CN111676276A - A method for rapid and accurate determination of gene editing mutation and its application - Google Patents

Abstract

The invention relates to a method and application for quickly and accurately determining gene editing mutation, and belongs to the technical field of gene detection. The method includes the following steps: 1) designing primers based on the target interval, amplifying, constructing a library, and sending high-throughput sequencing; 2) performing quality control on the sequencing data, removing sequences with a quality value less than 30; removing the "N" base content More than 10% of the sequences; 3) splicing at both ends to obtain long sequences, and counting and counting the number of different samples; 4) aligning the data obtained by S3 with the target genome sequence to obtain analysis results. The invention can quickly and efficiently detect the gene editing mutation of each sample, and the cost is low. In addition, 1G of sequencing data can be obtained in only 1-2 minutes, which greatly improves the analysis speed.

Description

A method for rapid and accurate determination of gene editing mutation and its application

【Technical field】

The invention relates to the technical field of gene detection, in particular to a method for rapidly and accurately determining the mutation status of gene editing and its application.

【Background technique】

Genome editing technology has been widely used in many fields such as basic research, gene therapy and genetic improvement. However, after the gene editing is completed, sequencing analysis is required to obtain the accurate mutation status and mutation type of the mutants. Sanger sequencing is not only expensive, but also has low throughput, and it is impossible to obtain the exact mutation type of the edited product through one sequencing result. High-throughput sequencing is not only low in cost, but also has been proven in relevant articles to achieve accurate identification of genotypes. However, the currently used high-throughput sequencing-based identification method for gene editing mutations is to re-comparison all sequencing reads. Back to the reference genome, and then perform mutation analysis and statistics. Open document "Hi-TOM: a platform for high-throughput tracking of mutations induced by CRISPR/Cassystems" (Liu Q et al. [J]. SCIENCE CHINA Life Sciences, Vol. 62, No. 1, pp. 1-7, 2019, 01 ) introduced the widely used Hi-TOM high-throughput mutation analysis platform, which can identify gene mutations in different species in large quantities. However, the analysis platform has the disadvantage of a long analysis process. Since it compares relatively short reads, it is likely to be compared to many places, so some sequences with high similarity cannot be compared to accurate results. 1G The analysis and processing process of the large amount of sequencing data may take more than an hour or longer, and sometimes the identification results cannot be obtained, and the analysis process and sequence design are relatively fixed, with poor flexibility, and cannot meet some specific analysis needs.

Therefore, this method intends to develop a set of analysis procedures, which can accurately obtain large-scale identification of mutant gene editing in a short period of time, and can design experiments and analysis procedures according to different specific needs. Therefore, it is necessary to take advantage of high-throughput sequencing to develop a cost-effective, rapid and precise method for determining gene editing mutations.

[Content of the invention]

Aiming at the deficiencies of the prior art, the present invention provides a method for determining gene editing mutations, which is based on the advantages of high-throughput sequencing to achieve the goals of low cost, rapidity and accuracy.

In order to solve the above problems, the present invention provides a method for quickly and accurately determining gene editing mutation, including the following steps:

1. Design primers based on the target interval, amplify, construct the library, and send it to high-throughput sequencing;

2. Quality control of sequencing data;

3. Splicing the reads at both ends of the quality-controlled sequencing data to obtain long sequences; then use statistical software or scripts to count samples with the same sequence as one, and count the number of different samples;

4. Compare the data obtained in step 3 with the target genome sequence, identify different base sequences, obtain analysis results, and determine gene mutation.

Further, when designing primer amplification in the step 1, the overlapping interval between the left and right ends of the sequencing data covering the target interval is greater than 8bp;

Further, the PE100 or PE250 method is used for sequencing in the step 1; the PE150 sequencing method is to construct a 100-260 bp library, and the PE250 sequencing method is used to construct a 100-460 bp library;

Further, the sequencing in the step 1 is based on the Illumina sequencing platform, and different barcode sequences are added to distinguish when multiple samples are mixed for sequencing.

Further, in the step 2, the quality control steps are: removing reads with a quality value less than 30; removing reads with an "N" base content that accounts for more than 10% of the total reads.

Further, in the step 3, according to the quality control of the obtained data, the reads that account for less than 5% of the total reads in the statistical count are removed.

Further, the target genome sequence in the step 4 includes the target interval in the step 1; the size of the target genome sequence is 500-1000bp;

Further, the comparison method in the step 4 is to first establish an index on the target genome sequence, and then align the data in the step 3 back to the target genome sequence.

Another object of the present invention is to provide a method for quickly and accurately determining gene editing mutations based on high-throughput sequencing in screening gene mutants.

Further, the gene mutants include mutants obtained by artificial methods and natural methods;

Further, the gene mutants include plant mutants, animal mutants and microbial mutants.

Compared with the prior art, the beneficial effects of the present invention are:

1) Fast: 1G second-generation sequencing data, the whole process only takes 1-2 minutes to obtain statistical results;

2) Accuracy: Based on the high-throughput advantage of next-generation sequencing technology, thousands of sequencing sequences can be obtained for each sample, which has significant statistical value;

3) Low cost: Multiple samples can be mixed for sequencing, and the mixed sequencing of PCR products amplified by more than 100 samples only needs 1G of data to meet the analysis requirements, which greatly reduces the cost of analysis.

【Description of drawings】

In order to illustrate the technical solutions of the embodiments of the present invention more clearly, the following briefly introduces the accompanying drawings used in the embodiments. It should be understood that the following drawings only show some embodiments of the present invention, and therefore do not It should be regarded as a limitation of the scope, and for those of ordinary skill in the art, other related drawings can also be obtained according to these drawings without any creative effort.

Figure 1 is a schematic diagram of the PCR amplification product of the present invention.

FIG. 2 is a schematic diagram of the data analysis process flow of the present invention.

FIG. 3 is a partial result comparison diagram of the target genome comparison of Example 1 of the present invention.

FIG. 4 is a comparison diagram of the comparison results of target genomes in Example 2 of the present invention.

【Detailed ways】

The following examples are intended to illustrate the present invention, but not to limit the scope of the present invention. Modifications or substitutions made to the methods, steps or conditions of the present invention without departing from the spirit and essence of the present invention all belong to the scope of the present invention.

Example 1

In the following examples, 7 rice edited seedlings with different targets for genome editing based on CRISPR-Cas9 technology were selected, a total of 96 samples were numbered A1-H12.

1. Primer Design

Since the specific primers and the high-throughput sequencing adapter primers are too long, in order to achieve the purpose of building the library, the multiplex PCR library building technology is chosen to complete the enrichment of the target region and the purpose of building the library through two rounds of PCR.

The first round of primers should include the following three parts: 1) specific amplification primer sequence; 2) sample tag sequence; 3) bridging sequence.

The second round of primers should include: 1) bridging sequence; 2) index sequence; 3) sequencing linker sequence. The schematic diagram of the PCR primers can be referred to as shown in Figure 1.

In this example, the PE150 sequencing method is selected for the design. The designed forward primer and reverse primer are between 70bp and 120bp from the target. The sequencing data at the left and right ends cover the target interval and the overlap interval is greater than 8bp. And the size after barcode is between 100bp-260bp.

2. Genome extraction and target fragment amplification

In order to achieve the purpose of rapidly extracting genomes in large quantities, the following quick genome extraction steps are adopted: Place 2mL centrifuge tubes corresponding to plant samples on a 2mL 96-well centrifuge tube rack, mark them well, and take 3-5cm of rice. The leaves of the mutant seedlings were added to the centrifuge tube. Add 50uL of 0.2mol KOH solution and 1-2 grinding beads to each well, respectively. At room temperature, transfer the 2mL centrifuge tubes to the adapters of the automatic grinder, and run for 30-60 seconds until the buffer turns green. 2uL of the supernatant was pipetted into a 96-well PCR plate using a discharge gun, and used as a template for PCR. Use the first round of primers for PCR amplification to obtain the target band. After PCR, take 3-5ul of the product for electrophoresis detection to ensure that the target band is amplified.

The first round PCR amplification system and amplification procedures are shown in Table 1 below.

Table 1: First-round PCR amplification system and amplification procedure

The second round of amplification was carried out with the first round product as the template, and the amplification system and amplification procedure are shown in Table 2.

Table 2: Second-round PCR amplification system and amplification procedure

After PCR amplification, electrophoresis detection, and finally mixing in equal proportions, high-throughput sequencing is sent. The amount of sequencing data is more than 1G. You can design any number of bases by yourself and add different barcode sequences to distinguish.

3. Analysis of sequencing results

After obtaining the sequencing data, analyze the data according to the data analysis process as shown in Figure 2. Generally, the sequencing company will deliver the clean data to the customer, which is the data that has undergone preliminary data quality control (you can also obtain the raw data and manually perform the preliminary data quality control. control). Obtain the sequencing data, split the samples according to the sub-sample tag sequence, obtain the sequencing data of 96 samples respectively, and then carry out the following analysis work:

3.1 Data quality control

The high-throughput data quality control software Fastp can be used to perform quality control processing on the sequencing data. According to the quality control requirements: the base quality value should be set to 30, and it is only qualified if it is greater than or equal to 30. Otherwise, the reads will be removed; "N" base reads.

3.2 Sequence splicing

The programmed script or sequence splicing software can be used to splicing the sequencing reads at both ends according to the characteristics of overlapping fragments of the sequencing reads at both ends to obtain a spliced sequence of higher quality and longer sequence.

3.3 De-redundancy and statistics

The existing deduplication statistical software or script can be used to keep only one duplicate data (same sequence) of each sample and perform statistical counting. Since the amplified fragments of the same target of the same sample have basically the same size, the sequence of the sequencing and splicing can be obtained. It is basically the same, so only one duplicate data is kept and counted, which can greatly reduce the amount of data for subsequent alignment back to the target sequence. In order to ensure the accuracy of the results, the filter value should be set to remove the sequences with a small number of counts. Generally, the counted reads should account for more than 5% of the total reads and can be kept and recorded. After deduplication processing, the amount of data will be greatly reduced.

3.4 Alignment with the target genome

In order to obtain the alignment results more quickly, the target genome is indexed first, and then the de-redundant sequences after the above processing are aligned back to the target genome. Based on the Burrow-Wheeler Aligner principle, if the alignment is different from the reference genome, it is determined that there is a mutation. In this process, it only takes a few seconds for 96 samples to be compared and analyzed at the same time (including the entire process time of the sequencing data processing process in this embodiment is 1-2 minutes) to obtain the comparison results shown in Table 3 and FIG. 3 . Not only can we intuitively understand the mutation status of each target site, but also the specific mutation status and the number of supported reads of each sample. The sequenced spliced sequences obtained for each sample can also be used for subsequent more in-depth analysis and research.

Table 3: Comparative analysis statistics of some samples (A01-B12)

Example 2

The Prime editor (PE) developed based on the CRISPR-Cas9 system can not only introduce insertions and deletions (indels) but also realize all 12 base-to-base conversions. It is a new generation editing tool. However, its editing efficiency is currently low, and it is difficult to accurately determine the editing effect using next-generation sequencing methods. Therefore, this example is based on the identification of editing efficiency after gene editing of 293T cells using the PE editing system.

1. Primer Design

Since the specific primers and the high-throughput sequencing adapter primers are too long, in order to achieve the purpose of building the library, the multiplex PCR library building technology is chosen to complete the enrichment of the target region and the purpose of building the library through two rounds of PCR. The first round of primers should include the following three parts: 1) specific amplification primer sequence; 2) sample tag sequence; 3) bridging sequence. The second round of primers should include: 1) bridging sequence; 2) index sequence; 3) sequencing linker sequence. The schematic diagram of the PCR primers can be referred to as shown in Figure 1.

In this example, the PE250 sequencing method is selected for the design. The designed forward primer and reverse primer are between 70bp and 120bp from the target. The sequencing data at the left and right ends cover the target interval and the overlap interval is greater than 8bp. The amplified fragment plus the sequencing adapter and The size after barcode is between 100-460bp.

2. Genome extraction and target fragment amplification

There is no need for additional genome extraction from cells and bacterial solutions, and only the edited cells or bacterial solutions need to be directly amplified by PCR. Use the first round of primers for PCR amplification to obtain the target band. After PCR, take 3-5ul of the product for electrophoresis detection to ensure that the target band is amplified.

The first round PCR amplification system and amplification procedures are shown in Table 4.

Table 4:

The first-round product was used as a template for the second round of amplification. The amplification system and amplification procedure are shown in Table 5.

table 5:

After the PCR amplification is completed, electrophoresis is performed, and after mixing in equal proportions, it is sent to high-throughput sequencing, and the amount of sequencing data is more than 1G.

3. Analysis of sequencing results

The data analysis process of this embodiment is the same as that of Embodiment 1, and the data analysis process is shown in FIG. 2 . Due to the low efficiency of PE editing, the filter value (the percentage of counted reads to the total number of reads) is not set here. The filter value can be set according to the experimental requirements, but the rest of the quality control conditions cannot be changed. Detailed analysis results can be obtained as shown in Table 6 and Figure 4, which can accurately understand the editing results and proportions of the PE system. It can be clearly observed that PE editing can achieve indels and point mutations in the genome, but the editing efficiency is low.

Table 6: Statistics of 293T cell comparison analysis

Comparative Example 1

Using the HI-TOM method (Hi-TOM: a platform for high-throughput tracking of mutations induced by CRISPR/Cas systems" Liu Q et al. [J]. SCIENCE CHINA LifeSciences, Vol. 62, No. 1, pp. 1-7, 2019, 01), based on CRISPR-Cas9 technology for genome editing of 7 different target rice edited seedlings for data processing and comparison analysis, a total of 96 samples, numbered A1-H12.

The obtained data was compared with the target genome. After more than one hour, it was found that all the results could not be obtained. The results are shown in Table 7, in which there is no data after A03 to A12 and B03.

Table 7:

The analysis and comparison results obtained in Comparative Example 1 are compared with those in Example 1, which proves that the method for determining gene editing mutation status based on high-throughput sequencing adopted in the present invention can obtain all results in a short time and is superior in terms of accuracy and speed. sex.

Comparative Example 2

Using the HI-TOM method (same as Comparative Example 1), the editing efficiency of 293T cells after gene editing was identified based on the PE editing system.

The obtained data was compared with the target genome, and no comparison result was obtained after more than one hour.

Compared with Example 2, the analysis and comparison results obtained in Comparative Example 2 prove that the method for determining gene editing mutation status based on high-throughput sequencing adopted in the present invention can obtain all results in a short period of time and is superior in accuracy and speed. sex.

The present invention is not limited only to what is described in the specification and embodiments, so that additional advantages and modifications can easily be realized by those skilled in the art, without departing from the spirit and scope of the general concept defined by the claims and equivalents However, the invention is not limited to the specific details, representative aspects and illustrative examples shown and described herein.

Claims (10)

1. A method for quickly and accurately determining a gene editing mutation, comprising the following steps: S1. Design primers based on the target interval, amplify, construct a library, and send for high-throughput sequencing; S2. Quality control the sequencing data; S3. Splicing the reads at both ends of the sequencing data after quality control to obtain long sequences, and counting them; the counting method is: using statistical software or scripts, counting samples with the same sequence as one, and counting the number of different samples and count; S4. Compare the data obtained in S3 with the target genome sequence, identify different base sequences, obtain analysis results, and determine gene mutation. 2 . The method for quickly and accurately determining gene editing mutations according to claim 1 , wherein, when designing primers to amplify in step S1 , the sequencing data at the left and right ends cover the target interval overlapped by more than 8 bp. 3 . 3. A kind of method for quickly and accurately determining gene editing mutation situation according to claim 1, it is characterized in that, described in the step S1, the sequencing uses one of PE150 sequencing method and PE250 sequencing method; using the PE150 sequencing method The sequencing method is to construct a 100-260 bp library, and when using the PE250 sequencing method, a 100-460 bp library is constructed; the sequencing is based on the Illumina sequencing platform, and when the sequencing is multi-sample mixed sample sequencing, different barcode sequences are added to distinguish. 4. The method for quickly and accurately determining gene editing mutations according to claim 1, wherein in the step S2, the quality control method is: removing reads with a quality value less than 30; removing "N" Reads with base content greater than 10%. 5. The method for quickly and accurately determining gene editing mutations according to claim 1, wherein the step S3 further comprises de-redundancy: removing the ratio of the total number of reads in the statistical count less than 5%. reads. 6 . The method for quickly and accurately determining gene editing mutations according to claim 1 , wherein the target genome sequence in step S4 includes the target interval in step S1 ; the target genome sequence Size is 500-1000bp. 7. The method for quickly and accurately determining gene editing mutations according to claim 1, wherein the comparison method in step S4 is to first establish an index on the target genome sequence, and then perform the step The data in S3 is aligned back to the target genome sequence. 8. The application of the method for rapidly and accurately determining gene editing mutations according to any one of claims 1-6 in screening gene mutants. 9 . The application according to claim 7 , wherein the gene mutants include mutants obtained by artificial methods and natural methods. 10 . 10. The application according to claim 7, wherein the gene mutants include plant mutants, animal mutants and microorganism mutants.

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