WO2016176846A1 - 检测染色体非整倍性的试剂盒、装置和方法 - Google Patents

检测染色体非整倍性的试剂盒、装置和方法 Download PDF

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WO2016176846A1
WO2016176846A1 PCT/CN2015/078421 CN2015078421W WO2016176846A1 WO 2016176846 A1 WO2016176846 A1 WO 2016176846A1 CN 2015078421 W CN2015078421 W CN 2015078421W WO 2016176846 A1 WO2016176846 A1 WO 2016176846A1
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chromosome
value
coverage
chromosomes
aneuploidy
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PCT/CN2015/078421
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English (en)
French (fr)
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陈重建
梁峻彬
玄兆伶
李大为
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安诺优达基因科技(北京)有限公司
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Publication of WO2016176846A1 publication Critical patent/WO2016176846A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/34—Measuring or testing with condition measuring or sensing means, e.g. colony counters
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids

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  • the present invention relates to the field of biomedicine, and in particular to a kit, device and method for detecting chromosome aneuploidy.
  • NIPT non-invasive prenatal testing methods
  • the non-invasive prenatal testing method has the following two advantages: The first aspect: NIPT does not need to bear any risk of miscarriage, but the clinical karyotype analysis by invasive methods such as amniocentesis and umbilical wear will bring about 1/ Abortion risk of 200, there are also studies showing that premature umbilical wear may also cause fetal position tilt; the first aspect: NIPT can be detected as early as 8 weeks of gestational age, giving risk judgment earlier, reducing induction of labor to pregnant women risks of.
  • the main object of the present application is to provide a kit, apparatus and method for detecting chromosomal aneuploidy to reduce the false positive rate of detection.
  • the first fetal concentration is recorded as f1
  • the second fetal concentration is recorded as f2
  • the difference in concentration of the fetus is normalized to obtain M f . If M f is less than or equal to 4, it is considered that it can be fitted; otherwise, it is considered that it cannot be fitted.
  • the method further comprises: calculating the coverage degree by dividing the windows into all the chromosomes in the sequencing data, Obtaining the pre-correction coverage of each chromosome; performing a Z-test on the number of single sequences of the pregnant women in each window to obtain a Z CNV value, and obtaining an abnormal copy number of the pregnant woman to be tested according to the magnitude of the Z CNV value;
  • the copy number abnormal fragment refers to a fragment of 300Kb or more in the sequencing data, and in the fragment of 300Kb or more, more than 80% of the windows have a Z CNV value of the chromosome fragment greater than or equal to 4 or less than or equal to -4;
  • the coverage is calculated in the form of a window that is divided into equal sizes for all chromosomes in the sequenced data, and the coverage of each chromosome is obtained.
  • each window has a size of 100 Kb, and the degree of overlap between adjacent windows is 50%.
  • the Z test is performed on the number of single sequences of the pregnant women to be tested in each window to obtain the Z CNV value
  • the step of obtaining the copy number abnormal fragments of the pregnant woman according to the Z CNV value includes: sequencing according to each sequence in the sequencing data Depth, the number of single sequences of each window is counted; the number of single sequences of each window is calculated according to the GC content and the ratio of each chromosome, and the pre-corrected coverage of the number of single sequences of each window is obtained; and for each window the number of unique sequences before correction coverage normalized to give a single number sequence Z CNV value of each window, and determines whether the pregnant woman measured copy number abnormalities according to the size of the fragment Z CNV value; when sequencing data There are more than 300Kb segments, and in the segment above 300Kb, more than 80% of the windows have a single sequence of Z CNV values greater than or equal to 4 or less than or equal to -4, then it is considered that the segment above 300Kb is the pregnant woman to be
  • the Za ⁇ o value is in accordance with To calculate, among them, Is the coverage value obtained by the known negative sample population according to the LOESS algorithm, and s is the negative sample population. Standard deviation.
  • a device for detecting chromosomal aneuploidy comprising: a detection module for performing high throughput on a sample to be tested for free DNA from a peripheral blood of a pregnant woman Sequencing, obtaining sequencing data containing all chromosomes; first judging module: for calculating all the chromosomes in the sequencing data into a window, obtaining the Z aneu value of each chromosome, and preliminary according to the Za ⁇ o value of each chromosome Determining whether there is aneuploidy in each chromosome; a first calculating module, configured to calculate a first concentration value of the fetal DNA in the sample to be tested according to the initially determined chromosome having aneuploidy; and a second calculating module, configured to The X chromosome or methylation method calculates the second concentration value of the fetal DNA in the sample to be tested; the second judgment module is used to determine the feta
  • the second determining module further includes: a fitting calculation sub-module: used according to the formula Standardizing the fetal DNA concentration difference to obtain M f ; wherein the first concentration value is f1 and the second concentration value is f2; the first fitting judgment sub-module: when M f is less than or equal to 4, Capable of fitting; second fitting judgment sub-module: in the case where M f is greater than 4, it is considered that the fitting cannot be performed.
  • a fitting calculation sub-module used according to the formula Standardizing the fetal DNA concentration difference to obtain M f ; wherein the first concentration value is f1 and the second concentration value is f2; the first fitting judgment sub-module: when M f is less than or equal to 4, Capable of fitting; second fitting judgment sub-module: in the case where M f is greater than 4, it is considered that the fitting cannot be performed.
  • the first determining module further includes: a first coverage calculation sub-module: configured to calculate coverage in a form of being divided into windows in all the chromosomes in the sequenced data, to obtain pre-correction coverage of each chromosome; and a single sequence calculator Module: Calculate the value of the Z aneu of the number of single sequences of the pregnant women in each window; the copy number abnormal segment query sub-module: used to query the fragments of 300Kb or more in the sequencing data, and in the fragments above 300Kb More than 80% of the chromosome fragments in the window have Z CNV values greater than or equal to 4 or less than or equal to -4; copy number abnormal segment determination sub-module: used for the above 300Kb fragments and will be obtained from the sequencing data.
  • a first coverage calculation sub-module configured to calculate coverage in a form of being divided into windows in all the chromosomes in the sequenced data, to obtain pre-correction coverage of each chromosome
  • a single sequence calculator Module Calcul
  • a fragment of a chromosome fragment having a Z CNV value greater than or equal to 4 or less than -4 is determined as a copy number abnormal fragment of the pregnant woman to be tested;
  • ⁇ first calculation sub-module for copying the parent's copy number in the fetus
  • the parameter ⁇ is calculated according to a calculation formula as shown in the formula (1), wherein the parameter ⁇ refers to an abnormal segment of the copy number of the pregnant woman. Effect of relaxing bodies before coverage;
  • ⁇ second calculation sub-module For the case of a chromosome having an abnormal copy number of the ismeeted mother of the fetus, the calculation formula parameter ⁇ is as shown in the formula (2):
  • m represents the effective length of the chromosome where the copy number abnormal fragment is located, and the unit is Mb; n represents the length of the pregnant woman in the copy number abnormal segment, the unit is Mb; cn represents the number of occurrences of the abnormal copy number of the pregnant woman; f represents the periphery of the pregnant woman to be tested
  • the correction sub-module for utilization Correcting the pre-correction coverage of each chromosome to obtain the corrected coverage of each chromosome; Representing the pre-correction coverage of each chromosome, x' represents the corrected chromosome coverage of each chromosome; the second coverage calculation sub-module is used to calculate the Z aneu value of each chromosome by using the corrected coverage of each chromosome;
  • the euploid preliminary judgment sub-module is configured to determine whether the chromosome has aneuploidy
  • the first coverage calculation sub-module further includes a chromosome window segmentation unit configured to slice all the chromosomes in the sequencing data into equal-sized windows; and the first coverage calculation unit is configured to be equal in size The coverage of the window is calculated to obtain the pre-correction coverage of each chromosome.
  • each window has a size of 100 Kb, and the coverage between adjacent windows is 50%.
  • the single sequence calculation sub-module includes a single sequence statistical unit for counting the number of single sequences of each window according to the sequencing depth of each sequence in the sequencing data; a single sequence correction unit for calculating the GC content of each chromosome And the ratio of the single sequence is corrected to obtain the pre-correction coverage of the number of each single sequence; the single sequence Za ⁇ o value calculation unit is used for standardizing the pre-correction coverage of the number of each single sequence, The Z aneu value of the number of each single sequence is obtained.
  • the Z aneu in the second coverage calculation sub-module is To calculate, among them, Is the pre-correction coverage value obtained by the negative sample population according to the LOESS algorithm, and s is the negative sample population. Standard deviation.
  • a kit for detecting chromosomal aneuploidy comprising: a detection reagent and a detection device for performing high-throughput sequencing of peripheral blood free DNA of a pregnant woman to be tested, Sequencing data containing all chromosomes; chromosomal aneuploidy preliminary judgment device: used to calculate all chromosomes in the sequencing data in a window-divided manner, to obtain the Z aneu value of each chromosome, and according to the Z neu of each chromosome The value is initially determined whether there is aneuploidy in each chromosome; the first computing device is used to calculate the first concentration value of the fetal DNA in the sample to be tested according to the preliminary determined aneuploidy chromosome; the second computing device: Calculating a second concentration value of fetal DNA in the sample to be tested according to the method of X chromosome or methylation; chromosomal aneuploidy judging device:
  • the chromosomal aneuploidy judging device further includes: a fitting calculation component: for using the formula Standardizing the fetal DNA concentration difference to obtain M f ; wherein the first concentration value is f1 and the second concentration value is f2; the first fitting determining component: when M f is less than or equal to 4, Fitting; second fitting determining component: in the case where M f is greater than 4, then it is considered that the fitting is impossible.
  • a fitting calculation component for using the formula Standardizing the fetal DNA concentration difference to obtain M f ; wherein the first concentration value is f1 and the second concentration value is f2; the first fitting determining component: when M f is less than or equal to 4, Fitting; second fitting determining component: in the case where M f is greater than 4, then it is considered that the fitting is impossible.
  • the chromosomal aneuploidy preliminary judgment apparatus further includes: a first coverage calculation unit: configured to calculate coverage in a form of being divided into windows in all the chromosomes in the sequencing data to obtain pre-correction coverage of each chromosome.
  • single sequence calculation component Calculated for the value of the single sequence of the pregnant woman in the window to be tested ;
  • copy number abnormal segment query component used to query the fragment of 300Kb or more in the sequencing data, and at 300Kb In the above fragment, more than 80% of the chromosome fragments in the window have Z CNV values greater than or equal to 4 or less than or equal to -4;
  • copy number abnormal fragment determining means for the 300Kb or more fragments to be queried from the sequencing data And in more than 80% of the windows, the Z CNV value of the chromosome fragment is greater than or equal to 4 or less than or equal to -4, and the fragment is determined as the copy number abnormal segment of the pregnant woman to be tested;
  • ⁇ first calculating component used for inheriting the maternal body in the fetus In the case of a copy number abnormal segment, the parameter ⁇ is calculated according to a calculation formula as shown in the formula (1), and the parameter ⁇ is a copy number abnormal segment of the pregnant woman. Effect of the front cover body color correction
  • ⁇ represents the effective length of the chromosome where the copy number abnormal fragment is located, and the unit is Mb; n represents the length of the abnormal number of copies of the pregnant woman, and the unit is Mb; cn represents the number of occurrences of the abnormal number of copy number of the pregnant woman; ⁇ second calculating part: used In the case of a chromosome in which the copy number of the ismeeted parent of the fetus is abnormal, the formula ⁇ is calculated as shown in the formula (2):
  • m represents the effective length of the chromosome where the copy number abnormal fragment is located, and the unit is Mb; n represents the length of the pregnant woman in the copy number abnormal segment, the unit is Mb; cn represents the number of occurrences of the abnormal copy number of the pregnant woman; f represents the periphery of the pregnant woman to be tested The concentration of fetal free DNA contained in blood free DNA and assuming that the concentration f of fetal free DNA is less than 50%; correcting component: for utilization Correcting the pre-correction coverage of each chromosome to obtain the corrected coverage of each chromosome; Representing the pre-correction coverage of each chromosome, x' represents the corrected chromosome coverage of each chromosome; and the second coverage calculation component is used to calculate the Z aneu value of each chromosome by using the corrected coverage of each chromosome; Pluricity preliminary judgment component: for judging whether the chromosome has aneuploidy according to whether the Za ⁇ or value is greater than or equal to
  • the first coverage calculation component further includes: a chromosome window segmentation component: configured to slice all the chromosomes in the sequenced data into equal-sized windows; and the first coverage calculation sub-component: for equal size The coverage is calculated in the form of a window to obtain pre-corrected coverage for each chromosome.
  • each window has a size of 100 Kb, and the coverage between adjacent windows is 50%.
  • the single sequence calculation component includes: a single sequence statistical component for counting the number of single sequences of each window according to the sequencing depth of each sequence in the sequencing data; a single sequence correction component: for the GC content according to each chromosome and The comparison rate corrects the number of each single sequence to obtain the pre-correction coverage of the number of each single sequence; the single-sequence Z-value calculation component: for normalizing the pre-correction coverage of the number of each single sequence, and obtaining each The number of Z aneu values for a single sequence.
  • Zaneu follows To calculate, among them, Is the pre-correction coverage value obtained by the negative sample population according to the LOESS algorithm, and s is the negative sample population. Standard deviation.
  • the method for calculating the fetal DNA concentration in the sample to be tested is further obtained by means of two commonly used methods for calculating the fetal DNA concentration in the sample to be tested. Whether the fetal DNA concentrations are equal to confirm the authenticity of the preliminary judgment results.
  • FIG. 1 is a flow chart showing a method of detecting chromosome aneuploidy according to an exemplary embodiment of the present application
  • FIG. 2 is a schematic structural view of a detecting device for chromosome aneuploidy according to an exemplary embodiment of the present application
  • FIG. 3A and 3B are views showing a result of sample detection in Embodiment 1 of the present application, wherein FIG. 3A is a scattergram, and FIG. 3B is a density profile;
  • FIG. 4 is a graph showing the results of the correction of aneuploidy on chromosome 21 of samples EK01875 and BD01462 according to Example 2 of the present application.
  • the Z value refers to the calculated value of the Z test in statistics, and is a method for the large value (ie, the sample size is greater than 30) mean difference test. It uses the theory of standard normal distribution to infer the probability of a difference occurring, and thus compares whether the difference between the two means is significant.
  • the alignment ratio refers to the ratio of the sequencing sequences within the window to the ratio of the genomic reference sequence. Since the sequencing sequences may be aligned to multiple positions on the genomic reference sequence at the same time, and may not be the only sequencing sequence, the sequencing sequence alignment ratio within the window is greater than the single sequence alignment ratio.
  • Normalized processing refers to the corrected value, doing (x-u)/sd(x-u), where x is the corrected value, u is the mean of x, and sd is the standard deviation.
  • the present application proposes a method for detecting chromosome aneuploidy.
  • the above method of the present application is further obtained by means of two commonly used methods for calculating fetal DNA concentration in a sample to be tested, which is initially judged to have chromosomal aneuploidy by a conventional method for detecting chromosomal aneuploidy. Whether the fetal DNA concentrations are equal to confirm the authenticity of the results of the preliminary judgment.
  • the determining step is: recording the first fetal concentration as f 1 and the second fetal concentration as f2, according to the formula
  • the Z value of each chromosome in the step of calculating the Z value of each chromosome by dividing into all the chromosomes in the sequencing data to obtain the Z value of each chromosome, the Z value of each chromosome can be calculated by the conventional method of the present application, but In order to make the Z value more accurately reflect the degree of aneuploidy of the chromosome, in a preferred embodiment of the present application, the step of the present application further comprises: calculating the coverage in the form of a window divided into all the chromosomes in the sequencing data.
  • the pre-correction coverage of each chromosome is obtained; the Z value of the number of single sequences of the pregnant women in each window is calculated, and the copy number abnormal number of the pregnant woman to be tested is obtained according to the Z CNV value; the copy of the pregnant woman to be tested
  • the number of abnormal fragments refers to fragments above 300Kb in the sequencing data, and in the fragments above 300Kb, the Z CNV values of the chromosome fragments in more than 80% of the windows are greater than or equal to 4 or less than or equal to -4;
  • m represents the effective length of the chromosome in which the copy number abnormal fragment is located, and the unit is Mb; n represents the length of the abnormal number of copy number of the pregnant woman to be tested, and the unit is Mb; The number of times the copy number abnormal segment appears; in the formula (2), f represents the concentration of fetal free DNA contained in the peripheral blood free DNA of the pregnant woman to be tested and assumes that the fetal free DNA concentration f is less than 50%; Correcting the coverage of each chromosome before correction, wherein Representing the pre-correction coverage of each chromosome, x' represents the corrected chromosome coverage of each chromosome.
  • the above method of the present application does not consider the direct removal of the maternal copy number abnormal segment in the sequencing data in the prior art when calculating the Z value of each chromosome (ignoring the ploidy of the chromosome of the fetus in the region), and By screening for a specific size copy number abnormality fragment existing on the female chromosome, and determining whether the chromosome has aneuploidy, the effect of the copy number abnormal fragment on calculating the coverage of each chromosome is removed, thereby obtaining each chromosome. The corrected coverage, such that the results of the chromosome aneuploidy detected by the method of the present application are more accurate.
  • the calculation method of the concentration f of fetal free DNA contained in the peripheral blood free DNA of the pregnant woman to be tested is a conventional calculation method in the art.
  • the concentration of fetal free DNA is as follows. Calculate, among them, Represents the ratio of the average single sequence number of the window on the X chromosome to the average single sequence number of all windows; and when the copy number abnormal segment is on chromosome 21, 18 or 13, the concentration of fetal free DNA is Calculate, among them, Represents the ratio of the average single sequence number of windows on chromosomes 21, 18, or 13 to the average single sequence number of all windows.
  • the fetus When the fetus is a female, it is necessary to perform methylation detection of a specific gene on the peripheral blood free DNA of the pregnant woman.
  • the principle is that certain specific genes differ in the form of methylation in maternal DNA and in fetal DNA.
  • the fetal and placenta-derived RASSF1A (Chromosome 3) gene is highly methylated, while the mother's own source of RASSF1A gene is unmethylated, using methylation-sensitive enzymes such as HhaI, BstUI (30U) and HpaII. Processing cfDNA, the unmethylated gene will be digested, and the methylated gene is not digested, so the fetal cfDNA content can be detected by Q-PCR.
  • the specific steps can be found in the literature PLOS ONE 9:71-7 ( 2014), Quantification of Cell-Free DNA in Normal and Complicated Pregnancies: Overcoming Biological and Technical Issues.
  • the requirement for the concentration of fetal DNA in the DNA of the sample to be tested is the same as the two methods for calculating the fetal DNA concentration mentioned in the foregoing steps, the only difference being that this step
  • the chromosome is not considered to be aneuploid, but either method can be used.
  • the calculated Z value does not affect the preliminary judgment of chromosome aneuploidy. That is, in this step, the concentration of fetal DNA in the DNA of the sample to be tested can be calculated by the X chromosome or methylation method, and it is not necessary to perform concentration calculation based on the chromosome in which aneuploidy is present.
  • the concentration of fetal DNA in the DNA of the sample to be tested can be calculated by the X chromosome or methylation method, and it is not necessary to perform concentration calculation based on the chromosome in which aneuploidy is present.
  • the coverage is calculated in the form of a split into a window, each window having a size of 100 Kb and a coverage between adjacent two windows of 50%. Controlling the size of each window to 100Kb and controlling the degree of overlap between two adjacent windows to 50%, not only can obtain a relatively robust chromosome coverage, but also increase the coverage between windows to improve the detection copy. The accuracy of several abnormal segments increases the detection efficiency of pregnant women's copy number abnormal segments.
  • the Z value of the single sequence number of the pregnant woman to be tested in each window is calculated, and the step of obtaining the copy number abnormal segment of the pregnant woman to be tested according to the Z value can be performed in a conventional calculation. Based on the method steps of the copy number abnormal segment, the conditions that should be satisfied by appropriately adjusting the copy number abnormal segment are obtained according to the quality of the sequencing data or the detection precision.
  • the Z CNV value of the single sequence number of the pregnant woman to be tested in each window is calculated, and the step of obtaining the copy number abnormal segment of the pregnant woman to be tested according to the Z CNV value includes: according to the sequencing data The sequencing depth of each sequence, the number of single sequences in each window is counted; the number of each single sequence is corrected according to the GC content and the ratio of each chromosome, and the pre-corrected coverage of each single sequence is obtained; The pre-correction coverage of the number of single sequences is normalized to obtain the value of the number of each single sequence, and according to the size of the Z CNV value, whether the pregnant woman has a copy number abnormality fragment; when there is more than 300 Kb in the sequencing data For the fragment, and the Z CNV value of the number of single sequences of more than 80% of the windows above 300 Kb is greater than or equal to 4 or less than -4, it is considered that the fragment above 300 Kb is the copy number abnormal fragment of the pregnant woman to be
  • the above-mentioned detection step of the application can detect a reliable abnormal copy number of the pregnant woman, and use these copy number abnormal fragments to correct the Z value of the chromosome in which the chromosome is located, thereby avoiding the error of the detection result of the abnormal copy number of the pregnant copy number. False negative judgment.
  • the step of calculating the Z aneu value of each chromosome using the corrected coverage of each chromosome is used.
  • the corrected Z aneu value calculated by the above formula can more accurately reflect the aneuploidy of the chromosome, so that the detection result is more accurate.
  • a device for detecting chromosomal aneuploidy comprising the following module: a detection module: for separating DNA from a source to a peripheral blood of a pregnant woman The sample to be tested is subjected to high-throughput sequencing to obtain sequencing data containing all chromosomes; the first determining module is configured to calculate all the chromosomes in the sequencing data into a window, to obtain the Z value of each chromosome, and according to The Z value of each chromosome preliminarily determines whether there is aneuploidy in each chromosome; the first calculation module is configured to calculate the first concentration value of the fetal DNA in the sample to be tested according to the preliminary determined aneuploid chromosome; a calculation module, configured to calculate a second concentration value of the fetal DNA in the sample to be tested according to the X chromosome or methylation method; and a second judgment module: when the preliminary determination
  • the above device of the present application performs high-throughput sequencing of a sample to be tested on the peripheral blood free DNA of a pregnant woman by performing a detection module to obtain sequencing data containing all chromosomes, and then executes a first judgment module for all chromosomes in the sequenced data.
  • the above device of the present application can statistically confirm whether the fetal DNA concentrations obtained by the two calculation methods are equal, and the detection accuracy is greatly improved.
  • the foregoing module of the present application may be operated as a part of the device in a computing terminal, and the processor provided by the computer terminal may be used to execute the foregoing detecting module, the first determining module, the first computing module,
  • the technical solutions implemented by the second computing module, the second determining module, the first determining module, and the second determining module are obviously that the computer terminal is a hardware implemented device, and the processor is also a hardware device for executing the program.
  • each of the functional modules provided by the present application can be operated in a mobile terminal, a computer terminal or the like, or can be stored as part of a storage medium.
  • the second determining module further includes: a fitting calculation sub-module: for using the formula Standardizing the fetal DNA concentration difference to obtain M f ; wherein the first concentration value is f1 and the second concentration value is f2; the first fitting judgment sub-module: when M f is less than or equal to 4, Capable of fitting; second fitting judgment sub-module: in the case where M f is greater than 4, it is considered that the fitting cannot be performed.
  • M f is a parameter that reflects the degree of dispersion and is used to characterize whether the difference between the two concentration values is significant.
  • the first determining module further includes: a first coverage calculation sub-module: configured to calculate coverage in a form of being divided into windows in all the chromosomes in the sequencing data, to obtain pre-corrected coverage of each chromosome.
  • Single sequence calculation sub-module used to calculate the Z value of the number of single sequences of pregnant women in each window; copy number abnormal segment query sub-module: used to query the fragment of 300Kb or more in the sequencing data, and at 300Kb In the above fragment, more than 80% of the chromosome fragments in the window have Z CNV values greater than or equal to 4 or less than or equal to -4; copy number abnormal fragment determination sub-module: used for more than 300Kb which will be queried from the sequencing data.
  • the Z CNV value of the chromosome fragment is greater than or equal to 4 or less than or equal to -4 is determined as the copy number abnormal segment of the pregnant woman to be tested;
  • ⁇ first calculation sub-module used for inheritance in the fetus
  • the parameter ⁇ is calculated according to the calculation formula shown in the formula (1), and the parameter ⁇ refers to the copy number abnormal segment of the pregnant woman. Effect of the front cover body color correction,
  • m represents the effective length of the chromosome where the copy number abnormal fragment is located, and the unit is Mb; n represents the length of the abnormal number of copies of the pregnant woman, and the unit is Mb; cn represents the number of occurrences of the abnormal copy number of the pregnant woman;
  • Correction sub-module for use Correcting the pre-correction coverage of each chromosome to obtain the corrected coverage of each chromosome; Representing the pre-correction coverage of each chromosome, x' represents the corrected chromosome coverage of each chromosome; second coverage calculation sub-module: used to calculate the Z aneu value of each chromosome by using the corrected coverage of each chromosome; The euploid preliminary judgment sub-module is used to judge whether the chromosome has aneuploidy according to whether the Za ⁇ or value is greater than or equal to 3; the chromosomal aneuploidy preliminary determination sub-module: for the case where the Za ⁇ o value is greater than or equal to 3 , to determine the chromosome has aneuploidy.
  • the copy number abnormal segment query submodule and the copy number abnormal segment determining submodule are executed to first confirm that the pregnant woman has a copy number abnormality segment in the sample to be tested, and adopts the ⁇ first calculation submodule And the ⁇ second calculation sub-module respectively calculates the ⁇ value in different cases, and then performs the correction sub-module, and corrects the influence of the female parent on the fetal chromosome coverage by ⁇ , thereby obtaining the coverage that is not affected by the female parent, and further The calculation result of the second coverage calculation sub-module is made more accurate, so that the obtained chromosome aneuploidy is also more accurate.
  • the foregoing module of the present application may be operated as a part of the device in a computing terminal, and the first coverage calculation submodule, the single sequence calculation submodule, and the copy may be executed by using the processor provided by the computer terminal.
  • the number of abnormal segment query submodule, the copy number abnormal segment determining submodule, the ⁇ first computing submodule, the ⁇ second computing submodule, the correcting submodule, the second coverage calculating submodule, the first preliminary determining submodule, and the second Initially determining the technical solution implemented by the sub-module it is obvious that the computer terminal is a hardware-implemented device, and the processor is also a hardware device for executing the program.
  • the above various functional sub-modules provided by the present application may be operated in a mobile terminal, a computer terminal or the like, or may be stored as part of a storage medium.
  • the first coverage calculation sub-module may be appropriately adjusted according to the difference of the sequencing data on the basis of the conventional calculation module.
  • the first coverage calculation sub-module includes: a chromosome window segmentation unit: configured to slice all the chromosomes in the sequenced data into equal-sized windows; the first coverage calculation unit Unit: Used to calculate coverage in the form of windows of equal size to obtain pre-corrected coverage for each chromosome.
  • each window has a size of 100 Kb, and the degree of overlap between adjacent windows is 50%.
  • the chromosome window segmentation unit divides each window into a form of a size of 100 Kb, and then performs a first coverage calculation sub-unit, which is advantageous for obtaining relatively robust coverage, and on the other hand, increasing the overlap between the windows to facilitate detection of copy number anomalies.
  • the accuracy of the fragment enhances the detection efficiency of pregnant women's copy number abnormal fragments.
  • the single sequence calculation sub-module includes: a single sequence statistical unit for counting the number of single sequences of each window according to the sequencing depth of each sequence in the sequencing data; a single sequence correction unit: for The GC content and the ratio of each chromosome are corrected for the number of each single sequence, and the pre-corrected coverage of the number of each single sequence is obtained; the single-sequence Z-value calculation sub-unit: pre-correction coverage for the number of each single sequence The degree is normalized to obtain the Z value of the number of each single sequence.
  • the above single sequence calculation sub-module of the present application counts the number of single sequences of each window according to the sequencing depth of each sequence in the sequencing data by first running a single sequence statistical unit, and then executes a single sequence correction unit according to the GC content of each chromosome. And correcting the ratio of the number of each single sequence to obtain the pre-corrected coverage of the number of each single sequence, and then performing a single-sequence Z-value calculation sub-unit, and normalizing the pre-corrected coverage of the number of each single sequence, The Z value of the number of each single sequence is obtained.
  • the above unit is an appropriate adjustment based on the conventional calculation and correction unit in the field, and is a basis and premise for confirming the sub-module abnormal segment query sub-module for query and copy number abnormal segment confirmation sub-module, so as to accurately determine The existence of the abnormal fragment of the maternal DNA copy number in the sample is provided to provide a basis for judgment.
  • the above-mentioned copy number abnormal segment query sub-module and the confirmation sub-module of the present application query the segment of the region of 300 kb or more in the sequencing data and 80% of the window Z CNV value in the region is greater than or equal to 4 or less than or equal to -4.
  • the above-mentioned copy number abnormality confirmation sub-module of the present application can confirm the detected trusted pregnant woman copy number abnormal segment, and use these copy number abnormal segments to correct the Z value of the chromosome in which the chromosome is located, thereby avoiding the copy number due to the pregnant woman. The detection result of the abnormal segment is wrong and the judgment of the false negative is caused.
  • the Z aneu value is among them, Is the pre-correction coverage value obtained by the negative sample population according to the LOESS algorithm, and s is the negative sample population. Standard deviation.
  • the corrected Z aneu value calculated by the above formula can more accurately reflect the aneuploidy of the chromosome, so that the detection result is more accurate.
  • each module, sub-module, and unit in the foregoing apparatus of the present application may be operated as a part of the device in a computing terminal, and the modules provided by the computer terminal may be used to execute the foregoing modules, sub-modules, and
  • the technical solution implemented by the unit is obvious that the computer terminal is a hardware-implemented device, and the processor is also a hardware device for executing a program.
  • the various functional modules, sub-modules and units provided by the present application can be operated in a mobile terminal, a computer terminal or the like, or can be stored as part of a storage medium.
  • a kit for detecting chromosomal aneuploidy comprising: a detection reagent and a detection device: Qualcomm for peripheral blood free DNA of a pregnant woman to be tested Sequencing to obtain sequencing data containing all chromosomes; chromosomal aneuploidy preliminary judgment device: used to calculate all chromosomes in the sequenced data into a window, to obtain the Z value of each chromosome, and according to each chromosome The Z value initially determines whether there is aneuploidy in each chromosome; the first computing device: used to calculate the first concentration value of the fetal DNA in the sample to be tested according to the preliminary determined aneuploidy chromosome; the second computing device : for calculating the second concentration value of fetal DNA in the sample to be tested according to the method of X chromosome or methylation; chromosomal aneuploidy judging device: for preliminarily determining the ane
  • the detection reagent and the detection device may include various reagents or chemicals used in each steps of DNA extraction, isolation, detection, library construction, etc.; the detection device may include 1.5 ml EP tube, PCR tube, and pipetting The 96-well plate used in the gun and the machine, and the high-throughput sequencer used in the machine; the chromosomal aneuploidy preliminary judgment device, the first calculation device, the second calculation device, the chromosome aneuploidy judgment device, and the chromosome non-
  • the euploid determination apparatus includes various hardware modules stored on a specific storage medium and using a computer terminal or a mobile terminal to perform the above calculation, judgment or confirmation functions.
  • the above kit of the present application further recognizes a device comprising a chromosome aneuploidy by means of a chromosomal aneuploidy when it is judged to have chromosomal aneuploidy using a conventional chromosomal aneuploidy preliminary judgment device, by means of two commonly used
  • the calculation device further confirms the authenticity of the judgment result of the preliminary judgment apparatus, and the above kit of the present application greatly improves the detection accuracy compared with the prior art.
  • the chromosome aneuploidy judging device further includes: a fitting calculation component: for using the formula Standardizing the fetal DNA concentration difference to obtain M f ; wherein the first concentration value is f1 and the second concentration value is f2; the first fitting determining component: when M f is less than or equal to 4, Fitting; second fitting determining component: in the case where M f is greater than 4, then it is considered that the fitting is impossible.
  • the above-described fitting technique component, the first fitting determining component, and the second fitting determining component as part of the instrument can perform the above-described calculation and determination functions individually or assembled into an instrument, and thus the above-described components are also a component of the instrument.
  • the chromosomal aneuploidy preliminary judgment apparatus further includes: a first coverage calculation unit for calculating coverage of all chromosomes in the sequenced data in a window-divided form to obtain each chromosome Pre-correction coverage; single sequence calculation component: used to calculate the Z value of the number of single sequences of pregnant women in each window; copy number abnormal segment query component: used to query the fragment of 300Kb or more in the sequencing data, And in the fragment above 300Kb, the Z CNV value of the chromosome fragment in more than 80% of the window is greater than or equal to 4 or less than or equal to -4; the copy number abnormal fragment determining component is used for 300Kb which is to be queried from the sequencing data.
  • the above fragment and in the window of 80% or more, the fragment having the Z value of the chromosome fragment greater than or equal to 4 or less than -4 is determined as the copy number abnormal fragment of the pregnant woman to be tested; the first first calculating part: used for inheriting in the fetus
  • the parameter ⁇ is calculated according to the calculation formula shown in the formula (1), and the parameter ⁇ is the copy number abnormal segment of the pregnant woman.
  • ⁇ represents the effective length of the chromosome where the copy number abnormal fragment is located, and the unit is Mb; n represents the length of the abnormal number of copies of the pregnant woman, and the unit is Mb; cn represents the number of occurrences of the abnormal number of copy number of the pregnant woman; ⁇ second calculating part: used In the case of a chromosome in which the copy number of the ismeeted parent of the fetus is abnormal, the formula ⁇ is calculated as shown in the formula (2):
  • m represents the effective length of the chromosome where the copy number abnormal fragment is located, and the unit is Mb; n represents the length of the pregnant woman in the copy number abnormal segment, the unit is Mb; cn represents the number of occurrences of the abnormal copy number of the pregnant woman; f represents the periphery of the pregnant woman to be tested The concentration of fetal free DNA contained in blood free DNA and assuming that the concentration f of fetal free DNA is less than 50%; correcting component: for utilization Correcting the pre-correction coverage of each chromosome to obtain the corrected coverage of each chromosome; Representing the pre-correction coverage of each chromosome, x' represents the corrected chromosome coverage of each chromosome; the second coverage calculation component is used to calculate the Z aneu value of each chromosome by using the corrected coverage of each chromosome; Pluricity preliminary judgment component: for judging whether the chromosome has aneuploidy according to whether the Za ⁇ or value is greater than or equal to 3
  • the chromosomal aneuploidy preliminary judgment device increases the copy number abnormal segment query component and the copy number abnormal slice confirming component and the correcting component, and the copy number abnormal segment confirming component of the present application is not adopted
  • the calculating component calculates the influence of the copy number abnormal segment of the female parent on the fetal calculation coverage, and then corrects the influence of the copy number abnormal segment on calculating the coverage of each chromosome by the correcting component, thereby making the chromosome of the present application aneuploid
  • the initial judgment of the device is more accurate in the preliminary judgment of the aneuploidy of the chromosome.
  • the fetal DNA concentration in the calculation formula of the parameter ⁇ is a conventional calculation method in the art, and is specifically described above and will not be described here
  • the first coverage calculation means can be appropriately adjusted based on the difference of the sequencing data on the basis of the conventional calculation means of the present invention.
  • the first coverage calculation component is configured to calculate coverage for all chromosomes in the sequenced data to be divided into equal-sized windows to obtain pre-correction coverage of each chromosome. . Computing with such computing components facilitates relatively robust coverage.
  • each window has a size of 100 Kb, and the degree of overlap between adjacent windows is 50%. Calculate each window into a size of 100Kb, which is beneficial to obtain relatively robust coverage.
  • increasing the coverage between windows can improve the accuracy of detecting abnormal segments of copy number, thereby improving the abnormal copy number of pregnant women. The detection efficiency of the fragment.
  • the single sequence calculation means of the present application can be obtained by appropriate adjustment based on the difference between the quality of the sequencing data and the detection accuracy on the basis of the conventional single sequence calculation means.
  • the single sequence calculation component further includes: a single sequence statistical component for counting the number of single sequences of each window according to the sequencing depth of each sequence in the sequencing data; a single sequence of correcting components : used to correct the number of each single sequence according to the GC content and the ratio of each chromosome, to obtain the pre-correction coverage of the number of each single sequence; a single-sequence Z-value calculation component: for the number of each single sequence The pre-correction coverage is normalized to obtain the Z value of the number of each single sequence.
  • the above single sequence calculation component of the present application counts the number of single sequences of each window according to the sequencing depth of each sequence in the sequencing data by first running a single sequence statistical component, and then executes a single sequence correction component according to the GC content of each chromosome and The comparison rate corrects the number of each single sequence, obtains the pre-correction coverage of the number of each single sequence, and then performs a single-sequence Z-value calculation component, and normalizes the pre-correction coverage of each single sequence to obtain each The Z value of the number of single sequences.
  • the above components are appropriately adjusted based on conventional calculation and correction components in the art, and are queried in the copy number abnormal segment query component. And the basis and premise of confirming the copy number abnormal segment confirmation component, and providing a judgment basis for accurately determining the existence of the abnormal copy number of the parent DNA copy number in the sample to be tested.
  • the above-mentioned copy number abnormal segment query component and the confirmation component of the present application make the present application by querying "a region of 300 kb or more in the sequencing data and 80% of the window Z value of the region is greater than or equal to 4 or less than -4".
  • the above-mentioned copy number abnormality confirmation sub-module can confirm the detected authentic copy number of pregnant women, and use these copy number abnormal fragments to correct the Z value of the chromosome in which the chromosome is located, thereby avoiding the abnormal fragment of the copy number of the pregnant woman. The test result is wrong and the judgment of false negative is caused.
  • the Za ⁇ o value is in accordance with To calculate, among them, Is the pre-correction coverage value obtained by the negative sample population according to the LOESS algorithm, and s is the negative sample population. Standard deviation.
  • the corrected Z aneu value calculated by the above calculation component can more accurately reflect the aneuploidy of the chromosome, so that the detection result is more accurate.
  • the components and components included in the above kit including a conventional electronic calculator or can be operated as a part of the device in a computing terminal, and the processor provided by the computer terminal is used to execute the above components
  • the computer terminal is a hardware-implemented device
  • the processor is also a hardware device for executing the program.
  • the various functional elements or components or instruments provided herein can be operated in a mobile terminal, computer terminal, or similar computing device, or can be stored as part of a storage medium.
  • Peripheral blood derived from 1000 pregnant women samples, 10 ml/person were used to extract free DNA from plasma using the kit QIAamp DNA Blood Mini Kit (Qiagen, Germany, catlog #51106), and were isolated using the library construction kit of Illmumina.
  • the plasma DNA was constructed by library, and the library was subjected to quality control.
  • the quality control library was sequenced by Illumina 2500, and sequenced into single-end sequencing. The sequencing data of each sample with a length of 35 bp was obtained, and the sequencing data was obtained. The average number of single sequences in the middle is 5.2M;
  • the MPS method counts the coverage of each chromosome and calculates the Z value.
  • the Z values of the sample CT00026 and the sample AC01466 are 4.39 and 6.12, respectively, both of which are greater than 3.
  • the initial judgment of the chromosome 18 of the sample CT00026 is aneuploidy. There is aneuploidy on chromosome 13 of AC01466;
  • the first concentration f1 of fetal DNA is calculated by chromosome 18 of CT00026
  • the second concentration f2 of fetal DNA is calculated by the method of X chromosome
  • the fetal DNA concentration difference (f2-f1) calculated by the two methods is standardized.
  • Pass Obtaining the M f value, if the absolute value of M f is greater than 4, it is considered that the positive result of the aneuploidy of the sample on chromosome 18 is not true, as shown in the scatter plot shown in FIG. 3A, the lower arrow The point (square) of the finger is obviously deviated from the curve y x; as can be seen from the density distribution graph shown in Fig. 3B, the sample CT00026 appears prominently at the edge position of the normal distribution map, that is, the point indicated by the right arrow. (Orthogonal triangle).
  • Peripheral blood from a sample of 6615 pregnant women, 10 ml/person was used to extract free DNA from the plasma using the QIAamp DNA Blood Mini Kit (Qiagen, Germany, catlog #51106), and was isolated using the library construction kit of Illmumina.
  • the plasma DNA was constructed by library, and the library was subjected to quality control.
  • the quality control library was sequenced by Illumina 2500, and sequenced into single-end sequencing. The sequencing data of each sample with a length of 35 bp was obtained, and the sequencing data was obtained.
  • the average number of single sequences in the middle is 5.2M;
  • the number of the single sequences of each window is counted according to the sequencing depth of each sequence in the sequencing data of each sample; the number of each single sequence is calculated according to the GC content and the comparison ratio of each chromosome, and the number of each single sequence is obtained.
  • Pre-correction coverage normalize the pre-correction coverage of the number of each single sequence to obtain the Z value of the number of each single sequence, and according to the size of the Z value, whether the pregnant woman to be tested has a copy number abnormality fragment
  • the segment above 300Kb is considered to be Is the copy number abnormal segment of the sample to be tested
  • the Z values of chromosome 21 of sample EK01875 and sample BD01462 were 4.66. and 3.87, respectively.
  • the Za ⁇ o values of chromosome 21 of sample EK01875 and sample BD01462 are 2.36. and 1.83, respectively. There is no chromosome aneuploidy in the sample;
  • the first concentration f1 of the fetal DNA is calculated for the chromosome 21 of the remaining sample which is presumed to be a certain sample having chromosomal aneuploidy
  • the second concentration f2 of the fetal DNA is calculated by the methylation method
  • a device for detecting aneuploidy of a chromosome comprising:
  • a detection module for performing high-throughput sequencing of a sample of the peripheral blood free DNA of the pregnant woman to obtain sequencing data including all chromosomes including an instrument for sequencing DNA in the maternal plasma sample, which may include Illumina cBot instrument and Illumina's Genome AnalXzer, HiSeq2000 sequencer or HiSeq2500 sequencer or ABI's SOLiD series of sequencers;
  • the first judging module is configured to calculate all the chromosomes in the sequencing data into a window, obtain the Z value of each chromosome, and preliminarily judge whether each chromosome has aneuploidy according to the Z value of each chromosome;
  • the window is split into equal-sized windows for calculation or the window is sliced into equal sizes and adjacent to each other.
  • the calculation is performed in the form of a certain degree of overlap between the windows, for example, in the form of a window divided into 100 kb size, and the overlap between the two adjacent windows is up to 50% for the Z value calculation;
  • a first calculating module configured to calculate a first concentration value of the fetal DNA in the sample to be tested according to the initially determined chromosome of aneuploidy; and calculate the fetal DNA in the sample to be tested according to the chromosome with aneuploidy
  • concentration value is Calculate, among them, The ratio of the average single sequence number of the window representing the chromosomes 21, 18 or 13 to the average single sequence number of all windows;
  • a second calculating module configured to calculate a second concentration value of the fetal DNA in the sample to be tested according to the method of X chromosome or methylation; wherein, according to The formula is calculated, Indicates the ratio of the average single sequence number of the window on the X chromosome to the average single sequence number of all windows; or the DNA of the sample to be tested by methylation sensitive enzymes such as HhaI, BstUI (30U) and HpaII, the non-A of the female parent
  • the geneized gene is digested, and the gene of the fetal methylation is not digested, thereby detecting the fetal DNA content in the sample by Q-PCR;
  • the first determining module determines whether the chromosome has aneuploidy according to the Z value
  • the Z value calculated by the prior art without the maternal copy number abnormal segment corrected chromosome coverage may be used. Judging, it is also possible to determine the Z aneu value calculated by the chromosome coverage corrected by the abnormal copy of the maternal copy number.
  • the first determining module uses the Z aneu value calculated by the chromosome coverage corrected by the parent copy number abnormal segment to determine, the first determining module further includes:
  • a first coverage calculation sub-module configured to calculate coverage of all chromosomes in the sequenced data in a window-divided manner to obtain pre-correction coverage of each chromosome
  • a single sequence calculation sub-module for calculating the Z value of the number of single sequences of pregnant women in each window
  • Copy number anomaly fragment query sub-module used to query the fragment of 300Kb or more in the sequencing data, and in the fragment above 300Kb, the Z CNV value of the chromosome fragment in more than 80% of the window is greater than or equal to 4 or less than or equal to -4.
  • a copy number abnormal segment determining submodule for determining a segment of 300 Kb or more obtained from the sequenced data and having a Z CNV value of the chromosome segment greater than or equal to 4 or less than -4 in more than 80% of the windows Measuring the copy number abnormal segment of the pregnant woman;
  • the ⁇ first calculation sub-module is configured to calculate the parameter ⁇ according to a calculation formula as shown in the formula (1) in the case where the fetus inherits the copy number abnormal segment of the mother, and the parameter ⁇ refers to the copy number abnormal segment of the pregnant woman.
  • m represents the effective length of the chromosome where the copy number abnormal fragment is located, and the unit is Mb; n represents the length of the abnormal number of copies of the pregnant woman, and the unit is Mb; cn represents the number of occurrences of the abnormal copy number of the pregnant woman;
  • the second second calculation sub-module is used to calculate the formula parameter ⁇ as shown in the formula (2) in the case of a chromosome having an abnormal copy number of the urgedeted mother of the fetus:
  • Correction submodule for use Correcting the pre-correction coverage of each chromosome to obtain the corrected coverage of each chromosome; Representing the pre-correction coverage of each chromosome, x' represents the corrected chromosome coverage of each chromosome;
  • a second coverage calculation sub-module for calculating a Z aneu value of each chromosome by using the corrected coverage of each chromosome
  • a preliminary sub-module module for chromosomal aneuploidy for determining whether a chromosome has aneuploidy according to whether the Z CNV value is greater than or equal to 3;
  • the first chromosome aneuploidy preliminary determination sub-module is configured to determine that the chromosome has aneuploidy when the Z CNV value is greater than or equal to 3;
  • a kit for detecting aneuploidy of a chromosome comprising:
  • Detection reagents and detection instruments high-throughput sequencing of peripheral blood free DNA of pregnant women to be obtained to obtain sequencing data containing all chromosomes, and detection reagents may include steps of DNA extraction, isolation, detection, library construction, and the like.
  • the detection device may include a 1.5 ml EP tube, a PCR tube, a pipetting gun, a 96-well plate for use on the machine, and a high-throughput sequencer for use on the machine;
  • Chromosome aneuploidy preliminary judgment device used to calculate all the chromosomes in the sequenced data into a window, to obtain the Z value of each chromosome, and to determine whether there is aneuploidy of each chromosome based on the Z value of each chromosome.
  • the first computing device is configured to calculate a first concentration value of the fetal DNA in the sample to be tested according to the initially determined chromosome having aneuploidy;
  • a second computing device for calculating a second concentration value of fetal DNA in a sample to be tested according to a method of X chromosome or methylation
  • chromosome aneuploidy preliminary judgment apparatus including various storage on a specific storage medium, and using a computer terminal Or a hardware module that the mobile terminal performs to perform the above calculation, judgment or confirmation function.
  • the chromosomal aneuploidy judging device further comprises:
  • the first fitting determining component is considered to be capable of fitting if M f is less than or equal to 4.
  • the second fitting determining component in the case where M f is greater than 4, is considered to be incapable of fitting.
  • the above-described fitting technical component, the first fitting determining component, and the second fitting determining component as part of the instrument can be separately executed or assembled into an apparatus to perform the above-described calculation, judgment, and determination functions, and thus the above-described components are also a component of the instrument.
  • the present application is based on the theoretical calculation of the current sequencing data based on when the sample is determined to be chromosomal aneuploidy.
  • the detection method of the present application is capable of correcting a judgment error caused by data fluctuations that may occur during the calculation process, and improving the accuracy of the detection.
  • modules, elements or steps of the present application described above may be implemented by a general-purpose computing device, which may be centralized on a single computing device or distributed across multiple computing devices. Alternatively, they may be implemented by program code executable by the computing device, such that they may be stored in the storage device for execution by the computing device, or they may be separately fabricated into individual integrated circuit modules, or Multiple of these modules or steps are fabricated as a single integrated circuit module. Thus, the application is not limited to any particular combination of hardware and software.

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Abstract

一种染色体非整倍性的检测方法、装置和试剂盒。其中，方法包括以下步骤：对来源于孕妇外周血游离DNA的待测样本进行高通量测序，得到包含所有染色体的测序数据；对所有染色体以切分成窗口的形式进行计算，得到各染色体的Z aneu值，并根据各染色体的Z aneu值初步判断各染色体是否存在非整倍性；若初步判断染色体存在非整倍性，则进一步判断待测样本中胎儿DNA的第一浓度值与第二浓度值能否被曲线y＝x所拟合；若能够拟合；则确认染色体存在非整倍性；待测样本中胎儿DNA的第一浓度值根据初步判断的存在非整倍性的染色体计算得到；第二浓度值根据X染色体或甲基化的方法计算得到。该检测方法检测染色体非整倍性的准确度更高。

Description

检测染色体非整倍性的试剂盒、装置和方法 技术领域
本发明涉及生物医药领域，具体而言，涉及一种检测染色体非整倍性的试剂盒、装置和方法。
背景技术
胎儿游离DNA(cff-DNA)自从1997被Lo发现至今已经有将近20个年头，正是这项发现为许多无创的产前检测方法(NIPT)提供了可能。无创的产前检测方法主要有以下两方面优势：第一方面：NIPT无需承担任何流产风险，而临床上通过羊水穿刺和脐穿等有创方式进行的染色体核型分析则会带来约1/200的流产风险，也有研究表明过早的脐穿还可能造成胎儿位置倾斜；第一方面：NIPT可以最早于孕周8周时进行检测，更早地给出风险判断，减少引产给孕妇带来的风险。
正是这些优势使得无创产前相关的研究方法日新月异，应用范围越来越广，现有的方法比如NIPT胎儿染色体非整倍性检测，NIPT胎儿单基因病检测，NIPT胎儿CNV检测，NIPT胎儿全基因组检测，NIPT胎儿亲子鉴定等等。
目前，在所有NIPT的应用中，应用最广泛也相对最成熟的当属胎儿染色体非整倍性检测。在对胎儿染色体非整倍性检测的众多算法中，Chui于2008年发明的基于高通量测序(MPS)的方法被认为临床使用中合适的，已经展现了它的稳健性。对于唐氏综合征，假阳性率(FPR)可以达到0.443％，假阴性率(FNR)低至0.004％；对于爱德华综合症，FPR则为0.22％，FNR为0.025％。
虽然上述方法已经达到一个极低的错误率，但仍存在判断错误的风险。因此，仍需要对现有的方法进行改进，以尽可能降低检测的错误率。
发明内容
本申请的主要目的在于提供一种检测染色体非整倍性的试剂盒、装置和方法，以降低检测的假阳性率。
为了实现上述目的，根据本申请的一个方面，提供了一种检测染色体非整倍性的方法，方法包括以下步骤：对来源于孕妇外周血游离DNA的待测样本进行高通量测序，得到包含所有染色体的测序数据；对测序数据中的所有染色体以切分成窗口的形 式进行计算，得到各染色体的Zaneu值，并根据各染色体的Zaneu值初步判断各染色体是否存在非整倍性；若初步判断染色体存在非整倍性，则进一步判断待测样本中胎儿DNA的第一浓度值与第二浓度值是否能被曲线y＝x所拟合；若能够拟合，则确认染色体存在非整倍性；其中，第一浓度值根据初步判断的存在非整倍性的染色体计算得到；第二浓度值根据X染色体或甲基化的方法计算得到。
进一步地，判断第一浓度值与第二浓度值是否能被曲线y＝x所拟合的步骤中，将第一胎儿浓度记为f1，第二胎儿浓度记为f2，按照公式
Figure PCTCN2015078421-appb-000001
对胎儿的浓度差进行标准化处理，得到Mf，若Mf小于等于4，则认为能够拟合；否则，认为不能拟合。
进一步地，对测序数据中的所有染色体以切分成窗口的形式进行计算，得到各染色体的Zaneu值的步骤中，还包括：对测序数据中的所有染色体以切分成窗口的形式计算覆盖度，得到各染色体的矫正前覆盖度；对待测孕妇在各窗口中的单一序列的数量进行Z检验，得到ZCNV值，并根据ZCNV值大小得到待测孕妇的拷贝数异常片段；待测孕妇的拷贝数异常片段是指在测序数据中300Kb以上的片段，且在300Kb以上的片段中，80％以上的窗口中染色体片段的ZCNV值都大于等于4或小于等于-4的片段；利用待测孕妇的拷贝数异常片段对各染色体的矫正前覆盖度的影响，对各染色体的矫正前覆盖度进行矫正，得到各染色体的矫正后覆盖度；以及利用各染色体的矫正后覆盖度对各染色体进行Z检验，得到Zaneu值，并根据Zaneu值的绝对值是否大于等于3来判断染色体是否具有非整倍性；当Zaneu值的绝对值大于等于3时，则染色体具有非整倍性；其中，待测孕妇的拷贝数异常片段对各染色体的矫正前覆盖度的影响用参数α表示，当胎儿遗传了母体的拷贝数异常片段时，参数α的计算公式如式(1)：
Figure PCTCN2015078421-appb-000002
当胎儿未遗传母体的拷贝数异常片段时，参数α的计算公式如式(2)：
Figure PCTCN2015078421-appb-000003
在式(1)和式(2)中，m表示拷贝数异常片段所在的染色体的有效长度，单位为Mb；n表示待测孕妇的拷贝数异常片段的长度，单位为Mb；cn表示孕妇的拷贝数 异常片段出现的次数；在式(2)中，f表示待测孕妇的外周血游离DNA中所含的胎儿游离DNA的浓度且假定胎儿游离DNA的浓度f小于50％。
进一步地，对测序数据中的所有染色体以切分成相等大小的窗口的形式计算覆盖度，得到各染色体的覆盖度。
进一步地，每个窗口的大小为100Kb，且相邻两个窗口之间的重叠度为50％。
进一步地，对待测孕妇在各窗口中的单一序列的数量进行Z检验，得到ZCNV值，并根据ZCNV值得到待测孕妇的拷贝数异常片段的步骤包括：根据测序数据中各序列的测序深度，统计各窗口的单一序列的数量；根据各染色体的GC含量和比对率对各窗口的单一序列的数量进行计算，得到各窗口的单一序列的数量的矫正前覆盖度；以及对各窗口的单一序列的数量的矫正前覆盖度进行标准化处理，得到各窗口的单一序列的数量的ZCNV值，并根据ZCNV值的大小判断待测孕妇是否具有拷贝数异常的片段；当在测序数据中存在300Kb以上的片段，且在300Kb以上的片段中80％以上的窗口的单一序列的数量的ZCNV值都大于等于4或小于等于-4时，则认为300Kb以上的片段是待测孕妇的拷贝数异常片段。
进一步地，利用各染色体的矫正后覆盖度对各染色体的进行Z检验，得到Zaneu值的步骤中，Zaneu值按照
Figure PCTCN2015078421-appb-000004
来计算，其中，
Figure PCTCN2015078421-appb-000005
是根据LOESS算法，通过已知阴性样本群体得到的覆盖度值，s表示阴性样本群体里
Figure PCTCN2015078421-appb-000006
的标准差。
为了实现上述目的，根据本申请的另一个方面，提供了一种检测染色体非整倍性的装置，装置包括：检测模块：用于对来源对孕妇外周血游离DNA的待测样本进行高通量测序，得到包含所有染色体的测序数据；第一判断模块：用于对测序数据中的所有染色体以切分成窗口的形式进行计算，得到各染色体的Zaneu值，并根据各染色体的Zaneu值初步判断各染色体是否存在非整倍性；第一计算模块，用于根据初步判断的存在非整倍性的染色体计算得到待测样本中胎儿DNA的第一浓度值；第二计算模块，用于根据X染色体或甲基化的方法计算得到待测样本中胎儿DNA的第二浓度值；第二判断模块：用于在初步判断染色体存在非整倍性的情况下，则判断待测样本中胎儿DNA的第一浓度值与第二浓度值是否被曲线y＝x所拟合；第一确定模块，用于在第一浓度值与第二浓度值能被曲线y＝x所拟合的情形下，确定染色体存在非整倍性。
进一步地，第二判断模块中还包括：拟合计算子模块：用于按照公式
Figure PCTCN2015078421-appb-000007
对胎儿的DNA浓度差进行标准化处理，得到Mf；其中，第一浓度值为f1，第二浓度值为f2；第一拟合判断子模块：在Mf小于等于4的情况下，则认为能够拟合；第二拟合判断子模块：在Mf大于4的情况下，则认为不能拟合。
进一步地，第一判断模块还包括：第一覆盖度计算子模块：用于对测序数据中的所有染色体以切分成窗口的形式计算覆盖度，得到各染色体的矫正前覆盖度；单一序列计算子模块：用于对待测孕妇在各窗口中的单一序列的数量的Zaneu值进行计算；拷贝数异常片段查询子模块：用于在测序数据中查询300Kb以上的片段，且在300Kb以上的片段中，80％以上的窗口中染色体片段的ZCNV值都大于等于4或小于等于-4的片段；拷贝数异常片段确定子模块：用于将从测序数据中查询得到的300Kb以上的片段且在80％以上的窗口中染色体片段的ZCNV值都大于等于4或小于等于-4的片段确定为待测孕妇的拷贝数异常片段；α第一计算子模块：用于在胎儿遗传了母体的拷贝数异常片段的情况下，按照如式(1)所示的计算公式计算参数α，其中，参数α是指孕妇的拷贝数异常片段对各染色体的矫正前覆盖度的影响；
Figure PCTCN2015078421-appb-000008
m表示拷贝数异常片段所在染色体的有效长度，单位为Mb；n表示孕妇在拷贝数异常片段的长度，单位为Mb；cn表示孕妇的拷贝数异常片段出现的次数；α第二计算子模块：用于在胎儿未遗传母体的拷贝数异常的染色体的情况下，按照如式(2)所示的计算公式参数α：
Figure PCTCN2015078421-appb-000009
m表示拷贝数异常片段所在染色体的有效长度，单位为Mb；n表示孕妇在拷贝数异常片段的长度，单位为Mb；cn表示孕妇的拷贝数异常片段出现的次数；f表示待测孕妇的外周血游离DNA中所含的胎儿游离DNA的浓度且假定胎儿游离DNA的浓度f小于50％；矫正子模块：用于利用
Figure PCTCN2015078421-appb-000010
对各染色体的矫正前覆盖度进行矫正，得到各染色体的矫正后覆盖度；其中，
Figure PCTCN2015078421-appb-000011
代表各染色体的矫正前覆盖度，x'代表各染色体的矫正后染色体覆盖度；第二覆盖度计算子模块，用于利用各染色体的矫正后覆盖度 来计算各染色体的Zaneu值；染色体非整倍性初步判断子模块，用于根据Zaneu值是否大于等于3来判断染色体是否具有非整倍性；初步确定子模块，用于在Zaneu值大于等于3的情况下，确定染色体具有非整倍性。
进一步地，第一覆盖度计算子模块中还包括，染色体窗口切分单元，用于对测序数据中的所有染色体以切分成相等大小的窗口；第一覆盖度计算单元，用于以相等大小的窗口的形式计算覆盖度，以得到各染色体的矫正前覆盖度。
进一步地，染色体窗口切分单元中，每个窗口的大小为100Kb，且相邻两个窗口之间的覆盖度为50％。
进一步地，单一序列计算子模块包括，单一序列统计单元，用于根据测序数据中各序列的测序深度，统计各窗口的单一序列的数量；单一序列的矫正单元，用于根据各染色体的GC含量和比对率对各单一序列的数量进行矫正，得到各单一序列的数量的矫正前覆盖度；单一序列Zaneu值计算单元：用于对各单一序列的数量的矫正前覆盖度进行标准化处理，得到各单一序列的数量的Zaneu值。
进一步地，第二覆盖度计算子模块中Zaneu按照
Figure PCTCN2015078421-appb-000012
来计算，其中，
Figure PCTCN2015078421-appb-000013
是根据LOESS算法，通过阴性样本群体得到的矫正前覆盖度值，s表示阴性样本群体里
Figure PCTCN2015078421-appb-000014
的标准差。
根据本发明的又一方面，提供了一种检测染色体非整倍性的试剂盒，该试剂盒包括：检测试剂和检测器械：用于对待测孕妇的外周血游离DNA进行高通量测序，以得到包含所有染色体的测序数据；染色体非整倍性初步判断器械：用于对测序数据中的所有染色体以切分成窗口的形式进行计算，得到各染色体的Zaneu值，并根据各染色体的Zaneu值初步判断各染色体是否存在非整倍性；第一计算器械：用于根据初步判断的存在非整倍性的染色体计算得到待测样本中胎儿DNA的第一浓度值；第二计算器械：用于根据X染色体或甲基化的方法计算得到待测样本中胎儿DNA的第二浓度值；染色体非整倍性判断器械：用于在初步判断染色体存在非整倍性的情况下，则判断待测样本中胎儿DNA的第一浓度值与第二浓度值是否被y＝x曲线所拟合；染色体非整倍性确定器械，用于在第一浓度值与第二浓度值能被曲线y＝x所拟合的情形下，确定染色体的拷贝数存在非整倍性。
进一步地，染色体非整倍性判断器械中还包括：拟合计算部件：用于按照公式
Figure PCTCN2015078421-appb-000015
对胎儿的DNA浓度差进行标准化处理，得到Mf；其中，第一浓度值为f1，第二浓度值为f2；第一拟合确定部件：在Mf小于等于4的情况下，则认为能够拟合；第二拟合确定部件：在Mf大于4的情况下，则认为不能拟合。
进一步地，染色体非整倍性初步判断器械中，还包括：第一覆盖度计算部件：用于对测序数据中的所有染色体以切分成窗口的形式计算覆盖度，以得到各染色体的矫正前覆盖度；单一序列计算部件：用于对待测孕妇在各窗口中的单一序列的数量的Zaneu值进行计算；拷贝数异常片段查询部件：用于在测序数据中查询300Kb以上的片段，且在300Kb以上的片段中，80％以上的窗口中染色体片段的ZCNV值都大于等于4或小于等于-4的片段；拷贝数异常片段确定部件：用于将从测序数据中查询得到的300Kb以上的片段且在80％以上的窗口中染色体片段的ZCNV值都大于等于4或小于等于-4的片段确定为待测孕妇的拷贝数异常片段；α第一计算部件：用于在胎儿遗传了母体的拷贝数异常片段的情况下，按照如式(1)所示的计算公式计算参数α，参数α为孕妇的拷贝数异常片段对各染色体的矫正前覆盖度的影响，
Figure PCTCN2015078421-appb-000016
m表示拷贝数异常片段所在染色体的有效长度，单位为Mb；n表示孕妇在拷贝数异常片段的长度，单位为Mb；cn表示孕妇的拷贝数异常片段出现的次数；α第二计算部件：用于在胎儿未遗传母体的拷贝数异常的染色体的情况下，按照如式(2)所示的计算公式参数α：
Figure PCTCN2015078421-appb-000017
m表示拷贝数异常片段所在染色体的有效长度，单位为Mb；n表示孕妇在拷贝数异常片段的长度，单位为Mb；cn表示孕妇的拷贝数异常片段出现的次数；f表示待测孕妇的外周血游离DNA中所含的胎儿游离DNA的浓度且假定胎儿游离DNA的浓度f小于50％；矫正部件：用于利用
Figure PCTCN2015078421-appb-000018
对各染色体的矫正前覆盖度进行矫正，得到各染色体的矫正后覆盖度；其中，
Figure PCTCN2015078421-appb-000019
代表各染色体的矫正前覆盖度，x'代表各染色体的矫正后染色体覆盖度；第二覆盖度计算部件：用于利用各染色体的矫正后覆盖度来计 算各染色体的Zaneu值；染色体非整倍性初步判断部件：用于根据Zaneu值是否大于等于3来判断染色体是否具有非整倍性；初步确定部件：用于在Zaneu值大于等于3的情况下，确定染色体具有非整倍性。
进一步地，第一覆盖度计算部件中还包括：染色体窗口切分元件：用于对测序数据中的所有染色体以切分成相等大小的窗口；第一覆盖度计算子元件：用于以相等大小的窗口的形式计算覆盖度，以得到各染色体的校正前覆盖度。
进一步地，染色体窗口切分元件中，每个窗口的大小为100Kb，且相邻两个窗口之间的覆盖度为50％。
进一步地，单一序列计算部件包括：单一序列统计元件：用于根据测序数据中各序列的测序深度，统计各窗口的单一序列的数量；单一序列的矫正元件：用于根据各染色体的GC含量和比对率对各单一序列的数量进行矫正，得到各单一序列的数量的矫正前覆盖度；单一序列Z值计算元件：用于对各单一序列的数量的矫正前覆盖度进行标准化处理，得到各单一序列的数量的Zaneu值。
进一步地，第二覆盖度计算部件中，Zaneu按照
Figure PCTCN2015078421-appb-000020
来计算，其中，
Figure PCTCN2015078421-appb-000021
是根据LOESS算法，通过阴性样本群体得到的矫正前覆盖度值，s表示阴性样本群体里
Figure PCTCN2015078421-appb-000022
的标准差。
应用本申请的技术方案，通过在利用常规的检测染色体非整倍性的方法初步判断为具有染色体非整倍性时，进一步借助于两种常用的计算待测样本中的胎儿DNA浓度的方法得到的胎儿DNA浓度是否相等，来确认初步判断的结果的真实性。在判断两种方法计算得到的胎儿DNA浓度是否相等时，通过能否被曲线y＝x所拟合来判断，当第一浓度和第二浓度不相等时，说明此时的数据波动较大，数据不可信，进而判断得到的染色体的拷贝数存在非整倍性的结论也不可信，进而从统计学意义上确认两种计算方法得到的胎儿DNA浓度是否相等，从而判断结果的可靠性较高。上述方法增加了确认判断的步骤，相比现有技术的染色体非整倍性的检测方法，准确度得到了大大提高。
附图说明
构成本申请的一部分的说明书附图用来提供对本发明的进一步理解，本发明的示意性实施例及其说明用于解释本发明，并不构成对本发明的不当限定。在附图中：
图1示出了根据本申请的一种典型实施方式中染色体非整倍性的检测方法的流程示意图；
图2示出了根据本申请的一种典型实施方式中染色体非整倍性的检测装置的结构示意图；
图3A和图3B示出了本申请的实施例1中样本检测结果示意图，其中，图3A是散点图，图3B是密度分布图；以及
图4示出了根据本申请的实施例2对样本EK01875和BD01462在21号染色体上的非整倍性的矫正结果示意图。
具体实施方式
需要说明的是，在不冲突的情况下，本申请中的实施例及实施例中的特征可以相互组合。下面将参考附图并结合实施例来详细说明本申请。
本申请中，Z值是指统计学中的Z检验的计算值，是用于大样本(即样本容量大于30)平均值差异性检验的方法。它是用标准正态分布的理论来推断差异发生的概率，从而比较两个平均数的差异是否显著。
比对率是指窗口内的测序序列比对到基因组参考序列上的比率。由于测序序列可能同时比对到基因组参考序列上的多个位置，可能并不是唯一的测序序列，所以，窗口内的测序序列比对率是大于单一序列的比对率的。
标准化处理指对矫正后的值，做(x-u)/sd(x-u)，其中x为矫正后的值，u为x的均值，sd为标准差。
本申请的申请人通过对现有方法进行大量分析，发现至少存在以下三种导致NIPT判断错误的可能性：
首先，Lo在1998年发现cff-DNA是来源于胎盘的，这意味着如果胎盘出现嵌合(CPM)时，我们将难以通过NIPT的结果准确地估计胎儿情况，结果容易失准；其次，如果孕妇自身存在一定的CNV的话，基于MPS统计coverage并转化为Z值的方法将失准。因为当孕妇存在Duplication时，比对到染色体上的相对Unique Reads数将变多，而coverage的升高则会使Z值变大，从而增加假阳性的风险。反之，如果孕妇存在deletion时，Z值将降低，增加假阴性的风险。而且在之前的一些研究中也表明，CPM 和孕妇CNV是造成假阳性判断的重要原因。最后，在计算染色体coverage或是利用GC含量矫正coverage的过程中可能出现的数据波动情况，导致产生误差。
为此，在对上述染色体非整倍性判断错误的原因进行综合分析的基础上，本申请提出了一种检测染色体非整倍性的方法，如图1所示，该方法包括以下步骤：对来源于孕妇外周血游离DNA的待测样本进行高通量测序，得到包含所有染色体的测序数据；对测序数据中的所有染色体以切分成窗口的形式进行计算，得到各染色体的Zaneu值，并根据各染色体的Zaneu值初步判断各染色体是否存在非整倍性；若初步判断染色体存在非整倍性，则进一步判断待测样本中胎儿DNA的第一浓度值与第二浓度值能否被曲线y＝x所拟合；若能够拟合；则确认染色体存在非整倍性；待测样本中胎儿DNA的第一浓度值根据初步判断的存在非整倍性的染色体计算得到；待测样本中胎儿DNA的第二浓度值根据X染色体或甲基化的方法计算得到。
本申请的上述方法，通过在利用常规的检测染色体非整倍性的方法初步判断为具有染色体非整倍性时，进一步借助于两种常用的计算待测样本中的胎儿DNA浓度的方法得到的胎儿DNA浓度是否相等，来确认初步判断的结果的真实性。在判断两种方法计算得到的胎儿DNA浓度是否相等时，通过能否被曲线y＝x所拟合来判断，当第一浓度和第二浓度不相等时，说明此时的数据波动较大，数据不可信，进而判断得到的染色体的拷贝数存在非整倍性的结论也不可信，进而从统计学意义上确认两种计算方法得到的胎儿DNA浓度是否相等，从而判断结果的可靠性较高。本申请的上述方法增加了确认判断的步骤，相比现有技术的染色体非整倍性的检测方法，准确度得到了大大提高。
本申请的上述方法，在判断两种方法计算得到的胎儿DNA的浓度能否被曲线y＝x所拟合时，在本申请的上述教导下，本领域技术人员选择合适的计算公式或方法来进行上述判断。在本申请中，优选上述判断步骤为：将第一胎儿浓度记为f1，第二胎儿浓度记为f2，按照公式
Figure PCTCN2015078421-appb-000023
对胎儿的浓度差进行标准化处理，得到Mf，若Mf小于等于4，则认为能够拟合；否则，认为不能拟合。通过上述标准化处理得到的Mf来判断能否被曲线y＝x所拟合判断更准确。
在本申请的上述方法中，对测序数据中的所有染色体以切分成窗口的形式进行计算，得到各染色体的Z值的步骤中，可以利用本申请常规的方法计算得到各染色体的Z值，但为了使Z值更准确地体现染色体的非整倍性程度，在本申请一种优选的实施例中，本申请的该步骤还包括：对测序数据中的所有染色体以切分成窗口的形式计算 覆盖度，得到各染色体的矫正前覆盖度；对待测孕妇在各窗口中的单一序列的数量的Z值进行计算，并根据ZCNV值大小得到待测孕妇的拷贝数异常片段；待测孕妇的拷贝数异常片段是指在测序数据中300Kb以上的片段，且在300Kb以上的片段中，80％以上的窗口中染色体片段的ZCNV值都大于等于4或小于等于-4的片段；利用待测孕妇的拷贝数异常片段对各染色体的矫正前覆盖度的影响，对各染色体的矫正前覆盖度进行矫正，得到各染色体的矫正后覆盖度；以及利用各染色体的矫正后覆盖度计算各染色体的Zaneu值，并根据Zaneu值是否大于等于3来判断染色体是否具有非整倍性；当Zaneu值大于等于3时，则染色体具有非整倍性；当Zaneu值小于3时，则染色体不具有非整倍性；其中，待测孕妇的拷贝数异常片段对各染色体的矫正前覆盖度的影响用参数α表示，当胎儿遗传了母体的拷贝数异常片段时，参数α的计算公式如式(1)：
Figure PCTCN2015078421-appb-000024
当胎儿未遗传母体的拷贝数异常片段时，参数α的计算公式如式(2)：
Figure PCTCN2015078421-appb-000025
在式(1)和式(2)中，m表示拷贝数异常片段所在的染色体的有效长度，单位为Mb；n表示待测孕妇的拷贝数异常片段的长度，单位为Mb；cn表示孕妇的拷贝数异常片段出现的次数；在式(2)中，f表示待测孕妇的外周血游离DNA中所含的胎儿游离DNA的浓度且假定胎儿游离DNA的浓度f小于50％；并利用
Figure PCTCN2015078421-appb-000026
对各染色体的矫正前的覆盖度进行矫正，其中，
Figure PCTCN2015078421-appb-000027
代表各染色体的矫正前覆盖度，x'代表各染色体的矫正后染色体覆盖度。
本申请的上述方法在计算各染色体的Z值时，不是像现有技术中将测序数据中母本有拷贝数异常片段直接去除不予考虑(忽略胎儿在该区域的染色体的倍性)，而是通过筛选母本染色体上存在的特定大小的拷贝数异常片段，并在判断染色体是否存在非整倍性时，将该拷贝数异常片段对计算各染色体的覆盖度的影响去除，从而得到各染色体矫正后的覆盖度，从而使得本申请的方法所检测得到的染色体非整倍性的结果更准确。
上述公式中，待测孕妇的外周血游离DNA中所含的胎儿游离DNA的浓度f的计算方法为本领域常规的计算方法。比如，当胎儿为男性时，且当拷贝数异常片段在X 染色体时，胎儿游离DNA的浓度按照
Figure PCTCN2015078421-appb-000028
进行计算，其中，
Figure PCTCN2015078421-appb-000029
表示X染色体上的窗口平均单一序列数和所有窗口平均单一序列数之比；而当拷贝数异常片段在21、18或13号染色体时，胎儿游离DNA的浓度按照
Figure PCTCN2015078421-appb-000030
进行计算，其中，
Figure PCTCN2015078421-appb-000031
表示21、18或13号染色体的窗口平均单一序列数和所有窗口平均单一序列数之比。当胎儿为女性时,需要对孕妇外周血游离DNA进行特定基因的甲基化检测。原理是，某些特定基因在孕妇DNA中和胎儿DNA中的是否甲基化的形式不同。例如，胎儿和胎盘来源的RASSF1A(3号染色体)基因是高度甲基化的，而母亲自身来源的RASSF1A基因是非甲基化的，利用甲基化敏感酶，如HhaI、BstUI(30U)和HpaII处理cfDNA，非甲基化的基因将被消化掉，而甲基化的基因未被消化，由此通过Q-PCR可以检测胎儿cfDNA的含量，具体步骤可参考文献PLOS ONE 9:71-7(2014)，Quantification of Cell-Free DNA in Normal and Complicated Pregnancies:Overcoming Biological and Technical Issues。
在本申请的上述计算各染色体的Z值时，对胎儿DNA占待测样本DNA的浓度的要求与前述步骤中所提及的两种计算胎儿DNA浓度的方法相同，唯一不同的是，该步骤中不考虑染色体是否为非整倍性，而是采用两种方法中的任一种方法都行，算出的Z值不会影响染色体非整倍性的初步判断结果。即在该步骤中，胎儿DNA占待测样本DNA的浓度可以通过X染色体或甲基化的方法进行计算，而不一定需要根据存在非整倍性的染色体来进行浓度计算。而在前述的两种计算胎儿DNA浓度的方法中，由于是利用初步判断为具有非整倍性的染色体进行计算，因而必须利用该方法进行计算。
在本申请的上述方法中，在对各染色体的矫正前覆盖度进行计算时，由于将染色体切分成窗口的形式进行计算，能够获得一个相对稳健的染色体覆盖度。因此，在本申请中，优选对测序数据中的所有染色体以切分成相等大小的窗口的形式计算覆盖度，得到各染色体的矫正前覆盖度。
在本申请一种更优选的实施例中，在切分成窗口的形式进行计算覆盖度，每个窗口的大小为100Kb，且相邻两个窗口之间的覆盖度为50％。将每个窗口的大小控制在100Kb且将相邻两个窗口之间的重叠度度控制为50％，不仅能够获得一个相对稳健的染色体覆盖度，而且增加窗口之间的覆盖度可以提升检测拷贝数异常片段的精准度，进而提升孕妇拷贝数异常片段的检出效率。
在本申请的上述优选实施例中，在对待测孕妇在各窗口中的单一序列数的Z值进行计算，并根据Z值得到待测孕妇的拷贝数异常片段的步骤中，可以在常规的计算拷贝数异常片段的方法步骤基础上，根据测序数据质量或检测精度的不同，通过适当地调整拷贝数异常片段所应该满足的条件得到。在本申请一种优选的实施例中，对待测孕妇在各窗口中的单一序列数的ZCNV值进行计算，并根据ZCNV值得到待测孕妇的拷贝数异常片段的步骤包括：根据测序数据中各序列的测序深度，统计各窗口的单一序列的数量；根据各染色体的GC含量和比对率对各单一序列的数量进行矫正，得到各单一序列的数量的矫正前覆盖度；以及对各单一序列的数量的矫正前覆盖度进行标准化处理，得到各单一序列的数量的值，并根据ZCNV值的大小片段待测孕妇是否具有拷贝数异常的片段；当在测序数据中存在300Kb以上的片段，且在300Kb以上的片段中80％以上的窗口的单一序列的数量的ZCNV值都大于等于4或小于等于-4，则认为300Kb以上的片段是待测孕妇的拷贝数异常片段。
本申请的上述检测待测孕妇的拷贝数异常片段的步骤中，通过设定“至少为300kb的区域和该区域中80％的窗口Z值大于等于4或小于等于-4”的条件，使得本申请的上述检测步骤能够检出可信的孕妇拷贝数异常片段，并利用这些拷贝数异常片段对其所在的染色体的Z值进行修正，进而可以避免因孕妇拷贝数异常片段的检测结果错误而造成假阴性的判断。
在本申请的上述方法中，在通过利用孕妇的拷贝数异常片段的影响对各染色的Z值进行矫正的步骤中，利用各染色体的矫正后覆盖度来计算各染色体的Zaneu值的步骤中，Zaneu值按照
Figure PCTCN2015078421-appb-000032
来计算，其中，
Figure PCTCN2015078421-appb-000033
是根据LOESS算法，通过阴性样本群体得到的矫正前覆盖度值，s表示阴性样本群体里
Figure PCTCN2015078421-appb-000034
的标准差。通过上述公式计算得到的矫正后的Zaneu值能更准确地反映染色体的非整倍性，使得检测结果更准确。
在本申请另一种典型的实施方式中，提供了一种检测染色体非整倍性的装置，如图2所示，该装置包括以下模块：检测模块：用于对来源对孕妇外周血游离DNA的待测样本进行高通量测序，得到包含所有染色体的测序数据；第一判断模块：用于对测序数据中的所有染色体以切分成窗口的形式进行计算，得到各染色体的Z值，并根据各染色体的Z值初步判断各染色体是否存在非整倍性；第一计算模块，用于根据初步判断的存在非整倍性的染色体计算得到待测样本中胎儿DNA的第一浓度值；第二计算模块，用于根据X染色体或甲基化的方法计算得到待测样本中胎儿DNA的第二浓度值；第二判断模块：用于当初步判断染色体存在非整倍性时，则判断待测样本中 胎儿DNA的第一浓度值与第二浓度值是否被曲线y＝x所拟合；第一确定模块，用于在第一浓度值与第二浓度值能被曲线y＝x所拟合的情形下，确定染色体的拷贝数存在非整倍性。
本申请的上述装置通过执行检测模块对来源对孕妇外周血游离DNA的待测样本进行高通量测序，得到包含所有染色体的测序数据，然后执行第一判断模块，对对测序数据中的所有染色体以切分成窗口的形式进行计算，得到各染色体的Z值，并根据各染色体的Z值初步判断各染色体是否存在非整倍性；接着执行第一计算模块，根据初步判断的存在非整倍性的染色体计算得到待测样本中胎儿DNA的第一浓度值；同时执行第二计算模块，根据X染色体或甲基化的方法计算得到待测样本中胎儿DNA的第二浓度值；然后执行第二判断模块，在初步判断染色体存在非整倍性的情况下，判断待测样本中胎儿DNA的第一浓度值与第二浓度值是否被曲线y＝x所拟合；然后执行第一确定模块，在第一浓度值与第二浓度值能被曲线y＝x所拟合的情形下，确定染色体的拷贝数存在非整倍性。
上述装置通过增加第一计算模块和第二计算模块，得到两种方式计算的待测样本中胎儿DNA的浓度值，并通过第二判断模块判断两个浓度值能否被曲线y＝x所拟合，并由第一确定模块来确定染色体存在非整倍性。本申请的上述装置借助于两种常用的计算待测样本中的胎儿DNA浓度的方法得到的胎儿DNA浓度能否被曲线y＝x所拟合来进一步判断初步判断结果的真实性。当第一浓度值和第二浓度值能被曲线y＝x所拟合时，说明用于计算的数据的波动性小，初步判断的结果是真实的，进而通过第一确定模块确定染色体确实存在非整倍性；但两个浓度值不能被曲线y＝x所拟合时，说明此时的数据波动较大，数据不可信，进而判断得到的染色体的拷贝数存在非整倍性的结论也不可信，但这并不表明该染色体一定不存在非整倍性。本申请的上述装置能够从统计学意义上确认两种计算方法得到的胎儿DNA浓度是否相等，大大提高检测准确度。
此处要说明的是，本申请的上述模块作为装置的一部分可以运行在一个计算终端中，可以利用该计算机终端所提供的处理器来执行上述检测模块、第一判断模块、第一计算模块、第二计算模块、第二判断模块、第一确定模块以及第二确定模块所实现的技术方案，显而易见的是该计算机终端是硬件实现的设备，处理器也是用于执行程序的硬件装置。而且本申请所提供的各个功能模块可以在移动终端、计算机终端或者类似的运算装置中运行，也可以作为存储介质的一部分进行存储。
本申请的上述装置中，第二判断模块还包括：拟合计算子模块：用于按照公式
Figure PCTCN2015078421-appb-000035
对胎儿的DNA浓度差进行标准化处理，得到Mf；其中，第一浓度值为f1，第二浓度值为f2；第一拟合判断子模块：在Mf小于等于4的情况下，则认为能够拟合；第二拟合判断子模块：在Mf大于4的情况下，则认为不能拟合。
此处的Mf是体现离散程度的参数，用来表征两个浓度值之间的差别是否显著。通过上述拟合计算子模块和两个拟合判断子模块能够准确地判断待测样本中的胎儿浓度值是否相等，从而便于确定初步判断的染色体存在非整倍性的情况是否真实。
在本申请的上述装置中，第一判断模块还包括：第一覆盖度计算子模块：用于对测序数据中的所有染色体以切分成窗口的形式计算覆盖度，得到各染色体的矫正前覆盖度；单一序列计算子模块：用于对待测孕妇在各窗口中的单一序列的数量的Z值进行计算；拷贝数异常片段查询子模块：用于在测序数据中查询300Kb以上的片段，且在300Kb以上的片段中，80％以上的窗口中染色体片段的ZCNV值都大于等于4或小于等于-4的片段；拷贝数异常片段确定子模块：用于将从测序数据中查询得到的300Kb以上的片段且在80％以上的窗口中染色体片段的ZCNV值都大于等于4或小于等于-4的片段确定为待测孕妇的拷贝数异常片段；α第一计算子模块：用于在胎儿遗传了母体的拷贝数异常片段的情况下，按照如式(1)所示的计算公式计算参数α，参数α是指孕妇的拷贝数异常片段对各染色体的矫正前覆盖度的影响，
Figure PCTCN2015078421-appb-000036
m表示拷贝数异常片段所在染色体的有效长度，单位为Mb；n表示孕妇在拷贝数异常片段的长度，单位为Mb；cn表示孕妇的拷贝数异常片段出现的次数；
α第二计算子模块：用于在胎儿未遗传母体的拷贝数异常的染色体的情况下，按照如式(2)所示的计算公式参数α：
Figure PCTCN2015078421-appb-000037
m表示拷贝数异常片段所在染色体的有效长度，单位为Mb；n表示孕妇在拷贝数异常片段的长度，单位为Mb；cn表示孕妇的拷贝数异常片段出现的次数；f表示待测 孕妇的外周血游离DNA中所含的胎儿游离DNA的浓度且假定胎儿游离DNA的浓度f小于50％；
矫正子模块：用于利用
Figure PCTCN2015078421-appb-000038
对各染色体的矫正前覆盖度进行矫正，得到各染色体的矫正后覆盖度；其中，
Figure PCTCN2015078421-appb-000039
代表各染色体的矫正前覆盖度，x'代表各染色体的矫正后染色体覆盖度；第二覆盖度计算子模块：用于利用各染色体的矫正后覆盖度来计算各染色体的Zaneu值；染色体非整倍性初步判断子模块：用于根据Zaneu值是否大于等于3来判断染色体是否具有非整倍性；染色体非整倍性初步确定子模块：用于在Zaneu值大于等于3的情况下，确定染色体具有非整倍性。
本申请的上述第一判断模块中，通过执行拷贝数异常片段查询子模块和拷贝数异常片段确定子模块来首先确认待测样本中孕妇存在拷贝数异常的片段，并通过α第一计算子模块和α第二计算子模块分别计算在不同情况下的α值，然后执行矫正子模块，通过α将母本对胎儿染色体覆盖度的影响进行矫正，从而得到不受母本影响的覆盖度，进而使得第二覆盖度计算子模块的计算结果更准确，从而判断得到的染色体非整倍性也更准确。
需要说明的是，本申请的上述模块作为装置的一部分可以运行在一个计算终端中，可以利用该计算机终端所提供的处理器来执行上述第一覆盖度计算子模块、单一序列计算子模块、拷贝数异常片段查询子模块、拷贝数异常片段确定子模块、α第一计算子模块、α第二计算子模块、矫正子模块、第二覆盖度计算子模块、第一初步确定子模块以及第二初步确定子模块所实现的技术方案，显而易见的是该计算机终端是硬件实现的设备，处理器也是用于执行程序的硬件装置。而且本申请所提供的上述各个功能子模块可以在移动终端、计算机终端或者类似的运算装置中运行，也可以作为存储介质的一部分进行存储。
在本申请的上述装置中，第一覆盖度计算子模块可以在本领常规的计算模块基础上，根据测序数据的不同经过适当调整得到。在本申请一种优选的实施例中，上述第一覆盖度计算子模块包括：染色体窗口切分单元：用于对测序数据中的所有染色体以切分成相等大小的窗口；第一覆盖度计算子单元：用于以相等大小的窗口的形式计算覆盖度，以得到各染色体的校正前覆盖度。通过本申请的上述单元进行计算，能够得到相对稳健的覆盖度。
而且，显而易见的是，上述单元也是用于可以在移动过终端、计算机终端或类似的运算装置中运行，也可以作为存储介质的一部分进行存储，因而也是用于执行程序的硬件单元。
在本申请一种更优选的实施例中，上述染色体窗口切分单元中，每个窗口的大小为100Kb，且相邻两个窗口之间的重叠度为50％。染色体窗口切分单元将每个窗口分成100Kb的大小的形式，然后执行第一覆盖度计算子单元，利于得到相对稳健的覆盖度，另一方面增加窗口之间的重叠度便于提升检测拷贝数异常片段的精准度，进而提升孕妇拷贝数异常片段的检出效率。
在本申请的上述装置中，单一序列计算子模块包括：单一序列统计单元：用于根据测序数据中各序列的测序深度，统计各窗口的单一序列的数量；单一序列的矫正单元：用于根据各染色体的GC含量和比对率对各单一序列的数量进行矫正，得到各单一序列的数量的矫正前覆盖度；单一序列Z值计算子单元：用于对各单一序列的数量的矫正前覆盖度进行标准化处理，得到各单一序列的数量的Z值。
本申请的上述单一序列计算子模块，通过首先运行单一序列统计单元，根据测序数据中各序列的测序深度，统计各窗口的单一序列的数量，然后执行单一序列矫正单元，根据各染色体的GC含量和比对率对各单一序列的数量进行矫正，得到各单一序列的数量的矫正前覆盖度，接着执行单一序列Z值计算子单元，对各单一序列的数量的矫正前覆盖度进行标准化处理，得到各单一序列的数量的Z值。上述单元是在本领域常规的计算和矫正单元的基础上进行的适当调整，是在拷贝数异常片段查询子模块进行查询和拷贝数异常片段确认子模块进行确认的根据和前提，为准确确定待测样本中母本DNA拷贝数异常片段的存在提供判断依据。
而本申请的上述拷贝数异常片段查询子模块和确认子模块，通过查询“测序数据中300kb以上的区域且该区域中80％的窗口ZCNV值大于等于4或小于等于-4”的片段，使得本申请的上述拷贝数异常确认子模块能够确认检出的可信的孕妇拷贝数异常片段，并利用这些拷贝数异常片段对其所在的染色体的Z值进行修正，进而可以避免因孕妇拷贝数异常片段的检测结果错误而造成假阴性的判断。
在本申请的上述第二覆盖度计算子模块中，Zaneu值按照
Figure PCTCN2015078421-appb-000040
其中，
Figure PCTCN2015078421-appb-000041
是根据LOESS算法，通过阴性样本群体得到的矫正前覆盖度值，s表示阴性样本群体里
Figure PCTCN2015078421-appb-000042
的标准差。通过上述公式计算得到的矫正后的Zaneu值能更准确地反映染色体的非整倍性，使得检测结果更准确。
需要说明的是，本申请的上述装置中的各模块、子模块和单元作为装置的一部分可以运行在一个计算终端中，可以利用该计算机终端所提供的处理器来执行上述各模块、子模块和单元所实现的技术方案，显而易见的是该计算机终端是硬件实现的设备，处理器也是用于执行程序的硬件装置。而且本申请所提供的各个功能模块、子模块和单元可以在移动终端、计算机终端或者类似的运算装置中运行，也可以作为存储介质的一部分进行存储。
在本申请再一种典型的实施方式中，提供了一种检测染色体非整倍性的试剂盒，该试剂盒包括：检测试剂和检测器械：用于对待测孕妇的外周血游离DNA进行高通量测序，以得到包含所有染色体的测序数据；染色体非整倍性初步判断器械：用于对测序数据中的所有染色体以切分成窗口的形式进行计算，得到各染色体的Z值，并根据各染色体的Z值初步判断各染色体是否存在非整倍性；第一计算器械：用于根据初步判断的存在非整倍性的染色体计算得到待测样本中胎儿DNA的第一浓度值；第二计算器械：用于根据X染色体或甲基化的方法计算得到待测样本中胎儿DNA的第二浓度值；染色体非整倍性判断器械：用于在初步判断染色体存在非整倍性的情况下，则判断待测样本中胎儿DNA的第一浓度值与第二浓度值是否被y＝x曲线所拟合；染色体非整倍性确定器械，用于在第一浓度值与第二浓度值能被曲线y＝x所拟合的情形下，确定染色体的拷贝数存在非整倍性。
本申请的上述试剂盒中，检测试剂和检测器械可以包括DNA提取、分离、检测、文库构建等各步骤所用到的各种试剂或化学药品；检测器械可以包括1.5mlEP管、PCR管、移液枪、上机所用的96孔板以及上机所用到高通量测序仪等；染色体非整倍性初步判断器械、第一计算器械、第二计算器械、染色体非整倍性判断器械和染色体非整倍性确定器械，包括各种存储在特定存储介质上，并利用计算机终端或移动终端来来执行上述计算、判断或确认功能的硬件模块。
本申请的上述试剂盒，通过在利用常规的染色体非整倍性的初步判断器械判断为具有染色体非整倍性时，进一步通过包含染色体非整倍性确认器械，该器械通过借助于两种常用的计算器械，来进一步确认初步判断器械的判断结果的真实性，本申请的上述试剂盒相比现有技术，大大提高检测准确度。
本申请的上述试剂盒中，染色体非整倍性判断器械中还包括：拟合计算部件：用于按照公式
Figure PCTCN2015078421-appb-000043
对胎儿的DNA浓度差进行标准化处理，得到Mf；其中，第一浓度值为f1，第二浓度值为f2；第一拟合确定部件：在Mf小于等于4的情况下，则认为能够拟合；第二拟合确定部件：在Mf大于4的情况下，则认为不能拟合。上 述拟合技术部件、第一拟合确定部件和第二拟合确定部件作为器械的一部分，能够单独或组装成器械执行上述计算和确定功能，因此上述部件也是器械的一个组成。
在本申请的上述试剂盒中，染色体非整倍性初步判断器械还包括：第一覆盖度计算部件：用于对测序数据中的所有染色体以切分成窗口的形式计算覆盖度，以得到各染色体的矫正前覆盖度；单一序列计算部件：用于对待测孕妇在各窗口中的单一序列的数量的Z值进行计算；拷贝数异常片段查询部件：用于在测序数据中查询300Kb以上的片段，且在300Kb以上的片段中，80％以上的窗口中染色体片段的ZCNV值都大于等于4或小于等于-4的片段；拷贝数异常片段确定部件：用于将从测序数据中查询得到的300Kb以上的片段且在80％以上的窗口中染色体片段的Z值都大于等于4或小于等于-4的片段确定为待测孕妇的拷贝数异常片段；α第一计算部件：用于在胎儿遗传了母体的拷贝数异常片段的情况下，按照如式(1)所示的计算公式计算参数α，参数α为孕妇的拷贝数异常片段对各染色体的矫正前覆盖度的影响
Figure PCTCN2015078421-appb-000044
m表示拷贝数异常片段所在染色体的有效长度，单位为Mb；n表示孕妇在拷贝数异常片段的长度，单位为Mb；cn表示孕妇的拷贝数异常片段出现的次数；α第二计算部件：用于在胎儿未遗传母体的拷贝数异常的染色体的情况下，按照如式(2)所示的计算公式参数α：
Figure PCTCN2015078421-appb-000045
m表示拷贝数异常片段所在染色体的有效长度，单位为Mb；n表示孕妇在拷贝数异常片段的长度，单位为Mb；cn表示孕妇的拷贝数异常片段出现的次数；f表示待测孕妇的外周血游离DNA中所含的胎儿游离DNA的浓度且假定胎儿游离DNA的浓度f小于50％；矫正部件：用于利用
Figure PCTCN2015078421-appb-000046
对各染色体的矫正前覆盖度进行矫正，得到各染色体的矫正后覆盖度；其中，
Figure PCTCN2015078421-appb-000047
代表各染色体的矫正前覆盖度，x'代表各染色体的矫正后染色体覆盖度；第二覆盖度计算部件：用于利用各染色体的矫正后覆盖度来计算各染色体的Zaneu值；染色体非整倍性初步判断部件：用于根据Zaneu值是否大于等于3来判断染色体是否具有非整倍性；初步确定部件：用于在Zaneu值大于等于3的情况下，确定染色体具有非整倍性。
本申请的上述试剂盒中，染色体非整倍性初步判断器械通过增加了拷贝数异常片段查询部件和拷贝数异常片度确认部件以及矫正部件，且本申请的拷贝数异常片段确认部件不是采用现有技术将母本有拷贝数异常片段直接去除不予考虑的确认部件，而是通过筛选母本染色体上存在的特定大小的拷贝数异常片段来进行确认，并用α第一计算部件和α第二计算部件计算得到母本的拷贝数异常片段对胎儿计算覆盖度的影响，然后通过矫正部件将该拷贝数异常片段对计算各染色体的覆盖度的影响进行矫正，从而使得本申请的染色体非整倍性初步判断器械对染色体的非整倍性的初步判断结果更准确。本申请的上述试剂盒中，参数α的计算公式中的胎儿DNA浓度为本领域常规的计算方法，具体如前述，此处不再赘述。
在本申请的上述试剂盒中，第一覆盖度计算部件可以在本领常规的计算部件基础上，根据测序数据的不同经过适当调整得到。在本申请一种优选的实施例中，上述第一覆盖度计算部件是用于对测序数据中的所有染色体以切分成相等大小的窗口的形式计算覆盖度，以得到各染色体的矫正前覆盖度。利用这种计算部件进行计算便于得到相对稳健的覆盖度。
在本申请一种更优选的实施例中，上述第一覆盖度计算部件中，每个窗口的大小为100Kb，且相邻两个窗口之间的重叠度为50％。将每个窗口分成100Kb的大小的形式进行计算，一方面利于得到相对稳健的覆盖度，另一方面增加窗口之间的覆盖度可以提升检测拷贝数异常片段的精准度，进而提升孕妇拷贝数异常片段的检出效率。
在本申请的上述试剂盒中，本申请的单一序列计算部件可以在常规的单一序列计算部件的基础上，根据测序数据质量和检测精度的不同，通过适当调整得到。在本申请一种优选的实施例中，上述单一序列计算部件还包括：单一序列统计元件：用于根据测序数据中各序列的测序深度，统计各窗口的单一序列的数量；单一序列的矫正元件：用于根据各染色体的GC含量和比对率对各单一序列的数量进行矫正，得到各单一序列的数量的矫正前覆盖度；单一序列Z值计算元件：用于对各单一序列的数量的矫正前覆盖度进行标准化处理，得到各单一序列的数量的Z值。
本申请的上述单一序列计算部件，通过首先运行单一序列统计元件，根据测序数据中各序列的测序深度，统计各窗口的单一序列的数量，然后执行单一序列矫正元件，根据各染色体的GC含量和比对率对各单一序列的数量进行矫正，得到各单一序列的数量的矫正前覆盖度，接着执行单一序列Z值计算元件，对各单一序列的数量的矫正前覆盖度进行标准化处理，得到各单一序列的数量的Z值。上述元件是在本领域常规的计算和矫正元件的基础上进行的适当调整，是在拷贝数异常片段查询部件进行查询 和拷贝数异常片段确认部件进行确认的根据和前提，为准确确定待测样本中母本DNA拷贝数异常片段的存在提供判断依据。
而本申请的上述拷贝数异常片段查询部件和确认部件，通过查询“测序数据中300kb以上的区域且该区域中80％的窗口Z值大于等于4或小于等于-4”的片段，使得本申请的上述拷贝数异常确认子模块能够确认检出的可信的孕妇拷贝数异常片段，并利用这些拷贝数异常片段对其所在的染色体的Z值进行修正，进而可以避免因孕妇拷贝数异常片段的检测结果错误而造成假阴性的判断。
在本申请的上述试剂盒中，在第二覆盖度计算部件中，Zaneu值按照
Figure PCTCN2015078421-appb-000048
来计算，其中，
Figure PCTCN2015078421-appb-000049
是根据LOESS算法，通过阴性样本群体得到的矫正前覆盖度值，s表示阴性样本群体里
Figure PCTCN2015078421-appb-000050
的标准差。通过上述计算部件计算得到的矫正后的Zaneu值能更准确地反映染色体的非整倍性，使得检测结果更准确。
需要说明的是，上述试剂盒中所包含的元件、部件，包括常规的电子计算器或者可以作为器械的一部分运行在一个计算终端中，并利用该计算机终端所提供的处理器来执行上述元件、部件、器械所实现的技术方案，显而易见的是该计算机终端是硬件实现的设备，处理器也是用于执行程序的硬件器械。而且本申请所提供的各个功能元件或部件或器械可以在移动终端、计算机终端或者类似的运算装置中运行，也可以作为存储介质的一部分进行存储。
下面将结合具体的实施例进一步说明本申请的有益效果。
实施例1
取来源于1000个孕妇样本的外周血，10ml/人，分别用试剂盒QIAamp DNA Blood Mini Kit(Qiagen，德国，catlog#51106)提取血浆中的游离DNA，采用Illmumina公司的文库构建试剂盒对游离血浆DNA进行文库构建，并对文库进行质控检测；利用Illumina2500对质控合格的文库进行上机测序，测序为单端测序，得到每条序列的长度为35bp的各样本的测序数据，测序数据中平均单一序列的数量为5.2M；
将各样本的测序数据中的所有染色体切分成等大小的窗口，每个窗口的大小为100kb，两个窗口之间的重叠度为50％，然后参照Chui于2008年申请的基于高通量测序的MPS方法统计各染色体的覆盖度，并计算Z值，其中，样本CT00026和样本AC01466的Z值分别为4.39和6.12，均大于3，初步判断样本CT00026的18号染色体存在非整倍性，样本AC01466的13号染色体存在非整倍性；
进一步，通过CT00026的18号染色体计算胎儿DNA的第一浓度f1，通过X染色体的方法计算计算胎儿DNA的第二浓度f2；对两种方法计算的胎儿DNA浓度差(f2-f1)进行标准化处理，即通过
Figure PCTCN2015078421-appb-000051
得到Mf值，如果Mf绝对值大于4，则认为该样本在18号染色体存在非整倍性的阳性结果是不真实的，如图3A所示的散点图中，偏下的箭头所指的点(方形)，明显偏离曲线y＝x；从图3B所示的密度分布曲线图中也可以看出，样本CT00026明显出现在正态分布图的边缘位置，即右边箭头所示的点(正三角形)。
同样，对样本AC01466进行上述计算，得到样本AC01466对应的Mf值。具体检测结果见附图3A的散点图中偏上的箭头所指的点(星形)和附图3B的密度分布曲线图中的正态分布图右侧的偏左的箭头所示的点(倒三角形)。
从附图3A和图3B上均可以看出，样本CT00026和样本AC01466两种计算方法得到的样本中的胎儿DNA的浓度被y＝x曲线拟合度都较差，说明这两个样本在初步判断得出存在染色体非整倍性时的数据波动较大，判断结果不可信。
实施例2
取来源于6615个孕妇样本的外周血，10ml/人，分别用试剂盒QIAamp DNA Blood Mini Kit(Qiagen，德国，catlog#51106)提取血浆中的游离DNA，采用Illmumina公司的文库构建试剂盒对游离血浆DNA进行文库构建，并对文库进行质控检测；利用Illumina2500对质控合格的文库进行上机测序，测序为单端测序，得到每条序列的长度为35bp的各样本的测序数据，测序数据中平均单一序列的数量为5.2M；
将各样本的测序数据中的所有染色体切分成等大小的窗口，每个窗口的大小为100kb，两个窗口之间的重叠度为50％，然后参照Chui于2008年申请的基于高通量测序的MPS方法统计各染色体的矫正前覆盖度；
根据每个样本的测序数据中各序列的测序深度，统计各窗口的所述单一序列的数量；根据各染色体的GC含量和比对率对各单一序列的数量进行计算，得到各单一序列的数量的矫正前覆盖度；对各单一序列的数量的矫正前覆盖度进行标准化处理，得到各单一序列的数量的Z值，并根据Z值的大小片段所述待测孕妇是否具有拷贝数异常的片段；当在测序数据中存在300Kb以上的片段，且在300Kb以上的片段中80％以上的窗口的单一序列的数量的ZCNV值都大于等于4或小于等于-4，则认为该300Kb以上的片段是该待测样本的拷贝数异常片段；
利用公式(1)或公式(2)所示的待测孕妇的拷贝数异常片段对各染色体的矫正前覆盖度的影响，和公式
Figure PCTCN2015078421-appb-000052
对各染色体的覆盖度进行矫正，得到各染色体的矫正后覆盖度；其中，
Figure PCTCN2015078421-appb-000053
代表各染色体的矫正前覆盖度，x'代表各染色体的矫正后染色体覆盖度；
利用各染色体的矫正后覆盖度对各染色体进行Zaneu值计算，并根据Zaneu值是否大于等于3来判断染色体是否具有非整倍性；当Zaneu值大于等于3时，则染色体具有非整倍性。具体矫正结果见附图4；
从附图4中看出，在利用母本的染色体拷贝数异常片段对染色体的Z值矫正前(修正前)，样本EK01875和样本BD01462的21号染色体的Z值分别为4.66.和3.87，均大于3，利用母本的染色体拷贝数异常片段对染色体的Z值矫正前(修正后)，样本EK01875和样本BD01462的21号染色体的Zaneu值分别为2.36.和1.83，则初步判断上述两个样本不存在染色体非整倍性；
进一步，对剩余的样本中初步推断为具有染色体非整倍性的某一样本的21号染色体计算胎儿DNA的第一浓度f1，通过甲基化的方法计算计算胎儿DNA的第二浓度f2；对两种方法计算的胎儿DNA浓度差(f2-f1)进行标准化处理，即通过
Figure PCTCN2015078421-appb-000054
得到Mf值，如果Mf绝对值大于4，则认为剩余样本中的某一样本在21号染色体存在非整倍性的阳性结果是不真实的。若经两种计算方法得到的剩余样本中的胎儿DNA的浓度都能很好地被y＝x曲线拟合，说明该某一样本在初步判断得出存在染色体非整倍性时的判断结果可信，是真实的。
实施例3
一种检测检测染色体非整倍性的装置，包括：
检测模块，用于对来源对孕妇外周血游离DNA的待测样本进行高通量测序，得到包含所有染色体的测序数据；其中包括了对母体血浆样本中的DNA进行测序的仪器，可以包括Illumina的cBot仪器和Illumina的Genome AnalXzer、HiSeq2000测序仪或HiSeq2500测序仪或ABI公司的SOLiD系列的测序仪；
第一判断模块，用于对测序数据中的所有染色体以切分成窗口的形式进行计算，得到各染色体的Z值，并根据各染色体的Z值初步判断各染色体是否存在非整倍性；可以将窗口切分相等大小的窗口的形式进行计算或者将窗口切分成相等大小且相邻两 个窗口之间保持一定重叠度的形式进行计算，比如可以是切分成100kb大小的窗口的形式，相邻两个窗口之间的重叠度高达50％的形式来进行Z值计算；
第一计算模块，用于根据初步判断的存在非整倍性的染色体计算得到待测样本中胎儿DNA的第一浓度值；根据存在非整倍性的染色体计算得到待测样本中胎儿DNA的第一浓度值的方式为按照
Figure PCTCN2015078421-appb-000055
进行计算，其中，
Figure PCTCN2015078421-appb-000056
表示21、18或13号染色体的窗口平均单一序列数和所有窗口平均单一序列数之比；
第二计算模块，用于根据X染色体或甲基化的方法计算得到待测样本中胎儿DNA的第二浓度值；其中，按照
Figure PCTCN2015078421-appb-000057
公式进行计算，
Figure PCTCN2015078421-appb-000058
表示X染色体上的窗口平均单一序列数和所有窗口平均单一序列数之比；或者利用甲基化敏感酶，如HhaI、BstUI(30U)和HpaII处理待测样本的DNA，将母本的非甲基化的基因消化掉，而胎儿的甲基化的基因未被消化，由此通过Q-PCR可以检测样本中胎儿DNA的含量；
第二判断模块，用于当初步判断染色体存在非整倍性时，进一步判断待测样本中胎儿DNA的第一浓度值与第二浓度值是否被曲线y＝x所拟合；此处的拟合是统计学意义上的相等，即根据第一浓度值和第二浓度值是否都落在曲线y＝x上来判断两个浓度值是否相等；
第一确定模块，用于在第一浓度值与第二浓度值能被曲线y＝x所拟合的情形下，确定染色体的拷贝数存在非整倍性，该模块通过第一浓度值和第二浓度值落在曲线y＝x两侧可接受的范围内时，就进一步确定染色体存是在非整倍性。
优选地，上述第一判断模块在根据Z值判断染色体是否存在非整倍性时，可以采用现有技术中的不经过母本拷贝数异常片段矫正过的染色体覆盖度进行计算得到的Z值进行判断，也可以采用先经母本拷贝数异常片段矫正过的染色体覆盖度进行计算得到的Zaneu值进行判断。其中，在上述第一判断模块采用经母本拷贝数异常片段矫正过的染色体覆盖度进行计算得到的Zaneu值进行判断的情形下，该第一判断模块还包括：
第一覆盖度计算子模块，用于对测序数据中的所有染色体以切分成窗口的形式计算覆盖度，得到各染色体的矫正前覆盖度；
单一序列计算子模块，用于对待测孕妇在各窗口中的单一序列的数量的Z值进行计算；
拷贝数异常片段查询子模块：用于在测序数据中查询300Kb以上的片段，且在300Kb以上的片段中，80％以上的窗口中染色体片段的ZCNV值都大于等于4或小于等于-4的片段；
拷贝数异常片段确定子模块，用于将从测序数据中查询得到的300Kb以上的片段且在80％以上的窗口中染色体片段的ZCNV值都大于等于4或小于等于-4的片段确定为待测孕妇的拷贝数异常片段；
α第一计算子模块，用于在胎儿遗传了母体的拷贝数异常片段的情况下，按照如式(1)所示的计算公式计算参数α，参数α是指孕妇的拷贝数异常片段对各染色体的矫正前覆盖度的影响，
Figure PCTCN2015078421-appb-000059
m表示拷贝数异常片段所在染色体的有效长度，单位为Mb；n表示孕妇在拷贝数异常片段的长度，单位为Mb；cn表示孕妇的拷贝数异常片段出现的次数；
α第二计算子模块，用于在胎儿未遗传母体的拷贝数异常的染色体的情况下，按照如式(2)所示的计算公式参数α：
Figure PCTCN2015078421-appb-000060
m表示拷贝数异常片段所在染色体的有效长度，单位为Mb；n表示孕妇在拷贝数异常片段的长度，单位为Mb；cn表示孕妇的拷贝数异常片段出现的次数；f表示待测孕妇的外周血游离DNA中所含的胎儿游离DNA的浓度且假定胎儿游离DNA的浓度f小于50％；
矫正子模块，用于利用
Figure PCTCN2015078421-appb-000061
对各染色体的矫正前覆盖度进行矫正，得到各染色体的矫正后覆盖度；其中，
Figure PCTCN2015078421-appb-000062
代表各染色体的矫正前覆盖度，x'代表各染色体的矫正后染色体覆盖度；
第二覆盖度计算子模块，用于利用各染色体的矫正后覆盖度来计算各染色体的Zaneu值；
染色体非整倍性初步判断子模块，用于根据ZCNV值是否大于等于3来判断染色体是否具有非整倍性；
第一染色体非整倍性初步确定子模块，用于在ZCNV值大于等于3的情况下，确定染色体具有非整倍性；
第二染色体非整倍性初步确定子模块，用于在ZCNV值小于3的情况下，确定染色体不具有非整倍性。
实施例4
一种检测检测染色体非整倍性的试剂盒，包括：
检测试剂和检测器械：用于对待测孕妇的外周血游离DNA进行高通量测序，以得到包含所有染色体的测序数据，检测试剂可以包括DNA提取、分离、检测、文库构建等各步骤所用到的各种试剂或化学药品；检测器械可以包括1.5mlEP管、PCR管、移液枪、上机所用的96孔板以及上机所用到高通量测序仪等；
染色体非整倍性初步判断器械：用于对测序数据中的所有染色体以切分成窗口的形式进行计算，得到各染色体的Z值，并根据各染色体的Z值初步判断各染色体是否存在非整倍性；
第一计算器械：用于根据初步判断的存在非整倍性的染色体计算得到待测样本中胎儿DNA的第一浓度值；
第二计算器械：用于根据X染色体或甲基化的方法计算得到待测样本中胎儿DNA的第二浓度值；
染色体非整倍性判断器械：用于在初步判断染色体存在非整倍性的情况下，则判断待测样本中胎儿DNA的第一浓度值与第二浓度值是否被y＝x曲线所拟合；
染色体非整倍性确定器械，用于在第一浓度值与第二浓度值能被曲线y＝x所拟合的情形下，确定染色体的拷贝数存在非整倍性。
上述染色体非整倍性初步判断器械、第一计算器械、第二计算器械、染色体非整倍性判断器械和染色体非整倍性确定器械，包括各种存储在特定存储介质上，并利用计算机终端或移动终端来来执行上述计算、判断或确认功能的硬件模块。
优选地，在染色体非整倍性判断器械中还包括：
拟合计算部件，用于按照公式
Figure PCTCN2015078421-appb-000063
对胎儿的DNA浓度差进行标准化处理，得到Mf；其中，第一浓度值为f1，第二浓度值为f2；
第一拟合确定部件，在Mf小于等于4的情况下，则认为能够拟合；
第二拟合确定部件，在Mf大于4的情况下，则认为不能拟合。
上述拟合技术部件、第一拟合确定部件和第二拟合确定部件作为器械的一部分，能够单独执行或组装成器械执行上述计算、判断和确定功能，因此上述部件也是器械的一个组成。
从以上的描述中，可以看出，本申请上述的实施例实现了如下技术效果：本申请通过基于当样本判定为染色体非整倍性时，根据理论上，利用目前的测序数据计算得到的待测样本中胎儿DNA的浓度应该是相同的，即可以被曲线y＝x很好的拟合这一思想，对检测结果为阳性的样本，通过上述两种方法计算得到的胎儿DNA浓度差进行标准化处理，并根据标准化处理后的离散程度来进一步确定染色体是否真的存在非整倍性。本申请的这种检测方法能够修正因在计算过程中可能出现的数据波动而导致的判断错误，提高检测的准确性。
而且，从上述实施例也可以看出，在对染色体的非整倍性进行初步判断的过程中，先将母本拷贝数异常片段的存在对染色体非整倍性判断的影响进行矫正，使得本申请在初步判断的结果相比现有技术更准确的基础上，进一步通过两种计算方法得到的待测样本中的胎儿DNA浓度是否相同，对初步判断为存在非整倍性的染色体进行再次确认，进一步提高了检测结果的准确度。
显然，本领域的技术人员应该明白，上述的本申请的一些模块、元件或一些步骤可以用通用的计算装置来实现，它们可以集中在单个的计算装置上，或者分布在多个计算装置所组成的网络上，可选地，它们可以用计算装置可执行的程序代码来实现，从而，可以将它们存储在存储装置中由计算装置来执行，或者将它们分别制作成各个集成电路模块，或者将它们中的多个模块或步骤制作成单个集成电路模块来实现。这样，本申请不限制于任何特定的硬件和软件结合。
以上所述仅为本申请的优选实施例而已，并不用于限制本申请，对于本领域的技术人员来说，本申请可以有各种更改和变化。凡在本申请的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本申请的保护范围之内。

Claims (21)

  1. 一种检测染色体非整倍性的方法，其特征在于，所述方法包括以下步骤：
    对来源于孕妇外周血游离DNA的待测样本进行高通量测序，得到包含所有染色体的测序数据；
    对所述测序数据中的所有染色体以切分成窗口的形式进行计算，得到各所述染色体的Zaneu值，并根据各所述染色体的Zaneu值初步判断各所述染色体是否存在非整倍性；
    若初步判断所述染色体存在非整倍性，则进一步判断所述待测样本中胎儿DNA的第一浓度值与第二浓度值是否能被曲线y＝x所拟合；
    若能够拟合，则确认所述染色体存在非整倍性；
    其中，所述第一浓度值根据所述初步判断的存在非整倍性的所述染色体计算得到；所述第二浓度值根据X染色体或甲基化的方法计算得到。
  2. 根据权利要求1所述的方法，其特征在于，判断所述第一浓度值与所述第二浓度值是否能被曲线y＝x所拟合的步骤中，将所述第一胎儿浓度记为f1，所述第二胎儿浓度记为f2，按照公式
    Figure PCTCN2015078421-appb-100001
    对胎儿的浓度差进行标准化处理，得到Mf，若Mf小于等于4，则认为能够拟合；否则，认为不能拟合。
  3. 根据权利要求1所述的方法，其特征在于，对所述测序数据中的所有染色体以切分成窗口的形式进行计算，得到各所述染色体的Zaneu值的步骤中，还包括：
    对所述测序数据中的所有染色体以切分成窗口的形式计算覆盖度，得到各所述染色体的矫正前覆盖度；
    对所述待测孕妇在各所述窗口中的单一序列的数量进行Z检验，得到ZCNV值，并根据所述ZCNV值大小得到所述待测孕妇的拷贝数异常片段；所述待测孕妇的拷贝数异常片段是指在所述测序数据中300Kb以上的片段，且在所述300Kb以上的片段中，80％以上的窗口中染色体片段的ZCNV值都大于等于4或小于等于-4的片段；
    利用所述待测孕妇的拷贝数异常片段对各所述染色体的矫正前覆盖度的影响，对各所述染色体的矫正前覆盖度进行矫正，得到各所述染色体的矫正后覆盖度；以及
    利用各所述染色体的矫正后覆盖度对各所述染色体进行Z检验，得到Zaneu值，并根据所述Zaneu值的绝对值是否大于等于3来判断所述染色体是否具有非整倍性；当所述Zaneu值的绝对值大于等于3时，则所述染色体具有非整倍性；
    其中，所述待测孕妇的拷贝数异常片段对各所述染色体的矫正前覆盖度的影响用参数α表示，
    当胎儿遗传了母体的所述拷贝数异常片段时，所述参数α的计算公式如式(1)：
    Figure PCTCN2015078421-appb-100002
    当胎儿未遗传母体的所述拷贝数异常片段时，所述参数α的计算公式如式(2)：
    Figure PCTCN2015078421-appb-100003
    在所述式(1)和式(2)中，m表示所述拷贝数异常片段所在的染色体的有效长度，单位为Mb；n表示所述待测孕妇的所述拷贝数异常片段的长度，单位为Mb；cn表示所述孕妇的所述拷贝数异常片段出现的次数；
    在所述式(2)中，f表示所述待测孕妇的外周血游离DNA中所含的胎儿游离DNA的浓度且假定所述胎儿游离DNA的浓度f小于50％；
    并利用
    Figure PCTCN2015078421-appb-100004
    对各所述染色体的矫正前覆盖度进行矫正，其中，
    Figure PCTCN2015078421-appb-100005
    代表各所述染色体的矫正前覆盖度，x'代表各所述染色体的矫正后染色体覆盖度。
  4. 根据权利要求1或3所述的方法，其特征在于，对所述测序数据中的所有染色体以切分成相等大小的窗口的形式计算覆盖度，得到各所述染色体的覆盖度。
  5. 根据权利要求4所述的方法，其特征在于，每个所述窗口的大小为100Kb，且相邻两个所述窗口之间的重叠度为50％。
  6. 根据权利要求3所述的方法，其特征在于，对所述待测孕妇在各所述窗口中的单一序列的数量进行Z检验，得到ZCNV值，并根据所述ZCNV值得到所述待测孕妇的拷贝数异常片段的步骤包括：
    根据所述测序数据中各序列的测序深度，统计各所述窗口的所述单一序列的数量；
    根据各所述染色体的GC含量和比对率对各所述窗口的所述单一序列的数量进行计算，得到各所述窗口的所述单一序列的数量的矫正前覆盖度；以及
    对各所述窗口的所述单一序列的数量的矫正前覆盖度进行标准化处理，得到各所述窗口的所述单一序列的数量的ZCNV值，并根据所述ZCNV值的大小判断所述待测孕妇是否具有所述拷贝数异常的片段；
    当在所述测序数据中存在300Kb以上的片段，且在所述300Kb以上的片段中80％以上的窗口的所述单一序列的数量的ZCNV值都大于等于4或小于等于-4时，则认为所述300Kb以上的片段是所述待测孕妇的拷贝数异常片段。
  7. 根据权利要求3所述的方法，其特征在于，利用各所述染色体的矫正后覆盖度对各所述染色体的进行Z检验，得到Zaneu值的步骤中，所述Zaneu值按照
    Figure PCTCN2015078421-appb-100006
    来计算，其中，
    Figure PCTCN2015078421-appb-100007
    是根据LOESS算法，通过已知阴性样本群体得到的矫正前覆盖度；s表示阴性样本群体里
    Figure PCTCN2015078421-appb-100008
    的标准差。
  8. 一种检测染色体非整倍性的装置，其特征在于，所述装置包括：
    测序数据检测模块：用于对待测孕妇的外周血游离DNA进行高通量测序，以得到包含所有染色体的测序数据；
    第一判断模块：用于对所述测序数据中的所有染色体以切分成窗口的形式进行计算，得到各所述染色体的Zaneu值，并根据各所述染色体的Zaneu值初步判断各所述染色体是否存在非整倍性；
    第一计算模块，用于根据所述初步判断的存在非整倍性的所述染色体计算得到待测样本中胎儿DNA的第一浓度值；
    第二计算模块，用于根据X染色体或甲基化的方法计算得到所述待测样本中胎儿DNA的第二浓度值；
    第二判断模块：用于在初步判断所述染色体存在非整倍性的情况下，判断所述待测样本中胎儿DNA的所述第一浓度值与所述第二浓度值是否被曲线y＝x所拟合；
    第一确定模块，用于在所述第一浓度值与所述第二浓度值能被曲线y＝x所拟合的情形下，确定所述染色体存在非整倍性。
  9. 根据权利要求8所述的装置，其特征在于，所述第二判断模块中还包括：
    拟合计算子模块：用于按照公式
    Figure PCTCN2015078421-appb-100009
    对胎儿的DNA浓度差进行标准化处理，得到Mf；其中，所述第一浓度值为f1，所述第二浓度值为f2；
    第一拟合判断子模块：在Mf小于等于4的情况下，则认为能够拟合；
    第二拟合判断子模块：在Mf大于4的情况下，则认为不能拟合。
  10. 根据权利要求8所述的装置，其特征在于，所述第一判断模块还包括：
    第一覆盖度计算子模块：用于对所述测序数据中的所有染色体以切分成窗口的形式计算覆盖度，得到各所述染色体的矫正前覆盖度；
    单一序列计算子模块：用于对所述待测孕妇在各所述窗口中的单一序列的数量进行Z值检验；
    拷贝数异常片段查询子模块：用于在所述测序数据中查询300Kb以上的片段，且在所述300Kb以上的片段中，80％以上的窗口中染色体片段的ZCNV值都大于等于4或小于等于-4的片段；
    拷贝数异常片段确定子模块：用于将从所述测序数据中查询得到的所述300Kb以上的片段且在80％以上的窗口中染色体片段的ZCNV值都大于等于4或小于等于-4的片段确定为待测孕妇的拷贝数异常片段；
    α第一计算子模块：用于在胎儿遗传了母体的拷贝数异常片段的情况下，按照如式(1)所示的计算公式计算参数α，其中，所述参数α是指孕妇的拷贝数异常片段对各所述染色体的矫正前覆盖度的影响；
    Figure PCTCN2015078421-appb-100010
    m表示所述拷贝数异常片段所在染色体的有效长度，单位为Mb；n表示所述孕妇在所述拷贝数异常片段的长度，单位为Mb；cn表示所述孕妇的所述拷贝数异常片段出现的次数；
    α第二计算子模块：用于在胎儿未遗传母体的拷贝数异常的染色体的情况下，按照如式(2)所示的计算公式所述参数α：
    Figure PCTCN2015078421-appb-100011
    m表示所述拷贝数异常片段所在染色体的有效长度，单位为Mb；n表示所述孕妇在所述拷贝数异常片段的长度，单位为Mb；cn表示所述孕妇的所述拷贝数异常片段出现的次数；f表示所述待测孕妇的外周血游离DNA中所含的胎儿游离DNA的浓度且假定所述胎儿游离DNA的浓度f小于50％；
    矫正子模块：用于利用
    Figure PCTCN2015078421-appb-100012
    对各所述染色体的矫正前覆盖度进行矫正，得到各所述染色体的矫正后覆盖度；其中，
    Figure PCTCN2015078421-appb-100013
    代表各所述染色体的矫正前覆盖度，x'代表各所述染色体的矫正后染色体覆盖度；
    第二覆盖度计算子模块：用于利用各所述染色体的矫正后覆盖度来计算各染色体的Zaneu值；
    染色体非整倍性初步判断子模块：用于根据所述Zaneu值是否大于等于3来判断所述染色体是否具有非整倍性；
    初步确定子模块：用于在所述Zaneu值大于等于3的情况下，确定所述染色体具有非整倍性。
  11. 根据权利要求10所述的装置，其特征在于，所述第一覆盖度计算子模块中还包括：
    染色体窗口切分单元：用于对所述测序数据中的所有染色体以切分成相等大小的窗口；
    第一覆盖度计算单元：用于以所述相等大小的窗口的形式计算覆盖度，以得到各所述染色体的矫正前覆盖度。
  12. 根据权利要求11所述的装置，其特征在于，所述染色体窗口切分单元中，每个所述窗口的大小为100Kb，且相邻两个所述窗口之间的覆盖度为50％。
  13. 根据权利要求10所述的装置，其特征在于，所述单一序列计算子模块包括：
    单一序列统计单元：用于根据所述测序数据中各序列的测序深度，统计各所述窗口的单一序列的数量；
    单一序列的矫正单元：用于根据各所述染色体的GC含量和比对率对各所述单一序列的数量进行矫正，得到各所述单一序列的数量的矫正前覆盖度；
    单一序列ZCNV值计算单元：用于对各所述单一序列的数量的矫正前覆盖度进行标准化处理，得到各所述单一序列的数量的ZCNV值。
  14. 根据权利要求10所述的装置，其特征在于，所述第二覆盖度计算子模块中所述Zaneu按照
    Figure PCTCN2015078421-appb-100014
    来计算，其中，
    Figure PCTCN2015078421-appb-100015
    是根据LOESS算法，通过阴性样本群体得到的矫正前覆盖度值，s表示阴性样本群体里
    Figure PCTCN2015078421-appb-100016
    的标准差。
  15. 一种检测染色体非整倍性的试剂盒，其特征在于，所述试剂盒包括：
    检测试剂和检测器械：用于对待测孕妇的外周血游离DNA进行高通量测序，以得到包含所有染色体的测序数据；
    染色体非整倍性初步判断器械：用于对所述测序数据中的所有染色体以切分成窗口的形式进行计算，得到各所述染色体的Zaneu值，并根据各所述染色体的Zaneu值初步判断各所述染色体是否存在非整倍性；
    第一计算器械：用于根据所述初步判断的存在非整倍性的所述染色体计算得到待测样本中胎儿DNA的第一浓度值；
    第二计算器械：用于根据X染色体或甲基化的方法计算得到所述待测样本中胎儿DNA的第二浓度值；
    染色体非整倍性判断器械：用于在初步判断所述染色体存在非整倍性的情况下，判断所述待测样本中胎儿DNA的第一浓度值与第二浓度值是否被y＝x曲线所拟合；
    染色体非整倍性确定器械，用于在所述第一浓度值与所述第二浓度值能被曲线y＝x所拟合的情形下，确定所述染色体的拷贝数存在非整倍性。
  16. 根据权利要求15所述的试剂盒，其特征在于，所述染色体非整倍性判断器械中还包括：
    拟合计算部件：用于按照公式
    Figure PCTCN2015078421-appb-100017
    对胎儿的DNA浓度差进行标准化处理，得到Mf；其中，所述第一浓度值为f1，所述第二浓度值为f2；
    第一拟合确定部件：在Mf小于等于4的情况下，则认为所述能够拟合；
    第二拟合确定部件：在Mf大于4的情况下，则认为不能拟合。
  17. 根据权利要求15所述的试剂盒，其特征在于，所述染色体非整倍性初步判断器械中，还包括：
    第一覆盖度计算部件：用于对所述测序数据中的所有染色体以切分成窗口的形式计算覆盖度，以得到各所述染色体的矫正前覆盖度；
    单一序列计算部件：用于对所述待测孕妇在各所述窗口中的单一序列的数量的Z值进行计算；
    拷贝数异常片段查询部件：用于在所述测序数据中查询300Kb以上的片段，且在所述300Kb以上的片段中，80％以上的窗口中染色体片段的ZCNV值都大于等于4或小于等于-4的片段；
    拷贝数异常片段确定部件：用于将从所述测序数据中查询得到的所述300Kb以上的片段且在80％以上的窗口中染色体片段的ZCNV值都大于等于4或小于等于-4的片段确定为待测孕妇的拷贝数异常片段；
    α第一计算部件：用于在胎儿遗传了母体的拷贝数异常片段的情况下，按照如式(1)所示的计算公式计算参数α，所述参数α为孕妇的拷贝数异常片段对各所述染色体的矫正前覆盖度的影响，
    Figure PCTCN2015078421-appb-100018
    m表示所述拷贝数异常片段所在染色体的有效长度，单位为Mb；n表示所述孕妇在所述拷贝数异常片段的长度，单位为Mb；cn表示所述孕妇的所述拷贝数异常片段出现的次数；
    α第二计算部件：用于在胎儿未遗传母体的拷贝数异常的染色体的情况下，按照如式(2)所示的计算公式所述参数α：
    Figure PCTCN2015078421-appb-100019
    m表示所述拷贝数异常片段所在染色体的有效长度，单位为Mb；n表示所述孕妇在所述拷贝数异常片段的长度，单位为Mb；cn表示所述孕妇的所述拷贝数异常片段出现的次数；f表示所述待测孕妇的外周血游离DNA中所含的胎儿游离DNA的浓度且假定所述胎儿游离DNA的浓度f小于50％；
    矫正部件：用于利用
    Figure PCTCN2015078421-appb-100020
    对各所述染色体的矫正前覆盖度进行矫正，得到各所述染色体的矫正后覆盖度；其中，
    Figure PCTCN2015078421-appb-100021
    代表各所述染色体的矫正前覆盖度，x'代表各所述染色体的矫正后染色体覆盖度；
    第二覆盖度计算部件：用于利用各所述染色体的矫正后覆盖度来计算各染色体的Zaneu值；
    染色体非整倍性初步判断部件：用于根据所述Zaneu值是否大于等于3来判断所述染色体是否具有非整倍性；
    初步确定部件：用于在所述Zaneu值大于等于3的情况下，确定所述染色体具有非整倍性。
  18. 根据权利要求17所述的试剂盒，其特征在于，所述第一覆盖度计算部件中还包括：
    染色体窗口切分元件：用于对所述测序数据中的所有染色体以切分成相等大小的窗口；
    第一覆盖度计算子元件：用于以所述相等大小的窗口的形式计算覆盖度，以得到各所述染色体的校正前覆盖度。
  19. 根据权利要求18所述的试剂盒，其特征在于，所述染色体窗口切分元件中，每个所述窗口的大小为100Kb，且相邻两个所述窗口之间的覆盖度为50％。
  20. 根据权利要求17所述的试剂盒，其特征在于，所述单一序列计算部件包括：
    单一序列统计元件：用于根据所述测序数据中各序列的测序深度，统计各所述窗口的单一序列的数量；
    单一序列的矫正元件：用于根据各所述染色体的GC含量和比对率对各所述单一序列的数量进行矫正，得到各所述单一序列的数量的矫正前覆盖度；
    单一序列ZCNV值计算元件：用于对各所述单一序列的数量的矫正前覆盖度进行标准化处理，得到各所述单一序列的数量的ZCNV值。
  21. 根据权利要求17所述的试剂盒，其特征在于，所述第二覆盖度计算部件中，所述Zaneu按照
    Figure PCTCN2015078421-appb-100022
    来计算，其中，
    Figure PCTCN2015078421-appb-100023
    是根据LOESS算法，通过阴性样本群体得到的矫正前覆盖度值，s表示阴性样本群体里
    Figure PCTCN2015078421-appb-100024
    的标准差。
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CN113012759A (zh) * 2020-12-09 2021-06-22 人和未来生物科技（长沙）有限公司 一种基于X染色体的男胎cffDNA含量计算方法
CN113012759B (zh) * 2020-12-09 2022-08-12 人和未来生物科技（长沙）有限公司 一种基于X染色体的男胎cffDNA含量计算方法
CN116240273A (zh) * 2023-04-19 2023-06-09 北京优迅医学检验实验室有限公司 一种基于低深度全基因组测序的判断母源污染比例的方法及其应用
CN116240273B (zh) * 2023-04-19 2023-08-15 北京优迅医学检验实验室有限公司 一种基于低深度全基因组测序的判断母源污染比例的方法及其应用

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