TWI640636B - A method for simultaneous performing gene locus, chromosome and linkage analysis - Google Patents

Abstract

The invention relates to a method for simultaneously performing gene locus, chromosome and linkage analysis, which particularly comprises obtaining embryonic cell sample, whole genome amplification, amplification of target gene mutation site, whole genome amplification product and target gene mutation site. The main steps of database establishment, high-throughput sequencing and data analysis, by using whole-genome amplification technology combined with high-throughput sequencing, complete multiple comprehensive detections in one step, avoiding the use of multiple methods, multiple steps to separate single genes Detection of genetic mutation sites, chromosomal disorders, and linkage analysis. The method provided by the invention provides favorable conditions for a small amount of samples, and can be used not only for PGD detection, but also for determining whether an embryo carries a disease-causing gene and an abnormality of chromosome replication; and is also suitable for genetic screening of abortion, older women's embryos, and realizing a The step completes a single sample multiple test. Because of its simple operation, short cycle and strong feasibility, it is conducive to promotion and application.

Description

A method for simultaneously performing gene locus, chromosome and linkage analysis

The invention relates to the field of genomic sequence analysis and the field of bioinformatics, and particularly relates to a method for realizing single-cell monogenic diseases, chromosomal diseases and linkage analysis by using MALBAC amplification technology combined with high-throughput sequencing.

Genetic diseases refer to diseases caused by changes in genetic material (gene sequences on chromosomes or mitochondrial DNA) in humans. In recent years, the incidence of genetic diseases has increased year by year. In China, as many as tens of thousands of children with chromosomal abnormalities are born each year, and children with chromosomal abnormalities are still incurable. At present, there are more than 7,000 single-gene genetic diseases, of which more than 4,000 have been identified. Although the incidence of single disease in single-gene genetic diseases is low, due to its wide variety, it is in the life of infants. The overall incidence and overall prevalence in the population are not low, and most monogenic genetic diseases are lethal, disabling or teratogenic, with the exception of a small number of corrections that can be corrected by some treatments, most There is no effective treatment to date. The fundamental way to solve the above problems is to pre-natal or pre-embryo diagnosis to avoid the birth of such children. Therefore, pre-implantation genetic diagnosis has an important science to prevent the occurrence and transmission of monogenic genetic diseases and chromosomal genetic diseases. And social significance.

Preimplantation genetic diagnosis (PGD) of genetic diseases is an important part of reproductive medicine. It can block the birth of embryos before implantation, avoiding and reducing the occurrence of genetic diseases, and reducing the economic burden of family and society. At present, preimplantation genetic diagnosis mainly uses FISH and single cell PCR technology, as well as CGH array and SNP array technology applied to clinical PGD in recent years. Among them, the application of FISH for embryo screening is limited by probes, and it is impossible to detect all chromosome states at the same time; single cell fixation is easy to cause loss of information, single-cell PCR technology is easy to cause allele loss (ADO), which causes misdiagnosis; CGH array can only Diagnosing changes in chromosome number and large fragment chromosome structure (non-equilibrium translocation, duplication, deletion), while SNP array can perform all CGH-diagnosed chromosomal abnormalities with higher resolution than CGH, but cannot simultaneously complete single-gene genetic diseases and chromosomes. Diagnosis of the disease.

It can be seen that the existing preimplantation genetic diagnosis technology still has various deficiencies, which cannot meet the actual needs, and is limited in use and promotion.

In addition, the existing linkage analysis method mainly uses two genetic markers, STR and SNP. Multiplex PCR technology combines multiplex PCR with fluorescent probes to detect STR typing. Since there are few STR linkage markers, there may be no STR sites available in specific cases, so a lot of pre-experimental work is needed before clinical testing. Most of the STR genetic markers are far away from the pathogenic site, which is prone to misdiagnosis due to chromosome recombination, and the detection cost is high. Therefore, the technique cannot perform batch detection. The method of capturing SNP by the wafer and the probe is higher in cost and longer in cycle than the conventional PCR capture SNP method.

In summary, even for samples of a large number of cells, the prior art cannot simultaneously perform single-gene genetic diseases, chromosomal abnormalities, and linkage analysis by one-step detection. In addition, for samples of a small number of cells (especially for micro-cells used for preimplantation genetic diagnosis), it is even more difficult to perform simultaneous single-gene genetic diseases, chromosomal abnormalities, and linkage analysis.

Therefore, there is an urgent need in the art to develop a PGD detection method that can simultaneously perform single gene genetic diseases, chromosomal abnormalities, and linkage analysis using only a small number of cells.

It is an object of the present invention to provide a PGD assay for the development of single gene genetic diseases, chromosomal abnormalities and linkage analysis based on only a small number of cells (e.g., 3-10 cells or less).

In a first aspect of the invention, there is provided a method for simultaneously performing gene locus, chromosome and linkage analysis, comprising the following steps: (1) obtaining an embryonic cell sample: obtaining a fertilized egg by single sperm injection, and culturing to a capsule The embryonic stage is isolated from the outer trophoblast cells to obtain a sample containing 3-10 cells; (2) Whole genome amplification: the lysate is added to the sample and placed in a PCR machine for lysis, inactivating protease, and the obtained cell lysis sample is subjected to Whole genome amplification; (3) Amplification of target gene mutation site: After completion of primer design and primer evaluation, the target gene mutation site is subjected to ordinary PCR amplification to obtain PCR product, and the PCR product and step of the same sample (2) The fragmented genomic DNA obtained in the mixture is mixed according to a certain ratio; (4) the whole genome amplification product and the target gene mutation site are used for database establishment; (5) the sample is sequenced by using a high-throughput sequencing platform; (6) Data analysis (6.1) The sequence results obtained in step (5) are removed from the adapter and low quality data, and the comparison software is used to compare the reference genome; (6.2) After the quality control, the data of the lower machine is compared to the reference sequence, the distribution of the alleles of the statistical locus and its frequency, and the mutation information of the target gene locus is detected; (6.3) The data of the lower machine is compared by the quality control. On the reference sequence, the sequence was GC-corrected according to a certain window, and the single-cell data of thousands of whole-genome amplifications were corrected for the database, and the chromosome replication number variation (CNV) was finally detected; (6.4) SNP-single-type type was used. Linkage analysis.

In another preferred embodiment, the method is non-diagnostic, non-therapeutic.

In another preferred embodiment, in the step (3), the template for ordinary PCR amplification is selected from the group consisting of: (i) nucleic acid of the cell lysis sample, and (ii) the product of the whole genome amplification reaction of the step (2). Or (iii) a mixed stencil formed by mixing (i) and (ii).

In another preferred embodiment, the common PCR amplified template is the product of the whole genome amplification reaction of step (2), or the nucleic acid of the cell lysis sample and the product of the whole genome amplification reaction of step (2). The mixture (wherein the mixing ratio of (ii) to (i) is from 10 to 10000:1, preferably from 100 to 1,000:1).

In another preferred embodiment, the whole genome amplification of step (2) is achieved by a multiple binding and loop-forming amplification cycle (MALBAC), including a first round of linear amplification and a second round of exponential amplification.

In another preferred embodiment, the conditions of the first round of linear amplification in the step (2) are: (1) 90-98 ° C reaction for 90 s - 5 min; (2) 15 - 25 ° C reaction for 30 s - 1 min; 25-35 ° C reaction 30s-1min; (4) 35-45 ° C reaction 20s-1min; (5) 45-55 ° C reaction 20s-1min; (6) 55-65 ° C reaction 20s-1min; (7) 65 -85 ° C reaction 2min-6min; (8) 90-98 ° C reaction 10-40s; (9) 45-65 ° C reaction 5-20s; (10) repeat steps (2) to step (9) 5 to 20 cycles .

In another preferred embodiment, the conditions of the first round of linear amplification in the step (2) are preferably: (1) reaction at 94 ° C for 3 min; (2) reaction at 20 ° C for 40 s; (3) reaction at 30 ° C for 40 s; 4) 40 ° C reaction for 30 s; (5) 50 ° C reaction for 30 s; (6) 60 ° C reaction for 30 s; (7) 70 ° C reaction for 4 min; (8) 95 ° C reaction for 20 s; (9) 58 ° C reaction for 10 s; (10) Repeat steps (2) through (9) for 8 cycles.

In another preferred embodiment, the conditions of the second round of exponential amplification in the step (2) are: (1) 90-98 ° C reaction for 10 s - 50 s; (2) 90-98 ° C reaction for 10 s - 40 s; 45-65 ° C reaction 20s-45s; (4) 65-80 ° C reaction 75s-5min; (5) repeat steps (2) to step (4) 10 to 30 cycles; (6) the product after amplification Store at 0-5 °C.

In another preferred embodiment, the condition of the second round of exponential amplification in the step (2) is preferably: (1) reaction at 94 ° C for 30 s; (2) reaction at 94 ° C for 20 s; (3) reaction at 58 ° C for 30 s; 4) React at 72 ° C for 3 min; (5) Repeat steps (2) to (4) for 17 cycles; (6) Store the amplified product at 4 ° C.

In another preferred embodiment, the product after MALBAC amplification is between 300-2000 bp or between 250-2000 bp.

In another preferred embodiment, the primer design in the step (3) is designed to have a length of 20-25 bp, and the size of the amplified fragment is adjusted according to the order of the read, and the single-ended cannot exceed the read length. The double end can't be more than 2 times longer.

In another preferred embodiment, the mixing ratio of the PCR product to the fragmented genomic DNA in the step (3) is 1: (1-100); preferably 1: (10-100); more preferably 1 :(20-50).

In another preferred embodiment, the high-throughput sequencing in the step (5) is performed only on a single gene pathogenic site and a chromosome replication number variation (CNV) analysis, and each sample has a minimum average order depth of the genome. 0.1 times; if SNP linkage information is to be obtained at the same time, the average depth of sequencing of each sample is at least 2 times that of the genome.

In another preferred embodiment, the step (6.4) uses a SNP-single set to perform linkage analysis as follows: (1) selecting a SNP region, defined within a range of 1 M upstream and downstream of the target gene; (2) obtaining a target with software The SNP of the region, the soft body is selected from the group consisting of: samtools mpileup, GATK, FreeBayes, VarScan; (3) SNP filtration: the lowest allergy heterozygosity difference is 10%, removing potentially erroneous SNPs; (4) screening differentiated SNPs, And construct the male and female SNP-single type: the same SNP locus, in the four allele bases of the male and female parent, one base is different from the other three to distinguish; Analysis of SNP-single-type: There are 2 embryonic SNP-single-type types, which are inherited from the father and the female, respectively. According to the distinguishing SNP and Mendelian genetic principle, it is judged which SNP-single type is specific. Parental inheritance, which is maternal inheritance; (6) Analysis of results: According to step (4) to determine the male and female SNP-single type, distinguish the single set of linkages with the pathogenic site, the SNP where the embryo is located Specific allelic comparison, according to Mendelian genetic principle, to determine whether the embryo carries a causative basis Site.

In another preferred embodiment, in the embryonic SNP-single set analysis of step (5), the minimum SNP is 10, and if there are more than 3 SNP errors, the embryo SNP-single set data is regarded as insufficient data. It is difficult to analyze.

It is to be understood that within the scope of the present invention, the various technical features of the present invention and the various technical features specifically described hereinafter (as in the embodiments) may be combined with each other to constitute a new or preferred technical solution. Due to space limitations, we will not repeat them here.

Detailed description of the preferred embodiment

The inventors have long-term extensive and in-depth research, through a large number of experiments, for the first time developed a micro-cellular sample (such as only 3-10 cells), based on MALBAC amplification technology combined with high-throughput sequencing, simple, fast A comprehensive method for comprehensive detection of embryonic monogenic genetic diseases, chromosomal diseases and linkage analysis in the same sample in one step. Specifically, the method of the present invention performs whole genome amplification (MALBAC amplification) on a trace sample (such as an embryo sample such as 3-10 trophoblast cells), and then uses a sample nucleic acid (preferably a whole genome amplification product) as a template. Ordinary PCR amplification (ie, PCR amplification of the mutation site), and the PCR amplification product of the mutation site from the same sample is mixed with the whole genome amplification product in a certain ratio [eg 1: (10-100)] to establish data together. The library then performs high-throughput sequencing and performs comparative analysis on the sequenced data to achieve multiple detections of embryonic monogenic genetic diseases, chromosomal diseases, and linkage analysis in one step. On the basis of this, the inventors completed the present invention. Detection method

The present invention provides a method of simultaneously performing gene locus, chromosome and linkage analysis as described in the first aspect.

Typically, a method for simultaneously performing gene locus, chromosome, and linkage analysis includes the following steps: (1) Obtaining an embryonic cell sample: obtaining a fertilized egg by single sperm injection, and culturing until blastocyst stage is obtained in an outer trophoblast cell. A sample comprising 3-10 cells, wherein the embryonic cells can be selected from a human or other mammal. (2) Whole genome amplification: lysate is added to the sample to be lysed, inactivated, and the whole cell is amplified. The obtained cell lysate sample is subjected to whole genome amplification, preferably using multiple binding and loop-forming amplification cycles (MALBAC). Implementation; specifically, a pre-amplification mixture is added to the cell lysis sample for the first round of linear amplification, and then the amplification mixture is added for the second round of exponential amplification to purify the amplified product; MALBAC expansion The increased product is between 300-2000 bp (or between 250-2000 bp). Typically, the first round of linear amplification conditions are: (1) 90-98 ° C reaction 90s-5min; (2) 15-25 ° C reaction 30s-1min; (3) 25-35 ° C reaction 30s-1min; 4) 35-45 ° C reaction 20s-1min; (5) 45-55 ° C reaction 20s-1min; (6) 55-65 ° C reaction 20s-1min; (7) 65-85 ° C reaction 2min-6min; (8) Reaction at 90-98 ° C for 10-40 s; (9) Reaction at 45-65 ° C for 5-20 s; (10) Repeat steps (2) to (9) for 5 to 20 cycles. The second round of exponential amplification conditions are: (1) 90-98 ° C reaction 10s-50s; (2) 90-98 ° C reaction 10s-40s; (3) 45-65 ° C reaction 20s-45s; (4) 65 -80 ° C reaction for 75 s - 5 min; (5) Repeat steps (2) to (4) 10 to 30 cycles; (6) Store the amplified product at 0-5 ° C. (3) Amplification of the target gene mutation site: After completing the primer design and the primer evaluation, the target gene mutation site is subjected to ordinary PCR amplification to obtain a PCR product, and the PCR product of the same sample and the fragment obtained in the step (2) are obtained. The genomic DNA is mixed in a mass ratio of 1: (1-100) [preferably 1: (10-100)]. In the design of the primer, the length of the primer is 20-25 bp, and the size of the amplified fragment is adjusted according to the order of the read. The single-ended can not exceed the length of the reads, and the double-ended can not exceed 2 times. (4) Construction of the gene pool by MALBAC amplification products and target gene mutation sites. (5) Using a high-throughput sequencing platform to sequence the samples; if only single-gene causative sites and chromosome replication number variation (CNV) analysis are performed, the average depth of sequencing of each sample is at least 0.1 times that of the genome; If you want to obtain SNP linkage information at the same time, each sample needs to be ordered to have a minimum depth of 2 times the genome. (6) Data analysis (6.1) The sequence results obtained in step (5) are removed from the adapter and low-quality data, and the comparison software is used to compare the reference genome; (6.2) The data of the lower machine is quality-controlled and compared. To the reference sequence, statistically distribute the alleles of the locus and their frequencies, and detect the mutation information of the target locus; (6.3) The data of the lower machine is compared with the reference sequence by quality control, and the sequence is performed according to a certain window. Correction, using thousands of MALBAC-expanded single-cell data as a database for correction, and finally detecting chromosome replication number variation (CNV); (6.4) SNP-single-type linkage analysis: (6.4.1) Selection of SNP regions , defined in the range of 1M upstream and downstream of the target gene; (6.4.2) obtaining the SNP of the target region by using software, including but not limited to at least one of samtools mpileup, GATK, FreeBayes, VarScan; (6.4.3) SNP Filtration: the lowest allele heterozygosity difference is 10%, remove the potential wrong SNP; (6.4.4) Screen the distinguishing SNP, and construct the male and female SNP-single type: the same SNP locus, in the male parent And 4 of the female parent Among the bases of the gene, one base is different from the other three; (6.4.5) Analysis of SNP-single type: There are 2 embryonic SNP-single type, which are inherited from the father and the female parent respectively. According to the distinguishing SNP and Mendelian genetic principle, it is judged which SNP-single type is the parental inheritance and which is the maternal inheritance; among them, the minimum SNP is 10, if there are more than 3 SNP errors The embryonic SNP-single-type data is regarded as insufficient data, which is difficult to analyze; (6.4.6) Analysis of results: According to step (6.4.4), the male and female SNP-single sets are determined, and the results are distinguished. A single set of disease-linked loci, comparing the specific alleles of the SNPs of the embryos, and judging whether the embryos carry the disease-causing gene loci according to the Mendelian genetic principle.

Preferably, the conditions of the first round of linear amplification in the step (2) are: (1) reaction at 94 ° C for 3 min; (2) reaction at 20 ° C for 40 s; (3) reaction at 30 ° C for 40 s; (4) reaction at 40 ° C 30s; (5) 50 ° C reaction 30s; (6) 60 ° C reaction 30s; (7) 70 ° C reaction 4min; (8) 95 ° C reaction 20s; (9) 58 ° C reaction 10s; (10) Repeat step (2) Go to step (9) for 8 cycles.

Preferably, the conditions of the second round of exponential amplification in the step (2) are: (1) reaction at 94 ° C for 30 s; (2) reaction at 94 ° C for 20 s; (3) reaction at 58 ° C for 30 s; (4) reaction at 72 ° C 3 min; (5) Repeat steps (2) through (4) for 17 cycles; (6) Store the amplified product at 4 °C.

A salient feature of the present invention is that a single measurement or diagnosis is obtained in a single use using only a small number of cells (3-10 cells), which helps to improve the accuracy and cost of PGD detection. To this end, the method of the present invention is optimized by combining parameters and steps, thereby ensuring the validity of conventional chromosome (such as CNV) detection for nucleic acid samples from a small number of cells, and fully expanding the frequency of occurrence. Low or very low monogenic genetic diseases and/or linkages for detection of effective and accurate analysis.

In the present invention, for nucleic acid samples derived from a small amount of cells, MALBAC amplification is first performed. Among them, the MALBAC amplification primer used may be an amplification primer existing in the art, such as the primer described in WO2012/166425. However, in order to achieve simultaneous detection of a plurality of cells for a plurality of times, the optimized MALBAC amplification parameters are as described above.

Furthermore, in order to efficiently perform a single gene genetic disease and/or linkage analysis, it is necessary to obtain a high quality (i.e., high fidelity and amplified coverage area) amplification product. For this reason, when performing conventional PCR amplification for a mutation site, although a high-quality nucleic acid extracted from a micro cell can be used as a template, it is preferred to use a partial MALBAC amplification product as a template.

Typically, in a PCR amplification reaction at a mutation site, the template is selected from the group consisting of: (i) a nucleic acid of a cell lysing sample, and (ii) a product of a whole genome amplification reaction of step (2) (ie, a fragmented genome) DNA), or (iii) a mixed stencil formed by mixing (i) and (ii). Preferably, the template is a product of the whole genome amplification reaction of step (2), or a mixture of the nucleic acid of the cell lysis sample and the product of the whole genome amplification reaction of step (2) [wherein (ii) and ( The mixing ratio of i) is 10-10000:1, preferably 100-1000:1].

Further, the whole genome amplification product obtained in the step (2) and the amplification product of the target gene mutation site obtained in the step (3) can be used in the present invention by mixing them in a certain ratio. Subsequent database establishment, sequencing and analysis will obtain comprehensive test results. In this way, it is possible to avoid the trouble of separately establishing a database and/or separately detecting.

In the present invention, in the mixing step, the mixing ratio of the PCR amplification product of the mutation site to the fragmented genomic DNA is 1: (1-100); preferably 1: (10-100); More preferably 1: (20-50).

The main advantages of the present invention include: (1) The method of the present invention requires only a small amount of samples (from 3 to 10 trophoblast cells from an embryo sample) to perform a test that would otherwise require a large number of samples in the art or that requires stepwise. The invention utilizes the MALBAC amplification technology to whole-genome amplification and combine with the mutation of the target gene to amplify, and then, through a high-throughput sequencing of the mixed gene library of the amplified product, completes the embryo single genetic disease in one step for the same sample. Comprehensive tests such as chromosomal diseases and linkage analysis. (2) The method of the invention has strong operability, low cost, simple and rapid operation, wide application range, and avoids detection of single gene genetic disease mutation sites, chromosomal diseases and linkage analysis by using multiple methods and multiple steps. (3) The method of the invention is widely used, and can be used not only for preimplantation genetic diagnosis (PGD) detection, but also for determining whether an embryo carries a disease-causing gene locus and an abnormality of chromosome replication; the same is applicable to abortion, adolescent women's embryos. Hereditary screening, simple, rapid and accurate completion of multiple and comprehensive detection of micro-cell samples. (4) The detection result of the invention is accurate, and the problem of allele tripping (ADO) can be effectively reduced.

The invention is further illustrated below in conjunction with specific embodiments. It is to be understood that the examples are not intended to limit the scope of the invention. The experimental methods in the following examples which do not specify the specific conditions are usually manufactured according to the conditions described in the conventional conditions, for example, Sambrook et al., Molecular Colonization: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturing conditions. The conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight and parts by weight. Reagents and equipment

Unless otherwise stated, all reagents and instruments are commercially available or prepared in a conventional manner.

The MALBAC primer is described in Example 1 of WO2012/166425. EXAMPLES Example 1

An integrated chromosomal genetic disease model with a disease-causing gene A. In this family, both the father and the father's father are carriers, carrying the Mutation mutation site (c.233delC). After analysis, embryos 9, 10, 11 and 12 are pathogenic embryos of the parent-derived mutation carrier, and embryos 7 and 8 do not carry the mutation site. 1 Take embryonic single cells: 1.1 sperm fertilization

For the MII phase of the egg microinjection of single sperm (ICSI), the egg was placed in the (G-MOPS) operating solution, transferred to the micromanipulator platform for micromanipulation. 1.2 Embryo culture in vitro

The fertilized embryos are cultured in G1 broth (Vitrolife) or Gm broth (Global) for about 72 hours to 5-8 cell phase, laser perforated on a zona pellucida, and transferred to the equilibrated G2 broth ( Vitrolife or Gm medium (Global) is continued to culture to the blastocyst, and the hatched blastocyst trophoblast cells are separated by a laser, containing 3-10 cells.

Among them, the embryonic cells may be selected from humans or other mammals. 2. MALBAC amplification 2.1 MALBAC amplification 2.1.1 Lysis cells

Embryonic cells were transferred to a PCR tube containing 5 μl of lysate and placed in a PCR machine for lysis at 50 ° C for 50 minutes. 2.1.2 Inactivating protease

The PCR tube was placed in a PCR machine and inactivated at 80 ° C for 10 minutes. 2.1.3 First round of linear amplification

Add 30 μL of pre-amplification mixture to 5 μl of cell lysate sample.

Temperature flow: 94 ° C for 3 minutes, 8 × (20 ° C - -40 seconds, 30 ° C - -40 seconds, 40 ° C - 30 seconds, 50 ° C - 30 seconds, 60 ° C - 30 seconds , 70 ° C - 4 minutes, 95 ° C - -20 seconds, 58 ° C - 10 seconds). 2.1.4 Second round of exponential amplification

Add 30 μl of the amplification mixture to the 35 μl preamplification mixture.

Temperature flow: 94 ° C - -30 sec, 17 × (94 ° C - -20 sec, 58 ° C - -30 sec, 72 ° C - - 3 min), stored at 4 ° C. 2.2 MALBAC amplification results

Single-cell or equivalent DNA can obtain 2-4 μg of amplified product ranging from about 300-2000 bp (ie, fragmented genomic DNA) from each 65 μL reaction system by single cell amplification. .

The product electrophoresis is shown in Figure 1. In the figure, M represents a 2K DNA ladder, E1, E2, E3, and E4 represent four embryo amplification products, and NC represents a negative control group. As can be seen from Fig. 1, the single cell amplification end product size ranges from about 250 to 2000 bp. 2.3 MALBAC product purification

Purification of the MALBAC product was carried out using a commercial purification kit. 3. Target gene mutation site amplification 3.1 Primer design

Use Oligo, Primer and other primer design software or primer professional design website for primer design. When designing, the length of the primer is about 20-25bp. The size of the amplified fragment is adjusted according to the order of the read. The single-ended can't exceed the length of the read. The end cannot be more than 2 times longer. 3.2 Introduction evaluation

After the primer design is completed, the NCBI or UCSC and other professional websites are used for the specific evaluation of the primers (such as mismatch rate, hairpin structure, etc.), and the primers are synthesized after the evaluation is passed. 3.3 Target gene mutation site amplification

The target gene mutation site was subjected to ordinary PCR amplification.

The PCR product from the same sample and the fragmented genomic DNA were mixed at a certain mass ratio, and the PCR product of the target gene mutation site: genomic DNA = 1: (10-100). 4. Establishment of a database of MALBAC products and target gene mutation sites

A database of MALBAC products and target gene mutation sites was established using a commercial database to establish a kit. 5. High-throughput sequencing

The sample is sequenced using a high-throughput sequencing platform. The sequencing platform is not particularly limited, and the second generation sequencing platform includes, but is not limited to, Illumina's GA, GAII, GAIIx, HiSeq1000/2000/2500/3000/4000, X Ten, X Five, NextSeq500/550, MiSeq, Applied Biosystems' SOLiD, Roche's 454 FLX, Thermo Fisher Scientific (Life Technologies)'s Ion Torrent, Ion PGM, Ion Proton I/II; third generation single molecule sequencing platform: including but not limited to Helicos BioSciences' HeliScope system, Pacific Bioscience's SMRT system, GridION, MinION of Oxford Nanopore Technologies. The sequencing type can be single-ended (Single End) or double-ended (Paired End) sequencing.

If only single-gene causative sites and chromosome replication number variation (CNV) analysis are performed, the average depth of each sample is only 0.1X; if SNP linkage information is to be obtained at the same time, each sample needs to be ordered to have an average depth of 2X. . 6. Data Analysis 6.1 Reference Sequence Alignment

The sequencing result is removed and the low quality data is removed and compared to the reference genome. The reference genome can be a whole genome, an arbitrary chromosome, a part of a chromosome, or a gene. The reference genome usually selects a sequence whose expression can be determined to be NCBI or UCSC as a reference sequence (eg, human Hg19, mouse mm10), or any chromosome. The comparison software can be used with any kind of free or commercial software, such as BWA (Burrows-Wheeler Alignment tool), SOAPaligner/soap2 (Short Oligonucleotide Analysis Package), Bowtie/Bowtie2. 6.2 Analysis of target gene mutation sites

The data of the lower machine was compared with the reference sequence (such as human Hg19, mouse mm10) after quality control, and the allele distribution and frequency of the statistical locus were detected to detect the mutation information of the target gene locus. 6.3 CNV analysis

The data of the lower machine was compared with the reference sequence (such as human Hg19, mouse mm10) after quality control, and the sequence was GC-corrected according to a certain bin (bin), and the database of thousands of MALBAC-expanded single cells was used as a database. Corrected, the number of chromosome copies was finally detected, see CNV analysis examples: Table 1 and Figure 2. In Fig. 2, the X coordinate indicates the chromosome number, the Y coordinate indicates the number of chromosome copies, and the arrow indicates the chromosome copy number abnormal chromosome. Table 1. CNV analysis results 6.4 Linkage analysis

In this method, a SNP-single-sleeve type is taken as an example. 6.4.1 SNP calling and SNP filtering

The SNP region was selected and defined in the range of 1M upstream and downstream of the target gene, and the heterozygosity was higher (the frequency in the individual database was more than 0.3); the smaller the SNP spacing, the smaller the recombination rate.

The SNP of the target area is obtained by software, including but not limited to at least one of samtools mpileup, GATK, FreeBayes, and VarScan.

SNP filtration, the lowest allergy heterozygosity difference is 10%, removing potentially erroneous SNPs. 6.4.2 Screen the distinguishing SNPs and construct the male and female SNP-single sets.

A distinguishing SNP, that is, the same SNP site, one of the four allele bases of the male parent and the female parent, one base is different from the other three. As shown in Table 2, A-SNP1, the paternal allele genotype is A/G, the maternal allele genotype is A/A, and the paternal father's individual allele genotype is A/A. It is indicated that the base A of the paternal locus and the causative locus are linked in the same single set and passed to the offspring; the base G of the paternal locus is linked to the normal allele in the same single set. This SNP site is a differentiated SNP, and the other sites are deduced by analogy. See Table 2 for an example. Table 2. Partial SNP genotypes in the A gene region Notes: 1. “_” in Table 2 indicates that the corresponding SNP data cannot be obtained (no data coverage or low depth); 2. The bold italic indicates the c.233delC pathogenic mutation carried; 3. The parental single set in Table 2 Type 1 indicates a single set of disease-causing mutations; 4. Mutation in Table 2 is a disease-causing mutation. 6.4.3 Analysis of embryonic SNPs - single set

There are 2 embryonic SNP-single-type types, which are inherited from the father and the female, respectively. According to the distinguishing SNP and Mendelian genetic principle, it is judged which SNP-single type is the parent source and which is the mother source. Genetic. In the SNP-single-type analysis of embryos, the minimum SNP of the differentiated SNP is 10. If there are more than 3 SNP errors, the SNP-single type analysis of this embryo is wrong. 6.4.4 Analysis of results

The male and female SNP-single sets were determined according to 6.4.2, and a single set of linkages to the pathogenic sites was distinguished, and the specific alleles of the SNPs of the embryos were compared. For example, in Table 2 A-SNP1, the alleles of embryos 7 and 8 all have a base G, indicating that the two embryos do not carry the pathogenic gene locus of the parent; embryos 9, 10, 11, and 12 The alleles have base A, indicating that these four embryos carry the pathogenic gene locus of the father. According to Mendelian genetic principle, it is judged whether the embryo carries a disease-causing gene locus. 7. Method feasibility verification 7.1 Sanger sequencing

Design specific primers for the target gene mutation site, perform common PCR amplification and Sanger sequencing, and compare with high-throughput sequencing results to verify the feasibility of the target gene mutation site detection method in this patent. See Figure 3. In Figure 3, the position marked in black is the target gene mutation site; in the Sanger sequence diagram, in addition to the four bases "A/T/C/G", there are other simple bases such as "R/S/M/W/V". The bases represent the A/G, G/C, A/C, and G/A/C heterozygous peaks caused by the mutation, respectively. As can be seen from Figure 3, the Sanger sequencing results are consistent with the high-throughput sequencing results in Table 2. 7.2 STR locus linkage analysis

The specific primers were designed for the two STR loci (STR1 and STR2) linked to the target gene, and the linkage analysis of STR loci was carried out to verify the feasibility of the high-throughput sequencing SNP-single-sleeve analysis method in the present invention. 4. Among them, “■” means a sick male, “●” means a sick female, “□” means a normal male, and “○” means a normal female. In the figure, the numbers "229", "235" and "238" indicate the STR1 locus allele, and "193", "195" and "197" indicate the STR2 locus allele. It can be seen from Fig. 4 that the results of the linkage analysis of the STR locus are consistent with the results of the SNP-single set analysis in Table 2.

In summary, the method for performing single gene, chromosome and linkage analysis in a single sequence in the present invention is feasible and accurate. discuss

In the present invention, for micro-cells (such as 3-10 cells or less), using MALBAC amplification technology combined with high-throughput sequencing, one-step comprehensive detection of embryonic monogenic genetic diseases, chromosomal diseases and linkage analysis The utility model has the advantages of strong operability, low cost, simple and rapid operation, wide application range, and avoids the detection of single gene genetic disease mutation sites, chromosomal diseases and linkage analysis by using multiple methods and multiple steps. The method provided by the invention provides favorable conditions for various detections of a small sample (such as an embryo sample), and is widely used, and can be used not only for PGD detection, but also whether the embryo carries a disease-causing gene locus and an abnormality of chromosome copy number; the same applies to repeated The genetic screening of abortion, older women's embryos, to achieve a single step to complete a single sample multiple tests. In summary, the present invention is simple in operation, short in work cycle, and has strong practical feasibility, especially using a high-throughput sequencing method, which is fast in detection and data analysis, and is advantageous for promotion and application.

All documents mentioned in the present application are hereby incorporated by reference in their entirety in their entireties in the the the the the the the the In addition, it should be understood that various modifications and changes may be made by those skilled in the art without departing from the scope of the invention.

Figure 1 is a product electrophoresis pattern of the results of MALBAC amplification of the present invention.

2 is a CNV analysis diagram of whole genome sequencing of embryos in Example 1 of the present invention.

Figure 3 is a Sanger sequence diagram of the mutation site of the target gene in Example 1 of the present invention.

4 is a diagram showing linkage analysis of STR sites in Example 1 of the present invention.

Claims (11)

A method for simultaneously performing gene locus, chromosome and linkage analysis, comprising the following steps: (1) obtaining embryonic cell samples: obtaining fertilized eggs by single sperm injection, and culturing to blastocyst stage in outer trophoblast cells Separate and obtain samples containing 3-10 cells; (2) Whole genome amplification: Add lysate into the sample to be lysed in the PCR instrument, inactivate the protease, and perform whole genome amplification on the obtained cell lysate sample. Fragmented genomic DNA; (3) Amplification of target gene mutation site: After completion of primer design and primer evaluation, the target gene mutation site is subjected to ordinary PCR amplification to obtain a PCR product, and the PCR product and step of the same sample are 2) The fragmented genomic DNA obtained in the mixture is mixed according to the mass ratio of 1:(1-100); (4) the whole genome amplification product and the target gene mutation site are used for database establishment; (5) high-throughput determination is adopted. Sequence platform, sequence the sample; (6) data analysis (6.1) remove the transfer result obtained from step (5) and the low quality data, compare the reference software to the reference genome; (6.2) Machine After the quality control, the data is compared to the reference sequence, the distribution of the alleles of the statistical locus and its frequency, and the mutation information of the target gene locus is detected; (6.3) the data of the lower machine is compared with the reference sequence by quality control. The sequence was GC-corrected according to a certain window, and the single-cell data of thousands of whole-genome amplifications were corrected for the database, and the chromosome replication number variation (CNV) was finally detected; (6.4) The SNP-single-type type was used for linkage analysis. The method of claim 1, wherein the whole genome amplification of the step (2) is achieved by a multiple binding and loop-forming amplification cycle (MALBAC), including a first round of linear amplification and a second round of exponential amplification. . The method of claim 2, wherein the first round of linear amplification in the step (2) is: (1) 90-98 ° C reaction for 90 s - 5 min; (2) 15 - 25 ° C reaction for 30 s - 1min; (3) 25-35 ° C reaction 30s-1min; (4) 35-45 ° C reaction 20s-1min; (5) 45-55 ° C reaction 20s-1min; (6) 55-65 ° C reaction 20s-1min; (7) 65-85 ° C reaction 2min-6min; (8) 90-98 ° C reaction 10-40s; (9) 45-65 ° C reaction 5-20s; (10) repeat steps (2) to step (9) 5 Up to 20 cycles. The method of claim 2, wherein the second round of exponential amplification in the step (2) is: (1) 90-98 ° C reaction for 10 s - 50 s; (2) 90-98 ° C reaction for 10 s - 40s; (3) 45-65 ° C reaction 20s-45s; (4) 65-80 ° C reaction 75s-5min; (5) repeat steps (2) to step (4) 10 to 30 cycles; (6) will expand The added product was stored at 0-5 °C. The method of claim 1, wherein in the step (3), the template for ordinary PCR amplification is selected from the group consisting of: (i) nucleic acid of the cell lysing sample, and (ii) whole genome expansion of the step (2) The product of the increased reaction, or (iii) a mixed stencil formed by mixing (i) and (ii). The method of any one of claims 2 to 5, wherein the product after MALBAC amplification is between 300-2000 bp or between 250-2000 bp. The method of claim 1, wherein the primer design in the step (3) is designed to have a length of 20-25 bp, and the size of the amplified segment is adjusted according to the order of the read, and the single-ended cannot More than the length of the read, the double end can not exceed 2 times longer. The method of claim 1, wherein the mixing ratio of the PCR product to the fragmented genomic DNA in the step (3) is 1: (10-100). The method of claim 1, characterized in that the high-throughput sequencing in the step (5) is performed only on a single gene pathogenic site and a chromosome copy number variation (CNV) analysis, and the average depth of each sample is determined. The lowest is 0.1 times of the genome; if you want to obtain SNP linkage information at the same time, the average depth of each sample is at least 2 times that of the genome. The method of claim 1, wherein the step (6.4) performs a linkage analysis using a SNP-single set of specific operations as follows: (1) selecting a SNP region, defined within a range of 1 M upstream and downstream of the target gene; (2) The SNP of the target region is obtained by software, which is selected from the group consisting of: samtools mpileup, GATK, FreeBayes, VarScan; (3) SNP filtration: the lowest allergy heterozygosity difference is 10%, the potential error SNP is removed; (4) screening Differentiate SNPs and construct a male and female SNP-single type: the same SNP locus. In the four allele bases of the male and female parent, one base is different from the other three. (5) Analysis of SNP-single-set type: There are 2 embryonic SNP-single-type types, which are inherited from the father and the female, respectively. According to the distinguishing SNP and Mendelian genetic principle, the SNP-single set is judged. Which type is parental inheritance and which is maternal inheritance; (6) Analysis of results: According to step (4), the male and female SNP-single sets are determined, and a single set that is linked to the pathogenic site is distinguished. Compare the specific alleles of the SNPs of the embryos, and judge whether the embryos carry the disease-causing gene according to the Mendelian genetic principle. point. The method of claim 10, characterized in that in the embryonic SNP-single-type analysis of step (5), the minimum SNP is 10, and if there are more than 3 SNP errors, the embryo SNP-single data is regarded as For the lack of data, it is difficult to analyze.