WO2023179766A1 - 制备dna文库和检测逆转录病毒整合位点的方法 - Google Patents

制备dna文库和检测逆转录病毒整合位点的方法 Download PDF

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WO2023179766A1
WO2023179766A1 PCT/CN2023/083705 CN2023083705W WO2023179766A1 WO 2023179766 A1 WO2023179766 A1 WO 2023179766A1 CN 2023083705 W CN2023083705 W CN 2023083705W WO 2023179766 A1 WO2023179766 A1 WO 2023179766A1
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pcr
round
sequence
cells
primers
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刘进
曹乾升
陈庆庆
张敏
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Nanjing Legend Biotechnology Co Ltd
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Nanjing Legend Biotechnology Co Ltd
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Priority to CN202380028831.7A priority Critical patent/CN118891374A/zh
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Priority to EP23774011.3A priority patent/EP4502177A4/en
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    • C12N2740/10011—Retroviridae
    • C12N2740/13011—Gammaretrovirus, e.g. murine leukeamia virus
    • C12N2740/13041—Use of virus, viral particle or viral elements as a vector
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    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the present disclosure belongs to the field of molecular biology, especially the field of gene analysis and detection technology. Specifically, it relates to a method for preparing a DNA library and a method for detecting retrovirus integration sites. In particular, it relates to a method for the absolute quantitative detection of integration sites of retroviruses (eg, lentiviruses).
  • retroviruses eg, lentiviruses
  • Gene and cell therapy is an effective means to treat genetic diseases and other malignant diseases, such as CD19 and BCMA CAR-T, which have been approved for clinical treatment of hematological tumors.
  • CAR-T products on the market around the world use viral vector transduction.
  • Novartis’ Kymriah is developed and produced based on lentiviral vectors
  • Gilead’s Kite’s Yescarta uses a gamma-retroviral vector transduction system. .
  • the Retroviridae family is divided into seven genera: Alpharetrovirus, Betaretrovirus, Gammaretrovirus, Deltaretrovirus, and Epsilon retrovirus.
  • the genus Epsilonretrovirus, the genus Lentivirus, and the genus Spumavirus are divided into 5 groups, including bovine lentivirus, equine lentivirus, feline lentivirus, sheep lentivirus, and human lentivirus.
  • Virome. HIV's position in the "big family" of viruses is the human immunodeficiency virus group in the genus Lentivirus of the family Retroviridae.
  • Lentiviruses are a subset of retroviruses and are one of the most common and useful virus types used in research. Lentiviruses can transduce both dividing and non-dividing cells without generating an overt immune response. These viruses can also stably integrate into the host genome, enabling long-term transgene expression. There are some safety considerations that need to be evaluated when using lentiviruses: These viruses are HIV-1 based and may require additional laboratory biosafety procedures.
  • this virus may inactivate tumor suppressor genes or activate proto-oncogene expression, leading to the risk of cancer (Hacein-Bey-Abina S et al., science, 2003, 302(5644) :415-419.; Six E et al., Molecular Therapy.50 HAMPSHIRE ST, FLOOR 5, CAMBRIDGE, MA 02139 USA: CELL PRESS, 2017, 25(5): 347-348; Bluebird bio reports second quarter financial results and provides operational update. News release. bluebirdbio. August 9, 2021).
  • Gamma-retroviruses are RNA viruses that consist of their genome and several structural and enzymatic proteins, including reverse transcriptase and integrase. Once inside the target cell, the virus uses two reverse transcriptase enzymes to produce a DNA provirus. This provirus is then integrated into the host's genome via the accompanying integrase protein.
  • gamma-retroviruses When scientists discuss retroviruses, they are usually referring to a subset of retroviruses called gamma-retroviruses. Gamma-retroviruses can encapsulate relatively large amounts of DNA (up to 8 kb), and infection results in long-term transgene expression. Some disadvantages of gamma-retroviruses are that they can only transduce dividing cells (this is because they can only enter the nucleus during mitotic rupture of the nuclear envelope). In addition, gamma-retroviruses integrate randomly into the host's genome, which can lead to tumorigenesis (called insertional mutations).
  • the detection technology of viral integration sites can also be used to track the clonal evolution of immune-engineered cells after cell therapy.
  • CAR-T-specific TCR (CAR-T therapy) detection methods to track the proportion of CAR-T cells
  • the detection method of viral integration sites has: 1) There is no need to identify and identify before serial time point tracking. Specific TCR sequence that distinguishes CAR-T and endogenous T cells; 2) No need to sort CAR-T in the sample; 3) No need for fresh blood/tissue samples; 4) Low cost, short experimental cycle, etc. .
  • Probe method which has low sensitivity and is difficult to implement in samples with low integration ratios
  • LAM Linear amplification-mediated
  • MGS Modified Genome Sequencing
  • Amplicon method this method is a method that combines non-restrictive endonuclease cleavage LAM-PCR and anchored PCR (Anchored multiplex PCR), which can be used to detect lentivirus integration sites.
  • the inventors After in-depth research and creative work, the inventors obtained a method for preparing a DNA library and the prepared DNA library, and further obtained a method for detecting retrovirus integration sites. The inventors surprisingly found that the detection method disclosed in the present disclosure has extremely high sensitivity and good application potential. The following invention is thereby provided:
  • One aspect of the present disclosure relates to a method of preparing a DNA library, comprising the steps of:
  • the linker is an asymmetric double-stranded linker including a long chain sequence and a short chain sequence, wherein the long chain sequence includes a fixed sequence, a random UMI sequence and an amplification primer binding sequence from the 5' end to the 3' end,
  • the short chain sequence includes a sequence complementary to the fixed sequence
  • step 4) Perform a second round of PCR on the first round PCR product using primers for the LTR sequence, primers for the internal reference gene sequence, and primers for the adapter sequence that are different from those in step 3) to obtain the second round PCR product;
  • adding the internal reference gene to the reaction system will allow the integration site to form a sufficient amount of DNA even when the content/frequency in the sample is particularly low (for example, 1% or less) to stabilize the reaction program.
  • the inventor found that when PCR is used to amplify and enrich integration sites, when the content/frequency of integration sites in the sample is particularly low, if the internal reference gene is not introduced, under a fixed number of PCR cycles, Insufficient DNA is not available for purification and as a template for the next step of PCR, which will make the detection step impossible. In this case, there are usually two solutions: 1.
  • the sequencing reads of the internal reference gene can be used as the standard for downsampling (downsampling, randomly extracting and reducing data) for subsequent bioinformatics analysis to ensure that the sequencing depth of each integration site in each experiment is as consistent as possible.
  • downsampling data based on a certain amount of internal reference genes
  • the sequencing depth can be kept as consistent as possible regardless of the copy number of the integration site, so that a cutoff value can be stabilized in each experiment (for example, only when ⁇ 3 reads are detected, the sequence is determined) for real results) to filter out background noise.
  • the internal reference gene of the present disclosure can be used as a calibration value to calibrate the copy number concentration (copy number/ ⁇ g gDNA) of each integration site in the sample of the present disclosure, thereby achieving absolute quantification of each integration site.
  • the preparation method wherein the retrovirus-infected cells are engineered immune cells
  • the engineered immune cells contain chimeric antigen receptors; preferably, the The engineered immune cells are selected from cytotoxic T cells, helper T cells, natural killer T cells, ⁇ T cells, NK cells, macrophages, B cells, antigen-presenting cells, dendritic cells and stem cell-induced immune cells.
  • the length of the DNA fragment is 500-1500bp, 800-1200bp, 900-1100bp or 1000bp.
  • the preparation method wherein in step 2), the length of the fixed sequence is 14-30 bases; and/or,
  • the length of the random UMI sequence is 5-12 bases, where each base is independently A, G, C or T; preferably, the length of the random UMI sequence is 5-9 bases.
  • the random UMI sequence is in the form (N)x, where N independently represents base A, G, C or T, and x is a natural number from 5 to 12 or 5 to 9, such as 5, 6, 7, 8, 9, 10, 11 or 12.
  • the preparation method wherein in step 2), the 5' end of the short chain sequence has a modification that prevents primer extension; preferably an inverted dT modification or a dideoxycytidine modification .
  • the preparation method wherein in step 4):
  • the primers of the LTR sequence in the second round of PCR are located downstream of the primers of the LTR sequence in the first round of PCR,
  • the primers of the internal reference gene sequence in the second round of PCR are located at the end of the internal reference gene sequence in the first round of PCR. downstream of the primer, and
  • the primers of the linker sequence in the second round of PCR cover the primers of the linker sequence in the first round of PCR and extend downstream.
  • the total number of cycles of the first round of PCR, the second round of PCR and the third round of PCR is 36-63, preferably 36-45 or 38-42.
  • the preparation method wherein the cycle number ratio of the first round of PCR and the second round of PCR is (1-3):2, such as 1:2, 3: 2. 1:1 or 3:4;
  • the cycle numbers of the first round of PCR and the second round of PCR are 30 and 20, 15 and 20, 10 and 20, or 15 and 15 respectively.
  • the preparation method wherein the number of cycles of the third round of PCR is 6-13, such as 6, 10 or 13;
  • the cycle numbers of the first round of PCR, the second round of PCR and the third round of PCR are 15, 20 and 6; 10, 20 and 6; or 15, 15 and 6 respectively.
  • the preparation method wherein:
  • the cycle number of the first round of PCR is 15-30, 15-20 or 15-18;
  • Cycle number for the second round of PCR is 15-20 or 18-22;
  • the cycle number of the third round of PCR is 6-10 or 6-8.
  • the preparation method wherein:
  • the step 3) also includes the following steps: screening to obtain the first round of PCR products with a fragment length less than 1000 bp;
  • the step 4) also includes the following steps: screening to obtain a second round of PCR products with a fragment length less than 1000 bp; and/or
  • the step 5) also includes the following step: screening to obtain the third round of PCR products with a fragment length less than 1000 bp.
  • the first round PCR product, the second round PCR product and/or the third round PCR product with a fragment length less than 1000 bp are obtained through magnetic bead screening.
  • the preparation method wherein the internal reference gene is a house-keeping gene, such as gene ATCB, gene GAPDH or gene ApoB.
  • the preparation method wherein the retrovirus is a lentivirus or a gamma-retrovirus.
  • DNA library which is prepared by the preparation method described in any one of the present disclosure; preferably, the DNA library is a sequencing library; more preferably, the DNA library is used for detecting reversal Sequencing library recording viral integration sites.
  • Yet another aspect of the present disclosure relates to a method for detecting retroviral integration sites, which includes the steps of sequencing the DNA library of the present disclosure and obtaining sequencing data;
  • the sequencing is second generation sequencing.
  • Yet another aspect of the disclosure relates to a method for detecting retroviral integration sites, including the method for preparing a DNA library according to any one of the disclosure, and further comprising the following steps:
  • the sequencing is second generation sequencing.
  • the detection method further includes the following steps:
  • the present disclosure sets the cutoff value to filter ultra-low frequency fragments, and adjusts the cutoff value to make the detection results after background removal as consistent as possible with the theoretical value/value verified by other methods such as ddPCR. Effectively reduce false positive fragments caused by PCR or sequencing errors; downsample sequencing data to keep the sequencing depth of internal reference genes and integration site fragments consistent; apply UMI deduplication to reduce result deviations caused by different fragment amplification efficiencies. .
  • the detection method is a quantitative detection method, preferably an absolute quantitative detection method.
  • the detection method wherein the retrovirus is a lentivirus or a gamma-retrovirus.
  • the flow of the detection method is shown in Figure 1.
  • TA is connected to a linker of known sequence
  • primers that specifically bind to the LTR sequence/ApoB (internal reference gene) sequence and primers that specifically bind to the adapter sequence are used. The primers perform the first round of amplification of the ligation product to enrich the sequence containing the LTR/ApoB fragment in the interrupted sequence;
  • the reagents and raw materials used in this disclosure are all commercially available.
  • Yet another aspect of the disclosure relates to a method for preparing engineered immune cells, which includes the step of detecting the engineered immune cells according to any of the detection methods described in the disclosure.
  • Some embodiments of the present disclosure also include identifying the integration sites and/or integration frequencies of retroviruses (such as lentiviruses or gamma-retroviruses), analyzing the characteristics of genome changes, and evaluating the safety associated with lentivirus integration sites. , and assess potential risks. After evaluation, the probability of abnormal cell behavior (for example, causing key gene mutations or activating proto-oncogenes, thereby increasing the risk of malignant tumors) is low or non-existent. Engineered immune cells that meet relevant safety standards can be released for follow-up. Expanded culture, preservation or combination with pharmaceutically acceptable carriers to obtain pharmaceutical compositions, etc.
  • retroviruses such as lentiviruses or gamma-retroviruses
  • the method for preparing engineered immune cells wherein the engineered immune cells are obtained by a method comprising the following steps:
  • step (3) Introduce the recombinant retroviral vector obtained in step (2) into immune cells.
  • methods of making engineered immune cells may include transfecting or transducing immune cells isolated from an individual such that the immune cells express the desired CAR or CARs.
  • Methods of preparing engineered immune cells for immunotherapy are described, for example, in WO2014/130635, WO2013/176916 and WO2013/176915, which patents are incorporated herein by reference.
  • Individual steps that can be used to prepare engineered immune cells are disclosed, for example, in WO2014/039523, WO2014/184741, WO2014/191128, WO2014/184744 and WO2014/184143, which patents are incorporated herein by reference.
  • a method for preparing engineered immune cells is provided.
  • the engineered immune cells are obtained by isolating immune cells, preparing a retroviral vector encoding CAR (Chimeric Antigen Receptor), and introducing the vector into immune cells.
  • engineered immune cells that meet the expected requirements are obtained.
  • the method of preparing engineered immune cells wherein the engineered immune cells are selected from the group consisting of cytotoxic T cells, helper T cells, natural killer T cells, ⁇ T cells, NK cells, Immune cells induced by macrophages, B cells, antigen-presenting cells, dendritic cells, and stem cells.
  • the engineered immune cells are CAR-T cells.
  • This disclosure also relates to any one or more of the following items 1 to 13:
  • An absolute quantitative detection method for lentivirus integration sites characterized in that it includes the following steps:
  • the adapter is an asymmetric double-stranded adapter including a long chain sequence and a short chain sequence, wherein the long chain sequence is from the 5' end to the 3' end.
  • the end includes a fixed sequence, a random UMI sequence and an amplification primer binding sequence in sequence, and the short chain sequence includes a sequence complementary to the fixed sequence;
  • the length of the UMI sequence is a sequence NNNNNN of 5-9 bases, and the N represents the base A, G, C or T.
  • the absolute quantitative detection method as described in item 1 or 2 characterized in that, in the second round of PCR The primer that binds the LTR sequence/internal reference gene sequence is located downstream of the primer that binds the LTR sequence/internal reference gene sequence in the first round of PCR, and the primer that binds the adapter sequence in the second round of PCR covers the binding adapter in the first round of PCR. sequence primer and extend downstream.
  • the cycle numbers of the first round of PCR and the second round of PCR are 30 and 20, or 15 and 20, or 10 and 20, or 15 and 15 respectively.
  • the times of the first round of PCR, the second round of PCR and the third round of PCR are 15, 20 and 6 respectively.
  • the internal reference gene is selected from the group consisting of gene ATCB, gene GAPDH and gene ApoB.
  • a method for preparing engineered immune cells characterized in that the preparation method includes performing an absolute quantitative detection method of lentiviral integration sites on engineered immune cells as described in any one of items 1 to 10 A step of.
  • step (3) Introduce the vector obtained in step (2) into immune cells.
  • the programmed immune cells are selected from cytotoxic T cells, helper T cells, natural killer T cells, ⁇ T cells and NK cells.
  • the sensitivity of the disclosed method has been verified to reach a single site copy number of 17 copies/ ⁇ g gDNA (as shown in Table 13). It is the first integrated site detection method with clear sensitivity, and its sensitivity can be truly applied to the detection of clinical samples;
  • the disclosed method reduces the number of amplification cycles as much as possible, thereby effectively reducing the ratio of soft-clip and the base deviation caused by amplification;
  • the method in this disclosure sets up an internal reference gene, which can be used to calibrate the actual amount of DNA in the library.
  • the absolute quantification of each integration site can be achieved (in a certain amount of DNA per microgram of DNA). How many copies of each integration site are there), instead of just getting the ratio between each integration site, and at the same time, making the product of each library construction within a certain controllable range, thereby stabilizing the operating steps (PCR cycles) in the library construction process parameter settings) to avoid repeated database creation;
  • the method in this disclosure is equipped with UMI deduplication, which can effectively reduce fragment amplification errors and amplification efficiency deviations of different fragments caused during the PCR process;
  • This disclosure improves the adapter sequence and modifies the end of the short chain to block amplification (inverted dT), greatly reducing the spontaneous amplification products of the first round of adapter primers;
  • This disclosure only uses ultrasonic interruption, PCR and magnetic bead purification methods, which not only avoids the preference caused by enzyme digestion, but also does not use streptavidin-labeled magnetic beads to pull out biotin-labeled target fragments. , compared with nrLAM PCR and other steps, the steps are simple and the cost is low.
  • This disclosure has broad application prospects and can be applied to the detection of lentiviral integration sites in all biological samples that can effectively extract DNA, including but not limited to various cell therapy products modified with lentivirus and patient clinical trials. Testing of cell samples. For example, as a safety quality control standard, it can be used in the QC release of CAR-T products; by testing patient clinical cell samples, the dynamic changes of CAR-T cells at different integration sites can be monitored in real time, and can be used to evaluate cell therapy products. efficacy and safety, etc.
  • Figure 1 Experimental flow chart of the example.
  • FIG. 1 Example bioinformatics analysis flow chart.
  • FIG. 3A Electrophoresis pattern exported by tapestation 4150.
  • FIG. 3B Electrophoresis pattern exported by tapestation 4150.
  • Figure 3A and Figure 3B show that using adapters with inverted dT results in better fragment enrichment than adapters without inverted dT.
  • Figure 4 Softclip ratio of library construction and sequencing results with different cycle numbers, showing the impact of different cycle number settings of three rounds of PCR on the soft-clip ratio of the final library.
  • Figure 5 A linear correlation dot plot drawn according to Table 13, showing the correlation between the detection results of the disclosed method and the qPCR method.
  • Figure 6 A dot plot of the number of identical initial molecules corresponding to the number of detected reads drawn based on the sequencing results, showing the distribution of the number of reads after library construction and amplification of the initial molecules before filtering.
  • Figure 7 Box plot of the number of detected reads corresponding to the initial molecules, showing the distribution of the number of reads after the initial molecules were amplified after library construction.
  • This disclosed test cell line gDNA for detecting lentivirus integration sites comes from a CAR-T positive single-copy cell line independently prepared and preserved by our company. The positive rate is close to 100%. It has been verified by qPCR and ddPCR, -20 degrees (°C )save. CAR-T cells themselves are the source of genomic DNA, so this disclosure is not limited to the above-mentioned cell lines and is applicable to any cells from which DNA can be obtained.
  • DNA used in the present disclosure is derived from CART cells, but the method is not limited to DNA derived from CART cells.
  • DNA can also be the international standard WHO 1st Reference Reagent 2019 for lentivirals vector integration site analysis, purchased from the following address:
  • test cell line gDNA for detecting retrovirus integration sites comes from two monoclonal cell lines (cell lines 1 and 2) independently prepared and preserved by our company and isolated after transfection with gamma-retrovirus. test certificate.
  • the present disclosure is not limited to the above-mentioned cell lines and is applicable to any cell from which DNA can be obtained.
  • Example 5 First round of PCR (taking lentivirus as an example)
  • the primer information used in this step is shown in Table 16 below.
  • Example 6 Second round of PCR (taking lentivirus as an example)
  • the primer information used in this step is shown in Table 16 below.
  • Example 8 Bioinformatics analysis process (taking lentivirus as an example)
  • the reference sequence is GRCh38.
  • the original bam file is further filtered to remove soft clip base numbers greater than 15, alignment quality less than 20, and read length less than 50 and reads containing hard clips, then count the number of unique umi reads based on UMI and the sequence of reads, and calculate the median value after alignment and deduplication, and remove reads with the number of umi reads less than 1% of the median value as background.
  • umi_tools to deduplicate UMI on both ends of the cleaned bam file, set the alignment quality to greater than 20, discard chimeric-pairs and unpaired-reads, and then use flag to extract all reverse sequencing reads.
  • Reverse dT modification on the short linker chain can avoid or reduce non-specific amplification in PCR reactions.
  • the first round of PCR is performed, and then the second round of PCR is performed with 0.8 ⁇ magnetic beads for purification.
  • the results are shown in Figure 3A and Figure 3B. The results show that the proportion of adapter target fragments ( ⁇ 1000bp) with inverted dT is higher than that without inverted dT.
  • the internal reference gene hApoB primer set When measuring the lentivirus integration site, the internal reference gene hApoB primer set is introduced.
  • the input amount of fragments after fragmentation can be known through the internal reference gene, so as to achieve absolute quantification of the copy number of each integration site and determine the sensitivity of the method.
  • Bioinformatics analysis is further optimized, and background noise can be effectively filtered by setting the cutoff value.
  • the method of the present disclosure can be used to detect integration sites with a copy number of a single site in a sample greater than or equal to 17 copies/ ⁇ g gDNA, and can maintain good linearity above 107 copies/ ⁇ g gDNA (Table 14). Therefore, it can be truly applied in the tracking and detection of clinical samples of genetically modified cell therapy based on lentiviral transfection technology.
  • the inventors changed the first and second rounds of primers to specific primers for the LTR of gamma-retroviruses (such as MSCV/MoMLV) and proved that This method is also applicable to the detection of integration sites of gamma-retrovirus (or vector).
  • the first and second rounds of primers were changed to specific primers for the LTR of ⁇ -retrovirus (Rvbd_F_1_3 and Rvbd_F_2_3 in Table 20), and the two strains were transfected with retrovirus
  • the monoclonal cell lines (cell lines 1 and 2) isolated later were constructed and analyzed by RIS to obtain the integration site information.
  • Specific primer/probe combinations were set based on the integration site information and ddPCR was performed for verification. The results are as follows:
  • the integration of viral/non-viral vectors for genome integration based on fixed integration sequences such as long terminal repeats (LTR) of retroviruses and inverted terminal repeats (ITRs) of adeno-associated viruses) Site, specific primers are designed through this fixed integration sequence, and the integration sites of various viral/non-viral vectors can be detected by cooperating with the method of the present disclosure.
  • fixed integration sequences such as long terminal repeats (LTR) of retroviruses and inverted terminal repeats (ITRs) of adeno-associated viruses

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Abstract

本公开属于分子生物学领域特别是基因分析检测技术领域，具体地，涉及一种制备DNA文库的方法和检测逆转录病毒整合位点的方法。具体地，所述制备DNA文库的方法，包括如下步骤：1)将被逆转录病毒感染的细胞的基因组DNA片段化，得到DNA片段；2)将DNA片段的末端修复加并连接上接头得到连接产物；其中，所述接头为包括长链序列和短链序列的不对称双链接头，其中所述长链序列自5'端至3'端依次包含固定序列、随机UMI序列以及扩增引物结合序列，所述短链序列包含与所述固定序列互补的序列；以及其它步骤。本公开的检测方法具有极高的灵敏度，具有良好的应用潜力。

Description

制备DNA文库和检测逆转录病毒整合位点的方法
相关申请的交叉引用
本申请要求2022年03月24日提交的中国专利申请号为202210303736.0的优先权权益，该专利申请的内容以引用方式整体并入本文。
技术领域
本公开属于分子生物学领域特别是基因分析检测技术领域，具体地，涉及一种制备DNA文库的方法和检测逆转录病毒整合位点的方法。特别地，涉及一种绝对定量检测逆转录病毒(例如慢病毒)整合位点的方法。
背景技术
基因和细胞治疗是一种治疗遗传病和其它恶性疾病的有效手段，如CD19和BCMA CAR-T已批准用于在血液肿瘤的临床治疗中。目前全球已上市的CAR-T产品均采用病毒载体转导的方式，例如诺华的Kymriah是基于慢病毒载体开发生产的，而吉利德旗下Kite的Yescarta则采用了γ-逆转录病毒载体转导系统。
按照目前国际病毒分类标准，逆转录病毒科分为7个属：α逆转录病毒属Alpharetrovirus、β逆转录病毒属Betaretrovirus、γ反转录病毒属Gammaretrovirus、δ反转录病毒属Deltaretrovirus、ε反转录病毒属Epsilonretrovirus、慢病毒属Lentivirus、泡沫病毒属Spumavirus、其中的慢病毒属又分为5个组，包括牛慢病毒组、马慢病毒组、猫慢病毒组、羊慢病毒组和人类慢病毒组。HIV在病毒“大家族”中的定位是逆转录病毒科慢病毒属中的人类免疫缺陷病毒组。
慢病毒是逆转录病毒的一个子集，是研究中最常见和最有用的病毒类型之一。慢病毒可以转导分裂细胞和非分裂细胞，而不产生明显的免疫反应。这些病毒还能稳定地整合到宿主基因组中，从而实现长期的转基因表达。在使用慢病毒时，有一些安全考虑需要评估:这些病毒是基于HIV-1的，可能需要额外的实验室生物安全程序。此外，由于这种病毒随机地整合到宿主基因组中，可能会失活抑癌基因或激活原癌基因表达，导致癌变的风险(Hacein-Bey-Abina S等人，science,2003,302(5644):415-419.；Six E等人，Molecular Therapy.50 HAMPSHIRE ST,FLOOR 5,CAMBRIDGE,MA 02139 USA:CELL PRESS,2017,25(5):347-348；Bluebird bio reports second quarter  financial results and provides operational update.News release.bluebirdbio.August 9,2021)。
γ-逆转录病毒是一种RNA病毒，由其基因组和几种结构蛋白和酶蛋白组成，包括逆转录酶和整合酶。一旦进入目标细胞，病毒就会利用两种逆转录酶产生DNA原病毒。这个原病毒然后通过伴随的整合酶蛋白整合到宿主的基因组中。
当科学家讨论逆转录病毒时，他们通常指的是逆转录病毒的一个子集，称为γ-逆转录病毒。γ-逆转录病毒可以包裹相对大量的DNA(高达8kb)，感染导致长期转基因表达。γ-逆转录病毒的一些缺点是它们只能转导正在分裂的细胞(这是因为它们只能在核膜有丝分裂破裂时进入细胞核)。此外，γ-逆转录病毒随机整合到宿主的基因组中，这可能导致肿瘤发生(称为插入突变)。
在临床前研究及临床应用中，评估病毒整合位点及相关的安全性是必须要进行的一项内容。如美国FDA和欧洲EMA均要求在基因和细胞治疗中检测病毒整合位点和整合频率，以明确该疗法的安全性(O'Leary M C等人，Clinical Cancer Research,2019,25(4):1142-1146；U.S.Department of Health and Human Services.Long Term Follow-Up After Administration of Human Gene Therapy Products；Guidance for Industry.Jan 30,2020.；European medicines agency.Guideline on the quality,non-clinical and clinical aspects of gene therapy medicinal products.22 March,2018.)。国内《基因修饰细胞治疗产品非临床研究技术指导原则(试行)》(2021年11月)也明确整合/插入位点风险评估为临床前安全性评价的一部分。
另外，病毒的整合位点的检测技术也能应用于细胞治疗后免疫工程细胞的克隆性演变的跟踪。相较于用CAR-T特异性的TCR(CAR-T疗法)的检测方法对CAR-T细胞的比例进行跟踪，病毒整合位点的检测方法拥有：1)无需在系列时间点跟踪之前鉴定和区分CAR-T和内源性T细胞的特异性TCR序列；2)无需对样本中的CAR-T进行分选；3)无需新鲜的血液/组织样本；4)成本低，实验周期短等优点。
目前病毒整合位点的检测方法有以下几种：1)探针法，该方法灵敏度比较低，很难在低整合比例的样本中实施；2)线性扩增法(Linear amplification-mediated(LAM)-PCR)，现有的病毒整合位点分析多基于该方法；3)Modified Genome Sequencing(MGS)-PCR方法，与LAM-PCR相比，该方法的流程相对更加简易；4)扩增子法，该方法是一种非限制性内切酶切割的LAM-PCR与锚定PCR(Anchored multiplex PCR)相结合的方法，可用于慢病毒整合位点的检测。
但上述方法都存在一些缺陷，例如灵敏度较低，准确性较差。此外，上述方法都无法实现对各整合位点的绝对定量，在整体拷贝数未知的情况下无法评估方法的灵敏度。因此，尚需要开发新的针对逆转录病毒整合位点的检测方法。
发明内容
本发明人经过深入的研究和创造性的劳动，得到了一种制备DNA文库的方法和制得的DNA文库，并进一步得到了一种检测逆转录病毒整合位点的方法。本发明人惊奇地发现，本公开的检测方法具有极高的灵敏度，具有良好的应用潜力。由此提供了下述发明：
本公开的一个方面涉及一种制备DNA文库的方法，包括如下步骤：
1)将被逆转录病毒感染的细胞的基因组DNA片段化(例如超声打断)，得到DNA片段；
2)将DNA片段的末端修复加并连接上接头，得到连接产物；
其中，所述接头为包括长链序列和短链序列的不对称双链接头，其中所述长链序列自5’端至3’端依次包含固定序列、随机UMI序列以及扩增引物结合序列，所述短链序列包含与所述固定序列互补的序列；
3)用LTR序列的引物、内参基因序列的引物和接头序列的引物对连接产物进行第一轮PCR，得到第一轮PCR产物；
4)用与步骤3)中不同的LTR序列的引物、内参基因序列的引物和接头序列的引物对第一轮PCR产物进行第二轮PCR，得到第二轮PCR产物；
5)用带有测序接头的引物对第二轮PCR产物进行第三轮PCR，得到第三轮PCR产物，为DNA文库。
不拘于理论的限制，加入内参基因的反应体系会使整合位点在样本中的含量/频率特别低的情况下(例如1％或者更低)也能形成足量的DNA，以稳定的反应程序完成检测。本发明人发现，应用PCR对整合位点进行扩增和富集时，在整合位点在样本中的含量/频率特别低的情况下，如果不引入内参基因，则在固定的PCR循环数下得不到足量的DNA用以纯化和作为模板进行下一步PCR，这将使检测步骤无法进行下去。在这种情况下，通常有两种解决方法：1.为了得到足够量的DNA以高循环数去进行扩增，但这会导致非特异性扩增增加，影响最终文库的有效数据及质量；2.用其它 方法预测整合位点总共的频率，灵活调整循环数以完成建库，但此时每一批数据的建库程序不一致，导致文库质量不一致，下游数据分析无法制定稳定的背景过滤阈值。
不拘于理论的限制，内参基因的测序read可以作为后续生物信息学分析的downsample(下采样，随机抽取缩小数据)的标准，以尽量保证每一次实验每个整合位点测序的深度保持一致。按照一定量的内参基因去downsample数据时，无论整合位点拷贝数为多少都可以尽量保持测序深度一致，如此在每一次实验中都可以稳定一个cutoff值(比如检测到大于等于3条reads才判定为真实结果)去过滤背景噪音。如果没有内参基因，因为每个样本中整合位点的频率不一致，按照一定的测序数据量去downsample时，不同样本实际每个整合位点的测序深度不一致，这会导致背景噪音的大小在不同样本里不一样，进而无法形成一个稳定的cutoff值去去除背景噪音。
此外，本公开的内参基因可以作为校准值校准本公开样本中每一种整合位点的在样本中的拷贝数浓度(拷贝数/μg gDNA)，进而实现对各整合位点的绝对定量。
在本公开的一些实施方式中，所述的制备方法，其中，所述的逆转录病毒感染的细胞为工程化免疫细胞，所述工程化免疫细胞包含嵌合抗原受体；优选地，所述工程化免疫细胞选自细胞毒性T细胞、辅助T细胞、自然杀伤T细胞、γδT细胞、NK细胞、巨噬细胞、B细胞、抗原呈递细胞、树突状细胞和干细胞诱导的免疫细胞。
在本公开的一些实施方式中，所述的制备方法，其中，步骤1)中，所述DNA片段的长度为500-1500bp、800-1200bp、900-1100bp或1000bp。
在本公开的一些实施方式中，所述的制备方法，其中，步骤2)中，所述固定序列的长度为14-30个碱基；和/或，
所述随机UMI序列的长度为5-12个碱基，其中各碱基独立地为A、G、C或T；优选地，所述随机UMI序列的长度为5-9个碱基。
所述随机UMI序列形式上为(N)x，所述N独立地代表碱基A、G、C或T，x为5-12或5-9的自然数，例如5、6、7、8、9、10、11或12。
在本公开的一些实施方式中，所述的制备方法，其中，步骤2)中，所述短链序列的5’端带有阻止引物延伸的修饰；优选为inverted dT修饰或者双脱氧胞苷修饰。
在本公开的一些实施方式中，所述的制备方法，其中，步骤4)中：
所述第二轮PCR中的LTR序列的引物位于第一轮PCR中的LTR序列的引物的下游，
所述第二轮PCR中的内参基因序列的引物位于第一轮PCR中的内参基因序列的 引物的下游，和
所述第二轮PCR中的接头序列的引物覆盖第一轮PCR中的接头序列的引物并延伸至下游。
在本公开的一些实施方式中，所述的制备方法，其中，第一轮PCR、第二轮PCR和第三轮PCR的循环数合计为36-63，优选为36-45或38-42。
在本公开的一些实施方式中，所述的制备方法，其中，所述第一轮PCR和所述第二轮PCR的循环数比值为(1-3):2，例如1:2、3:2、1:1或者3:4；
优选地，所述第一轮PCR和所述第二轮PCR的循环数分别为30和20、15和20、10和20、或者15和15。
在本公开的一些实施方式中，所述的制备方法，其中，所述第三轮PCR的循环数为6-13，例如6、10或者13；
优选地，所述第一轮PCR、第二轮PCR和第三轮PCR的循环数分别为15、20和6；10、20和6；或者15、15和6。
在本公开的一些实施方式中，所述的制备方法，其中：
第一轮PCR的循环数为15-30、15-20或15-18；
第二轮PCR的循环数为15-20或18-22；和/或
第三轮PCR的循环数为6-10或6-8。
在本公开的一些实施方式中，所述的制备方法，其中：
所述步骤3)中还包含如下步骤：筛选得到片段长度小于1000bp的第一轮PCR产物；
所述步骤4)中还包含如下步骤：筛选得到片段长度小于1000bp的第二轮PCR产物；和/或
所述步骤5)中还包含如下步骤：筛选得到片段长度小于1000bp的第三轮PCR产物。
在本公开的一些实施方式中，所述的制备方法，其中，通过磁珠筛选得到片段长度小于1000bp的第一轮PCR产物、第二轮PCR产物和/或第三轮PCR产物。
在本公开的一些实施方式中，所述的制备方法，其中，所述内参基因是看家基因(house-keeping gene)，例如基因ATCB、基因GAPDH或基因ApoB。
在本公开的一些实施方式中，所述的制备方法，其中，所述逆转录病毒为慢病毒或γ-逆转录病毒。
本公开的另一方面涉及一种DNA文库，其由本公开中任一项所述的制备方法制得；优选地，所述DNA文库为测序文库；更优选，所述DNA文库为用于检测逆转录病毒整合位点的测序文库。
本公开的再一方面涉及一种检测逆转录病毒整合位点的方法，包括对本公开的DNA文库进行测序以及得到测序数据的步骤；
优选地，所述测序为第二代测序。
本公开的再一方面涉及一种检测逆转录病毒整合位点的方法，包括本公开中任一项所述的制备DNA文库的方法，并且还包括如下步骤：
6)对得到的DNA文库进行测序，得到测序数据；
优选地，所述测序为第二代测序。
在本公开的一些实施方式中，所述的检测方法，还包括如下步骤：
7)对测序数据进行生物信息学分析，得到整合位点的数据。
在本公开的一些实施方式中，生物信息学分析的流程如图2所示。
在生物信息学分析流程中，本公开设置cutoff值过滤超低频率片段，调整cutoff值使去背景以后的检测结果与理论值/经ddPCR等其它方法验证的值尽量一致。有效减少因PCR或者测序错误导致的假阳性片段；对测序数据进行下采样(downsample)使得内参基因及整合位点片段的测序深度保持一致；应用UMI去重减少不同片段扩增效率导致的结果偏差。
在本公开的一些实施方式中，所述的检测方法，其为定量检测方法，优选为绝对定量检测方法。
在本公开的一些实施方式中，所述的检测方法，其中，所述逆转录病毒为慢病毒或γ-逆转录病毒。
在本公开的一些实施方式中，检测方法的流程如图1所示。
在本公开一个具体的实施方案中，绝对定量检测逆转录病毒整合位点的方法的大致流程如下：
1.使用超声打断仪将基因组DNA打断为1000bp左右的小片段；
2.末端修复加A后，TA连接上已知序列的接头；
3.用特异性结合LTR序列/ApoB(内参基因)序列的引物和特异性结合接头序列的 引物对连接产物进行第一轮扩增，富集打断序列中含有LTR/ApoB片段的序列；
4.磁珠筛选1000bp以下的产物片段，用另外一组特异性结合LTR序列/ApoB(内参基因)序列的引物和特异性结合接头序列的引物对第一轮扩增产物进行第二轮PCR(巢氏扩增)，以进一步富集打断序列中含有LTR/ApoB片段的序列；
5.磁珠筛选1000bp以下的产物片段，用带有测序接头片段的引物对第二轮PCR产物进行第三轮PCR扩增，加上测序接头；
6.磁珠筛选1000bp以下的产物片段，应用Illumina测序仪对筛选后的产物进行二代测序。
在符合本领域常识的基础上，上述各优选条件，可任意组合，即得本公开各实例。
本公开所用试剂和原料均市售可得。
本公开的再一方面涉及一种制备工程化免疫细胞的方法，包括对工程化免疫细胞按照本公开中任一项所述的检测方法进行检测的步骤。
本公开一些实施方案中，还包括明确逆转录病毒(例如慢病毒或γ-逆转录病毒)的整合位点和/或整合频率，分析基因组改变的特征，评估慢病毒整合位点相关的安全性，并评估潜在风险。经评估，发生异常细胞行为(例如：导致关键基因突变或者激活原癌基因，进而导致发生恶性肿瘤的风险增加)的概率较低或没有，符合相关安全标准的工程化免疫细胞可以放行，进行后续的扩大培养、保存或与药学上可接受的载剂组合以获得药物组合物等。
在本公开的一些实施方式中，所述的制备工程化免疫细胞的方法，其中，所述工程化免疫细胞通过包含如下步骤的方法获得：
(1)分离免疫细胞；
(2)构建包含编码嵌合抗原受体的核酸的重组逆转录病毒载体；
(3)将步骤(2)所得的重组逆转录病毒载体导入免疫细胞。
具体地，制备工程化免疫细胞的方法可包括转染或转导自个体分离的免疫细胞以使得所述免疫细胞表达所需的一种或多种CAR。制备用于免疫疗法的工程化免疫细胞的方法描述于例如WO2014/130635、WO2013/176916和WO2013/176915中，这些专利以引用的方式并入本文中。可用于制备工程化免疫细胞的个别步骤公开于例如WO2014/039523、WO2014/184741、WO2014/191128、WO2014/184744和WO2014/184143中，这些专利以引用的方式并入本文中。
例如，提供了一种工程化免疫细胞的制备方法，通过分离免疫细胞，制备编码CAR(Chimeric Antigen Receptor)的逆转录病毒载体，将该载体导入免疫细胞等步骤获得该工程化免疫细胞。对获得的工程化免疫细胞进行本公开中任意一项逆转录病毒整合位点的绝对定量检测的步骤。从而评估所制备的工程免疫细胞是否达到预期效果，没有潜在的安全风险。最终获得符合预期要求的工程化免疫细胞。
在本公开的一些实施方式中，所述的制备工程化免疫细胞的方法，其中，所述工程化免疫细胞选自细胞毒性T细胞、辅助T细胞、自然杀伤T细胞、γδT细胞、NK细胞、巨噬细胞、B细胞、抗原呈递细胞、树突状细胞和干细胞诱导的免疫细胞。
在本公开一具体实施方案中，所述工程化免疫细胞为CAR-T细胞。
本公开还涉及选自如下的1至13项中的任意一项或者几项：
1.一种慢病毒整合位点的绝对定量检测方法，其特征在于，其包括如下步骤：
1)将来自慢病毒感染细胞中的基因组DNA片段化；
2)将片段化DNA的末端修复加并连接上接头得到连接产物；所述接头为包括长链序列和短链序列的不对称双链接头，其中所述长链序列自5’端至3’端依次包含固定序列、随机UMI序列以及扩增引物结合序列，所述短链序列包含与所述固定序列互补的序列；
3)用特异性结合LTR序列/内参基因序列的引物和特异性结合接头序列的引物对连接产物进行第一轮PCR扩增，富集打断序列中含有LTR/内参基因片段的序列；
4)用另外一组特异性结合LTR序列/内参基因序列的引物和特异性结合接头序列的引物对第一轮PCR产物进行第二轮PCR扩增，以进一步富集打断序列中含有LTR/内参基因片段的序列；
5)用带有测序接头片段的引物对第二轮PCR产物进行第三轮PCR扩增，加上测序接头；
6)对筛选后的产物进行测序。
2.如第1项所述的绝对定量检测方法，其特征在于，所述固定序列的长度为14-30个碱基；
和/或，所述UMI序列的长度为5-9个碱基的序列NNNNNNNN，所述N代表碱基A、G、C或T。
3.如第1项或第2项所述的绝对定量检测方法，其特征在于，所述第二轮PCR中的 结合LTR序列/内参基因序列的引物位于第一轮PCR中的结合LTR序列/内参基因序列的引物的下游，所述第二轮PCR中的结合接头序列的引物覆盖第一轮PCR中的结合接头序列的引物并延伸至下游。
4.如第1至3项中任一项所述的绝对定量检测方法，其特征在于，步骤3)-5)中所述扩增的循环数共为36-63。
5.如第1至4项中任一项所述的绝对定量检测方法，其特征在于，所述第一轮PCR和所述第二轮PCR的循环数比值为(1-3):2，例如1:2、3:2、1:1或者3:4；
较佳地，所述第一轮PCR和所述第二轮PCR的循环数分别为30和20，或者15和20，或者10和20，或者15和15。
6.如第1至5项中任一项所述的绝对定量检测方法，其特征在于，所述第三轮PCR的循环数为6-13，例如6、10或者13；
较佳地，所述第一轮PCR、第二轮PCR和第三轮PCR的次数分别为15、20和6。
7.如第1至6项中任一项所述的绝对定量检测方法，其特征在于，所述短链序列的5’端带有阻止引物延伸的修饰；较佳地为inverted dT修饰片段或者双脱氧胞苷；
和/或，所述内参基因选自基因ATCB、基因GAPDH和基因ApoB。
8.如第1至7项中任一项所述的绝对定量检测方法，其特征在于，步骤1)中所述基因组DNA被打断为1000bp左右的小片段。
9.如第1至8项中任一项所述的绝对定量检测方法，其特征在于，步骤4)-6)中均筛选1000bp以下的产物片段，以分别进行第二轮PCR、第三轮PCR和测序。
10.如第1至9项中任一项所述的绝对定量检测方法，其特征在于，步骤6)中所述测序为第二代测序。
11.一种工程化免疫细胞的制备方法，其特征在于，所述制备方法包括对工程化免疫细胞进行如第1至10项中任一项所述的慢病毒整合位点的绝对定量检测方法的步骤。
12.如第11项所述的工程化免疫细胞的制备方法，其特征在于，所述工程化免疫细胞至少经如下步骤获得：
(1)分离免疫细胞；
(2)制备编码CAR(Chimeric Antigen Receptor)的慢病毒载体；
(3)将步骤(2)所得载体导入免疫细胞。
13.如第11项或第12项所述的工程化免疫细胞的制备方法，其特征在于，所述工 程化免疫细胞选自细胞毒性T细胞、辅助T细胞、自然杀伤T细胞、γδT细胞和NK细胞。
发明的有益效果
本公开取得了如下技术效果(1)至(8)中的任意一项或者多项：
(1)本公开方法的灵敏度经验证达到单一位点拷贝数17拷贝/μg gDNA(如表13)，是首个明确灵敏度的整合位点检测方法，其灵敏度可以真正应用于临床样本的检测；
(2)本公开的方法尽可能地减少了扩增循环数，进而有效降低了soft-clip的比例及扩增产生的碱基偏差；
(3)本公开的方法中使用的生物信息学分析流程经过多轮调整和验证，得到了过滤噪音的关键cutoff；
(4)本公开中的方法设置了内参基因，可以用以标定实际的成库DNA量，整合位点的检测结果与之相比可以实现各个整合位点的绝对定量(每微克DNA中在某个整合位点有多少拷贝数)，而非仅仅得到各个整合位点之间的比例，同时让每一次建库产物在可控制的一定范围，进而稳定了建库过程中的操作步骤(PCR循环数参数的设置)，避免重复建库；
(5)本公开中的方法设置了UMI去重，可以有效降低PCR过程中导致的片段扩增错误及不同片段的扩增效率偏差；
(6)本公开改善了接头序列，对短链的末端进行了阻断扩增的修饰(inverted dT)，使第一轮接头引物自发扩增的产物大大减少；
(7)本公开仅使用超声打断、PCR及磁珠纯化的方法，既避免了酶切带来的偏好性，也不使用链霉亲和素标记的磁珠拉取生物素标记的靶标片段，相对于nrLAM PCR等步骤简单，成本低。
(8)本公开具有广阔的应用前景，能够应用于所有能够有效提取DNA的生物样本中的慢病毒整合位点的检测，包括但不限于应用慢病毒进行改造的各类细胞治疗产品及患者临床细胞样本的检测。如作为安全性的质控标准，应用于CAR-T产品的QC放行；对患者临床细胞样本进行检测，可以实时监测不同整合位点的CAR-T细胞的动态变化，可以用来评估细胞治疗产品的疗效和安全性等。
附图说明
图1：示例的实验流程图。
图2：示例的生物信息学分析流程图。
图3A：tapestation 4150导出的电泳图谱。
图3B：tapestation 4150导出的电泳图谱。
图3A和图3B显示了使用带inverted dT比不带inverted dT的接头对片段富集效果更好。
图4：不同循环数建库测序结果的softclip比例，显示了三轮PCR不同循环数设置对终文库soft-clip占比的影响。
图5：根据表13绘制的线性相关性点状图，显示了本公开方法与qPCR方法检测结果的相关性。
图6：根据测序结果绘制的相同初始分子的个数对应检测reads数的点状图，显示了过滤前初始分子经过建库扩增后的reads数量分布情况。
图7：初始分子对应检测reads数的箱线图，显示了初始分子经过建库扩增后的reads数量分布情况。
具体实施方式
下面通过实施例的方式进一步说明本公开，但并不因此将本公开限制在所述的实施例范围之中。下列实施例中未注明具体条件的实验方法，按照常规方法和条件，或按照商品说明书选择。
本公开检测慢病毒整合位点的测试细胞系gDNA，来自本公司独立制备及保存的CAR-T阳性的单拷贝细胞系，阳性率接近100％，已通过qPCR及ddPCR验证，-20度(℃)保存。CAR-T细胞本身为基因组DNA的来源，故本公开并不限于上述细胞系，适用于任何的可获取得到DNA的细胞。
本公开所用基因组DNA来源于CART细胞，但该方法并不限于CART细胞来源的DNA。例如DNA还可以为国际标准品WHO 1st Reference Reagent 2019for lentivirals vector integration site analysis，购自如下地址：
https://www.nibsc.org/products/brm_product_catalogue/detail_page.aspx？catid＝18/144。
本公开检测逆转录病毒整合位点的测试细胞系gDNA，来自本公司独立制备及保存的两株经γ-逆转录病毒转染后分离的单克隆细胞系(细胞系1、2)，经ddPCR验 证。本公开并不限于上述细胞系，适用于任何的可获取得到DNA的细胞。
实施例1：DNA片段化
1.1取300～1000ng基因组DNA，使用Ultrapure water稀释到总体积为130μL，混合均匀。
1.2将130μL全部加入microTUBE AFA Fiber Snap-Cap管中。
1.3在Covaris M220上运行以下程序对基因组DNA进行打断：
表1
1.4打断完成后，立刻将打断产物转移至EP管中，进行下一步。
1.5向上一步打断产物加入230μL(1.8×)的SPRIselect beads,使用移液枪充分混合均匀，室温孵育5min。
1.6将EP管转移到磁力架上。待液体澄清后，吸出350μL液体，弃去。
1.7加入300μL 80％(v/v)乙醇，30s后，小心移去上清。
1.8加入200μL 80％乙醇，30s后，小心移去上清。
1.9离心EP管，将EP管转移到磁力架上，吸出残余液体。
1.10室温干燥2min，将EP管移除磁力架，立刻加入50.5μL EB buffer重悬磁珠。
1.11室温孵育5min，转移50μL产物至PCR管中。
实施例2：末端修复及纯化
向50μL的产物中加入15μL End Prep Mix 4(Vazyme N203-02)，混合均匀，离心，置于PCR仪上，运行以下程序：
表2
2.2震荡SPRIselect beads直至完全重悬。
2.3将52μL SPRIselect beads(0.8×)加入到每个65μL样品中，枪头混匀15次，室温孵育5min。
2.4将PCR管放置于磁力架上，直到上清澄清透明，小心移去弃掉上清。
2.5加入200μL 80％乙醇，30s后，小心移去上清。
2.6加入200μL 80％乙醇，30s后，小心移去上清。
2.7离心PCR管，将PCR管转移到磁力架上，吸出残余液体。
2.8室温干燥2min，将EP管移除磁力架，立刻加入21μL 1×IDTE Buffer(IDT，11-05-01-09)重悬磁珠。
2.9室温孵育5min，转移20μL产物至PCR管中。
实施例3：接头退火
3.1把待退火的DNA oligo用NFW(Nuclease-Free Water)配制成终浓度为50μM的溶液。溶解Annealing Buffer for DNA Oligos(5×)(碧云天D0251)，混匀备用。
3.2使用的具体接头信息见表16。
3.3设置如下的退火体系：
表3
3.4在ABI proflex PCR system仪器上运行如下程序：
表4
实施例4：片段-接头连接及纯化
4.1将2×Rapid Ligation buffer，T4DNA ligase(Vazyme N103)解冻后颠倒混匀，置于冰上备用。按照下表进行配制连接反应体系：
表5
4.2移液枪吹打混匀，简短离心，将上述混合好的体系置于ABI proflex PCR system仪器上运行下表程序：
表6
4.3震荡混匀SPRIselect beads，使用移液枪充分混合均匀。
4.4吸取48μL的SPRIselect beads(0.8×)至连接产物中，使用移液器充分混匀，室温孵育5min。
4.5将EP管转移到磁力架上。待液体澄清后，小心移去弃掉上清。
4.6加入200μL 80％乙醇，30s后，小心移去上清。
4.7加入200μL 80％乙醇，30s后，小心移去上清。
4.8离心PCR管，将PCR管转移到磁力架上，吸出残余液体。
4.9室温干燥2min，将EP管移除磁力架，立刻加入21μL 1×IDTE Buffer重悬。
4.10室温孵育5min，转移20μL产物至新PCR管中。
实施例5：第一轮PCR(以慢病毒为例)
5.1 Phanta Super-Fidelity DNA Polymerase(Vazyme P501)试剂盒中的各组分解冻后需充分摇匀，所有操作应于冰上进行。
5.2配制如下的第一轮PCR反应体系：
表7
该步骤使用引物信息见后面的表16。
5.3移液枪吹打，短暂离心，将上述混好的反应体系放置于ABI proflex PCR system仪器上，运行下表程序：
表8

5.4震荡SPRIselect beads直至完全重悬。
5.5吸取30μL SPRIselect beads(0.6×)加入到每个样品中，枪头混匀15次，室温孵育5min。
5.6将PCR管放置于磁力架上，直到上清澄清透明，将上清转移至新的PCR管中。
5.7吸取10μL SPRIselect beads(0.8×)加入到每个样品中，枪头混匀15次，室温孵育5min。
5.8将PCR管放置于磁力架上，直到上清澄清透明，小心移除上清。
5.9吸取200μL 80％乙醇加入到每个管中，静置30s，小心移去弃掉乙醇液体。
5.10吸取200μL 80％乙醇加入每个管中，静置30s，小心移去弃掉乙醇液体。
5.11瞬时离心PCR管，放在磁力架上移去弃掉残余乙醇，室温干燥2min。
5.12将PCR管从磁力架拿下，加入21μL的1×IDTE Buffer，枪头混匀吹打15次，室温孵育2min。
5.13将PCR管放置于磁力架上，直到上清澄清透明。
5.14转移20μL样品到新管中，样品在4℃保存72h或-20℃长期保存，或直接进行下一步实验。
实施例6：第二轮PCR(以慢病毒为例)
6.1试剂盒中的各组分解冻后需充分摇匀，所有操作应于冰上进行操作。
6.2配制如下的第二轮PCR反应体系：
表9

6.3该步骤使用的引物信息见后面的表16。
6.4移液枪吹打，短暂离心，将上述混好的反应体系放置于ABI Proflex PCR system仪器上运行下表程序：
表10
6.5重复5.4-5.14步骤。
实施例7：第三轮PCR
7.1试剂盒中的各组分解冻后需充分摇匀，所有操作应置于冰上进行。
7.2配制如下第三轮PCR反应体系：
表11

该步骤使用的引物信息见后面的表16。
7.3移液枪吹打，短暂离心，将上述混好的反应体系放置于ABI proflex PCR system仪器上运行下表程序：
表12
7.4重复5.4-5.13步骤。
7.5转移20μL样品到新管中，使用Qubit检测纯化后产物的浓度，用Tapestation4150检测片段大小，文库后续可以直接投入Illumina测序仪进行测序。
实施例8：生物信息学分析流程(以慢病毒为例)
本方法涉及的数据分析流程如下：
1.对测序的原始fastq数据使用fastqc查看质控信息，使用fastp对测序数据进行清洗，仅保留测序质量值大于25，reads长度大于80，N碱基小于等于3，最多允许5％的碱基未能正确匹配的reads，对清洗后的fastq数据使用fastqc再次确认质控信息。
2.对清洗的fastq数据使用umi_tools提取R1测序数据的前8个碱基作为UMI并添加至reads的坐标中，并根据坐标将UMI序列映射到R2测序数据中，根据LTR/ApoB的引物序列对数据进行拆分，容错率设置为不超过2个碱基。
3.对拆分后的ApoB fastq文件随机抽取3000000reads，并计算抽样比例，随后 抽取相同比例的LTR reads。
4.对抽样后的ApoB/LTR reads使用BWA比对至人基因组，参考序列为GRCh38，对原始的bam文件进一步进行过滤，除去soft clip碱基数目大于15，比对质量小于20，reads长度小于50以及含有hard clip的reads，然后根据UMI以及reads的序列统计唯一的umi reads数量并对齐去重后计算中位值，将umi reads数目小于1％中位值的reads作为背景去除。
5.对清洗后的bam文件使用umi_tools对UMI进行双端去重，设置比对质量大于20，并丢弃chimeric-pairs及unpaired-reads，随后利用flag抽取所有的反向测序reads。
6.使用bedtools对反向测序reads的5’端位置进行统计，并出具报告。
实验结果：
接头短链上进行反向dT修饰，可以避免或者减少PCR反应非特异性扩增。根据本公开描述的实验方法，分别用带inverted dT和不带inverted dT的接头对打断后的DNA进行连接和修复后，进行第一轮PCR后以0.8×磁珠纯化再进行第二轮PCR，使用Tapestation 4150自动化电泳仪对建库后样本进行片段大小及分布分析，结果如图3A和图3B所示。结果显示，用带inverted dT相较于不带inverted dT的接头靶标片段(<1000bp)比例更高。
尽可能地减少了扩增循环数，进而有效降低了soft-clip的比例及扩增产生的碱基偏差。通过调整三轮PCR的各自的循环数，经过上述本公开的生物信息学分析流程，抽取目标基因的reads后对soft-clip比例进行统计，结果显示soft-clip的比例随着总的循环数的减少而减少，如图4所示。
在测定慢病毒整合位点的同时引入内参基因hApoB引物组，可以通过内参基因获知打断后片段的投入量，以实现对各整合位点的拷贝数进行绝对定量及确定方法的灵敏度。
应用本方法对梯队稀释的已知插入位点的细胞系进行检测，结果如表13及图5所示，其中拷贝数检测结果(IS/μg gDNA)的计算公式为：
表13：本公开方法的灵敏度结果
生物信息学分析进一步优化，通过设定cutoff值，可以有效过滤背景噪音。以20211125-4-lib为例，选用10ng Jurkat clone10 P32(经ddPCR验证的单拷贝细胞系，其拷贝数为LTR:ApoB＝1:2)与990ng正常人PBMC制备1％质量比的模拟样本，也即LTR:ApoB的理论拷贝数比例为0.5％。经上文描述方法建库测序并分析，对原始的bam比对文件中初始分子进行统计，对初始分子的数目计算中位数，设置初始分子对应的read数量小于中位值的1％作为背景，绘图如下(图6)，经过过滤相较于不经过过滤步骤得到的结果明显更加接近理论值(表14)。
表14：是否通过设置cutoff值过滤超低频率reads对结果读出的影响
设置UMI去重，可以有效降低PCR过程中导致的片段扩增错误及不同片段的扩增效率偏差。对20211125-4-lib中靶标基因的初始分子进行reads数量分布统计，结果(如图7)显示不同初始分子经过扩增后的reads数量分布从几个到1000多个不等，如果不进行去重将会对结果造成极大干扰。
经过以上的优化，应用本公开的方法可以检出样本中单一位点拷贝数大于等于17拷贝/μg gDNA以上的整合位点，并且在107拷贝/μg gDNA以上还能维持较好的线性(表14)。因此能够真正应用于基于慢病毒转染技术的基因改造的细胞治疗的临床样本的跟踪检测中。以下为应用案例，对不同细胞系应用本方法进行检测后，所有检测位点均能被ddPCR方法验证，准确度100％，结果如表15所示。
表15：本公开方法的检测准确度
表16：本公开所涉及引物序列

/phos/：磷酸化修饰；invdT：反向dT修饰；*：硫代修饰(T)；N：随机碱基，A、T、C或G。
实施例9：γ-逆转录病毒的整合位点检测
除了适用于慢病毒(或载体)的整合位点检测以外，本发明人更改了第一、二轮的引物为针对γ-逆转录病毒(例如MSCV/MoMLV)的LTR的特异性引物后，证明该方法对γ-逆转录病毒(或载体)的整合位点检测同样适用。
1.准确度实验：
采用本专利的建库及分析流程，更改第一、二轮的引物为针对γ-逆转录病毒的LTR的特异性引物(表20中的Rvbd_F_1_3及Rvbd_F_2_3)，对两株经逆转录病毒转染后分离的单克隆细胞系(细胞系1、2)进行RIS建库并分析得到整合位点信息，并根据整合位点信息设置特异性引物/探针组合，进行ddPCR验证，结果如下：
由NGS方法和ddPCR法进行比较可知：以ddPCR法为参考，利用本专利的NGS检测流程的阴性符合率和阳性符合率均为100％，即本方法能特异地检出RIS位点。
2.灵敏度实验：
对细胞系1和细胞系2进行不同比例的混合，以细胞系2为背景DNA，混入1％和0.1％的细胞系1的DNA；同理，以细胞系1为背景DNA，混入1％和0.1％的细胞系2的DNA,制备成灵敏度样本1、2、3、4,进行RIS建库，测序完成后进行生物信息学，获得RIS位点比例信息，并与理论值进行比较，结果如下表：
结果可知：本方法的灵敏度(RIS/ALL)在理论比例0.167％～0.249％时,检出率为100％(10/10)，在理论比例0.0167％～0.025％时，检出率为90％(9/10)，初步确定本方法的灵敏度(RIS/ALL)在0.167％以下。
表19：RIS检测样本来源：
表20：引物序列

/phos/：磷酸化修饰；invdT：反向dT修饰；*：硫代修饰(T)；N：随机碱基，A、T、C或G。
根据上面的实施例可以预见，对于基于固定整合序列(比如逆转录病毒的长末端重复序列(LTR)、腺相关病毒的末端反向重复序列(ITR)进行基因组整合的病毒/非病毒载体的整合位点，通过这段固定整合序列设计特异性引物，配合本公开的方法可以实现对多种病毒/非病毒载体的整合位点的检测。
尽管本公开的具体实施方式已经得到详细的描述，本领域技术人员将会理解。根据已经公开的所有教导，可以对那些细节进行各种修改和替换，这些改变均在本公开的保护范围之内。本公开的全部范围由所附权利要求及其任何等同物给出。

Claims (18)

  1. 一种制备DNA文库的方法，包括如下步骤：
    1)将被逆转录病毒感染的细胞的基因组DNA片段化，得到DNA片段；
    2)将DNA片段的末端修复加并连接上接头，得到连接产物；
    其中，所述接头为包括长链序列和短链序列的不对称双链接头，其中所述长链序列自5’端至3’端依次包含固定序列、随机UMI序列以及扩增引物结合序列，所述短链序列包含与所述固定序列互补的序列；
    3)用LTR序列的引物、内参基因序列的引物和接头序列的引物对连接产物进行第一轮PCR，得到第一轮PCR产物；
    4)用与步骤3)中不同的LTR序列的引物、内参基因序列的引物和接头序列的引物对第一轮PCR产物进行第二轮PCR，得到第二轮PCR产物；
    5)用带有测序接头的引物对第二轮PCR产物进行第三轮PCR，得到第三轮PCR产物，为DNA文库。
  2. 根据权利要求1所述的制备方法，其中，步骤1)中，所述DNA片段的长度为500-1500bp、800-1200bp、900-1100bp或1000bp。
  3. 根据权利要求1至2中任一权利要求所述的制备方法，其中，步骤2)中，所述固定序列的长度为14-30个碱基；和/或，
    所述随机UMI序列的长度为5-12个碱基，其中各碱基独立地为A、G、C或T；优选地，所述随机UMI序列的长度为5-9个碱基。
  4. 根据权利要求1至3中任一权利要求所述的制备方法，其中，步骤2)中，所述短链序列的5’端带有阻止引物延伸的修饰；优选为inverted dT修饰或者双脱氧胞苷修饰。
  5. 根据权利要求1至4中任一权利要求所述的制备方法，其中，步骤4)中：
    所述第二轮PCR中的LTR序列的引物位于第一轮PCR中的LTR序列的引物的下游，
    所述第二轮PCR中的内参基因序列的引物位于第一轮PCR中的内参基因序列的 引物的下游，和
    所述第二轮PCR中的接头序列的引物覆盖第一轮PCR中的接头序列的引物并延伸至下游。
  6. 根据权利要求1至5中任一权利要求所述的制备方法，其中，第一轮PCR、第二轮PCR和第三轮PCR的扩增循环数合计为36-63，优选为36-45。
  7. 根据权利要求1至6中任一权利要求所述的制备方法，其中，
    所述第一轮PCR和所述第二轮PCR的循环数比值为(1-3):2，例如1:2、3:2、1:1或者3:4；
    优选地，所述第一轮PCR和所述第二轮PCR的循环数分别为30和20、15和20、10和20、或者15和15；
    优选地，所述第三轮PCR的循环数为6-13，例如6、10或者13；
    优选地，所述第一轮PCR、第二轮PCR和第三轮PCR的循环数分别为15、20和6；10、20和6；或者15、15和6；
    优选地，第一轮PCR的循环数为15-18，第二轮PCR的循环数为18-22，第三轮PCR的循环数为6-8。
  8. 根据权利要求1至7中任一权利要求所述的制备方法，其中，
    其中：
    所述步骤3)中还包含如下步骤：筛选得到片段长度小于1000bp的第一轮PCR产物；
    所述步骤4)中还包含如下步骤：筛选得到片段长度小于1000bp的第二轮PCR产物；和/或
    所述步骤5)中还包含如下步骤：筛选得到片段长度小于1000bp的第三轮PCR产物。
  9. 根据权利要求1至8中任一权利要求所述的制备方法，其中，所述逆转录病毒为慢病毒或γ-逆转录病毒。
  10. 根据权利要求1至9中任一权利要求所述的制备方法，其中，所述内参基因是看家基因，例如基因ATCB、基因GAPDH或基因ApoB。
  11. 一种DNA文库，其由权利要求1至10中任一权利要求所述的制备方法制得；优选地，所述DNA文库为测序文库；更优选，所述DNA文库为用于检测逆转录病毒整合位点的测序文库。
  12. 一种检测逆转录病毒整合位点的方法，包括权利要求1至10中任一权利要求所述的制备DNA文库的方法，并且还包括如下步骤：
    6)对得到的DNA文库进行测序，得到测序数据；
    优选地，所述测序为第二代测序。
  13. 根据权利要求12所述的检测方法，还包括如下步骤：
    7)对测序数据进行生物信息学分析，得到整合位点的数据。
  14. 根据权利要求12至13中任一权利要求所述的检测方法，其为定量检测方法，优选为绝对定量检测方法。
  15. 根据权利要求12至14中任一权利要求所述的检测方法，其中，所述逆转录病毒为慢病毒或γ-逆转录病毒。
  16. 一种制备工程化免疫细胞的方法，包括对工程化免疫细胞按照权利要求12至15中任一权利要求所述的检测方法进行检测的步骤。
  17. 根据权利要求16所述的制备工程化免疫细胞的方法，其中，所述工程化免疫细胞通过包含如下步骤的方法获得：
    (1)分离免疫细胞；
    (2)构建包含编码嵌合抗原受体的核酸的重组逆转录病毒载体；
    (3)将步骤(2)所得的重组逆转录病毒载体导入免疫细胞。
  18. 根据权利要求16至17中任一权利要求所述的制备工程化免疫细胞的方法，其中，所述工程化免疫细胞选自细胞毒性T细胞、辅助T细胞、自然杀伤T细胞、γδT细胞、NK细胞、巨噬细胞、B细胞、抗原呈递细胞、树突状细胞和干细胞诱导的免疫细胞。
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