Method for detecting human peripheral blood chromosome structure variation by using Hi-C technology

Abstract

The invention discloses a method for detecting human peripheral blood chromosome structure variation by using a Hi-C technology. The method comprises the following steps: separating lymphocytes and carrying out cross-linking treatment by using a formaldehyde solution; terminating the reaction after incubation, collecting a cross-linked product, and quickly freezing and storing; splitting the cross-linked lymphocytes, releasing cell nucleuses, and digesting and digesting chromatin; carrying out biotin labeling on the tail end of the DNA subjected to enzyme digestion, and carrying out blunt end ligation; carrying out de-crosslinking treatment on the connected DNA, and extracting and purifying the DNA; carrying out fragmentation treatment on the purified DNA, and constructing a library through PCR (Polymerase Chain Reaction) amplification; performing quality control on the constructed library; sequencing data is subjected to quality control and comparison, translocation breakpoint positions are calculated and screened, and a detection result is judged by setting a threshold value. According to the method, high-resolution, high-throughput, low-cost, rapid and comprehensive chromosome structure variation detection can be realized, and the limitation of the prior art is overcome.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular biology, and particularly relates to a method for detecting chromosomal structural variations in human peripheral blood using Hi-C technology. Background Art

[0002] Human peripheral blood chromosome karyotype analysis has been widely used clinically for nearly 50 years. The commonly used method is G-banding karyotype analysis, which labels mitotic (M-phase) condensed chromosomes by Giemsa staining and is commonly used to detect chromosomal numerical abnormalities (such as trisomy syndrome) and larger structural abnormalities (such as translocations and inversions). Although this method is a classical cytogenetic technique, it has many limitations. First, the resolution of chromosome karyotype analysis is relatively low, usually only able to detect chromosomal abnormalities greater than 5 Mb (million base pairs). For microdeletions or microduplications less than 5 Mb, karyotype analysis is difficult to detect.

[0003] Chromosome karyotype analysis requires professional equipment and technical personnel, and the experimental operation is complex. From cell culture to chromosome preparation, banding treatment, microscope observation, and image analysis, etc., all require skilled experimental techniques and rich experience. The result interpretation also requires in-depth professional knowledge. Especially when there are chromosomal structural abnormalities, it is necessary to accurately identify and judge various complex chromosomal variations. In addition, the cell culture process of chromosome karyotype analysis takes a long time, usually several days, and requires a large amount of manpower and material resources. This limits the application of this technology in some clinical scenarios that require rapid diagnosis.

[0004] Karyotype analysis mainly focuses on the number and structural changes of chromosomes and cannot detect minute mutations at the gene level (such as single nucleotide variations). Therefore, for some genetic diseases caused by gene point mutations, karyotype analysis cannot provide effective diagnostic information. When detecting chimeras, due to the limitations of cell culture and analysis, karyotype analysis may not be able to accurately judge the proportion and type of chimeras. For some complex chromosomal abnormalities, such as translocations and inversions of multiple chromosomes, the interpretation of karyotype analysis is difficult and may not be able to completely and accurately describe all abnormalities.

[0005] The results of karyotype analysis can only be obtained at a specific stage of cell division (such as metaphase) and rely on complete chromosomes and good staining effects. If problems occur during cell culture or staining, it may lead to inaccurate results or inability to interpret. Chromosome karyotype analysis can only provide chromosomal-level information and cannot perform a comprehensive scan of the entire genome. In contrast, some emerging molecular genetics techniques (such as high-throughput sequencing) can detect a wider range of genetic variations at the whole-genome level.

[0006] Therefore, the present invention proposes a method for detecting chromosomal structural variations in human peripheral blood using Hi-C technology to overcome the limitations of the prior art and provide a method for detecting chromosomal structural variations with high resolution, high throughput, low cost, rapidity, and comprehensiveness. Summary of the Invention

[0007] The object of the present invention is to propose a method for detecting chromosomal structural variations in human peripheral blood using Hi-C technology to solve the problems in the prior art.

[0008] To achieve the above object, the present invention adopts the following technical solution: A method for detecting chromosomal structural variations in human peripheral blood using Hi-C technology, comprising the following steps: Step S1: Isolate lymphocytes from human peripheral blood and perform cross-linking treatment using a formaldehyde solution. Step S2: After incubation at room temperature, add a glycine solution to terminate the reaction, collect the cross-linked product, and perform quick-freezing preservation. Step S3: Lyse the cross-linked lymphocytes to release cell nuclei, digest and digest chromatin, and cut genomic DNA using a restriction endonuclease. Step S4: Biotin-label the ends of the digested DNA and perform blunt-end ligation, using T4 DNA ligase to ligate DNA fragments that are spatially close. Step S5: Perform de-cross-linking treatment on the ligated DNA, extract the DNA, and purify it, using ethanol precipitation to extract the DNA. Step S6: Fragment the purified DNA, retrieve the biotin-labeled fragments, perform end repair, add A, add adapters, and construct a library by PCR amplification. Step S7: Perform quality control on the constructed library, measure the concentration and fragment size of the detected library, and perform PE150 sequencing using a sequencing platform to obtain paired-end sequencing reads. Step S8: Perform quality control, alignment, calculation of translocation breakpoint positions on the sequencing data, and screen for structural variations at a certain resolution, and judge the detection results through a set threshold.

[0009] Further, in step S1, the following sub-steps are further included: S1-1: Isolate lymphocytes from human peripheral blood and isolate 50,000 to 100,000 lymphocytes using density gradient centrifugation. S1-2: Suspend the isolated lymphocytes in a phosphate buffer solution, add a formaldehyde solution for cross-linking treatment to cross-link the chromatin in the lymphocyte nuclei.

[0010] Further, in step S2, the following sub-steps are further included: S2-1. Incubate the cross-linked lymphocytes at room temperature, where the room temperature ranges from 18°C to 25°C and the incubation time ranges from 8 to 15 minutes; S2-2. Add glycine solution to terminate the cross-linking reaction, and the termination reaction time is 1 to 3 minutes; S2-3. Wash the lymphocytes 3 times with phosphate buffer solution and centrifuge to ensure no residual cross-linking reagent on the lymphocyte surface. The centrifugation speed is 1000 rpm and the duration is 5 minutes; S2-4. Collect the cross-linked lymphocytes, perform quick freezing in liquid nitrogen and store at low temperature. The low temperature storage temperature is -70°C to -85°C.

[0011] Furthermore, in step S3, the following sub-steps are also included: S3-1. Use lysis buffer to lyse the cell membrane and release the cell nucleus. The lysis time is 10 to 20 minutes. The lysis buffer includes Tris-HCl, EDTA, and NP-40; S3-2. Add sodium dodecyl sulfate (SDS) solution to open chromatin and expose the restriction sites. The SDS treatment time is 10 to 15 minutes; S3-3. Add Triton X-100 to terminate the SDS reaction and reduce interference with the restriction reaction. The Triton X-100 treatment time is 5 to 10 minutes; S3-4. Use restriction endonuclease to cut genomic DNA. The DNA fragments after digestion have blunt ends or sticky ends, and the length distribution is between 100 and 1000 bp. The restriction endonuclease is MboI.

[0012] Furthermore, in step S4, the following sub-steps are also included: S4-1. Use biotin-labeled base C to fill in and label the ends of the digested DNA to make the DNA fragments carry biotin; S4-2. Use T4 DNA ligase to ligate two spatially adjacent DNA fragments with blunt ends.

[0013] Furthermore, in step S5, the following sub-steps are also included: S5-1. Use proteinase K for de-crosslinking treatment to digest the proteins connecting the DNA fragments. The digestion temperature is 65°C and the digestion time is 2 hours; S5-2. Use ethanol precipitation method to extract DNA and purify the extracted DNA; S5-3. Perform quality detection on the extracted DNA. Use a NanoDrop spectrophotometer to detect the DNA concentration, and remove the DNA that does not meet the concentration requirements. The concentration requirement is that the DNA concentration is between 10 - 50 ng / μL.

[0014] Further, in step S6, the following sub-steps are also included: S6-1. Fragment the DNA by ultrasonic waves. The length of the fragmented DNA fragments is 200 - 500 bp. S6-2. Use streptavidin magnetic beads to retrieve biotin-labeled DNA fragments, perform end repair and A-tailing, and prepare for ligation of adapters. S6-3. Ligate adapters to the processed fragments and construct a library by PCR amplification. The number of cycles of the PCR amplification is 10 cycles. S6-4. Purify the amplified library using magnetic beads to remove unbound primers and adapters. S6-5. Perform enzymatic digestion quality control on the purified library to detect the ligation situation of the library.

[0015] Further, in step S7, the following sub-steps are also included: S7-1. Quantify the library using a quantitative instrument to determine the library concentration. The quantitative instrument is Qubit3.0. S7-2. Detect the library fragment size using a fragment analyzer. The fragment analyzer is Agilent 5400. S7-3. Perform sequencing using the DNBSEQ T7 sequencing platform, run the PE150 sequencing strategy, and obtain 150-bp paired-end sequencing reads.

[0016] Further, in step S8, the following sub-steps are also included: S8-1. Use a software tool to perform quality control on the paired-end sequencing data, and remove low-quality reads and adapter-contaminated reads. The software tool is the filter module in fqtools_plus v3.0.0 software. The quality control standard is that the proportion of bases with Phred Quality Score ≥ 20 is ≥ 95%. S8-2. Use alignment software to perform single-end alignment of Reads1 and Reads2 with the human hg38 reference genome respectively to obtain single-end alignment results. The alignment software is the GLOBAL module in HiCPro V2.7 software. S8-3. Perform global alignment through alignment software to obtain the final alignment result. The alignment software is bowtie2v2.2.3. S8-4, using an analysis tool to input the alignment result file and calculate the translocation breakpoint position, the analysis tool is Hi-Cbreakfinder, and the confidence interval of the translocation breakpoint position is ±100000 bp; S8-5, screening the structural variations identified at 1M resolution, judging the detection results by the set threshold, if the score obtained by identification is higher than the threshold, it is reported as the detection result, otherwise the result is judged as a false positive and the detection result is not output.

[0017] The beneficial effects brought about by the technical solution provided by the present invention include at least: The present invention uses Hi-C technology to detect chromosome structural variations in human peripheral blood, thereby achieving a significant reduction in the amount of sequencing data, from the traditional 500G to 5G, which not only significantly reduces the detection cost, but also improves the detection efficiency, making large-scale genome structural variation detection more feasible and economical.

[0018] The present invention creatively performs single-end alignment and global alignment, can accurately calculate the positions of chromosome translocation breakpoints, can distinguish balanced genomic structural variations, significantly improves the accuracy and reliability of detection, and provides a new technical means for the comprehensive analysis of chromosome structural variations.

[0019] The method of the present invention screens structural variations (SVs) identified at 1M resolution, can identify small and unknown variations, significantly improves the sensitivity and specificity of detection, and provides more accurate detection results for clinical diagnosis and research.

[0020] The method of the present invention realizes automation of data analysis through software tools, reduces human errors, improves the repeatability and standardization of detection, and can provide standardized detection results by combining Hi-C technology with high-throughput sequencing and automated analysis tools, which is suitable for different laboratories and application scenarios. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the technical solutions and advantages in the embodiments of the present invention or the prior art, the drawings required for use in the embodiments or the prior art descriptions are briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0022] Figure 1 A flow chart of a method provided by an embodiment of the present invention; Figure 2 An experimental flow chart provided for an embodiment of the present invention; Figure 3 A schematic diagram of an experimental flow chart provided for an embodiment of the present invention. Detailed implementation manners

[0023] In order to further elaborate on the technical means and effects adopted by the present invention to achieve the intended invention purpose, the following combines the accompanying drawings and preferred embodiments to detail the specific implementation manners, structures, features, and effects of a method for detecting human peripheral blood chromosome structural variations using Hi-C technology proposed according to the present invention. In the following description, different "one embodiment" or "another embodiment" do not necessarily refer to the same embodiment. In addition, the specific features, structures, or characteristics in one or more embodiments can be combined in any suitable form.

[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which the present invention belongs.

[0025] The following embodiments are for illustrative purposes only and are not intended to limit the scope of the present invention.

[0026] The following specifically describes the specific solution of a method for detecting human peripheral blood chromosome structural variations using Hi-C technology provided by the present invention with reference to the accompanying drawings.

[0027] Embodiment 1 Please refer to Figure 1 , Figure 2 and Figure 3 , which show the method flow chart, experimental flow chart, and experimental process schematic diagram of a method for detecting human peripheral blood chromosome structural variations using Hi-C technology provided by an embodiment of the present invention. The method includes the following steps: Step S1: Isolate lymphocytes from human peripheral blood and perform cross-linking treatment using a formaldehyde solution; Among them, in step S1, the following sub-steps are further included: S1-1: Isolate lymphocytes from human peripheral blood and isolate 50,000 to 100,000 lymphocytes using density gradient centrifugation; S1-2: Suspend the isolated lymphocytes in a phosphate buffer solution, add a formaldehyde solution for cross-linking treatment to cross-link the chromatin in the lymphocyte nuclei.

[0028] It should be noted that density gradient centrifugation is a commonly used cell separation technique that can effectively separate cell populations with specific densities.

[0029] Selecting 50,000 to 100,000 lymphocytes is to ensure that there are enough cells for subsequent Hi-C experiments, and at the same time avoid excessive cells resulting in complex experimental operations and increased costs.

[0030] The concentration of the formaldehyde solution is 1%, and the cross-linking treatment time is 10 minutes. Formaldehyde cross-linking is a commonly used fixation method that can fix the proteins and DNA inside cells, keeping their structures stable.

[0031] The phosphate buffer solution can maintain the physiological environment of cells and ensure that cells are not damaged during the cross-linking process.

[0032] Step S2: After incubation at room temperature, add glycine solution to terminate the reaction, collect the cross-linked product, and perform quick-freezing preservation. Among them, in step S2, the following sub-steps are also included: S2-1: Incubate the cross-linked lymphocytes at room temperature, where the range of room temperature is 18 degrees to 25 degrees, and the incubation time range is 8 - 15 minutes. S2-2: Add glycine solution to terminate the cross-linking reaction, and the termination reaction time is 1 - 3 minutes. S2-3: Wash the lymphocytes 3 times with phosphate buffer solution, and make the lymphocyte surface free of residual cross-linking reagent by centrifugation. The centrifugation speed is 1000 rpm, and the duration is 5 minutes. S2-4: Collect the cross-linked lymphocytes, perform quick-freezing in liquid nitrogen and low-temperature preservation. The low-temperature preservation temperature is -70 degrees to -85 degrees.

[0033] It should be noted that incubation at room temperature is to allow the cross-linking reaction to proceed fully and ensure that the chromatin structure inside cells is fully fixed; controlling the incubation time within 8 - 15 minutes is to ensure the completion of the cross-linking reaction and avoid cell structure damage caused by too long a time.

[0034] The concentration of the glycine solution is 0.24M, and the termination reaction time is 1 - 3 minutes. Glycine can react with formaldehyde to neutralize the cross-linking effect of formaldehyde, thereby terminating the cross-linking reaction; controlling the termination reaction time within 1 - 3 minutes is to ensure the complete stop of the cross-linking reaction and avoid cell structure damage caused by too long a time.

[0035] Washing with phosphate buffer solution is to remove the residual cross-linking reagent on the cell surface, ensure the cell surface is clean, and provide good conditions for subsequent operations.

[0036] The centrifugation speed is 1000 rpm, and the duration is 5 minutes, which is to effectively remove the residual reagent in the supernatant without damaging the cell structure.

[0037] Quick-freezing in liquid nitrogen can rapidly reduce the temperature of cells, prevent the continuation of biochemical reactions inside cells, and thus maintain the cell structure and state.

[0038] The cryopreservation temperature is controlled between -70°C and -85°C to ensure the stability of cells during long-term preservation and avoid the degradation of cell structure.

[0039] Step S3: Lyse the cross-linked lymphocytes to release the cell nuclei, digest and enzymatically cleave the chromatin, and use a restriction endonuclease to cut the genomic DNA; In step S3, the following sub-steps are also included: S3-1: Use a lysis buffer to lyse the cell membrane and release the cell nuclei. The lysis time is 10 - 20 minutes. The lysis buffer includes Tris-HCl, EDTA, and NP-40; S3-2: Add a sodium dodecyl sulfate (SDS) solution to open the chromatin and expose the cleavage sites. The SDS treatment time is 10 - 15 minutes; S3-3: Add Triton X-100 to terminate the SDS reaction and reduce interference with the enzymatic cleavage reaction. The Triton X-100 treatment time is 5 - 10 minutes; S3-4: Use a restriction endonuclease to cut the genomic DNA. The DNA fragments after enzymatic cleavage have blunt ends or sticky ends, and the length distribution is between 100 - 1000 bp. The restriction endonuclease is MboI.

[0040] It should be noted that the main components of the lysis buffer include Tris-HCl, EDTA, and NP-40. Tris-HCl provides a buffering environment to maintain the stability of the pH value; EDTA can chelate metal ions, inhibit the activity of nucleases, and protect DNA from degradation; NP-40 is a non-ionic surfactant that can disrupt the structure of the cell membrane, cause the cell membrane to rupture, and release the cell nuclei; the lysis time of 10 - 20 minutes is to ensure the full rupture of the cell membrane while avoiding the destruction of the cell nucleus structure due to excessive time.

[0041] The sodium dodecyl sulfate (SDS) solution is a strong ionic surfactant that can disrupt the structure of chromatin, separate DNA and proteins in chromatin, and thus expose the cleavage sites; the SDS treatment time of 10 - 15 minutes is to ensure the full disruption of the chromatin structure while avoiding the degradation of DNA due to excessive time.

[0042] Triton X-100 is a non-ionic surfactant that can neutralize the strong ionic properties of SDS and terminate the SDS reaction; the treatment time of 5 - 10 minutes is to ensure the complete termination of the SDS reaction while avoiding the destruction of the cell structure due to excessive time.

[0043] The restriction endonuclease MboI is a commonly used enzyme digestion tool that can specifically recognize and cut DNA sequences. The length of the DNA fragments after enzyme digestion is distributed between 100 - 1000 bp, which can ensure the effective progress of subsequent Hi-C experiments. The enzyme digestion conditions are 37°C and the digestion time is 1 hour, which can ensure the high efficiency and specificity of the enzyme digestion reaction.

[0044] Step S4: Biotinylate the ends of the digested DNA and perform blunt-end ligation. Use T4 DNA ligase to ligate DNA fragments that are spatially close to each other. In step S4, the following sub-steps are also included: S4-1: Use biotin-labeled base C to fill in and label the ends of the digested DNA, so that the DNA fragments carry biotin. S4-2: Use T4 DNA ligase to perform blunt-end ligation of two DNA fragments that are spatially close to each other.

[0045] It should be noted that biotinylation is a commonly used molecular labeling technique that can achieve specific capture of DNA fragments through the high-affinity binding of biotin and streptavidin.

[0046] Using biotin-labeled base C to fill in and label the ends of the digested DNA can capture the biotin-bearing DNA fragments with streptavidin magnetic beads in subsequent steps.

[0047] T4 DNA ligase is an enzyme that can catalyze the ligation between DNA fragments and can ligate blunt-end DNA fragments. The ligation reaction temperature of T4 DNA ligase is 16°C and the ligation time is 1 hour, which can ensure the high efficiency and accuracy of the ligation reaction.

[0048] Step S5: Decrosslink the ligated DNA, extract the DNA and purify it. Use the ethanol precipitation method to extract the DNA. In step S5, the following sub-steps are also included: S5-1: Use proteinase K for decrosslinking treatment to digest the proteins that connect the DNA fragments. The digestion temperature is 65°C and the digestion time is 2 hours. S5-2: Use the ethanol precipitation method to extract the DNA and purify the extracted DNA. S5-3: Perform quality detection on the extracted DNA. Use a NanoDrop spectrophotometer to detect the DNA concentration and remove the DNA that does not meet the concentration requirements. The concentration requirement is that the DNA concentration is between 10 - 50 ng / μL.

[0049] It should be noted that Proteinase K is an enzyme capable of digesting proteins and can effectively remove the proteins linking DNA fragments; the digestion temperature is 65 degrees Celsius and the digestion time is 2 hours, which can ensure the complete digestion of proteins while avoiding DNA degradation caused by too long a time.

[0050] The ethanol precipitation method is a commonly used DNA extraction method that can precipitate DNA through a high-concentration ethanol environment, thereby achieving DNA extraction. The ethanol concentration used is 100%, which can ensure the sufficient precipitation and purification of DNA.

[0051] The NanoDrop spectrophotometer is an instrument capable of quickly detecting the DNA concentration. It determines the concentration by measuring the absorbance of DNA at a specific wavelength; the concentration requirement is between 10 - 50 ng / μL, which can ensure that the DNA concentration in subsequent experiments is moderate, neither too high to cause difficulties in experimental operations nor too low to affect the accuracy of experimental results.

[0052] Step S6: Fragment the purified DNA, retrieve the biotin-labeled fragments, perform end repair, add A, add adapters, and construct a library by PCR amplification; Among them, in step S6, the following sub-steps are also included: S6-1: Fragment the DNA by ultrasonic waves, and the length of the fragmented DNA fragments is 200 - 500 bp; S6-2: Use streptavidin magnetic beads to retrieve the biotin-labeled DNA fragments, perform end repair and A addition treatment, and prepare for adapter ligation; S6-3: Ligate adapters to the treated fragments and construct a library by PCR amplification. The number of PCR amplification cycles is 10 cycles; S6-4: Use magnetic beads to purify the amplified library and remove the unbound primers and adapters; S6-5: Perform enzymatic digestion quality control on the purified library to detect the ligation situation of the library.

[0053] It should be noted that ultrasonic fragmentation is a commonly used DNA fragmentation method that can fragment DNA into fragments of a specific length through the high-frequency vibration of ultrasonic waves; the length of the fragmented DNA fragments is 200 - 500 bp, which can ensure that the length of DNA fragments in subsequent experiments is moderate, neither too long to cause difficulties in experimental operations nor too short to affect the accuracy of experimental results.

[0054] Streptavidin has extremely high affinity with biotin and can specifically bind to biotin-labeled DNA fragments. Using streptavidin magnetic beads to retrieve biotin-labeled DNA fragments can, in subsequent steps, through end repair and A addition treatment, make the ends of DNA fragments suitable for adapter ligation.

[0055] Adapter ligation is a crucial step in constructing a sequencing library, enabling DNA fragments to be suitable for subsequent sequencing reactions.

[0056] The number of PCR amplification cycles is 10, which can ensure the construction efficiency and quality of the library while avoiding non-specific amplification caused by too many cycles.

[0057] Magnetic bead purification is a commonly used nucleic acid purification method that can remove unbound primers and adapters through the adsorption of magnetic beads, improving the purity and quality of the library.

[0058] Enzyme digestion quality control can detect the ligation efficiency and quality of DNA fragments in the library through enzyme digestion reactions. By ensuring good ligation of the library through enzyme digestion quality control, a high-quality library can be provided for subsequent sequencing experiments.

[0059] Step S7: Quality control the constructed library, detect the library concentration and fragment size, and perform PE150 sequencing using a sequencing platform to obtain paired-end sequencing reads; Among them, in step S7, the following sub-steps are also included: S7-1: Quantify the library using a quantitative instrument to determine the library concentration. The quantitative instrument is Qubit3.0; S7-2: Detect the library fragment size using a fragment analyzer. The fragment analyzer is Agilent 5400; S7-3: Perform sequencing using the DNBSEQ T7 sequencing platform, run the PE150 sequencing strategy, and obtain 150bp paired-end sequencing reads.

[0060] It should be noted that Qubit 3.0 is a high-sensitivity fluorescence quantitative instrument that can accurately measure the DNA concentration through the intensity of fluorescence signals. The detection range of Qubit 3.0 is 10-100 ng / μL, which can ensure the accurate measurement of the library concentration.

[0061] Agilent 5400 is a high-resolution fragment analyzer that can accurately measure the size of DNA fragments through capillary electrophoresis technology. By detecting the library fragment size using Agilent 5400, it is ensured that the length distribution of the library fragments meets the expectations.

[0062] DNBSEQ T7 is a high-throughput sequencing platform that can obtain 150bp paired-end sequencing reads through the PE150 sequencing strategy. By performing sequencing using the DNBSEQ T7 sequencing platform, it is ensured that the sequencing data is of high quality and high throughput.

[0063] Step S8: Perform quality control, alignment, calculate translocation breakpoint positions on the sequencing data, and screen for structural variations at a certain resolution, and judge the detection results through the set threshold; Among them, in step S8, the following sub-steps are also included: S8-1: Use software tools to perform quality control on the paired-end sequencing data, remove low-quality reads and adapter-contaminated reads. The software tool is the filter module in fqtools_plus v3.0.0 software. The quality control standard is that the base ratio with Phred Quality Score≥20≥95%; S8-2: Use alignment software to perform single-end alignment of Reads1 and Reads2 with the human hg38 reference genome respectively to obtain single-end alignment results. The alignment software is the GLOBAL module in HiCPro V2.7 software; S8-3: Perform global alignment through the alignment software to obtain the final alignment result. The alignment software is bowtie2 v2.2.3; S8-4: Use analysis tools to input the alignment result file and calculate the translocation breakpoint positions. The analysis tool is Hi-Cbreakfinder, and the confidence interval of the translocation breakpoint positions is ±100000bp; S8-5: Screen for the identified structural variations at 1M resolution, judge the detection results through the set threshold. If the identified score is higher than the threshold, report it as the detection result. Otherwise, judge the result as a false positive and do not output the detection result.

[0064] It should be noted that the filter module in fqtools_plus v3.0.0 software is a commonly used sequencing data quality control tool, which can remove low-quality reads and adapter-contaminated reads through the set quality control standard; the quality control standard that the base ratio with Phred Quality Score≥20≥95% is to ensure the quality of the sequencing data and remove low-quality data that may affect subsequent analysis.

[0065] The GLOBAL module in HiCPro V2.7 software is an efficient alignment tool, which can align sequencing reads with the reference genome to obtain alignment results, and obtain preliminary alignment results through single-end alignment, providing a basis for subsequent global alignment.

[0066] bowtie2 v2.2.3 is an efficient global alignment tool, which can perform global alignment of sequencing reads with the reference genome to obtain the final alignment result, ensuring the accuracy and integrity of the alignment result through global alignment, and providing high-quality alignment data for subsequent structural variation analysis.

[0067] Hi-C breakfinder is a tool specifically designed for analyzing Hi-C data. It can calculate the translocation breakpoint positions through the alignment result file. The confidence interval of the translocation breakpoint positions is ±100,000 bp to ensure the accuracy of the breakpoint positions, taking into account experimental errors and biological variability.

[0068] Example 2 Purpose of the experiment detection: Use Hi-C technology to detect the balanced translocation of chromosomes 1 and 19 in human peripheral blood.

[0069] Sample information: The sample name is zhanxiaojinAF7309, and the Latin name is Homo sapiens; the restriction enzyme used is MboI.

[0070] The experimental process includes: 1. Sample preparation: Isolate lymphocytes from human peripheral blood, perform cross-linking treatment with formaldehyde solution, then add glycine solution to terminate the reaction, collect the cross-linked products and store them frozen; 2. Library construction: Lyse the cross-linked lymphocytes to release the cell nuclei, digest and digest the chromatin, and cut the genomic DNA with a restriction enzyme (MboI); biotinylate the ends of the digested DNA and perform blunt-end ligation, using T4 DNA ligase to ligate DNA fragments that are close in spatial structure; perform de-cross-linking treatment on the ligated DNA, extract and purify the DNA, and use ethanol precipitation to extract the DNA; fragment the purified DNA, retrieve the biotinylated fragments, perform end repair, add A, add adapters, and construct a library by PCR amplification; perform quality control on the constructed library to ensure that the library concentration and fragment size meet the expectations, and use a sequencing platform for PE150 sequencing to obtain paired-end sequencing reads.

[0071] 3. Data analysis: Filter the original sequencing data to remove low-quality reads and adapter-contaminated reads to obtain high-quality Clean Reads; use the GLOBAL module in HiC-Pro (V2.7.8) software to perform single-end alignment of Reads1 and Reads2 with the human hg38 reference genome respectively to initially obtain the single-end alignment results; perform global alignment using bowtie2 (v2.2.3) to obtain the final alignment results; input the alignment result file through the Hi-C breakfinder tool to initially calculate the translocation breakpoint positions; screen the identified structural variations at 1M resolution, using 80 as the threshold. If the identified score is higher than this value, it will be reported as the detection result, otherwise it is judged that the result is a false positive and this detection result will not be output.

[0072] The detection results include: a. Translocation detection results: A balanced translocation between chromosome 1 and chromosome 19 was detected, and the breakpoint positions were chr19:41100000±100000 and chr1:5500000±100000 respectively.

[0073] The chromosome connection modes are chr19:0-41100000 → chr1:5500000-0, chr19:59128983-41100000 → chr1:5500000-249250621.

[0074] The variant karyotype result is 46,XX,t(1;19)(p36.31;q13.2).

[0075] b. CNV detection results: No pathogenic or likely pathogenic copy number variations above 100Kb were detected.

[0076] Example 3 The detection purpose of the experiment: To detect unbalanced translocations and copy number variations of chromosome 9 and chromosome 18 in human peripheral blood using Hi-C technology.

[0077] The sample information includes: The sample name is heqing; the Latin name of the species is Homo sapiens; the restriction enzyme used is MboI.

[0078] The experimental procedure includes: 1. Sample preparation: Isolate lymphocytes from human peripheral blood, perform cross-linking treatment with formaldehyde solution, then add glycine solution to terminate the reaction, collect the cross-linked products and perform quick-freezing preservation.

[0079] 2. Library construction: Lyse the cross-linked lymphocytes to release the cell nuclei, digest and digest the chromatin, cut the genomic DNA with a restriction enzyme (MboI); biotin-label the ends of the digested DNA, and perform blunt-end ligation, use T4 DNA ligase to ligate DNA fragments that are spatially close; perform de-cross-linking treatment on the ligated DNA, and extract and purify the DNA, use ethanol precipitation method to extract DNA; fragment the purified DNA, retrieve the biotin-labeled fragments, perform end repair, add A, add adapters, and construct a library by PCR amplification; perform quality control on the constructed library to ensure that the library concentration and fragment size meet the expectations, and use a sequencing platform for PE150 sequencing to obtain paired-end sequencing reads.

[0080] 3. Data analysis: Filter the original sequencing data to remove low-quality reads and adapter-contaminated reads, obtaining high-quality Clean Reads; use the GLOBAL module in HiC-Pro (V2.7.8) software to perform single-end alignment of Reads1 and Reads2 with the human hg38 reference genome respectively, initially obtaining the single-end alignment results; perform global alignment using bowtie2 (v2.2.3) to obtain the final alignment results; input the alignment result file through the Hi-C breakfinder tool to initially calculate the translocation breakpoint positions; screen the identified structural variations at 1M resolution, with a threshold of 80 points. If the identified score is higher than this value, it is reported as the detection result. Otherwise, the result is judged as a false positive and not output.

[0081] The detection results include: a. Translocation detection result: Unbalanced translocation was detected between chromosomes 9 and 18, and the breakpoint positions were chr9:55000000±16000000 and chr18:17000000±170000 respectively.

[0082] The chromosome connection mode is chr9:0 - 55000000 → chr18:17000000 - 78077248.

[0083] The variant karyotype result is 46,XX,der(18)t(9;18)(p10;q10) / 46,XX.

[0084] b. CNV detection result: A chimeric duplication was detected on chromosome 9, with the variant type of dup(9)(p24.3p23), the fragment size of 9.84 Mb, and the chimeric ratio of approximately 33%. The clinical significance is unknown.

[0085] A chimeric duplication was detected on chromosome 9, with the variant type of dup(9)(p23p13.1), the fragment size of 26.14 Mb, and the chimeric ratio of approximately 28%. It is pathogenic.

[0086] A chimeric deletion was detected on chromosome 18, with the variant type of del(18)(p11.32p11.1), the fragment size of 15.32 Mb, and the chimeric ratio of approximately 39%. It is pathogenic.

[0087] The above-described embodiments are only used to illustrate the technical solutions of the present application, rather than to limit them; although the present application has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the various embodiments of the present application, and should all be included within the protection scope of the present application.

Claims

1. A method for detecting chromosomal structural variations in human peripheral blood using Hi-C technology, characterized in that, The method includes: Step S1: Isolate lymphocytes from human peripheral blood and perform cross-linking treatment using a formaldehyde solution. Step S2: After incubation at room temperature, add a glycine solution to terminate the reaction, collect the cross-linked product, and perform quick-freezing preservation. Step S3: Lyse the cross-linked lymphocytes to release the cell nuclei, digest and enzymatically cut the chromatin, and cut the genomic DNA using a restriction endonuclease. Step S4: Biotin-label the ends of the enzymatically cut DNA and perform blunt-end ligation, using T4 DNA ligase to ligate DNA fragments that are spatially close. Step S5: Perform de-cross-linking treatment on the ligated DNA, extract the DNA, and purify it using the ethanol precipitation method. Step S6: Fragment the purified DNA, retrieve the biotin-labeled fragments, perform end repair, add A, add adapters, and construct a library by PCR amplification. Step S7: Perform quality control on the constructed library to detect the library concentration and fragment size, and perform PE150 sequencing using a sequencing platform to obtain paired-end sequencing reads. Step S8: Perform quality control, alignment, calculation of translocation breakpoint positions on the sequencing data, and screen for structural variations at a certain resolution, and judge the detection results through a set threshold.

2. A method for detecting chromosomal structural variations in human peripheral blood using the Hi-C technique according to claim 1, wherein: In step S1, the following sub-steps are further included: S1-1: Isolate lymphocytes from human peripheral blood and isolate 50,000 to 100,000 lymphocytes using density gradient centrifugation. S1-2: Suspend the isolated lymphocytes in a phosphate buffer solution, add a formaldehyde solution for cross-linking treatment to cross-link the chromatin in the lymphocyte nuclei.

3. A method for detecting chromosomal structural variations in human peripheral blood using the Hi-C technique according to claim 1, wherein: In step S2, the following sub-steps are further included: S2-1: Incubate the cross-linked lymphocytes at room temperature, where the room temperature ranges from 18°C to 25°C, and the incubation time ranges from 8 to 15 minutes. S2-2: Add a glycine solution to terminate the cross-linking reaction, and the termination reaction time is 1 to 3 minutes. S2-3: Wash the lymphocytes 3 times with a phosphate buffer solution and centrifuge to ensure that there is no residual cross-linking reagent on the lymphocyte surface. The centrifugation speed is 1000 rpm, and the duration is 5 minutes. S2-4: Collect the cross-linked lymphocytes, perform quick-freezing in liquid nitrogen and store at low temperature. The low-temperature storage temperature is -70°C to -85°C.

4. A method for detecting chromosomal structural variations in human peripheral blood using the Hi-C technique according to claim 1, wherein: In step S3, the following sub-steps are further included: S3-1: Use a lysis buffer to lyse the cell membrane and release the cell nuclei. The lysis time is 10 to 20 minutes. The lysis buffer includes Tris-HCl, EDTA, and NP-40. S3-2. Add sodium dodecyl sulfate (SDS) solution to open chromatin and expose restriction sites. The SDS treatment time is 10 - 15 minutes. S3-3. Add Triton X-100 to terminate the SDS reaction and reduce interference with the restriction reaction. The Triton X-100 treatment time is 5 - 10 minutes. S3-4. Use a restriction endonuclease to cut genomic DNA. The DNA fragments after digestion have blunt ends or sticky ends, and the length distribution is between 100 - 1000 bp. The restriction endonuclease is MboI.

5. A method for detecting chromosomal structural variations in human peripheral blood using the Hi-C technique according to claim 1, characterized in that: In step S4, the following sub-steps are further included: S4-1. Use biotin-labeled base C to fill in and label the ends of the digested DNA to make the DNA fragments carry biotin. S4-2. Use T4 DNA ligase to ligate two DNA fragments that are close in spatial structure with blunt ends.

6. A method for detecting chromosomal structural variations in human peripheral blood using the Hi-C technique according to claim 1, characterized in that: In step S5, the following sub-steps are further included: S5-1. Use proteinase K for de-crosslinking treatment to digest the proteins connecting the DNA fragments. The digestion temperature is 65 °C and the digestion time is 2 hours. S5-2. Use ethanol precipitation to extract DNA and purify the extracted DNA. S5-3. Perform quality detection on the extracted DNA. Use a NanoDrop spectrophotometer to detect the DNA concentration and remove DNA that does not meet the concentration requirements. The concentration requirement is that the DNA concentration is between 10 - 50 ng / μL.

7. A method for detecting chromosomal structural variations in human peripheral blood using the Hi-C technique according to claim 1, characterized in that: In step S6, the following sub-steps are further included: S6-1. Fragment the DNA by sonication. The length of the fragmented DNA fragments is 200 - 500 bp. S6-2. Use streptavidin magnetic beads to retrieve the biotin-labeled DNA fragments, perform end repair and A-tailing treatment, and prepare for ligation of adapters. S6-3. Ligate adapters to the treated fragments and construct a library by PCR amplification. The number of cycles of the PCR amplification is 10 cycles. S6-4. Use magnetic beads to purify the amplified library and remove unbound primers and adapters. S6-5. Perform enzymatic digestion quality control on the purified library to detect the ligation situation of the library.

8. A method for detecting chromosomal structural variations in human peripheral blood using the Hi-C technique according to claim 1, characterized in that: In step S7, the following sub-steps are further included: S7-1. Use a quantitative instrument to quantify the library and determine the library concentration. The quantitative instrument is Qubit 3.

0. S7-2. Use a fragment analyzer to detect the fragment size of the library. The fragment analyzer is Agilent 5400. S7-3, Sequencing was performed using the DNBSEQ T7 sequencing platform, running the PE150 sequencing strategy to obtain 150bp paired-end sequencing reads.

9. A method for detecting chromosomal structural variations in human peripheral blood using Hi-C technology according to claim 1, characterized in that: Wherein in step S8, the following sub-steps are further included: S8-1, Using software tools to perform quality control on the paired-end sequencing data, removing low-quality reads and adapter-contaminated reads. The software tool is the filter module in fqtools_plus v3.0.0 software, and the quality control standard is that the proportion of bases with PhredQuality Score ≥ 20 ≥ 95% S8-2, Using alignment software to perform single-end alignment of Reads1 and Reads2 with the human hg38 reference genome respectively to obtain single-end alignment results. The alignment software is the GLOBAL module in HiCPro V2.7 software; S8-3, Performing global alignment through alignment software to obtain the final alignment result. The alignment software is bowtie2v2.2.3; S8-4, Using an analysis tool to input the alignment result file and calculate the translocation breakpoint position. The analysis tool is Hi-Cbreakfinder, and the confidence interval of the translocation breakpoint position is ±100000bp; S8-5, Screening the identified structural variations at 1M resolution, judging the detection result through a set threshold. If the identified score is higher than the threshold, it is reported as the detection result. Otherwise, it is judged that the result is a false positive and the detection result is not output.

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