CN116004779A - Method for overcoming trace cell amplification allele tripping - Google Patents
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Abstract
The invention discloses a method for overcoming trace cell amplification allele tripping, which comprises the following steps: amplifying genome nucleic acid of the micro-cells; performing whole genome pooling on the amplified product; hybridizing and targeted capturing the constructed library by using a hybridization capturing probe pair; sequencing the library captured by hybridization, and analyzing the allele mutation frequency and sequencing depth of each SNP locus; designing at least one pair of primers and one inhibition probe for SNP loci with mutation frequency lower than 20%, and performing inhibition probe displacement amplification reaction; sequencing the inhibition probe substitution amplification product to obtain an enrichment result of the tripped allele. The invention also discloses a detection system for overcoming trace cell amplification allele tripping and application of the detection system in preparation of embryo genetics detection products before implantation. The method can directly and accurately enrich and detect the tripping position through the specificity enrichment mutation sequence, and obviously reduce the tripping phenomenon in PGT-M detection.
Description
Technical Field
The invention relates to the technical field of gene detection, in particular to a method for overcoming trace cell amplification allele tripping.
Background
The reduction of population birth defects is a national basic strategy, PGT-M (single gene genetic disease detection before embryo implantation) is an important means for blocking the transmission of severe genetic disease, and is also the only basis for embryo selection, and the existing single gene genetic disease detection (PGT-M) flow before embryo implantation is shown in figure 1. However, the phenomenon of Allele Drop-Out (ADO) has been one of the important reasons that plague PGT-M applications.
PGT assays use very small numbers of embryo biopsied cells, since the sample size is very small, and very small amounts of genetic material need to be amplified whole genome using molecular biology methods prior to detection, where the alleles may be asymmetrically amplified, i.e. where one allele is predominantly amplified, the other allele is not amplified or less amplified, resulting in detection of only one allele and the other allele not during later detection, which is known as "allele-tripping". If the allele is tripped during the PGT detection process, the single nucleotide variation detection result is false positive or false negative, which may lead to misdiagnosis and reduction of the number of available embryos, which is a limitation of the existing detection technology.
Current techniques for single cell whole genome amplification, such as MDA (Qiagen, GE), MALDBAC (YIkon), DOP-PCR (Sigma), MALDBAC-like (Rubicon), have no low release rates, on the order of 30% -80%.
Currently, embryo genotypes are diagnosed indirectly using linkage analysis methods: detecting SNP in 1-2M upstream and downstream of target genes of parents and embryos through NGS or chips, and judging whether the embryos carry gene mutation or not through linkage analysis. Considering the precious nature of embryo single cell materials and the serious influence of tripping phenomenon, PGT-M detection almost completely utilizes a mode of constructing haplotypes and directly detecting target sites to identify pathogenic site information, doctors can not only directly detect whether mutation which is the same as parents exists in embryo target genes, but also judge which chromosomes of family members are respectively provided with pathogenic mutation through family samples, are linked with which molecular markers (namely haplotypes and distinguish pathogenic chromosomes from nonpathogenic chromosomes), then judge whether embryos inherit parent pathogenic chromosomes according to the result of haplotype analysis, analyze whether embryos have risks of diseases in terms of genetic principle, and select embryos without risks of diseases and without pathogenic mutation sites for target gene sequencing for implantation. Even so, the number of molecular markers selected is small and still easily affected by ADO. Disadvantages of this PGT-M detection: high cost, long period and complex detection method.
Disclosure of Invention
The invention aims to solve the technical problem that the current clinical PGT-M detection has the trip phenomenon of about 20 percent at most, and provides a method for overcoming the trip of a trace cell amplification allele.
In order to solve the technical problems, the invention is realized by the following technical scheme:
in one aspect of the invention, a method is provided for overcoming trace cell expansion allele tripping, comprising the steps of:
amplifying genome nucleic acid of the micro-cells;
performing whole genome pooling on the amplified product;
hybridizing and targeted capturing the constructed library by using a hybridization capturing probe pair;
sequencing the library captured by hybridization, and analyzing the allele mutation frequency and sequencing depth of each SNP locus;
designing at least one pair of primers and one inhibition probe for carrying out a substitution amplification reaction of the inhibition probe on SNP loci with mutation frequency lower than 20%, wherein the oligonucleotide chain of the inhibition probe is in complementary combination with the base of a wild type DNA sequence corresponding to the SNP loci, and the complementary combination position of the inhibition probe on the wild type DNA sequence is partially overlapped with the complementary combination position of the primers on the wild type DNA sequence;
sequencing the inhibition probe substitution amplification product to obtain an enrichment result of the tripped allele.
The micro cells include one or several cells.
The genome nucleic acid amplification of the minicell comprises: the MDA method is adopted to carry out genome nucleic acid amplification on the micro cells.
In another aspect of the invention, there is also provided a detection system for overcoming trace cell expansion allele trip, the detection system comprising: at least one pair of primers designed for the SNP locus and an inhibition probe, wherein the oligonucleotide chain of the inhibition probe is complementary combined with the base of the wild type DNA sequence corresponding to the SNP locus, and the complementary combination position of the inhibition probe on the wild type DNA sequence is partially overlapped with the complementary combination position of the primers on the wild type DNA sequence.
The SNP locus is a SNP locus with the mutation frequency lower than 20 percent, and the detection system can detect the SNP locus with the mutation frequency lower than 1 percent through an enrichment result.
The detection system also comprises polymerase and genomic DNA, and is a PCR amplification reaction system.
In another aspect of the present invention, there is also provided a detection kit for overcoming trace cell amplification allele release, comprising at least one pair of primers designed for a SNP site and an inhibition probe, wherein the oligonucleotide strand of the inhibition probe is complementary to the base of a wild-type DNA sequence corresponding to the SNP site, and the complementary binding position of the inhibition probe on the wild-type DNA sequence overlaps with the complementary binding position of the primers on the wild-type DNA sequence.
In another aspect of the invention, the use of the above detection system for the preparation of a pre-implantation embryological detection product is also provided.
In another aspect of the invention, the application of the detection kit in preparing a single-gene genetic disease diagnosis product before embryo implantation is also provided.
The method for overcoming the trace cell amplification allele tripping, disclosed by the invention, can eliminate the influence of the tripping phenomenon on the application of PGT-M (polymerase chain reaction) while ensuring the accuracy and the specificity through the low-proportion variation of enrichment amplification detection, can directly detect the pathogenic site information in the single-cell whole-gene amplification product, and can avoid the construction of haplotypes, reduce the detection cost and enable the PGT-M to obtain the most visual result.
Drawings
The invention will be described in further detail with reference to the drawings and the detailed description.
FIG. 1 is a schematic diagram of a prior art single gene genetic disease detection (PGT-M) procedure prior to embryo implantation in accordance with the present invention;
FIG. 2 is a graph showing the results of LOD detection after BDA amplification of a part of SNP sites in example 1 of the present invention;
FIG. 3 is a graph showing statistical analysis of allele release rates of single-cell amplification products according to example 2 of the present invention;
FIG. 4 is a statistical chart of the proportion analysis of the heterozygous SNP locus of example 2 of the invention in the 1/3/6 cell amplification product;
FIG. 5 is a graph showing the results of mutation peaks at partial SNP sites, in which mutation peaks (> 20%) are visible by sanger sequencing after BDA amplification of 28 SNP sites according to example 3 of the present invention.
Detailed Description
Example 1 demonstrates the versatility of the technique of inhibition of probe displacement amplification (Blocker Displacement Amplification, BDA) in different sites and in different concentration ranges of ratios
1 screening of candidate Standard samples (bioinformatics analysis)
1.1 screening of suitable standard source samples: 14 volunteer samples (numbered abbreviation LJF, YHX, YYX, CLL, GX, ZW, BW, TR, LSS, WJP, LRQ, XX, CMM, DBH) were WES (human whole exon trap sequencing) sequenced and analyzed for SNP typing of the exon regions.
Experimental results: SNP typing of exon regions in these 14 cases of sequencing data was analyzed and compared, and finally a pair of paired samples (LJF, CMM) was screened out for subsequent standard preparation. The 320 SNP loci found in the paired samples can be used for subsequent BDA design, the 320 SNP loci are all homozygous SNP genotyping identical to the genome REF genotype in the LJF sample, and the 320 SNP loci are all homozygous SNP genotyping identical to the genome ALT genotype in the CMM sample. 320 SNP sites are shown in Table 1.
TABLE 1 320 SNP loci
1.2 analyzing exon region SNP locus information in WES data of an internal control sample, and finding 1000 heterozygous SNP loci from the exon region SNP locus information.
Experimental results: SNP typing information of exon regions in WES data of internal control samples LJF was analyzed, and 1247 heterozygous SNP sites with af=0.4-0.6 were found in total. 1247 SNP loci are shown in Table 2.
TABLE 2 1247 heterozygous SNP loci
2. Preparation of standards
2.1 extraction of DNA from Standard sample
Based on the analysis of WES data, a LJF and CMM paired sample was selected for subsequent standard preparation, with LJF as wild-type DNA sample and CMM as mutant DNA sample. 10mL of peripheral blood of LJF and CMM were collected for extraction of genomic DNA, and the method for blood genomic DNA extraction included the following steps:
1) 200. Mu.L of the blood sample is taken into a 2.0mL centrifuge tube, and if the blood sample is extracted to be less than 200. Mu.L, buffer GS can be added to complement to 200. Mu.L, and then the next experiment is carried out.
2) 200. Mu.L Buffer GB and 20. Mu.L protease K solution were added to the above samples and mixed well by shaking.
3) Incubation was carried out at 56℃for 10min, during which time the mixing was reversed several times, the solution was free of lumps, if necessary the lysis time was prolonged until the solution was free of lumps.
4) After standing at room temperature for 2-5min, 350 μl Buffer BD was added, and the mixture was thoroughly inverted and mixed, and the reaction mixture was collected to the bottom of the tube by brief centrifugation.
5) All liquid was transferred to a CG2 spin column (spin column placed in collection tube), spun at 12000rpm (12400×g) for 30sec, the waste liquid and collection tube discarded, and the spin column was nested into a new collection tube.
6) mu.L of Buffer GDB was added, centrifuged at 12000rpm (. About.12400 Xg) for 30sec, the waste liquid was discarded, and the column was returned to the collection tube.
7) 600. Mu.L of Buffer PWB was added, centrifuged at 12000rpm (. About.12400 Xg) for 30sec, the waste solution was discarded, and the column was returned to the collection tube.
8) The above steps are repeated.
9) Centrifuge at 12000rpm (12400 Xg) for 2min, discard the collection tube, transfer the column into a new 1.5mL centrifuge tube, leave it open at room temperature for about 5min, and thoroughly air dry the residual buffer on the adsorption membrane.
10 30. Mu.L of NFW was suspended in the center of the adsorption film of the column, and after 2min at room temperature, the column was centrifuged at 12000rpm (. About.12400 Xg) for 2min, and the eluate was collected in a centrifuge tube.
11 Repeating the previous step, and ending the centrifugation and discarding the column.
12 Agarose gel electrophoresis to detect DNA integrity and to determine DNA purity and concentration using NanoDrop and Qubit, respectively.
2.2 quantification and dilution of Standard sample DNA
The extracted LJF genomic DNA and CMM genomic DNA were each subjected to concentration measurement with Qubit, and diluted to 10 ng/. Mu.L with DNA diluent.
2.3 preparation of standards with different concentration gradients
2.3.1 mixing 900. Mu.L of LJF genomic DNA at 10 ng/. Mu.L and 100. Mu.L of CMM genomic DNA at 10 ng/. Mu.L, and vortexing to prepare 10 ng/. Mu.L of AF=10% standard DNA.
2.3.2 mixing 900. Mu.L of 10 ng/. Mu.L of AF=10% of standard DNA and 100. Mu.L of 10 ng/. Mu.L of CMM genomic DNA, and vortexing and mixing to prepare 10 ng/. Mu.L of AF=1% of standard DNA.
2.3.3 mixing 500. Mu.L of 10 ng/. Mu.L of AF=1% of standard DNA and 500. Mu.L of 10 ng/. Mu.L of CMM genomic DNA, and vortexing to prepare 10 ng/. Mu.L of AF=0.5% of standard DNA.
2.3.4 mixing 500. Mu.L of 10 ng/. Mu.L of AF=0.5% of standard DNA and 500. Mu.L of 10 ng/. Mu.L of CMM genomic DNA, and vortexing to prepare 10 ng/. Mu.L of AF=0.25% of standard DNA.
2.3.5 mixing 500. Mu.L of 10 ng/. Mu.L of AF=0.25% of standard DNA and 500. Mu.L of 10 ng/. Mu.L of CMM genomic DNA, and vortexing to prepare 10 ng/. Mu.L of AF=0.125% of standard DNA.
3. Studies inhibit LOD (Limit of detection, detection limit) of probe displacement amplification (BDA) technology in different SNP loci
3.1 ordering hybridization Capture probes
Hybridization capture probes were ordered from the biotechnology company Naonda (Nanj): the capture probes target 320 homozygous SNP sites and 1247 heterozygous SNP sites, including those mentioned in the standard preparation procedure above, and the probe package information is shown in table 3 below.
TABLE 3 genomic position information covered by probes
3.2 Detection of SNP in standard sample by BDA-sanger technology
3.2.1 random selection of 117 SNP sites from 320 homozygous SNP sites for BDA design: each SNP was designed with 1 pair of primers and 1 Blocker,351 oligos, see tables 4 and 5 below. BDA primers and blockers were designed automatically by NGSure software from the read genes company.
TABLE 4 BDA primers designed for 117 SNP loci
TABLE 5 Blocker (inhibition Probe) of BDA designed for 117 SNP loci
3.2.2 testing the effect of this 117 on BDA design: 10 ng/. Mu.L of AF=1% standard DNA was amplified with 117 pairs of BDA primers and Blockers using 3 concentration gradients of Blockers per pair (primers: blocker=1:3, 1:10, 1:30). And (3) carrying out Sanger sequencing on the BDA amplified products, and judging AF of the SNP locus to be detected in Sanger. AF of SNP loci to be detected in any 1 sanger result in 3 sanger concentration gradients is more than or equal to 20%, and BDA enrichment is considered successful. Otherwise, BDA enrichment fails. The BDA PCR system and amplification procedure were as follows: a polymerase (Thermo Fisher PowerUp SYBR Green Master Mix); primer concentration: forward primer 400nM (1 x), reverse primer 400nM (1 x), BDA inhibition probes (Blocker): 1.2uM (1:3), 4uM (1:10), 12uM (1:30); 30uL of the reaction system plus 30ng of genomic DNA, when samples with very low allele frequencies are processed, the total amount of added DNA needs to be increased to ensure that there are at least 20 copies of the allele molecule; the amplification procedure includes step 0:95 ℃ for 3min, step 1:95 ℃ for 30s, step 2:60 ℃ for 1min, step 3: repeating the steps 1 to 2 and 40 times, and step 4:4 ℃. Results: 91 pairs of BDA in 117 pairs of BDA designs were successfully enriched with a success rate of 77.78%.
3.2.3 second BDA designing and testing of SNP loci that were unsuccessful in the first BDA design, third BDA designing and testing of SNP loci that were unsuccessful in the second BDA design, and so on, for a single SNP locus, a maximum of five BDA designs and tests are performed. If the BDA design is unsuccessful for five times, stopping the design, and judging that the site design fails. The results were as follows: the enrichment of 12 SNP loci is successful in the second BDA design, and the success rate is 10.26%; the enrichment of 9 SNP loci is successful in the third BDA design, and the success rate is 7.69%; the enrichment of 3 SNP loci is successful in the fourth BDA design, and the success rate is 2.56%;1 SNP locus is successfully enriched in the fifth BDA design, and the success rate is 0.85 percent; none of the five attempts at BDA design at 1 SNP site was successful in enrichment, with a failure rate of 0.85%.
In summary, the success rate of BDA design for 117 SNP loci is shown in Table 6 below:
TABLE 6 success rate of BDA design for different primer pairs
Primer pair number | Number of sites | Proportion of | Ratio of |
1 | 91 | 77.78% | 77.78% |
2 | 12 | 10.26% | 88.03% |
3 | 9 | 7.69% | 95.72% |
4 | 3 | 2.56% | 98.29% |
5 | 1 | 0.85% | 99.14% |
Conclusion: 3 pairs of BDA primer design aiming at a single SNP can improve the success rate to more than 95 percent.
3.3 study of LOD at different SNP loci
3.3.1 for 116 SNP loci designed successfully for the BDA, BDA amplification is carried out by using the BDA primer designed successfully and a Blocker and standard substances (AF=1%, 0.5%,0.25% and 0.125%) with different concentrations of the Blocker, and the BDA amplified products are subjected to Sanger sequencing to judge AF of the SNP loci to be detected in Sanger. 4 replicates were performed for each concentration of standard. The AF of the SNP locus to be detected in 4 repeated sanger results of the standard substance of each concentration gradient is equal to or more than 20%, and the BDA reaction of the locus is considered to reach the concentration.
3.3.2 results: of the 116 SNP loci for which BDA design was successful, 88 loci had LOD of 0.125% and the ratio was 75.86%; LOD with 14 sites is 0.25% and the ratio is 12.07%; LOD at 5 sites was 0.50% in a ratio of 4.31%; the LOD at 9 sites was 1% and the ratio was 7.76%. The LOD of BDA at 117 SNP sites is shown in table 7, fig. 2 shows a graph of LOD detection results of BDA amplification at a part of SNP sites, and fig. 8 shows statistical results of BDA design at 117 SNP sites.
TABLE 7 LOD of BDA of 117 SNP loci
TABLE 8 statistical results of BDA design with 117 SNP loci
Example 2 demonstrates the universality of the BDA technique in the location of a new trip in MDA (whole genome isothermal amplification method) products
1. MDA amplification for peripheral blood single cells
Taking 1 cell and 3 cells from peripheral blood of an internal control sample (LJF) to respectively carry out MDA amplification; each was repeated 3 times. The MDA amplification kit uses the YIcon MDA kit and the Qiagen MDA kit respectively, wherein the Qiagen MDA kit is newly added with 3 repeats of 6 cells and 9 cells, and each single cell amplification operation step is shown in the kit operation instruction.
2. Pooling and targeted sequencing of single cell amplification products
2.1MDA amplification products were quantified with Qubit, 1. Mu.g of each MDA amplification product was broken down to 350bp using ultrasound.
2.2 library construction was performed on 1. Mu.g of MDA amplification product interrupted to 350bp using the Prism DNA library construction kit of IDT, and the library construction procedure was performed according to the instructions of xGen Prism DNA Library Prep Kit library preparation kit. The library-building kit can realize maximized library transformation, eliminate the formation of linker dimers, and remove various repeated data and correct errors by adding a single-molecule tag (UMI) sequence during single-chain ligation.
2.3 500ng of library each were mixed together and targeted capture was performed on the library using the hybridization capture probes tailored to example 1, hybridization capture procedure described below in 2.3.1 through 2.3.8.
2.3.1 library hybridization
1) Balancing VAHTS DNA Clean Beads at room temperature for 30min, and mixing under vortex vibration.
2) In a 1.5mL centrifuge tube, hybridization library mixtures were prepared according to Table 9 below:
TABLE 9 hybridization library mixture formulation System
Reagent name | Addition amount of |
Total library | 500ng/Lib |
Cot-1 DNA | 7.5μL |
VAHTS DNA Clean Beads | 1.8 times of the total volume of the two items |
3) Vortex mixing, standing at room temperature for 10min.
4) After the tube was briefly centrifuged, it was placed in a magnetic rack for about 5min, and after the solution was clarified, the supernatant in the tube was carefully removed.
5) The centrifuge tube was kept on a magnetic rack, 200. Mu.L of freshly prepared 80% ethanol was added, and left standing at room temperature for 30sec, taking care to remove the supernatant.
6) The above steps were repeated for a total of 2 rinses.
7) After the centrifuge tube is centrifuged briefly, the centrifuge tube is put back to a magnetic rack, the residual liquid is carefully removed, and the centrifuge tube is opened and air-dried until no ethanol residue is left (about 2-5min, taking care to avoid cracking of the magnetic beads).
8) In a centrifuge tube, hyb Mix hybridization reagents were prepared according to table 10 below, vortexed, and briefly centrifuged:
TABLE 10 Hyb Mix hybridization reagent formulation System
Reagent(s) | Reagent volume (mu L) |
|
9.5 |
xGen hybridization Buffer Enhancer | 3.0 |
xGen Lockdown Panel | 4.5 |
xGen Universal Blockers TS Mix | 2.0 |
Total volume of | 19 |
9) 19 mu L of Hyb Mix is added to the air-dried magnetic beads, and the mixture is vortexed and mixed uniformly, so that the magnetic beads are fully resuspended and kept stand at room temperature for 5min.
10 After the centrifuge tube is instantaneously centrifuged, the centrifuge tube is placed on a magnetic rack for 2min until the liquid is completely clarified.
11 mu.L of supernatant was pipetted into a new 0.2mL PCR tube.
12 0.2mL PCR tube was placed in a PCR instrument and the following hybridization procedure was run: denaturation at 95℃for 1min to 65℃for more than 16h, and thermal capping at 105 ℃.
2.3.2 reagent preparation
1) A 1x working fluid (which can be stored for two weeks at room temperature and can be scaled up) was prepared according to the following table 11 system:
TABLE 11 1X working fluid formulation System
2) Split charging 1 xWash Buffer I according to 110 μl/reaction, preheating in metal bath at 65deg.C for at least 30min before use, and standing the rest at room temperature.
3) 1X Stringent Wash Buffer was preheated for at least 30min in a 65℃metal bath prior to use.
2.3.3 magnetic bead Capture
1) Dynabeads M-270Streptavidin Beads was equilibrated at room temperature for 30min and vortexed at high speed for 15 sec.
2) 50. Mu.L of the equilibrated M-270 beads were dispensed per reaction in 1.5mL Lo-bind centrifuge tubes.
3) According to the amount of 100 mu L of each reaction, 1 XBead Wash Buffer is added into M-270 magnetic beads, the mixture is gently blown and sucked for 10 times by a pipette, the centrifuge tube is placed on a magnetic rack, and the mixture is kept stand for 1min until the solution is clarified, and then the supernatant is removed.
4) The above steps were repeated 2 times for a total of 3 rinses.
5) The magnetic bead suspensions were prepared in the proportions shown in Table 12 below:
TABLE 12 magnetic bead suspension formulation system
Reagent(s) | Reagent volume (mu L) |
|
8.5 |
xGen hybridization Buffer Enhancer | 2.7 |
NFW | 5.8 |
Total volume of | 17 |
6) The bead suspension was added to the M-270 beads in an amount of 17. Mu.L per reaction, and gently aspirated 10 times with a pipette and mixed well.
7) The hybridization PCR tube is kept in a PCR instrument, 17 mu L of M-270 magnetic beads which are suspended and uniformly mixed are added into a hybridization sample, and the mixture is gently blown and sucked for 10 times by a pipette for uniform mixing, so that air bubbles are avoided.
8) Continuing to incubate at 65 ℃ for 45min, and pulling down the cover of the PCR instrument, but not tightly covering the cover; incubating for 15min at 65 ℃, and gently blowing and sucking 10 times by a pipettor; incubating for 15min, and gently blowing and sucking for 10 times by a pipettor; incubation was carried out for 15min again, and the pipettor was gently blown and sucked 10 times.
2.3.465 ℃ thermal elution
1) The hybridization reaction PCR tube is kept in a PCR instrument, 100 mu L of 1 XWash Buffer I preheated at 65 ℃ is added into the PCR tube, and the gun head is gently blown and sucked for 10 times for uniform mixing, so that bubbles are avoided.
2) The PCR tube was placed on a magnetic rack and the supernatant was removed after the solution was clear.
3) 150. Mu.L of 1X Stringent Wash Buffer preheated at 65 ℃ is added into the PCR tube, the gun head is gently blown and sucked for 10 times and uniformly mixed, the PCR tube is placed on a magnetic frame after incubation for 5 minutes at 65 ℃, and the supernatant is removed after the solution is clarified.
4) The above steps are repeated.
2.3.5 elution at Room temperature
1) 150. Mu.L of 1 XWash Buffer I at room temperature is added into a PCR tube, vortex mixing is carried out, the mixture is placed at room temperature for 2min, the mixture is kept stand for 30s after vortex mixing is carried out for 30s, and the mixture is alternately carried out, so that full mixing is ensured.
2) The PCR tube was centrifuged briefly and returned to the magnetic rack, and the supernatant was removed after the solution was clarified.
3) 150. Mu.L of 1 XWash Buffer II at room temperature was added to the PCR tube, vortexed and mixed well, and left at room temperature for 2min, during which period vortexed and mixed well for 30s, and left to stand for 30s, alternately performed, to ensure sufficient mixing.
4) The PCR tube was centrifuged briefly and returned to the magnetic rack, and the supernatant was removed after the solution was clarified.
5) 150. Mu.L of 1 XWash Buffer III at room temperature was added to the PCR tube, vortexed and mixed well, and left at room temperature for 2min, after 30s of vortexing and mixing, left for 30s, and the mixture was alternated to ensure sufficient mixing.
6) The PCR tube was centrifuged briefly and returned to the magnetic rack, and the supernatant was removed after the solution was clarified.
2.3.6 post-hybridization PCR amplification
1) The PCR reaction solution was prepared in the following proportions of Table 13:
TABLE 13 PCR reaction solution preparation System
Reagent name | Reagent volume (mu L) |
Kapa Hifi mix | 25 |
P5 Primer(10μM) | 2.5 |
P7 Primer(10μM) | 2.5 |
|
20 |
2) Adding the PCR reaction liquid into the reaction tube of the previous step according to the quantity of 50 mu L of each reaction, and gently blowing and sucking for 10 times by using a pipettor to mix uniformly;
3) The PCR tubes were placed in a PCR instrument and the following table 14 reactions were performed:
TABLE 14 PCR reaction procedure
Temperature (temperature) | Time | |
98℃ | 45sec | |
98 | 15sec | |
60℃ | 30sec | |
72℃ | 30sec | |
72℃ | 1min |
4) After the reaction, 1. Mu.L of the PCR product was subjected to 2% agarose gel electrophoresis (120V, 30 min).
2.3.7 post-PCR magnetic bead purification
1) VAHTS DNA clean beads the mixture was equilibrated at room temperature for 30min and vortexed.
2) mu.L of beads was added to the PCR product, and the mixture was vortexed and allowed to stand at room temperature for 5min.
3) After the PCR tube was briefly centrifuged, it was placed in a magnetic rack for 2min, and after the solution was clarified, the supernatant was carefully removed with a pipette.
4) The PCR tube was kept on a magnetic rack, 200. Mu.L of freshly prepared 80% ethanol was added, and left standing at room temperature for 30sec, and the supernatant was carefully removed with a pipette.
5) The above steps were repeated 1 time for a total of 2 washes.
6) After short centrifugation, the tube was returned to the magnetic rack, the residual liquid was carefully removed with a pipette, and the lid was air-dried until no ethanol remained (about 2-5min, care was taken to avoid magnetic bead cracking).
7) Add 21. Mu.L NFW heavy suspension beads, vortex mix well and stand at room temperature for 2min.
8) The tube was centrifuged briefly and placed on a magnetic rack for 2min, and 20 μl of supernatant was transferred to a new centrifuge tube for use.
2.3.8 library QC
1) The library was quantified with Qubit.
2) The library was kept at 4℃for later use and long-term storage was required at-20 ℃.
2.4 the hybridization captured libraries were sent out to open reading frames for 2*150bp pair end NGS sequencing, with 6G of sequencing data per library expected.
3. Analysis of NGS sequencing data
3.1 analysis of the individual SNP loci for 1247 heterozygous SNP loci using the Deduplication method-B protocol of the analytical flow of Prism DNA library kit (allele mutation frequency) and sequencing depth (calculated as UMI counts).
3.2, the SNP locus mutation molecules are judged to be tripped by taking UMI count=0 as a standard, and the tripping rate of each single cell amplification product is counted to be as shown in fig. 3, the tripping rate of the Qiagen MDA kit is lower, the tripping rate of 3 cells is 2.09,0.08%, the tripping rate of 4.57% and the tripping rate of more than 6 cells is 0 respectively. The Yicon MDA 3 cell release rates were 0.08%,11.55%,3.61%, respectively.
3.3 further analysis of VAF for each SNP site for 3 replicates of 1/3/6 cells of Qiagen MDA kit, results as shown in fig. 4, the proportion of heterozygous SNP sites in 1 cell amplification product, <20% of sites averaged 29%; the proportion of heterozygous SNP sites in the 3-cell amplification product, <20% of sites on average account for 26%; the proportion of heterozygous SNP sites in the 6-cell amplification product was <20% of sites on average 2%.
Example 3 BDA design and verification for trip position
1. SNP loci with VAF lower than 1<% are selected from 3 cell amplification products of Qiagen MDA to design BDA design, and 28 SNP loci are selected for BDA design. The selected SNP sites and BDA primer and probe designs are shown in tables 15 and 16 below.
TABLE 15 BDA primer designed for 28 selected SNP loci
TABLE 16 BDA Probe designed for 28 selected SNP loci
BDA validation results of 2.28 SNP loci are shown in Table 17 below:
table 17 BDA verification results of 28 SNP loci
Conclusion: in the BDA design of 28 SNP loci with VAF lower than 1%, mutation peaks (> 20%) are visible in the sanger sequencing of 27 SNP loci, the success rate is 96.42%, and a partial SNP locus mutation peak result diagram of the mutation peaks (> 20%) of the sanger sequencing of 27 SNP loci after BDA amplification is shown in FIG. 5. The sequence of BDA product sanger with 1 site among 28 SNP sites appears a sleeve peak.
The foregoing examples merely illustrate embodiments of the invention and are described in more detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.
Claims (10)
1. A method of overcoming trace cell amplification allele tripping, comprising the steps of:
amplifying genome nucleic acid of the micro-cells;
performing whole genome pooling on the amplified product;
hybridizing and targeted capturing the constructed library by using a hybridization capturing probe pair;
sequencing the library captured by hybridization, and analyzing the allele mutation frequency and sequencing depth of each SNP locus;
designing at least one pair of primers and one inhibition probe for carrying out a substitution amplification reaction of the inhibition probe on SNP loci with mutation frequency lower than 20%, wherein the oligonucleotide chain of the inhibition probe is in complementary combination with the base of a wild type DNA sequence corresponding to the SNP loci, and the complementary combination position of the inhibition probe on the wild type DNA sequence is partially overlapped with the complementary combination position of the primers on the wild type DNA sequence;
sequencing the inhibition probe substitution amplification product to obtain an enrichment result of the tripped allele.
2. The method of claim 1, wherein the micro-cells comprise one or several cells.
3. The method of claim 1, wherein said amplifying genomic nucleic acid from a minicell comprises: the MDA method is adopted to carry out genome nucleic acid amplification on the micro cells.
4. A detection system for overcoming a trace cell amplification allele trip, the detection system comprising: at least one pair of primers designed for the SNP locus and an inhibition probe, wherein the oligonucleotide chain of the inhibition probe is complementary combined with the base of the wild type DNA sequence corresponding to the SNP locus, and the complementary combination position of the inhibition probe on the wild type DNA sequence is partially overlapped with the complementary combination position of the primers on the wild type DNA sequence.
5. The detection system according to claim 4, wherein the SNP site is a SNP site having a mutation frequency of less than 20%.
6. The detection system of claim 4, further comprising a polymerase and genomic DNA.
7. The detection system of claim 4, wherein the detection system is a PCR amplification reaction system.
8. A detection kit for overcoming the release of a micro-cell amplification allele, which is characterized by comprising at least one pair of primers designed for SNP loci and an inhibition probe, wherein an oligonucleotide chain of the inhibition probe is in complementary combination with a wild-type DNA sequence base corresponding to the SNP loci, and the complementary combination position of the inhibition probe on the wild-type DNA sequence is partially overlapped with the complementary combination position of the primers on the wild-type DNA sequence.
9. Use of the test system according to any one of claims 4-7 for the preparation of a pre-implantation embryological test product.
10. Use of the detection kit of claim 8 for the preparation of a single genetic disease diagnostic product prior to embryo implantation.
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