Kit, library construction method thereof and target variation detection and analysis method

Abstract

The invention relates to the technical field of high-throughput sequencing, and discloses a kit and a library construction method and a target variation detection and analysis method thereof, the kit comprises a pre-library construction reagent and a target region liquid phase hybridization capture reagent; wherein the pre-library construction reagent comprises a repair reagent, a joint, a connection reagent, an amplification reagent and purified magnetic beads; the target area liquid phase hybridization capture reagent comprises a hybridization reagent, a cleaning reagent, an amplification reagent, a purification magnetic bead, a target gene probe and a CNV / LOH probe. The invention provides a rapid, accurate and low-cost construction method of a library containing a targeting gene probe and a CNV / LOH probe and application of the library, through one-time library construction and sequencing, the advantages of a high-throughput targeting sequencing technology and a CNV-Seq technology are integrated; multi-gene point mutation, insertion and deletion, gene copy number variation, large-fragment copy number variation and heterozygosity deletion can be detected at the same time.

Description

Technical Field

[0001] The present invention relates to the technical field of high-throughput sequencing, and in particular to a kit and a library construction method thereof, and a target variation detection and analysis method. Background Art

[0002] High-throughput sequencing technology (NGS) uses a parallel sequencing strategy to simultaneously determine the sequences of hundreds of thousands to millions of DNA molecules, thereby significantly improving the sequencing throughput. Compared with the classic Sanger sequencing method, NGS technology, with its high throughput, low cost and fast sequencing speed, makes it possible to conduct a comprehensive and in-depth analysis of the transcriptome and genome of an organism.

[0003] The key steps of high-throughput sequencing technology include: sample preparation, library construction, sequencing reaction and data analysis. The sample preparation stage involves cutting DNA or RNA samples into fragments of appropriate size; in the library construction stage, these fragments are connected by adding specific adapters and then amplified; in the sequencing reaction stage, the synthesis-by-sequencing method is used to determine the base sequence of DNA using fluorescently labeled nucleotides; finally, in the data analysis stage, the raw data is processed by applying bioinformatics tools and converted into genetic information for research. This information can be used to analyze genetic variations such as single nucleotide polymorphisms (SNPs), insertions or deletions (InDels), and copy number variations (CNVs).

[0004] Gene mutation is the core driving factor of tumor occurrence and development. Cancer driver genes can significantly affect the regulation of cell proliferation and division, thereby giving cells the potential for unlimited proliferation, as well as the ability to escape immune surveillance and promote tumor metastasis. The types of mutations in cancer driver genes include but are not limited to point mutations, insertions or deletions, increases or amplifications of gene dosage, and structural rearrangements of chromosomes. These mutations may activate proto-oncogenes or inactivate tumor suppressor genes. For example, the TP53 gene, one of the most critical genes in oncology, has mutations associated with the occurrence of more than 50 different types of cancer. In addition to mutations in single genes, copy number variations, structural variations, gene fusion events, and epigenetic modifications in the tumor genome also play a vital role in the progression of tumors. Copy number variations, such as gain of chromosome 1q and loss of heterozygosity (LOH) of 1p and 16q, have been shown to have important predictive value in the prognostic analysis of a variety of tumors.

[0005] The role of genetic testing in the field of oncology is multifaceted. It not only plays a vital role in tumor classification and prognosis prediction, but also guides the formulation of clinical treatment decisions. By deeply analyzing the genetic mutations of tumor cells, specific subtypes of tumors can be identified, providing patients with more personalized treatment options. For example, relevant literature mentions that lung cancer patients with driver gene mutations such as EGFR, ALK, and KRAS usually have a poor prognosis. In addition, the NCCN guidelines point out that neuroblastoma patients with high copy number amplification of the MYCN gene are risk graded as high risk. Patients with Wilms tumor with loss of heterozygosity (LOH) of 1p and 16q often have a poor prognosis. Therefore, it is of great significance to simultaneously detect point mutations, gene copy number variations, chromosome CNVs, and LOH in tumor gene testing. These tests can provide important information for early diagnosis, prognosis assessment, and personalized treatment strategies of tumors.

[0006] In tumor genomics research, high-throughput sequencing technology has become an important means to detect tumor gene mutations. This technology usually adopts a targeted sequencing strategy, including two main methods: liquid hybridization capture and multiplex amplification. Liquid hybridization capture technology uses specific probes to hybridize with target DNA sequences to enrich and capture regions of interest in the genome, while multiplex amplification methods amplify specific regions through designed primers. The amplification method is mainly suitable for the detection of a small number of genes due to the number of primer pairs. In contrast, liquid hybridization capture technology is not limited by the number of target genes and is suitable for targeted sequencing of large-scale genes. The panel for tumor gene detection often contains hundreds of genes. The capture probe design is mainly designed in a tiled or shingled style for the target region and the reference genome sequence, which can effectively solve various types of gene mutations such as point mutations, small insertions and deletions, and gene copy number variations. Large-fragment copy number variation (CNV) or loss of heterozygosity (LOH) has a large region, and the direct design of tiled probes has a high design cost and a high detection cost. Therefore, the existing conventional capture probe design mode is not suitable for large-fragment copy number variation or loss of heterozygosity detection.

[0007] For the detection of large-segment CNVs, Chromosomal Microarray Analysis (CMA) and CNV-seq based on high-throughput sequencing are the two main technologies. CMA is a comparative genomic hybridization technology based on a microarray platform that can detect CNVs across the entire genome, and is particularly suitable for detecting large-segment genomic variations, such as chromosome deletions and duplications. However, CMA technology has limitations in detecting point mutations or small insertions and deletions (InDels), especially for the detection of new mutations. CNV-seq uses high-throughput sequencing technology to detect copy number variations in the genome. By comparing the sequence of sample DNA with that of the reference genome, the increase or decrease in the number of DNA copies in the sample can be identified. CNV-seq technology is capable of detecting large-segment CNVs and has the potential to discover new CNV events. This technology performs low-depth sequencing of the entire genome, but it is also unable to detect other types of genetic variations besides CNVs.

[0008] Therefore, it becomes an urgent problem to effectively use a single method to simultaneously solve point mutations, small insertions and deletions, gene copy number variations, large fragment copy number variations and heterozygosity loss detection. Summary of the invention

[0009] In view of the problems in the related art, the present invention proposes a kit and a library construction method thereof and a target variation detection and analysis method to overcome the above-mentioned technical problems existing in the existing related art.

[0010] To this end, the specific technical solution adopted by the present invention is as follows: According to one aspect of the present invention, a kit is provided, including a pre-library construction reagent and a target region liquid phase hybridization capture reagent, etc.; Wherein, the pre-library construction reagents include repair reagents, adapters, ligation reagents, amplification reagents and purification magnetic beads, etc.; The target region liquid phase hybridization capture reagent includes hybridization reagent, cleaning reagent, amplification reagent, purification magnetic beads, targeted gene probe and CNV / LOH probe, etc.; The molar concentration ratio of the targeted gene probe to the CNV / LOH probe is 1:1-1000:1, preferably 1:1-10:1.

[0011] Furthermore, the specific design principles of CNV / LOH probes are as follows: New probes are designed to analyze target regions, including but not limited to regional SNP polymorphism analysis, population frequency analysis, regional GC content analysis, and regional homology analysis; SNPs are selected with high population frequency (MAF) sites, with MAF between 0.2-0.8, preferably between 0.4-0.6; Based on the above target region analysis data, continuously spaced probes were designed with probe spacing of 5-5000 kb, preferably 50-100 kb probe spacing; The probe spacing can be equidistant but is not limited to equidistant. The probe length is designed to be 50-120 nt (i.e., the length of the targeted gene probe and the CNV / LOH probe are both 50-120 nt), preferably 60-110 nt. For covering SNP sites or SV structural variations, the probes cover the variation site and both sides of it respectively; For SNP sites or SV structural variation positions, the synthetic bases of the probe include but are not limited to modified bases, with modified bases being preferred; modified bases include but are not limited to one or more of LNA modifications, non-natural bases, and merged bases, with non-natural bases being preferred.

[0012] According to another aspect of the present invention, a library construction method is provided, wherein the above-mentioned kit is used to construct a library, and the library construction method comprises the following steps: The DNA of the sample to be tested is fragmented by using an ultrasonic crusher, and the fragmented product is end-repaired by using a repair reagent; Connect the adapter to the fragmented products after terminal repair, purify the connection products, and perform PCR amplification on the connection products, and purify them to obtain a pre-library; Based on the pre-library, hybridization was performed overnight using target gene probes and CNV / LOH probes mixed in a preset ratio; The hybridization products were captured and cleaned with streptavidin magnetic beads, and PCR enrichment amplification was performed based on the captured products to obtain the final library.

[0013] Further, the size of the target fragment after fragmentation treatment is 200bp-300bp. The adapter used in the process of connecting the adapter to the fragmented product after end repair is a universal adapter, and the purification includes purification using magnetic beads. The mixing ratio of the target gene probe and the CNV / LOH probe is 5:1.

[0014] According to another aspect of the present invention, a method for detecting and analyzing a target variation is provided, which is implemented by using a library constructed by the above-mentioned library construction method, wherein the target variation type includes but is not limited to one or more of point mutation, insertion and deletion (InDel), gene copy number variation, large fragment copy number variation (CNV) and loss of heterozygosity (LOH); the detection method includes but is not limited to a new probe design method for specific large fragment copy number variation or loss of heterozygosity; and the same set of probes is used for CNV / LOH detection in the detection method; The target variation detection analysis method comprises the following steps: The hybridized library is sequenced on a machine, and low-quality data and adapter sequences in the sequencing data are filtered out; The filtered data are aligned to the hg19 reference genome using software (e.g., software BWA-mem, v0.7.17) to mark repeated sequences; point mutations and insertions and deletions (InDels) in the target region are detected using analysis software (e.g., software GATK4.0 or VarDict, v1.8.2); Based on the normalized sequencing depth ratio of the target sample and the control sample in the target region, the copy number variation (CNV) was analyzed, and the heterozygosity loss and start and end positions were analyzed according to the distribution of SNP site variation frequency values ​​(VAF) in the region.

[0015] Furthermore, the normalized sequencing depth ratio of the target sample to the control sample in the target region includes: Calculate the average sequencing depth of the target region of the sample and divide it by the average sequencing depth of the entire autosomal region of the sample, and normalize the regional sequencing depth of all tested samples to eliminate the impact of the amount of sequencing data on the analysis; Calculate the ratio of the normalized sequencing depth of the target region of the sample to be tested to the normalized sequencing depth of the control sample (or reference set) in the target region; When the sequencing depth ratio is greater than 1.3, it is judged as a copy number duplication, and when the sequencing depth ratio is less than 0.7, it is judged as a copy number deletion.

[0016] Specifically, the target sample can select a fixed multi-sample reference set without copy number variation, or a dynamic multi-sample reference set sequenced in the same batch; tumor sample analysis can adopt the method of synchronous analysis of the target sample and its own normal sample as the control sample.

[0017] Furthermore, the analysis of heterozygosity loss and start and end positions according to the distribution of SNP site variation frequency (VAF) values ​​in the region includes: Select all SNV sites in the target region with a sequencing depth greater than 50X and a quality value greater than 30, and calculate the distance between the site VAF and 0.5, that is, calculate the absolute value of VAF-0.5. In theory, the distance value of the heterozygous site in a normal sample is 0, and the distance value of the homozygous site is 0.5; The chromosome is divided into 1Mb bins in a sliding window manner, with each sliding window step of 20Kb. The mean and standard deviation of the VAF of all SNV sites in each bin are calculated, and the score value is calculated based on the ratio of the standard deviation to the mean. When the average value is greater than 0.1 and the score value is less than 0.25, the diploid region is determined to be a loss of heterozygosity (LOH) region. It should be noted that the threshold can be adjusted according to the test results of multiple samples; Determine the first bin and the last bin of the region where the loss of heterozygosity appears continuously, and calculate and determine the start and end positions of the loss of heterozygosity (LOH) based on the determination results; In the first bin and the last bin, the mean, standard deviation and score value of VAF of 10 loci were calculated in a sliding window manner, and the loss of heterozygosity (LOH) region was determined according to the threshold; Based on the heterozygous loss region, the first site of the first bin was determined as the starting position of the heterozygous loss, and the last site of the last bin was determined as the ending position of the heterozygous loss.

[0018] The beneficial effects of the present invention are: 1) The present invention provides a fast, accurate and low-cost library construction method comprising targeted gene probes and CNV / LOH probes and its application. Through one-time library construction and sequencing, it combines the advantages of high-throughput targeted sequencing technology and CNV-Seq technology, and can simultaneously detect multi-gene point mutations, insertions and deletions (InDel), gene copy number variation, large fragment copy number variation (CNV) and loss of heterozygosity (LOH).

[0019] 2) The present invention uses a mixture of targeted gene probes and CNV / LOH probes during liquid hybridization capture to simultaneously capture high-depth gene regions and low-depth CNV / LOH analysis regions, and can simultaneously analyze point mutations, insertions and deletions (InDel), gene copy number variations, as well as large-fragment copy number variations (CNV) and loss of heterozygosity (LOH) in gene regions. The present invention provides a new method for determining loss of heterozygosity (LOH), which can identify the region and start and end positions where LOH occurs by calculation, rather than by manually determining the VAF distribution map. While maintaining low costs, the present invention covers a wider genomic region and can efficiently, sensitively and accurately detect a variety of genetic information including single gene variations, large-fragment copy number variations (CNV) and loss of heterozygosity (LOH). BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be 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.

[0021] Figure 1Schematic diagram of the distribution and dosage of targeted gene probes and CNV / LOH probes according to an embodiment of the present invention; Figure 2 This is a graph showing the copy number deletion detection result of the chromosome region of the PT24020204 sample according to an embodiment of the present invention; Figure 3 is a VAF scatter plot of the PT24082108 sample according to an embodiment of the present invention; Figure 4 is a VAF scatter plot of chromosome 3 of sample PT24082108 according to an embodiment of the present invention; Figure 5 is a scatter plot of the VAF and 0.5 distance value of chromosome 3 of the PT24082108 sample according to an embodiment of the present invention; Figure 6 is a scatter plot of VAF mean calculated by bin region for chromosome 3 of sample PT24082108 according to an embodiment of the present invention; Figure 7 It is a scatter plot of score values ​​calculated by bin region for chromosome 3 of sample PT24082108 according to an embodiment of the present invention. DETAILED DESCRIPTION

[0022] To further illustrate each embodiment, the present invention provides drawings, which are part of the disclosure of the present invention and are mainly used to illustrate the embodiments. They can be used in conjunction with the relevant descriptions in the specification to explain the operating principles of the embodiments. With reference to these contents, ordinary technicians in this field should be able to understand other possible implementation methods and advantages of the present invention.

[0023] 1. Prelibrary construction. The specific steps of prelibrary construction are briefly described as follows: 1.1 DNA fragmentation: Take 200 ng of DNA from each sample to be tested and use Covaris LE220 non-contact ultrasonic crusher for fragmentation. The expected target fragment size is 200 bp-300 bp. The ultrasonic parameters are: Peak Power 450 W, Duty Factor 20%, Cycles / Burst 1000 cycles, Time 80 s.

[0024] 1.2. DNA end repair and adding "A (i.e. A base)": 1.2.1) Add 50 μL of fragmentation product and 15 μL of repair reagent into a 0.2 mL PCR tube.

[0025] 1.2.2) Use a pipette to gently pipette to mix, and centrifuge briefly to collect the reaction solution at the bottom of the tube.

[0026] 1.2.3) Place the PCR tube in a thermal cycler and run the following program (heat cover at 105°C): 20°C, 15 min; 65°C, 15 min; 4°C, hold.

[0027] 1.3. Connector connection and purification: 1.3.1) Take out Rapid Ligation Buffer, Rapid DNA Ligase and Universal Adapter, thaw and mix on ice, centrifuge briefly to collect to the bottom of the tube, and place on ice for later use; take out VAHTS DNA Clean Beads and place them at room temperature for at least 30 minutes.

[0028] 1.3.2) After the reaction in step 1.2.3) is finished, take out the 0.2 mL PCR tube and place it on ice for later use.

[0029] 1.3.3) Take a new 1.5mL EP tube and mark it as "ligation mix", add 25μL DNA ligation buffer and 5μL DNA ligase, prepare the ligation pre-set solution, and flick until completely mixed (about 10S).

[0030] 1.3.4) Add 5 μL of universal adapter to the 0.2 mL PCR tube in step 1.2.3), vortex to mix, centrifuge briefly to collect the liquid at the bottom of the tube, and place on ice.

[0031] 1.3.5) Add 30 μL of ligation pre-setting solution to the PCR tube in step 1.3.4), flick the tube to mix thoroughly (about 10 seconds), centrifuge to collect the liquid at the bottom of the tube, and place on ice.

[0032] 1.3.6) Place the PCR tube in a thermal cycler and run the following program (heat cover at 105°C): 20°C, 15 min; 4°C, hold.

[0033] 1.3.7) After the program is completed, centrifuge the PCR tube briefly to collect the adapter ligation products at the bottom of the tube.

[0034] 1.3.8) Vortex the purified magnetic beads that have been equilibrated to room temperature to mix them thoroughly.

[0035] 1.3.9) Pipette 60 μL of magnetic beads into 100 μL of adapter ligation product and mix by gently pipetting.

[0036] 1.3.10) Incubate at room temperature for 5 min.

[0037] 1.3.11) Briefly centrifuge the PCR tube and place it on a magnetic rack to separate the beads and liquid (approximately 3 minutes). After the solution is clear, carefully remove the supernatant, avoiding contact with the beads.

[0038] 1.3.12) Keep the PCR tube on the magnetic rack, add 200 μL of freshly prepared 80% ethanol to rinse the magnetic beads, incubate at room temperature for 30 seconds, and carefully remove the supernatant.

[0039] 1.3.13) Repeat step 1.3.12) for a total of two rinses.

[0040] 1.3.14) Keep the PCR tube on the magnetic rack at all times, open the lid and let it dry at room temperature until it becomes matte (about 5-10 minutes).

[0041] 1.3.15) Remove the PCR tube from the magnetic rack, add 22.5 μL Nuclease-free water, vortex at medium speed to fully suspend the magnetic beads (about 20 seconds), and leave at room temperature for 5 minutes.

[0042] 1.3.16) Centrifuge the PCR tube briefly and place it on a magnetic stand. Once the solution is clear, carefully pipette 20 μL of the supernatant into a new labeled 0.2 mL PCR tube without touching the magnetic beads.

[0043] 1.4. Library amplification and sorting: 1.4.1) Take out the PCR primers and PCR enrichment amplification mix, thaw on ice and mix, centrifuge briefly to collect the liquid to the bottom of the tube, and place on ice for later use; take out the VAHTS DNA Clean Beads and place them at room temperature for at least 30 minutes.

[0044] 1.4.2) Take the PCR primers with corresponding numbers, sort and check the sequence, and add 2.5 μL of PCR primers to the 0.2 mL PCR tubes in step 1.3.16).

[0045] 1.4.3) Add 25 μL of PCR enrichment and amplification mix to each of the above tubes, pipette gently to mix 10 times, cover the tube tightly and flick to mix thoroughly, centrifuge briefly to collect the reaction solution at the bottom of the tube.

[0046] 1.4.4) Place the PCR tube in a PCR instrument and run the following program (heat cover at 105°C): 95°C, 3 min; 98°C, 20 sec; 60°C, 15 sec; 72°C, 30 sec; 72°C, 5 min; 4°C, hold. PCR amplification for 7 cycles.

[0047] 1.4.5) After the PCR reaction is completed, add 50 μL of Nuclease-free water to the amplified product to make up the volume to 100 μL. Vortex and mix the purified magnetic beads that have been equilibrated to room temperature to fully suspend them.

[0048] 1.4.6) Accurately pipette 55 μL of magnetic beads into 100 μL of amplified product and mix by gently pipetting.

[0049] 1.4.7) Incubate at room temperature for 5 min.

[0050] 1.4.8) Briefly centrifuge the PCR tube and place it on a magnetic rack to separate the magnetic beads and liquid. After the solution is clear, carefully transfer the supernatant (about 166μL) to a new PCR tube and discard the magnetic beads.

[0051] 1.4.9) Pipette 20 μL of magnetic beads into the supernatant and mix by gently pipetting.

[0052] 1.4.10) Incubate at room temperature for 5 min.

[0053] 1.4.11) Briefly centrifuge the PCR tube and place it on a magnetic rack to separate the magnetic beads and liquid. After the solution is clear (about 3 minutes), carefully remove the supernatant and avoid contact with the magnetic beads.

[0054] 1.4.12) Keep the PCR tube on the magnetic rack, add 200 μL of freshly prepared 80% ethanol to rinse the magnetic beads, incubate at room temperature for 30 seconds, and carefully remove the supernatant.

[0055] 1.4.13) Repeat step 1.4.12) for a total of two rinses.

[0056] 1.4.14) Keep the PCR tube on the magnetic rack at all times, open the lid and air-dry the magnetic beads for 5-10 minutes until no ethanol remains.

[0057] 1.4.15) Remove the PCR tube from the magnetic rack, add 22.5 μL Nuclease-free water, mix gently by pipetting, and leave at room temperature for 5 minutes.

[0058] 1.4.16) Centrifuge the PCR tube briefly and place it on a magnetic rack. After the solution is clear, carefully pipette 20 μL of the supernatant into a new EP tube without touching the magnetic beads.

[0059] 1.4.17) Use Qubit to measure the concentration of the supernatant in step 1.4.16), i.e. the concentration of the pre-library.

[0060] 2. Liquid phase hybridization capture. The specific steps of liquid phase hybridization capture are briefly described as follows: 2.1. Hybridization system configuration: 2.1.1) Take out Hyb Human Block, Blocking Oligo (for ILM) and RNaseBlock from -20℃, place them on ice to dissolve, mix thoroughly and place on ice or at 4℃ for later use.

[0061] 2.1.2) 500 ng of each pre-library was added, and the corresponding volume of library was added to the bottom of the 0.2 ml EP tube according to the concentration of the pre-library (note that the total amount of pooling library in each tube should not exceed 6000 ng).

[0062] 2.1.3) Mark the liquid level of each tube and place it in a vacuum concentrator, making sure the opening is facing the center. Concentrate at 45°C under vacuum until no liquid remains (approximately 30 minutes).

[0063] 2.1.4) Add 2.6 µL Nuclease-free water to the bottom of the tube in step 2.1.3) and place in the EP tube rack for later use.

[0064] 2.1.5) Prepare hybridization mixture by adding 13μL Hyb Buffer, 5μL Hyb Human Block, 2μL Blocking Oligo, 5μL RNase Block, 2μL targeted gene probe, and 0.4μL CNV / LOH probe.

[0065] 2.1.6) Add 27.4ul of hybridization mixture from step 2.1.5) to the EP tube from step 2.1.4) and mix by pipetting 3 times. Centrifuge and place on ice for later use.

[0066] 2.2. Probe hybridization: 2.2.1) Turn on the PCR instrument and run the hybridization program. The PCR instrument parameters are as follows (heat cover 85℃): 80℃, 5min; 50℃, overnight (16h-24h).

[0067] 2.2.2) Quickly place the tube from step 2.1.6) into the PCR instrument, make sure the tube cap is tightly closed, and start the program immediately.

[0068] 2.3. Preparation of capture magnetic beads: 2.3.1) Remove the capture beads from 4°C, equilibrate at room temperature for at least 30 minutes, and vortex at medium speed (speed 6) until the capture beads are fully resuspended (about 1 minute).

[0069] 2.3.2) Take a new 1.5 mL centrifuge tube, label the tube cap, dispense 50 µL of captured magnetic beads to the bottom of the tube, place it on a magnetic rack and let it stand for 1 min. Wait for the solution to become clear and discard the supernatant.

[0070] 2.3.3) Add 180µL Binding Buffer to each tube and vortex at medium speed (speed 6) to fully resuspend the magnetic beads. Collect the liquid at the bottom of the tube by centrifugation, place on a magnetic stand until it becomes clear (about 30S), and discard the supernatant.

[0071] 2.3.4) Repeat step 2.3.3) twice, washing the magnetic beads a total of 3 times.

[0072] 2.3.5) Remove the 1.5mL centrifuge tubes from the magnetic rack, add 180µL Binding Buffer to each tube, vortex at medium speed (speed 6) to fully resuspend the magnetic beads, and place at room temperature for later use.

[0073] 2.4. Post-hybridization cleaning: 2.4.1) Keep the hybridization tube on the PCR instrument, take an appropriate amount (about 100µL) of the resuspended capture beads in step 2.3.5) and add it to the corresponding numbered hybridization tube, pipetting and mixing 6 times. Then transfer all the liquid from the hybridization tube back to the corresponding 1.5mL centrifuge tube, cover the tube tightly, place it on the LCD digital control rotary mixer, set the speed to 10r / min, and incubate at room temperature for 30min.

[0074] 2.4.2) Take 1.5mL centrifuge tubes and dispense 1mL of Wash Buffer into each tube. Place the tubes on a constant temperature oscillating mixer and incubate at 53℃ for later use. Invert and mix for 5 minutes before use. Set the oscillation frequency to 600r / min (if the metal bath temperature control is accurate, set it to 50℃).

[0075] 2.4.3) Remove the 1.5 mL centrifuge tube from the LCD digital control rotary mixer, collect the liquid at the bottom of the tube by centrifugation, and place it on a magnetic rack until it becomes clear (about 30 seconds), and carefully discard the supernatant.

[0076] 2.4.4) Add 150 µL of Wash Buffer 1, vortex at low speed (speed 4), and incubate at room temperature for 15 min on an LCD digital rotary mixer.

[0077] 2.4.5) Remove the 1.5 mL centrifuge tube from the LCD digital control rotary mixer, collect the liquid at the bottom of the tube, and place it on a magnetic rack until it becomes clear (about 30 seconds). Carefully aspirate and discard the supernatant.

[0078] 2.4.6) Place the above centrifuge tube on a constant temperature oscillating mixer and immediately start the Wash Buffer step.

[0079] 2.4.7) Take 150µL of the 53℃ preheated Wash Buffer from step 2.4.2) into the 1.5mL centrifuge tubes in the above step, and mix by gently pipetting 20 times. Immediately place on a constant temperature oscillating mixer and incubate at 53℃ for 10 minutes, maintaining the oscillation frequency at 600r / min.

[0080] 2.4.8) Remove the 1.5 mL centrifuge tube from the constant temperature oscillating mixer and collect the liquid at the bottom of the tube. Place it on a magnetic rack until it becomes clear (about 30 seconds) and carefully discard the supernatant.

[0081] 2.4.9) Repeat steps 2.4.7)-2.4.8) three times, for a total of 4 washes. Completely remove the Wash Buffer for the last wash.

[0082] 2.4.10) Quickly centrifuge and collect the liquid to the bottom of the tube. Place the 1.5mL centrifuge tube on the magnetic rack and use a 10µL pipette to remove as much residual supernatant as possible, being careful not to touch the magnetic beads.

[0083] 2.4.11) Keep the 1.5mL centrifuge tube on the magnetic stand and add 400µL 80% ethanol to the opposite side of the magnetic beads. Let it stand for 30 seconds and then completely remove the ethanol solution.

[0084] 2.4.12) Centrifuge the tube to collect the liquid to the bottom, place the 1.5mL centrifuge tube on the magnetic stand, and use a 10µL pipette to remove the remaining supernatant as much as possible, being careful not to touch the magnetic beads. Dry at room temperature until matte.

[0085] 2.4.13) Add 24 µL Nuclease-free water to the 1.5 mL centrifuge tube, remove the 1.5 mL centrifuge tube from the magnetic rack, gently tap the tube several times to resuspend the magnetic beads adsorbed on the tube wall, and place it at room temperature for later use.

[0086] 2.5. PCR enrichment reaction: 2.5.1) Preparation before the experiment: 2.5.1.1) Take out the PCR enrichment amplification mix and PCR universal primer (25μM, for ILM) reagents from the kit stored at -20℃ in advance, place them on an ice box to dissolve, and place them on an ice box for later use after dissolving and mixing.

[0087] 2.5.1.2) Take a new 0.2mL PCR tube, label the tube cap, add 25µL PCR enrichment amplification mix and 1µL PCR universal primer (25μM, for ILM) to prepare the PCR enrichment reaction system.

[0088] 2.5.1.3) Quickly collect the liquid to the bottom of the tube.

[0089] 2.5.1.4) Add all the resuspended magnetic beads from step 2.4.13) to the tube by pipetting and mixing, and gently pipette and mix 6 times to ensure that all the liquid is at the bottom of the tube and no bubbles remain. Then immediately place it on the PCR instrument.

[0090] 2.5.2) PCR enrichment amplification experiment: 2.5.2.1) Run the PCR program (heat cover 105℃): 95℃, 1min; 98℃, 20sec; 60℃, 30sec; 72℃, 30sec; 72℃, 5min; 4℃, hold. PCR amplification for 10 cycles.

[0091] 2.5.2.2) Remove the purified magnetic beads from 4°C and equilibrate them at room temperature for at least 30 minutes.

[0092] 2.5.2.3) After the PCR is completed, remove the reaction system from the PCR instrument and centrifuge to collect the liquid at the bottom of the tube.

[0093] 2.5.2.4) Vortex the purified magnetic beads for 1 min.

[0094] 2.5.2.5) Take a new 1.5mL centrifuge tube, label the cap, and add 55µL of purified magnetic beads to each centrifuge tube.

[0095] 2.5.2.6) Mix the PCR product by pipetting and transfer to the above centrifuge tube, and vortex at medium speed to mix.

[0096] 2.5.2.7) Incubate at room temperature for 10 minutes, place the centrifuge tube on a magnetic rack and let it stand until the solution clarifies (about 3 minutes), then discard the supernatant.

[0097] 2.5.2.8) Keep the centrifuge tube on the magnetic stand, add 400µL 80% ethanol to the opposite side of the magnetic beads, let it stand for 30 seconds, and discard the supernatant.

[0098] 2.5.2.9) Repeat step 2.5.2.8) once, for a total of 2 washes.

[0099] 2.5.2.10) Collect the liquid at the bottom of the tube by centrifugation, place the centrifuge tube on the magnetic rack, use a 10µL pipette to remove the remaining supernatant as much as possible, being careful not to touch the magnetic beads, and let it dry at room temperature until it becomes matte (about 10 minutes).

[0100] 2.5.2.11) Add 25 µL of Nuclease-free water, remove the centrifuge tube from the magnetic stand, vortex at medium speed to fully resuspend the magnetic beads, and incubate at room temperature for 10 minutes.

[0101] 2.5.2.12) Collect the liquid at the bottom of the tube by centrifugation, place the centrifuge tube on a magnetic rack and let it stand for 2 minutes until the solution becomes clear.

[0102] 2.5.2.13) Take a new 1.5ml centrifuge tube, label it, and use a pipette to transfer 23µL of the supernatant to the corresponding numbered 1.5ml centrifuge tube.

[0103] 2.5.2.14) Take 1µL of library and use Qubit dsDNA HS Assay Kit for quantification and record the library concentration. The library concentration is required to be between 5-40ng / µL.

[0104] 2.5.2.15) Take another 1µL library and use QIAxcel Advanced System for fragment analysis. The library length is required to be between 315bp and 450bp.

[0105] 3. Sequencing: The hybridized library was sequenced using the BGI T7 sequencing platform. The sequencing mode was PE150. The specific operations were performed according to the instructions for use of the T7 sequencer.

[0106] 4. Sequencing data analysis: Sequencing data were quality controlled and filtered, including adapter sequence filtering, low-quality data filtering, etc. The filtered data were aligned to the hg19 reference genome using the software BWA-mem (v0.7.17) to mark repeated sequences. The software VarDict (v1.8.2) was used to detect point mutations and indels in the target region.

[0107] The results of sequencing data quality control are shown in Table 1. The average depth of the targeted gene probe capture region is 2300X~4400X, and the corresponding average depth of the CNV / LOH probe capture region is 250X~450X. The ratio between the two is (8~10): 1. The average sequencing depth fluctuates due to the difference in the amount of sequencing data of a single sample, but the ratio of the average depth of the targeted gene probe capture region to the CNV / LOH probe capture region tends to be stable. This medium-to-low depth sequencing strategy for the CNV / LOH probe capture region saves a lot of data and reduces sequencing costs.

[0108] Table 1 Sequencing data quality control results:

[0109] The results of chromosome region copy number variation are shown in Tables 2 and Figure 2 The results showed that there were copy number deletions in multiple chromosome segments of the whole genome of the PT24020204 sample, such as a 42.69Mb copy number deletion in the p24.3p12 region of chromosome 9, with a detected copy number of 1.12, and a 16.72Mb copy number deletion in the p13.3p11.2 region of chromosome 17, with a detected copy number of 1.14.

[0110] Table 2 Copy number deletion detection results

[0111] The results of the loss of heterozygosity analysis showed that there was a loss of heterozygosity (LOH) region of approximately 41.5 Mb and 103.3 Mb in the 840260-42334157 region and the 80507771-183852861 region of chromosome 3 of sample PT24082108, respectively. The VAF distribution scatter plot is attached. Figure 3 and attached Figure 4 , the numerical scatter plot of the calculation process is shown in Figure 5 , Figure 6 , Figure 7 Some calculation results are shown in Tables 3 and 4.

[0112] Table 3 Partial locus information of chromosome 3 of sample PT24082108 for LOH analysis: ; ; ; ;

[0113] Table 4 Calculation results of chromosome 3 of sample PT24082108 by bin sliding window: ;

[0114] In summary, in this embodiment, the sequencing library is obtained by using targeted gene probes and CNV / LOH probes together for liquid phase hybridization capture, which combines the advantages of both common targeted sequencing and CNV-Seq (low-depth whole genome sequencing) strategies, and can simultaneously detect gene variations (point mutations, insertions and deletions, copy number variations) and chromosome region variations (copy number variations, heterozygosity loss). One experiment replaces the previous two experiments, which is more convenient to operate and less costly.

[0115] The targeted gene probes cover all exon regions of the target gene; the CNV / LOH probes are evenly distributed in the regions outside the target gene on the genome at intervals of 50Kb~1000Kb, and the probe coverage area includes SNP sites with a population frequency of 0.4~0.6.

[0116] The concentration of targeted gene probes is higher than that of CNV / LOH probes. The depth of the target gene region in the sequencing results is about 10 times that of the CNV / LOH probe region. The targeted gene region is sequenced at a high depth for accurate analysis of point mutations, insertions and deletions, and gene copy number variations. The CNV / LOH probe capture region is sequenced at a medium to low depth to reduce the amount of sequencing data and reduce costs. This area is mainly used for the analysis of large-fragment CNV / LOH.

[0117] The loss of heterozygosity (LOH) calculation method can easily and accurately determine the region where the LOH variation occurs, and determine the location of the LOH variation by quantitative means rather than manually observing the VAF distribution map.

[0118] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A kit, characterized in that: It includes pre-library construction reagents and target region liquid phase hybridization capture reagents; Wherein, the pre-library construction reagents include repair reagents, adapters, ligation reagents, amplification reagents and purification magnetic beads; The target region liquid phase hybridization capture reagent includes a hybridization reagent, a cleaning reagent, an amplification reagent, purification magnetic beads, a targeted gene probe and a CNV / LOH probe.

2. A kit according to claim 1, characterized in that The molar concentration ratio of the targeted gene probe to the CNV / LOH probe is 1:1-1000:1, and the lengths of the targeted gene probe and the CNV / LOH probe are both 50-120 nt.

3. A method for constructing a library, using the kit described in any one of claims 1-2 to construct a library, characterized in that: The library construction method comprises the following steps: The DNA of the sample to be tested is fragmented by using an ultrasonic crusher, and the fragmented product is end-repaired by using a repair reagent; Connect the adapter to the fragmented products after terminal repair, purify the connection products, and perform PCR amplification on the connection products, and purify them to obtain a pre-library; Based on the pre-library, hybridization was performed overnight using target gene probes and CNV / LOH probes mixed in a preset ratio; The hybridization products were captured and cleaned with streptavidin magnetic beads, and PCR enrichment amplification was performed based on the captured products to obtain the final library.

4. A library construction method according to claim 3, characterized in that, The target fragment size after fragmentation is 200bp-300bp.

5. A library construction method according to claim 3, characterized in that, The adapter used in the process of connecting the fragmented products after end repair to the adapter is a universal adapter, and the purification includes purification using magnetic beads.

6. A library construction method according to claim 3, characterized in that, The mixing ratio of the target gene probe to the CNV / LOH probe is 5:

1.

7. A library construction method according to claim 3, characterized in that, The CNV / LOH probes are uniformly and continuously designed at intervals of 5 Kb to 5000 Kb; the probe coverage area includes SNP sites with a population frequency of 0.2 to 0.8; To cover SNP sites or SV structural variations, the probes cover the variation site and both sides of it respectively; For the SNP site or SV structural variation position, the probe synthetic bases include modified bases, and the modified bases include any one or more of LNA modification, non-natural bases, and degenerate bases.

8. A method for detecting and analyzing target variation, implemented by using a library constructed by the library construction method described in any one of claims 3 to 7, characterized in that: The target variation detection analysis method comprises the following steps: The hybridized library is sequenced on a machine, and low-quality data and adapter sequences in the sequencing data are filtered out; The filtered sequencing data were aligned to the hg19 reference genome and the repeated sequences were marked; point mutations and indels in the target region were detected using analysis software; Analyze copy number variation based on the normalized sequencing depth ratio of target samples and control samples in the target region; The loss of heterozygosity and the start and end positions were analyzed based on the distribution of SNP site variation frequency values ​​in the region.

9. A target variation detection and analysis method according to claim 8, characterized in that: The normalized sequencing depth ratio based on the target sample and the control sample in the target region includes: The average sequencing depth of the target region of the sample was calculated and divided by the average sequencing depth of the entire autosomal region of the sample to normalize the regional sequencing depth of all tested samples; Calculate the ratio of the normalized sequencing depth of the target region of the sample to be tested to the normalized sequencing depth of the target region of the control sample; When the sequencing depth ratio is greater than 1.3, it is judged as a copy number duplication, and when the sequencing depth ratio is less than 0.7, it is judged as a copy number deletion.

10. A target variation detection and analysis method according to claim 8, characterized in that: The analysis of heterozygosity loss and start and end positions according to the distribution of SNP site variation frequency values ​​in the region includes: Select all SNV sites with a sequencing depth greater than 50X and a quality value greater than 30 in the target region, and calculate the distance value between the site VAF and 0.5; The chromosome is divided into 1Mb bins in a sliding window manner, and the bins are shifted by 20Kb each time. The mean and standard deviation of the VAF of all SNV sites in each bin are calculated, and the score value is calculated based on the ratio of the standard deviation to the mean. When the average value is greater than 0.1 and the Score value is less than 0.25, the diploid region is determined to be a heterozygous loss region; Determine the first bin and the last bin of the region where the heterozygous loss occurs continuously, and calculate and determine the start and end positions of the heterozygous loss based on the determination results; In the first bin and the last bin, the mean, standard deviation and score value of VAF of 10 loci were calculated in a sliding window manner, and the heterozygous loss region was determined according to the threshold; Based on the heterozygous loss region, the first site of the first bin was determined as the starting position of the heterozygous loss, and the last site of the last bin was determined as the ending position of the heterozygous loss.

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