CN114196736B - Full-chromosome gene parting chip for synchronously detecting various birth defect genetic diseases, method and application thereof - Google Patents

Abstract

The invention provides a whole chromosome gene parting chip for synchronously detecting various birth defect genetic diseases, a method and application thereof. The chip is used for detecting various birth defect genetic diseases caused by gene copy number variation and single nucleotide variation. The invention reduces or does not design probes in the unimportant region of the genome on the basis of the gene copy number variation probes covered at the whole genome level, encrypts relevant haploid dose deficiency and triploid dose sensitivity pathogenic genes in a targeted manner, designs probes at relevant mononucleotide variation sites of relevant common birth defect genetic diseases, and increases the probe density of corresponding gene regions, thereby increasing the detection of mononucleotide variation of pathogenic genes while reducing the detection of clinically significant gene copy number variation, extending the high performance of the traditional chip, improving the detection effectiveness, and improving the diagnosis, treatment, prognosis and genetic consultation levels of clinically relevant gene copy number variation and mononucleotide variation diseases of birth defects.

Description

Full-chromosome gene parting chip for synchronously detecting various birth defect genetic diseases, method and application thereof

Technical Field

The invention belongs to the field of comprehensive prevention and treatment of birth defects, and particularly relates to a whole chromosome gene parting chip for synchronously detecting various birth defect genetic diseases, a method and application thereof.

Background

About 90 ten thousand cases of birth defects infants are newly increased in China every year, about 80% of the birth defects infants are caused by genome variation, the types of variation are more, the phenotype symptoms are complex, the clinical diagnosis is difficult, and the birth defects infants are one of the main causes of disability and death of postpartum children. Of these, about 24.3% are microdeletion microreplication syndromes due to gene Copy Number Variation (CNV), including many unexplained dysnoesia, growth retardation, autism, and multiple congenital anomalies; about 17.4% are severe monogenic genetic diseases whose pathogenesis is Single Nucleotide Variation (SNV), broadly involving point mutations and small fragment insertion/deletion (InDel). The cumulative infant mortality due to gene Copy Number Variation (CNV) and Single Nucleotide Variation (SNV) was about 19.1% and the cumulative pediatric hospitalization was about 18%. It can be seen that accurate synchronous detection of gene Copy Number Variation (CNV) and Single Nucleotide Variation (SNV) has important clinical significance for early discovery, early prevention and control, and early treatment of birth defect genetic diseases.

The main detection methods of gene Copy Number Variation (CNV) are G banding, fluorescence in situ hybridization (Fluorescence In Situ Hybridization, FISH) and chromosomal microarray analysis (Chromosome Microarray Analysis, CMA). G banding and FISH can detect partial chromosome regions, but are difficult to provide variation information with higher resolution at the genome level, and have the disadvantages of complex operation, low flux and 3% of the overall positive detection rate. The CMA technology comprises comparison of genome hybridization (Array Comparative Genomic Hybridization, aCGH) and single nucleotide polymorphism microarray chip (Single Nucleotide Polymorphism-based Array, SNP Array), and is widely developed by various national hospitals as a first-line clinical diagnosis strategy of gene Copy Number Variation (CNV) for prenatal diagnosis and dysplasia detection of postpartum unknown reasons, and the detection rate of positive gene Copy Number Variation (CNV) at the whole genome level can reach 31.6%. However, neither G-banding nor FISH nor CMA can be used to detect Single Nucleotide Variation (SNV) that causes monogenic genetic diseases, which is currently detected by Sanger sequencing, quantitative PCR, long PCR, second generation sequencing, and other methods, and these techniques cannot detect genetic diseases caused by gene Copy Number Variation (CNV), and the current clinical strategy is to detect CNV first and then SNV, so developing a method for simultaneously detecting CNV and SNV has important significance for early discovery, early prevention and control, early treatment and cost reduction of birth defect genetic diseases.

It is reported that in the contrast detection of 113 cases of children, the detection rate of CNV (computerized numerical control) is 81.4% and the detection rate of Array-CGH is 67.3% [1]; carrying out rapid prenatal diagnosis on the common chromosome aneuploidy of 270 fetuses by adopting a fluorescent quantitative PCR (polymerase chain reaction) combined with a whole genome copy number variation sequencing (CNV-Seq) technology and detecting the whole genome copy number variation of more than 100kb, wherein the total detection rate of the fluorescent quantitative PCR is 19, and the detection rate is 7.03%; 43 cases of CNV-Seq detection abnormality, the detection rate is 15.9% [2]; performing amniotic fluid puncture on 52 pregnant women, performing prenatal diagnosis by CMA technology, and detecting 15 copy number variations consistent with NIPT result, wherein the detection rate reaches 28.85% [3]; 206 cases (12.31%) of G band karyotype analysis anomalies in 1674 pregnant woman samples in the middle gestation period, wherein 73 cases of CMA do not detect anomalies; CMA analysis showed 147 cases (8.81%), of which 26 cases were normal with karyotype analysis, mostly small fragment repeats or deletions [4].

In addition, many mutation hotspots of monogenic genetic diseases include both gene Copy Number Variation (CNV) and Single Nucleotide Variation (SNV), e.g., about 70% of mutations in the duchenne muscular dystrophy DMD gene are large fragment exon deletions/duplications, and about 23% are caused by Single Nucleotide Variation (SNV) in the DMD internal exon region and flanking regions. Under the condition that point mutation is not detected clinically in the disease, methods such as Multiplex Ligation Probe Amplification (MLPA), polymerase chain restriction fragment length polymorphism analysis (PCR-RFLP) and the like are additionally used for detecting gene Copy Number Variation (CNV) in the gene, so that the detection time cost and the detection economic cost are greatly increased.

In summary, due to the limitations of the prior art, it is often necessary to combine various technical means, such as CMA combined with MLPA and PCR combined with Sanger, to detect gene Copy Number Variation (CNV) and Single Nucleotide Variation (SNV). The gene Panel for predicting the neoantigen load and detecting the genome mutation disclosed in CN110592213A is mainly used for detecting DNA mutations of a plurality of sites of tumor cells at one time based on second-generation sequencing, can also be used for measuring SNV, inDel, CNV, HLA and other various types of mutations in the tumor immunity field, but can only measure mutation frequencies (VAFs) of various mutation types of the tumor cells, and cannot be used for diagnosis and detection of birth defect genetic diseases.

Reference is made to:

[1] qin Qian, liu Bo, yang Lin, etc. establishment and application of a copy number variation screening assay procedure based on high throughput sequencing technology [ J ]. J.China journal of evidence-based pediatrics, 2018,08 (13): 275.

[2] Li Qin, wei Zhaolian, qiao Jin, et al, application of high throughput sequencing technology in fetal chromosomal copy number variation detection [ J ]. J. International health on reproduction/family planning journal, 2020,39 (6): 450-455.

[3] Jiang Nan, zhang Yinshuai, song Lijie, etc. the use of high throughput sequencing techniques in fetal chromosomal copy number variation detection [ J ]. J.J.China journal of medical genetics, 2020,37 (7): 779-784.

[4] Qian, linghu Keyan, zhuo Zhaozhen, etc. 1674 pregnant women amniotic fluid cells G are subjected to nuclear and chromosome microarray results comparison analysis [ J ]. J.J.of family planning, 2021,29 (05): 1046-1049.

Disclosure of Invention

In view of the problems in the diagnosis and detection of the existing genetic birth defect diseases, the invention provides a whole chromosome genotyping chip and a method for synchronously detecting a plurality of genetic diseases with birth defects, aiming at gene Copy Number Variation (CNV) and Single Nucleotide Variation (SNV) of the genetic diseases with birth defects. Based on SNV-array technology, genome_frame probe sets are designed for 16 single genes and 350 pathogenicity single-dose deficiency (HI) genes and triple dose sensitivity (TS) genes which are disclosed, meanwhile, on the basis of the genome_frame probe sets, specific_gene probe sets are further designed for pathogenicity Single Nucleotide Variation (SNV) of 190 important birth defect genetic genes, and through the design, the defect that an existing detection method can only detect gene Copy Number Variation (CNV)/Single Nucleotide Variation (SNV) singly is overcome, and synchronous detection of gene Copy Number Variation (CNV) and Single Nucleotide Variation (SNV) which cause birth defect genetic diseases is realized.

The invention aims to provide a whole chromosome genotyping chip for synchronously detecting a plurality of birth defect genetic diseases, which is used for synchronously detecting gene Copy Number Variation (CNV) and Single Nucleotide Variation (SNV), and can realize multiple results by only one experiment, thereby effectively reducing the economic burden of families of partial patients and improving the clinical economic applicability, and the chip comprises a genome_frame probe set and a specific_gene probe set;

the genome_frame probe group covers the whole genome of the chromosome, the total number of probes is 642338, and the average distribution density of the probes is 1/5 Kb; the number of chromosome genome-wide coverage probes is shown in table 1 below;

TABLE 1 genome-wide coverage Probe number (genome version: hg 38)

The genome_frame work probe set comprises skeleton probes which are designed according to a certain interval aiming at autosomes and aiming at pertinence chromosomes; the backbone probe reduces/removes probes in the head and tail end regions of each chromosome, in the centromere region and in other unrelated bond gene regions; non-important genomic regions are not or less probed to reduce clinically insignificant Copy Number Variation (CNV) regions of the gene,

the genome_frame work probe set comprises probes designed for the gene copy number variation region of 16 single gene levels, and the average distribution density of the probes is more than or equal to 15/5 Kb; the probe density is highly encrypted, and the related diseases reported by the CNV in the single gene are set according to the genetic characteristics of each disease;

The 16 monogenes include VHL, QDPR, SMN1, CYP21A2, HBB, PTS, PAH, ATP7B, GCH1, HBA2, NF1, PHEX, DMD, OTC, MECP2 as shown in table 2 below;

TABLE 2 16 important monogenic horizontal CNV regions covering the number of probes and the average distribution Density of probes (genome version: hg 38)

The genome_frame work probe set comprises probes designed for the gene copy number variation regions of pathogenic single-dose deficiency genes and triple-dose sensitive genes, and the average distribution density of the probes is set to be more than or equal to 1/5 Kb for moderate encryption and exceeds the average distribution density of the whole genome coverage probes according to Clingen databases and literature reports; the pathogenic single dose deficiency (HI) genes and triple dose sensitivity (TS) genes are 350, including AHDC1, ARID 11 1, 26, LHX4, 1, 3B4, SLC2A1, ZBTB18, BAG3, BMPR 12, GATA3, KAT6 11, PAX2, PTEN, RPS24, WAC, ZMYND11, ALX4, ARCN1, ATM, CDKN 12, FZD4, HMBS, KCNQ1OT1, KMT2 5, MEN1, MYBPC3, PAX6, PHF21 2, SDHD, SHANK2, WT1, ACVRL1, ARID2, CDKN 12, KMT2 3, MED13, RPS26, SLC17A8, SOX5, TBX3, TBX1, KYBQ 1, KCNQ1, KMT2, MEN1, MYBPC3, PAX6, PHF21, SDHD, SHANK2, WT1, ACVRL1, ARID2, CDKN 12, KMT2, MED13, RPS26, SLC17A8, SOX5, TBX3 as shown in Table 3 below BRCA2, CHAMP1, ZIC2, BMP4, CHD8, DICER1, FOXG1, GPHN, PAX9, AAGAB, CHD2, FBN1, IGF 12, RPS17, SIN3, SPRED1, TCF12, UBE3 11, CDH1, 2, FOXF1, PKD1, SALL1, SETD 1B1, TSC2, AXIN2, BRCA1, BRIP1, COL1A1, EFTUD2, FLCN, HNF1, PAFAH1B1, PMP22, RAD51 1, RNF135, TBX4, TP53, ASXL3, DSC2, GATA6, SETBP1, SMT 4 BRCA2, CHAMP1, ZIC2, BMP4, CHD8, DICER1, FOXG1, GPHN, PAX9, AAGAB, CHD2, FBN1, IGF 12, RPS17, SIN3, SPRED1, TCF12, UBE3 11, CDH1, 2, FOXF1, PKD1, SALL1 SETD1 2B1, TSC2, AXIN2, BRCA1, BRIP1, COL1A1, EFTUD2, FLCN, HNF1, PAFAH1B1, PMP22, RAD51 1, RNF135, TBX4, TP53, ASXL3, DSC2, GATA6, SETBP1, SMAD4, FLCN, HNF1, PAFAH1B1, PMP22, RAD51 1, RNF135, TBX4, ASXL3, DSC2, GATA6, SETBP1, SMAD4, PAFAH1B1, PAFAH1, PMP 1, TRPS1, ZFPM2, COL5A1, DMRT1, EHMT1, ENG, HNRNPK, LMX1B, NR A1, PTCH1, STXBP1, TGFBR1, TSC1, ZNF462, ABCD1, actsl 4, AFF2, ANOS1, AP1S2, AR, ARHGEF9, ARX, ATP7A, ATRX, AVPR2, BCOR, BRWD3, BTK, CASK, CD LG, CDKL5, CHM, CHRDL1, CLCN4, CLCN5, CNKSR2, COL4A5, CUL4B, CYBB, DCX, DDX3X, DLG, EBP, EDA, EFNB1, F8, F9, FANCB, FGD1, FLNA, FMR1, FRMD7, FTSJ1, GDI1, GK, GLA, GPC3, GRIA3, HCCS, HDAC8, HPRT1, HDAC IDS, IKBKG, IL1RAPL1, IQSEC2, KDM5C, KDM6A, L1CAM, LAMP2, MAGT1, MID1, MTM1, NDP, NHS, NR B1, NSDHL, NYX, OCRL, OFD1, OPHN1, PAK3, PCDH19, PDHA1, PGK1, PHF6, PIGA, PLP1, PORCN, PQBP1, PRPS1, PTCHD1, RAB39B, RP2, RPS6KA3, RS1, SH2D1A, SLC16A2, SLC35A2, SLC6A8, SLC9A6, SMC1A, SMS, STS, SYN1, TBX22, TIMM8A, TRAPPC2, TSPAN7, UBE2A, UPF3B, USP9X, WDR45, XIAP, XIST, ZC H2, ZDHC 9, ZIC3, ZNF711, SHOX, SRY;

TABLE 3 coverage of 350 pathogenic HI and TS Gene regions the number of probes and the average distribution Density of the probes (genome version: hg 38)

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The special_gene probe set covers 37083 single nucleotide variation sites of 190 important birth defect genetic genes, the total number of corresponding probes is 330272, and 9 repeated probes are designed for each single nucleotide variation site on average;

the 190 important birth defect genetic disease genes are selected according to the principles of allele carrying rate, epiratio, morbidity, patient attack time, clinical preventive and therapeutic measures and the like aiming at the field of prevention and treatment of birth defect postnatal genetic disease in combination with the literature report of clinical laboratories, the relevant loci of the 190 important birth defect genetic disease genes are counted and screened by ClinVar, HGMD, gnomAD and a database of a local clinical laboratory, the high-incidence SNV loci of Chinese population of each gene are confirmed, in order to increase the accuracy and positive detection rate of the result of the corresponding SNV loci, the probes are randomly placed at different positions of the chip, and the minimum 4 repeated probes of each SNV locus are ensured, the 190 important birth defect genetic disease genes are ABCB1, ABCC8, ABCD1, ABCD4, ABCG5, ABCG8, ACADM, ACADS, ACADVL, ACAT1, ACE, ACSF3, AGL, AMH, AMHR2, APOA5, APOB, APOC3, AR, ARG1, ARSA, ARSB, ASS1, ATP7A, ATP7B, BCKDHA, BCKDHB, BMP1, BTD, CBS, CD320, CLCN5, COCH, COL1A1, COL1A2, COL2A1, COMP, CPS1, CPT1A, CPT2, CRHR1, CYP11B1, CYP17A1, CYP1A1, CYP21A2, CYP2C19, CYP2D6, CYP3A5, DBT, DIABLO, DLD, DMD, DMP1 DNAJC12, DRD2, DSPP, ELN, ENPP, EPHX1, ETFA, ETFDH, FAH, FGF, FGFR1, FGFR2, FGFR3, FKBP5, G6PC, G6PD, GAA, GALC, GALE, GALK1, GALNS, GALT, GBA, GBE1, GCDH, GCH1, GCK, GJB2, GJB3, GLA, GLB1, GNPTAB, GNPTG, GNS, GUSB, GYS2, HADH, HBA1, HBA2, HBB, HEXA, HEXB, HGSNAT, HLA _ B, HLCS, HNF4A, HPD, HSD B2, HTR2C, HYAL1, IDS, IDUA, IFITM, IFNL4, IVD, KCNJ11, LDLR, LHCGR, LIPI, LMBRD1, LPL, MC4R, MCCC1, MCCC2, MCEE, MCOLN1, MLYCD, MMAA, MMAB, MMACHC, MMADHC, MMUT, MT-CO1, MT-RNR1, MT-TH, MT-TL1, MT-TS1, MTHFR, MTR, MYO15A, MYO A A, NAGLU, NDN, NPC1, NPC2, NR0B1, NR5A1, OTC, PAH, PCBD1, PCCA, PCCB, PHEX, PHKA2, PHKB, PHKG2, PLA2G4A, PLP1, POLG, PPIB, PRPS1, PTS, PYGL, PYGM, QDPR, RP1, SDHA, SGSH, SLC A1, SLC22A5, SLC25A13, SLC25A15, SLC25A20, SLC26A4, SLC34A3, SLC37A4, SLCO2B1, SMN1/SMN2, SMPD1, SNRPN, SPR, SPRN, SRD A2, SRY, STAR, SUMF1, TAT, TBX1, TECTA, TMC1, TPMT, TYRP1, UCP2, UGT1A, UGT A4, VKORC1, WFS1; the 37083 personal pathogenicity SNV statistics of the chip against the 190 important birth defect genetic disease genes are shown in table 4 below.

TABLE 4 SNV number of 190 genes of birth defect inheritance disease

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Preferably, the specific_gene probe set gives consideration to increasing the probe distribution density of the gene_frame probe set corresponding to the gene region.

Preferably, the nucleotide sequences of the genome_frame work probe set and the specific_gene probe set include the probe sequences shown in SEQ ID No.1 to SEQ ID No.720, but the partial sequences are only a small example.

Preferably, the genome_frame probe set calculates the per probe copy number CN for variations of the 16 single genes, the pathogenic single dose deficiency genes, and the triple dose sensitive genes; the autosomal and female X chromosomes are normal, the probe copy number CN1 is 2, the variation condition of the probe copy number CN1 is calculated to be 2, wherein the probe copy number CN1 is 0 or 1 belongs to deletion, and 3 or 4 belongs to repetition; the normal probe copy number CN2 of male X and Y chromosomes is 1, the number of the probe copy number CN2 variation is calculated to be 2, wherein the probe copy number CN2 is 0 and belongs to deletion, and the probe copy number CN2 is 2 and belongs to repetition;

when the probe copy number CN abnormal condition of 50 continuous probes occurs, judging that the gene copy number is variant;

The calculated copy number metric value is log2 Ratio, where Ratio = observed value per probe copy number +.a reference value per probe copy number, a reference value per probe copy number is a set of data from a normal population:

cn=0 when log2 Ratio is approximately-1.5±0.05;

cn=1 when log2 Ratio is approximately-1±0.05;

cn=2 when log2 Ratio is approximately 0±0.05;

cn=3 when log2 Ratio is approximately 0.58±0.05;

cn=4 when log2 Ratio is approximately 1±0.05.

Preferably, the specific_gene probe set detects the corresponding SNV loci of the 190 important birth defect genetic diseases genes, and the result is interpreted based on the detection value of the part of probes by using a Genotype_SNV and ES clustering algorithm.

The Genotype_SNV method is realized by carrying out bicolor fluorescent labeling on an SNV array probe, wherein the probe can obtain two fluorescence detection values A and B, and a genotyping result is obtained by calculating the A and B values, and the calculation formula is as follows:

t(snv)＝(A-B)/(A+B)

when t (snv) is approximately equal to 1+/-0.05, the typing result is AA;

when t (snv) is approximately equal to-1+/-0.05, the parting result is BB;

when t (snv) is approximately equal to 0+/-0.05, the typing result is AB;

genotyping results included three, AA, AB and BB, where AA and BB represent pure and types, one wild type, one homozygote, and wild type and homozygote were judged based on clustering results, and AB represents heterozygote.

The ES clustering algorithm is used for clustering the genotyping result, and the calculation formula is as follows:

t(x)＝log2(A/B)；

t(y)＝[log2(A)+log2(B)]/2；

and clustering by using t (x) as a horizontal axis and t (y) as a vertical axis in the ES clustering algorithm, and judging the positive or negative of the detection result according to the sample typing result and the clustering number.

Preferably, the chip further comprises a denaturing mixture, an amplification mixture, a fragmenting mixture, a precipitation mixture, and a hybridization mixture.

The denatured mixed solution was 10 Xdenatured solution diluted 10-fold with pure water.

The amplification mixed solution is prepared by mixing 1 volume of amplification enzyme and 45 volumes of amplification solution.

The fragmentation mixture was prepared by mixing 1 volume of the fragmented enzyme, 10.3 volumes of the fragmentation diluent, and 45.7 volumes of the 10 x fragmentation buffer.

The precipitation mixed solution is prepared by mixing 1 volume of precipitation solution 2 with 119 volumes of precipitation solution 1.

The hybridization mixture is prepared by mixing 1 volume of hybridization solution 1, 18 volumes of hybridization solution 2 and 140 volumes of hybridization buffer.

Preferably, the chip is onThe detection is performed in a microarray scanner.

The saidThe matched reagent of the microarray scanner comprises a dye mixture A, a dye mixture B, a stabilizing mixture, a connecting mixture A and a connecting mixture B.

The dye mixture A is prepared by mixing 1 volume of dyeing liquid 1-A, 1 volume of dyeing liquid 1-B, 2 volumes of dyeing buffer solution and 96 volumes of washing liquid A.

The dye mixture B is prepared by mixing 1 volume of dyeing liquid 2-A, 1 volume of dyeing liquid 2-B, 2 volumes of dyeing buffer solution and 96 volumes of washing liquid A.

The stabilizing mixture is formed by diluting 1 volume of stabilizing solution and 8 volumes of fixed diluent with pure water by 7.9 times.

The ligation mixture A was prepared by mixing 1 volume of ligation solution 1, 0.4 volume of ligation solution 2, and 5 volumes of ligation buffer.

The ligation mixture B was prepared by mixing 1 volume of ligase, 6.6 volumes of probe mixture 1, 6.6 volumes of probe mixture 2 and 52 volumes of ligation mixture A.

It is another object of the present invention to provide a method for simultaneously detecting a plurality of genes of birth defects,

the method comprises the steps of,

s1, collecting a sample;

s2, sample preparation and quality control;

s3, detection reaction:

s31, DNA amplification: 100ng of DNA sample (5 ng/. Mu.L) was taken, 2. Mu.l of 10 Xdenaturing mixture and 18. Mu.l of pure water were added and incubated at room temperature for 10min; adding 130 μl of the neutralization solution, uniformly mixing, adding 225 μl of the amplification mixed solution and 5 μl of the amplification enzyme, incubating at 37deg.C for 23+ -1 h, transferring to 65deg.C for 20min, and continuing to incubate at 37deg.C for 45min;

S32, DNA fragmentation and precipitation: adding 45.7 mu l of 10 Xfragmentation buffer, 10.3 mu l of fragmentation diluent and 1.0 mu l of fragmentation enzyme into the sample incubated in the step S31, uniformly mixing, briefly centrifuging, incubating at 37 ℃ for 30min, adding 19 mu l of stop solution at room temperature, vibrating, uniformly mixing and centrifuging; adding 238 mu l of precipitation solution 1, 2 mu l of precipitation solution 2 and 600 mu l of isopropanol at room temperature, fully and uniformly mixing the materials up and down by a pipette, and freezing the materials at-20 ℃ for 16-24 hours;

s33, drying, resuspension and quality control: centrifuging the sample treated in the step S32 at 3200rpm at 4 ℃ for 40min, pouring out the waste liquid, then placing the waste liquid at 37 ℃ for airing for 20min, adding 35 mu l of heavy suspension buffer solution, shaking and mixing uniformly, shaking for 10min, adding 70.5 mu l of hybridization buffer solution, 0.5 mu l of hybridization solution 1 and 9 mu l of hybridization solution 2, shaking and mixing uniformly, and centrifuging to obtain 115 mu l of modified hybridization solution; quality control using 10. Mu.l of the denatured hybridization solution, including absorbance measurement with Nanodrop and gel electrophoresis, wherein the gel-loading dye is Invitrogen ^TM Trackit Cyan/Orange Loading Buffer;25bp Invitrogen Ladder; the Gel electrophoresis system is Invitrogen E-Gel ^TM 48agarose gels 4％，G8008-04；

S34, denaturation and hybridization: the denatured hybridization solution produced in step S33 is placed on a hybridization plate, and then placed in a PCR instrument, and a denaturation program (modification program) is started: denaturation at 95℃for 10min and denaturation at 48℃for 3min, cooling. The remaining 105. Mu.l of denatured hybridization solution was carefully transferred to a hybridization plate with a pipette and loaded A microarray chip scanner, continuously hybridizing for 23.5-24 hours;

s35, detecting by a chip scanner: performing dye-disc corresponding split charging on the mixture generated in the step S34: wherein 2 dye trays of dye mixture A, wherein 100.8. Mu.l of wash solution A, 2.1. Mu.l of staining buffer, 1.05. Mu.l of staining solution 1-A and 1.05. Mu.l of staining solution 1-B were prepared in advance; 1 dye tray of dye mixture B, in which 100.8. Mu.l of wash solution A, 2.1. Mu.l of staining buffer, 1.05. Mu.l of staining solution 2-A and 1.05. Mu.l of staining solution 2-B were prepared in advance; 1 dye tray for stabilizing mixture, wherein 93.2. Mu.l of pure water, 10.5. Mu.l of fixed dilution and 1.3. Mu.l of stabilizing solution were prepared in advance; 1 dye disc of ligation mixture, wherein 66.2. Mu.l ligation buffer, 13.1. Mu.l ligation buffer 1, 3.1. Mu.l ligation buffer 2, 10.5. Mu.l probe mix 1, 10.5. Mu.l probe mix 2 and 1.6. Mu.l of ligase currently withdrawn at-20℃were prepared in advance; and then directly loading the sample into a chip scanner, and simultaneously adding 150 mu l of fixed buffer solution into each hole of each scanning disk to avoid contacting the bottom of the scanning disk. Aligning the notch angle of the cover with the notch edge of the scanning disc, covering the scanning disc, then placing the scanning disc on the top of the workbench, and finally starting a scanning detection flow;

s4, data quality control and analysis;

S5, analyzing and annotating results.

The invention also provides application of the chip in synchronous detection of various birth defect genetic diseases CNV and SNV.

The whole chromosome genotyping chip for synchronously detecting the genetic diseases of various birth defects can synchronously detect the copy number variation and the single nucleotide variation of genes, is simple to operate and high in diagnosis efficiency, and can realize targeted detection of CNV in a whole genome range and SNV loci related to 190 common genetic diseases of birth defects by only one experiment.

The method for synchronously detecting the genes of various birth defects adopts the whole chromosome genotyping chip, can obviously improve the standardization degree of clinical detection of the birth defect related CNV and single gene diseases by doctors, is a molecular diagnosis method with high timeliness, high accuracy and high operability, and can obviously improve the diagnosis, treatment, prognosis and genetic consultation level of the diseases.

Drawings

FIG. 1 is a flow chart of the method detection of the present invention;

FIG. 2 is a hybridization signal diagram of a chip of the present invention;

FIG. 3-1 is a graph of gene copy number deletion results of the present invention, with the ordinate representing log2Ratio;

FIG. 3-2 is a graph of the gene copy number repeat result of the present invention, with the ordinate representing log2Ratio;

FIG. 4-1 is a graph showing the positive results of single nucleotide variation;

FIG. 4-2 is a graph showing the positive results of single nucleotide variation;

FIG. 5-1 is a graph showing a negative result of single nucleotide variation;

FIG. 5-2 is a graph showing a negative result of single nucleotide variation.

Detailed Description

The design, positive sample verification and result analysis of the present invention are further described below in conjunction with specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. Further, it is understood that various changes and modifications may be made by those skilled in the art after reading the teachings of the present invention, and such equivalents are intended to fall within the scope of the claims appended hereto.

Example 1

A whole chromosome genotyping chip for simultaneous detection of multiple birth defect genetic diseases in this embodiment includes a genome_frame work probe set and a specific_gene probe set.

genome_frame probe sets include backbone probes designed for autosomes and for sex chromosomes; the backbone probe reduces/removes the head and tail end regions of each chromosome, the centromere region and other non-genetic region probes;

genome_frame probe set comprising probes designed for gene Copy Number Variation (CNV) regions at 16 single gene levels, 16 single genes comprising VHL, QDPR, SMN1, CYP21A2, HBB, PTS, PAH, ATP7B, GCH1, HBA2, NF1, PHEX, DMD, OTC, MECP2;

The genome_frame probe set includes probes designed for the gene Copy Number Variation (CNV) region of 350 pathogenic single dose deficiency (HI) genes and triple dose sensitivity (TS) genes, pathogenic single dose deficiency (HI) genes and triple dose sensitivity (TS) genes include AHDC1, ARID 11 1, 2, LHX4, 1, 3B4, SLC2A1, ZBTB18, BAG3, BMPR 12, GATA3, KAT6 11, PAX2, PTEN, RPS24, WAC, ZMYND11, ALX4, ARCN1, ATM, CDKN1, FZD4, HMBS, KCNQ1OT1, KMT2 5, MEN1, MYBPC3, PAX6, PHF21 2, SDHD, SHANK2, WT1 ACVRL1, ARID2, CDKN 12 2, KMT2 3, MED13 11, RPS26, SLC17A8, SOX5, TBX3, BRCA2, CHAMP1, ZIC2, BMP4, CHD8, DICER1, FOXG1, GPHN, PAX9, AAGAB, CHD2, FBN1, IGF 12, RPS17, SIN3, SPRED1, TCF12, UBE3, CDH1, 2, FOXF1, PKD1, SALL1, SETD 1B1, TSC2, AXIN2, BRCA1, BRIP1, COL1A1, EFTUD2, FLCN ACVRL1, ARID2, CDKN 12 2, KMT2 3, MED13 11, RPS26, SLC17A8, SOX5, TBX3, BRCA2, CHAMP1, ZIC2, BMP4, CHD8, DICER1, FOXG1, GPHN, PAX9, AAGAB, CHD2, CHD 3, CHC 1, CHC 2, CHC 1, ZIC2, CHD2, CHC 2, CHN FBN1, IGF 12, RPS17, SIN3, SPRED1, TCF12, UBE3 11, CDH1, 2, FOXF1, PKD1, SALL1, SETD 1B1, TSC2, AXIN2, BRCA1, BRIP1, COL1A1, EFTUD2, FLCN, NKX2-5, PURA, RASA1, SPINK1, TRIO, ARID1B, EYA, FOXC1, HIVEP2, PHIP, SYNGAP1, TFAP2B, AUTS2, CAMK2B, ELN, GLI3, KCNH2, KMT2C, MNX1, PMS2, SGCE, SHH, TWIST1, CHD7, EXT1, GATA4, KAT6A, TRPS1, ZFPM2, COL5A1, DMRT1, EHMT1, ENG, HNRNPK, LMX1B, NR A1, PTCH1, STXBP1, TGFBR1, TSC1, ZNF462, ABCD1, ACSL4, AFF2, ANOS1, AP1S2, ARAR, ARHGEF9, ARX, ATP7A, ATRX, AVPR, KCOR, BRWD3, BTK, CASK, CD LG, CDKL5, CHM, CHRDL1, CLCN4, CLCN5, CNKSR2, COL4A5, CUL B, CYBB, DCX, DDX, 793, STRUL 3, STRUL 9, EBP, EDA, EFNB, 328; FANCB, FGD1, FLNA, FMR1, FRMD7, FTSJ1, GDI1, GK, GLA, GPC3, GRIA3, HCCS, HDAC8, HPRT1, IDS, IKBKG, IL1RAPL1, IQSEC2, KDM5C, KDM6A, L CAM, LAMP2, MAGT1, MID1, MTM1, NDP, NHS, NR0B1, NSDHL, NYX, OCRL, OFD1, OPHN1, PAK3, PCDH19, PDHA1, PGK1, PHF6, PIGA, PLP1, PORCN, PQBP1, PRPS1, PTCHD1, RAB39 NSDHL, NYX, OCRL, OFD 2, RPS6KA3, RS1, SH2D1 NSDHL, NYX, OCRL, OFD A2, SLC35A2, SLC6A8, SLC9A6, SMC1 NSDHL, NYX, OCRL, OFD1, TBX22, TIMM8 NSDHL, NYX, OCRL, OFD 2, AN7, UBE2 NSDHL, NYX, OCRL, OFD 3 NSDHL, NYX, OCRL, OFD, 5245, NSDHL, NYX, OCRL, OFD H2, ZHC 9, ZIC3, SHSRS 3, ZSRY, TSPbY;

The special_gene probe set covers 37083 single nucleotide variation sites of 190 important birth defect genetic diseases, the total number of corresponding probes is 330272, and 9 repeated probes are designed for each single nucleotide variation site on average;

the 90 important birth defect genetic genes are ABCB1, ABCC8, ABCD1, ABCD4, ABCG5, ABCG8, ACADM, ACADS, ACADVL, ACAT, ACE, ACSF3, ACADM, ACADS, ACADVL, ACAT 2, APOA5, APOB, APOC3, AR, ARG1, ACADM, ACADS, ACADVL, ACAT1, ATP7 ACADM, ACADS, ACADVL, ACAT 7 ACADM, ACADS, ACADVL, ACAT1, ACADM, ACADS, ACADVL, ACAT 320, CLCN5, COCH, COL1A1, COL1A2, COL2A1, COMP, CPT1 ACADM, ACADS, ACADVL, ACAT 2, CRHR1, CYP11B1, CYP17A1, CYP1A1, CYP21A2, CYP2C19, CYP2D6, CYP3A5, ACADM, ACADS, ACADVL, ACAT1, DNAJC12, DRD2, ACADM, ACADS, ACADVL, ACAT1, EPC 1, ACADM, ACADS, ACADVL, ACAT, FGFR1, FGFR2, FGFR3, FKBP5, G6PC, G6 ACADM, ACADS, ACADVL, ACAT, 52JB 1, GCDH, GCH1, GCK, G2, G3, GJB 1, GLB1, DH, GLB 37, HAB 2, HAB 37, and combinations thereof HBA1, HBA2, 5237_5237 4 ACADM, ACADS, ACADVL, ACAT B2, HTR2 ACADM, ACADS, ACADVL, ACAT1, ACADM, ACADS, ACADVL, ACAT 5, IFNL4, IVD, KCNJ11, ACADM, ACADS, ACADVL, ACAT1, LPL, MC4 ACADM, ACADS, ACADVL, ACAT1, MCC 2, MCEE, MCOLN1, ACADM, ACADS, ACADVL, ACAT-CO 1, MT-RNR1, MT-TH, MT-TL1, MT-TS1, ACADM, ACADS, ACADVL, ACAT 15 ACADM, ACADS, ACADVL, ACAT 7 ACADM, ACADS, ACADVL, ACAT1, NPC2, NR0B1, NR5A1, ACADM, ACADS, ACADVL, ACAT 2, PHKB, PHKG2 PLA2G4 ACADM, ACADS, ACADVL, ACAT1, ACADM, ACADS, ACADVL, ACAT 16A1, SLC22A5, SLC25A13, SLC25A15, SLC25A20, SLC26A4, SLC34A3, SLC37A4, SLCO2B1, SMN1/SMN2, SMPD1, ACADM, ACADS, ACADVL, ACAT A2, ACADM, ACADS, ACADVL, ACAT1, TAT, TBX1, TECTA, TMC1, TPMT, TYRP1, UCP2, UGT1 ACADM, ACADS, ACADVL, ACAT A4, VKORC1, WFS1.

The specific_gene probe set gives consideration to the improvement of the probe distribution density of the corresponding gene region of the genome_frame work probe set.

Wherein, genome_frame work probe set is designed for gene Copy Number Variation (CNV), and the basic design principle is as follows:

(1) The whole genome coverage must be ensured to realize basic detection performance, so that skeleton probes are respectively designed for autosomes and sex chromosomes according to a certain interval, and the total number of probes is 642338;

(2) The unimportant genome region is not designed or is less designed with probes so as to reduce clinically-undefined gene Copy Number Variation (CNV) regions, which are mainly located in the head and tail end regions of each chromosome, the centromere region and other unrelated key gene regions;

(3) According to the genetic characteristics of each disease, the related diseases reported by gene Copy Number Variation (CNV) in single gene are highly encrypted, so that the average distribution density of probes is more than or equal to 15/5 Kb;

(4) According to the Clingen database and literature reports, known 350 pathogenic single dose deficiency (HI) genes and triple dose sensitivity (TS) genes are moderately encrypted, so that the average distribution density of probes in the areas is more than or equal to 1/5 Kb, namely the average distribution density of probes covered by the whole genome is exceeded.

The specific_gene probe set was designed for Single Nucleotide Variation (SNV), the basic design principle is as follows:

according to the literature report of a clinical laboratory, 190 genes are selected according to the principles of allele carrying rate, exon rate, morbidity, patient attack time, clinical preventive and treatable measures and the like aiming at the field of postnatal genetic disease prevention and treatment of birth defects, population frequency statistics and screening are carried out on related loci of the genes through a ClinVar, HGMD, gnomAD database and a local clinical laboratory, high-incidence SNV loci of Chinese population of each gene are confirmed, and in order to increase the result accuracy and positive detection rate of corresponding SNV loci, the probes are randomly placed at different positions of a chip, and the minimum 4 repeated probes of each SNV locus are ensured;

the nucleotide sequences of the genome_frame and specific_gene probe sets include the probe sequences shown in SEQ ID No.1 through SEQ ID No.720, as set forth in Table 5 below, but this partial sequence is only a small example.

TABLE 5 nucleotide sequences of partial genome_frame work probe set and specific_gene probe set

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The genome_frame probe set calculates the copy number CN for each probe for variation of 16 single genes, 350 pathogenic single dose deficiency (HI) genes, three times dose sensitivity (TS) genes; the normal probe copy number CN1 of autosomes and female X chromosomes is 2, the variation condition of the probe copy number CN1 is calculated to be 2, wherein the probe copy number CN1 is 0 or 1, the deletion is 3 or 4, and the repetition is calculated; the normal probe copy number CN2 of male X and Y chromosomes is 1, the number of the variation of the probe copy number CN2 is calculated to be 2, wherein the probe copy number CN2 is 0 and belongs to deletion, and the probe copy number CN2 is 2 and belongs to repetition; when the probe copy number CN of 50 continuous probes is abnormal, judging that the gene copy number is changed;

The calculated copy number metric value is log2 Ratio, where Ratio = observed value per probe copy number +.a reference value per probe copy number, a reference value per probe copy number is a set of data from a normal population:

cn=0 when log2 Ratio is approximately-1.5±0.05;

cn=1 when log2 Ratio is approximately-1±0.05;

cn=2 when log2 Ratio is approximately 0±0.05;

cn=3 when log2 Ratio is approximately 0.58±0.05;

cn=4 when log2 Ratio is approximately 1±0.05.

The specific_gene probe set detects corresponding SNV loci of 190 important birth defect genetic genes, and the result interpretation is realized based on the detection value of the part of probes by using a Genotype_SNV and ES clustering algorithm.

The Genotype_SNV method is realized by carrying out bicolor fluorescent labeling on an SNV array probe, wherein the probe can obtain two fluorescence detection values A and B, and a genotyping result is obtained by calculating the A and B values, and the calculation formula is as follows:

t(snv)＝(A-B)/(A+B)

when t (snv) is approximately equal to 1+/-0.05, the typing result is AA;

when t (snv) is approximately equal to-1+/-0.05, the parting result is BB;

when t (snv) is approximately equal to 0+/-0.05, the typing result is AB;

genotyping results included three, AA, AB and BB, where AA and BB represent pure and types, one wild type, one homozygote, and AB represents heterozygote, as judged by clustering results for wild type and homozygote.

The ES clustering algorithm clusters the genotyping results, and the calculation formula is as follows:

t(x)＝log2(A/B)；

t(y)＝[log2(A)+log2(B)]/2；

the ES clustering algorithm clusters by taking t (x) as a horizontal axis and t (y) as a vertical axis, and the positivity or negativity of the detection result is judged according to the sample typing result and the clustering number, as shown in fig. 4-1 and fig. 4-2, the detection result belongs to single nucleotide variation positivity, as shown in fig. 5-1 and fig. 5-2, and the detection result belongs to single nucleotide variation negativity.

The components of the chip also comprise denaturation mixed solution, amplification mixed solution, fragmentation mixed solution, precipitation mixed solution and hybridization mixed solution.

The denatured mixed solution was 10 Xdenatured solution diluted 10-fold with pure water.

The amplification mixed solution is prepared by mixing 1 volume of amplification enzyme and 45 volumes of amplification solution.

The fragmentation mixture was prepared by mixing 1 volume of the fragmenting enzyme, 10.3 volumes of the fragmenting dilution, and 45.7 volumes of 10 Xfragmenting buffer.

The precipitation mixture is prepared by mixing 1 volume of precipitation solution 2 with 119 volumes of precipitation solution 1.

The hybridization mixture was prepared by mixing 1 volume of hybridization solution 1, 18 volumes of hybridization solution 2 and 140 volumes of hybridization buffer.

Chip onDetection in microarray scanner >The matched reagent of the microarray scanner comprises a dye mixture A, a dye mixture B, a stabilizing mixture, a connecting mixture A and a connecting mixture B.

Dye mixture A was prepared by mixing 1 volume of dye solution 1-A, 1 volume of dye solution 1-B, 2 volumes of dye buffer and 96 volumes of wash solution A.

Dye mixture B was prepared by mixing 1 volume of dye solution 2-A, 1 volume of dye solution 2-B, 2 volumes of dye buffer and 96 volumes of wash solution A.

The stabilizing mixture was prepared by diluting 1 volume of stabilizing solution and 8 volumes of fixed diluent with pure water 7.9 times.

Ligation mixture A was prepared by mixing 1 volume of ligation solution 1, 0.4 volume of ligation solution 2, and 5 volumes of ligation buffer.

Ligation mixture B was prepared by mixing 1 volume of ligase, 6.6 volumes of probe mix 1, 6.6 volumes of probe mix 2 and 52 volumes of ligation mixture A.

The reagents used in the kit may be selected from the following products (available from Jiangsu Yuanlong medical science, inc.), as shown in Table 6 below:

TABLE 6 reagent names and goods numbers

Example 2

One method for synchronously detecting a plurality of birth defect genetic diseases CNV and SNV in this embodiment includes the steps of, as shown in FIG. 1,

S1: collecting a sample: a total of 96 samples, 54 men and 42 women, of which 48 were already validated patients with CNV and 48 were already validated patients with SNV, as positive validation samples of the invention.

S2: sample preparation and quality control, verification of sample DNA extraction, quality control by gel electrophoresis, nanodrop and the like, and ensuring that each DNA sample has no degradation, no impurity pollution and high purity. Samples which have Optical Density (OD) 260/280nm ratio of 1.8-2.0 and OD 260/230nm ratio of 1.5-2.0 and do not meet any condition need to be purified and the like; wherein the gel loading dye is Invitrogen ^TM Trackit Cyan/Orange Loading Buffer (Invitrogen P/N10482-028); 25bp Invitrogen Ladder (Invitrogen P/N10488-022); the Gel electrophoresis system is Invitrogen E-Gel ^TM 48agarose gels 4％，G8008-04。

S3: detection reaction:

s31, DNA amplification: 100ng of DNA sample (5 ng/. Mu.L) was taken, 2. Mu.l of 10 Xdenaturing mixture and 18. Mu.l of pure water were added and incubated at room temperature for 10min; adding 130 μl of the neutralization solution, adding 225 μl of the amplification mixed solution and 5 μl of the amplification enzyme after mixing, incubating at 37deg.C for 23+ -1 h, transferring to 65deg.C for 20min, and finally continuously incubating 45min100ng DNA sample (5 ng/. Mu.L) at 37deg.C, adding 20 μl of the denaturation mixed solution, and incubating at room temperature for 10min; adding 130 μl of the neutralization solution, shaking and mixing, adding 230 μl of the amplification mixed solution, shaking and mixing, and centrifuging at 1000rpm for 1min; incubating for 23+/-1 h in an incubator at 37 ℃; incubating for 20min in an incubator at 65 ℃; incubating in an incubator at 37 ℃ for 45min;

S32, DNA fragmentation and precipitation: adding 45.7 mu l of 10 Xfragmentation buffer, 10.3 mu l of fragmentation diluent and 1.0 mu l of fragmentation enzyme into the sample incubated in the step S31, uniformly mixing, briefly centrifuging, incubating at 37 ℃ for 30min, adding 19 mu l of stop solution at room temperature, vibrating, uniformly mixing and centrifuging; adding 238 mu l of precipitation solution 1, 2 mu l of precipitation solution 2 and 600 mu l of isopropanol at room temperature, fully and uniformly mixing the materials up and down by a pipette, freezing the mixture at-20 ℃ for 16-24 hours, adding 57 mu l of fragmented mixed solution, uniformly mixing, and carrying out short centrifugation; incubating for 30min at 37 ℃, adding 19 μl of stop solution at room temperature, shaking, mixing, and centrifuging; adding 240 mu l of precipitation mixed solution and 600 mu l of isopropanol at room temperature, and fully and uniformly mixing; placing for 16-24 h at the temperature of minus 20 ℃;

s33, drying, resuspension and quality control: centrifuging the sample treated in the step S32 at 3200rpm at 4 ℃ for 40min, pouring out the waste liquid, then placing the waste liquid at 37 ℃ for airing for 20min, adding 35 mu l of heavy suspension buffer solution, shaking and mixing uniformly, shaking for 10min, adding 70.5 mu l of hybridization buffer solution, 0.5 mu l of hybridization solution 1 and 9 mu l of hybridization solution 2, shaking and mixing uniformly, and centrifuging to obtain 115 mu l of modified hybridization solution; centrifuging at 3200rpm at 4deg.C for 40min, removing waste liquid, air drying in a hybridization oven at 37deg.C for 20min, adding 35 μl of heavy suspension buffer, shaking for 10min, adding 80 μl of hybridization mixture, shaking for mixing, and centrifuging to obtain 115 μl of modified hybridization solution;

Quality control using 10. Mu.l of the denatured hybridization solution, including absorbance measurement with Nanodrop and gel electrophoresis, wherein the gel-loading dye is Invitrogen ^TM Trackit Cyan/Orange Loading Buffer;25bp Invitrogen Ladder; the Gel electrophoresis system is Invitrogen E-Gel ^TM 48agarose gels 4％，G8008-04；

S34, denaturation and hybridization: the denatured hybridization solution produced in step S33 is placed on a hybridization plate, and then placed in a PCR instrument, and a denaturation program (modification program) is started: denaturation at 95℃for 10min and denaturation at 48℃for 3min, cooling. The remaining 105. Mu.l of denatured hybridization solution was carefully transferred to a hybridization plate with a pipette and loadedThe microarray chip scanner, continue hybridizing for 23.5 h-24 h, put the said hybridization plate into PCR instrument to denature the procedure: denaturation at 95℃for 10min; denaturation at 48℃for 3min; cooling; transfer 105. Mu.l of denatured hybridization solution to hybridization plate using pipettor, load +.>The microarray chip processor continuously hybridizes for 23.5-24 hours;

s35, detecting by a chip scanner: performing dye-disc corresponding split charging on the mixture generated in the step S34: wherein 2 dye trays of dye mixture A, wherein 100.8. Mu.l of wash solution A, 2.1. Mu.l of staining buffer, 1.05. Mu.l of staining solution 1-A and 1.05. Mu.l of staining solution 1-B were prepared in advance; 1 dye tray of dye mixture B, in which 100.8. Mu.l of wash solution A, 2.1. Mu.l of staining buffer, 1.05. Mu.l of staining solution 2-A and 1.05. Mu.l of staining solution 2-B were prepared in advance; 1 dye tray for stabilizing mixture, wherein 93.2. Mu.l of pure water, 10.5. Mu.l of fixed dilution and 1.3. Mu.l of stabilizing solution were prepared in advance; 1 dye disc of ligation mixture, wherein 66.2. Mu.l ligation buffer, 13.1. Mu.l ligation buffer 1, 3.1. Mu.l ligation buffer 2, 10.5. Mu.l probe mix 1, 10.5. Mu.l probe mix 2 and 1.6. Mu.l of ligase currently withdrawn at-20℃were prepared in advance; and then directly loading the sample into a chip scanner, and simultaneously adding 150 mu l of fixed buffer solution into each hole of each scanning disk to avoid contacting the bottom of the scanning disk. Aligning the notch angle of the cover with the notch edge of the scanning disc, covering the scanning disc, then placing the scanning disc on the top of the workbench, and finally starting a scanning detection process, wherein 2 dye discs of dye mixture A are arranged, and 105 mu l of each hole is formed; 1 dye disc of dye mixture B, 105 μl per well; 1 dye disc of the stable mixture, 105 μl per well; 1 dye disc of mixture B was attached, 105 μl per well; loading the split-packed dye discs into a chip scanner, simultaneously adding 150 mu l of fixed buffer solution into each hole of the dye discs, covering the dye discs, and placing the dye discs on the top of a workbench for detection;

S4: data quality control and analysis: as shown in fig. 2, after the imaging is completed, the original fluorescence signal data of each hybridization plate is obtained, and quality control and analysis are performed on the original fluorescence signal data by using automatic analysis software, wherein the quality control and analysis comprise the processes of whole plate quality control, sample quality control, probe sequence identification, sample comparison, bioinformatics analysis and the like, so that a mutation site information result file of the corresponding sample can be obtained.

S5: result analysis and annotation: the results contain copy number variation information and gene mutation sites detected by each sample, and the information is subjected to further annotation analysis, including mutation rating and medical interpretation, so as to obtain clear and real site information finally.

Sample site information for this gene Copy Number Variation (CNV) validation is shown in table 7 below; the results of the gene Copy Number Variation (CNV) analysis are shown in Table 8 below.

TABLE 7 verification of sample site information for gene Copy Number Variation (CNV)

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TABLE 8 analysis of Copy Number Variation (CNV) of genes

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Note that: NA indicates no result, noise indicates that the density map for judging the authenticity of CNV has a disordered signal, and cannot be judged. The measured size refers to the range of CNV lengths that the software analyzed.

The invention detects 48 positive CNV samples and 62 CNV samples, 60 CNV samples are detected, and the CNV detection rate is 96.8%. Further analysis revealed that CNV-42 samples showed a Noise density pattern, as shown in FIG. 3-1 and FIG. 3-2, CNV-47 detected CNV with a probe number less than 50 (threshold 50), CNV-48 samples failed QC (possibly the problem of experiment or sample itself), and CNV of the remaining 45 positive samples was effectively detected, and the CNV positive verification rate was 93.75%.

Sample site information for this Single Nucleotide Variation (SNV) validation is shown in table 9 below; the Single Nucleotide Variation (SNV) analysis results are shown in table 10 below.

Table 9 Single Nucleotide Variation (SNV) validation of sample site information

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TABLE 10 Single Nucleotide Variation (SNV) analysis results

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Note that: normal indicates that the SNV site is not detected as a mutation; affilected indicates that heterozygous or homozygous mutation was detected and consistent with the patient genotype; carrier means Carrier; not designed means that no probes are designed on the chip.

According to the invention, verification tests are carried out on 48 cases of positive SNV loci, and the result shows that CNV-09 loci are not provided with probes, other sample loci are detected, the SNV detection rate reaches 97.9%, as shown in fig. 4-1, 1 sample is interpreted as AB (heterozygote), the carrier is considered, the other 94 samples are BB, and the wild type sample is considered; as shown in fig. 4-2, 1 sample was interpreted as BB (homozygote), considered as a carrier, and the remaining 84 samples were all BB, considered as wild type. Further analyzing the accuracy of the results, finding that the results of the detection of the CNV-19 and CNV-30 samples by the kit are homozygous and the interpretation result is Normal, namely that the corresponding mutation is not detected, and the three samples are all heterozygous mutations and are corresponding patients with diseases, so that the two samples can be considered to fail to verify. The results of the detection of the CNV-32 and CNV-38 samples by the kit provided by the invention are all homozygous and the interpretation result is marked, which indicates that homozygous mutation is detected instead of heterozygous mutation patients provided by the original results. Here, both the original results were considered to be correct, and CNV-32 and CNV-38 sample results were also considered to be verification failures, so the SNV positive verification rate was 91.5%.

In conclusion, the product provided by the invention verifies 96 positive samples, including 48 CNV samples and 48 SNV samples, wherein the CNV detection rate is 96.8% and the positive rate is 93.75%. The SNV detection rate is 97.9% and the positive rate is 91.5%. The probes designed by the product can synchronously detect the CNV and SNV related to the birth defect genetic disease genes, fully illustrates the advantages of the chip, and mainly comprises the following steps:

(1) Clinical versatility: the CNV and SNV related to the birth defect genetic disease genes are synchronously detected, the operation is simple, the diagnosis is efficient, the CNV and 190 single gene diseases in the whole genome range can be detected by only one experiment, and the method is suitable for most clinical genetic detection.

(2) Effectiveness is as follows: a brand new probe design method is adopted, and the traditional scheme is to design skeleton probes to realize whole genome coverage. The scheme reduces or removes probes of unimportant regions such as the head region, the tail region, the centromere region, other non-gene regions and the like of each chromosome on the basis of skeleton probes, encrypts 350 pathogenicity single dose deficiency (HI) genes and three times dose sensitivity (TS) genes which are disclosed, reduces the detection of CNV with unknown clinical significance, and simultaneously remarkably increases the detection of the pathogenicity CNV. In particular, 16 single-gene internal CNV regions are highly encrypted, so that the detection rate of the CNV of the small fragments in the single gene is ensured, and the effectiveness of the product can be ensured to the greatest extent.

(3) Creative: by adding 190 single-gene disease-related SNVs, 37083 in total, and combining an improved genotyping algorithm and a clustering method, the detection point mutation and InDel of a full-chromosome genotyping chip are creatively realized, and the SNV-array technology is derived.

It should also be noted that the above-listed embodiment is only one specific embodiment of the present invention, and only some of the identified CNV patients and SNV patients are selected, and more CNVs and 37083 SNVs can be detected. Other persons skilled in the art can use one experiment to synchronously detect CNV at the whole genome level and 37083 SNVs described above from the disclosure of the present invention, but such design concept should be considered as the scope of the present invention. The present invention is not limited to the above-described embodiments and examples, and various changes may be made within the knowledge of those skilled in the art without departing from the spirit of the present invention.

Claims (7)

1. A whole chromosome genotyping chip for synchronously detecting a plurality of birth defect genetic diseases caused by gene copy number variation and single nucleotide variation, characterized in that:

the chip comprises a genome_frame work probe set and a specific_gene probe set;

The genome_frame probe group covers the whole genome of the chromosome, the total number of probes is 643228, and the average distribution density of the probes is 1/5 Kb;

the genome_frame work probe set includes a backbone probe designed for an autosome and for a sex chromosome; the backbone probe reduces/removes the head and tail end regions of each chromosome, centromere region and other non-genetic region probes;

the genome_frame work probe set comprises probes designed for the gene copy number variation region of 16 single gene levels, and the average distribution density of the probes is more than or equal to 15/5 Kb; the 16 monogenes are VHL, QDPR, SMN1, CYP21A2, HBB, PTS, PAH, ATP7B, GCH1, HBA2, NF1, PHEX, DMD, OTC and MECP2;

the genome_frame work probe set comprises probes designed for the gene copy number variation regions of pathogenic single-dose deficiency genes and triple-dose sensitive genes, and the average distribution density of the probes is more than or equal to 1/5 Kb; the pathogenic single dose deficiency gene and three times dose sensitivity gene are 350, AHDC1, ARID 11 1, 26, LHX4, 1, 3B4, SLC2A1, ZBTB18, BAG3, BMPR 12, GATA3, KAT6 11, PAX2, PTEN, RPS24, WAC, ZMYND11, ALX4, ARCN1, ATM, CDKN 12, FZD4, HMBS, KCNQ1OT1, KMT2 5, MEN1, MYBPC3, PAX6, PHF21 2, SDHD, SHANK2, WT1, ACL 1, ARID2, CDKN 12, KMT2 3, MED13, RPS26, SLC17A8, SOX5, TBX3, BRCA2, CHAMP1, ZIC2, CHAMP1, CHAID 1, CHARQ 1, CHARC 2, CHARQ 1, CHARC 1, ZYK 1, CHARC, ZYK 1, ZYMX, ZYX, ZX, ZMX, ZYX TBX, ZYX, ZYMX, ZYX, CHJYBYBYBYBY, BMP4, CHD8, DICER1, FOXG1, GPHN, PAX9, AAGAB, CHD2, FBN1, IGF 12, RPS17, SIN3 3, SPRED1, TCF12, UBE3 11, CDH1, 2, FOXF1, PKD1, SALL1, SETD 1B1, TSC2, AXIN2, BRCA1, BRIP1, COL1A1, EFTUD2, FLCN, HNF1, PAFAH1B1, PMP22, RAD51 1, RNF135, TBX4, TP53, ASXL3, DSC2, GATA6, SETBP1, SMAD4, TCF4, TGIF1, CACNA 12 19, SMARCA4 BMP4, CHD8, DICER1, FOXG1, GPHN, PAX9, AAGAB, CHD2, FBN1, IGF 12, RPS17, SIN3 3, SPRED1, TCF12, UBE3 11, CDH1, 2, FOXF1, PKD1, SALL1, SETD 1B1, TSC2, AXIN2, shi BRCA1, BRIP1, COL1A1, EFTUD2, FLCN, HNF1, PAFAH1B1, PMP22, RAD51 1, RNF135, TBX4, TP53, ASXL3, DSC2, GATA6, SETBP1, SMAD4, TCF4, TGIF1, CACNA 12 19, SMARCA4, SMART 4, NANO 2, NANO 1, NANO 2, NANO 1, NAC 4, and NAC 4, LAC 1, LAG 1, and/4, and/1, TGFBR1, TSC1, ZNF462, ABCD1, ACSL4, AFF2, ANOS1, AP1S2, AR, ARHGEF9, ARX, ATP7A, ATRX, AVPR2, BCOR, BRWD3, BTK, CASK, CD LG, CDKL5, CHM, CHRDL1, CLCN4, CLCN5, CNKSR2, COL4A5, CUL4B, CYBB, DCX, DDX3X, DLG3, EBP, EDA, EFNB1, F8, F9, FANCB, FGD1, FLNA, FMR1, FRRAPD 7, FTSJ1, GDI1, GK, GLA, GPC, GRIA3, HCCS, HDAC8, HPRT1, IDS, IKBKG, IL 1L 1, IQSEC2, KDM5C, KDM and A, L CAM 1 LAMP2, MAGT1, MID1, MTM1, NDP, NHS, NR B1, NSDHL, NYX, OCRL, OFD, OPHN1, PAK3, PCDH19, PDHA1, PGK1, PHF6, PIGA, PLP1, PORCN, PQBP1, PRPS1, PTCHD1, RAB39B, RP2, RPS6KA3, RS1, SH2D1A, SLC A2, SLC35A2, SLC6A8, SLC9A6, SMC1A, SMS, STS, SYN1, TBX22, TIMM8A, TRAPPC2, TSPAN7, UBE2A, UPF B, USP9X, WDR45, XIAP, XIST, ZC H2, zdhc 9, ZIC3, ZNF711, SHOX and SRY;

The special_gene probe set covers 37083 single nucleotide variation sites of 190 important birth defect genetic genes, the total number of corresponding probes is 330272, and 9 repeated probes are designed for each single nucleotide variation site on average;

the 190 important birth defect genetic disease genes are ABCB1, ABCC8, ABCD1, ABCD4, ABCG5, ABCG8, ACADM, ACADS, ACADVL, ACAT1, ACE, ACSF3, ACADM, ACADS, ACADVL, ACAT 2, APOA5, APOB, APOC3, AR, ARG1, ACADM, ACADS, ACADVL, ACAT1, ATP7 ACADM, ACADS, ACADVL, ACAT 7 ACADM, ACADS, ACADVL, ACAT, ACADM, ACADS, ACADVL, ACAT 320, CLCN5, COCH, COL1A1, COL1A2, COL2A1, COMP, CPS1, CPT1 ACADM, ACADS, ACADVL, ACAT 2, CRHR1, CYP11B1, CYP17A1, CYP1A1, CYP21A2, CYP2C19, CYP2D6, CYP3A5, ACADM, ACADS, ACADVL, ACAT1, DNAJC12, DRD2, ACADM, ACADS, ACADVL, ACAT, EPHX1, ACADM, ACADS, ACADVL, ACAT, FGFR1, FGFR2, FGFR3, FKBP5, G6PC, G6 ACADM, ACADS, ACADVL, ACAT, GCDH, GCH1, GCK, G2, GB 3, GLB1, GLDH, GLB1, HA37, HADH HBA1, HBA2, 5237_5237 4 ACADM, ACADS, ACADVL, ACAT B2, HTR2 ACADM, ACADS, ACADVL, ACAT1, ACADM, ACADS, ACADVL, ACAT 5, IFNL4, IVD, KCNJ11, ACADM, ACADS, ACADVL, ACAT1, LPL, MC4 ACADM, ACADS, ACADVL, ACAT1, MCC 2, MCEE, MCOLN1, ACADM, ACADS, ACADVL, ACAT-CO 1, MT-RNR1, MT-TH, MT-TL1, MT-TS1, ACADM, ACADS, ACADVL, ACAT 15 ACADM, ACADS, ACADVL, ACAT 7 ACADM, ACADS, ACADVL, ACAT1, NPC2, NR0B1, NR5A1, ACADM, ACADS, ACADVL, ACAT 2, PHKB, PHKG2 PLA2G4 ACADM, ACADS, ACADVL, ACAT1, ACADM, ACADS, ACADVL, ACAT 16A1, SLC22A5, SLC25A13, SLC25A15, SLC25A20, SLC26A4, SLC34A3, SLC37A4, SLCO2B1, SMN1/SMN2, SMPD1, ACADM, ACADS, ACADVL, ACAT A2, ACADM, ACADS, ACADVL, ACAT1, TAT, TBX1, TECTA, TMC1, TPMT, TYRP1, UCP2, UGT1 ACADM, ACADS, ACADVL, ACAT A4, VKORC1 and WFS1;

The nucleotide sequences of the genome_frame work probe set and the specific_gene probe set comprise probe sequences shown as SEQ ID No. 1-SEQ ID No. 720.

2. The chip of claim 1, wherein:

the specific_gene probe set gives consideration to improving the probe distribution density of the corresponding gene region of the genome_frame work probe set.

3. The chip of claim 1, wherein:

the genome_frame work probe set calculates the copy number CN of each probe for variations of the 16 single genes, the pathogenic single dose deficiency genes, and the triple dose sensitive genes; the autosomal and female X chromosomes are normal, the probe copy number CN1 is 2, the variation condition of the probe copy number CN1 is calculated to be 2, wherein the probe copy number CN1 is 0 or 1 belongs to deletion, and 3 or 4 belongs to repetition; the normal probe copy number CN2 of male X and Y chromosomes is 1, the number of the probe copy number CN2 variation is calculated to be 2, wherein the probe copy number CN2 is 0 and belongs to deletion, and the probe copy number CN2 is 2 and belongs to repetition; when the probe copy number CN abnormal condition of 50 continuous probes occurs, judging that the gene copy number is variant;

The calculated copy number metric value is log2 Ratio, where Ratio = observed value per probe copy number +.a reference value per probe copy number, a reference value per probe copy number is a set of data from a normal population:

cn=0 when log2 Ratio is approximately-1.5±0.05;

cn=1 when log2 Ratio is approximately-1±0.05;

cn=2 when log2 Ratio is approximately 0±0.05;

cn=3 when log2 Ratio is approximately 0.58±0.05;

cn=4 when log2 Ratio is approximately 1±0.05.

4. The chip of claim 1, wherein:

the specific_gene probe set detects corresponding SNV loci of the 190 important birth defect genetic diseases genes, the result interpretation is realized based on the detection value of the part of probes, and the adopted method is a Genotype_SNV and ES clustering algorithm;

the Genotype_SNV method is realized by carrying out bicolor fluorescent labeling on an SNV array probe, wherein the probe can obtain two fluorescence detection values A and B, and a genotyping result is obtained by calculating the A and B values, and the calculation formula is as follows:

t(snv)=(A-B)/(A+B)

when t (snv) is approximately equal to 1+/-0.05, the typing result is AA;

when t (snv) is approximately equal to-1+/-0.05, the parting result is BB;

when t (snv) is approximately equal to 0+/-0.05, the typing result is AB;

the genotyping results comprise three types, AA, AB and BB, wherein the AA and BB represent pure types, one represents wild types, one represents homozygotes, the wild types and homozygotes are judged according to the clustering results, and the AB represents heterozygotes;

The ES clustering algorithm is used for clustering the genotyping result, and the calculation formula is as follows:

t(x)=log2(A/B)；

t(y)=[log2(A)+log2(B)]/2；

and clustering by using t (x) as a horizontal axis and t (y) as a vertical axis in the ES clustering algorithm, and judging the positive or negative of the detection result according to the sample typing result and the clustering number.

5. The chip of claim 1, wherein:

the chip further comprises a denaturation mixture, an amplification mixture, a fragmentation mixture, a precipitation mixture and a hybridization mixture.

6. The chip of claim 1, wherein:

the chip is detected and used in a CNVPLUS micro-array chip scanner.

7. Use of the chip of claim 1 for the preparation of a product for simultaneous detection of a plurality of birth-defect genetic diseases CNV and SNV.