WO2020199127A1

WO2020199127A1 - Design of sequencing primers and pcr-based method for sequencing whole genome

Info

Publication number: WO2020199127A1
Application number: PCT/CN2019/081029
Authority: WO
Inventors: 夏志强; 邹枚伶
Original assignee: 中国热带农业科学院热带生物技术研究所
Priority date: 2019-04-02
Filing date: 2019-04-02
Publication date: 2020-10-08

Abstract

Disclosed are a sequencing primer set, comprising a universal upstream primer, a universal downstream primer, and a promoter-enriched downstream primer; and a PCR-based method for sequencing a whole genome, the method comprising: by using the universal upstream primer and the universal downstream primer, performing first PCR amplification on the DNA of a sample to be sequenced, so as to obtain a first amplification product, and by using the universal upstream primer and the universal downstream primer, performing second PCR amplification on the DNA of the sample to be sequenced, so as to obtain a second amplification product; and simultaneously performing sequencing on the two amplification products. According to the above-mentioned method, the genome size and the sample amount can be adjustable, and the requirements for DNA quality are not high; there is no need for known SNP markers and for pre-construction of chips; and the method can meet requirements for the detection and screening of the whole genome.

Description

Design of sequencing primers and PCR-based whole-genome sequencing method

Technical field

The present invention relates to the field of gene sequencing, in particular to the design of sequencing primers and a PCR-based whole genome sequencing method.

Background technique

As genotyping is a series of genetic analysis, including the use of high-throughput sequencing technology for molecular marker discovery and genotyping, one of the most powerful applications is in the field of plant breeding, opening up new ideas for plant breeding and plant genetics research. Possibility. It provides a cost-effective whole genome scanning and multiple sequencing platform. Simplified sequencing technology is a new application of high-throughput sequencing for mining single nucleotide polymorphisms (SNPs) in species populations and genotyping them. Bioinformatics tools are needed to analyze and parse simplified sequencing data sets. It is a low-cost technology and an excellent MAS tool. Simplified sequencing has been successfully applied to genome-wide association analysis, molecular marker mining, genetic linkage map, genomic genetic selection, and population genomic diversity research in large-scale plant molecular breeding strategies.

Simplified genome sequencing is developed on the basis of second-generation sequencing. It uses enzyme digestion technology, sequence capture chip technology or other experimental methods to reduce the complexity of the species genome, sequence specific regions of the genome, and then reflect the structural information of part of the genome sequence. Sequencing technology. Simplified genome sequencing currently developed includes: polymorphic sequence sequencing with reduced complexity, DNA sequencing related to restriction enzyme sites, and genotyping sequencing.

A simple, fast and cost-effective system has been used for sequencing in non-model organisms. Among them, the most widely used is the sequencing technology of restriction enzyme site-related DNA, which uses restriction enzymes to digest the genome, generate fragments of a certain size, construct a sequencing library, and limit the restriction generated after digestion. Restriction site marking for high-throughput sequencing. Since restriction enzyme site markers are genome-wide small fragments of DNA tags near specific restriction sites, which represent the sequence characteristics of the entire genome, the restriction enzyme site markers can be sequenced in most Thousands of single nucleotide polymorphism markers are obtained in biology.

Sequencing-based genotyping is a new application of high-throughput sequencing solutions for the discovery and genotyping of single nucleotide polymorphisms for crop improvement. The low cost of simplified sequencing makes it an attractive method to completely map and propagate populations through high-density single nucleotide polymorphism markers. The continuous improvement of sequencing and base calling software will enable high-throughput sequencing technology to provide higher sequencing throughput for each run, thereby achieving deeper multiplexing to obtain a fixed average sequencing depth for each sample. With the continuous increase in the quantity and quality of sequence information generated by each run, which makes the plex of each sample higher and lower in cost, simplified sequencing has become a cost-competitive alternative to other whole-genome genotyping platforms. Plant breeders will be able to sequence the genomes of large crops and build high-density genetic linkage maps from breeding populations. The future application of simplified sequencing in crop improvement may allow plant breeders to mark new germplasm or species without first developing any previous molecular tools. Since sequence-based genotyping can be used for entire genome research, simplified sequencing will become one of the main components of plant genetics and breeding.

The identification of high-density single-nucleotide polymorphism markers through simplified sequencing to construct a genetic map is of great value for many applications in plant breeding.

Genotyping chips are designed to design probes using the sequences flanking the known SNPs. After the probe is fixed on the chip, the DNA of the sample to be tested hybridizes with the chip and the hybridization fluorescence signal is scanned to identify the genotype of these probe sites (sites of single nucleotide polymorphism). The most representative brands are illumina and affymetrix.

Single nucleotide polymorphism chips have become an important tool for crop genetic improvement. The era of molecular breeding has come in an all-round way. In order to solve the shortcomings of molecular marker detection methods, traditional breeding cycles are long and unpredictable, relying on the experience of breeders and visual screening. The field can be moved to the laboratory for large-scale precision screening, and more than 95% of the individual plants can be excluded, and a small number of individual plants can be planted in the field, which greatly reduces the workload of the field. The original method has an average breeding cycle of 8 to 10 years for a variety, and this method can be completed in 3 to 5 years.

At present, the chip has very high practical value in the rapid identification of breeding materials, genome selection, germplasm resource analysis, variety improvement, QTL mapping, genetic analysis, and variety authenticity identification, and has great cost advantages. However, the current use of chips has some difficulties that limit the wide application of chips in crop genetic improvement. The main difficulties are: (1) At present, the cost of early-stage development of the chip is relatively high, and it needs to have a sequenced reference genome and known SNP markers; only known SNP sites can be detected, and the number of markers obtained is small; only existing SNP markers can be detected , It is impossible to find new SNP sites; the detection methods and instruments are highly resistant; currently only a few breeding model species have corresponding chips; (2) the simplified sequencing cost is not low enough, because the library construction is complicated and the efficiency is not high enough. The detection of large breeding samples is difficult to be widely used; due to the need for enzyme digestion methods, simplified sequencing requires high DNA quality, and it is not possible to use directly de-sorted DNA (direct PCR); the amount of sequencing between samples is uneven during library construction Features need to be sequenced; there is no enrichment effect of gene regions.

technical problem

The embodiment of the present invention provides a sequencing primer set and a PCR-based whole-genome sequencing method, which aims to solve the technical problems of slow speed, high requirements, and complicated operations in screening genomes of SNP sites in the existing chip.

Technical solutions

The embodiments of the present invention are implemented in this way. In the first aspect, a sequencing primer set is provided, and the sequencing primer set includes a universal upstream primer, a universal downstream primer, and an enriched promoter downstream primer;

The sequence of the universal upstream primer includes: 5'-T[barcode]CAAAXXXXNNN-3';

The sequence of the universal downstream primer includes: 5'-GACTGCGTACGZZZZNNN-3';

The sequence of the primer downstream of the enriched promoter includes: 5'-GACTGCGTACYYNCTATA-3';

Wherein, the length of "XXXX" is 4 bases, and the base is selected from at least one of A, T, C and G; the length of "ZZZZ" is 4 bases, and the base is selected From at least one of A, T, C and G; the length of the "barcode" is 4-6 bases, and the base is selected from at least one of A, T, C and G; the "N "Is any one of bases A, T, C, and G, and the "Y" is base C or T.

In the second aspect, a PCR-based whole genome sequencing method is provided, which includes the following steps:

Prepare the DNA of the sample to be tested;

The sequencing primer set of any one of the above is provided, the first PCR amplification is performed on the DNA of the test sample using the universal upstream primer and the universal downstream primer to obtain the first amplification product, and the universal upstream primer and The downstream primer of the enriched promoter performs a second PCR amplification on the DNA of the test sample to obtain a second amplification product;

The first amplification product and the second amplification product are respectively subjected to sequencing analysis.

Beneficial effect

The sequencing primer set provided by the present invention includes a universal upstream primer with a unique sequence, a universal downstream primer, and an enriched promoter downstream primer. The universal upstream primer and the universal downstream primer include four bases: "XXXX" and "ZZZZ" ( The bases are selected from at least one of A, T, C and G in combination), these eight bases are the core base region of the universal primer, which can be changed according to the changes in the genome of the sample to be tested. Different, so that it can amplify different sequence fragments on different genomes, the primer scheme can be optimized according to the sequence of the genome, and the size of the genome and the size of the sample can be adjusted arbitrarily; at the same time, the design has a starter by simulating the base sequence of the upstream region of eukaryotes The primers that are characteristic of the Pormoter region are the downstream primers of the enriched promoter, so that the universal upstream primer and the downstream primer of the enriched promoter can effectively amplify the sequence near the promoter. Such a primer set is used for whole-genome amplification and sequencing, not only has a good enrichment effect, but also makes library construction and sequencing more concise and accurate, and reduces sequencing costs.

At the same time, the present invention provides a PCR-based whole-genome sequencing method, which amplifies the DNA of the test sample by using the above-mentioned sequencing primer set to obtain the PCR product of the test sample, and then performs sequencing analysis. The above-mentioned PCR-based whole-genome sequencing method takes into account the characteristics of "discovering" and "detecting" single nucleotide polymorphism (SNP) sites, and can detect and screen the whole genome while new hair styles are marked. Therefore, There is no need to build a chip in the early stage, and no need to mark the known single nucleotide polymorphism (SNP) sites; at the same time, using this method for whole genome sequencing does not require high sample DNA quality, and the library construction is more concise and convenient , At the same time, it has high coverage, gene enrichment effect, short sequencing time, and can be widely used in genome sequencing of different samples.

Description of the drawings

Figure 1 shows the results of agarose electrophoresis analysis of the PCR products of 5 samples with universal primers provided in Example 1 of the present invention.

Fig. 2 is the agarose electrophoresis analysis result of five samples enriched in Pormoter region primer PCR products provided in Example 1 of the present invention.

Figure 3 is a graph of the genome coverage analysis of sequencing reads obtained by the universal primer method.

Figure 4 is a graph of genome coverage analysis of sequencing reads obtained by enriching Pormoter region primers.

Figure 5 shows the enrichment of PCR products in the gene region.

Embodiments of the invention

In order to make the objectives, technical solutions, and advantages of the present invention clearer, the following further describes the present invention in detail with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described here are only used to explain the present invention, but not to limit the present invention.

An example of the present invention provides a sequencing primer set, which includes a universal upstream primer, a universal downstream primer, and an enriched promoter downstream primer;

Wherein, the length of "XXXX" is 4 bases, and the base is selected from at least one of A, T, C and G; the length of "ZZZZ" is 4 bases, and the base is selected From at least one of A, T, C and G; the length of the "barcode" is 5 bases, and the base is selected from at least one of A, T, C and G; the "N" is Any one of bases A, T, C, and G, and the "Y" is base C or T.

Specifically, the length of "XXXX" is 4 bases, and the base is selected from at least one of A, T, C, and G; the length of "ZZZZ" is 4 bases, and the base is At least one selected from A, T, C, and G. These eight bases are the core base region of the universal primer, which can be changed according to the changes in the genome of the sample to be tested, so it can amplify different sequence fragments on different genomes according to the difference of bases. Specifically, the "barcode" is designed on the universal upstream primer, and its length is 5 bases, and it is any combination of bases A, T, C, and G. Preferably, the "barcode" is preferably 5 bases, and based on any combination of bases A, T, C, and G, a total of 384 "barcodes" are available for selection. In a preferred embodiment of the present invention, any number of "barcode" sequences can be added according to different samples to further distinguish the samples. The added "barcode" changes according to the change of the genome of the sample to be tested. The purpose of adding "barcode" is to distinguish each different sample, that is, to distinguish each DNA sequence;

Specifically, the downstream primer of the enriched promoter: 5'-GACTGCGTACYYNCTATA-3'. Designing an enriched promoter downstream primer, the principle is to design a primer with the characteristics of the promoter region by simulating the base sequence of the upstream region of eukaryotes, that is, the enriched promoter downstream primer, so that the primer can effectively amplify the vicinity of the promoter region The sequence to further achieve the purpose of gene enrichment, which is conducive to subsequent experiments.

Preferably, in the aforementioned universal upstream primer, universal downstream primer and enriched promoter downstream primer, the "N" is any one of bases A, T, C, and G, and the "Y" is base C Or T. By further designing the "N" and "Y" bases on the primers, on the one hand, the coverage of the primers can be increased, and the primers can amplify the entire genomic DNA to a greater extent; on the other hand, the design method of mixed bases is adopted. It can further improve the binding ability of the primer and the sample to be tested, and achieve the purpose of gene enrichment. "N" is a random base. Several random bases are designed at the end of the universal primer, which can eliminate the bias effect of PCR enrichment and is used to restore the real genome. It has great advantages in copy number analysis; preferably, the "N "It can be designed to any number of bases from 3 to 8 for greater simplification of the genome. It can be used in complex or large genomes, and it can also be adjusted freely to reduce costs (larger samples, reduced SNP).

In a preferred embodiment of the present invention, cassava is used as the detection object. In the sequencing primer set, the sequence of the universal upstream primer is: 5'-T[barcode]CAAACCGGNNN-3', and the sequence of the universal downstream primer is : 5'-GACTGCGTACGAATTNNN-3'; or,

The sequence of the universal upstream primer is: 5'-T[barcode]CAAACGCGNNN-3', and the sequence of the universal downstream primer is: 5'-GACTGCGTACGTATANNN-3'.

By analyzing the gene sequence with higher frequency in the cassava gene, it is further determined that the core base "XXXX" of the universal upstream primer is "CCGG" or "CGCG"; the core base "ZZZZ" of the universal downstream primer is determined as "AATT" "Or "ATAT". Choosing the bases with a higher frequency in the cassava gene as the core bases of the primers can further increase the coverage of the amplification and have a good enrichment effect.

In another preferred embodiment of the present invention, cassava is used as the detection object, and the "barcode" is selected from at least one of CTTAT, GTAGA, CCTCG, GAACT and TTACT. According to different cassava test samples, a 5-base-length "barcode" is designed on the universal upstream primers to distinguish each DNA sequence, while ensuring more uniform amplification, higher coverage of the whole genome, and more comprehensive .

Therefore, compared with the prior art, the sequencing primer set provided by the present invention includes a universal upstream primer with a unique sequence, a universal downstream primer, and an enriched promoter downstream primer, wherein the universal upstream primer and the universal downstream primer each include four bases : "XXXX" and "ZZZZ" (the bases are all selected from at least one of A, T, C and G in combination), these eight bases are the core base regions of the universal primer, which can be based on the sample genome According to the different bases, it can amplify different sequence fragments on different genomes. The primer scheme can be optimized according to the sequence of the genome, and the genome size and sample size can be adjusted arbitrarily; at the same time, by simulating eukaryotes For the base sequence of the upstream region, design a primer with the characteristics of the promoter region, that is, the downstream primer of the enriched promoter, so that the universal upstream primer and the downstream primer of the enriched promoter can effectively amplify the sequence near the promoter. Such a primer set is used for whole-genome amplification and sequencing, not only has a good enrichment effect, but also makes library construction and sequencing more concise and accurate, and reduces sequencing costs.

Correspondingly, an embodiment of the present invention also provides a PCR-based whole genome sequencing method, which includes the following steps:

S01. Prepare the DNA of the sample to be tested;

S02. Provide the sequencing primer set, use the universal upstream primer and the universal downstream primer to perform the first PCR amplification on the DNA of the test sample to obtain the first amplification product, and use the universal upstream primer and the The downstream primer of the enriched promoter performs a second PCR amplification on the DNA of the test sample to obtain a second amplification product;

S03. The first amplification product and the second amplification product are respectively subjected to sequencing analysis.

Specifically, in the above step S01, the DNA of the sample to be tested is prepared. In a preferred embodiment of the present invention, the method for preparing the DNA of the sample to be tested is selected from any one of a whole genome DNA extraction method, a PCR kit amplification method, and a lysis solution preparation method. Preferably, the ordinary whole-genome DNA extraction method mainly includes the use of cetyltrimethylammonium bromide method (CTAB method). This method mainly uses cationic detergents to precipitate nucleic acids and acidity from low ionic strength solutions. Polysaccharides. In a solution with high ion concentration, cetyltrimethylammonium bromide forms complexes with proteins and polysaccharides, but does not precipitate nucleic acids. After extraction with organic solvents to remove impurities such as proteins, polysaccharides, and phenols, ethanol precipitation is added, even if the nucleic acid is separated, the whole genome DNA of the sample is obtained; or SDS (sodium dodecylbenzene sulfonate) is used to lyse the cells to make Chromosome segregation and protein denaturation, while SDS combines with protein and polysaccharides to form a complex, releasing nucleic acid. Secondly, it can also be directly prepared by the PCR kit amplification method; or the whole genomic DNA can be obtained by preparing a lysis solution and lysing tissue. Since subsequent experiments do not require high DNA quality, the direct PCR method described in the embodiments of the present invention can quickly enrich the whole genomic DNA of the sample. At the same time, the DNA requirements are not high, the method is simple and fast, and the library preparation time is greatly shortened. , Improve test efficiency.

Specifically, in the above step S02, the sequencing primer set is provided, the first PCR amplification is performed on the DNA of the test sample using the universal upstream primer and the universal downstream primer to obtain the first amplification product, and the The universal upstream primer and the downstream primer of the enriched promoter perform a second PCR amplification on the DNA of the test sample to obtain a second amplification product;

Preferably, wherein the first PCR amplification step includes:

Advanced use of universal upstream primers and universal downstream primers bound to the DNA of the sample to be tested for binding amplification, and then enrichment amplification of the enrichment target region.

Among them, the general PCR system (20μL system) is as follows:

DNA sample (20ng) (20ng) 1.0μL

2× NEB Taq Master Mix 10μL

5μM Primer universal upstream primer (corresponding to 5 samples) 0.6μL

5μM Primer Universal Downstream Primer 0.6μL

ddH ₂ O 7.8μL;

The binding amplification includes the following reaction program at 94°C for 5 minutes and 5 cycles: 94°C, 1 minute; 35°C, 1 minute; 72°C, 1.5 minutes; and

The enrichment amplification includes 35 cycles of the following reaction procedures: 94°C, 1 minute; 50-58°C, 1 minute; 72°C, 1.5 minutes.

72°C, 7 minutes

Preferably, the second PCR amplification step includes:

Advanced use of universal upstream primers and enrichment promoter downstream primers are bound to the DNA of the sample to be tested for binding amplification, and then enrichment amplification of the enriched target region is performed.

Among them, the PCR system (20μL system) for enriching the Pormoter zone is as follows:

DNA sample (20ng) (20ng) 1.0μL

2× NEB Taq Master Mix 10μL

5μM Primer universal upstream primer (corresponding to 5 samples) 0.6μL

5μM Primer Universal Downstream Primer 0.6μL

ddH ₂ O 7.8μL;

72°C, 7 minutes

Among them, the main purpose of combined amplification is to first combine the aforementioned primers with the sample DNA. Among them, the number of PCR reaction cycles combined with amplification is 5, and each primer can bind to the sample DNA strand under this number of cycles. According to the bases of "XXXX", "ZZZZ", "N" and "Y" designed on the primer, the annealing temperature is 35℃, mainly through this pre-amplification step, setting a lower annealing temperature , So that the "XXXX", "N" and "Y" designed on the primer can be accurately and quickly combined with the sample DNA, and the primer is positioned on the sample DNA, which is conducive to subsequent enrichment and amplification.

Preferably, the main purpose of enrichment and amplification is to enrich the gene to produce an enrichment effect and facilitate subsequent detection. In a preferred embodiment of the present invention, the number of cycles of the PCR reaction of the enrichment amplification is 35, and the number of cycles is increased to 35, mainly to be able to amplify a large amount of sample DNA and increase the number of sample DNA enriched fragments . Moreover, according to the length of each primer, the annealing temperature is set to 50~58℃. A suitable temperature can increase the rate of DNA amplification and increase the amount of DNA enrichment.

Specifically, in the foregoing step S03, the first amplification product and the second amplification product are respectively subjected to sequencing analysis. In a preferred embodiment of the present invention, agarose gel electrophoresis is used to detect the first amplification product and the second amplification product respectively to ensure that the product can be obtained by PCR amplification. Preferably, the first amplification product is quantified to a concentration of 180-200 ng/μL, and the second amplification product is quantified to a concentration of 180-200 ng/μL, and the concentration of each PCR product is kept consistent. If the PCR product concentration is too low, the amount of PCR amplification product obtained is small, and it is easy to be interfered by impurities in the subsequent test and detection process; if the concentration is too high, the amount of PCR amplification product is too much. High, unclear analysis, unclear sequencing results.

Preferably, after adjusting the first amplification product to the same concentration, all samples are mixed to obtain a first mixture, and after adjusting the second amplification product to the same concentration, all samples are mixed to obtain a second mixture. Preferably, the mixing method is selected from any one of suction mixing and centrifugal mixing. In the specific embodiment of the present invention, the single samples are respectively quantified and then mixed for sequencing, which can overcome the problem of the uniformity of the data output from the common phenomenon of simplified sequencing to a certain extent.

Preferably, the second-generation library construction sequencing is performed on the first mixture and the second mixture, and the sequencing results are analyzed. Preferably, the second-generation library construction sequencing analysis usually requires illumina sequencing. In a preferred embodiment of the present invention, the obtained sequencing results are analyzed, and the [barcode] of different primers designed can be used to distinguish samples for analysis, not only for detection but also for mining population variation sites.

Compared with the prior art, the present invention provides a PCR-based whole-genome sequencing method that amplifies the DNA of the test sample by using the sequencing primer set to obtain the PCR product of the test sample, and then performs sequencing analysis. The above-mentioned PCR-based whole-genome sequencing method takes into account the characteristics of "discovering" and "detecting" single nucleotide polymorphism (SNP) sites, and can detect and screen the whole genome while new hair styles are marked. Therefore, There is no need to build a chip in the early stage, and no need to mark the known single nucleotide polymorphism (SNP) sites; at the same time, using this method for whole genome sequencing does not require high sample DNA quality, and the library construction is more concise and convenient , At the same time, it has high coverage, gene enrichment effect, short sequencing time, and can be widely used in genome sequencing of different samples. The description is given below in conjunction with specific embodiments.

Example 1

A PCR-based whole-genome sequencing method, using different varieties of cassava as an example to test,

It includes the following steps:

Step 1: Prepare the DNA of the sample to be tested;

Genome DNA was extracted from different varieties of cassava that had the same growth under the same growth conditions, the same growth period, the same part, and no pests and diseases. Long-term storage of samples requires liquid nitrogen or a refrigerator below -70°C. Adopt DNeasy 96 Plant Kit (QIAGEN) kit for extraction of genomic DNA.

Quality detection and quantification of extracted genomic DNA: agarose gel detection is marked with λmarker, take 1μL of DNA, add 2μL of l0× bromophenol blue loading buffer, mix well, and spot containing 0.5μg/ml In a 0.8% agarose gel with Goldview dye, use 1× TAE buffer, 90 V for 40 min electrophoresis; gel imaging analysis system (Tanon4100) to observe DNA bands.

Take 1-2μL DNA sample and use NANODROP 2000C to detect genomic DNA. Calculate the DNA concentration based on the light absorption value at 260nm, and determine the presence or absence of impurities such as polysaccharides, proteins, and RNA based on the ratio of OD260/OD280 and OD260/OD230 to determine the purity of DNA.

Step 2: Provide the sequencing primer set, use the universal upstream primer and universal downstream primer to perform the first PCR amplification on the DNA of the test sample to obtain the first amplification product, and use the universal upstream primer and the universal The downstream primer of the enriched promoter performs a second PCR amplification on the DNA of the test sample to obtain a second amplification product;

Among them, 5 universal upstream primers are designed, namely

SEQ ID NO.1 (5’-TCTTATCAAACCGGNNN-3’),

SEQ ID NO.2(5’-TGTAGACAAACCGGNNN-3’),

SEQ ID NO.3(5’-TCCTCGCAAACCGGNNN-3’),

SEQ ID NO.4 (5’-TGAACTCAAACCGGNNN-3’),

SEQ ID NO.5 (5'-TTTACTCAAACCGGNNN-3'), where the [barcode] region is a different sequence, the "XXXX" sequence in the universal upstream primer is "GGCC", because "GGCC" is a higher frequency in cassava Sequence, so choose "GGCC".

A universal downstream primer sequence was designed as:

SEQ ID NO.6 (5'-GACTGCGTACGAATTNNN-3'), where the "ZZZZ" sequence is "AATT". Since "AATT" is a sequence with a higher frequency in cassava, "AATT" is selected.

Designed a downstream primer of enriched promoter:

SEQ ID NO. 7 (5'-GACTGCGTACYYNCTATA-3').

The first PCR amplification is performed on the DNA of the sample to be tested using the universal upstream primer and the universal downstream primer, and the first PCR amplification step includes:

Among them, the general PCR system (20μL system) is as follows:

DNA sample (20ng) (20ng) 1.0μL

2× NEB Taq Master Mix 10μL

5μM Primer universal upstream primer (corresponding to 5 samples) 0.6μL

5μM Primer Universal Downstream Primer 0.6μL

ddH ₂ O 7.8μL;

72°C, 7 minutes

The second PCR amplification is performed on the DNA of the test sample by using the universal upstream primer and the downstream primer of the enriched promoter, and the second PCR amplification step includes:

The PCR system for enriching the Pormoter zone (20μL system) is as follows:

DNA sample (20ng) (20ng) 1.0μL

2× NEB Taq Master Mix 10μL

5μM Primer universal upstream primer (corresponding to 5 samples) 0.6μL

5μM Primer Universal Downstream Primer 0.6μL

ddH ₂ O 7.8μL;

72°C, 7 minutes

Step 3: Perform sequencing analysis on the first amplification product and the second amplification product respectively.

Take 8 μL of the first amplification product and 8 μL of the second amplification product, and 2% agarose gel for detection. The test results are shown in Figure 1 and Figure 2. Figure 1 is a graph of the detection results of the first amplification product of 5 samples, and Figure 2 is a graph of the detection results of the second amplification product of 5 samples. To quantify all PCR products separately, it needs to be homogenized and accurately quantified to 100ng/μL. Take 2ul of each sample of the quantified first amplification product and the second amplification product and mix them into the first mixed product and the second mixed product. Purify and recover 200bp-700bp size fragments. Sent to a third-party sequencing company, using Hiseq2500 sequencing, the sequencing length is double-ended 150bp. The total data volume of original reads is 5Gb. The sequencing results are shown in Figure 3, Figure 4, and Figure 5. Figure 3 is the genome coverage of the sequencing reads obtained by the universal primer method, Figure 4 is the genome coverage of the sequencing reads obtained by the enrichment Pormoter region primer method, and Figure 5 is the enrichment in the gene region. As shown in Figure 5, 70% of the PCR products are Enriched in non-gene region (Non-gene region), 17% of PCR products are enriched in intron region (Intron), 7% of PCR products are enriched in CDS region, 6% of PCR products are enriched in UP_2000 region . Based on the above three figures, it can be analyzed that the PCR method of the present invention is used to perform whole-genome sequencing, which has a very high coverage rate on the genome, and the result is extremely reliable. At the same time, this method is more concise and convenient to build a database, at the same time, it has high coverage, has a gene enrichment effect, and has a short sequencing time. It can be widely used in genome sequencing of different samples.

The above-mentioned embodiments are only used to illustrate the technical solutions of the present invention, not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that it can still implement the foregoing The technical solutions recorded in the examples are modified, or some of the technical features are equivalently replaced; these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention, and should be included in Within the protection scope of the present invention.

Claims

A sequencing primer set, characterized in that the sequencing primer set includes a universal upstream primer, a universal downstream primer, and an enriched promoter downstream primer;

The sequence of the universal upstream primer includes: 5'-T[barcode]CAAAXXXXNNN-3';

The sequence of the universal downstream primer includes: 5'-GACTGCGTACGZZZZNNN-3';

The sequence of the primer downstream of the enriched promoter includes: 5'-GACTGCGTACYYNCTATA-3';

Wherein, the length of "XXXX" is 4 bases, and the base is selected from at least one of A, T, C and G; the length of "ZZZZ" is 4 bases, and the base is selected From at least one of A, T, C and G; the length of the "barcode" is 4-6 bases, and the base is selected from at least one of A, T, C and G; the "N "Is any one of bases A, T, C, and G, and the "Y" is base C or T.
The sequencing primer set of claim 1, wherein in the sequencing primer set, the sequence of the universal upstream primer is: 5'-T[barcode]CAAACCGGNNN-3', and the sequence of the universal downstream primer It is: 5'-GACTGCGTACGAATTNNN-3'.
The sequencing primer set of claim 1, wherein in the sequencing primer set, the sequence of the universal upstream primer is: 5'-T[barcode]CAAACGCGNNN-3', and the sequence of the universal downstream primer It is: 5'-GACTGCGTACGTATANNN-3'.
The sequencing primer set of claim 1, wherein the "barcode" is selected from at least one of CTTAT, GTAGA, CCTCG, GAACT and TTACT.
The sequencing primer set of claim 1, wherein the universal upstream primer is SEQ ID As shown in NO.1~5, the universal downstream primer is as SEQ ID NO.6 shown.
The sequencing primer set of claim 1, wherein the universal upstream primer is SEQ ID No. 1 to 5, the downstream primer of the enriched promoter is shown in SEQ ID NO.7.
A PCR-based whole-genome sequencing method, which includes the following steps:

Prepare the DNA of the sample to be tested;

Provide the sequencing primer set according to any one of claims 1 to 6, using the universal upstream primer and universal downstream primer to perform first PCR amplification on the DNA of the sample to be tested to obtain a first amplification product, and using the The universal upstream primer and the downstream primer of the enriched promoter perform a second PCR amplification on the DNA of the test sample to obtain a second amplification product;

The first amplification product and the second amplification product are mixed and subjected to sequencing analysis.
The PCR-based whole genome sequencing method according to claim 7, wherein the first PCR amplification step comprises:

Advanced use of universal upstream primers and universal downstream primers bound to the DNA of the sample to be tested for binding amplification, and then enrichment amplification of the enrichment target region.
The PCR-based whole-genome sequencing method according to claim 8, wherein the combined amplification comprises 94°C, 5 minutes; 5 cycles of the following reaction program: 94°C, 1 minute; 35°C, 1 minute ; 72°C, 1.5 minutes.
The PCR-based whole-genome sequencing method of claim 8, wherein the enrichment and amplification includes 35 cycles of the following reaction procedures: 94°C, 1 minute; 50~58°C, 1 minute; 72°C , 1.5 minutes; 72°C, 7 minutes.
The PCR-based whole-genome sequencing method according to claim 7, wherein the second PCR amplification step comprises:

Advanced use of universal upstream primers and enrichment promoter downstream primers are bound to the DNA of the sample to be tested for binding amplification, and then enrichment amplification of the enriched target region is performed.
The PCR-based whole-genome sequencing method according to claim 11, wherein the combined amplification comprises 94°C, 5 minutes; 5 cycles of the following reaction procedures: 94°C, 1 minute; 35°C, 1 minute ; 72°C, 1.5 minutes.
The PCR-based whole-genome sequencing method according to claim 11, wherein the enrichment and amplification includes 35 cycles of the following reaction procedures: 94°C, 1 minute; 50~58°C, 1 minute; 72°C , 1.5 minutes; 72°C, 7 minutes.
The PCR-based whole-genome sequencing method according to claim 7, wherein the method for preparing the DNA of the sample to be tested is selected from the group consisting of whole-genome DNA extraction method, PCR kit amplification method, and preparation of lysis solution and tissue lysis method Any of them.
The PCR-based whole genome sequencing method of claim 7, wherein the first amplification product is quantified to a concentration of 180-200 ng/μL, and the second amplification product is quantified to a concentration of After 180-200ng/μL, sequencing analysis was performed respectively.