (specific variant unknown). (Flagged as an inversion because the SCR may have been missed and the orientation of the segment found on the opposing sister chromatid is unknown.) 2e: Chromosome 2 with either an SCR or an inversion event (specific variant unknown). (Flagged as an SCR because the inversion may have been missed and the orientation of the segment found on the opposing sister chromatid is unknown.) [Figure 2-1]An example of an interchromosomal rearrangement (translocation between two different chromosomes) is illustrated by comparing banded and monochrome dGH paints. 1a: Normal chromosome 2 prepared for dGH and hybridized with the multicolored banded chromosome 2 dGH paint. 1b: Normal chromosome 4 (uncolored for illustration purposes). [Figure 2-2] 2a: Normal chromosome 2 prepared for dGH and hybridized with monochrome chromosome 2 dGH paint. 2b: Normal chromosome 4 (unpainted for illustration purposes). [Figure 2-3] 1c: Derivative chromosome A (a reciprocal translocation product) in which material from chromosome 2 (bands 1–11) has fused with material from chromosome 4 (uncolored). 1d: Derivative chromosome B (another reciprocal translocation product) in which material from chromosome 2 (bands 12–19) has fused with material from chromosome 4 (uncolored). [Figure 2-4] 2c: Material from chromosome 2 fused with material from chromosome 4 (not colored) (fusion coordinates unknown), derivative chromosome A (reciprocal translocation product). 2d: Material from chromosome 2 fused with material from chromosome 4 (not colored) (fusion coordinates unknown), derivative chromosome B (another reciprocal translocation product). [Figure 3-1] An example of an interchromosomal allelic rearrangement (translocation between two homologs of the same chromosome) is illustrated. Comparison of banded and monochrome dGH paints. 1a: Normal chromosome 2 homolog 1 prepared for dGH and hybridized with multicolored banded chromosome 2 dGH paint. 1b: Normal chromosome 2 homolog 2 prepared for dGH and hybridized with multicolored banded chromosome 2 dGH paint. [Figure 3-2] 2a: Normal chromosome 2 homolog 1 prepared for dGH and hybridized with monochromatic chromosome 2 dGH paint. 2b: Normal chromosome 2 homolog 2 prepared for dGH and hybridized with monochromatic chromosome 2 dGH paint. [Figure 3-3]1c: Derivative chromosome A (a reciprocal translocation product between homologs) in which material from chromosome 2 homolog 1 has been swapped with material from chromosome 2 homolog 2 at the same breakpoint (between bands 11 and 12). The statistical probability of two SCEs at the exact same location on each homolog is very unlikely compared to the highly likely allelic translocation events, especially in cells edited at a single location (two DSBs per homolog). 1d: Derivative chromosome B (a reciprocal translocation product between homologs) in which material from chromosome 2 homolog 1 has been swapped with material from chromosome 2 homolog 2 at the same breakpoint (between bands 11 and 12). The statistical probability of two SCEs at the exact same location on each homolog is very unlikely compared to the highly likely allelic translocation events, especially in cells edited at a single location (two DSBs per homolog). [Figure 3-4] 2c: Derivative chromosome A (product of a reciprocal translocation between homologs) in which material from chromosome 2 homolog 1 was swapped with material from chromosome 2 homolog 2 at an unknown breakpoint. The statistical probability of two SCEs at the exact same location on each homolog is extremely unlikely compared to the highly likely allelic translocation events, especially in cells edited at a single location (two DSBs per homolog), but cannot be confirmed by monochrome paint due to the lack of genomic coordinate specificity. 2d: Derivative chromosome B (product of a reciprocal translocation between homologs) in which material from chromosome 2 homolog 1 was swapped with material from chromosome 2 homolog 2 at an unknown breakpoint. The statistical probability of two SCEs at the exact same location on each homolog is extremely unlikely compared to the highly likely allelic translocation events, especially in cells edited at a single location (two DSBs per homolog), but cannot be confirmed by monochrome paint due to the lack of genomic coordinate specificity. [Figure 4-1]An example of a complex chromosomal rearrangement is illustrated. The first image shows both chromosome 2 homologs from a blood-borne lymphocyte cell recently exposed to ionizing radiation for prostate cancer treatment. Complex structural mutations are present on the right homolog, which can be visualized after hybridization with the banded dGH paint described in Table 1. The graphic provided after the image illustrates how this complex rearrangement appears using the multicolor banded dGH paint compared to the monochrome dGH paint. [Figure 4-2] 1a: Normal chromosome 2 prepared for dGH and hybridized with chromosome 2 dGH paint with multicolored bands. [Figure 4-3] 1b: Chromosome 2 with multicolored bands. Chromosome 2 with complex structural rearrangements hybridized with dGH paint. A large pericentric inversion is present, with one breakpoint occurring between bands 1 and 2 on 2p and the other occurring at bisected band 18 on 2q. An additional, smaller paracentric inversion is present near the centromere on 2q, with the first breakpoint occurring between bands 9 and 10 and the second between bands 10 and 11. There is also a large sister chromatid exchange event between bands 9 and 11, which shares the same proximal breakpoint as the small paracentric inversion, and can be verified by the order of the bands, which still appear in the correct numerical order but are now on the sister chromatid (left sister chromatid) opposite the primary paint (right sister chromatid). [Figure 4-4] 2a: Normal chromosome 2 prepared for dGH and hybridized with monochrome chromosome 2 dGH paint. [Figure 4-5] 2b: Monochrome chromosome 2. Chromosome 2 with complex structural rearrangements hybridized with dGH paint. Without colored bands to provide segment order, the rearrangements could not be identified or explained by coordinates. In reality, the chromosome appeared to have small terminal SCEs or inversions (p-arm) and large inversions (q-arm), and the true classification of the structural rearrangements present would have been misidentified. [Figure 5-1]An example of a targeted probe dGH assay for SV detection is illustrated: 1a: Normal chromosome 2 prepared for dGH and hybridized with four targeted probes surrounding the locus of interest. [Figure 5-2] 1b: Chromosome 2 with a deletion of part of the locus of interest (spanning the genomic coordinates covered by target probe 2). [Figure 5-3] 1c: Chromosome 2 with a sister chromatid recombination event, where target probes 2 and 3 were found on the sister chromatid opposite target probes 1 and 4, and the probes were maintained in the order 1, 2, 3, 4 from the telomeric to the centromeric side. [Figure 5-4] 1d: Chromosome 2 with an inversion event, where target probes 2 and 3 are found on sister chromatids opposite target probes 1 and 4, reversing the order of probes 2 and 3. The probes appear in the order 1, 3, 2, 4 from telomeric to centromeric. [Figure 6] 1 illustrates example images of monochromatic dGH paint labeling chromosomes 1, 2, and 3 in rearranged cells from a radiation-exposed blood-derived lymphocyte sample prepared for dGH. [Figure 7-1] Images A and B show chromosome 2 homolog pairs from two separate normal metaphase cells in which no structural mutations are present (normal immortalized human fibroblast cell line BJ-5ta). [Figure 7-2] Images C and D show chromosome 2 homologs from two separate metaphase cells (normal immortalized human fibroblast cell line BJ-5ta) showing structural mutations in one homolog resulting from sister chromatid exchange (color order is maintained, but signals are on opposite sister chromatids). [Figure 8A-1] Hybridization, probe distribution, and fluorescence wavelength intensity of normal chromosome 2 are shown. [Figure 8A-2] Hybridization, probe distribution, and fluorescence wavelength intensity of normal chromosome 2 are shown. [Figure 8A-3] Hybridization, probe distribution, and fluorescence wavelength intensity of normal chromosome 2 are shown. [Figure 8B-1]Hybridization, probe distribution, and fluorescence wavelength intensity of SCE detected on chromosome 2 are shown. [Figure 8B-2] Hybridization, probe distribution, and fluorescence wavelength intensity of SCE detected on chromosome 2 are shown. [Figure 8B-3] Hybridization, probe distribution, and fluorescence wavelength intensity of SCE detected on chromosome 2 are shown. [Figure 9] Three separate ladder assays hybridized to chromosomes are described. The first ladder measures the limit of detection for the number of oligos contributing to each signal and is spaced approximately 20 MB apart on the p arm of chromosome 2 (labeled Ladder 1 in the image). The second ladder (chromosome 2q) assesses the target size that a fixed amount of oligos can spread, is also spaced approximately 20 MB apart, and also measures the limit of detection (labeled Ladder 2 in the image). The third ladder (seen below, hybridized to chromosome 1q) spaces probes close together and further apart, allowing assessment of the resolution of two closely spaced spots in any given metaphase spread (labeled Ladder 3 in the image). DETAILED DESCRIPTION OF THE INVENTION

[0149] Unless otherwise stated, technical terms are used according to conventional usage.The definition of common terms in molecular biology can be found in Benjamin Lewin, Genes V, published by Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); Robert A. Meyers (ed.), and Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).Unless otherwise stated, all technical and scientific terms used herein have the same meaning as that commonly understood by those skilled in the art to which this disclosure belongs. The singular terms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. "Comprising A or B" means including A, or B, or A and B. It is further understood that all base sizes or amino acid sizes and all molecular weights or molecular weight values ​​given for nucleic acids or polypeptides are approximate and are provided for illustrative purposes.

[0150] It is further understood that ranges provided herein are shorthand for all values ​​within that range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or subrange from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 27, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 (and fractions thereof unless the context clearly dictates otherwise). Unless otherwise indicated, any concentration range, percentage range, ratio range, or integer range is understood to include any integer value within the recited range, and, where appropriate, fractions thereof (e.g., tenths and hundredths of integers, etc.). Also, any numerical range recited herein relating to any physical characteristic, such as polymer subunits, size, or thickness, is understood to include any integer within the recited range, unless otherwise indicated. As used herein, "about" or "consisting essentially of" means ±20% of the mean of the indicated range, value, or structure, unless otherwise indicated. As used herein, the terms "include" and "comprise" are open-ended and are used interchangeably.

[0151] Although methods and materials similar or equivalent to those described herein can be used to implement or test this disclosure, suitable methods and materials are described below.All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety.In case of conflict, the present specification, including explanations of terms, will prevail.In addition, materials, methods and examples are only illustrative and are not intended to be limiting.

[0152] As used herein, a "band" refers to a chromosomal region hybridized with probes labeled with a similar light emission signature (eg, probes of the same color).

[0153] As used herein, "breathing" refers to the optical emission signature of one band that partially overlaps with or otherwise appears partially above at least one other band.

[0154] As used herein, "color" refers to a wavelength of light radiation that can be detected as separate and distinct from other wavelengths.

[0155] As used herein, "chromosomal segment" refers to a DNA region defined by start and end coordinates of a genome (e.g., bp 12900-14900 of human chromosome 2) or known sequence content (e.g., the sequence of a gene or mobile element). A chromosomal segment can be as small as two base pairs or as large as an entire chromosome.

[0156] As used herein, "color channel" refers to a region of the light spectrum that includes visible, infrared, and ultraviolet light. A color channel may be specified as a broad or narrow set of wavelengths useful to an individual practicing the methods disclosed herein.

[0157] As used herein, "directional genomic hybridization" or "dGH" refers to a sample preparation method combined with a probe hybridization method, whereby (1) a DNA analog (BrdU) is provided to actively dividing cells for one replication cycle and selectively incorporated into newly synthesized daughter strands, (2) metaphase spreads are prepared, (3) the incorporated analog is photolytically targeted to create DNA nicks that are selectively used to enzymatically digest and degrade the newly synthesized strands, and (4) the single-stranded metaphase spreads are hybridized in situ with a unidirectional probe designed to a unique sequence in a reference genome such that only one single-stranded sister chromatid of the metaphase chromosome is labeled with the unique target site(s).

[0158] As used herein, "episome" or "episomal DNA" refers to a segment of DNA that resides in the cytoplasm of a cell and is capable of autonomous replication.

[0159] As used herein, "extrachromosomal DNA" or "ecDNA" refers to any DNA found on chromosomes either inside or outside the nucleus of a cell. In certain embodiments, ecDNA may be harmful and may carry amplified oncogenes. In some embodiments, harmful ecDNA may be 100-1,000 times larger than the kilobase-sized circular DNA found in healthy somatic tissues. In certain embodiments, ecDNA includes episomal DNA and vector-integrated DNA.

[0160] As used herein, the terms "feature node" and "node" are used interchangeably to refer to a numerical value, including a set of numerical values, that represents any region of interest on an oligonucleotide or polynucleotide strand. A node can be a specific locus, a set of loci, a gene, multiple genes, a band, or an entire chromosome. Nodes can be configurable and vary in size to allow for different levels of granularity during analysis. As a non-limiting example, a node can represent a normal or abnormal feature of a target DNA strand. Also, as a non-limiting example, a node can provide a numerical value for spectral profile data from labeled probe hybridization to a control DNA strand, where the node represents either a normal or abnormal structural feature of the control DNA strand.

[0161] As used herein, a "feature lookup table" refers to a table of numbers that represent one or more feature nodes.

[0162] As used herein, "probe" refers to a labeled oligonucleotide designed to be complementary to a target DNA sequence of interest so that, when coupled in a hybridization reaction, it will bind to and detect the target.

[0163] As used herein, "single-stranded chromatid" refers to the product of a process in which a DNA analog (e.g., BrdU) is provided to actively dividing cells over a single replication cycle, then selectively incorporated into newly synthesized daughter strands to prepare metaphase spreads, and the incorporated analog is photolytically targeted to create DNA nicks that are selectively used to enzymatically digest and degrade the newly synthesized strands, resulting in single-stranded products. When the terms Watson and Crick are used to describe the 5' to 3' and 3' to 5' strands of a double-stranded DNA complex, an untreated metaphase chromosome will have one sister chromatid with a parent Watson / daughter Crick and one sister chromatid with a daughter Watson / parent Crick. In chromosomes prepared according to the above method, one sister chromatid will consist solely of the parent Watson strand and the other sister chromatid will consist solely of the parent Crick strand.

[0164] As used herein, "sister chromatid exchange" or "SCE" refers to the error-free exchange (crossover) of identical, precisely matched DNA strands. Although sister chromatid exchange is not a structural variant, it is associated with increased rates of genomic instability due to the increased probability that alternative template strands, such as repetitive elements adjacent to the break site, will cause unequal exchanges that result in structural variants.

[0165] As used herein, "sister chromatid recombination" or "SCR" refers to the homologous recombination process involving identical sister chromatids, resulting in a unidirectional non-crossover event, otherwise known as a gene conversion event. This is thought to occur when a homologous recombination intermediate known as a double Holliday junction is resolved in a manner that results in non-crossover. SCR can be used by cells to resolve both single-stranded DNA lesions (with corresponding replication fork collapse) and double-stranded breaks. Gene conversion between sister chromatids is not usually associated with reciprocal exchange and is therefore distinct from SCE.

[0166] As used herein, "spectral profile" refers to a graphical representation of the variation in light intensity of a material(s) at one or more wavelengths.

[0167] As used herein, "structural feature" broadly refers to any aspect of the sequence of bases in an oligonucleotide or polynucleotide, including normal or abnormal characteristics of the sequence. For example, structural features include, but are not limited to, genetic elements selected from protein coding regions, regions that affect transcription, regions that affect translation, regions that affect post-translational modification, and any combination thereof. As a further non-limiting example, structural features include genetic elements selected from exons, introns, 5' untranslated regions, 3' untranslated regions, promoters, enhancers, silencers, operators, terminators, poly(A) tails, inverted terminal repeats, mRNA stability elements, and any combination thereof.

[0168] As used herein, "trained" or "training" refers to the creation of a model that is trained based on training data and can then be used to process additional data. Types of models that can be used for training include, but are not limited to, artificial neural networks, decision trees, support vector machines, regression analysis, Bayesian networks, and genetic algorithms.

[0169] As used herein, "structural variant" or "chromosomal structural variant" or "SV" refers to a DNA region that has undergone genomic alterations resulting in copy number, structural, and content changes of a segment size greater than 50 bp. The term SV is used as the operational boundary between single-nucleotide variants / INDELs and segmental copy number variants. These alterations include deletions, novel sequence insertions, mobile element insertions, tandem and interspersed segmental duplications, inversions, breaks, and translocations in the test genome compared to the reference genome.

[0170] As used herein, "target DNA" refers to a DNA region defined by the start and end coordinates of a reference genome (e.g., bp 12900-14900 of human chromosome 2) or a known sequence content to be detected (e.g., the sequence of a gene or mobile element).

[0171] As used herein, "target enrichment" refers to the use of additional probes for a particular target region of interest beyond the probes used for banding to track any changes to that target region. In certain embodiments, the target region of interest may be smaller than a band. In certain embodiments, the target region of interest may be limited to a portion of a band, cover an entire band, or span part or all of two or more bands.

[0172] As used herein, "vector-incorporated DNA" refers to any vector that serves as a vehicle for DNA insertion. These may be cloning vectors, expression vectors, or plasmid vectors that are introduced into cells, including, but not limited to, artificial chromosome vectors, phage and phagemid vectors, shuttle vectors, and cosmid vectors.

[0173] A method for detecting structural variations in chromosomes is disclosed, by labeling single-stranded chromatids with probes of different colors.The hybridization patterns of the labeled probes generate spectral profiles that allow high-resolution detection of structural variations, making it easy to distinguish between benign variations and harmful structural variations.Furthermore, the spectral profiles provide information about complex structural variations that may result from more than one rearrangement of chromosome segments.

[0174] Single-stranded chromatids can be generated by any means known in the art, including, but not limited to, CO-FISH techniques.

[0175] Probes capable of hybridizing to single-stranded chromatids can be of any functional length. Without being limited to any particular embodiment, the probes can be 10-100 nucleotides in length, 15-90 nucleotides in length, 25-75 nucleotides in length, 30-50 nucleotides in length, 37-43 nucleotides in length, or any combination thereof.

[0176] In certain aspects, the number of probes in a labeled probe set used in the methods disclosed herein can range from smaller probe sets that target specific chromosomal regions on one or more chromosomes, providing locus-specific banding on a limited number of chromosomal regions (e.g., one or more chromosomal regions), to larger probe sets that provide an array of probes targeting chromosomal regions across the genome.

[0177] In certain embodiments, the number of probes in the labeled probe set used in the method disclosed herein can range from a small probe set that targets one or more genes of interest to a larger probe set that targets all known genes in the organism under study.In one embodiment, the probe targets can be relatively evenly distributed throughout the genome.In another embodiment, the probe targets can be more concentrated in a certain region of genome, and more distributed in other regions of genome.

[0178] In certain embodiments, labeled probe sets can be designed to target the loci in genome that are known to affect or cause disease states.In one embodiment, probe sets can be designed to target genes that are known to be associated with the development or existence of lung cancer.Similarly, probe sets can be designed and utilized using the methods disclosed herein for any disease or condition of interest.

[0179] In certain embodiments, labeled probe sets can be designed to target loci within the genome known to correlate with different states of a particular disease, hi one embodiment, probe sets can be designed to indicate states of disease progression in, for example, a neurodegenerative disease.

[0180] In certain embodiments, labeled probe sets can be designed to target loci within the genome known to be correlated with genetic disorders, hi one embodiment, probe sets can be designed as prenatal diagnostic tools for genetic disorders.

[0181] In certain embodiments, labeled probe sets can be designed to target loci within a genome to provide a diagnostic tool for any disease or condition of interest, hi certain embodiments, the disease or condition can be selected from respiratory tract diseases, musculoskeletal disorders, neurological disorders, skin diseases, gastrointestinal diseases, and various types of cancer.

[0182] In certain embodiments, labeled probe sets can be designed to target specific classes of genes within the genome, hi one embodiment, probes can be designed to target genes for different types of kinases.

[0183] In certain embodiments, labeled probe sets can be designed to focus on a research area of ​​interest. In one embodiment, probes can be designed to test almost any hypothesis related to genomic DNA sequences in biomedical sciences.

[0184] In certain embodiments, a labeled probe set can be designed to provide bands that flank the centromere of one or more chromosomes, and such probes can function as a single probe panel or a multi-probe panel for chromosome identification and enumeration. In certain embodiments, the bands on either side of the centromere of each chromosome can be labeled with different colors to further differentiate the p and q arms.

[0185] In certain embodiments, the labeled probe set can be designed to provide bands targeting the telomere periphery and / or telomere region of one or more chromosomes. In some embodiments, the p-arm and q-arm end bands of the probe set can serve as separate probe panels or multiple probe panels for tracking the telomere periphery and / or telomere region of one or more chromosomes. In certain embodiments, the probes targeting the telomere periphery and / or telomere region of one or more chromosomes provide not only structural information of the target chromosome, but also structural information of the specific arm of the target chromosome. The application of the probes to the telomere periphery and / or telomere region of the band provides information for detecting structural rearrangement events involving the targeted telomere periphery and / or telomere region.

[0186] Any individual band may cover part or all of a gene, and any particular gene may be covered by all or part of one or more bands.

[0187] In certain embodiments, a target enrichment strategy may be used in which additional probes are used to detect features of the target region of interest beyond the probes used for banding the target region of interest. In certain embodiments, the target region of interest may be smaller than a band. In certain embodiments, the target region of interest may be limited to a portion of a band, cover an entire band, or extend over a portion or entirety of two or more bands. In certain embodiments, the probes used for target enrichment may be labeled with the same or different fluorophores as the bands to which the target enrichment probes hybridize. In embodiments in which the same fluorophore is used in the target enrichment probes, the intensity of the fluorescent signal is boosted within that channel. In embodiments in which different fluorophores are used in the target enrichment probes, combinatorial fluorescent signals are generated.

[0188] In certain embodiments, the oligonucleotide probes designed for target enrichment have the same or different design parameters as the probes used for banding painting.Using the same design parameters results in competitive hybridization, while using different design parameters results in a mixture of competitive and non-competitive hybridization.Target enrichment improves the detection limit and improves the ability to track specific chromosome loci.

[0189] Any reference spectral profile can be used as a standard for comparing the spectral profile of the chromosome under study. The reference spectral profile can be that of a chromosome with a known abnormality, a chromosome considered normal, a corresponding sister chromatid, a statistically determined normal profile, a database containing reference data for chromosomes considered to have a normal or abnormal profile, or any combination thereof. In addition, the distribution of probes designed for a reference genome or sequence (i.e., the density pattern of probes across unique or repetitive sequences in silico) relative to the reference spectral profile (increased brightness in areas with more probes and decreased brightness in areas with fewer probes) can be used to identify and explain structural variations in test samples when deviations in the expected spectral profile of the target exist.

[0190] The structural variation determined by this method can be any type of structural variation from normal, including, but not limited to, a change in the copy number of a chromosomal segment, an inversion, a translocation, a breakage, a sister chromatid recombination, a micronucleus formation, a chromosomal fragmentation event, or any combination thereof. The change in copy number of a segment can be a deletion, an amplification, or any combination thereof.

[0191] The labeled probe can be labeled by any means known in the art. The probe can also contain any number of different types of labels. The combination of probes can also have any number of different types of labels, with the labels differing between probes. The label on the probe can be fluorescent. The light emitted by the label on the probe can be detectable in the visible light spectrum, the infrared light spectrum, the ultraviolet light spectrum, or any combination thereof. The light emitted from the probe can be detected in false color or otherwise assigned a color different from the actual light emitted by the probe.

[0192] In one embodiment, the probe set used for hybridization comprises probes in which different probes are labeled with different colors.Probe set can be differently labeled probes, and these probes are labeled with two different colors (i.e., the first probe of the first color and the second probe of the second color), three different colors, four different colors, five different colors, six different colors, seven different colors, eight different colors, nine different colors, ten different colors, eleven different colors, twelve different colors, thirteen different colors, fourteen different colors, fifteen different colors, sixteen different colors, seventeen different colors, eighteen different colors, nineteen different colors, twenty different colors, twenty one different colors, twenty two different colors, twenty three different colors, twenty four different colors, twenty five different colors, twenty six different colors, twenty seven different colors, twenty eight different colors, twenty nine different colors, thirty different colors, or more than thirty different colors.

[0193] The position of the label on the hybridization probe can be any position on the probe that can support the binding of a label. The probe can be labeled at the end of the probe, at the side of the probe, in the body of the probe, or any combination thereof. The label on the body of the probe can be on the sugar or amidite functional group of the probe.

[0194] The detection of the probe can be carried out by any means known in the art. Any means can be used to filter the light signal from the probe, including but not limited to narrow band filters. Any means can be used to process the light signal from the probe, including but not limited to computational software. In some embodiments, only a certain part of the light signature from the probe is used to analyze chromosomal structural variants.

[0195] The method disclosed herein can be combined with other techniques for detecting chromosomal abnormalities.In one embodiment, the method disclosed herein can be combined with chromosome staining techniques, including but not limited to, DAPI, Hoechst 33258, actinomycin D, or any combination thereof.

[0196] Directed genomic hybridization (dGH) is a technique that can be applied to measure both the rate of misrepair and the identity of a specific misrepair. This method can be used to detect both novel SVs in metaphase chromosomes in individual cells or to evaluate SVs associated with specific genomic loci. In the above embodiment, detection of orientation changes (inversions), sister chromatid exchanges, and non-crossover sister chromatid recombinations, as well as balanced allelic translocations, will be visualized as the same signal pattern changes in a single cell using a single method. These SVs are detected alongside and in addition to SVs visible with standard chromosome-based cytogenetic analysis methods (unbalanced and balanced nonallelic translocations, ploidy changes, large inversions, large insertions, and large duplications). However, unless targeted methods are used, it is often impossible to distinguish orientation-changed SVs (high risk) from transient repair intermediates resulting from SCE and SCR events (low risk) and balanced translocations between two homologous chromosomes (relatively low risk). In recent years, additional types of misrepair and their relative contributions to carcinogenesis and genome instability have been described, further illustrating the need for more precise resolution of events visible via dGH, beyond the obvious need for more precise mapping of breakpoints and description of the genomic regions involved in SVs detected by dGH. Most of the research on the molecular mechanisms of SCE formation discussed herein involves work in yeast, which is far more advanced than our knowledge of mammals. We do not claim that the mechanisms are identical, but to the extent that the processes are similar, the approach described in this application will help further such understanding.

[0197] Because DNA misrepair can lead to cell death or pose risks to patients, novel techniques for both measuring misrepair rates and identifying SVs de novo are essential. The present invention combines dGH techniques with unique dGH hybridization probe designs and unique image analysis methodologies to identify and characterize SVs with significantly increased resolution. Because this characterization includes location and orientation data, it can be combined with publicly available bioinformatic data on genes, promoters, and genomic regions to assess the risk of genotoxicity caused by misrepair(s) for individual cells, as well as proteomic and transcriptomic data to inform patient diagnosis.

[0198] Directed genomic hybridization (dGH) can be performed either as a novel method capable of detecting structural variants against a reference (normal) genome, or as a targeted method to evaluate structural variants at specific target regions, such as editing sites (Figure 5). In both of these embodiments, the dGH method is designed to be qualitative and provide definitive data regarding the prevalence or occurrence of one or more structural variants in individual cells. When using the targeted embodiment, the presence of a specific target can be inferred because the assay is designed as a binary test of the target. However, while this novel embodiment can detect almost all SVs without a prior target hypothesis, it can only provide a rough identity of the variant (e.g., a putative telomeric inversion in the p-arm of C3 at approximately 7 Mb) and cannot provide definitive data regarding the variant's rearrangement type, orientation, size, location, or sequence.

[0199] Chromosome banding, using light and dark or multicolor bands, is a widely used technique for distinguishing normal karyotypes from structurally rearranged karyotypes. Each banding method has its advantages and disadvantages. G-banding, inversion (or R-banding with DAPI), and chromomycin staining are the most widely used techniques for producing differential light and dark banding of chromosomes. They are suitable for detecting a subset of simple structural variants, including numerical variants (variations in the number of whole chromosomes or large portions of chromosomes), simple translocations, and some large inversions (depending on the degree of disruption of the banding pattern). These are rapid, cost-effective DNA staining methods and are the current industry standard for karyotyping in clinical diagnostics. While they provide basic karyotypic information, these techniques have very limited utility in detecting smaller numerical variants (deletions and insertions) and small inversions, and they often cannot be used to explain complex rearrangements. They do not provide any locus-specific information beyond describing the observed light / dark band disruption, including the general region of interest. In the case of translocations, they also have significant blind spots. When chromosome banding patterns are presented in an alternating "...light-dark-light-dark..." sequence, as seen in G-banding, the resolution of exchange breakpoint locations will be inherently poorer than the same pattern presented in an alternating color sequence, e.g., "...RGBY...". These staining-based methods are subject to "three-band uncertainty" in the localization of translocation breakpoints (Savage 1977) when applied to the first (light-dark) situation. In addition, these methods do not detect balanced translocations, which are equivalent exchanges between two homologous chromosomes with breakpoints at the same or nearby loci, nor do they detect sister chromatid exchange / sister chromatid recombination (gene conversion) events.

[0200] Because each chromosome (two copies of each chromosome per normal cell) is labeled with a different color, whole-chromosome FISH painting techniques such as SKY and MFISH can be used to provide a more precise description of the observed structural variants. While these techniques identify which chromosomes are involved in the observed rearrangement, they cannot provide breakpoint coordinates or identify which genomic segments of the chromosome are included or missing as the product of the rearrangement. For example, much like monochrome dGH painting, deletions or amplifications cannot be attributed to any specific region or locus of a particular chromosome by SKY, MFISH, or similar methods.

[0201] Band-specific multicolor labeling strategies (the most well-known method is mBAND), including identifying which segments of a particular chromosome are involved in the rearrangement, can provide a more resolved picture of a particular complex event, limited by the resolution of the assay. The resolution of the mBAND assay is determined by how modest (small) the band sizes are in any given region and how well the sample is able to resolve bands for both their presence and their relative order (e.g., how long and elongated the chromosome is). However, like all other FISH-based techniques, mBAND cannot detect balanced translocations between homologous chromosomes, small inversions, or sister chromatid exchange / sister chromatid recombination events (gene conversions), no matter how high the resolution. Furthermore, these bands are generated by amplifying and differentially labeling portions of needle-microdissected chromosomes via DOP-PCR to create overlapping libraries of probes, and evaluating these bands in normal karyotypes against high-resolution G-banding and / or reverse DAPI banding to estimate the location of each band. Therefore, the exact start and end coordinates of each band are unknown and can only be inferred by comparison with highest resolution G-banding of metaphase cells with a normal karyotype.

[0202] "Oligocoloring," as referred to in U.S. Patent Application Publication No. 2010 / 0304994, offers an advantage over mBAND in that bands can be precisely designed to known genomic coordinates using synthetic oligos. The exact start and end of each band are known genomic coordinates, not estimates based on comparison with light and dark banding in normal karyotypes. However, like all other FISH-based techniques, it cannot detect balanced translocations between homologous chromosomes, small inversions, or sister chromatid exchange / sister chromatid recombination events (gene conversions).

[0203] The disclosed method for detecting structural mutations provides missing elements from the monochromatic dGH paint, provides specific genomic coordinates, and distinguishes true inversion events (involving reordering of genome segments) from sister chromatid exchange events (which do not alter the order of genome segments but cannot be distinguished from inversions using monochromatic dGH paint). It is important for clinicians to understand the risks associated with these two events: inversions are high risk, while SCEs are low risk (because they are essentially correct repairs and do not alter the order or copy number of genome segments). There is a risk of loss of heterozygosity associated with sister chromatid exchange (where one good copy of a gene is replaced by a bad copy, resulting in a disease phenotype), but this should be distinguished from true inversion events in terms of risk and patient outcome. CRISPR Cas9 and other gene editing systems that rely on DNA cleavage and DNA cleavage repair require accurate risk profiling. The disclosed method allows for the distinction of these SCE / SCE "false positives" from potentially genotoxic events (inversions). The order of the genome segments is visible, as is the orientation of the signals on either the primary or opposite sister chromatids (see schematic). As a technique, K-banding is distinguished from other multicolor banding methods by the required sample preparation method, which involves removal of the newly synthesized DNA daughter strand from the sister chromatid complex, providing a single-stranded template that allows for chromatid-specific labeling.

[0204] In the context of gene editing, detecting and identifying structural variants generated during genome manipulation and modification is a priority for patient health. The need to measure inversions and sister chromatid exchanges as an important part of the repair equilibrium, along with deletions, amplifications, and translocations at high resolution in single cells, is widely recognized as a necessity in the diagnostic community and among regulatory agencies. The disclosed method can provide structural variant data missed by sequencing and inaccessible using other differential banding or FISH-based banding methods. As outlined in the preceding description, the sample preparation component of the assay, combined with the unidirectionality of the oligo probes, allows for the evaluation of events undetectable by other banding techniques, providing an important additional level of structural variant data. Because enzyme-directed gene editing processes hijack and suppress cellular synthesis and repair machinery, they introduce an additional level of complexity into an otherwise highly complex process. Sequencing approaches to confirm editing, as well as to evaluate the remainder of the genome for unintended effects, often rely on the presence of intact target sequences to generate data. However, when excisions and deletions occur in the target sequence region, amplification of the region for sequence analysis becomes impossible. Furthermore, in pooled DNA formats, this information is lacking, which is a concern when screening for structural variants that contain copy number variations and have increased risk of genotoxicity. Evaluating complex structural variants by sequencing is also extremely difficult. Thus, the most genomically unstable and dangerous structural variants are most likely to be missed by sequencing. In the context of metaphase spreads, the entire genome of each cell is available for measurement and assessment of the presence of structural variants without any amplification or sequence analysis. Not only de novo rearrangements but also rearrangements to the intended target can be measured, allowing the edited cell population to be monitored over time for unintended, spontaneous, stable structural variants (such as cancer-driving fusion genes) that may be of concern, as well as the stability of the desired edit over time.Using the genomic coordinate specificity provided by the methods of the present disclosure, sequencing can be used to interrogate observed structural variants in a deeper, base pair specific manner. These two techniques can be used together to enable more accurate detection and characterization of edited genomes. Analysis of extrachromosomal DNA (ECDNA)

[0205] The biological sample comprising cellular DNA is prepared to facilitate contacting the sample with the oligonucleotide probe that is single-stranded, unique, and complementary to at least a portion of DNA.In certain embodiments, the biological sample comprising cellular DNA further comprises ECDNA.Both ECDNA and chromosomal DNA can be hybridized with the probe that has the same nucleic acid sequence and fluorescent signature.In the embodiment where ECDNA and chromosomal DNA are labeled similarly, it can determine where ECDNA originates on chromosome.

[0206] The oligonucleotide probes used to band the chromosome under investigation can be selected to specifically locate the chromosomal source or origin of the DNA found in the ECDNA. In certain embodiments, spectral analysis of the hybridization pattern of the oligonucleotide probe to chromosomal DNA allows for the identification of the chromosomal source of the DNA in the ECDNA. Comparison of spectral signatures, in certain embodiments, examination of the similarity of the spectral signatures between the chromosome and the ECDNA, leads to the identification of specific chromosomal DNA as the source of the amplified region of DNA incorporated into the ECDNA. In certain embodiments, analysis of the banding pattern resulting from the hybridization of the probe to chromosomal DNA leads to the identification of the gene and region of interest in the chromosome under investigation. In certain embodiments, when multiple bands are identified as the source material for integration into the ECDNA to further characterize the sequences present in the ECDNA, the band(s) identified as of interest in the chromosome under investigation can be used to inform the design of a specific probe or panel of probes.

[0207] The method for analyzing ECDNA can be applied to episomal DNA, vector-integrated DNA, as well as any other DNA in the cell that is not chromosomal.

[0208] Spectral analysis In certain embodiments, spectral imaging and analysis captures information about all fluorophores in one image. In some embodiments, bands are so close to each other that adjacent bands appear to bleed each other. Breathing over can be used as an additional marker to improve the localization of events within a band based on the presence of bleed over from adjacent bands and the ratio of bleed over signal to band signal.

[0209] Directed genomic hybridization (dGH) expansion In certain embodiments, magnification microscopy (Asano et al. (2018) Current Protocols in Cell Biology e56, Volume 80) can be applied to dGH samples to improve the spatial resolution of dGH. In certain embodiments, magnification microscopy involves embedding the sample in an expandable hydrogel and then chemically binding the sample to the hydrogel. The sample can then be labeled, swelled, and imaged. The process of swelling the sample increases the spatial (x, y, z) resolution to a level comparable to the confocal or super-resolution of an unmagnified sample. Thus, improved ability to localize events, such as structural mutations, is achieved.

[0210] Node Analysis Disclosed herein are methods for identifying one or more structural features of a target DNA strand. In certain embodiments, such methods are implemented on a processor. In one embodiment, the method for identifying one or more structural features includes receiving a spectral profile representing at least one sequence of base pairs of the target DNA strand, the spectral profile including frequency data corresponding to the sequence of bases of the target DNA strand. The frequency data can be divided into at least two color channels. In a different embodiment, various data, including but not limited to position data and intensity data, are included in the color channels. The spectral profile can be converted into a data table including the position data, intensity data, and other data determined to be of interest in the at least two color channels. The data table thus generated for the target DNA strand can be compared to a reference feature lookup table including one or more feature nodes representing normal and / or abnormal features of a corresponding control DNA strand to identify one or more normal and / or abnormal features of the target DNA strand. In one embodiment, the feature nodes are defined by color bands representing base subsequences of the control DNA strand starting from the starting base and ending at the terminal base.

[0211] Node analysis, in which the spectral profile information of the target DNA sequence is converted into a numerical form for comparison with a control or reference DNA sequence, can be performed in conjunction with the directional genomic hybridization method disclosed herein, or can be used in conjunction with other methods that provide polynucleotide sequence data that can be converted into a numerical form.In certain embodiments, the reference or control lookup table is a single value table or multiple value tables.In some embodiments, different reference or control lookup tables provide values ​​corresponding to different genomic regions.In certain embodiments, the comparison of the lookup table from the target DNA with the reference or control lookup table is performed by machine learning and / or AI algorithms.The value of the spectral profile data from the target DNA strand can be associated with a specific node through the analysis of the control or reference lookup table.Then, a set of nodes can be subjected to node analysis to find related or affected pathways, where the relationship between nodes is previously known or determined by analysis.

[0212] In certain embodiments, the spectral profile data from the target DNA strands can be stored in memory for later comparison and analysis to determine structural features of interest. In some embodiments, the spectral profile data can be stored in a relational database, a graph database, a lookup table, or any other bioinformatic database format.

[0213] In some embodiments, the target characteristic of the subject DNA strand can be characterized as a normal characteristic corresponding to a characteristic of a healthy control DNA strand. In some embodiments, the target characteristic of the subject DNA strand can be characterized as an abnormal characteristic corresponding to a characteristic of a reference DNA strand that exhibits at least one abnormality.

[0214] In certain embodiments, spectral profile data is analyzed from DNA regions that are not spatially aligned. In some embodiments, the spectral profile data originates from spatially adjacent DNA regions. In certain embodiments, the spectral profile data is linked by a set of keys based on probe sequence, spectrum, oligonucleotide density, chromosome, chromosome arm, band ID, band orientation, and band coverage (e.g., gene region). In some embodiments, genomic features can be defined by band, band spectrum, band sequence, band orientation, and nearest neighbor of the band, or by probe, probe spectrum, probe orientation, and nearest neighbor of the probe.

[0215] In certain embodiments, a feature, chromosome arm, or sequence spanning a chromosome can be defined by analyzing the band spectrum, size, and coverage of each band, starting from the 5' end of the probe, band, or region of interest and then moving sequentially toward the 3' end. In some embodiments, these features are converted into keys, which can be compared to a database to determine the location and characteristics of aberrations or abnormalities, and by extension, which nodes in the database are affected by those aberrations or abnormalities. Some combinations of aberrations or abnormalities indicate specific rearrangement events; for example, a truncated band in one region combined with extra signals in the same spectrum in a different region would indicate a translocation event.

[0216] Spectral profile data can be analyzed or meta-analyzed using any statistical analysis tool, including, but not limited to, graph theory, node analysis, artificial intelligence, machine learning (including k-nearest neighbors, principal component analysis, etc.), and neural networks.

[0217] The method disclosed herein can be combined with the method of incorporating multiple types of data into database for analysis.In certain embodiments, data from other sources include but are not limited to sequencing, genomics, transcriptomics, proteomics and metabolomics.In certain embodiments, inversion, sister chromatid exchange and other dGH specific data are analyzed against sequencing data.Comparison can be performed with known publicly available sequencing data or with new or unpublished data.

[0218] In some embodiments, the data generated by the methods disclosed herein is summarized in a report with automatically generated ideograms showing unique recursive rearrangements and analyses, meta-analyses, or nodal analyses, both at the sample level and at the cohort or experiment level. [Example]

[0219] The following examples are presented to more fully illustrate some embodiments of the present invention. However, they should in no way be construed as limiting the broad scope of the present invention. Those skilled in the art can easily adopt the principles underlying this discovery to design various compounds without departing from the spirit of the present invention.

[0220] Example 1 Figure 6 provides an example image of monochromatic dGH paint labeling chromosomes 1, 2, and 3 in a rearranged cell derived from a radiation-exposed blood-derived lymphocyte sample prepared for dGH. Images were acquired on an ASI scanning microscope system and viewed using GenASIS cytogenetics software. Chromosomes from selected metaphases were organized into karyograms in the software (the original image of the entire metaphase spread displays the chromosomes in a vertical orientation and organizes them into homolog pairs). The labeled chromosomes 1, 2, and 3 homolog pairs were then cropped and enlarged from the original metaphase spread image. The entire metaphase spread is provided below the cropped and enlarged karyogram. While there is a clear rearrangement involving the painted chromosomes (chromosomes 1, 2, and 3) in this cell, without reference to the segment order at the position where the signal switch is observed relative to the unpainted sister chromatid, it is impossible to confirm the presence of a true structural variant for the sister chromatid exchange event, nor is it possible to determine the genomic coordinates of the observed events on each chromosome.

[0221] Example 2 Chromosome 2 dGH multicolor banding pilot experiment using the BJ-5ta normal human fibroblast cell line. Experiment description: Nineteen unique sequence oligo probe pools were labeled with five different fluorophores in an alternating color pattern. Each probe pool had the same number of oligos, except for the last probe pool at the end of chromosome 2, which had approximately 1.6-fold more oligos. Depending on the distribution of available unique sequences across chromosome 2, the oligo pools spanned longer or shorter stretches of DNA, creating a unique "fingerprint pattern" for chromosome 2. See Table 1 for the start-to-end position (band) in bp of each labeled pool, the total target size in bp of each labeled pool, the number of oligos per labeled pool, and the density distribution of fluorophores across the target region of DNA. The table also includes a pseudocolor assignment for each fluorophore (because some fluorophores lie outside the visible spectrum and / or have visually similar colors to each other in overlay, each color channel was assigned a color that allows visualization of bands separate from each other). The order of colors in the table and template strand assignments (Watson and Crick corresponding to each sister chromatid) are depicted. The color assigned to the "Crick" sister chromatid is blue, and the color assigned to the "Crick" sister chromatid is blue. The color reflects the DNA staining, as do the telomere, peritelomere, and centromeric regions, which were not labeled with probes in this experiment. The band color and strand assignment reflect the genomic coordinates of normal metaphase chromosome 2 (prepared for dGH). In this preliminary experiment, band sizes ranged from 9 to 15 million base pairs (MB). In this experiment, several control probe spots were included on both chromosome 8 and chromosome 1 to confirm resolution and hybridization quality. Note that the images included in all experiments involving this multicolored paint were converted to black and white, and the full color spectrum must be inferred using the table and the order of band appearance. [Table 1]

[0222] Images A and B in Figure 7 show chromosome 2 homolog pairs from two separate normal metaphase cells without structural mutations (normal immortalized human fibroblast cell line BJ-5ta). Images were acquired on an ASI scanning microscope system and viewed using GenASIS cytogenetics software. Chromosomes from selected metaphases were organized into karyograms in the software (which display the chromosomes vertically from the original metaphase spread and organize them into homolog pairs), and labeled chromosome 2 homolog pairs were cropped and enlarged from the original metaphase spread.

[0223] In addition, two cells (from the same experiment using the same cell line) showing abnormal signal patterns were imaged and analyzed. Images C and D in Figure 7 show chromosome 2 homologs from two separate metaphase cells (normal immortalized human fibroblast cell line BJ-5ta) showing a structural mutation in one homolog resulting from sister chromatid exchange (the color order is maintained, but the signal is on opposite sister chromatids). Note that when using monochromatic paint, a telomere or peritelomere probe is required to distinguish between large inversions (misduplications) and sister chromatid exchange (complete duplication) events. Classification of this type of event can be confounded using a monochromatic paint + telomere / peritelomere approach if additional sister chromatid recombination events are present in the telomeric or peritelomere regions. This novel embodiment allows both the detection and accurate classification of structural rearrangement events. In image C, the right-hand chromosome 2 homolog has an SCE at the breakpoint of SV bisection band number 13, while the right-hand homolog is normal. In image D, the homolog on the left has an SCE at the breakpoint occurring between bands 9 and 10, while the homolog on the right is normal.

[0224] Example 3 Chromosome 2 dGH multicolor banding pilot experiment using blood-derived lymphocytes recently exposed to ionizing radiation for the treatment of prostate cancer.

[0225] Using the assay described in Example 1, a dGH assay consisting of 19 unique sequence oligoprobe pools (each ranging from 9 to 15 MB) labeled with an alternating color pattern, such that the color order corresponds to the genomic coordinates of normal metaphase chromosome 2, was performed on radiation-exposed blood-derived lymphocyte samples prepared for dGH. Figure 4 shows a chromosome 2 homolog pair from a metaphase cell with identified SVs that would otherwise be impossible to characterize. A large pericentric inversion (potentially detectable by current cytogenetic techniques but potentially missed due to the nature of the band break occurring at the very distal end of the chromosome) is present, along with a smaller paracentric inversion near the centromere (magenta probes out of order on the opposite sister chromatid from the majority of the labeled pool on the q arm), and a larger sister chromatid exchange event in close proximity to the smaller paracentric inversion, all of which can be accounted for using the alternating colors as a frame of reference. Rearrangements that are difficult to visualize in the color combination overlay (shown) can be confirmed by viewing the signal in each separate color channel. Note that if this cell had been labeled with monochrome chromosome 2 dGH paint, as shown in Figure 4(2a, 2b), this chromosome would have been labeled with a small terminal SCE or inversion (p arm) and a large inversion (q arm), and the true classification of the structural rearrangement present would have been missed. Images of the hybridized chromosome pair are shown in Figure 4, with the corresponding ideographs labeled 1a and 1b, and the same ideograph depicting the dGH monochrome paint is shown on the right. Figure 4(1a) shows a normal chromosome 2 homolog prepared for dGH and hybridized with the multicolored banded chromosome 2 dGH paint. Figure 4(1b) shows a second chromosome 2 homolog from the same cell with a complex structural rearrangement. A large pericentric inversion is present, with one breakpoint occurring between bands 1 and 2 of 2p and the other occurring in bisected band 18 of 2q. There are additional smaller paracentric inversions near the centromere of 2q, with the first breakpoint between bands 9 and 10 and the second breakpoint between bands 10 and 11.There is also a large sister chromatid exchange event between bands 9 and 11, which shares the same proximal breakpoint as the small paracentric inversion, and can be verified by the order of the bands, which still appear in the correct numerical order but now on the sister chromatid opposite the primary paint (the left sister chromatid) (the right sister chromatid).

[0226] Without colored bands to provide segment order, rearrangements cannot be identified or explained by coordinates. In fact, using the schematic diagram on the right for visual reference, the chromosomes appear to have small terminal SCEs or inversions (p-arms) and large inversions (q-arms), and the true classification of the structural rearrangements present would have been misidentified.

[0227] Example 4 Using the assay, cell line, and imaging method of Example 2, spectral intensities along each sister chromatid were measured and plotted along with the oligo density distribution across chromosome 2. Figure 8A shows the hybridization, probe distribution, and fluorescence wavelength intensity of normal chromosome 2. Sister chromatids are depicted as "Watson" and "Crick." Both sister chromatid color channels were measured. For sister chromatid Crick, the signal intensity shown represents background noise on each channel; actual signal intensity peaks are visible in Watson. The signal intensity peaks are consistent with both the oligo distribution plot and the chromosome image overlay. The ideogram for chromosome 2 is provided in genomic context in Figure 8A. Figure 8B shows the hybridization, probe distribution, and fluorescence wavelength intensity of SCE detected on chromosome 2. Sister chromatids are depicted as "Watson" and "Crick." Both sister chromatid color channels were measured. The ideogram for chromosome 2 is provided in genomic context in Figure 8B. For sister chromatid Watson and Crick, the presence or absence of signal peaks on the spectral profile corresponds perpendicularly to the visible signal on each sister chromatid. The SCE breakpoint was estimated to bisect band 14 (shown in orange). The signal intensity peak is consistent with both the oligo distribution plot and the chromosome image overlay. The ideogram for chromosome 2 is provided in genomic context in Figure 8B. The breakpoint region ID was estimated in Figure 8B.

[0228] Example 5 Ladder Images - Introduction: Chromosome condensation (compact vs. long) in metaphase spread preparations varies between cells and cell preparations. This material variability must be accounted for in prior evaluations to determine the resolution of SV detection by the dGH assay. For example, in longer, more elongated chromatin configurations, hybridization signals from closely spaced probes can be resolved as separate signals, whereas in more compact, condensed chromatin, hybridization signals from closely spaced probes appear as a single integrated signal. In the metaphase spread shown in Figure 9, three separate ladder assays were hybridized to the chromosomes. The first ladder measures the detection limit for the number of oligos contributing to each signal and is spaced approximately 20 MB apart on the p arm of chromosome 2 (labeled Ladder 1 in the image). The second ladder (chromosome 2q) assesses the target size that a fixed amount of oligos can spread, also spaced approximately 20 MB apart, and similarly measures the detection limit (labeled Ladder 2 in the image). The third ladder (seen below, hybridized to chromosome 1q) spaces the probes close together and further apart, allowing assessment of the resolution of two closely spaced spots in any given metaphase spread (labeled ladder 3 in the image). These ladders were designed against the opposite DNA strand from the banding paint and can be used as an internal control for the resolution of the assay in each spread.

[0229] Example 6 Assays involving probe hybridization of Alu repeats associated with fragile sites in single-color banding dGH paint and multicolor banding dGH paint in other colors can be performed on metaphase samples prepared for dGH. Alu repeats (characterized and mapped in reference genomes) can be displayed and detected as unique banding patterns that are strongly associated with known fragile sites and regions known to be important for gene regulation, allowing the proximity of observed known or novel rearrangements to be compared with known fragile regions. Because structural variants present in rearranged chromosomes visualized by the assay are associated with known high-risk regions of the genome, they can be used to correlate phenotypes with genotypes.

[0230] Example 7 Multicolor banding paints can be combined with two specific color bands assigned to regions flanking the target of interest and run on metaphase samples prepared for dGH. In the same field of view, these two colors flanking the target of interest can be displayed in interphase cells (nuclei) as a "cleavage" assay, where selective analysis of specific color channels reveals specific region activity distinct from the remaining chromosome paint. This allows for the analysis of cells in the G1, S, and G2 phases of the cell cycle, along with cells that have passed all cellular checkpoints and successfully entered metaphase. Often, more interphase nuclei than metaphase nuclei are present in the sample at the time of slide preparation, and any nuclei present will hybridize with the probe simultaneously with the metaphase spread. Several types of data can be provided by a single assay when combined with specific imaging methods for visualizing regions of the genome separately, layered, and relative to each other in samples containing both metaphase and interphase cells.

[0231] The cancer cell line of Example 8A, which has large visible ecDNA of unknown origin, can be hybridized with dGH whole-chromosome paints, each with a unique color for each human chromosome. Chromosomal DNA amplified and included in the ecDNA contains signals of the same color(s) as the chromosome of origin. Once identified, specific chromosomes known to contain genetic material also present in the ecDNA can be subjected to sequential hybridization with banding paint(s) corresponding to the previously identified chromosome of origin. Because the labeled ecDNA corresponds to the specific band color(s) in the banded chromosome, regions or DNA coordinates can be identified. The coordinates can be further refined using specific target probes for the identified region of origin that appear in both the ecDNA and the corresponding chromosome and can be used to track and describe potentially deleterious changes to the genome.

[0232] Incorporation by Reference and description of transformations All references throughout this application, e.g., patent documents, including issued or granted patents or equivalents, patent application publications, and non-patent literature or other source materials, are incorporated herein by reference in their entirety, as if individually incorporated by reference, unless each reference is at least partially inconsistent with the present disclosure of this application (e.g., a partially inconsistent reference is incorporated by reference except for the partially inconsistent portion of the reference).

[0233] The terms and expressions used herein are used as terms of description, not of limitation, and there is no intention in the use of such terms and expressions to exclude any equivalents of the features shown and described, or portions thereof, although it is recognized that various modifications are possible within the scope of the invention as claimed. Thus, while the present invention has been specifically disclosed by preferred embodiments, exemplary embodiments, and optional features, it should be understood that modifications and variations of the concepts disclosed herein may be employed by those skilled in the art, and that such modifications and variations are considered to be within the scope of the invention as defined by the appended claims. The specific embodiments provided herein are examples of useful embodiments of the invention, and it will be apparent to those skilled in the art that the invention may be implemented using a wide variety of devices, device components, methods, and steps described in this description. As will be apparent to those skilled in the art, methods and devices useful for the present methods can include numerous optional compositions and processing elements and steps.

[0234] All patents and publications mentioned herein are indicative of the level of skill of those skilled in the art to which this invention pertains.The references cited herein are incorporated herein by reference in their entirety to indicate the state of the art as of their publication date or filing date, and it is intended that this information can be used herein, if necessary, to exclude certain aspects that are in the prior art.For example, when a composition is claimed, it should be understood that compounds that were known and available in the art prior to the applicant's invention, including compounds whose enabling disclosures are provided in the references cited herein, are not intended to be included in the composition claimed herein.

[0235] As used herein, "comprising" is synonymous with "including," "containing," or "characterized by" and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. As used herein, "consisting of" excludes any element, step, or ingredient not specified in the claim element. As used herein, "consisting essentially of" does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim. In each instance herein, any of the terms "comprising," "essentially consisting of," and "consisting of" may be replaced with either of the other two terms. The invention illustratively described herein may suitably be practiced in the absence of any element(s) or limitation(ies) not specifically disclosed herein.

[0236] Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosed disclosure belongs. The singular terms "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. "Comprising" means "including," so "comprising A or B" means "including A" or "including B" or "including A and B." All references cited herein are incorporated by reference.

[0237] Those skilled in the art will understand that starting materials, biological materials, reagents, synthetic methods, purification methods, analytical methods, assay methods, and biological methods other than those specifically exemplified can be used in the practice of the present invention without resort to undue experimentation. All art-known functional equivalents of any such materials and methods are intended to be encompassed by this invention. The terms and expressions used are used as terms of description, not of limitation, and there is no intention in the use of such terms and expressions to exclude any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention as claimed. Thus, while the present invention has been specifically disclosed by preferred embodiments and optional features, it should be understood that modifications and variations of the concepts disclosed herein may be employed by those skilled in the art, and that such modifications and variations are considered to be within the scope of the invention as defined by the appended claims.

Claims

1. 1. A method for detecting at least one structural mutation and / or repair event in a chromosome, comprising: a) generating a pair of single-stranded sister chromatids from the chromosome, wherein the sister chromatids comprise two or more target DNA sequences; b) contacting the single-stranded sister chromatid with two or more directed genomic hybridization (dGH) probes, each of the dGH probes comprising a pool of single-stranded oligonucleotides complementary to at least a portion of one of the two or more target DNA sequences, each of the dGH probes comprising a fluorescent label, and the dGH probes complementary to the target DNA sequences on the single-stranded sister chromatids comprising fluorescent labels of at least two different colors; c) generating a fluorescence pattern from the single-stranded sister chromatids using fluorescence detection, the fluorescence pattern being based on the hybridization pattern of the two or more dGH probes to the single-stranded sister chromatids; d) comparing the fluorescence pattern of said single-stranded sister chromatid with a reference fluorescence pattern; and determining the presence or absence of the at least one structural mutation and / or repair event in the chromosome based on at least one difference between the fluorescence pattern of the single-stranded sister chromatid and the reference fluorescence pattern.

2. The method described in claim 1, wherein each of the two or more dGH probes containing at least two different colored fluorescent labels hybridizes to adjacent DNA targets, producing a pattern of alternating fluorescent colors in at least a portion of the sister chromatids.

3. 3. The method of claim 1 or 2, wherein the structural mutation is selected from the group consisting of a change in the copy number of a segment of the chromosome, a change in the copy number of the chromosome, an inversion, a translocation, a sister chromatid recombination, a micronucleus formation, a chromosomal thrombus formation event, and any combination thereof.

4. 4. The method of claim 1, wherein the structural variation is a change in the copy number of a segment of the chromosome, and the change is selected from the group consisting of amplification, deletion, and any combination thereof.

5. contacting one or both single-stranded sister chromatids with a stain after step (a) and before step (c); detecting the staining pattern of one or both single-stranded sister chromatids after step (b), Step (d) further comprises comparing either or both staining patterns to a reference staining pattern representing a control; 5. The method of claim 1, wherein the determining step (e) further comprises determining the presence of the at least one structural variant based on at least one staining difference between either or both staining patterns and the reference staining pattern.

6. After step (a), contacting one or both single-stranded sister chromatids with oligonucleotide markers complementary to a repeated sequence on said single-stranded sister chromatid that is not the target DNA sequence, wherein each of said oligonucleotide markers comprises at least one label; detecting a marker hybridization pattern of one or both single-stranded sister chromatids after step (b), Step (d) further comprises comparing the marker hybridization pattern to a reference marker hybridization pattern representing a control; 6. The method of any one of claims 1-5, wherein the determining of step (e) further comprises determining the presence of the at least one structural variant based on at least one marker hybridization pattern difference between either or both marker hybridization patterns and the reference marker hybridization pattern.

7. The method of any one of claims 1 to 6, wherein the probes of the set of probes are 25 to 75 nucleotides in length.

8. The method of any one of claims 1 to 7, wherein the probes of the set of probes are 30 to 50 nucleotides in length.

9. The method of any one of claims 1 to 8, wherein the probes of the set of probes are 37 to 43 nucleotides in length.

10. 10. The method of any one of claims 1 to 9, wherein the probes complementary to the target DNA sequence on each single-stranded sister chromatid comprise labels of at least two different colors.

11. 11. The method of any one of claims 1 to 10, wherein the probes complementary to the target DNA sequence on each single-stranded sister chromatid comprise labels of at least three different colors.

12. 12. The method of any one of claims 1 to 11, wherein the probes complementary to the target DNA sequence on each single-stranded sister chromatid comprise labels of at least four different colors.

13. 13. The method of any one of claims 1 to 12, wherein the probes complementary to the target DNA sequence on each single-stranded sister chromatid comprise labels of at least five different colors.

14. 14. The method of any one of claims 1 to 13, wherein the at least one label is selected from the group consisting of a label detectable in the visible light spectrum, a label detectable in the infrared light spectrum, a label detectable in the ultraviolet light spectrum, and any combination thereof.

15. 14. The method of any one of claims 1 to 13, wherein the at least one label is selected from the group consisting of a label on the end of the probe, a label on the side of the probe, one or more labels on the body of the probe, and any combination thereof.

16. The method of any one of claims 1 to 15, wherein the at least one label is a backbone label on a sugar or amidite functional group of the probe.

17. The method described in claim 1, wherein each of the two or more dGH probes containing fluorescent labels of different colors hybridizes to adjacent DNA labels, producing a pattern of alternating fluorescent colors in at least a portion of the sister chromatids.

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