CN117248030A

CN117248030A - PKD1 variant molecule detection method based on single-cell whole genome amplification and application thereof

Info

Publication number: CN117248030A
Application number: CN202310363378.7A
Authority: CN
Inventors: 肖敏; 雷彩霞; 奚燕萍; 孙晓溪
Original assignee: Shanghai Jiai Genetics And Infertility Diagnosis And Treatment Center Co ltd; Obstetrics and Gynecology Hospital of Fudan University
Current assignee: Shanghai Jiai Genetics And Infertility Diagnosis And Treatment Center Co ltd; Obstetrics and Gynecology Hospital of Fudan University
Priority date: 2023-04-07
Filing date: 2023-04-07
Publication date: 2023-12-19

Abstract

The invention discloses a PKD1 variation molecular detection method based on single-cell whole genome amplification and application thereof, wherein cells to be detected carry out whole genome DNA amplification, PKD1 gene variation and pseudogene locus information thereof are searched, primers are designed, short segments with the length of 150-250bp are amplified, and deep targeted second generation sequencing is carried out to distinguish reads of PKD1 and pseudogenes, so that PKD1 variation locus information is obtained; and (3) carrying out genome copy number variation analysis after obtaining genome SNP information by whole genome SNP chip or second generation sequencing, and simultaneously carrying out family haplotype linkage analysis by combining mutation information and SNP genotyping, judging whether an embryo carries a haplotype where PKD1mutation is located in a family, and detecting the genome DNA total amount before embryo implantation under the condition that the sequencing concentration or the DNA continuous fragment length is below 500 bp.

Description

PKD1 variant molecule detection method based on single-cell whole genome amplification and application thereof

Technical Field

The invention belongs to the field of genetic detection before embryo implantation, and particularly relates to a PKD1 variant molecule detection method based on single-cell whole genome amplification and application thereof.

Background

In the second generation sequencing technology, it is often impossible to determine whether the mutation itself is located in the target gene region or in the non-target homologous/pseudogene region for genes having other highly homologous regions, such as PKD 1. Although some SNPs exist on these genes, it is possible to align to the respective specific regions by reference to the gene sequences, since the genomic DNA is randomly broken by the second generation sequencing, the broken fragments may be caused by the uneven sequencing result of the specific SNPs during the sequencing process, and the actual regions of the target variation cannot be clearly distinguished in the subsequent bioinformatics analysis process due to the too low frequency in the original data.

Taking the PKD1 gene as an example, the gene is on chromosome 16 and has 6 highly homologous pseudogenes (PKD 1P1, PKD1P2, PKD1P3, PKD1P4, PKD1P5 and PKD1P 6) (the homology of a coding region is about 97.49% -98.81%), after the common second generation sequencing result, a Long chain polymerase chain reaction (Long-range PCR) method is required to amplify the mutation site of PKD1 containing low frequency population, and then the combined method of san sequencing or second generation sequencing can be performed on the Long fragment polymerase product.

In 2021, university of Beijing third Hospital [ A comprehensive PGT-M strategy for ADPKD patients with de novo PKD1mutations using affected embryo or gametes as proband Wang, Y et al journal of Assisted Reproduction and genetics.03May 2021] and university of Zhengzhou affiliated first Hospital [ Anovel monogenic preimplantation genetic testing strategy for sporadic polycystic kidney caused by de novo PKD1mutation RUNNING TITLE: A novel PGT-M strategy for ADPKD.Shi, H.et.al.Clin Genet.2021Feb;99 (2) after genome-wide amplification of small number of embryo cells by the team of 250-258], sanger sequencing by Long-range PCR method, and determining whether mutation is located in a specific haploid by combining polymorphism (SNP) provided by second generation sequencing (NGS), as a strategy for performing preimplantation gene detection (PGT-M) of single gene/single gene disease, to screen healthy embryo implantation in utero, so that patients can develop children not carrying PKD1 (suspected) pathogenic mutation.

The above strategies all need to adopt a method of combining Long-range PCR with Sanger sequencing to judge whether embryos carry (suspected) pathogenic variation, however, the structure of PKD1 genes is quite complex, and the GC content in the gene sequences is up to 70-80%, so that the execution difficulty of Long-range PCR is increased. In addition, the whole genome DNA of the embryo cells cannot be amplified to ensure that amplified product fragments of each embryo can generate enough DNA amplified fragments as templates (templates) for subsequent Long-range PCR, because after the fertilized eggs are formed in vitro, the fertilized eggs can only be cultured in vitro for 5-6 days (blasts), 3-8 cells are biopsied from blasts trophectoderm for genetic detection, so the number of cells capable of extracting the genome DNA is quite rare, partial fracture conditions of the genome DNA cannot be ensured in the process of lysis and amplification, once the fractured parts of the genome are near pathogenic sites, templates suitable for the length or sequence interval of Long-range PCR cannot be obtained in the process of amplification, thus the failure rate of Long-range PCR is improved, and the problem of healthy child growth of patients cannot be solved by adopting the original strategy.

In the above-mentioned Zhengzhou university literature, it is mentioned that the amplification products of 2 embryos of 39 embryos used cannot successfully obtain the Long-range PCR product, but in the actual use process, the father of the embryo also carries out molecular diagnosis by combining Long-range PCR with sanger, but when the same primers are used for operating the genome amplification products of embryo cells, the genome amplification products of 10 embryos cannot obtain the Long-range PCR product, which indicates that the original method may have extremely high failure probability, and a new detection method needs to be developed urgently.

Disclosure of Invention

Aiming at the defects existing in the prior art, the main purpose of the invention is to provide a PKD1 variant molecule detection method based on single-cell whole genome amplification, which uses the genome DNA amplification products of single sperm, embryo or polar body to verify the existence of (suspected) pathogenic variation and haploid type linked with the (suspected) pathogenic variation, and can obtain healthy embryo without adopting a Long-range PCR method with higher failure rate.

Another object of the present invention is to propose the use of the above-described PKD1 variant molecule detection method based on single cell whole genome amplification in molecular biology detection for non-diagnostic purposes.

The above object of the present invention is achieved by the following technical solutions:

the invention provides a PKD1 variant molecule detection method based on single-cell whole genome amplification for non-diagnostic purposes, which is based on germ cells or embryonic cells of a male sperm or a couple after in-vitro fertilization of the male sperm or the couple as detection objects, and comprises the following steps:

s1: extracting germ cells or embryo cells of a male sperm or in vitro fertilization of a couple of a subject as cells to be detected, and carrying out full genome DNA amplification to obtain a full genome DNA amplification product;

s2: searching PKD1 gene mutation sites and pseudogene site information and designing primers;

s3: amplifying short fragments with the length of 150-250bp in the whole genome DNA amplification product, and carrying out deep targeting second generation sequencing to distinguish reads of PKD1 and pseudogenes to obtain PKD1 variation site information;

s4: detecting the whole genome DNA amplification product by adopting a whole genome SNP chip or second generation sequencing to obtain whole genome SNP information which comprises PKD1 genes and SNP information in 2Mb upstream and downstream of the PKD1 genes, so as to perform genome copy number variation analysis;

s5: and simultaneously, carrying out SNP genotyping by combining mutation information and SNP information, and then carrying out family haplotype linkage analysis to judge whether embryos subjected to in vitro fertilization of both couples of the subjects carry haplotypes in which PKD1mutation is located in families.

Preferably, in step S1, the test cells are 3-8 blastocyst biopsies or polar bodies or single sperm cultured after in vitro fertilization.

Preferably, in the step S2, the PKD1 gene can be specifically amplified by human beings, and the primer comprises a primer pair with the upstream and downstream sequences shown as SEQ ID NO. 1-30 respectively, wherein the design principle is to distinguish the region from the interference region of the pseudogene as far as possible, and the primer pair is used for targeted amplification of the mutation site of the PKD1 and the region within 500bp upstream and downstream of the mutation site.

Preferably, in step S5, the method for detecting SNP genotype includes: the samples obtained by the whole genome DNA amplification products in the step S2 are grouped according to families, the samples detected positive by the targeted second-generation sequencing in the step S3 are used as a precursor reference, the samples are respectively grouped with the whole genome amplification samples of the couples of the subjects, SNP genotyping is carried out by combining SNPs information obtained by the whole genome SNP chip or the second-generation sequencing, and embryo molecular karyotype and family haplotypes are constructed, so that the copy number variation condition of the embryo genome and the carrying state of the embryo genetic variation are respectively identified.

More preferably, in step S5, the method of haplotype analysis comprises: the gene mutation carrier is heterozygous, the mating is wild type, the target second-generation sequencing detection reference sample is SNP locus of the carrier heterozygous, information SNPs covering the gene mutation point region is selected from 2Mb upstream and downstream of the mutation point, at least 1 information SNP is selected in each Mb of the chromosome, the determined information SNPs locus is subjected to family linkage analysis to obtain a haplotype covering the whole chromosome of the gene mutation region, and the set of different chromosome haplotypes is family haplotype.

The invention also provides a detection product before embryo implantation, which is characterized by comprising a primer pair with the upstream and downstream sequences shown as SEQ ID NO. 1-30 respectively.

Preferably, the detection product is a detection kit.

The invention also provides application of the PKD1 variant molecule detection method based on single-cell whole genome amplification for non-diagnosis purpose in molecular biological detection of a cell to be detected with the total genome DNA reaching less than sequencing concentration or DNA continuous fragment length below 500bp, wherein the cell to be detected is a germ cell or embryo cell of a male sperm of a subject before embryo implantation or after in-vitro fertilization of a couple.

When the total genome DNA of the cell to be detected is not up to the sequencing concentration or the quality is lower (the continuous DNA fragment is degraded to below 500 bp), a Long-Range PCR combined Sanger sequencing method cannot be adopted to determine whether a specific low-frequency locus is positioned on the PKD1 gene or 6 pseudogenes which are highly homologous to the PKD1 gene.

The traditional method for combining Long-range-PCR with sanger sequencing has higher quality requirements on the amplification of the genome DNA, and is easy to cause the increase of failure rate. The invention aims at monogenic disease patients with pseudogene or homologous region sequence interference diagnosis in genome, especially patients carrying (suspected) pathogenic variation to new variation of couples or incapable of obtaining parental gene information of the patients and not having child-related or lacking forerunner samples (especially genetic variation easily diagnosed by pseudogene interference), single-cell or small-cell amplification parallel deep targeting second-generation sequencing detection is carried out by embryo biopsy cells after single sperm, polar body or in vitro insemination of the patients (but PCR products for distinguishing PKD1 and the pseudogene are not required to be produced firstly), family monomer linkage analysis is carried out by utilizing germ cells or embryo cells after single sperm or in vitro insemination of the patients and partners of men (deep targeting second-generation sequencing detection is carried or not carried with samples), embryos with single-gene disease variation and normal embryos of wild type can be rapidly, simply and accurately distinguished, chromosome copy number variation of the embryos can be screened simultaneously, clinical rate can be improved, and genetic transfer of single-gene diseases is blocked down to the next generation before embryo transplantation is realized.

In addition, by adopting a short-fragment PCR targeted enrichment (suspected) pathogenic site signal combined with deep targeted sequencing combined somatic mutation analysis method, germ cells or embryo cells carrying (suspected) pathogenic variation and not carrying (suspected) pathogenic variation can be distinguished, and the individual haplotypes are found out by combining SNP chip analysis on the germ cells, so that whether all embryos to be detected (suspected) pathogenic variation has high correlation with a specific haplotype or not is further analyzed, and the haplotypes not carrying the pathogenic variation are obtained, thereby being beneficial to selecting healthy embryos for implantation.

Compared with the prior art, the invention has the following beneficial effects:

1. the invention is based on molecular biological detection of single-cell whole genome amplification, is used for genetic diagnosis and human assisted reproduction as embryo implantation pre-detection, can detect only by the existence of a short fragment (-200 bp) of a genome DNA amplification product of an embryo, a sperm or a polar body, verifies the existence of (suspected) pathogenic variation and combines SNP family linkage to obtain a haploid type linked with the (suspected) pathogenic variation, thereby reducing the difficulty of embryo selection and further improving the probability of healthy child generation of ADPKD patients.

2. The length of the short fragment amplified by the whole genome of the single cell is 150-250bp, the cost requirement of PCR economy and time is low, enrichment can be completed in 1 hour generally, the Long-range PCR economy and cost are high, and the time for operating one round is at least 4-8 hours. In addition, the short fragment PCR is not easily affected by DNA quality or secondary structure, and the success rate is high; the Long-range PCR is easily affected by DNA quality or secondary structure, and the failure rate is high.

3. The cost of analyzing haploid by utilizing SNP chips is lower than the time and economic cost of providing haploid by using second generation sequencing.

4. According to the invention, the enriched short-fragment PCR product is used for deep targeted second-generation sequencing and combined with somatic mutation analysis, the reads of the proprietary PKD1 and the reads on the pseudogene are directly distinguished by comparing the reference sequences, and the homologous region of the PKD1 and the pseudogene cannot be directly distinguished by the conventional sanger analysis, so that the sequencing by using the Long-range PCR product with higher failure rate is forced.

5. The detection technology of the invention can be directly carried out on the basis of the embryo cell amplification product, and the operation steps are easier than the original method.

Drawings

FIG. 1 is a flow chart of a method for detecting PKD1 variant molecules based on single-cell whole genome amplification in the example.

FIG. 2 is a gel diagram of primer tests performed on negative blood samples in the examples.

FIG. 3 shows the library construction result in the embodiment.

FIG. 4 is a gel diagram of a positive sample for secondary primer testing in the examples.

FIG. 5 is a gel diagram of the test of the 7 th pair of new primers in the example.

FIG. 6 is a gel diagram of six-site PCR purification after changing new primers in the example.

FIG. 7 shows the library construction result in the example.

FIG. 8 shows the genotyping results for each embryo PKD1 gene haplotype in the examples.

FIG. 9 shows the results of analysis of genome-wide copy number variation of embryos in the examples.

FIG. 10 is a gel diagram of amplification of the PKD1 gene variation sites c.74115 dup, c.10821+1dup and c.11880_11950del in the examples.

Detailed Description

The invention is further illustrated below in connection with specific examples which are provided solely for the purpose of illustration and are not intended to limit the scope of the invention.

Example 1

1. Material

Autosomal dominant hereditary polycystic kidney disease (ADPKD) is the most common hereditary kidney disease, with patients mostly developing after adulthood, with a 1/1000 incidence in the population.

ADPKD is caused by mutations in protein kinase D1 (PKD 1) (about 85% of cases) or protein kinase D2 (PKD 2) (about 15% of cases).

The PKD1 gene is located on the short arm of chromosome 16 (16p13.3) and consists of 51 exons, and the cDNA coding region consists of 12912 nucleotides (NM-001009944.2) and encodes 4303 amino acids.

Polycystic kidney caused by PKD1 gene mutation is inherited by autosomal dominant, the generation of offspring of patients has 50% probability of inheritance and pathogenicity, and the generation of polycystic kidney of offspring can be prevented by prenatal diagnosis or preimplantation genetic diagnosis of the patients.

2. Purpose(s)

The embryo transfer sequence was diagnosed by detecting whether the sample was mutated at the corresponding detection site in accordance with the method shown in FIG. 1, in combination with SNP linkage analysis (haplotype analysis).

3. Method of

3.1 objects

Taking one family as an example, a couple of couples of Shanghai collection inheritance and sterility diagnosis and treatment centers of a gynaecology and obstetrics hospital affiliated to the compound denier university are recruited, wherein analysis of semen detection results of men shows that azoospermia is caused, polycystic kidney disease is caused, and gene detection by Shanghai's medical test suggests PKD1, NM_001009944, c.74115 dup (p.S2475Lfs multiplied by 26), het, P.and AZFc microdeletion is detected by other men, the deletion size is about 1.5Mb (chrY: 24872470-25170492; 25665496-27120666), VUS. Male father has gone, mother's body is healthy, brother is healthy, and other family members have no polycystic kidney disease history. The male and mother performed PKD1 site verification, and the result suggested that the mother was negative for the site, and the male PKD1, NM-001009944, c.74115 dup (p.S2475Lfs. Times.26), het, P. Site source was unknown. The couples require PGT-M for this site, informing AZFc microdeletion that it may lead to oligospermia in adult males, and the correlation with azoospermia in this male is unknown, and the couples indicate that it is not required to do PGT-M for AZFc microdeletion. The polycystic kidney caused by PKD1 is an autosomal dominant genetic disease, the male genotype corresponds to a phenotype, the genetic variation is a frameshift mutation, the pathogenic variation is 50% of risk, PGT-M is suggested, and the PGT-M is developed for the purpose of selecting embryos which are not inherited to the variation and are euploids for transplantation. The written informed consent was signed and the study protocol was approved by the human subject ethics committee of the Shanghai Jiai genetics and sterile diagnostic center of the complex university gynaecology and obstetrics hospital.

3.2. Blastocyst biopsy and Whole Genome Amplification (WGA)

3.2.1. In vitro fertilization

In Vitro Fertilization (IVF) was performed on the recruited home, following methods conventional in the art, and subsequent biopsies and mutation site analysis were performed on blasts obtained from this family.

3.2.2. Blastocyst biopsy and whole genome amplification

Taking the embryo at the blastula stage, and removing 3 to 8 cells from the trophectoderm on the 5 th or 6 th day of embryo development. The biopsied cells were placed in PCR tubes with PBS buffer and subjected to Whole Genome Amplification (WGA) by the Multiple Displacement Amplification (MDA) method. Isothermal DNA amplification (REPLI-g single cell whole genome amplification kit, cat. Nos. 1503443 or 150345,QIAGEN GmbH,Hilden,Germany) was performed with phi 29DNA polymerase according to the method described in the kit specification, and the specific procedure is as follows:

1) Pretreatment: buffer DLB resuspension:

buffer DLB was added 500. Mu. L H ₂ Mixing O sc, centrifuging and storing at-20 ℃ for 6 months;

2) Buffer D2 preparation

DTT，1M 3μL

Buffer DLB(reconstituted)33μL

Total volume 36μL

Buffer D2 is frozen at-20deg.C for no more than 3 months;

3) Taking 4 mu L of biopsy cell lysis sample, uniformly mixing with 3 mu L of Buffer D2, and reacting for 10min at 65 ℃;

4) Adding 3 mu L of stop solution into the reaction solution in the step 3) to stop the reaction;

5) Master mix formulation:

6) Adding 40 mu L of Master mix into each reaction obtained in the step 4), and adding 50 mu L of the total reaction system;

7) Placing the reaction system in the step 6) at 30 ℃ for reaction for 8 hours, and stopping the reaction at 65 ℃ for 3 minutes;

8) The reaction products were subjected to agarose gel electrophoresis and stored at-20 ℃.

3.3. Primer design

3.3.1 primers were designed for the desired detection sites based on the mutation sites as shown in Table 1.

TABLE 1

3.3.2 sample mixing amplicon pooling to differentiate sequencing data for different samples, AC modifications and different i5 and i7Barcode were added before primers to differentiate different samples, as shown in table 2.

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3.4. Primer test

3.4.1 testing of primers with negative blood samples, ensuring primer amplification efficiency, wherein:

input amount: 20ng.

The pectin diagram is shown in FIG. 2, and shows that the amplification effect of other primers is good except that the amplification efficiency of the seventh pair of primers is slightly low (but accords with the lowest concentration of the library establishment); the library construction result is shown in fig. 3, and shows that the library construction result is good.

3.4.2 use of positive samples for secondary testing of primers, ensuring that the concentration of amplified product is sufficient for subsequent experiments, wherein:

input amount: 20ng.

The gel chart results are shown in FIG. 4, and shows that the results after amplification are good except for the seventh pair of primers (new primers are designed later, and the new primers are shown in the primer table).

Test2: a new primer test of the 7 th pair, wherein:

input amount: 20ng.

The results are shown in FIG. 5, which shows that the seventh pair of new primers had good amplification results.

3.5. Formal experiments

3.5.1PCR amplification: positive samples and corresponding NTC amplification experiments were performed:

input amount: ext> 20ext> ngext> (ext> 10ext> ngext> forext> 4ext> sampleext> inputsext> atext> positionsext> C.786ext> -ext> 787ext> insCext> Pext> 22000612ext> (ext> kext> -ext> 14ext> -ext> 3ext>)ext>,ext> C.8311ext> Gext> -ext> Aext> Pext> 22000613ext> (ext> kext> -ext> 15ext> -ext> 53ext>)ext>,ext> C.9388_9393ext> delext> Pext> 22000592ext> (ext> Pext> -ext> 5136ext> -ext> 8#cext>)ext> andext> Pext> 22000619ext> (ext> Kext> -ext> 20ext> -ext> 0502ext>)ext>)ext>,ext> theext> gelext> resultsext> areext> shownext> inext> FIG.ext> 6ext>:ext> Six-site PCR purification gel map after new primer exchange: the PCR procedure was the same as 3.2.1, with good overall amplification (where c.6424C > P22000577 (P-3671-1#c) samples at the T site, P22000593 (P-5136-16#c) at the c.9388_9393del site, and P22000602 (P-5352-16#c) at the c.12031C > T site failed amplification).

3.5.2 after the first amplification step is completed, the samples are divided into two groups, amplicon library construction is carried out, and quality inspection is carried out:

amplicon pooling: 73 samples were pooled in two groups.

Input amount: 4ng per sample;

library 1-1: ext> containsext> sitesext> c.11241_ext> 11255ext> delext>,ext> c.9547Cext> >ext> Text>,ext> c.10821+1ext>,ext> c.9388_9393ext> delext>,ext> c.6424Cext> >ext> Text>,ext> c.2534Text> >ext> Cext>,ext> c.8311Gext> -ext> Aext> (ext> 36ext> samplesext> totalext>)ext>.ext>

Library 2-1: comprising positions c.786-787insC, c.1938G > A, c.12031C > T, c.12494-12501 del, c.6132C > G (total of 37 samples).

And (3) library building flow:

1. sample dilution and mixing

The PCR purified products in both libraries were diluted to 2 ng/. Mu.L (concentration below 2 ng/. Mu.L without dilution of direct input 4 ng), input 4ng per sample and mixed.

2. Terminal Repair +A [ End Repair ]

PCR system:

Reagent	Vol for 1Lib(uL)
		End Repair&A-Tailing Buffer	7
End Repair&A-Tailing Enzyme Mix	3
		Total Volume	10

PCR procedure:

20℃	30min
		65℃	30min
4℃	Hold

3. joint-up [ Ligation ] -Ligation Adaptors

PCR system:

Reagent	Vol for 1Lib(uL)
		Ligation Buffer	30
DNA Ligase	10
		NFW	5
Total Volume	45

PCR procedure:

20℃	15min
		4℃	Hold

4. library purification

5. Library amplification

PCR system:

Reagent	Vol for 1Lib(uL)
		KAPA HiFi HotStart ReadyMix(2x)	25
Adapter-ligation library	20
		Library Amplification Primer Mix(10x)	5
Total Volume	50

PCR procedure:

6. library purification

Library results are shown in fig. 7: the upper panel is library 1-1 and the lower panel is library 2-1.

Library 1-1 concentration was 5.42 ng/. Mu.L;

library 2-1 concentration was 4.04 ng/. Mu.L;

the library construction results were good.

3.5.3 on-machine sequencing

3.5.4 data processing

1. Data filtering

The Illumina platform NovaSeq 6000 sequencer sequenced the sample library in high throughput in PE150 mode, and two 150bp sequence fragments were obtained for each target fragment. During the preparation of the sequencing library, the DNA sequence is randomly fragmented, possibly resulting in an insert of less than 150bp, resulting in a situation of sequencing, the linker sequence being detected at the 3' end of the sequencing sequence.

Ensuring the accuracy of subsequent mutation analysis requires processing of the sequence containing the sequencing adapter. The sequencing adaptors that may occur in the original sequencing data (Raw reads) are sheared and removed using software fastp (v0.22.0) (Chen shift el at., 2018), while low quality base reads and reads with a higher proportion of ambiguous bases in the original sequence are removed, and at the same time, the quality control is required to have a read length of at least 75bp, and the remaining high quality data (Clean reads) are used in the subsequent analysis flow of the biological messages.

2. Obtaining optimal alignment

Since there are 6 pseudogenes in the target gene, it is also necessary to remove the sequence derived from the pseudogene. The sequencing data was aligned to the reference genome (hg 19) using the MEM (maximal exact matched) (Li heng, 2013) algorithm in BWA (v0.7.17) software to obtain an alignment file in bam format, and the sequence optimally aligned to the PKD1 gene was extracted for subsequent analysis.

3. Identification of mutation sites

Mutation detection and genotyping of SNV and InDel was performed using sendeon (202010.01) (Donald Freed et al, 2016).

4. Mutation site validation

The bam file was checked for confirmed variation using igvbam (v 2.12.3) (James t.robinson el at, 2011).

3.5.5 sequencing data QC are exemplified as follows:

3.6. sequencing result determination examples are as follows:

SNP genotyping and haplotyping

SNP genotyping

SNP genotyping was performed using a Illumina human Karyomap-12V1.0 microarray. Each Karyomap-12 chip contains approximately 300,000 SNPs, which can cover the entire human 23 pairs of chromosomes. The samples obtained by performing embryo biopsy whole genome amplification in the examples are grouped according to families, one of the target second-generation sequencing detection samples (a sample which is detected as positive is generally selected) is used as a precursor, and the samples are respectively grouped with the whole genome amplification samples of couples of family patients, so that microarray SNP genotype detection and analysis are performed, and specific reference is made to the specification.

3.7.1. Genomic copy number variation and SNP linkage (haplotype) analysis

After all SNPs information detected by the chip is obtained, embryo molecular karyotype and family haplotype are constructed and are respectively used for identifying the copy number variation condition of the whole genome of the embryo and identifying the carrying state of the genetic variation of the embryo, and the specific operation method is as follows:

A. construction of family haplotypes:

1) Genotyping of samples: SNP genotyping of both patient couples, target second generation sequencing positive samples (forerunner reference), and embryos (blastula);

2) Determining information SNPs sites: the selection criteria for the information SNPs were: a SNP site that is heterozygous in the carrier of the genetic variation, homozygous in its partner, and homozygous in the targeted second-generation sequencing test reference sample; selecting information SNPs covering a gene variation point region, and selecting at least 1 information SNP in a chromosome per Mb range; in the coverage gene mutation region, the information SNPs is selected from the range of 2Mb at the upstream and downstream of the mutation point;

3) Constructing a family haplotype: collecting the information SNPs loci determined in the step 2) to obtain haplotypes covering the whole chromosome of the gene mutation region through family linkage analysis, wherein the collection of different chromosome haplotypes is called family haplotypes.

3. Taking one of the families and embryo thereof as an example to display embryo genetic variation carrying condition screening result

Taking the result of each embryo PKD1 gene haplotype as an example (taking the targeting second-generation sequencing positive embryo 6#c as a reference), as shown in FIG. 8, the whole genome copy number variation of the embryo is shown in FIG. 9, and the summary report of diagnosis of 10 embryos to be transferred through whole genome copy number variation and family haplotype linkage analysis is shown in Table 3.

TABLE 3 Table 3

The previous description of the embodiments is provided to facilitate a person of ordinary skill in the art in order to make and use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above-described embodiments, and those skilled in the art, based on the principle of the present invention, should make improvements and modifications without departing from the scope of the present invention.

Claims

1. A method for detecting a PKD1 variant molecule based on single cell whole genome amplification for non-diagnostic purposes, comprising the steps of:

2. The method for detecting PKD1 variant molecules based on single-cell whole genome amplification for non-diagnostic purposes according to claim 1, wherein in step S1, the test cells are 3-8 or polar bodies or single sperm of blastocyst trophoblast biopsies cultured after in vitro fertilization.

3. The method for detecting PKD1 variation molecules based on single-cell whole genome amplification for non-diagnostic purposes according to claim 1, wherein in the step S2, the PKD1 gene is human-specifically amplified PKD1 gene, and the primers comprise a primer pair with the upstream and downstream sequences shown in SEQ ID NOs 1-30, respectively.

4. The method for detecting PKD1 variant molecules based on single cell whole genome amplification for non-diagnostic purposes according to claim 1, wherein in step S5, the method for SNP genotype detection comprises: the samples obtained by the whole genome DNA amplification products in the step S2 are grouped according to families, the samples detected positive by the targeted second-generation sequencing in the step S3 are used as a precursor reference, the samples are respectively grouped with the whole genome amplification samples of the couples of the subjects, SNP genotyping is carried out by combining SNPs information obtained by the whole genome SNP chip or the second-generation sequencing, and embryo molecular karyotype and family haplotypes are constructed, so that the copy number variation condition of the embryo genome and the carrying state of the embryo genetic variation are respectively identified.

5. The method for detecting a PKD1 variant molecule based on single cell whole genome amplification for non-diagnostic purposes according to claim 4, wherein in step S5, the method for haplotype analysis comprises: the gene mutation carrier is heterozygous, the mating is wild type, the target second-generation sequencing detection reference sample is SNP locus of the carrier heterozygous, information SNPs covering the gene mutation point region is selected from 2Mb upstream and downstream of the mutation point, at least 1 information SNP is selected in each Mb of the chromosome, the determined information SNPs locus is subjected to family linkage analysis to obtain a haplotype covering the whole chromosome of the gene mutation region, and the set of different chromosome haplotypes is family haplotype.

6. Use of the method for detecting PKD1 variant molecules based on single cell whole genome amplification for non-diagnostic purposes according to any one of claims 1 to 5 in molecular biology detection of test cells with total genomic DNA of less than a sequencing concentration or a DNA continuous fragment length of 500bp or less, wherein the test cells are germ cells or embryonic cells of a subject after in vitro fertilization of either single sperm of a male subject or of a couple prior to embryo implantation.

7. A detection product before embryo implantation is characterized by comprising a primer pair with the upstream and downstream sequences shown as SEQ ID NO. 1-30 respectively.

8. The pre-embryo implantation test product of claim 7, wherein the test product is a test kit.