Method for simultaneously detecting foreground DNA molecular markers and background DNA molecular markers and its application

Abstract

The present invention provides a method and application for simultaneously detecting foreground and background DNA molecular markers, relating to the technical field of genetic engineering. This method utilizes specific amplification triggered by a perfect primer-template match as the foreground marker. Simultaneously, and despite technical bias, it utilizes nonspecific amplification triggered by an imperfect primer-template match as the genome-wide background marker screening. This method enables simultaneous screening of foreground and background markers, improving both detection efficiency and accuracy, simplifying experimental procedures, and reducing costs, thus possessing significant application value.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and in particular to a method for simultaneously detecting foreground DNA molecular markers and background DNA molecular markers (FBI-seq) and its application. Background Art

[0002] In the fields of scientific research, agricultural breeding and clinical testing, it is often necessary to detect the genotype of individual genes or the whole genome of individual organisms. For example, in animal and plant breeding research, the transmission of genetic information between parents is analyzed by analyzing the genotype of the individual, screening for favorable genotypes and eliminating non-target genotypes; with the positioning and cloning of more and more QTLs and genes that control different agronomic traits, genetic linkage markers or functional markers based on these QTLs or genes are widely used in molecular marker-assisted selection breeding. PCR (Polymerase Chain Reaction) is a commonly used technique in molecular biology research, and the PCR-based molecular marker method provides a mature tool for the screening of target genes (foreground marker detection). In order to evaluate the pedigree of breeding materials, whole genome genotype detection (background marker detection) is often required.

[0003] Methods for whole-genome genotyping primarily include DNA arrays and various next-generation sequencing (NGS)-based approaches, such as genome-wide targeted enrichment (Guo et al., 2019), multiplex PCR (Onda et al., 2018), and various types of restriction site-associated DNA sequencing (RAD-seq). With the exception of RAD-seq, which randomly sequences a portion of the genome (generally 1%-10%), the other three methods can target any target fragment within the genome. In theory, these methods could be used to develop tools that simultaneously screen for both foreground and background markers. However, different researchers and breeding programs require screening for different foreground genes. Therefore, to meet these needs, DNA arrays, genome-wide targeted probes, or multiplex PCR primers must be redesigned and produced. The high initial costs make such strategies difficult to implement in practice. In addition, the time-of-flight mass spectrometry biochip system (Sequenom MassArray) and competitive allele-specific PCR (Kompetitive Allele Specific PCR) (Ertiro et al., 2015) are also commonly used methods for genotyping. However, these two methods are more suitable for projects with large sample sizes and dozens of detection sites and are not suitable for whole-genome molecular marker detection.

[0004] Therefore, there is still a lack of effective tools that can simultaneously screen samples for both foreground markers and background markers genome-wide. For example, when improving the grain shape of the Y58S sterile line, BGI first used an indel marker to screen for foreground genes and then used RAD-seq to screen for background markers (Hou Junliang et al., 2018). Therefore, developing new technologies that can be used across different organisms and simultaneously screen for foreground and background markers has important application value. Summary of the Invention

[0005] In view of this, the present invention is dedicated to studying the application of PCR amplification with incomplete primer-template matching in detecting genetic background markers, and has developed a method that can be used in different organisms and can simultaneously detect foreground DNA molecular markers and background DNA molecular markers (Fore-and Background Integrated genotyping by sequencing, abbreviated as FBI-seq).

[0006] A first aspect of the present invention provides a method for simultaneously detecting foreground DNA molecular markers and background DNA molecular markers (FBI-seq), comprising the following steps:

[0007] (a) Design PCR primers (FBI-SP) for the target gene based on the sample genomic sequence, and add DNA elements for high-throughput sequencing at the 5' end of the primers;

[0008] (b) fragmenting the sample genomic DNA and adding DNA adapters to the ends of the DNA fragments;

[0009] (c) PCR amplification using FBI-SP primers, during which amplification products are generated both when the FBI-SP primers are completely matched with the template and when they are not completely matched with the template;

[0010] (d) Statistical data analysis after PCR product sequencing, including the genetic information obtained by amplification of primer-template perfect match and incomplete match.

[0011] In step (a), the DNA element added to the 5' end of the FBI-SP primer is not specifically limited, and different DNA elements can be selected according to different sequencing platforms. For example, the required DNA elements can be added according to sequencing platforms such as Illumina, BGI, and Thermofisher.

[0012] In a preferred embodiment of the present invention, a DNA element for Illumina platform sequencing is added to the 5' end of the FBI-SP primer.

[0013] In step (b), the method for fragmenting the sample genomic DNA is not particularly limited, and can be, for example, fragmenting the DNA using a transposase complex, fragmenting the DNA using ultrasound, or fragmenting the DNA using restriction endonucleases.

[0014] The addition of DNA linkers to the ends of DNA fragments can be selected according to the method of DNA interruption, such as using a transposase complex to interrupt DNA and connect DNA linkers; ultrasonic interruption of DNA and connection of DNA linkers with ligase; restriction endonuclease digestion and connection of DNA linkers with ligase.

[0015] In step (c), a perfect match between the FBI-SP primers and the template binds to the target gene, amplifying a PCR product of the target gene for detecting the foreground gene. Incomplete matches between the FBI-SP primers and the template bind to sites on the genome with sequence similarity to the target gene, amplifying a mismatched PCR product for detecting the background gene.

[0016] Furthermore, primer-template incomplete matching is not suppressed and / or enhanced during PCR amplification.

[0017] Furthermore, during the PCR amplification process, the primer concentration is increased, and / or the annealing temperature is raised, and / or the cycle number is changed.

[0018] In one embodiment of the present invention, during the PCR amplification process, the primer concentration is increased to 400 nM, and / or two annealing temperatures of 60°C and 65°C are used for amplification respectively, and / or the number of cycles of the first round of PCR is changed to 10 annealing cycles at 65°C + 3 annealing cycles at 60°C, and the number of cycles of the second round of PCR is changed to 8 annealing cycles at 60°C.

[0019] During the PCR reaction, primers anneal / pair on the DNA template, and DNA polymerase then initiates the synthesis of a new DNA strand at the 3' end of the primer. This annealing / pairing of primers on the DNA template follows the kinetics of DNA denaturation and renaturation. Therefore, both theoretically and practically, there can be both perfect primer-template annealing (PTPA) and mismatched primer-template annealing (PTMA). To prevent experimental interference from nonspecific amplification caused by PTMA, researchers often reduce the risk of nonspecific amplification caused by PTMA by increasing primer length, raising the annealing temperature during PCR cycling, and optimizing the PCR reaction system.

[0020] The applicants of the present invention have discovered that during the LM-PCR process, imperfect primer matching (PTMA) on the genomic template can trigger stable and reproducible amplification of tens of thousands of sites on the genome. The PTMA amplified sites are relatively evenly distributed on the chromosomes and can therefore serve as background markers for the entire genome.

[0021] The present invention overcomes technical bias and not only does not reduce the nonspecific amplification caused by PTMA according to conventional means, but tends to increase the probability of PTMA occurrence, and further verifies that the amplification caused by PTMA is stable and repeatable. The nonspecific amplification caused by PTMA is used as a whole-genome background marker, realizing the simultaneous detection of gene foreground markers and background markers, simplifying the experimental process, improving the accuracy of marker detection, reducing costs, and having important application value.

[0022] The FBI-seq developed in the present invention uses NGS to perform high-throughput sequencing of numerous PCR amplification products triggered by PTPA and PTMA during the LM-PCR process. Requiring only a single common primer that does not require special design and optimization, FBI-seq can simultaneously detect foreground markers and background markers at tens of thousands of sites. Furthermore, FBI-seq can be easily switched to different foreground genes and different species, especially species with less accumulated genomic information. This method holds important application prospects in whole-genome genotyping.

[0023] Compared with DNA chips, genome-targeted enrichment, and multiplex PCR, FBI-seq requires only one primer, has no expensive upfront costs, and does not require large batches of samples to dilute the cost of a single sample for the user. Therefore, FBI-seq can be very conveniently used to screen different prospect genes and whole-genome background genes in different species and any number of samples.

[0024] In one embodiment of the present invention, LM-PCR (ligation-mediated PCR, also called adapter ligation PCR) is used for PCR amplification with incomplete primer-template matching, combined with NGS (Next Generation Sequencing) to simultaneously detect sample foreground markers and background markers.

[0025] Ligation-mediated PCR (LM-PCR) is a variant of the PCR method, commonly used to isolate flanking sequences of a known sequence. During the PCR process, researchers generally use nested primers to reduce the interference of nonspecific amplification.

[0026] By utilizing PTPA and PTMA in the LM-PCR process, the PCR amplification products initiated by PTPA are used for foreground marker screening, and the PCR amplification products initiated by PTMA are used for background marker screening. Taking advantage of the ability of NGS to sequence multiple products simultaneously, the PCR products of PTPA and PTMA are sequenced simultaneously, achieving foreground and whole-genome background screening of the sample using a primer designed based on the sequence of the foreground gene to be tested.

[0027] Furthermore, the FBI-seq method comprises the following steps:

[0028] (1) Fragmentation of sample genomic DNA: The sample genomic DNA is fragmented using a transposase complex, and DNA adapters are added to the fragmented DNA fragments;

[0029] (2) First-round PCR reaction: Amplification is performed using primer 2 and FBI-SP. During the amplification process, amplifications triggered by both perfect and incomplete matches between the FBI-SP primer and the template occur simultaneously, without inhibiting and / or enhancing incomplete primer-template matches. DNA fragments containing the target gene that are perfectly matched and amplified DNA fragments triggered by incomplete matches are enriched to obtain the first-round PCR products.

[0030] The primer 2 binds to the DNA linker; the FBI-SP primers that completely match the template bind to the target gene, and their amplified products are used to detect the foreground gene; the FBI-SP primers that do not completely match the template bind to sites on the genome that have sequence similarity to the target gene, and their amplified products are used to detect the background gene;

[0031] (3) Second round of PCR reaction: Using the first round PCR product as a template, primers 1 and PPMi7 were used for PCR amplification. The ends of the target PCR product fragment were added with the adapter sequences required for sequencing on the sequencing platform;

[0032] The primer 1 binds to the 5' end of the FBI-SP primer; PPMi7 binds to the 5' end of Primer 2.

[0033] (4) Enriched fragment sequencing: The enriched fragments are sequenced after fragment selection, and the data analysis obtains the genetic information of the primer-template complete match and incomplete match amplification products.

[0034] In one embodiment of the present invention, the FBI-seq gene detection method, referring to Figure 1 The following steps are involved:

[0035] (1) Cutting the sample genomic DNA: Using the Tn5 transposase complex to cut the sample genomic DNA, the Tn5 adapter (Adaptor1 and Adaptor2) serves as the reaction substrate of the Tn5 transposase. The Tn5 adapter is connected to the breakpoint while Tn5 cuts the DNA. Adaptor2 will serve as the binding site for one of the primers (Primer 2) in the subsequent PCR reaction.

[0036] All the broken DNA fragments are divided into fragments containing the target gene and fragments without the target gene;

[0037] (2) First round PCR reaction: The first round PCR reaction is performed using Primer 2 paired with FBI-SP. Both Primer 2 and FBI-SP contain the DNA element sequence required for Illumina sequencing at the 5' end.

[0038] In addition to amplifying at primer-perfect matching sites (PTPA) in genomic templates, FBI-SP can also amplify from tens of thousands of incomplete matching sites (PTMA);

[0039] After the first round of PCR, DNA fragments containing the target site are enriched through PCR amplification. One end of the PCR product is the FBI-SP primer (PTPA amplification product) or a portion thereof (PTMA amplification product). This end is added with a partial P5 adapter sequence required for DNA sequencing on the Illumina sequencing platform, and the other end is added with a P7 adapter sequence required for DNA sequencing on the Illumina sequencing platform.

[0040] Enrich the DNA fragments containing the target gene that are completely matched and amplified by FBI-SP primers, as well as the DNA fragments that are not completely matched and amplified;

[0041] (3) Second round of PCR reaction: Using the first round PCR product as a template, PCR amplification was performed using primers Primer 1 (binding to the 5' end of the FBI-SP primer) and PPMi7 (binding to the 5' end of Primer 2). Both ends of the target PCR product fragment were added with all the adapter sequences at the P5 and P7 ends required for DNA sequencing on the Illumina sequencing platform;

[0042] (4) Enriched fragment sequencing: After two rounds of PCR amplification, the sequences near the PTPA and PTMA sites of the FBI-SP primers are enriched. Fragments with a length of 300-700 bp are selected from the enriched DNA for high-throughput sequencing. The SNP information contained in the obtained amplified fragments reflects the DNA polymorphism of the corresponding region of the genome.

[0043] Furthermore, the 5' end of the primer 2 is a PPMi7 sequence, the middle is an index sequence (usually 6-10 random bases N), and the 3' end is a complementary sequence of the Tn5 adapter; and / or

[0044] The 3' end of the primer 1 is a PPMi5 sequence, the middle is an index sequence (usually 6-10 random bases N), and the 5' end is a complementary sequence of the Tn5 linker.

[0045] The FBI-seq gene detection method combines traditional LM-PCR with high-throughput sequencing, simplifying the traditional LM-PCR experimental process. It also cleverly utilizes amplification triggered by incomplete primer-template matches, improving LM-PCR efficiency. This method is simple to use, requiring only a single foreground-labeled primer, making it highly adaptable for screening for different foreground genes in different species. It can also simultaneously perform foreground and whole-genome background detection on different samples.

[0046] FBI-seq can amplify tens of thousands of gene loci using a single primer. Therefore, other technologies that can perform genotyping on a few to tens of thousands of loci, such as chips, multiplex PCR, simplified genome sequencing, whole genome sequencing, etc., can also be done by FBI-seq.

[0047] Furthermore, based on the technical solution provided by the present invention, the step (c) further comprises random amplification products of the library construction primers. The data analysis in the step (d) further comprises genetic information obtained by random amplification of the library construction primers.

[0048] For genetic information obtained by PCR amplification or random amplification with incomplete primer-template matching, those skilled in the art would usually discard this part of the data. However, the present invention does just the opposite. When analyzing the data, the genetic information obtained by incomplete PCR amplification and random amplification is used as the whole genome background marker. This is completely unexpected and can save experimental procedures without the need for additional separate screening of background markers.

[0049] There is no specific limitation on how to analyze the data from genetic testing, and it can be determined according to the specific method used. However, regardless of the method used to analyze the data, it includes analyzing the genetic information obtained by PCR amplification with incomplete primer-template matching and / or the genetic information obtained by random amplification, and the genetic information includes but is not limited to, for example, SNP markers, RAPD markers, SSR markers, SSLP markers, AFLP markers, etc.

[0050] The second aspect of the present invention provides the application of the above-mentioned FBI-seq method in whole genome detection.

[0051] Furthermore, it includes applications in biological genotype detection, and / or diagnosis or auxiliary diagnosis of biological genetic diseases, and / or amplification of unknown sequences next to known sequences.

[0052] For example, the application of the FBI-seq method in biological genotype detection can include detecting the genotype of all living organisms with DNA sequences, such as plants, animals, or microorganisms. For example, the type of genotype can be identified, or the homozygous state of the genotype can be identified.

[0053] Illustratively, the application of the FBI-seq method in the diagnosis or auxiliary diagnosis of genetic diseases can include the diagnosis or auxiliary diagnosis of human (or other animal and plant) genetic diseases, such as the diagnosis of genetic diseases caused by gene mutations, gene recombination, etc.

[0054] Furthermore, the application of the FBI-seq method in genetic breeding preferably includes the selection of superior varieties, seed authenticity identification, seed purity identification, and parental tracing.

[0055] The genetic information obtained by sequencing PCR amplification products with incomplete primer-template matching can be used as a background marker to compare with the genomic data of the parents, and to screen for excellent animal and plant varieties with excellent traits and background genomes that meet expectations; or to identify the authenticity and purity of seeds, reducing the cost of crop variety identification; or to trace the parentage, more intuitively determining which parent the variety to be tested originated from.

[0056] Furthermore, based on the technical solution provided by the present invention, it also includes the application of PCR amplification with incomplete primer-template matching in paternity testing, forensic identification, and food safety testing.

[0057] The detection and analysis of DNA genetic markers is the theoretical basis of paternity testing. During the PCR amplification process, in addition to certain specific genetic marker sites that are completely matched and amplified, the background genetic markers reflected by PCR amplification with incomplete primer-template matching can also be used to directly compare with the genomes of the parents' samples to determine whether a parent-child relationship exists.

[0058] In judicial identification, individual characteristic analysis and judgment of the DNA molecules of the specimen samples can be used as forensic evidence. The present invention can simultaneously detect foreground DNA molecular markers and background DNA molecular markers, thereby being able to more accurately and comprehensively detect the genetic information of the DNA molecules of the specimen samples and obtain DNA information more quickly and accurately.

[0059] In the field of food safety testing, some genetically modified or bacterial and viral contaminated foods are involved. The method of the present invention for simultaneously detecting foreground DNA molecular markers and background DNA molecular markers can be used. This method can not only detect unqualified foods, but also trace the source background of the food based on the gene database.

[0060] A third aspect of the present invention provides reagents and / or kits comprising the components used in the FBI-seq method;

[0061] Furthermore, the components include FBI-SP, primer 1, primer 2, PPMi7 primer, and optionally DNA elements and DNA linkers.

[0062] Furthermore, the components also include reagents and / or consumables commonly used in PCR amplification, such as DNA polymerase and its buffer, N5XX, N7XX, etc.

[0063] The present invention adopts the above technical solution to achieve the following beneficial effects:

[0064] (1) The FBI-seq method provided by the present invention overcomes technical bias and uses the nonspecific amplification triggered by PTMA as the whole-genome background marker screening. When analyzing data, the genetic information obtained by incompletely matched PCR amplification is used as the whole-genome background marker. There is no need to screen the background marker separately, which saves experimental procedures and realizes the simultaneous screening of foreground markers and background markers. It not only improves the efficiency of detection, but also improves the accuracy of detection, simplifies the experimental process, reduces costs, and has important application value.

[0065] (2) The FBI-seq detection method provided by the present invention uses NGS to perform high-throughput sequencing of numerous PCR amplification products initiated by PTPA and PTMA during the LM-PCR process, thereby achieving the purpose of using a primer that specifically amplifies the foreground gene to be detected to screen the sample for foreground and whole-genome background. It can be very conveniently used to screen different foreground genes and whole-genome background genes in different species.

[0066] (3) The present invention provides an application of the FBI-seq method in whole genome detection, which can be used in the fields of genetic breeding, paternity testing, forensic identification, food safety testing, etc. It has a wide range of applications and important application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] Figure 1 Shown is a schematic diagram of the experimental process of FBI-seq of the present invention.

[0068] Figure 2 The figure shows the distribution of sequencing data of the FBI-seq library in Example 1 on the genome browser.

[0069] Figure 3 The figure shows the number of reads of different soft-clipped bases on the genome using the FBI-Pi2-1 primer in Example 1. The bar graph shows the number of reads of PTMA with different numbers of bases.

[0070] Figure 4 Shown is the base composition of the FBI-Pi2-1 primer at the PTMA site on the genome in Example 1 (primer binding site base composition diagram). 7 soft-clipped bases represents the portion of the FBI-Pi2-1 primer from the Pi2 gene. Seven bases 5' of the primer are truncated (soft-clipped) by the NGS sequence alignment software due to lack of genomic homology. The base compositions of the primer PTMA sites for five conditions (7, 8, 9, 10, and 12) were statistically analyzed.

[0071] Figure 5The figures show the PTPA and PTMA amplification results of different primers FBI-qsh1, FBI-Alk, and FBI-qGL3 in Example 2.

[0072] Figure 6 Shown are the enrichment multiples of two rounds of PCR under 5+N cycle conditions in Example 3.

[0073] Figure 7 Shown are the enrichment multiples of two rounds of PCR under 10+N cycle conditions in Example 3.

[0074] Figure 8 Shown is a comparison chart of different cycling conditions and PCRfree data in Example 3.

[0075] Figure 9 Shown are the shared tags in three technical replicates of eggplant in Example 4.

[0076] Figure 10 The figure shows the distribution of PTPA amplified fragments of the two primers (FBI-GW5 and FBI-PSTOL1) in the FBI-seq of Example 5 on the genome browser.

[0077] Figure 11 Shown is a genotype detection diagram of a rice backcross breeding material in Example 6.

[0078] Figure 12 Shown are the enrichment multiples of two cycles with different primer concentrations in Example 7. DETAILED DESCRIPTION

[0079] Unless otherwise defined, all scientific and technical terms used in the present invention have the same meanings as commonly understood by one of ordinary skill in the art to which the present invention relates.

[0080] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0081] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0082] The present invention will be described in detail below with reference to specific embodiments. These embodiments are intended to help you understand the present invention but are not intended to limit it.

[0083] In the embodiments of the present invention, the sequencing platform used is Illumina. After replacing the relevant primer sequences, it can also be used for other sequencing platforms such as BGI and Thermofisher.

[0084] Example 1 Verification of FBI-seq's ability to simultaneously detect gene foreground markers and background markers

[0085] Experimental purpose: Use a primer from the rice blast resistance gene Pi2 to achieve simultaneous amplification of the Pi2 gene locus (foreground marker) and background markers.

[0086] Experimental procedure: The rice variety Shuhui 498 (high-quality genome sequence available, see DO1:10.1038, http: / / www.mbkbase.org / rice / help / download.html) was used as the material, and the primer FBI-Pi2-1, derived from the rice blast resistance gene Pi2, was used to test the feasibility of the FBI-seq method. Figure 1 The experimental process.

[0087] 1. Planting of Rice Materials, DNA Extraction and Quantification

[0088] The seeds of Shuhui 498 were soaked and germinated according to conventional methods, and then sown in a culture dish covered with filter paper. They were placed in a light culture room and grown for about 7 days. Then, leaves of about 2 cm were taken and DNA was extracted using the CTAB method. Then, a fluorescent dye-based method was used, such as The dsDNA High Sensitivity Kit accurately measures DNA concentration.

[0089] 2. Use the transposase complex to break the rice genomic DNA and add DNA adapters to the broken DNA. In this example, the TruePrepTM DNA Library Prep Kit V2 for Prepare the Tn5 transposase complex (TTE Mix V50) in the kit using the ingredients in Table 1 in a sterile PCR tube. Place the tube in a PCR instrument and run the reaction program in Table 2.

[0090] Table 1. Transposase complex reaction system

[0091]

[0092] Note: x depends on the DNA concentration. The total amount of DNA is fixed at 20 ng.

[0093] Table 2 Reaction procedure

[0094]

[0095] 3. After the reaction is completed, add 1 μL of 1.3% sodium dodecyl sulfate to terminate the reaction.

[0096] 4. Perform the first round of LM-PCR reaction to amplify the Pi2 gene locus and the PTMA site using the FBI-Pi2-1 primer.

[0097] The FBI-Pi2-1 primer sequence is as follows:

[0098] (SEQ ID NO.1) In this embodiment, Illumina sequencing is used. The italicized part is the DNA element required for Illumina sequencing, and the underlined part is the sequence from the rice Pi2 gene. Other similar primers in this article all use this description method.

[0099] Primer 2 sequence: (SEQ ID NO.2) (The straight underlined portion is the PPMi7 sequence, i7 is an index sequence consisting of 8 random bases N, and the wavy underlined portion is the complementary sequence of the DNA linker).

[0100] The PCR reaction is performed as follows: (1) Place the PCR tube on ice and set up the reaction according to the system in Table 3:

[0101] Table 3 First round PCR configuration system

[0102]

[0103] *TruePrep TM Index Kit V2 for (Vazyme#TD202) provides 8 N5XX and 12 N7XX types, which can be selected according to the number of samples and index matching strategy.

[0104] (2) Use a pipette to gently pipette 5 times to mix thoroughly;

[0105] (3) Place the sample in the PCR instrument, and the program is shown in Table 4:

[0106] Table 4 Reaction procedures for the first round of PCR

[0107]

[0108] 6. Use VAHTS DNA Clean Beads from Nanjing Novozyme Biotechnology Co., Ltd. to purify the PCR amplified product according to the reagent instructions and dissolve it in 20 μL ultrapure water.

[0109] 7. Perform a second round of PCR amplification on the first PCR product to further enrich the target fragment and add the DNA sequence elements and index required for sequencing.

[0110] Primer 1 sequence: (SEQ ID NO.3) (The straight underlined portion is the PPMi5 sequence, [i5] is the index sequence composed of 8 random bases N, and the wavy underlined portion is the complementary sequence of the DNA linker).

[0111] PPMi7 primer sequence: 5'-CAAGCAGAAGACGGCATACGAGAT-3' (SEQ ID NO.4) .

[0112] The PCR reaction was performed as follows: Place the PCR tube on ice and prepare the reaction system according to Table 5:

[0113] Table 5 Second round PCR configuration system

[0114]

[0115] Use a pipette to gently pipette and mix thoroughly. Place the sample in a PCR instrument. The program is as shown in Table 6:

[0116] Table 6 Reaction procedures for the second round of PCR

[0117]

[0118] 8. Purify the PCR amplified product

[0119] The PCR amplified product was purified using VAHTS DNA Clean Beads from Nanjing Novozymes Biotechnology Co., Ltd. according to the instructions of the reagent and dissolved in 20 μL of ultrapure water.

[0120] 9. Clip Selection

[0121] FBI-seq amplification products are DNA fragments ranging from 200 to 1000 bp, requiring sorting of DNA fragments suitable for sequencing. In this example, a Sage ELF instrument from Sage Science was used to perform fragment selection on an FBI-seq library. The library was first mixed with 6× loading buffer and then subjected to DNA extraction using a 2% agarose gel cassette in the time mode to recover fragments of approximately 460 to 600 bp.

[0122] 10. Library quality testing

[0123] In this example, Agilent Bioanalyzer 2100 was used for library quality control.

[0124] 12. Sequencing

[0125] According to the concentration of the recovered library and the requirements of the relevant Illumina instruments, the sequencing was performed.

[0126] 13. Data Analysis

[0127] (1) After locating the 250Mb sequencing data of FBI-seq to the reference genome sequence of rice variety Shuhui 498 using the second-generation sequencing data alignment software BWA-mem, it was found that the sequencing data of the FBI-seq library can be divided into three categories: Figure 2 ): First, reads that fully match the FBI-Pi2-1 primers. These fragments are amplified by the primer PTPA. There are 22 reads in total, totaling approximately 6.5 kb, a negligible proportion of the total 250 Mb of sequencing data. Second, reads that do not fully match the FBI-Pi2-1 primers. These fragments are amplified by the primer PTMA, accounting for 32% of the sequencing data. Third, other reads that do not contain the FBI-Pi2-1 primer sequence and are randomly amplified during the FBI-seq library construction process account for 68% of the sequencing data.

[0128] (2) PTPA amplification is indeed triggered by the perfect match of the FBI-Pi2-1 primer on the genome.

[0129] The distribution of reads amplified by the FBI-Pi2-1 primer at its perfect match site in this example was analyzed. It was found that there were 19 reads at this site, all of which started at the position of the FBI-Pi2-1 primer on the genome ( Figure 2 ), indicating that during the FBI-seq library preparation process, the perfect match of the FBI-Pi2-1 primer on the genome can trigger the amplification of the target site.

[0130] (3) PTMA amplification was indeed triggered by the incomplete match of the FBI-Pi2-1 primer on the genome.

[0131] In this example, we analyzed the distribution characteristics of reads that meet the following three conditions: i) there are more than three reads at a certain site; ii) one end of these reads starts at the same position; iii) the other end of these reads ends at different positions (the site where the transposase randomly breaks the DNA and connects to the DNA adapter). These reads form the following patterns on the IGV genome browser: Figure 2The distribution characteristics shown in (b) form a sequence tag. According to the sequence of the reference genome, the sequence of the starting position of these tags is extracted, and the number of soft-clipped reads of different base lengths is counted ( Figure 3 ), and the base composition of these sequences was counted, and it was found that they had 5-15bp sequences that were homologous to the FBI-Pi2-1 primer sequence ( Figure 4 ), indicating that this type of read is indeed triggered by an incomplete match of the FBI-Pi2-1 primer on the genome. We obtained a total of 23,163 such tags. Further analysis showed that these tags are evenly distributed across the genome, allowing for identification of background genotypes.

[0132] (4) Whole-genome genotyping of rice using PTMA-amplified reads

[0133] The FBI-seq data of Shuhui 498 were extracted, and the reads described in step (3) of the data analysis were used to obtain the whole-genome SNPs of Shuhui 498 with the genome sequence of the rice variety Nipponbare as a reference. To verify the accuracy of the genotype analysis results, Shuhui 498 was subjected to approximately 50× whole-genome sequencing. Based on the results of the whole-genome sequencing, the whole-genome SNPs of Shuhui 498 were obtained with the genome sequence of Nipponbare as a reference. The results of the comparison between the two showed that of the 25,258 SNPs obtained by FBI-seq, 25,232 (99.9%) were consistent with the results of the whole-genome sequencing.

[0134] (5) Whole-genome genotyping of rice using randomly amplified reads

[0135] The whole-genome SNPs of Shuhui 498 were obtained using the genome sequence of the rice variety Nipponbare as a reference for reads with whole-genome coverage less than or equal to 3. These SNPs were then compared with the SNPs obtained based on whole-genome sequencing. The results of the comparison showed that among the 49,495 SNPs obtained in the FBI-seq data using this method, 49,445 (99.9%) were consistent with the results of whole-genome sequencing.

[0136] (6) The results of whole-genome genotyping of rice using PTMA and randomly amplified reads can verify each other, improving the accuracy of the test results.

[0137] In summary, the PTPA-amplified reads in the FBI-seq library can directly detect foreground markers; at the same time, the PTMA-amplified reads and randomly amplified reads can both independently detect the whole genome background. At this time, the PTMA-amplified reads can detect the same site of different individuals in a genetic population, and their detection results are equivalent to the results of chips, targeted enrichment, simplified genome sequencing or multiplex PCR, while the detection results of randomly amplified reads are essentially whole genome low coverage sequencing. PTMA amplification is essentially LM-PCR amplification; and the essence of random amplification is whole genome resequencing library amplification. Although the two are carried out in the same reaction, they are independent of each other, and the results of the two can be verified by each other; the Bin map made according to the background detection results can also be used to verify the results of foreground marker detection, thereby improving the accuracy of foreground detection (see Example 6). Therefore, the FBI-seq technology achieves simultaneous detection of foreground markers and background markers through one library preparation, and is equivalent to detecting both foreground markers and background markers twice, which not only improves the efficiency of detection, but also improves the accuracy of detection.

[0138] Example 2 Different primers have the ability to trigger amplification using PTMA

[0139] In Example 1, the primer FBI-Pi2-1 was used to initiate amplification of PTPA and PTMA in FBI-seq, thereby successfully achieving simultaneous detection of foreground markers and background markers, which is equivalent to detecting both foreground markers and background markers twice.

[0140] To verify whether the PTMA-induced amplification ability in FBI-seq is a capability shared by different primers, three primers for FBI-seq were designed based on the sequences of the qsh1, Alk, and qGL3 genes of the rice variety Shuhui 498. They are FBI-qsh1, FBI-Alk, and FBI-qGL3, respectively. FBI-seq library construction, sequencing, and bioinformatics analysis were performed according to the operating procedures of Example 1.

[0141] FBI-qsh1 primer sequences (SEQ ID NO.5) :

[0142] FBI-Alk primer sequences (SEQ ID NO.6) :

[0143] FBI-qGL3 primer sequences (SEQ ID NO.7) :

[0144] The experimental results showed that when these primers were used for FBI-seq, they all had the ability to trigger amplification of PTPA and PTMA. Analysis of the sequencing data revealed that in 500Mb of data, the number of reads generated by PTPA using primers FBI-qsh1, FBI-Alk, and FBI-qGL3 were 86, 155, and 74, respectively. Figure 5 The left side shows the distribution of PTPA amplified reads of the three primers on the genome browser); the number of reads generated by PTMA of these primers are 478,421, 287,980, and 597,539 respectively ( Figure 5 The middle right side shows the distribution of PTMA amplified reads of three prospects on the genome browser).

[0145] In Example 2, three different primers were used in FBI-seq to trigger amplification of both PTPA and PTMA, successfully enabling simultaneous detection of foreground and background markers using different primers, demonstrating that all primers are capable of priming amplification using PTMA. Thus, the FBI-seq developed by this invention enables foreground and genome-wide background detection of different genes with a single primer change, whereas similar technologies require redesign, production, and optimization of the chip, target enrichment probes, and primers for multiplex PCR.

[0146] Example 3: Changing PCR amplification conditions to increase the probability of PTMA amplification

[0147] To optimize PCR amplification conditions and improve the amplification efficiency of PTMA, we redesigned two prospective target primers, FBI-GW5 and FBI-PSTOL1, based on the gene sequence of the rice variety Shuhui 498. We used these two prospective primers to optimize amplification conditions. PCR amplification experiments were performed according to the operational process framework of Example 1.

[0148] FBI-GW5 primer sequences (SEQ ID NO.8) :

[0149] FBI-PSTOL1 primer sequences (SEQ ID NO.9) :

[0150] In the first round of PCR, the annealing temperature was set at 65°C for five cycles, and two gradients of 10 and 15 cycles were set at 60°C. The results showed that five cycles of annealing at 65°C followed by ten cycles of annealing at 60°C produced 1008.0 ng of PCR product, while five cycles of annealing at 65°C followed by 15 cycles of annealing at 60°C produced only 777.0 ng of PCR product. The total amount of PCR product did not increase with increasing cycle number, indicating that the number of cycles was too high, exceeding the exponential amplification period of the PCR amplification process. Furthermore, the 1000 ng of PCR product exceeded the required amount, and over-amplification could affect the quality of library construction.

[0151] To further optimize the PCR reaction conditions, the first-round PCR was set at 65°C for five cycles, and the annealing temperature was set at 60°C for two gradients: five and eight cycles. The results showed that the two different cycle settings of five annealing cycles at 65°C followed by five annealing cycles at 60°C, and five annealing cycles at 65°C followed by eight annealing cycles at 60°C, produced 140.4 ng and 332.5 ng of PCR product, respectively. The total amount of PCR product continued to increase rapidly with increasing cycle number, indicating that the PCR amplification was still in the exponential amplification phase and that the total amount of PCR product obtained with the five annealing cycles at 65°C followed by eight annealing cycles at 60°C was reasonable.

[0152] On this basis, the second round of PCR was set to 7 and 9 cycles. Q-PCR was used to detect the enrichment of foreground genes under different conditions.

[0153] Figure 6 Experimental results showed that in the first round of PCR, five annealing cycles at 65°C followed by eight annealing cycles at 60°C achieved the highest enrichment. The degree of enrichment in the second round of PCR also depended on the enrichment level of the first round. Sequencing data analysis also showed that a PCR cycle system of five annealing cycles at 65°C followed by N annealing cycles at 60°C enriched both PTPA and PTMA, enabling simultaneous foreground and background detection.

[0154] Based on the results of the above series of experiments, we hypothesized that increasing the number of 65°C annealing cycles and reducing the number of 60°C annealing cycles might yield more PTPA and PTMA. Therefore, we optimized the cycling conditions of the first step and set up three gradient PCR cycles: 10 65°C annealing cycles followed by 3 60°C annealing cycles, 10 65°C annealing cycles followed by 5 60°C annealing cycles, and 10 65°C annealing cycles followed by 7 60°C annealing cycles. We then conducted the same experiments and analyses.

[0155] Figure 7Experimental results show that 10 cycles of annealing at 65°C followed by N cycles of annealing at 60°C enrich for foreground gene fragments at approximately twice the rate of 5 cycles of annealing at 65°C followed by N cycles of annealing at 60°C. Furthermore, analysis of sequencing data revealed that PTMA still generates tens of thousands of tags, ensuring the amplification of foreground gene fragments while fully meeting the requirements for background tag detection.

[0156] The sequencing results of all different PCR reaction programs were compared with the PCR free data ( Figure 8 ), it can be seen that the sequencing data of 10 65℃ annealing + 3 60℃ annealing are most significantly enriched for the genomic region.

[0157] Based on all the experimental results in Example 3, we determined that FBI-seq requires two rounds of PCR amplification. The most suitable PCR cycle setting is: first round: 10 65°C annealing + 3 60°C annealing, second round: 8 60°C annealing. Under these conditions, the foreground gene fragment (PTPA) can be enriched hundreds of times, and tens of thousands of tags generated by PTMA (tag, read depth > 3) can be generated for background genotype detection.

[0158] Example 4: The same FBI-seq primers can be used to detect background markers in different species

[0159] When using microarrays, targeted enrichment, or multiplex PCR to genotype samples, each probe / primer targets only a single locus. Therefore, whole-genome testing requires the design and production of microarrays or a large number of probes and primers, and these are not universally applicable to different species. FBI-seq, on the other hand, requires only a single primer, and through its PTMA-triggered amplification, it can genotype tens of thousands of loci across the entire genome. Therefore, FBI-seq only requires a single primer design for different species, and PTMA only requires a 5-15bp sequence to trigger amplification. Therefore, we speculate that when only whole-genome background testing is required, any single primer can achieve whole-genome testing across different species.

[0160] To verify that the same FBI-seq primers can be used in different species, the inventors used two FBI-seq primers designed based on rice Shuhui 498, FBI-Pi2-3 and FBI-Waxy.

[0161] Primer sequence FBI-Pi2-3 (SEQ ID NO.10):

[0162] Primer sequence FBI-Waxy (SEQ ID NO.11):

[0163] FBI-seq was performed on two samples of eggplant (Solanum melongena var.ser-pentinum) and cowpea (Vigna unguiculatassp.sesquipedalis) with three technical replicates.

[0164] The results showed that these two primers designed based on the rice genome sequence can be used for whole-genome genotype detection of eggplant and cowpea. Figure 9 The following table shows the results of three replicates of FBI-Pi2-3 primers on eggplant DNA. All three replicates showed a large number of PTMA-generated tags. PTMA was present at the same sites across all three replicates. The analysis also showed that the number of tags generated by FBI-Waxy primers in the three replicates of eggplant samples was 43,314, 43,383, and 59,310, respectively. For long-haired beans, the number of tags generated was 53,100, 53,546, and 60,016, respectively.

[0165] The results of Example 4 confirmed that FBI-seq can achieve whole-genome detection of different species using any primer, and the generated PTMA can be used for background genome detection.

[0166] Example 5: Simultaneous detection of two foreground gene markers and other background markers

[0167] Sometimes, breeders need to detect multiple target genes (foreground markers) to screen multiple foreground genes together with background genes. Using the two primers FBI-GW5 and FBI-PSTOL1 designed in Example 3, a joint screening experiment of the two primers was performed. Using the FBI-seq experimental method optimized in Example 3, the library construction and sequencing of the two foreground gene markers were completed. Using the same data analysis method, it was found that both foreground sites were enriched in large quantities ( Figure 10 ).

[0168] The number of PTMA tags was further counted, as shown in Table 7 below, which shows the number of PTMA tags of the two primers under different sequencing data amounts.

[0169] Table 7 Number of tags under different data amounts

[0170]

[0171] The results of Example 5 confirm that the FBI-seq provided by the present invention can be used to simultaneously detect two foreground genes and tens of thousands of background sites.

[0172] Example 6 Application of PTMA Amplification in Genetic Breeding

[0173] To further illustrate that FBI-seq can perform foreground and whole-genome background detection on breeding samples, a rice backcross population was used as an example to illustrate that FBI-seq can perform foreground and whole-genome background detection on breeding samples.

[0174] Using the FBI-seq experimental conditions optimized in Example 3, two backcross BC2F4 rice populations (WYG33×VE6219 and CNG1×VE6219, WYG33 and CNG1 are both susceptible to rice blast, while VE6219 contains the rice blast resistance gene Pi2 on chromosome 6) were used to test the ability of FBI-seq to simultaneously detect foreground and background genes.

[0175] We designed the FBI-Pi2-2 primers using the sequence of the disease resistance gene Pi2. (SEQ ID NO.12) FBI-seq was used to screen for Pi2 foreground genes and background genes in 9 individual plants, and finally a breeding strain with smaller introduced fragments and higher background recovery rate was obtained.

[0176] We used the Bin Map to determine the recombination breakpoints and background information of the progeny strains by calling SNPs in the obtained parental and progeny data. The results showed that FBI-seq can successfully detect the foreground and background genotypes of each individual strain ( Figure 11 Furthermore, for the foreground gene, the reads obtained by PTPA amplification of FBI-Pi2-2 can directly detect the genotype of the Pi2 gene. In addition, the Bin Map diagram drawn based on the reads obtained by PTMA amplification can further determine whether the region where the Pi2 gene locus is located comes from the donor parent or the recipient parent. This is equivalent to testing the Pi2 gene twice, which can improve the detection accuracy.

[0177] The experimental results of Example 6 show that the FBI-seq method can detect the foreground and whole-genome background of breeding samples and efficiently complete breeding work.

[0178] Example 7 Simultaneous detection of six foreground gene markers and background markers

[0179] Sometimes, breeders need to detect more foreground genes simultaneously, such as those controlling yield, disease resistance, taste quality, and lodging resistance. After testing the simultaneous detection of two foreground genes using two primers in FBI-seq, the number of foreground genes was increased to 6, hoping to achieve the goal of simultaneously detecting more foreground genes. In the PCR system, the presence of multiple primers will interfere with each other, resulting in low amplification efficiency. Therefore, this example attempts to reduce the concentration of each primer, setting up two primer final concentration tests of 100nM and 400nM. Similarly, Q-PCR was used to detect the two rounds of products, and the enrichment of the foreground gene fragments in the first and second rounds of PCR products was calculated.

[0180] To reduce the workload, only three of the six foreground genes were randomly selected for enrichment fold testing. The experimental results showed that in the first round of PCR, a primer concentration of 100 nM produced approximately three times the total amount of PCR product compared to a primer concentration of 400 nM. This suggests that higher primer concentrations do not produce more PCR product, but rather interfere with each other, hindering PCR amplification.

[0181] The results of Q-PCR showed that when the primer concentration was 100nM and 400nM, the foreground gene fragments were enriched to varying degrees ( Figure 12 ), however, compared to the two, the enrichment of foreground gene fragments was higher when the primer concentration was 400 nM. Compared with the two-primer experiment in Example 5, when there were six foreground primers, the overall enrichment efficiency was lower than that of the two-primer case regardless of the concentration in the experiment. However, there was still significant enrichment of foreground genes and it could meet the requirements of subsequent analysis. Therefore, we ultimately decided to maintain the final primer concentration at 400 nM when testing six foreground genes.

[0182] The sequencing results also showed that the fragments of the six prospect genes were enriched. In addition, the number of PTMA reads amplified by primers of different concentrations for the six prospect genes was counted in this example, as shown in Table 8 below.

[0183] Table 8 PTMA read status of 6 prospects

[0184]

[0185] Based on all the above experimental results, we determined that FBI-seq can achieve co-enrichment of multiple prospects and produce PTPA- and PTMA-induced amplification, completing the optimization and summary of the FBI-seq method.

[0186] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention. SEQUENCE LISTING <110> Shenzhen Institute of Agricultural Genomics, Chinese Academy of Agricultural Sciences <120> Method for simultaneously detecting foreground DNA molecular markers and background DNA molecular markers and its application <160> 12 <170> PatentIn version 3.5 <210> 1 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> FBI-Pi2-1 primer <400> 1 cgtcggcagc gtcagatgtg tataagagac agtccaaacc cgttgttgca c 51 <210> 2 <211> 47 <212> DNA <213> Artificial Sequence <220> <223> primer 2 <220> <221> misc_feature <222> (25)..(32) <223> n is a, c, g, or t <400> 2 caagcagaag acggcatacg agatnnnnnn nngtctcgtg ggctcgg 47 <210> 3 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> primer 1 <220> <221> misc_feature <222> (30)..(37) <223> n is a, c, g, or t <400> 3 aatgatacgg cgaccaccga gatctacacn nnnnnnntcg tcggcagcgt c <210> 4 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> PPMi7 advertisement <400> 4 24th annual acggcatacg <210> 5 <211> 54 <212> DNA <213> Artificial Sequence <220> <223> FBI-qsh1 infrastructure <400> 5 tcgtcggcag cgtcagatgt gtataagaga cagttctgga ggtccgcgaa cgag <210> 6 <211> 54 <212> DNA <213> Artificial Sequence <220> <223> FBI-Alk Office <400> 6 tcgtcggcag cgtcagatgt gtataagaga cagagcgcct cgatgagctt gtgc <210> 7 <211> 57 <212> DNA <213> Artificial Sequence <220> <223> FBI-qGL3 infrastructure <400> 7 tcgtcggcag cgtcagatgt gtataagaga cagccgtttg attgggccgc aggaaaa <210> 8 <211> 54 <212> DNA <213> Artificial Sequence <220> <223> FBI-GW5 Instrument <400> 8 tcgtcggcag cgtcagatgt gtataagaga cagagggagt aggcagaagg agga <210> 9 <211> 54 <212> DNA <213> Artificial Sequence <220> <223> FBI-PSTOL1 ENVIRONMENT <400> 9 tcgtcggcag cgtcagatgt gtataagaga cagcatggct tgggcattat tcag <210> 10 <211> 56 <212> DNA <213> Artificial Sequence <220> <223> FBI-Pi2-3 kit <400> 10 tcgtcggcag cgtcagatgt gtataagaga cagaggggag gaggagatga aataga <210> 11 <211> 56 <212> DNA <213> Artificial Sequence <220> <223> FBI-Waxy wax <400> 11 tcgtcggcag cgtcagatgt gtataagaga caggttgtca gtaacaaacc ggaagg <210> 12 <211> 53 <212> DNA <213> Artificial Sequence <220> <223> FBI-Pi2-2 kit <400> 12 tcgtcggcag cgtcagatgt gtataagaga cagcctccga acaacgccaa ctg

Claims

1. A method for simultaneous FBI-seq detection of foreground DNA molecular markers and background DNA molecular markers, characterized in that: The following steps are involved: (a) Design the target gene PCR primer FBI-SP based on the sample genomic sequence, and add a DNA element for high-throughput sequencing to the 5' end of the primer; (b) fragmenting the sample genomic DNA and adding DNA adapters to the ends of the DNA fragments; (c) performing PCR amplification using FBI-SP primers, including a first-round PCR reaction and a second-round PCR reaction, wherein amplification products initiated by both perfect matches and incomplete matches between the FBI-SP primers and the template coexist during the amplification process; First-round PCR reaction: Amplification is performed using primer 2 and FBI-SP as primers. During the amplification process, amplifications are triggered by both perfect matches and incomplete matches between the FBI-SP primer and the template. DNA fragments containing the target gene that are perfectly matched and amplified DNA fragments that are triggered by incomplete matches are enriched to obtain the first-round PCR products. The primer 2 binds to the DNA adapter. Second round of PCR reaction: Using the first round PCR product as a template, primers Primer 1 and PPMi7 were used for PCR amplification. The ends of the target PCR product fragment were added with the adapter sequences required for sequencing on the sequencing platform; Primer 1 binds to the 5' end of the FBI-SP primer; PPMi7 binds to the 5' end of Primer 2; The 5' end of the primer 2 is the PPMi7 sequence, the middle is the index sequence, and the 3' end is the complementary sequence of the DNA adapter; The 5' end of the primer 1 is the PPMi5 sequence, the middle is the index sequence, and the 3' end is the complementary sequence of the 5' end of the FBI-SP primer; The index sequence is 6-10 random bases N; (d) Statistical data analysis after PCR product sequencing, including the genetic information obtained by amplification of primer-template perfect match and incomplete match.

2. The method according to claim 1, characterized in that During PCR amplification, it does not inhibit and / or enhance amplification initiated by primer-template incomplete matching.

3. The method according to claim 2, characterized in that Increase the primer concentration, and / or raise the annealing temperature, and / or change the number of cycles during PCR amplification.

4. The method according to any one of claims 1 to 3, characterized in that LM-PCR combined with NGS sequencing was used to simultaneously detect foreground DNA molecular markers and background DNA molecular markers.

5. The method according to any one of claims 1 to 3, characterized in that In step (b), the sample genomic DNA is sheared using a transposase complex, and DNA adapters are added to the sheared DNA fragments; In the step (d), the PCR product fragments are enriched, the enriched PCR product fragments are sequenced after fragment selection, and the data is analyzed to obtain the genetic information of the primer-template complete match and incomplete match amplification products.

6. The method according to any one of claims 1 to 3, characterized in that The step (c) further comprises randomly amplifying products of the library construction primers.

7. The method according to any one of claims 1 to 3, characterized in that The data analysis in step (d) also includes genetic information obtained by random amplification of library construction primers.

8. Application of the FBI-seq method according to any one of claims 1 to 7 in whole genome detection; said application includes application in biological genotype detection, and / or amplification of unknown sequences flanking known sequences; said application is for non-disease diagnosis and treatment purposes.

9. The use according to claim 8, characterized in that The applications include applications in genetic breeding; the applications in genetic breeding include applications in selecting superior varieties, identifying seed authenticity, identifying seed purity, and tracing parental origins.

10. The use according to claim 8, characterized in that The applications include paternity testing, forensic identification, and food safety testing.

11. Reagents and / or kits, characterized in that comprising the components used in the FBI-seq method according to any one of claims 1 to 7; The components include FBI-SP, primer 1, primer 2, PPMi7 primer, DNA elements and DNA linkers.

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