CN109266729B - Large fragment deletion detection method based on genome second-generation sequencing - Google Patents
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- 238000012217 deletion Methods 0.000 title claims abstract description 38
- 230000037430 deletion Effects 0.000 title claims abstract description 38
- 239000012634 fragment Substances 0.000 title claims abstract description 37
- 238000012163 sequencing technique Methods 0.000 title claims abstract description 25
- 238000001514 detection method Methods 0.000 title abstract description 8
- 238000000034 method Methods 0.000 claims abstract description 22
- 238000011160 research Methods 0.000 claims abstract description 6
- 238000012216 screening Methods 0.000 claims description 15
- 238000012070 whole genome sequencing analysis Methods 0.000 claims description 6
- 230000001105 regulatory effect Effects 0.000 claims description 4
- 238000012268 genome sequencing Methods 0.000 claims description 3
- 238000012545 processing Methods 0.000 claims description 2
- 230000000717 retained effect Effects 0.000 claims description 2
- 238000011331 genomic analysis Methods 0.000 claims 2
- 238000004458 analytical method Methods 0.000 abstract description 12
- 108090000623 proteins and genes Proteins 0.000 abstract description 4
- 241000196324 Embryophyta Species 0.000 description 25
- 241000209094 Oryza Species 0.000 description 15
- 235000007164 Oryza sativa Nutrition 0.000 description 15
- 235000009566 rice Nutrition 0.000 description 15
- 231100000350 mutagenesis Toxicity 0.000 description 9
- 238000002703 mutagenesis Methods 0.000 description 9
- PLUBXMRUUVWRLT-UHFFFAOYSA-N Ethyl methanesulfonate Chemical compound CCOS(C)(=O)=O PLUBXMRUUVWRLT-UHFFFAOYSA-N 0.000 description 6
- 230000035772 mutation Effects 0.000 description 4
- 230000005251 gamma ray Effects 0.000 description 3
- 230000005855 radiation Effects 0.000 description 3
- 238000009331 sowing Methods 0.000 description 3
- 108020004414 DNA Proteins 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 238000003306 harvesting Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 230000003505 mutagenic effect Effects 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 241001470017 Laodelphax striatella Species 0.000 description 1
- 240000008467 Oryza sativa Japonica Group Species 0.000 description 1
- 108020005120 Plant DNA Proteins 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
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- 238000002474 experimental method Methods 0.000 description 1
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- 239000003471 mutagenic agent Substances 0.000 description 1
- 231100000707 mutagenic chemical Toxicity 0.000 description 1
- 238000012113 quantitative test Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
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Abstract
The invention discloses a large fragment deletion detection method based on genome second-generation sequencing. The method provided by the invention is adopted to detect the deletion of large fragments (fragments larger than 100 bp), the comparison speed is high, the working efficiency is improved, and the analysis of the deletion of the large fragments of the whole genome can be completed within about 1 hour. The method lays a foundation for later analysis of candidate genes in the deletion segment and research of a regulation and control network. The invention fills the blank of the technology for detecting large fragment deletion of genome by a second-generation sequencing method.
Description
Technical Field
The invention relates to a large fragment deletion detection method based on genome second-generation sequencing.
Background
In order to quickly obtain the ideal mutant of the plant, a regulation network corresponding to the important phenotype of the plant is researched, and the seed of the plant can be mutagenized by physical or chemical factors and the like, so that the mutation time of the plant is shortened. Chemical mutagenesis, such as the mutagen Ethyl Methanesulfonate (EMS), can cause single base mutations, physical mutagenesis involving various radiation, etc., such as gamma radiation, can cause fragment deletions to plant DNA bases. With the maturity of the second-generation genome sequencing technology and the further reduction of the price, the method for analyzing cloned genes by using mixed packet analysis (BSA) based on the second-generation sequencing technology is easy to implement, the experimental efficiency of the method is greatly improved compared with the traditional map-based cloning technology, the single base mutation comparison technology generated by the second-generation sequencing is mature at present, but the deletion of large fragments of a genome caused by gamma rays is still difficult due to the technical limitation of the second-generation sequencing, the size of the fragments generated by the second-generation sequencing is generally 150bp at present, the analysis of the deletion of the fragments smaller than 150bp by using the traditional analysis software based on the second-generation sequencing is easy, but the analysis of the deletion of the fragments with the length larger than 150bp and even more than dozens of kbp is not easy, and no software can directly analyze the specific position information of the large fragment deletion so far.
Disclosure of Invention
The invention aims to provide a method for detecting large fragment deletion based on genome second-generation sequencing.
The invention firstly protects a method for detecting genome fragment deletion in plant mutants, which comprises the following steps (1) and (2):
(1) carrying out whole genome sequencing on the mutant to be detected;
(2) after the step (1) is completed, the following steps (a) to (d) are carried out on the sequencing result:
(a) cutting off 2/3-length sequences from the 5 '-3' direction of the complete sequences of all reads, aligning the rest parts to a plant reference genome, screening reads which can be aligned to the plant reference genome by 100%, and recording the alignment position of each read;
(b) cutting off 2/3-length sequences from 3 '-5' direction of the complete sequences of all reads obtained by screening in the step (a), aligning the rest parts to a plant reference genome, screening reads which can be aligned to the plant reference genome by 100%, and recording the aligned position of each read;
(c) comparing the complete sequences of all reads obtained by screening in the step (b) to a plant reference genome, removing the reads which can be completely compared to the plant reference genome by 100%, and reserving the rest of the reads;
(d) processing all reads retained in step (c) as follows: subtracting the position of the same read aligned to the genome in the steps (a) and (b), and if the value is greater than 2/3 of the sequencing read length, determining that the fragment is deleted;
the plant reference genome is a reference genome of a plant to which the mutant plant belongs.
The length of the deletion fragment is more than 100 bp.
The genome sequencing can specifically adopt second-generation sequencing. The second generation sequencing may be specifically 454 technology by Roch corporation, Solexa and Hiseq technology by illumina corporation, and Solid technology by ABI corporation.
The step (2) is realized by using sequence alignment software; the sequence alignment software was bowtie 2.
The step (2) further comprises a step (e): and (d) sequencing all sites with fragment deletion according to the size through the analysis of the step (d), and calculating the number of deletion sites of the whole genome.
The invention also provides a system for detecting deletion of a genome fragment in a plant mutant, which comprises a whole genome sequencing instrument, sequence alignment software and an instruction recorded with any one of the methods. The sequence alignment software may specifically be bowtie 2.
The length of the deletion fragment is more than 100 bp.
Any of the above mutants can be obtained by various methods, for example, by purchasing a mutant having the target phenotype from an existing mutant library, or by screening a mutant having the target phenotype by using EMS (ethyl methane sulfonate) mutagenesis, gamma ray mutagenesis, natural mutation, or the like.
The invention also protects the application of any one of the methods in the genome analysis of the plant mutant.
The invention also protects the application of any one of the systems in the genome analysis of the plant mutant.
The invention also protects the application of any one of the methods in the whole genome regulatory network research.
The invention also protects the application of any one of the systems in the whole genome regulatory network research.
Any of the above plant mutants may specifically be a rice mutant.
Any one of the plant reference genomes described above may specifically be a rice nipponica reference genome.
The method provided by the invention is adopted to detect the deletion of large fragments (fragments larger than 100 bp), the comparison speed is high, the working efficiency is improved, and the analysis of the deletion of the large fragments of the whole genome can be completed within about 1 hour. Can analyze the candidate gene in the deletion segment and lay the foundation for the research of the regulation and control network. The invention fills the blank of the technology for detecting large fragment deletion of genome by a second-generation sequencing method.
Drawings
FIG. 1 shows phenotypic observations of rice kittake and mutants.
Fig. 2 shows the result of the deletion detection verification.
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.
Rice kittake (japonica rice): has a multi-tillering phenotype; reference documents: the expression of rice defense related genes under the stress of Laodelphax striatellus [ J ] crop report, 2012,38(9): 1625-; the public is available from the institute of genetics and developmental biology, academy of Chinese sciences.
Example 1 obtaining of mutagenic Material
1. Taking rice kittake seeds as raw materials, and carrying out gamma ray mutagenesis (taking the seeds and carrying out radiation treatment on the seeds by adopting gamma rays with the intensity of 25.00 c/kg).
2. Sowing and cultivating the seeds obtained in the step 1 into plants, and harvesting the seeds after the plants are selfed (M)1Seed generation).
3. Sowing and cultivating the seeds obtained in the step 2 into plants, and harvesting the seeds after the plants are selfed (M)2Seed generation).
4. And (4) sowing the seeds obtained in the step (3) and cultivating the seeds into plants, screening out one mutant strain with the tillering angle of more than 20 from the plants, and naming the mutant strain as a mutant Mu 1.
A photograph of the phenotypes of rice kittake and mutant Mu1 is shown in FIG. 1.
In practice, mutants can be obtained by any conventional method, for example, mutants can be purchased from a conventional mutant library, and desired mutants can be obtained by screening by methods such as EMS (ethyl methane sulfonate) mutagenesis, gamma ray mutagenesis, or natural mutagenesis.
Example 2 detection and analysis of Large fragment deletions
1. The mutant Mu1 obtained in example 1 was subjected to whole genome sequencing using a second generation sequencing technique. The whole genome sequencing may be performed by a second-generation sequencing technique such as 454 by Roch corporation, Solexa by illumina corporation, Hiseq technique, and Solid technique by ABI corporation.
2. After the step 1 is completed, the bowtie2 comparison software is adopted for comparison, and the method comprises the following steps:
(1) cutting off sequences of 2/3 length from 5 '-3' direction of all complete reads obtained by sequencing in the step 1, comparing the rest partial sequences to the Nipponbare reference genome of rice, screening all the reads sequences which can be compared on the genome of the rice by 100%, and recording the comparison position of each read;
(2) comparing all the complete reads obtained by screening in the step (1) from the 3 '-5' direction to 2/3-length sequences, comparing the rest sequences to the Nipponbare reference genome of rice, screening all the reads sequences which can be compared to the genome of the rice by 100%, and recording the comparison position of each read;
(3) comparing the complete sequences of all reads obtained by screening in the step (2) with the rice genome, removing the reads which can be compared with the rice genome by 100%, eliminating false positives, and reserving the rest of the reads;
(4) and (4) performing the following treatment on all reads reserved in the step (3): subtracting the position of the same read aligned to the genome in the steps (1) and (2), and if the value is greater than 2/3 of the sequencing read length, determining that the deletion exists;
(5) through the analysis of the step (4), all sites with deletion can be sorted according to size, and the number of deletion sites of the whole genome can be calculated.
Through the analysis, 39353bp of sequence is deleted in the No. 3 chromosome position 1848K-1888K interval of the mutant Mu 1.
3. To verify the results of steps 1 and 2, the genomic DNA of mutant Mu1 and the genomic DNA of rice kittake were PCR amplified using a primer pair consisting of primers 2f and 2r (designed for the deleted 39353bp fragment), respectively, and the amplified products were analyzed by electrophoresis.
2f：5’-TCATGTACTTTTAGCAGTA-3’；
2r：5’-TTTACGAGTAATATTTCC-3’。
Reaction system of PCR amplification: taq enzyme (Takara) 0.1. mu.l, 10 XBuffer 1. mu.l, dNTP (10 Mm/L): 1. mu.l, template 0.5. mu.l, primer 2f (10. mu.M/L) 0.5. mu.l, primer 2r (10. mu.M/L) 0.5. mu.l, water: 6.4 microliter.
Reaction conditions for PCR amplification: first 94 ℃ for 4 minutes, then 94 ℃ for 1 minute, 50 ℃ for 1 minute, 72 ℃ for 1 minute for 35 cycles, and finally 72 ℃ for 5 minutes.
If a deletion is true, a 150bp fragment can be amplified in the mutant Mu1(mutant), while in the control (rice kittake, WT), the PCR product size is 39353bp due to the absence of the deletion, and a band cannot be observed by electrophoresis due to the fact that the fragment is too large to be amplified. The results are shown in FIG. 2. The result shows that the large fragment deletion detection method based on the second-generation sequencing is correct and reliable.
The large fragment deletion detection is carried out by using commercially available mutants or mutants obtained by other mutagenesis methods, and the large fragment deletion condition of the mutants can be correctly detected through multiple experimental verification, so that the universality of the method is verified.
Claims (7)
1. A method for detecting genome fragment deletion in plant mutants comprises the following steps (1) and (2):
(1) carrying out whole genome sequencing on the mutant to be detected;
(2) after the step (1) is completed, the following steps (a) to (d) are carried out on the sequencing result:
(a) cutting off 2/3-length sequences from the 5 '-3' direction of the complete sequences of all reads, aligning the rest parts to a plant reference genome, screening reads which can be aligned to the plant reference genome by 100%, and recording the alignment position of each read;
(b) cutting off 2/3-length sequences from 3 '-5' direction of the complete sequences of all reads obtained by screening in the step (a), aligning the rest parts to a plant reference genome, screening reads which can be aligned to the plant reference genome by 100%, and recording the aligned position of each read;
(c) comparing the complete sequences of all reads obtained by screening in the step (b) to a plant reference genome, removing the reads which can be completely compared to the plant reference genome by 100%, and reserving the rest of the reads;
(d) processing all reads retained in step (c) as follows: subtracting the position of the same read aligned to the genome in the steps (a) and (b), and if the value is greater than 2/3 of the sequencing read length, determining that the fragment is deleted;
the plant reference genome is a reference genome of a plant to which the mutant plant belongs; the length of the deletion fragment is more than 100 bp;
the genome sequencing adopts second-generation sequencing.
2. The method of claim 1, wherein: the step (2) is realized by using sequence alignment software; the sequence alignment software was bowtie 2.
3. A system for detecting genomic fragment deletions in plant mutants comprising a whole genome sequencing instrument, sequence alignment software and instructions describing the method of claim 1 or 2; the length of the deletion fragment is more than 100 bp.
4. Use of the method of claim 1 or 2 for genomic analysis of plant mutants.
5. Use of the system of claim 3 for the genomic analysis of plant mutants.
6. Use of the method of claim 1 or 2 in genome-wide regulatory network research.
7. Use of the system of claim 3 in genome-wide regulatory network research.
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