Method and device for detecting fetal triploid based on CNVSeq

Abstract

The invention provides a method and a device for detecting a fetal triploid based on CNVSeq. The method comprises the following steps: respectively preprocessing CNVSeq sequencing data of a negative sample and a sample to be detected to obtain first preprocessed data and second preprocessed data; respectively calculating the autosomal fetal concentration FFsnps and the sex chromosome fetal concentration FFsex of the negative sample and the to-be-detected sample and the fetal chromosome average copy number of the to-be-detected sample by using the first preprocessed data and the second preprocessed data; judging whether the to-be-detected sample is a triploid or not according to the first autosomal fetal concentration FFsnps, the first sex chromosome fetal concentration FFsex and the average copy number of fetal chromosomes; wherein the sample to be detected is a prenatal diagnosis sample. The result obtained by judging according to the parameters is more accurate, misjudgment possibly caused by a single index is avoided, the detection cost can be reduced, and the experimental period of triploid detection can be shortened.

Description

Technical Field

[0001] The present invention relates to the technical field of chromosome detection, and in particular to a method and device for detecting fetal triploidy based on CNVSeq. Background Art

[0002] Triploidy is a rare chromosomal abnormality. Triploidy means that each chromosome has an extra copy, which causes the normal 46 chromosomes in humans to change to 69. Triploidy has an extra set of haploid chromosomes compared to normal diploidy, and has three sex chromosomes, which usually leads to early pregnancy miscarriage. Triploidy syndrome is one of the common chromosomal abnormalities in human pregnancy and the most common polyploidy in prenatal diagnosis. Triploidy accounts for 1-3% of all human pregnancies and 7-10% of spontaneous abortions in early pregnancy. 99% of triploid fetuses cannot survive, and most of them miscarry at 10-20 weeks of gestation.

[0003] There are three types of triploid karyotypes, namely 69XXY, 69XXX, and 69XYY, accounting for 60%, 37%, and 3% respectively. The mechanisms of triploidy mainly include diandrogenic fertilization and difemale fertilization. If the extra set of chromosomes comes from the mother, it is called diandrogenic triploidy, which is often caused by the fertilization of a diploid egg (2n) with a haploid sperm (n). If the extra set of chromosomes comes from the father, it is called diandrogenic triploidy, which is often caused by the fertilization of two haploid sperm (n+n) with a haploid egg (n); in a few cases, it is also seen in the fertilization of a diploid sperm (2n) with a haploid egg (n). Given that this phenomenon is quite common in preimplantation embryos, pregnancies, and miscarriages, and is unfavorable to obstetric outcomes, it is of clinical significance to develop technologies that can detect this abnormality and potentially avoid pregnancy with a triploid karyotype.

[0004] There are many technologies for detecting chromosomal abnormalities, such as karyotype analysis, fluorescence in situ hybridization (FISH), and chromosome microarray analysis (CMA). Karyotype analysis can be used to detect polyploidy, chromosomal aneuploidy, and large-segment copy number variation, but the resolution of copy number variation is low, usually ≥10Mb. Fluorescence in situ hybridization (FISH) can verify the location of chromosomal abnormalities found by CMA and CNVSeq, but cannot detect new chromosomal abnormalities. The CMA method can be used for polyploidy, chromosomal aneuploidy, and copy number variation, but the throughput is low and the detection cost is high. CNVSeq technology based on low-depth whole genome sequencing has been widely used in prenatal diagnosis due to its high throughput and low cost, but it is usually used to detect chromosomal aneuploidy and copy number variation, and STR methods are used to assist in judging polyploidy, maternal contamination, etc. However, since STR technology requires additional experimental processes and a long experimental cycle, it increases the cost of clinical testing and requires a higher sample volume. Therefore, how to detect chromosomal abnormalities more efficiently while CNVSeq is being tested is of great significance for clinical applications. Summary of the invention

[0005] The main purpose of the present invention is to provide a method and device for detecting fetal triploidy based on CNVSeq, so as to solve the problem of low detection efficiency of chromosomal abnormalities in low-depth whole genome sequencing in the prior art.

[0006] In order to achieve the above object, according to a first aspect of the present invention, a method for detecting fetal triploidy based on CNVSeq is provided, the method comprising: preprocessing CNVSeq sequencing data of a negative sample and a sample to be tested, respectively, to obtain first preprocessed data and second preprocessed data; using the first preprocessed data and the second preprocessed data, respectively, to calculate the autosomal fetal concentration FF of the negative sample and the sample to be tested. snps and sex chromosome fetal concentration FF sex , record the autosomal fetal concentration FF of the sample to be tested snps FF is the concentration of the first autosome snps , the fetal sex chromosome concentration of the sample to be tested is FF sex FF is the concentration of the first sex chromosome in the fetus sex ; Calculate the average copy number of fetal chromosomes of the sample to be tested using the first preprocessed data and the second preprocessed data; Use the first autosome fetal concentration FF snps , fetal concentration of first sex chromosome FF sex and the average copy number of fetal chromosomes to determine whether the sample to be tested is triploid; wherein the sample to be tested is a prenatal diagnosis sample.

[0007] Furthermore, the autosomal fetal concentration FF was calculated snpsIncluding: According to the genotypes of the mother and fetus in the sample, the frequency of allele A in the CNVSeq sequencing data is used to calculate the autosomal fetal concentration FF as follows snps ,

[0008]

[0009] Wherein, A and B in the mother's genotype + fetus' genotype represent two alleles at one SNP site, respectively, and e represents the sequencing error rate; preferably, the sex chromosome fetal concentration FF is calculated. sex Includes: BinX the sequence number of all windows in chromosomes X and Y in the sample readcount and BinY readcount Substituting into formula Ⅰ, we can calculate BinY dosage and BinX dosage , Where N is X or Y; BinY dosage and BinX dosage Substituting into formula II, we can calculate chrY dosage and chrY dosage ;chrN dosage =Mean(BinN dosage ) Formula II, wherein N is X or Y; chrY dosage and chrY dosage Substitute into formula III to calculate the sex chromosome fetal concentration FF sex ; FF sex =2*chY dosage / (chrX dosage +chrY dosage )Formula III.

[0010] Furthermore, calculating the average copy number of fetal chromosomes in the sample to be tested includes: using the first preprocessing data to calculate the average sequence number of each window in the negative sample, recorded as the first average value u; calculating the difference multiple f between the negative sample and the sample to be tested, the sequence number of a single window in the sample to be tested in the second preprocessing data is M, f=M / u; using the difference multiple f to calculate the average chromosome copy number of the autosomes and sex chromosomes in the sample to be tested.

[0011] Further, calculating the average chromosome copy number of the autosomes and sex chromosomes in the sample to be tested using the difference multiple f includes: calculating the chromosome copy number of a single window in the sample to be tested using the difference multiple f, wherein the chromosome copy number of the single window of the autosome and the X chromosome of the female fetus is 2f; the chromosome copy number of the single window of the X chromosome and the Y chromosome of the male fetus is f; fitting the chromosome copy number of different single windows on the chromosome using a trend fitting algorithm to obtain a chromosome copy number fitting curve, and calculating the average chromosome copy number of the autosomes and sex chromosomes in the sample to be tested.

[0012] Furthermore, the criteria for determining whether the sample to be tested is triploid include: comparing the autosomal fetal concentrations of the negative sample and the sample to be tested with the FF snps and sex chromosome fetal concentration FF sex Linear regression fitting was performed to obtain the fetal concentration fitting curve; if the first autosomal fetal concentration FF snps >0 and the first sex chromosome fetal concentration FF sex If the average chromosome copy number of the Y chromosome is distributed between 2 / 3±0.1 or 4 / 3±0.1 of the horizontal coordinate in the fetal concentration fitting curve, the sample to be tested is triploid; preferably, the judgment criterion of whether the sample to be tested is triploid also includes: the average chromosome copy number of the Y chromosome is distributed between 2 / 3±0.1 or 4 / 3±0.1 of the horizontal coordinate in the chromosome copy number fitting curve, then the sample to be tested is triploid.

[0013] In order to achieve the above-mentioned object, according to a second aspect of the present invention, a device for detecting fetal triploidy based on CNVSeq is provided, the device comprising: a preprocessing module, configured to preprocess the CNVSeq sequencing data of a negative sample and a sample to be tested, respectively, to obtain first preprocessing data and second preprocessing data; a fetal concentration calculation module, configured to calculate the autosomal fetal concentration FF of the negative sample and the sample to be tested, respectively, using the first preprocessing data and the second preprocessing data. snps and sex chromosome fetal concentration FF sex , record the autosomal fetal concentration FF of the sample to be tested snps FF is the first autosomal fetal concentration snps , the fetal sex chromosome concentration of the sample to be tested is FF sex FF is the concentration of the first sex chromosome in the fetus sex The average fetal chromosome copy number calculation module is configured to calculate the average fetal chromosome copy number of the sample to be tested using the first preprocessing data and the second preprocessing data; the judgment module is configured to use the first autosome fetal concentration FF snps , fetal concentration of first sex chromosome FF sexand the average copy number of fetal chromosomes to determine whether the sample to be tested is triploid; wherein the sample to be tested is a prenatal diagnosis sample.

[0014] Furthermore, the fetal concentration calculation module includes: autosomal fetal concentration FF snps The calculation unit is set to calculate the autosomal fetal concentration FF based on the genotypes of the mother and fetus in the sample using the frequency of allele A in the CNVSeq sequencing data as follows: snps ,

[0015] The frequency of allele A in the mother's genotype + fetus' genotype sequencing data

[0016]

[0017] Wherein, A and B in the mother's genotype + fetus' genotype respectively represent two alleles at one SNP site, and e represents the sequencing error rate; preferably, the fetal concentration calculation module also includes: sex chromosome fetal concentration FF sex The calculation unit includes: a window sequence number calculation submodule, which is configured to calculate the sequence number BinX of all windows in the sample neutral chromosomes X and Y readcount and BinY readcount Substituting into formula Ⅰ, we can calculate BinY dosage and BinX dosage , Where N is X or Y; the chromosome dose calculation submodule is set to BinY dosage and BinX dosage Substituting into formula II, we can calculate chrY dosage and chrY dosage ;chrN dosage =Mean(BinN dosage ) Formula II, wherein N is X or Y; fetal concentration FF sex The calculation submodule is set to convert chrY dosage and chrY dosage Substitute into formula III to calculate the sex chromosome fetal concentration FF sex ; FF sex =2*chY dosage / (chrX dosage +chrY dosage )Formula III.

[0018] Furthermore, the module for calculating the average copy number of fetal chromosomes includes: a first average value calculation unit, which is configured to use the first preprocessing data to calculate the average sequence number of each window in the negative sample, recorded as the first average value u; a difference multiple calculation unit, which is configured to calculate the difference multiple f between the negative sample and the sample to be tested, and the sequence number of a single window in the sample to be tested in the second preprocessing data is M, f=M / u; the chromosome average copy number calculation unit is configured to use the difference multiple f to calculate the average chromosome copy number of the autosomes and sex chromosomes in the sample to be tested.

[0019] Further, the chromosome average copy number calculation unit includes: a single window calculation unit, which is configured to calculate the chromosome copy number of a single window in the sample to be tested using the difference multiple f, wherein the chromosome copy number of the single window of the autosome and the X chromosome of the female fetus is 2f; the chromosome copy number of the single window of the X chromosome and the Y chromosome of the male fetus is f; a chromosome copy number fitting unit, which is configured to fit the chromosome copy numbers of different single windows on the chromosome using a trend fitting algorithm to obtain a chromosome copy number fitting curve, and calculate the chromosome average copy number of the autosomes and sex chromosomes in the sample to be tested.

[0020] Furthermore, the judgment module includes: a fetal concentration fitting unit, which is configured to fit the autosomal fetal concentration FF of the negative sample and the sample to be tested. snps and sex chromosome fetal concentration FF sex Linear regression fitting is performed to obtain a fetal concentration fitting curve; the fetal concentration judgment unit is set to if the first autosomal fetal concentration FF snps >0 and the first sex chromosome fetal concentration FF sex If the average chromosome copy number of the Y chromosome is distributed between 2 / 3±0.1 or 4 / 3±0.1 of the horizontal coordinate in the fetal concentration fitting curve, then the sample to be tested is output as a triploid; preferably, the judgment module also includes: a chromosome copy number judgment unit, and the average chromosome copy number of the Y chromosome is distributed between 2 / 3±0.1 or 4 / 3±0.1 of the horizontal coordinate in the chromosome copy number fitting curve, then the sample to be tested is output as a triploid.

[0021] In order to achieve the above-mentioned purpose, according to the third aspect of the present invention, a computer-readable storage medium is provided, the storage medium includes a stored program, wherein when the program is running, the device where the storage medium is located is controlled to execute the above-mentioned CNVSeq-based method for detecting fetal triploidy.

[0022] In order to achieve the above objective, according to a fourth aspect of the present invention, a processor is provided, and the processor is used to run a program, wherein the program runs the above CNVSeq-based method for detecting fetal triploidy.

[0023] Applying the technical solution of the present invention, the autosomal fetal concentration FF of negative samples and samples to be tested is calculated using CNVSeq sequencing data. snps , sex chromosome fetal concentration FF sex and the average copy number of fetal chromosomes. The use of the above parameters to judge whether the fetus has triploidy is more accurate, avoiding the misjudgment that may be caused by a single indicator, and can reduce the detection cost and shorten the experimental cycle of triploidy detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The drawings constituting a part of the present application are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0025] Figure 1 The fetal concentration fitting curve in Example 1 of the present application is shown. DETAILED DESCRIPTION

[0026] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present invention will be described in detail below with reference to the accompanying drawings and in combination with the embodiments.

[0027] As mentioned in the background technology, the existing technology for detecting chromosomal abnormalities has a low resolution for copy number variation, usually ≥10Mb. And the CNVSeq technology based on low-depth whole genome sequencing requires the use of STR methods to assist in determining polyploidy. However, since STR technology requires additional experimental procedures, the experimental cycle is long, the cost of clinical testing is increased, and a higher sample volume requirement is required. Therefore, the present application intends to provide a method for fetal triploid detection based on CNVSeq sequencing data.

[0028] In a first typical embodiment of the present application, a method for detecting fetal triploidy based on CNVSeq is provided, the method comprising: preprocessing the CNVSeq sequencing data of the negative sample and the sample to be tested, respectively, to obtain first preprocessed data and second preprocessed data; using the first preprocessed data and the second preprocessed data to calculate the autosomal fetal concentration FF of the sample to be tested. snps and sex chromosome fetal concentration FF sex ; record the autosomal fetal concentration FF of the sample to be tested snps FF is the concentration of the first autosome snps , the fetal sex chromosome concentration of the sample to be tested is FF sex FF is the concentration of the first sex chromosome in the fetus sex ; Calculate the average copy number of fetal chromosomes of the sample to be tested using the first preprocessed data and the second preprocessed data; Use the first autosome fetal concentration FFsnps , fetal concentration of first sex chromosome FF sex and the average copy number of fetal chromosomes to determine whether the sample to be tested is triploid; wherein the sample to be tested is a prenatal diagnosis sample.

[0029] In order to further obtain a more accurate autosomal fetal concentration FF snps In a preferred embodiment, the autosomal fetal concentration FF is calculated snps Including: According to the genotypes of the mother and fetus in the sample, the frequency of allele A in the CNVSeq sequencing data is used as shown in Table 1 below to calculate the autosomal fetal concentration FF snps ,

[0030] Table 1:

[0031]

[0032]

[0033] Among them, A and B in the mother's genotype + the fetus's genotype respectively represent two alleles at one SNP site, and e represents the sequencing error rate.

[0034] Since the factors that affect the joint probability of genotypes include at least one of the following: the inbreeding coefficient of the mother, the inbreeding coefficient of the offspring, the sequencing error rate or the allele frequency information of the population, the fetal concentration of different genotypes has different differences. By analyzing the joint probability of different genotypes, it can be calculated according to the formula in the above table. Specific genotypes and autosomal fetal concentration FF snps The association derivation is as in Chinese patent document CN116240273B.

[0035] In order to further obtain a more accurate fetal sex chromosome concentration FF sex In a preferred embodiment, the sex chromosome fetal concentration FF is calculated sex Includes: BinX the sequence number of all windows in chromosomes X and Y in the sample readcount and BinY readcount Substituting into formula Ⅰ, we can calculate BinY dosage and BinX dosage , BinY dosage and BinX dosage Substituting into formula II, we can calculate chrY dosage and chrY dosage ; chrY dosage and chrY dosage Substitute into formula III to calculate the sex chromosome fetal concentration FF sex ;

[0036] Wherein, N is X or Y;

[0037] oeLh dosage =Mean(BinN dosage ) Formula II, wherein N is X or Y;

[0038] FF sex =2*chY dosage / (chrX dosage +chrY dosage )Formula III.

[0039] In order to further obtain an accurate average chromosome copy number, in a preferred embodiment, calculating the average fetal chromosome copy number of the sample to be tested includes: using the first preprocessing data to calculate the average sequence number of each window in the negative sample, recorded as the first average value u; calculating the difference multiple f between the negative sample and the sample to be tested, the sequence number of a single window in the sample to be tested in the second preprocessing data is M, f=M / u; using the difference multiple f to calculate the average chromosome copy number of the autosomes and sex chromosomes in the sample to be tested.

[0040] In order to further obtain an accurate average chromosome copy number, in a preferred embodiment, using the difference multiple f to calculate the average chromosome copy number of the autosomes and sex chromosomes in the sample to be tested includes: using the difference multiple f to calculate the chromosome copy number of a single window in the sample to be tested, wherein the chromosome copy number of the single window of the autosomes and the X chromosome of the female fetus is 2f; the chromosome copy number of the single window of the X chromosome and the Y chromosome of the male fetus is f; using a trend fitting algorithm to fit the chromosome copy number of different single windows on the chromosome to obtain a chromosome copy number fitting curve, and calculate the average chromosome copy number of the autosomes and sex chromosomes in the sample to be tested. Among them, the trend fitting algorithm includes the hidden Markov algorithm (HMM), the circular binary segmentation algorithm (CBS), the Fused Lass algorithm (Fused Lass algorithm), the fused ..., the fused Lass algorithm, the fused Lass algorithm, the fused Las o algorithm.

[0041] In order to be able to use the first autosomal fetal concentration FF snps , single sex chromosome fetal concentration FF sex The average chromosome copy number can be used to more accurately determine the triploidy. In a preferred embodiment, the criteria for determining whether the sample to be tested is triploid include: comparing the autosomal fetal concentration FF of the negative sample and the sample to be tested. snps and sex chromosome fetal concentration FF sex Linear regression fitting was performed to obtain the fetal concentration fitting curve; if the first autosomal fetal concentration FF snps >0 and the first sex chromosome fetal concentration FF sexIf the average chromosome copy number of the Y chromosome is distributed between 2 / 3±0.1 or 4 / 3±0.1 of the horizontal coordinate in the fetal concentration fitting curve, the sample to be tested is triploid; preferably, the judgment criterion of whether the sample to be tested is triploid also includes: the average chromosome copy number of the Y chromosome is distributed between 2 / 3±0.1 or 4 / 3±0.1 of the horizontal coordinate in the chromosome copy number fitting curve, then the sample to be tested is triploid.

[0042] In order to further determine the triploid karyotype of the fetus, in a preferred embodiment, if the concentration of the first sex chromosome fetus is FF sex If the fetal concentration is between 2 / 3±0.1 of the horizontal axis in the fetal concentration fitting curve, the fetal karyotype is XXY; if the fetal concentration of the first sex chromosome is FF sex If the distribution is between 4 / 3±0.1 of the horizontal axis in the fetal concentration fitting curve, the fetal karyotype is XYY.

[0043] Any sample capable of detecting fetal DNA is suitable for the present application. In a preferred embodiment, the sample to be tested includes any one of chorionic villus, amniotic fluid, umbilical cord blood or abortion products, or a combination of at least two thereof.

[0044] In order to screen and organize the data in the CNVSeq sequencing data, in a preferred embodiment, the preprocessing includes: performing human genome alignment, data bias correction and data normalization processing on the CNVSeq sequencing data of the negative sample and the sample to be tested in sequence;

[0045] In a preferred embodiment, human genome alignment includes: aligning the genomes of negative samples and samples to be tested with human reference genome sequences, dividing the genomes of samples to be tested into multiple one-to-one corresponding windows of the same length; preferably, data bias correction includes: GC content correction and / or Mappability correction. Preferably, data normalization processing includes: correcting the total number of sequences of all samples to a uniform data volume, such as the actual data volume of the samples to be tested is n, the data volume of each window is m, and the uniform data volume is N, then the data volume M of the unified window is: M = N / n*m.

[0046] In a second typical embodiment of the present application, a device for detecting fetal triploidy based on CNVSeq is provided, the device comprising: a preprocessing module, configured to preprocess the CNVSeq sequencing data of the negative sample and the sample to be tested, respectively, to obtain first preprocessing data and second preprocessing data; a fetal concentration calculation module, configured to calculate the autosomal fetal concentration FF of the sample to be tested using the first preprocessing data and the second preprocessing data snps and sex chromosome fetal concentration FF sex , record the autosomal fetal concentration FF of the sample to be tested snpsFF is the concentration of the first autosome snps , the fetal sex chromosome concentration of the sample to be tested is FF sex FF is the concentration of the first sex chromosome in the fetus sex The average fetal chromosome copy number calculation module is configured to calculate the average fetal chromosome copy number of the sample to be tested using the first preprocessing data and the second preprocessing data; the judgment module is configured to use the autosomal fetal concentration FF snps , sex chromosome fetal concentration FF sex and the average copy number of fetal chromosomes to determine whether the sample to be tested is triploid; wherein the sample to be tested is a prenatal diagnosis sample.

[0047] In order to further obtain a more accurate autosomal fetal concentration FF snps In a preferred embodiment, the fetal concentration calculation module includes: autosomal fetal concentration FF snps The calculation unit is set to calculate the autosomal fetal concentration FF based on the genotypes of the mother and fetus in the sample using the frequency of allele A in the CNVSeq sequencing data as follows: snps ,

[0048] Table 1:

[0049]

[0050]

[0051] Among them, A and B in the mother's genotype + fetus's genotype respectively represent two alleles at one SNP site, and e represents the sequencing error rate;

[0052] In order to further obtain a more accurate fetal sex chromosome concentration FF sex In a preferred embodiment, the fetal concentration calculation module also includes: sex chromosome fetal concentration FF sex The calculation unit includes: a window sequence number calculation submodule, which is configured to calculate the sequence number BinX of all windows in the sample neutral chromosomes X and Y readcount and BinY readcount Substituting into formula Ⅰ, we can calculate BinY dosage and BinX dosage , the chromosome dosage calculation submodule, is set to BinY dosage and BinX dosage Substituting into formula II, we can calculate chrY dosage and chrY dosage ; Fetal concentration FF sex The calculation submodule is set to convert chrY dosage and chrY dosageSubstitute into formula III to calculate the sex chromosome fetal concentration FF sex ;

[0053] Wherein, N is X or Y;

[0054] oeLh dosage =Mean(BinN dosage ) Formula II, wherein N is X or Y;

[0055] FF sex =2*chY dosage / (chrX dosage +chrY dosage )Formula III.

[0056] In order to further obtain an accurate average chromosome copy number, in a preferred embodiment, the fetal chromosome average copy number calculation module includes: a first average value calculation unit, which is configured to use the first preprocessing data to calculate the average sequence number of each window in the negative sample, recorded as the first average value u; a difference multiple calculation unit, which is configured to calculate the difference multiple f between the negative sample and the sample to be tested, the sequence number of a single window in the sample to be tested in the second preprocessing data is M, f=M / u; the chromosome average copy number calculation unit is configured to use the difference multiple f to calculate the average chromosome copy number of the autosomes and sex chromosomes in the sample to be tested.

[0057] In order to further obtain an accurate average chromosome copy number, in a preferred embodiment, the chromosome average copy number calculation unit includes: a single window calculation unit, which is configured to calculate the chromosome copy number of a single window in the sample to be tested using the difference multiple f, wherein the chromosome copy number of the single window of the autosome and the X chromosome of the female fetus is 2f; the chromosome copy number of the single window of the X chromosome and the Y chromosome of the male fetus is f; a fitting unit, which is configured to fit the chromosome copy number of different single windows on the chromosome using a trend fitting algorithm to obtain a chromosome copy number fitting curve, and calculate the average chromosome copy number of the autosome and sex chromosome in the sample to be tested. Among them, the trend fitting algorithm includes a hidden Markov algorithm (HMM), a circular binary segmentation algorithm (CBS), and a Fused Lasso algorithm.

[0058] In order to be able to use the above autosomal fetal concentration FF snps , sex chromosome fetal concentration FF sex and the average chromosome copy number to more accurately determine the triploidy. In a preferred embodiment, the determination module includes: a fetal concentration fitting unit, which is configured to fit the autosomal fetal concentration FF of the negative sample and the sample to be tested. snps and sex chromosome fetal concentration FF sexLinear regression fitting is performed to obtain a fetal concentration fitting curve; the fetal concentration judgment unit is set to if the first autosomal fetal concentration FF snps >0 and the first sex chromosome fetal concentration FF sex If the average chromosome copy number of the Y chromosome is distributed between 2 / 3±0.1 or 4 / 3±0.1 of the horizontal coordinate in the fetal concentration fitting curve, then the sample to be tested is output as a triploid; preferably, the judgment module also includes: a chromosome copy number judgment unit, and the average chromosome copy number of the Y chromosome is distributed between 2 / 3±0.1 or 4 / 3±0.1 of the horizontal coordinate in the chromosome copy number fitting curve, then the sample to be tested is output as a triploid.

[0059] In order to further judge the triploid karyotype of the fetus, in a preferred embodiment, the judgment module also includes: a karyotype analysis unit, which is configured to: if the first sex chromosome fetal concentration FFsex is distributed between 2 / 3±0.1 of the horizontal coordinate in the fetal concentration fitting curve, the fetal karyotype is XXY; if the first sex chromosome fetal concentration FFsex is distributed between 4 / 3±0.1 of the horizontal coordinate in the fetal concentration fitting curve, the fetal karyotype is XYY.

[0060] Any sample capable of detecting fetal DNA is suitable for the present application. In a preferred embodiment, the sample to be tested includes any one of chorionic villus, amniotic fluid, umbilical cord blood or abortion products, or a combination of at least two thereof.

[0061] In order to screen and organize the data in the CNVSeq sequencing data, in a preferred embodiment, the preprocessing module includes: a human genome alignment unit, a data bias correction unit and a data normalization processing unit.

[0062] In a preferred embodiment, the human genome comparison unit includes: comparing the genomes of negative samples and samples to be tested with the human reference genome sequence, and dividing the genome of the sample to be tested into multiple one-to-one corresponding windows with the same length; preferably, the data preference correction unit includes: a GC content correction unit and / or a Mappability correction unit. Preferably, the data normalization processing unit includes: correcting the total number of sequences of all samples to a unified data volume, such as the actual data volume of the sample to be tested is n, the data volume of each window is m, and the unified data volume is N, then the data volume M of the unified window is: M = N / n*m.

[0063] In a third typical embodiment of the present application, a computer-readable storage medium is provided, the storage medium including a stored program, wherein when the program is executed, the device where the storage medium is located is controlled to execute the above-mentioned CNVSeq-based method for detecting fetal triploidy.

[0064] In a fourth typical embodiment of the present application, a processor is provided, and the processor is used to run a program, wherein the program runs the above-mentioned CNVSeq-based method for detecting fetal triploidy.

[0065] The present application is further described in detail below in conjunction with specific embodiments. These embodiments should not be construed as limiting the scope of protection claimed in the present application.

[0066] Example 1

[0067] The samples to be tested in this example are 45 clinical prenatal diagnosis CNVSeq test samples with unknown chromosome copy number abnormalities, including sample types such as amniotic fluid or abortion products, and the negative samples are 1369 clinical CNVSeq samples with no reported chromosome copy number abnormalities.

[0068] 1. Preprocessing: Extract DNA from negative samples and test samples respectively, perform CNVSeq sequencing, and preprocess the sequencing results, including: human genome alignment, data bias correction, and data normalization.

[0069] Among them, human genome alignment includes: aligning the genomes of negative samples and samples to be tested with the human reference genome sequence, dividing the genome of the sample to be tested into multiple one-to-one corresponding windows with the same length; data bias correction includes: GC content correction and / or Mappability correction, and the correction method is loess algorithm or spline algorithm. Data volume normalization includes: correcting the total number of sequences of all samples to be tested to a unified data volume. Assuming that the actual data volume of the test sample is n, the data volume of each window is m, and the unified data volume is N, then the data volume M of the unified window is: M = N / n*m.

[0070] 2. Autosomal fetal concentration FF snps Calculation: Using the pre-processed data of the negative sample and the sample to be tested, i.e., the first pre-processed data and the second pre-processed data, according to the genotypes of the mother and the fetus of the sample, the frequency of allele A in the sequencing data is used to calculate the autosomal fetal concentration FF as shown in Table 1 above snps .

[0071] Among them, A and B in the genotype of mother + fetus represent two alleles at one SNP site, and e represents the sequencing error rate.

[0072] 3. Sex chromosome fetal concentration FF sex Calculation: BinX the sequence number of all windows in chromosomes X and Y in the sample readcount and BinY readcount Substituting into formula Ⅰ, we can calculate BinY dosage and BinX dosage , BinYdosage and BinX dosage Substituting into formula II, we can calculate chrY dosage and chrY dosage ; chrY dosage and chrY dosage Substitute into formula III to calculate the sex chromosome fetal concentration FF sex ;

[0073] Wherein, N is X or Y;

[0074] oeLh dosage =Mean(BinN dosage ) Formula II, wherein N is X or Y;

[0075] FF sex =2*chY dosage / (chrX dosage +chrY dosage )Formula III.

[0076] 4. Calculation of the average copy number of fetal chromosomes in the sample to be tested: Using the data after preprocessing of the negative sample, i.e., the first preprocessing data, the average number of sequences in each window in the negative sample is calculated, recorded as the first average value u; the difference multiple f between the negative sample and the sample to be tested is calculated, and the number of sequences in a single window in the sample to be tested in the second preprocessing data is M, f=M / u; the chromosome copy number of a single window in the sample to be tested is calculated using the difference multiple f, wherein the chromosome copy number of a single window of an autosome and a female fetus X chromosome is 2f; the chromosome copy number of a single window of a male fetus X chromosome and a Y chromosome is f; the chromosome copy number of different single windows on the chromosome is fitted using a trend fitting algorithm to obtain a chromosome copy number fitting curve, and the average chromosome copy number of the autosome and sex chromosome in the sample to be tested is calculated.

[0077] 5. Triploidy determination:

[0078] The autosomal fetal concentrations of negative samples and samples to be tested are FF snps and sex chromosome fetal concentration FF sex Linear regression fitting was performed to obtain the fetal concentration fitting curve (such as Figure 1 As shown, type 1 is FF snps Samples with >0.5, type 2 is FF snps ≤0.5 for samples); first autosomal fetal concentration FF snps Significantly greater than 0 and sex chromosome fetal concentration FF sex Distributed in Figure 1If the average chromosome copy number of the Y chromosome is between 2 / 3±0.1 or 4 / 3±0.1 of the horizontal coordinate, the sample to be tested is triploid. If the fetus is a male fetus, it is necessary to further observe that the average chromosome copy number of the Y chromosome is distributed between 2 / 3±0.1 or 4 / 3±0.1 of the horizontal coordinate of the chromosome copy number fitting curve, the sample to be tested is triploid. The above method is used to judge the samples S01-S05 to be tested, and the results are shown in Table 2 below:

[0079] Table 2:

[0080] Sample No. <![CDATA[FF sex ]]> <![CDATA[FF snps ]]> Karyotype inference Y chromosome copy number S01 0.67 0.19 69XXY 0.7 S02 0.64 0.19 69XXY 0.68 S03 0.66 0.20 69XXY 0.69 S04 0.62 0.25 69XXY 0.67 S05 1.30 0.10 69XYY 1.37

[0081] 6. The samples S01-S05 were verified using the WGA SNP Array chip method. The results are shown in Table 3 below.

[0082] Table 3:

[0083] Sample No. WGA Verification Is it consistent? S01 69XXY Consistency S02 69XXY Consistency S03 69XXY Consistency S04 69XXY Consistency S05 69XYY Consistency

[0084] From the above description, it can be seen that the above embodiments of the present invention achieve the following technical effects: the autosomal fetal concentration FF of the negative sample and the sample to be tested obtained by the calculation method of the present application snps , sex chromosome fetal concentration FF sex and the average copy number of fetal chromosomes to determine whether the fetus has triploidy. This can obtain more accurate results, avoid the misjudgment that may be caused by a single indicator, and can reduce the detection cost and shorten the experimental cycle of triploidy detection.

[0085] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for detecting fetal triploidy based on CNVSeq, characterized in that: The method comprises: Preprocessing the CNVSeq sequencing data of the negative sample and the sample to be tested respectively to obtain first preprocessed data and second preprocessed data; The autosomal fetal concentration FF of the negative sample and the sample to be tested is calculated using the first preprocessed data and the second preprocessed data. snps and sex chromosome fetal concentration FF sex ; The autosomal fetal concentration FF of the sample to be tested is recorded as snps FF is the concentration of the first autosome snps , the sex chromosome fetal concentration FF of the sample to be tested sex FF is the concentration of the first sex chromosome in the fetus sex ; Calculating the average copy number of the fetal chromosome of the sample to be tested using the first preprocessed data and the second preprocessed data; Using the first autosomal fetal concentration FF snps , the first sex chromosome fetal concentration FF sex and the average copy number of the fetal chromosomes to determine whether the sample to be tested is triploid; Wherein, the sample to be tested is a prenatal diagnosis sample.

2. The method according to claim 1, characterized in that Calculate the autosomal fetal concentration FF snps include: According to the genotypes of the mother and fetus in the sample, the autosomal fetal concentration FF is calculated as follows using the frequency of allele A in the CNVSeq sequencing data: snps , Wherein, A and B in the mother's genotype + fetus' genotype respectively represent two alleles at one SNP site, and e represents the sequencing error rate; Preferably, the sex chromosome fetal concentration FF is calculated sex include: BinX is the sequence number of all windows in chromosomes X and Y in the sample. readcount and BinY readcount Substituting into formula Ⅰ, we can calculate BinY dosage and BinX dosage , Wherein, N is X or Y; The BinY dosage And the BinX dosage Substituting into formula II, we can calculate chrY dosage and chrY dosage ; ne dosage =Mean(BinN dosage ) Formula II, wherein N is x or Y; The chrY dosage and the chrY dosage Substitute into formula III to calculate the sex chromosome fetal concentration FF sex ; FF sex = 2 * chY dosage / (chrX dosage + chrY dosage ) Formula III.

3. The method according to claim 1, characterized in that Calculating the average copy number of the fetal chromosome of the sample to be tested includes: The first preprocessed data is used to calculate the average value of the number of sequences in each window in the negative sample, which is recorded as a first average value u; Calculating the difference multiple f between the negative sample and the sample to be tested, the number of sequences in a single window in the sample to be tested in the second preprocessed data is M, and f=M / u; The difference multiple f is used to calculate the average chromosome copy number of the autosomes and sex chromosomes in the sample to be tested.

4. The method according to claim 3, characterized in that The method of calculating the average chromosome copy number of the autosomes and sex chromosomes in the sample to be tested by using the difference multiple f includes: The chromosome copy number of a single window in the sample to be tested is calculated using the difference multiple f. The chromosome copy numbers of the single windows of the autosomes and the X chromosome of the female fetus are both 2f; the chromosome copy numbers of the single windows of the X chromosome and the Y chromosome of the male fetus are both f; The chromosome copy numbers of different single windows on the chromosome are fitted using a trend fitting algorithm to obtain a chromosome copy number fitting curve, and the average chromosome copy numbers of the autosomes and sex chromosomes in the sample to be tested are calculated.

5. The method according to claim 4, characterized in that The criteria for determining whether the sample to be tested is triploid include: The autosomal fetal concentration FF of the negative sample and the sample to be tested snps and sex chromosome fetal concentration FF sex Linear regression fitting was performed to obtain the fetal concentration fitting curve; If the first autosomal fetal concentration FF snps >0 and the first sex chromosome fetal concentration FF sex If the sample is distributed between 2 / 3±0.1 or 4 / 3±0.1 of the horizontal coordinate in the fetal concentration fitting curve, the sample to be tested is triploid; Preferably, the criterion for judging whether the sample to be tested is triploid also includes: if the average chromosome copy number of the Y chromosome is distributed between 2 / 3±0.1 or 4 / 3±0.1 of the horizontal coordinate in the chromosome copy number fitting curve, then the sample to be tested is triploid.

6. A device for detecting fetal triploidy based on CNVSeq, characterized in that: The device comprises: A preprocessing module is configured to preprocess the CNVSeq sequencing data of the negative sample and the sample to be tested respectively to obtain first preprocessed data and second preprocessed data; A fetal concentration calculation module is configured to calculate the autosomal fetal concentration FF of the negative sample and the sample to be tested using the first preprocessed data and the second preprocessed data. snps and sex chromosome fetal concentration FF sex , The autosomal fetal concentration FF of the sample to be tested is recorded as snps FF is the concentration of the first autosome snps , the sex chromosome fetal concentration FF of the sample to be tested sex FF is the concentration of the first sex chromosome in the fetus sex ; a fetal chromosome average copy number calculation module, configured to calculate the fetal chromosome average copy number of the sample to be tested using the first preprocessed data and the second preprocessed data; The determination module is configured to use the first autosomal fetal concentration FF snps , the first sex chromosome fetal concentration FF sex and the average copy number of the fetal chromosomes to determine whether the sample to be tested is triploid; Wherein, the sample to be tested is a prenatal diagnosis sample.

7. The device according to claim 6, characterized in that The fetal concentration calculation module includes: autosomal fetal concentration FF snps The calculation unit is set to calculate the autosomal fetal concentration FF according to the genotypes of the mother and fetus in the sample using the frequency of allele A in the CNVSeq sequencing data as follows: snps , The frequency of allele A in the mother's genotype + fetus' genotype sequencing data Wherein, A and B in the mother's genotype + fetus' genotype respectively represent two alleles at one SNP site, and e represents the sequencing error rate; Preferably, the fetal concentration calculation module also includes: sex chromosome fetal concentration FF sex Computing unit, including: The window sequence number calculation submodule is configured to calculate the sequence number BinX of all windows in the neutral chromosomes X and Y of the sample. readcount and BinY readcount Substituting into formula Ⅰ, we can calculate BinY dosage and BinX dosage , Wherein, N is X or Y; The chromosome dosage calculation submodule is set to convert the BinY dosage And the BinX dosage Substituting into formula II, we can calculate chrY dosage and chrY dosage ; ne dosage =Mean(BinN dosage ) Formula II, wherein N is x or Y; Fetal concentration FF sex The calculation submodule is configured to convert the chrY dosage and the chrY dosage Substitute into formula III to calculate the sex chromosome fetal concentration FF sex ; FF sex = 2 * chY dosage / (chrX dosage + chrY dosage ) Formula III.

8. The device according to claim 6, characterized in that The fetal chromosome average copy number calculation module comprises: A first average value calculation unit is configured to calculate the average value of the sequence number of each window in the negative sample using the first preprocessed data, recorded as a first average value u; A difference multiple calculation unit is configured to calculate a difference multiple f between the negative sample and the sample to be tested, wherein the number of sequences in a single window in the sample to be tested in the second preprocessed data is M, and f=M / u; The chromosome average copy number calculation unit is configured to calculate the average chromosome copy number of the autosomes and sex chromosomes in the sample to be tested using the difference multiple f.

9. The device according to claim 8, characterized in that The chromosome average copy number calculation unit comprises: A single window calculation unit is configured to calculate the chromosome copy number of a single window in the sample to be tested using the difference multiple f. The chromosome copy numbers of the single windows of the autosomes and the X chromosome of the female fetus are both 2f; the chromosome copy numbers of the single windows of the X chromosome and the Y chromosome of the male fetus are both f; The chromosome copy number fitting unit is configured to fit the chromosome copy numbers of different single windows on the chromosome using a trend fitting algorithm to obtain a chromosome copy number fitting curve, and calculate the average chromosome copy numbers of the autosomes and sex chromosomes in the sample to be tested.

10. The device according to claim 9, characterized in that The judging module comprises: A fetal concentration fitting unit is configured to fit the autosomal fetal concentration FF of the negative sample and the sample to be tested. snps and sex chromosome fetal concentration FF sex Linear regression fitting was performed to obtain the fetal concentration fitting curve; The fetal concentration determination unit is configured to determine if the first autosomal fetal concentration FF snps >0 and the first sex chromosome fetal concentration FF sex If the sample is distributed between 2 / 3±0.1 or 4 / 3±0.1 of the horizontal coordinate in the fetal concentration fitting curve, it is output that the sample to be tested is triploid; Preferably, the judgment module further includes: a chromosome copy number judgment unit, and if the average chromosome copy number of the Y chromosome is distributed between 2 / 3±0.1 or 4 / 3±0.1 of the horizontal coordinate in the chromosome copy number fitting curve, then the sample to be tested is output as a triploid.

11. A computer-readable storage medium, characterized in that: The storage medium includes a stored program, wherein when the program is executed, the device where the storage medium is located is controlled to execute the method for detecting fetal triploidy based on CNVSeq according to any one of claims 1 to 5.

12. A processor, characterized in that: The processor is used to run a program, wherein the program runs the method for detecting fetal triploidy based on CNVSeq described in any one of claims 1 to 5.

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