Gene extraction method for AAV third-generation sequencing

Abstract

The invention provides a gene extraction method for AAV third-generation sequencing, and particularly provides a simple and efficient rAAV gene extraction method for third-generation sequencing, which comprises the following steps: firstly, performing shell breaking on rAAV purified virus by using protease K to obtain an initial reaction solution; performing alkali denaturation on the initial reaction solution, neutralizing, purifying by using a DNA product purification kit, and eluting by using nuclease-free water to obtain denatured virus genome DNA; and finally, incubating and annealing the denatured virus genome DNA to finally form the complete double-stranded DNA. Compared with a traditional AAV genome extraction method, the method provided by the invention is simpler and more efficient, and the extracted DNA is higher in integrity.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a gene extraction method for AAV third-generation sequencing. Background Art

[0002] Adeno-associated virus (AAV) vectors contain single-stranded DNA, are non-enveloped, and belong to the B19 family of parvoviruses that rely on helper viruses. AAV replication in the host requires the assistance of host cells or other viruses, such as adenovirus and herpes virus, and is therefore called adeno-associated virus. The AAV genome is a linear single-stranded positive or negative strand DNA with a length of approximately 4,700 nucleotides. The genome of the adeno-associated virus contains two open reading frames, rep and cap, with terminal repeat sequences (ITRs) at both ends.

[0003] There are natural wild-type adeno-associated viruses in nature, and the AAV vector used in experiments is a plasmid that has been artificially modified based on the wild adeno-associated virus, so it is also called recombinant adeno-associated virus (rAAV). When not otherwise specified, the abbreviation AAV generally refers to the modified AAV vector. Recombinant adeno-associated viruses are widely used in the field of biology. They can be used for gene delivery or directly used to produce plasmid DNA vaccines. They are also the basis for packaging viral vectors in gene therapy and cell therapy. Therefore, accurate sequencing of plasmid sequences is very necessary for the production of high-quality viral vectors and other clinical treatment products.

[0004] Sanger sequencing, also known as the first-generation sequencing technology, as the gold standard for sequencing, promoted the drawing of the "working framework map" of the human genome. However, Sanger sequencing relies on pure consensus sequence templates and cannot simultaneously sequence and quantify mixed sequences. Therefore, it is not suitable for high-throughput large-scale sequencing, nor can it perform quantitative analysis on each sequence in a mixed sequence. The disadvantages of low throughput and high cost of Sanger sequencing limit its further large-scale application. With the rise of the next generation of high-throughput sequencing technology, that is, the second-generation sequencing technology, also known as the next-generation sequencing technology, it has been widely used in basic research and clinical diagnosis and treatment due to its advantages of high throughput and low cost. Although the second-generation sequencing effectively solves the problem of high-throughput simultaneous sequencing and quantitative analysis of mixed sequences, its read length is short (tens to hundreds of bases), and a single read length often cannot cover the length of a single sequence in the sample. Multiple read sequences need to be spliced ​​to obtain a longer complete sequence. Therefore, the second-generation sequencing cannot directly quantify the complete long sequence, and the length quality control requirements of rAAV require accurate quantitative evaluation of DNA sequences of different lengths that may exist in rAAV, which requires a high-throughput sequencing method with a read length that can cover sequences of different lengths in the sample at one time. With the emergence of the third-generation sequencing technology, due to its long read length advantage, it provides new technical means for the analysis of complex repetitive regions in gene sequences, full-length sequencing of long fragment sequences, accurate quantification, and high-quality genome assembly.

[0005] In recent years, the third-generation sequencing technology has been further developed and improved, and its clinical application value has gradually been reflected in related fields such as tumors, immunity, and reproduction. However, for the sequencing of AAV genome, the library construction method of PCR amplification after interruption is generally used, and the conventional method of extracting AAV genome results in low-quality DNA sample fragments and poor integrity (Kung SN, TranNT, Manokaran S, He R, et al. Direct ITR-to-ITR Nanopore Sequencing of AAVVector Genomes. Hum Gene Ther. 2022 Nov;33(21-22):1187-1196.). The conventional viral genome extraction steps are as follows: 10 10 The rAAV particles were resuspended in 100 µl DNase I buffer (20 mM Tris HCl, 1 mM MgCl 2, pH 8.0), 100 U of DNase I per 1 ml, incubated at 37 °C for 1 h, and then treated with 200 µl of proteinase K buffer (10 mM Tris HCl, 10 mM EDTA, 5% sodium dodecyl sulfate, pH 8.0) containing 2 mg / ml proteinase K at 50 °C for 1 h. Viral DNA was extracted by the sodium iodide method using a DNA extraction kit (Wako Chemicals USA, Richmond, Va.) (HIROYUKI NAKAI et al. Recruitment of Single-Stranded Recombinant Adeno-Associated Virus Vector Genomes and Intermolecular Recombination Are Responsible for Stable Transduction of Liver In Vivo, JOURNAL OF VIROLOGY, Oct. 2000, p. 9451–9463). The conventional denaturation and annealing steps for positive and negative single-stranded rAAV genomes are as follows: rAAV DNA is denatured with 0.2 N sodium hydroxide base, neutralized with ammonium acetate, ethanol precipitated, and resuspended in 1XSSC (0.15 M NaCl + 0.015 M sodium citrate). The denatured rAAV DNA is incubated at 70 °C for 1 h and slowly cooled (1 °C / min) to 20 °C. (Berns, KI, and JA Rose. 1970. Evidence for a single-stranded adenovirusassociated virus genome: isolation and separationof complementary single strands. J. Virol. 5:693–699.).

[0006] In addition, during the sequencing process, due to the randomness of the base forward speed, base recognition is easily missed or sequencing is terminated prematurely when the interval is too short, resulting in systematic errors in subsequent analysis and limiting the value of nanopore sequencing results. Therefore, optimizing the detection system and developing analysis algorithms to correct nanopore sequencing results are very meaningful research directions. Summary of the invention

[0007] The present invention also provides a gene extraction method for AAV third-generation sequencing, comprising the following steps: S1a: Use proteinase K to break the shell of the purified rAAV virus to obtain the initial reaction solution.

[0008] rAAV is composed of an icosahedral protein capsid with a diameter of about 26 nm and a single-stranded DNA genome. The protein capsid of rAAV is digested and broken by proteinase K to obtain an initial reaction solution containing the single-stranded DNA genome.

[0009] S2a: The initial reaction solution is subjected to alkaline denaturation, and after neutralization, it is purified using a DNA product purification kit to obtain denatured viral genomic DNA.

[0010] The single strand of the AAV genome is a positive strand or a negative strand. The genome includes inverted terminal repeats (ITRs) at both ends of the DNA strand. Alkaline denaturation can effectively open the complementary secondary structure of the ITR and denature it into a pure single strand. In the subsequent annealing process, the positive and negative strands can form effective double-stranded ends, which is conducive to adding sequencing adapters and assembling the motor protein required for nanopore sequencing during library construction. In some embodiments, the alkaline denaturation is sodium hydroxide alkaline denaturation. In some embodiments, ammonium acetate is used for neutralization after denaturation.

[0011] S3a: Incubate and anneal the denatured viral genomic DNA to form double-stranded DNA.

[0012] Preferably, the viral genomic DNA denatured in step S2a is not contacted with the sodium citrate reagent.

[0013] Preferably, the S2a step does not include suspending the denatured viral genomic DNA with an SSC solution.

[0014] Preferably, step S2a does not include ethanol precipitation.

[0015] Preferably, the DNA product purification kit uses a centrifugal adsorption column to purify denatured viral genomic DNA.

[0016] Preferably, the centrifugal adsorption column is washed with nuclease-free water.

[0017] The positive strand or negative strand of the AAV genome can be complementary. Through the alkaline denaturation in the previous step, the positive strand or negative strand has been denatured into a pure single strand, and then incubated and annealed to finally form double-stranded DNA. In some embodiments, the incubation temperature is 45-70°C. Specifically, the temperature can be 45, 50, 55, 60, 65 or 70°C. In some embodiments, the incubation time is 1 h.

[0018] Beneficial effects: Compared with the traditional AAV genome extraction method, the method provided by the present invention is simpler and more efficient, and the extracted DNA has a higher integrity. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to better understand the present invention and more clearly show how to implement the present invention, the features of the solution implemented according to the present invention are now described by way of example and with reference to the accompanying drawings, in which: Figure 1 : Flow chart of the quality control procedure analysis method.

[0020] Figure 2 : Capillary electrophoresis detection of extracted genomic DNA integrity.

[0021] Figure 3 : Capillary electrophoresis detection diagram of the integrity of extracted genomic DNA after step optimization.

[0022] Figure 4 : NanoPlot analysis results: A: Length distribution diagram, the horizontal axis represents the corresponding base sequence length of the Reads sequence, and the vertical axis represents the number of Reads in the corresponding sequence length interval; B: Weighted length distribution diagram: the horizontal axis represents the corresponding base sequence length of the Reads sequence, and the vertical axis represents the number of bases in the corresponding sequence length interval; C: Logarithmically transformed length distribution diagram; D: Logarithmically transformed weighted length distribution diagram.

[0023] Figure 5 : Analysis results of the Virus_QC quality control length distribution ratio of AAV genome DNA third-generation sequencing.

[0024] Figure 6 : Capillary electrophoresis detection of rAAV genomic DNA treated with different annealing conditions.

[0025] Figure 7 : Capillary electrophoresis detection diagram of the integrity of extracted genomic DNA after further optimization of the steps.

[0026] Figure 8 : Pie chart of the proportion of the number of reads in the Virus_QC quality control comparison of the third-generation sequencing of AAV genome DNA.

[0027] Fig. 9 : Pie chart of the proportion and classification of the number of bases compared by Virus_QC quality control in the third generation sequencing of AAV genome DNA.

[0028] Fig.10 : Analysis results of the length distribution ratio of Virus_QC quality control positive control in the third-generation sequencing of AAV genome DNA.

[0029] Fig.11 : Analysis results of the length distribution ratio of AAV genome DNA third-generation sequencing samples before Virus_QC quality control correction.

[0030] Fig.12 : Analysis results of the length distribution ratio of AAV genome DNA third-generation sequencing samples after Virus_QC quality control correction.

[0031] Fig.13 : Visual analysis results of Virus_QC quality control sequence alignment of AAV genome DNA third-generation sequencing. DETAILED DESCRIPTION

[0032] definition: In order to provide a clear and consistent understanding of the terms used in the specification of the present invention, some definitions are provided below. In addition, unless otherwise specified, all technical and scientific terms used in the present invention have the same meaning as commonly understood by ordinary technicians in the field to which the present invention belongs.

[0033] When used in conjunction with the term "comprising" in the claims and / or the specification, the use of the word "a" may mean "one", but it is also known with the meanings of "one or more", "at least one" and "one or more than one". Similarly, the word "another" may mean at least a second or many.

[0034] As used in this specification and claims, the words "comprising" (and any forms of comprising, such as "including" and "comprising"), "having" (and any forms of having, "having", "including" and "containing") are inclusive and open-ended, and do not exclude additional, unrecited elements or processing steps.

[0035] The third generation sequencing is used in many applications of single-molecule long sequence sequencing. During the sequencing process, due to the randomness of the base forward speed, base recognition is easily missed or sequencing is terminated prematurely when the interval is too short, resulting in systematic errors in subsequent analysis and limiting the value of nanopore sequencing results. The inventors of the present invention have invented a concise and efficient third generation sequencing quality control program analysis method. Specifically, referring to Figure 1 , including the following steps: S1b: The extracted gene sequence is sequenced to obtain the POD5 text of the initial data.

[0036] The present invention obtains data by directly reading the DNA sequence of the full length. Any relatively complete double-stranded DNA sequence can be used for data analysis by the method provided by the present invention. In some embodiments, the DNA or RNA sample is mainly sequenced by Nanopore equipment, and the collected data is usually in a long-read format, with a high base error rate, but can cover complex regions of the genome such as repetitive sequences. The initial format of the data is POD5 text, which is a file format used by Oxford Nanopore to store raw current signals. The data contains unprocessed sequencing information, including the Q (Qualities) value of DNA, which is a standard indicator for evaluating the accuracy of base reading during sequencing, and the higher the score, the better the base quality. Without limitation, the data read out by other third-generation sequencing devices that can read double-stranded DNA sequences can also be analyzed using the quality control program analysis method provided by the present invention.

[0037] In some embodiments, the third-generation sequencing is the third-generation sequencing of rAAV, and the extracted genes are genes of rAAV.

[0038] S2b: Preprocess the initial data, convert the POD5 text into a FASTQ file, and remove the Adapter data to obtain the second data.

[0039] This processing step is the basis for ensuring data purity and high quality. FASTQ files are a common data format for bioinformatics analysis, which contain the base sequence of sequencing reads and the quality score of each base. The specific tasks of this preprocessing step include: Adapter Removal: Adapters are non-biological sequences added during the sequencing process to guide the sequencing process. These redundant adapter sequences must be removed before analysis to ensure that the analysis is based only on the biological sequence of the sample.

[0040] Data format conversion: Convert raw sequencing data from POD5 text format to FASTQ format to facilitate subsequent analysis using other tools.

[0041] In some embodiments, Dorado is selected to process the initial data and obtain the second data. Dorado is a data processing tool officially recommended by Oxford Nanopore, which is mainly used to convert the POD5 text format into the standard FASTQ format.

[0042] S3b: Perform quality assessment on the second data to obtain clean, non-redundant Reads data.

[0043] In some embodiments, NanoPlot is used to perform quality assessment on the second data. NanoPlot is a quality assessment and visualization tool for long-read sequencing data such as Oxford Nanopore and PacBio. Its main function is to generate visual charts so that researchers can quickly assess the quality of data sets. It is currently widely used as a conventional quality assessment software. The charts generated by NanoPlot include: Read length distribution graph: Displays the length distribution of each read. Researchers can use this graph to understand whether the length of reads in the dataset is within the expected range and whether there are abnormal reads that are shorter or longer.

[0044] Quality score distribution graph: Generate a Q value quality score distribution graph to evaluate the overall quality of reads.

[0045] Other visualizations: NanoPlot can also generate a variety of charts such as base composition, quality score variation with position, and correlation between read length and quality score.

[0046] Through these charts, researchers can quickly identify potential problems in the data, such as low-quality reads or abnormal length distribution, so as to adjust subsequent analysis strategies in a timely manner.

[0047] S4b: Compare and analyze the Reads data with the target base sequence of theoretical length to obtain the first statistical result.

[0048] In some embodiments, Minimap2 is used to compare the filtered reads with the target base sequence of theoretical length. Minimap2 is a sequence alignment tool designed specifically for processing long read length data. Unlike traditional short read length alignment tools, Minimap2 has higher speed and accuracy when processing long read lengths, and is particularly suitable for Nanopore and PacBio sequencing data. Its core functions include: Whole genome alignment: Minimap2 can efficiently align long reads to the reference genome and generate alignment results in SAM / BAM file format. The alignment results not only include the mapping positions of the reads to the reference genome, but also include detailed information such as the alignment quality and mismatches of each site.

[0049] Variant detection: Since long read data can cover longer genomic regions, Minimap2 is very suitable for detecting complex genomic structural variations (SVs), such as large-scale insertions, deletions, inversions, etc. These variations are difficult to identify in short read data, and long read data has a wider coverage, making Minimap2 perform well in variant detection.

[0050] Molecular biology research support: Minimap2's alignment results can be used not only for genome reconstruction, but also for RNA-seq, transcriptome analysis, etc. For diverse biological research, Minimap2 provides a reliable and efficient alignment solution.

[0051] In addition, Minimap2 has adjustable parameters that allow researchers to adjust the accuracy and speed of the alignment according to specific needs.

[0052] S5b: According to the first statistical result, the second data is further screened to filter out impurities to obtain pure third data.

[0053] In some embodiments, the impurities are long sequences greater than 10,000 bp, short sequences less than 500 bp, and low-quality sequences with a Q value less than 10, and the pure third data are data of sequences with a length within 500-10,000 bp and a Q value greater than or equal to 10.

[0054] Q (Quality, Q) is an important indicator to measure the accuracy of reads alignment to the reference genome. Usually, Q ≥ 10 is considered to be a more reliable alignment result. In this step, high-quality reads are screened and only reads with Q ≥ 10 are retained to ensure that subsequent analysis is based on high-quality data and reduce the impact of low-quality alignment results on the analysis.

[0055] The present invention adopts the conventional value 10 as the critical value of Q to judge the quality of the detection fragment. However, in some embodiments, other values ​​can be selected as the critical value to judge the quality of the detection fragment according to actual needs, such as 8, 9, 11, 12, 13, etc.

[0056] In some embodiments, Nanofilt is used for filtering. Nanofilt is a filtering tool for Nanopore data that allows researchers to filter reads based on multiple dimensions such as quality score, length, GC content, etc. Nanofilt can filter out short fragment reads or low-quality reads to ensure that the remaining data meets the needs of subsequent analysis.

[0057] This step provides a high-quality, reliable data set for subsequent analysis, thereby improving the accuracy and stability of the final analysis results.

[0058] S6b: Perform sequence length and sequence alignment analysis on the pure third data to obtain a detection result.

[0059] This stage is to further analyze the filtered reads in detail. In some embodiments, a combination of NanoPlot, Minimap2 and Pysamstats is used. The specific functions of each tool are as follows: NanoPlot: Used to visualize the length distribution, quality score and other indicators of reads, providing a reference for sequence length analysis.

[0060] Minimap2: In this step, the alignment results of Minimap2 obtained in the previous step will be used for further genome annotation, variation analysis and other tasks. Its alignment information can not only provide the mapping position of Reads, but also assist in gene expression, structural variation detection and other studies.

[0061] Pysamstats: A Python-based SAM / BAM file analysis tool that can extract detailed statistical information of alignment data. Its features include: 1) Coverage analysis of each site: Count the number of reads covering each site to help researchers determine the coverage of the genome.

[0062] 2) Mutation frequency statistics: Pysamstats can calculate the frequency of mismatched bases to help researchers identify potential mutation sites.

[0063] 3) Insertion / deletion analysis: It can also detect insertion or deletion events that occur during sequencing and help analyze the structural characteristics of reads.

[0064] The combination of these tools provides researchers with detailed data characterization, facilitating in-depth quality control and characterization of data from multiple perspectives.

[0065] The present invention also provides a method for rAAV third-generation sequencing, comprising: S1c: Extract gene sequence.

[0066] S2c: Use any of the third-generation sequencing data processing methods described above to read and process the gene sequence to obtain the test results.

[0067] In some embodiments, the third-generation sequencing is the third-generation sequencing of rAAV, and the extracted genes are genes of rAAV.

[0068] Furthermore, it also includes denoising the test results, that is, by setting a positive control to detect noise, and then removing the noise of the test results with reference to the noise detected by the positive control, so as to obtain a corrected result. In some embodiments, the positive control is a 5' ITR-target sequence-3' ITR linearized fragment obtained after enzyme digestion of the vector plasmid, and the positive controls are all complete DNA sequences. The signal of the incomplete sequence that appears during the detection process is the background noise of the instrument or the detection system itself. The background noise of the positive control and the object to be detected should be basically the same under the same environment. With reference to the background noise detected by the positive control, the noise of the object to be detected is filtered out, thereby obtaining a purer test result.

[0069] After the above steps, researchers can generate a quality control report containing information from multiple dimensions. The report will include: 1) Read length distribution and quality score of the dataset.

[0070] 2) Statistics of coverage and mutation frequency of each site.

[0071] 3) Visual display of sequence length and alignment quality.

[0072] By interpreting the report, researchers can better understand the characteristics of sample data and provide support for subsequent biological analysis or experimental verification. The entire process, from raw data acquisition, preprocessing, quality assessment, comparison analysis to final report generation, forms a highly optimized and reliable bioinformatics analysis process. NanoPlot provides convenient quality control and visualization methods, Minimap2 excels in long-read data processing with its fast and accurate comparison function, and Pysamstats provides powerful support for detailed statistical analysis after comparison. The combination of this series of tools makes the entire analysis process efficient and robust, suitable for complex bioinformatics problems and long-read data processing.

[0073] The present invention also provides a computer program product, which may be a software comprising one or more stored instruction sequences, which are accessible to a processor and, when executed by the processor, cause the processor to implement the steps of any of the above-described sequencing methods.

[0074] In some embodiments, the computer program product includes: a first unit, a second unit, a third unit, a fourth unit, a fifth unit, and a sixth unit, wherein: The first unit can be used to perform third-generation sequencing on the gene sequence of rAAV and generate POD5 sequencing data after sequencing on the machine; The second unit is used for initial filtering and data conversion, which can filter the Adapter data and convert the POD5 text into FASTQ files; The third unit can evaluate the quality of the data in the FASTQ file and obtain the data of the Reads distribution; In the fourth unit, the Reads data can be compared and analyzed with the theoretical reference sequence; The fifth unit can be used for data classification and screening, and screens out abnormal data and data that do not meet the quality requirements according to the comparison analysis results. In some embodiments, the data that do not meet the quality requirements are fragments with a Q value less than 10; The sixth unit can perform sequence length analysis and sequence alignment analysis on the filtered data and output the final results.

[0075] The present invention also provides a computer-readable medium, which carries one or more instruction sequences of the computer program product.

[0076] Unless specifically stated otherwise, it should be understood that throughout this specification, discussions using terms such as "compute," "calculate," "generate," or similar terms refer to the actions and / or processes of a computer or computing system or similar electronic computing device that manipulate data represented as physical (e.g., electronic) quantities within the computing system's registers and / or memories and / or transform them into other data similarly represented as physical quantities within the computing system's memories, registers, or other such information storage, transmission, or display devices.

[0077] Embodiments of the present invention may include apparatus for performing the operations described herein. The apparatus may be specially constructed for the desired purpose, or the apparatus may include a general purpose computer or a digital signal processor (DSP) selectively activated or reconfigured by a computer program stored in the computer. Such a computer program may be stored in a computer readable storage medium, such as but not limited to any type of disk, including a floppy disk, an optical disk, a CD-ROM, a magneto-optical disk, a read-only memory (ROM), a random access memory (RAM), an electrically programmable read-only memory (EPROM), an electrically erasable programmable read-only memory (EEPROM), a magnetic or optical card, or any other type of medium suitable for storing electronic instructions and capable of being coupled to a computer system bus.

[0078] The process and display mode proposed herein are not originally relevant to any specific computer or other equipment. Various general purpose systems can be used together with the program according to the teaching herein, or it can prove to be very convenient to build a more special-purpose device to perform the desired method. The desired structure of various these systems will be understood from the above description. In addition, embodiments of the present invention are not described with reference to any specific programming language. It will be appreciated that various programming languages ​​can be used to realize the teaching of the present invention as described herein.

[0079] Example

[0080] The present invention will be more readily understood by reference to the following examples, which are provided to illustrate the present invention and are not to be construed as limiting the scope of the present invention in any way.

[0081] Unless otherwise defined or the context clearly dictates otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. It should be understood that any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention.

[0082] Although the present invention has been described in detail with reference to the embodiments of the present invention, these embodiments are provided to illustrate rather than limit the present invention. Other embodiments that can be obtained according to the principles of the present invention all belong to the scope defined by the claims of the present invention.

[0083] Experimental methods not specifically described in the present invention are all carried out according to the specific methods in the book "Molecular Cloning: A Laboratory Manual (Fourth Edition)" by Joseph. Sambrook, or according to the instructions of relevant products. When used in this article, unless otherwise specified, all terms in the present invention should be understood according to their common meanings known in the art. The biological reagents and instruments used in the present invention can be obtained from commercial channels unless otherwise specified.

[0084] instrument: MinION Mk1B, Oxford Nanopore Technologies; Bio-Fragment Analyzer, Qsep1-Plus, C100001-P.

[0085] Material: Proteinase K solution (20 mg / ml) was purchased from Sangon Biotech; Common DNA product purification kit, purchased from TIANGEN; Ligation Sequencing Kit V14, purchased from Oxford Nanopore Technologies; The chip FLO-MIN114 R10.4.1 was purchased from Oxford Nanopore Technologies.

[0086] Example 1: rAAV genome extraction Purified rAAV virus (10 10 AAV virus particles), add 20 µl 20 mg / ml proteinase K (SangonBiotech), and incubate at 50 ℃ for 1 h. Denature with 0.2 N sodium hydroxide (incubate at room temperature for 5 min), neutralize with 5 M ammonium acetate, precipitate with ethanol, and elute with nuclease-free water. Purify using a common DNA product purification kit (DP204, TIANGEN), and resuspend in 1×SSC (0.15 M NaCl+0.015 M sodium citrate). After incubating rAAV DNA at 70 ℃ for 1 h, slowly cool (1 ℃ / min) to 20 ℃ to denature and anneal to form double-stranded AAV genomic DNA.

[0087] The AAV genomic DNA obtained by the above experimental steps was tested by capillary electrophoresis. The results are shown in Figure 2 , this test uses a 20 nt marker. The thin arrow indicates the actual length of AAV genomic DNA detected by capillary electrophoresis. The theoretical length of AAV genomic DNA sample is 4493 bp (SEQ ID NO: 1). Figure 2 The test results showed that the integrity of the AAV genome DNA obtained in this example was poor.

[0088] Among them, the brief operation process of purifying DNA with a common DNA product purification kit is as follows: 1. Column equilibration step: Add 500 µl of equilibration solution BL to the adsorption column CB2 (the adsorption column is placed in the collection tube), centrifuge at 12,000 rpm for 1 min, pour out the waste liquid in the collection tube, and put the adsorption column CB2 back into the collection tube (please use the column that has been processed on the same day).

[0089] 2. Estimate the volume of PCR reaction solution or enzyme digestion reaction solution, add 5 times the volume of Binding Buffer PB to it, and mix thoroughly.

[0090] 3. Add the solution obtained in the previous step to an adsorption column CB2 (the adsorption column is placed in a collection tube), place it at room temperature for 2 minutes, centrifuge it at 12,000 rpm for 30-60 seconds, pour out the waste liquid, and place the adsorption column CB2 back into the collection tube.

[0091] 4. Add 600 μl of washing solution PW to the adsorption column CB2 (please check whether anhydrous ethanol has been added before use), centrifuge at 12,000 rpm for 30-60 sec, discard the waste liquid, and place the adsorption column CB2 in the collection tube.

[0092] 5. Repeat step 4.

[0093] 6. Place the adsorption column CB2 back into the collection tube and centrifuge at 12,000 rpm for 2 min to remove as much rinsing solution as possible. Place the adsorption column CB2 at room temperature for several minutes to dry thoroughly to prevent residual rinsing solution from affecting the next step of the experiment.

[0094] 7. Place the adsorption column CB2 in a clean centrifuge tube and drop elution buffer ddH2O into the middle of the adsorption membrane. 2 O (30-50µl, not less than 30µl), let it stand at room temperature for 2 min, and centrifuge it at 12,000 rpm for 2 min to collect the DNA solution.

[0095] Example 2: Optimization of rAAV genome extraction steps Purified rAAV virus (10 10 AAV virus particles), 20 µl 20mg / ml proteinase K (SangonBiotech) was added, and incubated at 50°C for 1 h. Denatured with 0.2N sodium hydroxide (incubated at room temperature for 5 min), neutralized with 5 M ammonium acetate, and no ethanol precipitation and elution with nuclease-free water were performed. Purification was performed directly using a common DNA product purification kit (DP204, TIANGEN), and then resuspended in 1×SSC (0.15 M NaCl+0.015 M sodium citrate). rAAV genomic DNA was incubated at 70°C for 1 h and then slowly cooled (1°C / min) to 20°C to denature and anneal to form double-stranded AAV genomic DNA.

[0096] The AAV genomic DNA obtained by the above experimental steps was tested by capillary electrophoresis. The results are shown in Figure 3 This test uses a 20 bp-5000 bp marker. The thin arrow indicates the actual length of AAV genomic DNA detected by capillary electrophoresis. The theoretical length of AAV genomic DNA sample is 4493 bp. Figure 3The test results showed that the AAV genomic DNA extracted under such optimized conditions would degrade, and the integrity of the genomic DNA was still low.

[0097] Example 3: Analysis software NanoPlot Compared with the second-generation Illumina sequencing data, the third-generation Nanopore sequencing data has the characteristics of longer read length, high error rate, and uneven length distribution. NanoPlot analysis software is a sequence length analysis software provided by Nanopore. It provides a visualization tool that can be used for long-read sequencing data and alignment results. It aims to provide a visual statistical and graphical report on the quality of the sequencing data itself, helping users understand whether the test process is normal and whether the obtained sequencing data has reached the expected abundance, accuracy and coverage. It can also be used as a preliminary reference for AAV length quality control. NanoPlot is mainly based on linux system commands. The specific instructions are "NanoPlot - t12 -- fastq . / SQK-NBD114-24_barcode04.fastq.gz -- maxlength 40000 -- plotsdot -- legacy hex". Take the AAV genomic DNA sample in Example 2, and get the analysis results after running NanoPlot. Figure 4 .

[0098] Depend on Figure 4 It can be seen that the length analysis results of NanoPlot can only analyze the visualization of the length distribution of the different length regions of the sequenced reads, and the visualization results cannot display detailed information regardless of whether weight conversion or log conversion is performed. It can only be used as a rough quality assessment of the sequencing data itself (rather than quality control analysis for AAV virus particles). It can neither customize appropriate analysis parameters (such as the width setting of each data bar) according to the specific length and specific type of AAV virus samples, nor can it use control samples for proportion correction and calculate the proportion of other non-AAV genome sequences. Since NanoPlot is not a software specially designed for the quality control of AAV products, its analysis results are too simple, have serious defects and limitations, are not accurate enough, and are misleading, and cannot be used for accurate evaluation of AAV product quality.

[0099] Example 4: Quality Control Program Analysis Software Virus_QC A computer program software product Virus_QC for quality control program analysis includes: a first unit Nanopore, which sequences DNA or RNA samples through Nanopore equipment, collects original POD5 sequencing data of AAV (adeno-associated virus) virus samples, and sequences through Nanopore equipment and performs preliminary collection of sequencing data and comprehensive evaluation of data formats; a second unit Dorado, which mainly includes operations such as removing sequencing adapters and data format conversion, is used for initial filtering and data conversion, can filter adapter data, and convert POD5 text into FASTQ files; a third unit NanoPlot, which can perform quality evaluation on the data of the FASTQ file to obtain data of Reads distribution; a fourth unit Minimap2, which can compare and analyze the Reads data with theoretical reference sequences; a fifth unit Nanofilt, which can be used for data classification and screening, and screens out abnormal data and data with Q < 10 according to the comparison analysis results; a sixth unit NanoPlot, Minimap2 and Pysamstats combination unit, which can perform sequence length analysis and sequence alignment analysis on the screened data, and output the final results.

[0100] Example 5: Nanopore sequencing library construction and sequencing 1 μg of the AAV genomic DNA sample in Example 2 was taken and the library was constructed using the Ligation Sequencing Kit V14 (ONT, SQK-LSK114) kit of Oxford Nanopore, UK. The constructed library was sequenced on the MinION Mk1B device, the chip code hole type was FLO-MIN114 R10.4.1, and the running time was set to 72 h. The sequencing results were analyzed using the Virus_QC quality control program designed in Example 4, as shown in FIG. Figure 5 As shown in the figure, the number of reads with a theoretical length of 4493 bp in the AAV genomic DNA sample accounted for only 1.35%, indicating that the degraded AAV genomic DNA during the experiment had a greater impact on the sequencing results.

[0101] Example 6: Effects of different annealing temperatures on double-stranded DNA Referring to the steps of Example 2, rAAV purified virus (10 10AAV virus particles), add 20 µl 20 mg / ml proteinase K (Sangon Biotech), and incubate at 50 ℃ for 1 h. Denature with 0.2 N sodium hydroxide (incubate at room temperature for 5 min), neutralize with 5 M ammonium acetate, purify using a common DNA product purification kit (DP204, TIANGEN), and resuspend in 1×SSC (0.15 MNaCl+0.015 M sodium citrate). Six groups of equal amounts of rAAV genomic DNA were selected and annealed as follows: 45 ℃ for 1 h, 50 ℃ for 1 h, 55 ℃ for 1 h, 60 ℃ for 1 h, 65 ℃ for 1 h, and 70 ℃ for 1 h, and finally slowly cooled (1 ℃ / min) to 20 ℃. These 6 treated samples were tested by capillary electrophoresis, and the test results are shown in Figure 6 The results showed that the capillary electrophoresis detection results of samples treated with different annealing conditions were consistent, so the annealing temperature did not have a significant impact on the detection results.

[0102] Example 7: Further optimization of rAAV genome extraction steps Purified rAAV virus (10 10 AAV virus particles), add 20 µl 20 mg / ml proteinase K (SangonBiotech), and incubate at 50 ℃ for 1 h. Denature with 0.2 N sodium hydroxide (incubate at room temperature for 5 min), neutralize with 5 M ammonium acetate, purify using a common DNA product purification kit (DP204, TIANGEN), elute with nuclease-free water, incubate at 70 ℃ for 1 h, then slowly cool (1 ℃ / min) to 20 ℃ to denature and anneal to form double-stranded AAV genomic DNA. The results of capillary electrophoresis detection are shown in Figure 7 As shown, the theoretical length of the AAV genomic DNA sample is 4493 bp. The capillary electrophoresis detection results show that the integrity of the AAV genomic DNA is good, and the sample DNA peaks are rich and far higher than the 20 bp Marker peak.

[0103] Example 8: Third-generation sequencing to detect the integrity of rAAV genomic DNA The genome of the AAV virus sample was extracted and sequenced according to the optimized method in Example 7. The Reads sequence was obtained after sequencing. The impurity ratio analysis of the AAV genome sequence based on Virus_QC and the minimap2 sequence alignment showed the proportion of each Reads sequence component. Figure 8 and Fig. 9, of which AAV genome sequences account for more than 85%, packaging plasmids remain in approximately 5%, and there are some other components (such as random connection of adapters generated during library construction, misidentified sequences of the Basecalling algorithm, or residual host cell genome fragments in the sample).

[0104] Example 9: Positive Control Calibration Set up a positive control, i.e., a linearized plasmid fragment with ITRs at both ends (labeled ctr), and process AAV and the positive control (double-stranded DNA fragment of a linearized plasmid with ITRs at both ends) simultaneously according to the steps in Example 6. The length of the positive control fragment is 3558 bp (SEQ ID NO: 2). Fig.10 As shown in the figure, through the length analysis of the Virus_QC quality control program, the sequencing results of the positive control ctr data are good, and the detection sequence of the theoretical length of 3558 bp is relatively concentrated, and the proportion distributed in the theoretical length area is 46.09%. This data can be used as a control for sample correction in the actual AAV group, and the short fragments caused by possible accidental interruptions in the sequencing process can be removed through correction. Fig.11 As shown in Figure 2, the target sequence length before correction accounts for 51.32%; after correction by the data of the positive control group, as shown in Figure 2 Fig.12 As shown in the figure, the length of the target sequence accounts for 86.38%, which is a significant improvement compared with before correction.

[0105] Fig.13 This is the visualization result of the consistency analysis of the AAV genome sequence after correction. The overall matching rate of the AAV genome sequence is about 95%, indicating that the genomic DNA sequence consistency of this sample is also good.

[0106] The present invention provides a third-generation sequencing method, which can not only display the length analysis graph corresponding to the target length of the gene sequence, but also display the quality value (Q value) of the reads sequence in the target length region, as well as the sequence comparison graph of the target region where the sequence is located with the reference genome sequence. The present invention also provides a concise and efficient method for obtaining complete viral genomic DNA from recombinant adeno-associated virus (rAAV) particles. Researchers can directly measure the complete rAAV gene sequence to better understand the characteristics of the sample data and provide support for subsequent biological analysis or experimental verification. The entire process, from raw data acquisition, preprocessing, quality assessment, comparison analysis to final report generation, forms a highly optimized and reliable bioinformatics analysis process.

[0107] The above-described embodiments only express several embodiments of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the present invention. It should be pointed out that a person skilled in the art can make various changes, modifications and substitutions without departing from the spirit of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the attached claims.

[0108] Although the present invention has been described in detail with reference to the embodiments of the present invention, these embodiments are provided to illustrate rather than limit the present invention. Other embodiments that can be obtained according to the principles of the present invention all belong to the scope defined by the claims of the present invention.

Claims

1. A gene extraction method for AAV third-generation sequencing, characterized in that: The method is carried out according to the following steps: S1a: using proteinase K to break the shell of the purified rAAV virus to obtain the initial reaction solution; S2a: subjecting the initial reaction solution to alkaline denaturation, neutralization, and purification using a DNA product purification kit to obtain denatured viral genomic DNA; and S3a: incubating and annealing the denatured viral genomic DNA to form double-stranded DNA.

2. The gene extraction method according to claim 1, characterized in that: The denatured viral genomic DNA does not come into contact with the sodium citrate reagent in the S2a step.

3. The gene extraction method according to claim 1, characterized in that: The S2a step does not include suspending the denatured viral genomic DNA with an SSC solution.

4. The gene extraction method according to claim 1, characterized in that: The S2a step does not include ethanol precipitation.

5. The gene extraction method according to claim 1, characterized in that: The DNA product purification kit uses a centrifugal adsorption column to purify the denatured viral genomic DNA.

6. The gene extraction method according to claim 5, characterized in that: The centrifugal adsorption column was eluted with nuclease-free water.

7. The gene extraction method according to claim 1, characterized in that: The alkaline denaturation in the step S2a is sodium hydroxide alkaline denaturation.

8. The gene extraction method according to claim 1, characterized in that: The neutralization in the step S2a is ammonium acetate neutralization.

9. The gene extraction method according to claim 1, characterized in that: The incubation temperature in step S3a is 45°C-70°C.

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