CN115747319A - Method for simplifying genome sequencing and related application - Google Patents
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Abstract
The invention provides a method for simplifying genome sequencing and related application. The method for simplified genome sequencing of the invention comprises: after the heavy sequencing library is subjected to restriction enzyme digestion, fragments with target lengths and without enzyme digestion sites are selected for sequencing, and accordingly simplified genome sequencing is achieved. According to the invention, on the basis of a whole genome sequencing library, restriction enzyme digestion is carried out before sequencing, and a fragment containing a restriction enzyme digestion site cannot be sequenced because of being cut off, so that the fragment containing the restriction enzyme digestion site on a genome is reversely selected, and the simplification of the genome is realized.
Description
Technical Field
The invention relates to a method for simplifying sequencing of a genome and related application, and belongs to the technical field of genetic engineering.
Background
Reduced Representation Genome Sequencing (RRGS) is a method of taking out a part of the DNA sequence (usually about 10%) of the genome to sequence, and using the polymorphism of this part of the genomic DNA to represent the polymorphism at the whole genome level. Simplified genome sequencing because only about 10% of the genomes were selected for sequencing, the sequencing cost and subsequent data analysis cost was reduced by about 90% compared to Whole Genome Sequencing (WGS).
In the fields of agriculture, medicine, biology and the like, the conventional simplified genome sequencing technologies include SLAF-seq (specific-localized amplified fragment sequencing), RAD-seq (Restriction-site-associated DNA sequencing), ddRAD-seq (Double digest-site-associated DNA sequencing), 2bRAD-seq and the like, and the basic principle of the technologies is to select only a part of a genome for sequencing so as to achieve the purpose of reducing the sequencing cost. And after the SLAF-seq sequentially breaks the genome DNA by using two restriction enzymes respectively and connects the two restriction enzymes with a joint, screening a fragment with a specific length for high-throughput sequencing to obtain the SLAF fragment with sequencing depth and quality meeting the requirements to represent the whole genome information of the sample. RAD-seq firstly breaks the genome through restriction endonuclease, connects with a joint, then randomly breaks through ultrasonic waves, then connects with another joint, screens the length of DNA fragments, screens the fragments with proper length and carries out high-throughput sequencing. And (3) carrying out enzyme digestion on the genome by using two enzymes simultaneously by the ddRAD, screening out a target fragment with a proper length, and then carrying out high-throughput sequencing on the target DNA fragment after connecting a sequencing joint. 2bRAD is to cut out DNA fragments with equal length in a genome by using II B type restriction endonuclease, then to screen the fragments, and then to add adapters for sequencing at both ends of the fragments for high-throughput sequencing.
However, the simplified genome sequencing technologies have the following disadvantages: 1. the methods comprise the steps of DNA enzyme digestion, joint connection, PCR and the like, and the operation process is complicated; 2. the operation flow of the methods comprises once or even twice molecular biology operation steps of 'connecting sequencing joints after enzyme digestion', and the enzyme digestion efficiency and the DNA joint connection efficiency of different DNA fragments are different, so that the number of DNA joints successfully connected with different DNA fragments is different. These differences are exponentially amplified by the later PCR amplification, so that some fragments in the sequencing data are sequenced for multiple times to cause data waste, and some fragments which should be sequenced are not detected to cause data loss at the site.
Disclosure of Invention
The invention aims to provide a novel simplified genome sequencing method to solve the ubiquitous limitation of complicated operation procedures in the conventional simplified genome sequencing method.
It is another object of the present invention to provide related applications of the simplified genome sequencing method of the present invention.
The invention provides a novel genome simplification strategy, namely on the basis of a Whole genome sequencing library (WGS), carrying out restriction enzyme digestion on the WGS library before sequencing, wherein fragments containing enzyme digestion sites cannot be sequenced because of being cut off, so that the fragments containing the enzyme digestion sites on a genome are subjected to Inverse selection (Inverse RAD-seq, iRAD-seq) to realize the simplification of the genome. The method of the invention combines AIO-seq constructed by a high-throughput WGS library, can simplify the complicated experimental steps of traditional RAD-seq library construction, and reduces the experimental cost. By selecting different types and quantities of restriction enzymes, the quantity of sequencing fragments can be controlled, and the flexibility of fragment screening is increased.
In particular, the invention provides a method of simplified genomic sequencing, the method comprising: after the heavy sequencing library is subjected to restriction enzyme digestion, fragments with target length and without enzyme digestion sites are selected for sequencing, and accordingly simplified genome sequencing is achieved.
According to a specific embodiment of the present invention, the resequencing library in the method for simplified genome sequencing according to the present invention may be a resequencing library established by any feasible method in the prior art. For example, libraries can be constructed using AIO-seq library construction methods, true-seq library construction methods, or other methods of constructing re-sequencing libraries. For example, in some embodiments of the present invention, the traditional simplified genome sequencing workflow can be further simplified by transposase construction of a re-sequencing library, wherein the process of constructing the re-sequencing library comprises:
(1) Randomly breaking the genomic DNA by using a Tn5 transposase complex, and connecting a Tn5 joint to a breaking position while the transposase complex cuts the DNA;
(2) Taking a product obtained after the Tn5 transposase complex is randomly broken as a template, adding a joint primer used for sequencing to construct a PCR reaction system, and carrying out PCR amplification to obtain a re-sequencing library; or selectively mixing and purifying PCR amplification products to obtain the re-sequencing library.
According to a specific embodiment of the present invention, the restriction enzyme digestion of the resequencing library is mainly to simplify the genome in the method for simplifying genome sequencing of the present invention. Specifically, in the present invention, the fragment containing the cleavage site is preferably cleaved to achieve a simplification level of 60% to 95%, more preferably 75% to 95%, and still more preferably 80% to 90%.
In the present invention, the simplification refers to the proportion of the genome part that is not sequenced to the whole genome, and is defined as: reduced level (P) Reduced level ) = 1-total length of 300bp or more fragment (bp)/total length of genome (bp)) × 100%.
According to a specific embodiment of the present invention, in the method for simplifying genome sequencing of the present invention, any feasible restriction enzyme can be used for digesting the genome DNA of the re-sequencing library to achieve the desired simplification level. According to a specific embodiment of the present invention, the cleavage result of a predetermined restriction enzyme can be evaluated for a specific species using electron cleavage to screen restriction enzymes for a specific species. Thus, the invention can control the number of fragments with target length and total length by selecting the type and number of restriction enzymes, so that simplified sequencing of genome becomes more flexible and efficient.
According to a specific embodiment of the present invention, in the method for simplifying genome sequencing according to the present invention, in general, when the heavy sequencing library is digested, the heavy sequencing library is digested with 1-4 restriction enzymes, preferably with 2 or 3 restriction enzymes.
In some embodiments of the invention, the methods of the invention for simplified genome sequencing are used to sequence the genome of a plant species such as rice, soybean, maize or wheat.
According to a specific embodiment of the present invention, the restriction enzymes in the method for simplified genomic sequencing of the present invention include, but are not limited to, one or more of Mse I, msp I, hindIII, alu I II, dpn2, hinP1I, etc.
According to a specific embodiment of the present invention, after restriction enzyme digestion is performed on the resequencing library, fragments of a target length that do not contain a cleavage site can be selected for sequencing. Specifically, the target length of the fragment can be determined according to the sequencing platform selected for subsequent sequencing. According to some embodiments of the invention, the fragments of interest that are sorted in the method for simplified genomic sequencing of the invention are fragments of 430 to 580bp (comprising a sequencing linker). Specifically, methods of sorting fragments of a target length include, but are not limited to: sorting was performed using an ELF or Sage HT apparatus of Sage Science, sorting was performed using a gel cutting method, or sorting was performed using a biomagnetic bead method.
According to a specific embodiment of the present invention, the method for sequencing the sorted fragments in the method for simplified genome sequencing according to the present invention includes, but is not limited to: sequencing was performed using the huada sequencing platform, illumina sequencing platform, life Technology sequencing platform, or other sequencing platform.
The invention also provides application of the simplified genome sequencing method in whole genome genotype detection.
According to a particular embodiment of the invention, the method of simplified genomic sequencing of the invention is used in whole genome genotype testing, comprising: the method comprises one or more of genotype detection of breeding materials, variety authenticity identification, seed purity detection, species molecular identification, genotype detection of genetic groups, genetic map construction, QTL positioning, molecular marker development and whole genome association analysis in the breeding process of animals, plants and/or microorganisms.
In conclusion, the present invention provides a novel simplified genome sequencing method, reverse simplified genome sequencing (Inverse RAD-seq, iRAD-seq). In the prior art, the conventional RAD-seq firstly selects a part to be detected of a genome, then constructs a library for sequencing, and carries out forward selection and sequencing on fragments beside an enzyme cutting site on the genome. On the basis of condensing different RAD-seq principles, the iRAD-seq method provided by the invention designs a reverse RAD-seq genome simplification strategy of' building a library first, then selecting and carrying out reverse selection on fragments containing enzyme cutting sites (namely selecting fragments without enzyme cutting sites for sequencing). Different from the simplified genome sequencing method in the prior art, the simplified genome sequencing method firstly utilizes restriction enzyme to carry out enzyme digestion on a re-sequencing library, and then selects a fragment with a target length of, for example, 430-580bp for sequencing, the fragment containing the enzyme digestion site cannot be detected because of being cut off, and the fragment which does not contain the enzyme digestion site but has the length of not in the range of 430-580bp cannot be detected, so that the aim of selecting only about 10-20% of the genome for sequencing is fulfilled. Furthermore, the iRAD-seq can control the number and total length of the obtained fragments by controlling the number of restriction enzymes, so that simplified sequencing of the genome becomes more flexible and efficient.
Drawings
FIG. 1 is a schematic diagram of the main flow of iRAD-seq of the present invention.
FIG. 2 is a distribution diagram of the number of fragments of 300bp, 400 bp, 500 bp, 600 bp and 700bp obtained after the genome of soybean, rice, corn and wheat is electronically digested by different restriction enzyme combinations.
FIG. 3 shows the ratio of the total length of the fragments of 300bp, 400 bp, 500 bp, 600 bp and 700bp to the genome obtained after the soybean, rice, corn and wheat genomes are electronically digested by different restriction enzyme combinations.
FIG. 4 is a distribution diagram of fragments of 300bp or more obtained by electron-digesting wheat and soybean genomic DNAs with different combinations of restriction enzymes on different chromosomes.
FIG. 5 is a distribution diagram of fragments of 300bp or more obtained by electron-digesting genomic DNAs of rice and maize with different combinations of restriction enzymes on different chromosomes.
FIG. 6 shows the result of agarose gel electrophoresis of corn DNA.
FIG. 7 is a graph of the distribution of iRAD _ Example1 sequencing sequences in IGV.
FIG. 8 is a genetic map of the CML496/GEMS41 and CIMBL83/GEMS41 populations in iRAD _ Example 1.
FIG. 9 is a genetic map of the CML496/GEMS41 and CIMBL83/GEMS41 populations in iRAD _ Example 2.
FIG. 10 shows the QTL mapping results for the leaf angle trait of CML496/GEMS41 and CIMBL83/GEMS41 populations in iRAD _ Example 1.
FIG. 11 shows the QTL mapping results for the leaf angle trait of CML496/GEMS41 and CIMBL83/GEMS41 populations in iRAD _ Example 2.
FIG. 12 shows statistics of genomic coverage for iRAD-seq and whole genome low coverage sequencing data.
Detailed Description
For a clearer understanding of the technical features, objects, and advantages of the present invention, reference will now be made in detail to the present technical solutions with reference to specific embodiments, and it should be understood that these examples are only intended to illustrate the present invention and should not be construed as limiting the scope of the present invention. In the examples, each raw reagent material is commercially available, and the experimental method not specifying the specific conditions is a conventional method and a conventional condition well known in the art, or a condition recommended by an instrument manufacturer.
Unless specifically defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the relevant art.
The principle and operational flow of the method for simplifying genome sequencing according to the present invention are shown in FIG. 1. Wherein, picture a: the enzyme cutting positions of two restriction enzymes on the genome; picture B: the fragments after the transposase is broken correspond to the distribution on the genome and the restriction enzyme cutting sites; picture C: fragment length screening to obtain high-throughput sequencing-feasible fragments.
In some embodiments of the invention, the methods of the invention for simplified genome sequencing comprise:
constructing a re-sequencing library;
carrying out restriction enzyme digestion on the constructed re-sequencing library by using restriction enzymes, and cutting a fragment containing a digestion site to achieve a simplified level of 60-95%, preferably a simplified level of 75-95%;
the 430-580bp fragments are sorted and sequenced.
Preferably, in the method for simplifying genome sequencing, the library is constructed by using an AIO-seq library construction method, a True-seq library construction method or other methods for constructing a re-sequencing library.
Preferably, in the method for simplifying genome sequencing, when the re-sequencing library is digested, the restriction enzymes are digested by 1-4 restriction enzymes, preferably by 2 or 3 restriction enzymes.
Preferably, in the method for simplifying genome sequencing, the simplification level of 80-90% is achieved when the heavy sequencing library is subjected to enzyme digestion.
Preferably, in the method for simplifying genome sequencing according to the present invention, the restriction enzyme cleavage result of the predetermined restriction enzyme may be evaluated for a specific species by using electronic cleavage to screen the restriction enzyme for the specific species. Specifically, the fa2 cpap _ multi _ color. Pl script in the Solve software (https:// bionogenomics. Com /) can be used for carrying out electronic enzyme digestion on the reference genome sequence of the species to be sequenced, then the find _ enzyme. Pl script is used for carrying out statistics on enzyme digestion fragments with different lengths, and the length distribution of the DNA fragments of the genome subjected to electronic enzyme digestion by the combination of the preset restriction enzymes is analyzed, so that the proper restriction enzymes (which can reach the preset simplified level) are selected. The amount of each restriction enzyme used may be determined by the usual molecular biology experiments, and usually, the amount of each restriction enzyme used is 10U per enzyme per 1. Mu.g of DNA.
In some embodiments of the invention, the methods of the invention for simplified genome sequencing are used to sequence rice, soybean, corn, or wheat genomes.
Preferably, wherein the restriction enzyme includes but is not limited to one or more of Mse I, msp I, hindIII, alu I II, dpnII, hinP 1I.
In some more specific embodiments of the invention, the restriction enzymes are:
a combination of Mse I and Msp I;
a combination of Mse I, msp I and Alu I;
a combination of Mse I, msp I and DpnII;
a combination of Mse I, msp I and HinP 1I; or alternatively
Combinations of Mse I, msp I and HindIII, and the like.
In some more specific embodiments of the invention, the results of the enzymatic cleavage with different restriction enzymes for rice, soybean, corn or wheat are shown in table 1.
TABLE 1
| Endonuclease combinations | Corn (corn) | Rice (Oryza sativa L.) with improved resistance to stress | Soybean | Wheat (Triticum aestivum L.) |
| MseⅠ+MspⅠ | 44％ | 33％ | 33％ | 45％ |
| MseⅠ+MspⅠ+AluⅠ | 19％ | 14％ | 17％ | 23％ |
| MseⅠ+MspⅠ+ |
21％ | 15％ | 20％ | 21％ |
| MseⅠ+MspⅠ+HinP1I | 31％ | 24％ | 29％ | 34％ |
Note: the data in Table 1 are the percentage of the total length of the genome in total length of fragments of 300bp or more after the genome is electronically digested with the corresponding restriction enzymes. The amount of each restriction enzyme used was 10U per enzyme per 1. Mu.g of DNA.
Preferably, in the method for simplified genome sequencing according to the present invention, the method for sorting fragments of a target length includes, but is not limited to: sorting was performed by the eage Science ELF or eage HT apparatus, sorting by the gel cutting method, or sorting by the biomagnetic bead method.
Preferably, in the method for simplified genome sequencing of the present invention, the method for sequencing the sorted fragments includes, but is not limited to: sequencing was performed using the huada sequencing platform, illumina sequencing platform, life Technology sequencing platform, or other sequencing platform.
In some embodiments of the invention, the methods of the invention for simplified genome sequencing comprise:
(1) Random disruption of genomic DNA using the Tn5 transposase complex, with the Tn5 adaptor ligated to the disruption site at the same time as the transposase complex cleaves the DNA, allows the Tn5 adaptor to serve as a primer binding site for subsequent PCR amplification reactions.
(2) And (3) taking a product obtained after the Tn5 transposase complex is randomly broken as a template, adding a joint primer used for sequencing to construct a PCR reaction system, and carrying out PCR amplification. By amplification, adapters for sequencing were added to both ends of the template, facilitating the final sequencing with Illumina sequencer.
(3) And purifying the obtained PCR product, and adding the selected restriction enzyme to cut the DNA fragment after purification. The DNA fragment containing the enzyme cutting site is cut to be short, and the DNA fragment does not contain the influence of the enzyme cutting site not limited by restriction enzyme and is completely reserved.
(4) The digested DNA library was subjected to fragment length screening using a Sage ELF or HT apparatus from Sage Science, a 2% agarose gel DNA recovery gel box (Cassette) and larger DNA fragments (fragments of about 430-580bp in size containing the sequencing linker and about 300-440bp in size without the sequencing linker) were recovered. By the step, the DNA fragments smaller than 300bp in the library are removed, and larger DNA fragments are left, so that the aim of removing the DNA fragments in the region with dense enzyme cutting sites is fulfilled. Only when the distance between two adjacent enzyme cutting sites is larger than or equal to 300bp, the DNA fragments can be kept in the library to be sequenced, thus achieving the aim of simplifying genome sequencing, and the fragment spacing and the number of the genome sequencing can be controlled by the selection of the type and the number of restriction enzymes and the selection of the fragment size.
(5) DNA fragments between adjacent and far enough enzyme cutting sites are enriched after PCR reaction, and the obtained DNA fragments are sequenced by using an Illumina sequencing platform to obtain a DNA sequence.
Example 1: electronic enzyme digestion and result analysis of various crop genome DNA
This example evaluates the theoretical feasibility of the iRAD-seq solution of the present invention.
In this embodiment, firstly, the fa2 cmp _ multi _ color. Pl script in the solution software (https:// bionogenics. Com /) is used to perform electronic enzyme digestion on the reference genome sequences of soybean, rice, corn and wheat, and then the find _ enzyme. Pl script is used to perform statistics on enzyme digestion fragments with different lengths. Finally, analyzing the length distribution of the DNA fragments of the genome subjected to the electron enzyme digestion by different restriction enzyme combinations.
The results show that, when genomic DNAs of soybean, rice, corn and wheat were electronically digested with different combinations of enzymes such as MseI + MspI, mseI + MspI + AluI, mseI + MspI + Dpn II and MseI + MspI + HinP1I, the length distribution of the fragments obtained is shown in FIG. 2, and the ratio of the total length of fragments having a length greater than the different lengths to the genome size (representing the degree of simplification of the genome using different enzyme combinations) is shown in FIG. 3. Therefore, after the genomic DNA of rice, corn, wheat and soybean is subjected to enzyme digestion by different restriction enzyme combinations, the total lengths of the fragments which are larger than or equal to 300bp (PE 150 sequencing is adopted, and the maximum validity of sequencing data is ensured by a threshold value of 300 bp) respectively account for different percentages of the genome length, and the result shows that the total length of the genome to be sequenced can be controlled by controlling different combinations of the restriction enzymes and the insertion length of the DNA fragments of a sorting sequencing library, so that the density of the obtained Tag can be controlled.
This example predicts the distribution of fragments of 300bp or more obtained by electronically digesting rice genomic DNA with the above different combinations of restriction endonucleases on the chromosome, and the results are shown in FIGS. 4 and 5. The results show that the fragments are distributed more evenly on different chromosomes.
Example 2: and performing whole genome genotype detection and QTL positioning on two corn recombinant inbred line populations CML496/GEMS41 and CIMBL83/GEMS41 through iRAD-seq by using the enzyme digestion combination of Msp I + Mse I + HindIII.
This example verifies the application of the iRAD-seq method.
DNA quantification
The genomic DNA of the sample was obtained by first using a NanoDrop2000 spectrophotometer (NanoDrop 2000Spectrophotometer, thermo)
3.0fluorometer (
3.0Fluorometer, invitrogen) were tested for concentration (Table 2, only a portion of the sample concentration is shown). And screening all low concentration (lower than 17.1 ng/. Mu.L) and partial random selection high concentration DNA (higher than 41 ng/. Mu.L) according to the detection result to carry out DNA integrity detection. The quality integrity of the extracted corn genomic DNA (part of the extracted corn genomic DNA) is detected by using 1% agarose gel electrophoresis, the result is shown in figure 6, wherein the picture A is a low-concentration DNA electrophoresis result, and the picture B is a high-concentration DNA electrophoresis result, all DNA bands are found to be complete, which shows that the DNA integrity is better, and the requirement of library construction is met.
Table 2: DNA concentration detection
Note: the concentration 1 is the detection result of NanoDrop2000, and the concentration 2 is the detection result of Qubit.
2. Breaking corn genome DNA by transposase complex and adding Tn5 joint at breakpoint
Breaking corn genome DNA by using Tn5 transposase complex, configuring a reaction system in a sterilized PCR tube according to the components in the table 3, and sequentially adding H 2 O, corn genome DNA, 5 XTnp buffer and Tn5 transposase, and placing the reaction tube into a PCR instrument after uniformly mixing, and operating the following reaction program: hot cover at 105 ℃ and reverse at 55 ℃Hold should be at 10min,4 ℃.
Table 3: transposase complex reaction system
| Composition (I) | Volume (mu L) |
| 5×Tnp buffer | 1.6 |
| Maize genomic DNA | V1(30-50ng) |
| Tn5 transposase | V2(2-3μL) |
| H 2 O | 6.4-V1-V2 |
| Total volume | 8 |
Note: the result of the preliminary experiment finally determines that the using amount of the Tn5 transposase complex is 2-3 mu L; both Tn5 buffer and Tn5 transposase were derived from TruePrep DNA Library Prep Kit V2for Illumina Kit (Vazyme, TD 501-01/02).
3. Immediately after completion of the interrupt reaction, the DNA library was purified using magnetic beads.
4. Sequencing adapters were added to the disruption products after the transposase reaction by PCR and the whole genome was amplified, the PCR reaction system and PCR program as in tables 4 and 5.
Table 4: PCR reaction system
| Composition (I) | Volume (μ L) |
| Breaking of products | 9 |
| |
10 |
| |
1 |
| PPM mixed primer | 3 |
| N5 | 3 |
| N7 | 3 |
| H 2 O | 21 |
| |
50 |
Note: n7 and N5 are primers containing sequencing joint and barcode, and can be respectively combined with two joints of Tn5 to amplify DNA fragments randomly interrupted by Tn5 transposase, and different N5 and N7 contain different barcode sequences. The PPM mixed primer is an equal amount of mixed primers of PPMi5 and PPMi7, and can be used for enriching target fragments. 5 XTAB buffer and TAE enzymeFrom
DNA Library Prep Kit V2for Illumina Kit (Vazyme, TD 501-01/02).
Table 5: reaction procedure for PCR
And 5, after the PCR is finished, equally mixing PCR products, and then purifying by using magnetic beads, and performing the step 3. And (3) carrying out concentration detection on the library by utilizing the Qubit, and carrying out distribution detection on the library fragments by utilizing the Qsep 100.
6. The purified PCR reaction product was cleaved with restriction enzymes, and the reaction system is shown in Table 6.
Table 6: restriction enzyme digestion reaction system
| Composition (I) | Volume (μ L) |
| Purified PCR product | V1(1μg) |
| |
1 |
| |
1 |
| |
1 |
| Cutsmart buffer | 5 |
| H 2 O | 43- |
| Total volume | |
| 50 |
The enzyme digestion conditions are as follows: 37 ℃ for 4h. The Msp I enzyme cutting site is C ^ CGG (# R0106V); mse I enzyme cutting site is TT ^ AA (# R0525V); the HindIII cleavage site is A ^ AGCTT (# R0104V). All three enzymes were purchased from NEB and 100% of all enzyme activities were found in CutSmart buffe.
7. Segment selection
The cleaved library was subjected to fragment selection using the Sage HT apparatus of Sage Science, and fragments of about 430 to 580bp in size were recovered using a 2% agarose gel DNA recovery gel Cassette (Cassette).
8. Sequencing
And (4) performing machine sequencing according to the concentration of the recovered library and the requirements of an Illumina related instrument.
9. Data analysis
Analyzing and processing the off-line data by utilizing software such as FastQC, trimmomatic, fastUniq, BWA, GATK, samtools, picard and the like; and drawing by using R language and AI.
The read obtained by sequencing was mapped to the genome and the results of the mapping of the read were presented on an IGV genome browser (FIG. 7). As can be seen in panel A of FIG. 7, the genomic region containing the cleavage site is not covered by read, and it is expected that the restriction enzyme will cleave the fragment containing the cleavage site, rendering it incapable of being sequenced, and eventually leading to no read coverage. As can be seen in panel B of fig. 7, although a read occurs at the reference genomic cleavage site, it can be found that there is a SNP on the read sequence corresponding to the reference genomic cleavage site, indicating that the sample genome does not contain a cleavage site at this site, which eventually occurs in panel B of fig. 7. In summary, the data satisfy the expected experimental results.
Finally, through analysis and identification, 107,661 SNPs (CML 496/GEMS41 population) and 276,003 SNPs (CIMBL 83/GEMS41 population) are obtained from each filial generation of the two populations on average, and the number of SNPs in each sample is shown in the table 7 and the table 8. According to SNP information, two groups finally obtain a group genetic linkage map, and a picture A and a picture B in figure 8 are the genetic maps of a CML496/GEMS41 group and a CIMBL83/GEMS41 group respectively, and each group has 515 markers and 1,750 markers. Using iRAD sequencing data from the maize population in combination with phenotypic data, it was finally successful to locate 3 leaf angle controlling QTLs in the CML496/GEMS41 population (Panel A and Table 9 in FIG. 10) and 5 QTLs in the CIMBL83/GEMS41 population (Panel B and Table 9 in FIG. 10).
Table 7: statistics of SNP number of CML496/GEMS41 population sample
Table 8: statistics of SNP number of CIMBL83/GEMS41 population sample
Table 9: QTL positioning information table
Example 3: and performing whole genome genotype detection and QTL positioning on two corn recombinant inbred line populations CML496/GEMS41 and CIMBL83/GEMS41 by using enzyme digestion combination of Msp I, mse I and AluIII and iRAD-seq.
The corn population in the example2 is used for carrying out iRAD-seq experiment under the condition of the combination of Msp I + Mse I + AluIII enzyme digestion, after sequencing data are obtained, SNP variation information is searched by using the same analysis process, and finally high-quality SNPs126,464 and 150,842 SNPs are averagely obtained for each sample of the two populations CML496/GEMS41 and CIMBL83/GEMS41, wherein the specific number of SNPs in each sample is shown in the table 10 and the table 11. Finally, genetic linkage maps containing 1,611 markers (panel A in FIG. 9) and 2,822 markers (panel B in FIG. 9) were obtained from the SNP information CML496/GEMS41 and CIMBL83/GEMS41 populations, respectively. Using iRAD sequencing data from the maize population in combination with phenotypic data, it was finally successful to locate 4 leaf angle controlling QTLs in the CML496/GEMS41 population (Panel A and Table 12 in FIG. 11) and 8 QTLs in the CIMBL83/GEMS41 population (Panel B and Table 12 in FIG. 11). Comparison of the two methods revealed that the number of SNPs obtained by the combination of iRAD _ Example2 (Msp I + Mse I + Alu I) restriction enzymes with higher simplification was significantly reduced compared to iRAD _ Example1 (Msp I + Mse I + HindIII restriction enzyme combination), but the major QTL interval was mapped in the final mapping result. In both methods, 1 QTL is located on the No. 2 staining of the CML496/GEMS41 population, and a QTL site is located on each of the chromosomes 1, 3 and 5 of the CIMBL83/GEMS41 population. The distribution trend of the LOD values in the location map is also consistent, which also indicates the stability of the method (FIG. 11). Therefore, the simplified genome sequencing method can well locate the QTL locus under different simplification degrees.
Table 10: statistics of SNP number of CML496/GEMS41 population samples
| Sample numbering | Number of SNPs | Sample numbering | Number of SNPs | Sample numbering | Number of SNPs | Sample numbering | Number of SNPs |
| 3 | 91,064 | 22 | 135,485 | 42 | 75,787 | 65 | 140,048 |
| 4 | 132,454 | 23 | 111,472 | 43 | 72,560 | 66 | 137,212 |
| 5 | 138,107 | 24 | 131,807 | 45 | 126,259 | 67 | 156,199 |
| 6 | 127,310 | 25 | 121,688 | 50 | 147,131 | 68 | 138,346 |
| 7 | 128,611 | 27 | 145,491 | 51 | 71,656 | 69 | 143,614 |
| 8 | 144,305 | 28 | 109,493 | 52 | 191,027 | 70 | 145,051 |
| 9 | 132,950 | 29 | 134,584 | 53 | 122,737 | 71 | 153,064 |
| 10 | 131,380 | 30 | 143,415 | 54 | 5,291 | 72 | 157,200 |
| 12 | 137,814 | 31 | 132,258 | 55 | 128,985 | 73 | 108,874 |
| 13 | 67,943 | 32 | 135,959 | 56 | 132,532 | 74 | 143,684 |
| 14 | 132,097 | 33 | 137,665 | 57 | 128,754 | 75 | 87,214 |
| 15 | 128,252 | 34 | 136,297 | 58 | 129,921 | 76 | 159,413 |
| 16 | 129,388 | 35 | 89,555 | 59 | 137,701 | 77 | 145,978 |
| 17 | 129,622 | 36 | 127,945 | 60 | 166,704 | 79 | 131,164 |
| 18 | 127,654 | 37 | 120,900 | 61 | 100,007 | 80 | 174,047 |
| 19 | 104,233 | 38 | 76,663 | 62 | 179,331 | 81 | 151,323 |
| 20 | 133,277 | 39 | 119,796 | 63 | 143,765 | 82 | 86,647 |
| 21 | 136,662 | 40 | 8,883 | 64 | 163,041 | 83 | 150,666 |
Table 11: SNP number statistics of CIMBL83/GEMS41 population samples
Table 12: QTL positioning results
Example 4: coverage comparison of iRAD-seq with Low coverage sequenced genomes
This example infers the simplification capability of different combinations of restriction enzymes.
1. Genome coverage statistics
Library construction was performed using the library construction method of example2, and the pooled DNA library was divided into two parts before digestion, one part for whole genome low coverage sequencing (lcWGS) and the other part for iRAD library construction by restriction enzyme digestion. Two different enzyme cleavage combinations have been tried, iRAD _ Example1 (Example 2) and iRAD _ Example2 (Example 3). The sequencing data of 8 identical samples were randomly selected among the sequencing data of the above three methods for genome coverage analysis. In order to ensure the consistency of data quantity, firstly, seqkit software is used to randomly extract 2Gb data from original data, then the data are compared to a target reference genome, then qualimap software is used to analyze a BAM file, further, base coverage information in a result file of the previous analysis is extracted through a script, and finally, the obtained result is shown in fig. 12. The electronic enzyme cutting simplification degree of the combination of the Msp I + Mse I + HindIII enzyme cutting is 58.9%, and the Msp I + Mse I + Alu I electronic enzyme cutting simplification degree is 81.19%, and it can be found from FIG. 12 that the read coverage depth of the data inclines to high coverage along with the improvement of the simplification degree. This indicates that the genome simplification efficiency meets the situation of electronic enzyme digestion prediction, and different enzyme digestion combinations can obtain different genome simplification effects.
Claims (10)
1. A method of simplifying genome sequencing, the method comprising: after the heavy sequencing library is subjected to restriction enzyme digestion, fragments with target length and without enzyme digestion sites are selected for sequencing, and accordingly simplified genome sequencing is achieved.
2. The method of claim 1, comprising:
constructing a re-sequencing library;
carrying out restriction enzyme digestion on the constructed re-sequencing library by using restriction enzymes, and cutting a fragment containing an enzyme digestion site to achieve a simplification level of 60-95%; preferably to a simplification level of 75% to 95%;
the 430-580bp fragments are sorted and sequenced.
3. The method of claim 1 or 2, wherein the re-sequencing library is digested with 1-4 restriction enzymes, preferably 2 or 3 restriction enzymes.
4. The method of claim 1 or 2, wherein the re-sequencing library is digested to a level of 80% to 90% simplification.
5. The method of claim 1 or 2, wherein the re-sequencing library is constructed using an AIO-seq library construction method, a True-seq library construction method or other methods of constructing a re-sequencing library.
6. The method of claim 1 or 2, wherein constructing the resequencing library comprises:
(1) Randomly breaking the genomic DNA by using a Tn5 transposase complex, and connecting a Tn5 joint to a breaking position while the transposase complex cuts the DNA;
(2) Taking a product obtained after the Tn5 transposase complex is randomly broken as a template, adding a joint primer used for sequencing to construct a PCR reaction system, and carrying out PCR amplification to obtain a re-sequencing library; or selectively mixing and purifying PCR amplification products to obtain a re-sequencing library.
7. The method of claim 1 or 2, wherein the method of sorting fragments of a target length includes, but is not limited to: sorting by using an ELF or Sage HT instrument of Sage Science, sorting by using a gel cutting method or sorting by using a biological magnetic bead method;
preferably, methods of sequencing the sorted fragments include, but are not limited to: sequencing was performed using the huada sequencing platform, illumina sequencing platform, life Technology sequencing platform, or other sequencing platform.
8. The method according to claim 1 or 2, which is for sequencing a rice, soybean, maize or wheat genome;
preferably, wherein the restriction enzyme includes but is not limited to one or more of Mse I, msp I, hind III, alu I II, dpnII, hinP 1I.
9. The method of claim 8, wherein the restriction enzyme is:
a combination of Mse I and Msp I;
a combination of Mse I, msp I and Alu I;
a combination of Mse I, msp I and DpnII;
a combination of Mse I, msp I and HinP 1I; or
Combination of Mse I, msp I and Hind III.
10. Use of the method of any one of claims 1-9 for whole genome genotype detection;
preferably, the whole genome genotype detection comprises: the method comprises one or more of genotype detection of breeding materials, variety authenticity identification, seed purity detection, species molecular identification, genotype detection of genetic groups, genetic map construction, QTL positioning, molecular marker development and whole genome association analysis in the breeding process of animals, plants and/or microorganisms.
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