Method for efficiently identifying exogenous DNA insertion site in genome by using high-throughput sequencing technology

Abstract

The invention belongs to the technical field of gene sequencing, and particularly relates to a method for efficiently identifying an exogenous DNA insertion site in a genome by using a high-throughput sequencing technology. The method comprises the following steps: (a) extracting a sample, building a database and sequencing; (b) data quality control; (c) comparing data; (d) filtering comparison results; (e) preliminary screening; and (f) screening and identifying the positions of the exogenous insertion elements. According to the method, the high-throughput sequencing data and the genome and exogenous fragment sequences are compared and analyzed to obtain the candidate positions of the insertion sites, so that the test efficiency can be remarkably improved, manual judgment and screening work can be effectively replaced, and the result accuracy can be improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of gene sequencing, and in particular relates to a method for efficiently identifying exogenous DNA insertion sites in a genome using high-throughput sequencing technology. Background Art

[0002] The exogenous fragment insertion site refers to the location where the exogenous fragment sequence of the vector is inserted into the chromosome in the transgenic plant. In the study of plant functional genomics, it is very important to clarify the information of this site for the smooth development of related research. In the application of transgenic production, the exogenous fragment insertion site is the label of each new transgenic material, and the screening and identification of transgenic materials require the clarification of the exogenous fragment insertion site.

[0003] With the maturity of sequencing technology and the decrease in time and sequencing costs, high-throughput sequencing technology has been applied to the identification of insertion sites. High-throughput sequencing is used to obtain sample genome sequence information, and screening and identification are performed based on the comparison information with the reference genome and the exogenous insertion fragment sequence, ultimately obtaining the candidate position of the exogenous insertion fragment.

[0004] Prior art CN116189772A discloses a method for rapid identification of exogenous insertion sequences based on whole genome sequencing data. This method can provide the insertion site, insertion direction, insertion copy number, flanking sequence and gene function of the region surrounding the insertion site. However, this invention patent uses second-generation data for assembly to determine the flanking sequence, and the assembly will introduce certain errors. This method greatly reduces the accuracy of the flanking sequence, and also lacks the accuracy determination of the comparison result.

[0005] However, the existing technology has many shortcomings, such as:

[0006] a). There is a lot of manual judgment and screening work, which is time-consuming and labor-intensive, and has low accuracy.

[0007] After obtaining the comparison information, it is necessary to manually judge whether the position is logical, which is a huge manual workload, time-consuming, and very easy to make mistakes. The present invention uses an algorithm to make logical judgments, outputs results in one click, reduces manual operation, saves time, and improves the accuracy of results.

[0008] b) Based on the third generation sequencing test, the sample quality requirements are high and the sequencing cost is high

[0009] The third-generation long-read sequencing technology can detect the location of the insertion site and sequence information, but it requires high-quality samples and high-quality DNA to be extracted for effective sequencing, and the sequencing cost of the third-generation platform is high. This method is based on the DNBSEQ-T7 platform, and sequencing analysis can be performed by constructing a regular small fragment library. The sample quality requirements are low, and the sequencing cost of the domestic sequencing platform is low.

[0010] Based on the data results of high-throughput sequencing, the present invention determines the insertion position, direction, copy number and flanking sequence information of the exogenous insert fragment on the genome by comparing it with the genome and the exogenous insert fragment sequence. The present invention aims to improve the accuracy and efficiency of position judgment based on the reasonable chromosome position and direction of paired reads. Summary of the invention

[0011] In order to solve the deficiencies of the prior art, the first object of the present invention is to first provide a method for efficiently identifying exogenous DNA insertion sites in a genome using high-throughput sequencing technology, the method comprising the following steps:

[0012] (a) Sample extraction, library construction and sequencing;

[0013] (b) Data quality control;

[0014] (c) data comparison;

[0015] (d) filtering the comparison results;

[0016] (e) preliminary screening;

[0017] (f) Screening and identification of the location of exogenous inserted fragments:

[0018] In certain embodiments, the screening and identification of the position of the exogenous insertion fragment comprises:

[0019] (f1) The bam files were filtered and reads were retained, where read1 and read2 were simultaneously aligned to the same chromosome and the distance between the two reads was less than 400 bp, and the alignment was determined to be accurate; or reads where one read was aligned to the reference genome chromosome and the other was aligned to the exogenous insertion sequence;

[0020] (f2) retain and output the reads with logical optimal and suboptimal alignment directions. The specific situation of logical alignment is: read1 partially aligns to the target sequence and partially aligns to the insertion sequence. The alignment directions of the two parts need to be consistent. At the same time, the direction of read2 to align to the target sequence or the insertion sequence must be opposite to the alignment direction of read1 and the alignment position distance is within 400bp; determine the output exogenous sequence insertion position according to the reads support number, including the chromosome number and the starting position, and display it visually;

[0021] (f3) Determine the flanking sequence of the site based on the read coverage depth within 1Kbp upstream and downstream of the insertion site, and output the base sequence for subsequent experimental verification.

[0022] In certain embodiments, the sample extraction, library construction and sequencing uses automated equipment for DNA extraction, library construction and sequencing, and the sequencing depth is at least 30X data.

[0023] In certain embodiments, the data quality control uses fastp software or fastqc software to perform quality control on the high-throughput sequencing data results to obtain clean data.

[0024] In certain embodiments, the data comparison is as follows: first, the genome sequence of the edited sample is merged with the sequence of the exogenous insert to construct a reference genome sequence for analysis, and the clean data is compared therewith using BWA software.

[0025] In certain embodiments, the comparison result filtering is performed by using samtools software to sort the comparison result bam file obtained in the previous step, and then removing redundancy to obtain a valid comparison result file, and constructing an index for it.

[0026] In certain embodiments, the preliminary screening: uses extractSplitReads_BwaMem in lumpy software to identify split reads in bam files and outputs them as new split bams; uses samtools software to propose reads in split bams that are aligned to exogenous insertion sequences as new bams, and converts flags in split bams, converting flag numbers into different letter combinations, in order to reflect the specific properties of reads alignment.

[0027] The second object of the present invention is to provide an application of the above method, which is to locate the position where the exogenous fragment sequence of the vector is inserted into the chromosome in the transgenic plant.

[0028] The third object of the present invention is to provide a product for implementing the above method, wherein the product is a computer program, or a carrier containing a computer program, or a calculator, or a computing module device.

[0029] In certain embodiments, the product comprises a sample extraction, library construction and sequencing module, a sequencing data processing module, an exogenous insertion fragment position screening and identification module and a result display module.

[0030] The fourth object of the present invention is to provide an application of the above-mentioned product, characterized in that the application is to locate the position where the exogenous fragment sequence of the vector is inserted into the chromosome in the transgenic plant.

[0031] Compared with the prior art, the present invention has at least the following beneficial effects:

[0032] (1) This device uses high-throughput sequencing technology, with short sequencing cycle, low cost and large data volume;

[0033] (2) This device only needs to perform one comparison, saving time;

[0034] (3) This device is based on software judgment, the results are accurate, and the process is efficient;

[0035] (4) One-click output of candidate site location, sequence information, and insertion direction reduces manual operation steps and saves manpower. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 is the flow chart of the device;

[0037] Figure 2 It is the split reads graph;

[0038] Figure 3 This is an example graph of reads with logical alignment directions;

[0039] Figure 4 Visualization of insertion position and coverage;

[0040] Figure 5 Insertion position information and flanking sequence diagram for output;

[0041] Figure 6 It is an alignment diagram of exogenous T-DNA fragments; the green part is the genome sequence, the yellow part is the exogenous T-DNA fragment, and the framed part is the primer designed to verify the sequence. DETAILED DESCRIPTION

[0042] In order to make the technical problems, technical solutions and advantages to be solved by the present invention clearer, the following will be described in detail with reference to the accompanying drawings and specific embodiments. The materials, reagents, etc. used in the following embodiments, unless otherwise specified, can be obtained from commercial sources.

[0043] Example 1: A method for efficiently identifying exogenous DNA insertion sites in a genome using high-throughput sequencing technology

[0044] The method flow chart is as follows Figure 1 As shown, the following steps are included:

[0045] (a) Sample extraction, library construction and sequencing: DNA was extracted and library constructed using domestic automated equipment. High-throughput sequencing was performed using the PE150 sequencing strategy using the T7 sequencer manufactured by MGI, a domestic platform, with a sequencing depth of at least 30X data;

[0046] (b) Data quality control: Use fastp software to perform quality control on high-throughput sequencing data results to obtain clean data;

[0047] (c) Data alignment: First, the genome sequence of the edited sample was merged with the sequence of the exogenous insert to construct a reference genome sequence for analysis, and the clean data was aligned with it using BWA software;

[0048] (d) Filtering the alignment results: Use samtools software to sort the alignment result bam files obtained in the previous step, and then remove redundancy to obtain a valid alignment result file, and then build an index for it;

[0049] (e) Preliminary screening: Use extractSplitReads_BwaMem in lumpy software to identify split reads in bam files and output them as new split bams. Figure 2 ) can accurately determine the breakpoint of structural variation in the genome. When it is detected that part of the reads is aligned to a region, and the other part is aligned to a distant region or another chromosome, the position of the break can be determined. The samtools software is used to propose the reads aligned to the exogenous insertion sequence in the split bam as a new bam, and the flag in the split bam is converted to a different letter combination, aiming to reflect the specific properties of the reads alignment.

[0050] (f) Exogenous insertion fragment position screening and identification device:

[0051] (f1) The bam files were filtered and reads were retained, where read1 and read2 were simultaneously aligned to the same chromosome and the distance between the two reads was less than 400 bp, and the alignment was determined to be accurate; or reads where one read was aligned to the reference genome chromosome and the other was aligned to the exogenous insertion sequence;

[0052] (f2) Keep and output the reads with the best and suboptimal alignments in a logical direction. The specific situation of logical alignment is: read1 partially aligns to the target sequence and partially aligns to the inserted sequence. The two alignment directions need to be consistent. At the same time, read2 needs to align to the target sequence or the inserted sequence in a direction opposite to the alignment direction of read1 and the alignment position distance is within 400bp (see Figure 3 ). According to the reads support number, the insertion position of the output exogenous sequence is determined, including the chromosome number and the starting position, and a visual display is performed (see Figure 4 );

[0053] (f3) Determine the flanking sequence of the site based on the read coverage depth within 1Kbp upstream and downstream of the insertion site, and output the base sequence for subsequent experimental verification.

[0054] Example 2 A method for efficiently identifying exogenous DNA insertion sites in black bean genome using high-throughput sequencing technology

[0055] (a) Sample extraction, library construction and sequencing: DNA was extracted and library constructed from black bean leaf samples using domestic automated equipment. High-throughput sequencing was performed using the PE150 sequencing strategy using the T7 sequencer manufactured by MGI, a domestic platform, with a sequencing depth of 62X data.

[0056] (b) Data quality control: fastp (v0.23.0) software was used to perform quality control on the high-throughput sequencing data results (parameters: fastp--qualified_quality_phred 5--unqualified_percent_limit 50--n_base_limit 15) to obtain clean data;

[0057] (c) Data alignment: First, the genome sequence of the edited sample (Glycine-max Wm82.a4.v1) was merged with the exogenous insert sequence (T-DNA) to construct the reference genome sequence used for analysis, and the clean data was aligned with it using BWA (v2.2.1) software (parameters: bwa mem);

[0058] (d) Alignment result filtering: Use samtools (v1.15) software to sort the alignment result bam file obtained in the previous step, and then remove redundancy to obtain a valid alignment result file, and then build an index for it (parameters: samtoolssort, samtools markdup, samtools index);

[0059] (e) Preliminary screening: Use extractSplitReads_BwaMem (parameters: default) in lumpy (v0.2.13) software to identify split reads in the bam file and output them as new split bams. Figure 2) can accurately determine the breakpoint of structural variation in the genome. When it is detected that part of the reads is aligned to a region, and the other part is aligned to a distant region or another chromosome, the position of the break can be determined. Use samtools software to propose the reads aligned to the exogenous insertion sequence in the split bam as a new bam (parameter: samtools view T-DNA_id), and convert the flag in the bam, converting the flag number to a different letter combination (parameter: samtoolsview-X), in order to reflect the specific properties of the reads alignment.

[0060] (f) Exogenous insertion fragment position screening and identification device: A Python program written according to the screening logic screens and identifies the position of the exogenous insertion fragment to obtain the insertion position and flanking sequence;

[0061] (g) Experimental verification: Primers were designed based on the flanking sequences, PCR amplification was performed, and the amplified products were verified by first generation sequencing results. The output insertion position information and flanking sequences are as follows Figure 5 As shown. Figure 6 As shown, the green part is the genome sequence, the yellow part is the exogenous T-DNA fragment, and the framed part is the primer designed to verify the sequence.

[0062] Results: The existing detection methods did not further screen the insertion position according to logic, and the subsequent positions obtained were mostly (176), which were cumbersome to verify and had false positives.

[0063] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for efficiently identifying exogenous DNA insertion sites in a genome using high-throughput sequencing technology. It is characterized in that The method comprises the following steps: (a) Sample extraction, library construction and sequencing; (b) Data quality control; (c) data comparison; (d) filtering the comparison results; (e) preliminary screening; (f) Screening and identification of the location of exogenous inserted fragments.

2. The method according to claim 1, characterized in that The sample extraction, library construction and sequencing uses automated equipment for DNA extraction, library construction and sequencing, and the sequencing depth is at least 30X data.

3. The method according to claim 1, characterized in that The data quality control uses fastp software or fastqc software to perform quality control on the high-throughput sequencing data results to obtain clean data.

4. The method according to claim 1, characterized in that The data comparison is as follows: first, the genome sequence of the edited sample is merged with the sequence of the exogenous inserted fragment to construct a reference genome sequence for analysis, and the clean data is compared with it using BWA software.

5. The method according to claim 1, characterized in that The comparison result filtering: use samtools software to sort the comparison result bam file obtained in the previous step, and then remove redundancy to obtain a valid comparison result file, and construct an index for it.

6. The method according to claim 5, characterized in that The preliminary screening: use extractSplitReads_BwaMem in lumpy software to identify split reads in bam files and output them as new split bams; use samtools software to propose reads in split bams that are aligned to exogenous insertion sequences as new bams, and convert the flags in split bams to convert the numbers in the flags into different letter combinations, in order to reflect the specific properties of the reads alignment.

7. Use of the method according to any one of claims 1 to 6, characterized in that: The application is to locate the position where the exogenous fragment sequence of the vector is inserted into the chromosome in the transgenic plant.

8. A product implemented by the method according to any one of claims 1 to 6, characterized in that: The product is a computer program, or a carrier containing a computer program, or a calculator, or a computing module device.

9. The product according to claim 8, characterized in that The product comprises a sample extraction, library construction and sequencing module, a sequencing data processing module, an exogenous insertion fragment position screening and identification module and a result display module.

10. Use of the product according to any one of claims 8 to 9, characterized in that: The application is to locate the position where the exogenous fragment sequence of the vector is inserted into the chromosome in the transgenic plant.

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