CN113549682A - Detection method of mitosis recombination hot spot of yeast - Google Patents
Detection method of mitosis recombination hot spot of yeast Download PDFInfo
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Abstract
The invention relates to the technical field of synthetic biology, in particular to a detection method of mitotic recombination hot spots of yeast. The PCRTag tag combination provided by the invention can be used for rapidly detecting mitotic recombination hot spots of eukaryotic genome. The mitotic recombination hotspot of homologous chromosomes can be quickly identified by utilizing the label combination through the specific watermark label, and a convenient method is provided for researching the mitotic recombination event of eukaryotes.
Description
Technical Field
The invention relates to the technical field of synthetic biology, in particular to a detection method of mitotic recombination hot spots of yeast.
Background
Homologous recombination between DNA molecules refers to a biological process in which DNA sequences undergo double strand breaks, recombinational repair, and chromosome separation processes to achieve recombination between sequences, forming novel DNA molecules. Homologous recombination can not only repair double-stranded break (DSB) of DNA, maintain genome stability, but also realize cross exchange of genetic information, generate daughter cell genetic diversity and accelerate the biological evolution process.
Homologous recombination comprises both meiotic and mitotic recombination, and naturally, genetic material in a cell is more predisposed to meiotic recombination, and mitotic recombination occurs at a rate 10 times less than meiotic recombination events4～105Multiple, and thus one-to-meiosisRecombination events are more extensively studied. However, mitotic recombination also has important roles in eukaryotes, including repair of spontaneously occurring DSBs, restarting arrested replication forks, providing an alternative pathway for telomere replication in telomerase-deficient cells, and facilitating genome evolution by generating new chromosomal rearrangements. Chromosomal rearrangements during cell mitosis lead to loss of heterozygosity (LOH) at tumor suppressor sites, which in turn induces malignant growth of cells and induces cancer cells, although LOH causes are numerous, about half of the LOH events are associated with mitotic recombination in a study on retinoblastoma, and thus, the study on mitotic recombination mechanism can also provide guidance for the mechanism of cancer induction.
In the early period, only a single recombination hotspot region in yeast was explored, and a great deal of research was carried out to characterize the chromosome rearrangement rate by constructing a meiosis-specific integration plasmid, which can be integrated into the genome during meiosis to cause ectopic recombination of homologous regions, and the research finds that the meiosis recombination rate of a DNA fragment close to THR4 at 2-2.5cM/kb of chromosome III is 6 times higher than the average recombination rate of chromosomes, and the site is a strong DSB site. Using a similar approach, the following recombination hotspots for saccharomyces cerevisiae were also located: HIS2, HIS4, ARG4, CYS3, DED81, ARE1/IMG1, CDC19, LEU2-CEN 3. By sequence analysis of the above hot spots, most of the recombination hot spots are DSB sites, usually tandem repeats sensitive to DNase I, but there are exceptions, for example, the recombination hot spot of ade6-M26 is not located at the DSB site, but located in the sequence 500bp upstream of the DSB site, and the site can be combined with the transcription factor Atf1/Pcr1, and White and colleagues also need to combine with the transcription factors Bas1, Bas2 and Rap1 through the hot spot of experiment HIS4, and the hot spot which needs to be combined with the transcription factor is called 'alpha' hot spot, and the hot spot which does not need to be combined with the transcription factor is called 'beta' hot spot. The above experiments indicate that the DSB site is the first site for recombination fragmentation, but the DSB site is not the only factor affecting the recombination rate, and the recombination rate is also closely related to the chromatin structure and the environment of chromosomes.
With the development of DNA sequencing technology, people are beginning to focus on the exploration of recombination hot spots and cold spots in the whole genome. Gerton et al performed microarray analysis on 6200 ORFs by using a DNA probe method, ranked the region with the recombination rate of 12.5% as the recombination hotspot region, and finally detected 177 hotspots in total. As a result, all chromosomes have at least one hot spot, the significant correlation exists between the size of the chromosomes and the number of the hot spots, the comparative analysis of all recombination hot spots shows that no single significant characteristic exists in all hot spot regions, and the factor with the strongest correlation among the hot spots is high GC content. Mancera et al genotyped the parental strain S288c/YJM789 heterozygote and its 204 spores produced by 51 meioses using ssGengenotyping, and mapped the high resolution map of meiotic recombination in the yeast genome. Studies have shown that on average each meiosis produces 90.5 CO and 46.2 NCO, and that the amount of CO is linear with chromosome length. By counting the number of strains appearing at each marker, the recombination rate of each locus of the genome is characterized, and 84% of recombination hotspot regions are found to be overlapped with the promoter. Most of the genomes have differences in the distribution ratio of CO/NCO, and the differences can reach the maximum CO/NCO event ratio of 14: 0 and 0: 7. by analyzing the GC content of the recombination hotspot region, it was found that chromosomes are prone to fragmentation recombination at high GC content. Charles utilizes a SNP microarray method to research the mitotic recombination rule of a yeast chromosome IV, finds that the region where an inverted repeat sequence is located is very easy to become a breaking hot spot region, because the inverted repeat sequence is frequently subjected to intrachain pairing during recombination, the formed hairpin structure prevents the movement of a replication fork, a DSB gap is formed, a cross-shaped structure similar to a Hodgy knot is formed sometimes, and the cross-shaped structure is mistakenly identified and cut by endonuclease, and researches show that the inverted Ty element pair (YDRWTy2-3 and YDRCTy1-3) positioned between 981-992kb is obviously related to the recombination rate of a hot spot HS 4. Chromosomal genomic recombination is more likely to occur at positions in the yeast such as palindromic sequences, tandem repeats, G4 DNA sequences, tRNA's, intron-containing gene sequences, ARS elements, Ty elements and long terminal repeats thereof, highly transcribed genes, replication termination regions, high gamma-H2 AX regions, high GC content regions, Rrm3p termination sites, and the like, as determined by characterizing chromosomal fragile sites. Yang et al explored the chromosomal rearrangement hotspot during spontaneous mitosis in diploid yeast and found that large deletions/duplications and translocations were often mediated by homologous recombination between repeated sequences such as Ty element or delta element, where the break point of I-LOH (Interstinal LOH) in the yeast genome was significantly associated with the replication termination (Ter) sequence and regions with abnormal GC content, while T-LOH (terminal LOH) was mostly located in the G4 quadruplex sequence, the region with high gamma-H2 AX content, the region with high GC content, and the non-coding RNA gene.
Although a great deal of research has been conducted on the genome variation law of yeast mitotic recombination, the research on the hot and cold spot regions of yeast recombination is not sufficient because it is limited to the construction of mitotic libraries and the screening of recombinant strains. The SCRAMBLE system in the synthetic yeast can quickly construct a genome recombinant library, and secondly, the introduction of the PCRtag watermark provides a simple and efficient method for positioning the recombination hotspot region of the genome recombinant strain. The process of random disorganization and recombination of the synthetic chromosomes is very similar to the process of forming cancers, so that a theoretical model is hopefully provided for determining target genes related to human chromosome diseases by researching the recombination rule of the yeast synthetic chromosomes in the process of artificially inducing rearrangement, and a reference is provided for target screening and designing of antitumor drugs. The method is beneficial to analyzing the influence of genome doubling on genome recombination by comparing and analyzing the rules of synthetic diploid yeast and artificially constructed tetraploid yeast with doubled whole genome in artificially induced rearrangement, and has guiding significance on polyploidization and chromosome fragmentation research in cancer. However, no suitable PCRtag has been found for the study of mitotic recombination hotspots.
Disclosure of Invention
In view of the above, the present invention provides a method for detecting a yeast mitotic recombination hotspot.
The method adopted by the invention can be suitable for the exploration of all chromosome rearrangement hot spots containing specific watermark labels. However, due to the specificity of the chromosome tags of different species, the watermark tag corresponding to the chromosome needs to be selected when the chromosome is explored. The invention aims at the research of saccharomyces cerevisiae, and in the research, a yeast strain containing artificially synthesized chromosomes is adopted. Each synthetic chromosome is introduced with different numbers of specific watermark tags at the beginning of design, such as 532, 186, 339, 164, 490 and 681 pairs of PCRTag tags introduced into synII, synIII, synV, synVI, synX and synXII respectively, and the tags can be used for screening genome recombinant strains. However, in the process of detecting the mitotic recombination hotspot, the watermark label should be selected according to the length of the chromosome, and the aim is to finally obtain a good detection effect.
The invention researches on the chromosome V of yeast, and the PCRTag tag combination provided by the invention comprises: at least two of YEL038W _ amp1, YEL013W _ amp1, YER026C _ amp1, YER056C _ amp1, YER086W _ amp1, YER113C _ amp2, YER144C _ amp2, YER170W _ amp1, YEL032W _ amp2, YEL029C _ amp1, YEL023C _ amp1, YEL019C _ amp 1. In some embodiments, the PCRTag tag combination of the present invention includes: YEL038W _ amp1, YEL013W _ amp1, YER026C _ amp1, YER056C _ amp1, YER086W _ amp1, YER113C _ amp2, YER144C _ amp2, and YER170W _ amp 1.
In the PCRTag tag combinations provided by the present invention, YEL038W _ amp1, YEL013W _ amp1, YER026C _ amp1, YER056C _ amp1, YER086W _ amp1, YER113C _ amp2, YER144C _ amp2, and YER170W _ amp1 are evenly distributed on the synV chromosome, wherein 2 pcrtags are evenly distributed on the left arm of the V chromosome, and the remaining 6 are evenly distributed on the right arm of the V chromosome. More mitotic recombination events of chromosomes can be observed relative to other combinations of PCRTag tags.
In addition, for the strains with chromosome breakage between YEL038W _ amp 1-YEL 013W _ amp1, 10kb refinement analysis was also performed, and one PCRTag was reselected every 10kb to verify the genome loss of the above samples at YEL032W _ amp2, YEL029C _ amp1, YEL023C _ amp1, YEL019C _ amp1, respectively.
The invention also provides application of the label combination in mitotic recombination hotspot detection.
The invention realizes mitotic recombination of homologous chromosomes through manual intervention, and quickly constructs a recombinant strain library through a specific watermark label, thereby providing a convenient method for researching mitotic recombination events. In the present invention, the mitosis is a mitosis of a eukaryote. The eukaryote is a yeast, and in some embodiments, the eukaryote is saccharomyces cerevisiae.
The strains involved in the research of the invention comprise diploid saccharomyces cerevisiae, triploid saccharomyces cerevisiae and tetraploid saccharomyces cerevisiae.
The diploid s.cerevisiae contains one wild type chromosome V (wtV) and one synthetic type chromosome V (synV). Triploid Saccharomyces cerevisiae contains one wild type chromosome V (wtV) and two synthetic type chromosomes V (synV). Tetraploid Saccharomyces cerevisiae contains two wild type V chromosomes (wtV) and two synthetic type V chromosomes (synV).
The invention also provides a reagent for detecting the mitotic recombination hot spot of the yeast, which comprises a primer for detecting the label combination. The primer of the invention comprises:
an amplification primer of YEL038W _ amp1, the nucleic acid sequence of which is shown in SEQ ID NO. 1-2;
an amplification primer of YEL013W _ amp1, the nucleic acid sequence of which is shown in SEQ ID NO. 3-4;
an amplification primer of YER026C _ amp1, the nucleic acid sequence of which is shown in SEQ ID NO. 5-6;
an amplification primer of YER056C _ amp1, the nucleic acid sequence of which is shown in SEQ ID NO. 7-8;
an amplification primer of YER086W _ amp1, the nucleic acid sequence of which is shown in SEQ ID NO. 9-10;
an amplification primer of YER113C _ amp2, the nucleic acid sequence of which is shown in SEQ ID NO. 11-12;
an amplification primer of YER144C _ amp2, the nucleic acid sequence of which is shown in SEQ ID NO 13-14;
the nucleic acid sequence of the amplification primer of YER170W _ amp1 is shown in SEQ ID NO. 15-16.
In the present invention, the reagent further includes a PCR reaction reagent. The PCR reagent comprises Taq enzyme.
The invention also provides a recombinant hot spot method for detecting the mitosis of the yeast, which is to detect the yeast by using the reagent.
The invention takes chromosome V as a research object to research the mitosis recombination hotspot of the yeast.
The yeast comprises diploid saccharomyces cerevisiae, triploid saccharomyces cerevisiae and tetraploid saccharomyces cerevisiae. Wherein the diploid s.cerevisiae contains one wild type V chromosome (wtV) and one synthetic type V chromosome (synV). Triploid Saccharomyces cerevisiae contains one wild type chromosome V (wtV) and two synthetic type chromosomes V (synV). Tetraploid Saccharomyces cerevisiae contains two wild type V chromosomes (wtV) and two synthetic type V chromosomes (synV).
Before amplification detection, the yeast is subjected to induced rearrangement to construct a rearrangement library. The induction was with estradiol. The concentration of estradiol in the induced medium was 1. mu. mol/L.
The detection method comprises the following steps: the amplification primer of the invention is used for amplifying the microzyme. The system for amplification comprises: an upstream primer, a downstream primer, Taq enzyme, template DNA and water.
Based on comparing the amplification results of the three strains, genomic mitotic diversity was obtained. The method mainly comprises the steps of counting the number of breakpoints between adjacent PCRTag after rearrangement, and obtaining the mutation condition of the sample. For example: the presence of the PCRTag band of the synthetic type V chromosome indicates that at least one copy of the DNA fragment of all the synthetic type V chromosomes in the test strain is present. The PCRTag bands of the synthetic type V chromosome were not detected at all, indicating that the synthetic type V chromosome was lost in the test strain at all. The presence of the PCRTag band portion of the synthetic V chromosome indicates that both copies of the partially synthetic V chromosome DNA region were lost in the triploid test strain. If the number of amplification breakpoints is large, the segment is unstable and is a recombination hot spot region. And the small number of times of the amplified breakpoint indicates that the part of the genome is not easy to lose during recombination and is a recombination cold spot region.
In addition, the rearranged libraries were sequenced and statistically analyzed. The sequencing method comprises the following steps: whole genome sequencing reads obtained by specific extraction paired-end sequencing. The statistical analysis comprises: analyzing the copy number variation of homologous chromosomes of cells in the process of genome rearrangement by using the difference of PCRTag sequences on a synthetic V chromosome and a wild V chromosome; sequencing the number of synthetic V chromosomes and wild type V chromosomes in the deeply responsive cells; counting the mode and position of ectopic recombination, and analyzing unequal repetition/deletion caused by ectopic recombination among sister chromatids; and (4) counting the quantity of SNP sites of homologous chromosomes and analyzing the phenomenon of interstitial heterozygosity deletion.
The invention provides a PCRTag tag combination and provides application thereof in rapidly detecting mitotic recombination hot spots of eukaryotic genomes. The mitotic recombination hotspot of homologous chromosomes can be quickly identified by utilizing the label combination through the specific watermark label, and a convenient method is provided for researching the mitotic recombination event of eukaryotes.
Drawings
FIG. 1 shows a process for constructing polyploid strains of different karyotypes;
FIG. 2 shows a schematic of the PCRTag detection of synthetic, wild-type chromosomes;
FIG. 3 shows the PCRTag detection of chromosome V presence pattern of the triploid strain;
FIG. 4 shows the existence pattern and proportions of the triploid SCRaMbLE mutant synV;
FIG. 5 shows statistics of the number of occurrences of breakpoint regions between adjacent PCRTags in a diploid;
FIG. 6 shows statistics of 10kb thinning breakpoint times between diploid synV 70.5 kb-118.1 kb;
FIG. 7 shows a method for detecting the ORF to which it belongs using a PCRtag-specific CDS read;
FIG. 8 shows a genome-wide sequencing depth map of a triploid synV completely deleted yeast strain;
FIG. 9 shows the phenomenon of unequal duplication/deletion due to ectopic recombination between diploid yeast sister chromatids;
FIG. 10 shows I-LOH resulting from diploid mitotic recombination.
Detailed Description
The invention provides a method for detecting mitotic recombination hot spots of yeast, and a person skilled in the art can realize the detection by appropriately improving process parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
The test materials adopted by the invention are all common commercial products and can be purchased in the market. In the invention, the adopted strains are all from Tianjin university. The designation of the PCRtag is found in "Perfect" designer chromosome V and behavor of a ring derivative (Science,2017,355(1046): eaaf4704.) and http:// synthetic layer. The invention is further illustrated by the following examples:
examples
1. Construction of rearranged Strain libraries
1.1 strains
The diploid s.cerevisiae strain yLHM120, which is a starting strain, contains a wild-type chromosome V (wtV) and a synthetic chromosome V (synV) and is available from Tianjin university.
② the triploid saccharomyces cerevisiae strain yXZX1892 as an original strain, which contains a wild type V chromosome (wtV) and two synthetic type V chromosomes (synV) from Tianjin university.
③ the tetraploid saccharomyces cerevisiae strain yLHM086, which contains two wild type V chromosomes (wtV) and two synthetic type V chromosomes (synV), was used as the starting strain from the university of tianjin.
1.2 Induction of rearrangement:
from the SC-His plate, a single colony carrying pCRE4 was picked, inoculated into 3mL of SC-His medium, and cultured with shaking at 30 ℃ for 24 hours.
Centrifugation was carried out at 5000rpm for 2min at room temperature, the supernatant was aspirated, the cells were collected and washed three times with sterile water.
The cells in step 2 were transferred to 3mL complete Synthesis (SC) medium containing no histidine and glucose, containing 2% glycerol as a carbon source, and cultured with shaking overnight at 30 ℃.
Centrifugation was carried out at 5000rpm for 2min at room temperature, the supernatant was aspirated, the cells were collected and washed three times with sterile water.
Resuspend cells in 3mL complete Synthesis (SC) Medium without histidine and glucose, containing 2% glycerol and 2% galactose as carbon sources, to give initial OD600Estradiol was added to a concentration of 1. mu.M (0.6 to 1.0), and the mixture was cultured with shaking at 30 ℃ for 8 hours. (inducing pCRE4 plasmid in Saccharomyces cerevisiae cell to generate CRE recombinase once in cell cycle, so that DNA recombination occurs between loxPsym sites of synthetic type V chromosome in cell, generating structural variation such as DNA deletion, insertion and inversion, FIG. 1)
Cells were appropriately diluted with sterile water and plated on SC-His plates to ensure that similar numbers of colonies were observed on control and experimental (treated with galactose and estradiol) plates, and cultured at 30 ℃ for 72 hours, and the colony numbers and colony sizes were observed and counted.
2. Screening of genomic mitotic recombinant strains
The strain libraries were analyzed using a synthetic polymerase chain reaction tag (PCRTag). In 177 segments divided by loxPsym or loxP sites (a total of 174 loxPsym recombination sites and 2 loxP recombination sites on the synV chromosome, 176 recombination sites dividing the chromosome into 177 regions, numbering 1-177 from the start position of the left end of the chromosome). In 103 segments, there is at least one non-essential gene, and at least one pair of PCRtags is designed in the gene.
8 sets of non-redundant PCRtags in which the distribution is uniform were selected for analysis of chromosome V. The 8 PCRTag sequences belong to genes YEL038W, YEL013W, YER026C, YER056C, YER085W, YER113C, YER144C and YER170W, respectively. Of these, 2 pcrtags are evenly distributed on the left arm of chromosome V, and the remaining 6 are evenly distributed on the right arm of chromosome V (fig. 2).
TABLE 1 PCRtag primer List
TABLE 2 Saccharomyces cerevisiae colony PCRtag assay reaction System (10. mu.L)
| Reaction reagent | Volume (μ L) |
| PrimerF(c＝10μΜ) | 0.25 |
| PrimerR(c＝10μΜ) | 0.25 |
| 2×Rapid |
5 |
| Template(Boiled yeast) | 1 |
| ddH2O | 3.5 |
The results of the PCRTag detection of the triploid control strain yXZX1892 show that both the synthetic PCRTag detection band and the wild-type PCRTag detection band exist. Indicating that both synthetic V chromosome and wild-type V chromosome are present in the cell. The PCRTag test result shows that the mitotic variation of the genome rearrangement library presents diversity, and the existence form of the synthetic chromosome V is as follows:
1) the PCRTag bands of the synthetic type V chromosome were detected to be present in their entirety, as shown by the detection result of the sample yXZX2468 in FIG. 3. This indicates that at least one copy of the DNA fragment of all synthetic type V chromosomes was present in the test strain.
2) The PCRTag bands of the synthetic type V chromosome were not detected at all, as shown by the detection result of the sample yXZX1105 in FIG. 3. Indicating that the synthetic type V chromosome is totally lost in the test strain.
3) The presence of the PCRTag band portion of the synthetic chromosome V was examined. As shown by the results of the detection of the sample yXZX1937 in FIG. 3, only two synthetic PCRTags (PCRTag of genes YEL038W and YER 026C) were present. Indicating that both copies of the partially synthesized type V chromosomal DNA region were lost in the triploid test strain.
Then, the genotype statistical analysis is carried out on the experimental group strains with different existence forms of the synthetic type V chromosome in the genome rearrangement library. In the research, 274 samples are screened from the triploid saccharomyces cerevisiae genome rearrangement library, and the number and the corresponding proportion of different genotype strains in the screened library are respectively displayed. The classification statistics shows that 68 samples of the synthetic type V chromosome exist completely, and the total samples account for 24.82% of the total samples of the triploid. There were 10 samples of synthetic type V chromosome part, which accounted for 3.65% of the total sample of triploids. The total number of samples with complete loss of synthetic type V chromosome is 196, which accounts for 71.53% of the total samples. This indicates that the triploid Saccharomyces cerevisiae control strain yXZX1892 has different probabilities of forming different genotypes after genomic rearrangement. The highest probability of the occurrence of the synthetic type V chromosome loss event promoted the formation of aneuploid s.cerevisiae cells (FIG. 4).
3. Identification result of mitotic hotspot of chromosome
The strain libraries were analyzed using a synthetic polymerase chain reaction tag (PCRTag). In 177 segments divided by loxPsym or loxP sites (a total of 174 loxPsym recombination sites and 2 loxP recombination sites on the synV chromosome, 176 recombination sites dividing the chromosome into 177 regions, numbering 1-177 from the start position of the left end of the chromosome). In 103 segments, there is at least one non-essential gene, and at least one pair of PCRtags is designed in the gene.
The number of breakpoints occurring between adjacent pcrtags after rearrangement in the yhlm 120 was counted and classified into 7 different types (fig. 5). Among them, 33 samples showed a breakpoint in a certain segment between 70.5kb and 118.1kb, accounting for 33% of the total sample amount of partial deletion, and yFJ1779 is used as an example to illustrate Type 1, and PCR results show that the strain has synV chromosome loss in the 0-79.3 kb segment and chromosome breakage between YEL033W and YEL032W (B in FIG. 5). 39% of the strains had a breakpoint region between 118.1kb and 193.1kb, in which yFJ1809 the synthetic type V chromosome was broken near the centromere and the right chromosome of the centromere was lost. The two segments have a large proportion of chromosomal fragmentation and recombination, and in addition, 15% of the samples are fragmented between 193.1kb and 251.7kb, 12% between 251.7kb and 312.7kb, 8% between 312.7kb and 372.0kb, 6% between 372.0kb and 433.4kb, and 17% between 433.4kb and 494.0 kb.
These samples were verified for genome loss at YEL032W _ amp2, YEL029C _ amp1, YEL023C _ amp1, YEL019C _ amp1 (FIG. 6) for 33 strains where the yLHM120 breakpoint occurred between 70.5kb and 118.1 kb. The results showed that 17 strains showed breakpoints between 70.5kb and 79.3kb, 6 strains showed breakpoints between 79.3kb and 87.2kb, 9 strains showed breakpoints between 99.4kb and 109.2kb, 11 strains showed breakpoints between 109.2kb and 118.1kb, and none of the strains showed breakpoints between 87.2kb and 99.4kb, accounting for 52%, 18%, 27%, 33%, and 0% of the total sample size, respectively. The statistical result shows that 70.5 kb-79.3 kb are relative recombination hot spot regions of the segment, and the DNA sequence between 86.9 kb-99.4 kb is relatively stable, is not easy to lose during genome recombination and is a recombination cold spot region.
4. Establishment of homologous chromosome copy number detection method
Whole genome sequencing refers to double-ended sequencing of the genome of the strain using MGISEQ-2000. It is difficult to accurately identify the sequencing result of a synthetic V chromosome from whole genome sequencing data of a triploid saccharomyces cerevisiae carrying both the synthetic V chromosome and a wild-type V chromosome during the research process. To overcome this difficulty, a method for high throughput analysis of genomic sequencing data was developed in the present study. I.e. whole genome sequencing reads (reads) obtained by specific extraction paired-end sequencing (figure 7). The depth of coverage of the homologous regions of the synthetic V chromosome and the wild type V chromosome is then defined as the number of reads of the PCRTag number in the same segment. The difference in the PCRTag sequences on the synthetic V chromosome and the wild-type V chromosome was used to analyze the homologous chromosome copy number variation (CopyNumber, CN) that occurs in cells during genomic rearrangement.
5. Method for detecting homologous chromosome loss and duplication
The PCRTag is used for detecting that 71.53% of triploid strains in the rearranged strains have completely lost the synthetic V chromosome, so the occurrence of the chromosome variation event is never an accidental phenomenon. The present study showed that the number of chromosomes V of polyploid saccharomyces cerevisiae changed after chromosomal rearrangement based on the depth of coverage of whole genome sequencing reads, as shown in fig. 8. In the figure, blue represents the sequencing depth of the synthetic type V chromosome, and orange represents the sequencing depth of the wild type V chromosome. The abscissa represents the position of the segment of the ORF to which the corresponding PCRtag read belongs on the V chromosome, and the number of synthetic V chromosomes and wild-type V chromosomes in the deeply responsive cells by sequencing.
From the sequencing depth map, it can be seen that the sequencing depth of the synthetic V chromosome in the triploid control strain yXZX1892 was about 140, and the sequencing depth of the wild type V chromosome was about 70. The sequencing depth of the synthetic V chromosome was 2 times that of the wild type V chromosome. Therefore, the copy number of the synthetic V chromosome in the cell is2, the copy number of the wild V chromosome is 1, and no chromosome variation event occurs in the cell. The sequencing depth of the synthetic V-shaped chromosome in the experimental group strain yXZX2471 is 0, so that the copy number of the synthetic V-shaped chromosome in the cell is 0, namely the synthetic V-shaped chromosome is completely lost, and the correctness of the primary detection result of the PCRTag is verified. Further analysis shows that the sequencing depth of the corresponding wild type V chromosomes in the cells of the experimental group is changed to about 140, namely the wild type V chromosomes are amplified by 1 time, namely the triploid rearrangement strain contains two wild type V chromosomes to form 3n-1 type aneuploid yeast cells. This indicates that complete loss of synthetic type V chromosome does occur in triploid yeast cells after genomic rearrangement and that this chromosomal variation event is high frequency and stably exists.
6. Unequal repeat/deletion phenomena caused by ectopic recombination between sister chromatids
Translocation across sister chromatids results in unequal chromosomal crossovers, with partial deletion or replication of one homologous chromosome and the other homologous chromosome remaining unchanged (a in figure 9). We further divided ectopic recombination between sister chromatids into four types (B in fig. 9) based on the way and location of ectopic recombination, the first being interstitial deletion, i.e. deletion of part of the fragment in the middle of synV, while wtV remained unchanged (yFJ 1803). 20 samples in the diploid sequencing sample are subjected to interstitial deletion, the positions and the frequency of breakpoints of the interstitial deletion of the samples are counted, a hot spot distribution diagram shown as C in figure 9 is drawn, breakpoints with the occurrence frequency of more than 4 are defined as deletion fracture hot spot regions, and 4 hot spot regions are detected in total and are respectively located at 105.4 kb-106.8 kb, 118.6 kb-122.3 kb, 446.4 kb-446.7 kb and 518.0 kb-519.1 kb. As the literature reports that the GC content influences gene conversion and further influences the distribution of genome recombination hot spots, the GC content of the areas 105.4 kb-106.8 kb and 446.4 kb-446.7 kb is determined by using SnapGene software, and the GC content of the areas is 1 percent and 4 percent higher than the average value (38 percent) of synV, and the GC content of the areas 118.6 kb-122.3 kb and 518.0 kb-519.1 kb is 1 percent and 2 percent lower than the average value of the synV respectively.
If the deletion occurs at the end of the chromosome, we define it as an end deletion. As shown in FIG. 9, yFJ1865 had chromosome breaks at both left and right ends of 31.9kb to 33.2kb and 233.5kb to 235.8kb, and had two terminal deletion break sites. The end deletion of synV of 20 strains in total of the diploid occurs, and the positions of the breakpoints in the sample are counted, so that 23 percent of the strains generate chromosome rearrangement at 75.1-77.5 kb and 135.9-140.0 kb, and therefore the two regions are regarded as high-frequency fracture sites of the diploid strains in the end deletion, wherein the GC content of 75.1-77.5 kb is 3 percent higher than the average value, and the GC content of 135.9-140.0 kb is 3 percent lower than the average value. 135.9kb to 140.0kb flank the centromere, and this region contains a large number of repeated sequences and promotes ectopic recombination between sister chromatids.
The third is interstitial duplication, where the synV middle segment is replicated in one copy, wtV unchanged. Only 1 strain of the diploid test strain showed interstitial repeat (yFJ1798), which occurred in multiple segments with synV interstitial repeat, mainly consisting of 4 segments: 123.5 kb-124.0 kb, 130.4 kb-131.0 kb, 131.8 kb-133.8 kb, 191.8 kb-192.6 kb, except that the GC content of the 131.8 kb-133.8 kb segment is less than 3% of the average value, the GC content of the 130.4 kb-131.0 kb segment is equal to that of the control, and the remaining two segments are 4% and 2% higher than the average value, respectively.
If the repeats occur on both sides of the telomere, they are called terminal repeats, 1 sample in the diploid has terminal repeats (yFJ1843), synV is broken at 105.4kb to 106.8kb, the sequence between 0kb to 106.8kb on the left side is duplicated, and terminal deletions occur between 145.7kb to 536.0kb on the right side.
6. Loss of interstitial heterozygosity (I-LOH)
If recombination occurs between homologous chromosomes, a mitotic gene switch/cross-over event occurs that mediates a SNP to 0 in one homologous chromosome portion and a doubling of the original level in the homologous portion in the other chromosome, resulting in LOH. I-LOH is usually produced when DSB at the stage of G2 is repaired, and is the product of gene conversion in a small region (FIG. 10). I-LOH phenomenon is observed in diploid strains yFJ1805, yFJ1815 and yFJ1836, wherein SNPs of synV are reduced to 0 at 321.6 kb-339.9 kb and 344.1 kb-357.7 kb of yFJ1805 and yFJ1836 respectively, wtV is correspondingly increased, gene transformation is carried out at 438.8 kb-456.1 kb and at 481.9 kb-507.8 kb of yFJ1815, and the length is not more than 30 kb.
We found that in I-LOH, 3 breakpoints were located inside the gene, which were not detected when the sister chromatids were ectopically recombined, and all the samples that underwent ectopic recombination underwent chromosomal breaks between the two ORF frames. It has been reported that most of the ectopic recombination is mediated by homologous recombination between repeated sequences. LoxPsym is a reversed palindrome sequence which is 34bp outside the ORF frame, Cre acts on the loxPsym to generate a DSB gap during induction of rearrangement, and the gap can be repaired between any two loxPsym through homologous recombination between repeated sequences, so that ectopic recombination tends to occur between the loxPsym outside the ORF frame, and chromosome breakage does not easily occur inside the gene. Compared to the mitotic gene conversion process, ectopic repeats between sister chromatids place higher demands on the repeat sequence.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that it is obvious to those skilled in the art that various modifications and improvements can be made without departing from the principle of the present invention, and these modifications and improvements should also be considered as the protection scope of the present invention.
Sequence listing
<110> Tianjin university
<120> detection method of mitotic recombination hot spot of yeast
<130> MP21013007
<160> 16
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<213> Artificial Sequence (Artificial Sequence)
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tccagctcac gacagcttag acttaaac 28
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<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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aactggagcg ttgcctggtc tactg 25
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<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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tagcttgtta agcagtactg acccagac 28
<210> 4
<211> 28
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<213> Artificial Sequence (Artificial Sequence)
<400> 4
aactaagtgt ggtaaaccgc cggctcta 28
<210> 5
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
tgatttgctg atcataccgc aaccgtga 28
<210> 6
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
cggcaagcca cactatgttc agagagct 28
<210> 7
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
aaaagccaag ccggcgacca aactaaag 28
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<213> Artificial Sequence (Artificial Sequence)
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<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
tcgttacgtc catgtcccat caggtcgt 28
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Claims (10)
1. The detection method of the mitotic recombination hotspot of the yeast comprises the following steps: and constructing a rearrangement library of the saccharomyces cerevisiae, detecting and sequencing the PCRTag on the saccharomyces cerevisiae chromosome.
2. The method of claim 1, wherein the Saccharomyces cerevisiae chromosome is chromosome V.
3. The detection method according to claim 1, wherein the saccharomyces cerevisiae comprises:
diploid Saccharomyces cerevisiae: contains a wild type V chromosome and a synthetic type V chromosome;
triploid saccharomyces cerevisiae: contains a wild type V chromosome and two synthetic type V chromosomes;
tetraploid saccharomyces cerevisiae: contains two wild type V chromosomes and two synthetic type V chromosomes.
4. The assay of claim 3 wherein the specific watermark tags detected comprise at least two of YEL038W _ amp1, YEL013W _ amp1, YER026C _ amp1, YER056C _ amp1, YER086W _ amp1, YER113C _ amp2, YER144C _ amp2, YER170W _ amp1, YEL032W _ amp2, YEL029C _ amp1, YEL023C _ amp1, YEL019C _ amp 1.
5. The detection method according to claim 3, wherein the detected specific watermark label comprises: YEL038W _ amp1, YEL013W _ amp1, YER026C _ amp1, YER056C _ amp1, YER086W _ amp1, YER113C _ amp2, YER144C _ amp2, and YER170W _ amp 1.
6. The detection method according to claim 1, wherein the primer for detection comprises:
an amplification primer of YEL038W _ amp1, the nucleic acid sequence of which is shown in SEQ ID NO. 1-2;
an amplification primer of YEL013W _ amp1, the nucleic acid sequence of which is shown in SEQ ID NO. 3-4;
an amplification primer of YER026C _ amp1, the nucleic acid sequence of which is shown in SEQ ID NO. 5-6;
an amplification primer of YER056C _ amp1, the nucleic acid sequence of which is shown in SEQ ID NO. 7-8;
an amplification primer of YER086W _ amp1, the nucleic acid sequence of which is shown in SEQ ID NO. 9-10;
an amplification primer of YER113C _ amp2, the nucleic acid sequence of which is shown in SEQ ID NO. 11-12;
an amplification primer of YER144C _ amp2, the nucleic acid sequence of which is shown in SEQ ID NO 13-14;
the nucleic acid sequence of the amplification primer of YER170W _ amp1 is shown in SEQ ID NO. 15-16.
7. The assay of claim 1 wherein said rearrangement library is induced using estradiol, and the concentration of estradiol in the medium in which rearrangement is induced is 1 μmol/L.
8. The detection method according to claim 1, wherein the sample mutation condition is obtained according to the number of breakpoints occurring between adjacent pcrtags after rearrangement.
9. The detection method of claim 1, wherein the sequencing comprises: whole genome sequencing reads obtained by specific extraction paired-end sequencing.
10. The detection method according to claim 9, wherein after the sequencing:
counting the difference of the PCRTag sequences on the synthetic V chromosome and the wild V chromosome, and analyzing the copy number variation of homologous chromosomes of the cell in the process of genome rearrangement;
sequencing the number of synthetic V chromosomes and wild type V chromosomes in the deeply responsive cells;
counting the mode and position of ectopic recombination, and analyzing unequal repetition/deletion caused by ectopic recombination among sister chromatids;
and (4) counting the quantity of SNP sites of homologous chromosomes and analyzing the phenomenon of interstitial heterozygosity deletion.
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| US20040033494A1 (en) * | 2002-07-19 | 2004-02-19 | Alain Nicolas | Methods for inducing targeted stimulation of meiotic recombination and kits for performing said methods |
| CN113046255A (en) * | 2021-03-09 | 2021-06-29 | 天津大学 | Yeast with large-scale gene rearrangement and construction method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US20040033494A1 (en) * | 2002-07-19 | 2004-02-19 | Alain Nicolas | Methods for inducing targeted stimulation of meiotic recombination and kits for performing said methods |
| CN113046255A (en) * | 2021-03-09 | 2021-06-29 | 天津大学 | Yeast with large-scale gene rearrangement and construction method thereof |
Non-Patent Citations (2)
| Title |
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| JORDAN ST. CHARLES等: "High-Resolution Mapping of Spontaneous Mitotic Recombination Hotspots on the 1.1 Mb Arm of Yeast Chromosome IV", 《PLOS GENETICS》 * |
| 李云祥: "酵母基因组重排研究杂合性缺失", 《中国优秀博硕士学位论文全文数据库（硕士） 基础学科辑》 * |
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| CN114592048A (en) * | 2022-03-10 | 2022-06-07 | 天津大学温州安全(应急)研究院 | Method for predicting coagulation dysfunction of recurrent abortion population in pregnancy |
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