WO2011130880A1 - Procédé de détection de l'aneuploïdie chromosomique fœtale - Google Patents

Procédé de détection de l'aneuploïdie chromosomique fœtale Download PDF

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WO2011130880A1
WO2011130880A1 PCT/CN2010/000568 CN2010000568W WO2011130880A1 WO 2011130880 A1 WO2011130880 A1 WO 2011130880A1 CN 2010000568 W CN2010000568 W CN 2010000568W WO 2011130880 A1 WO2011130880 A1 WO 2011130880A1
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chromosome
window
base position
type
base
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PCT/CN2010/000568
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李英睿
李松岗
杨广霞
蒋馥蔓
张秀清
玄兆伶
陈芳
林静蓉
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深圳华大基因科技有限公司
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Priority to CN201080032859.0A priority Critical patent/CN102753703B/zh
Priority to PCT/CN2010/000568 priority patent/WO2011130880A1/fr
Publication of WO2011130880A1 publication Critical patent/WO2011130880A1/fr

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    • 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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6881—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
    • 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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the present invention relates to the field of medical detection, and in particular to a method for detecting aneuploidy of a fetal chromosome, the value of the molecular weight of the nucleic acid combined by the first type of chromosome window and the amount of the nucleic acid molecule of the corresponding window on the second type of chromosome, and A functional relationship between them to detect prenatal fetal chromosome aneuploidy.
  • a fetal aneuploid chromosome refers to an abnormality in the amount of a chromosome or a certain region of a chromosome.
  • the amount of this abnormality can be abnormally high, such as the fetal No. 21 trisomy, which is a chromosome 21 more than the normal diploid fetus; or abnormally low, such as Turner syndrome patients are missing one or part of X chromosome.
  • Non-cellular circulating fetal DNA is found in maternal plasma and serum, providing unlimited possibilities for non-invasive prenatal diagnosis.
  • Rossa W. LChiu et al., PNAS 2008, 105: 20458-20463, article "Using large-scale, high-throughput sequencing for non-invasive prenatal diagnosis of aneuploid chromosomes" The method was used to diagnose the aneuploidy of non-invasive fetuses, and pointed out that the information of nucleic acid molecules in peripheral blood can be obtained by means of sequencing, especially the nucleic acid molecules derived from fetal chromosomes contained in the extracted maternal samples. In the case where the amount of background parent nucleic acid molecules is relatively small (Rossa WK Chiu, et al.
  • the method comprises the following steps: first extracting nucleic acid molecules in the plasma of the peripheral blood of the pregnant woman, and then performing sequencing by a large-scale high-throughput second-generation sequencer to obtain sequence information of the DNA fragment and position information on which chromosome the sequence falls on. . Based on this basic information, Dennis Lo et al. then counted the amount of nucleic acid molecules derived from medically meaningful chromosomes (such as chromosomes 21, 18, and 13) and the amount of nucleic acid molecules derived from the background chromosome.
  • the experimental conditions for each batch of samples required by this method are strict. If the experimental conditions are large, it will be difficult to obtain a more reliable normal control value or sample. value.
  • the experimental conditions include pre-sample preparation, batch and type of reagents used, sequencing GC bias, system temperature, etc. In other words, changes in various experimental conditions should be controlled within a relatively small range, otherwise each negative will result.
  • the variance of the values between the control samples is increased, or the values of the respective negative control samples are greatly deviated from each other, and the values of some of the negative samples may even fall out of the normal value range and become outliers. Similarly, this method also depends on the accuracy of the value of the sample to be tested.
  • the preparation of the sample and the reagents used and sequencing conditions should be performed with negative control samples. Try to be consistent. Only in this way can it be more ideal and credible to count the anomalies of the distribution of abnormal samples on the chromosomes studied, and to ensure that their values are credible.
  • the inventors found that there is a correlation between the amounts of nucleic acids corresponding to each other between chromosomes, and according to this correlation, a method for detecting aneuploidy of fetal chromosomes according to the data acquisition and processing method of the present invention is provided. Sex.
  • the inventor's method includes the steps of:
  • a method of detecting an individual aneuploidy of an individual comprising:
  • the whole genome is sequenced, and the chromosome to be studied as the research object is defined as a chromosome of the same type, and the remaining chromosomes in the whole genome are defined as the second type chromosome; the correlation between the second type of chromosome fragment and the behavioral value of the first-class chromosome fragment Sex, build mathematical models on the whole genome;
  • the whole genome is sequenced, and then the behavior values of the first type chromosome and the second type chromosome of the individual to be detected are obtained. If the behavior value at a certain confidence level does not satisfy the aforementioned mathematical model, the test can be determined.
  • the chromosome of an individual is aneuploidy.
  • the entire chromosome is cut into overlapping or non-overlapping segments, each segment is defined as a window; the amount of nucleic acid molecules in each window of the chromosome from a normal individual sample without chromosomal defects is counted by sequencing, and the amount is defined as a behavior value. ;
  • a mathematical correlation method is used to find a window or a combination thereof which has strong correlation with the first-class chromosome on the second chromosome, and a functional relationship is established;
  • the above "confidence interval” includes the confidence interval of each window and the confidence interval of the entire chromosome.
  • the above "different actual conditions” mainly include inconsistent sequencing GC content, different reagent conditions, and different experimental preparation methods.
  • the present invention provides a method of using a single sample derived from a pregnant woman and determining whether the fetal chromosome is aneuploid based on its chromosomal data. In this way, the invention ultimately obtains a set of chromosome-like window combinations, and a combination of analog windows that are strongly correlated with the second type of chromosome, and a functional relationship that is corrected at a certain confidence level. Through these window and function relationships, it can be determined whether the first type of chromosome of a particular sample is aneuploid.
  • the method of the invention can be used for prenatal diagnosis of aneuploidy.
  • the method of the invention is free from the strong dependence of the original method for strictly controlling the experimental conditions, and can be applied to correct outlier samples due to changes in the actual conditions, thereby reducing waste.
  • the method of the present invention can be controlled by the sample itself. Without relying on the values of other samples, an important basis for determining whether a fetal chromosome is aneuploidy is provided; the method of the present invention also avoids the risk of invasive methods.
  • the method of the invention is based on sequencing not only for diagnosing whether a chromosome is aneuploidy, but also for chromosomal aneuploidy diagnosis of an isolated sample obtained under unknown experimental conditions or under poor control of experimental conditions, and Used in combination with the methods in the background art, mutual verification, thereby further improving the accuracy of the test, and from a large influence, can effectively control the birth rate of the aneuploid chromosome fetus including the No. 21 trisomy.
  • the method of the invention establishes a data processing method, which is flexible and can simulate chromosome 21, that is, finds the inner control window combination consistent with the optimal behavior value of chromosome 21, and can also be extended to all interested chromosomes. For example, chromosome 18, chromosome 13 and other first chromosomes, and even some chromosome fragments of interest, thereby increasing the scope of application of the method.
  • Figure 1 is a flow chart showing the method of the present invention for finding and determining a combination of simulation windows that are strongly correlated with each window combination of the first type of chromosome in the whole genome (except for the first type of chromosome to be studied), Method 100.
  • Figure 2 A flow chart showing the method of the present invention for diagnosing whether a fetus in a pregnant woman is chromosomal aneuploidy, as method 200.
  • Figure 3 A scatter plot showing the linear correlation between windows.
  • the magnitude of the nucleic acid molecule (89 behavioral values yi) uses a statistical correlation analysis, calculates the correlation coefficient between the two windows, and the relationship function, the R 2 shown on the graph is 0. 9724, indicating the correlation between the two windows well.
  • Figure 4 A scatter plot showing the linear correlation between windows.
  • the magnitude of the molecule (89 behavioral values yi ) using statistical Correlation analysis, calculate the correlation coefficient R between the two windows, because the correlation coefficient R 2 is only 0.6. From the figure, it can be seen that the linear relationship between the two windows is not good.
  • Figure 5 A scatter plot showing the linear correlation between windows.
  • the magnitude of the nucleic acid molecule (53 behavioral values yi) using statistical correlation analysis, calculates the correlation coefficient between the two windows, and the relationship function.
  • the R 2 shown on the graph is 0. 9816, indicating that the two windows are well correlated.
  • Figure 6 A scatter plot showing the linear correlation between windows.
  • the magnitude of the numerator (53 behavioral values yi) uses the statistical correlation analysis, calculates the correlation coefficient R between the two windows. Since the correlation coefficient R 2 is only 0. 0043, the two windows are linear. bad relationship.
  • Figure 7 A graph showing the relationship between the true values of normal and abnormal samples and their respective confidence intervals for analog values. Specifically, four nucleic acid samples from pregnant women's peripheral blood were displayed. The true values of samples 1 and 2 were outside the respective confidence interval of the simulated value, and the sample was aneuploidy of chromosome 21; and the true values of samples 5 and 6 were Within the confidence interval of each analog value, the sample is normal.
  • Figure 8 A graph showing the relationship between the true values of normal and abnormal samples and their respective simulated value confidence intervals, showing four nucleic acid samples from pregnant women's peripheral blood.
  • the true values of samples 3 and 4 are in their respective simulated confidence intervals.
  • the sample is aneuploidy on chromosome 21; and the true values of samples 7 and 8 are within the respective confidence intervals of the simulated values, and the samples are normal.
  • Figure 9 A graph showing the relationship between the true values of normal and abnormal samples and their respective confidence intervals for analog values. Specifically, four nucleic acid samples from pregnant women's peripheral blood were sampled. Sample 1, the true values of samples 2 and 3 were within the respective confidence intervals of the simulated values, and the samples were normal for chromosome 18; and the true values of sample 4 were at their respective simulated values. Beyond the confidence interval, chromosome 18 is aneuploidy.
  • Figure 10 A graph showing the relationship between the true values of normal and abnormal samples and their respective simulated value confidence intervals. Two nucleic acid samples from pregnant women's peripheral blood are shown. The true values of samples 5 and 6 are in their respective simulated confidence intervals. In addition, the sample is chromosome 18 aneuploidy.
  • Aneuploid chromosome or "chromosomal aneuploidy” means that a chromosome has a whole or partial deletion or redundancy relative to the chromosome on the normal diploid genome. Usually, there is an extra one chromosome or one of the chromosomes is missing. The most common case of chromosomal aneuploidy is the trisomy, which has an additional chromosome. For example, chromosome 13 is a chromosomal abnormality found in the cell for the third chromosome 21.
  • Bio sample means a sample containing a nucleic acid molecule of interest, such as plasma, serum or other biological sample containing nucleic acid molecules, derived from a subject, such as a pregnant woman.
  • Normal sample refers to a peripheral blood sample of a pregnant woman who does not have any aneuploidy abnormality on any chromosome.
  • Nucleic acid molecule refers to a deoxyribonucleotide molecule contained in an extracted biological sample, and herein refers to a nucleic acid molecule contained in a biological sample derived from human's 300 million genomic base pairs.
  • nucleic acid fragment molecule refers to a fragment nucleic acid molecule which can obtain its sequence information and its position information on the genome by sequencing means after the preparation and the like, and can represent the nucleic acid molecule corresponding thereto.
  • Unique nucleic acid molecule means that post-sequencing information analysis indicates that the nucleic acid molecule has a position on the human genome and has only one distribution.
  • Window means that a chromosome or a chromosomal region can be segmented into a segment having overlapping regions or no overlapping regions according to an artificially defined unit nucleic acid sequence length, and each window corresponds to a unique segment of the chromosome or chromosome region, the position Information is unique.
  • the "amount of nucleic acid molecule”, also called the behavior value, is based on the position of the nucleic acid fragment molecule based on sequencing to the specific position of each chromosome, and the number of nucleic acid fragments located in this region can be statistically reflected or how many nucleic acid molecules can be distributed.
  • the value in this region for example, the number of nucleic acid fragment molecules that can be located in this region, or the number of nucleic acid molecules from this region divided by the number of all statistically or partially counted nucleic acid molecules. A ratio obtained or a cumulative value of the molecular length of the nucleic acid fragment, etc.
  • the value reflects the amount of distribution of the nucleic acid molecule in the window, this value This can be referred to as the behavioral value or the amount of nucleic acid molecule derived from the window.
  • the amount of nucleic acid molecule derived from a certain window refers to the number of nucleic acid fragment molecules that can be located in this region after the nucleic acid fragment molecule is located at the specific position of each chromosome based on sequencing. Or a value that reflects how many nucleic acid molecules are distributed in this region, such as the number of nucleic acid fragment molecules that can be located in this region, or the number of nucleic acid molecules from the same region divided by all statistics. Or a fraction of the number of nucleic acid molecules counted or the cumulative amount of the length of the nucleic acid fragment, etc. In summary, for an experimental sample, as long as this value reflects the amount of distribution of the nucleic acid molecule in this window This value can be called the behavior value or the amount of nucleic acid molecules derived from the window.
  • Consistent behavior values refers to two behavioral values, one of which changes in behavior, and the other behavioral value that occurs simultaneously with a functional relationship.
  • Real value refers to the amount of nucleic acid molecule derived from the window of the first type of chromosome.
  • First type of chromosome refers to all or part of a nucleic acid sequence of clinical research value, or medically, the imbalance of these chromosomes is commonly used to detect certain abnormal symptoms. For example, chromosome 21 sequence, chromosome 18 sequence, chromosome 1 sequence, X chromosome sequence or Y chromosome sequence.
  • “Second type chromosome” refers to other chromosomes on the genome from which the first type of chromosome is removed, and in the present invention refers to other chromosomes in which the first type of chromosome to be studied is removed in a specific embodiment.
  • the first type of chromosome window combination refers to a combination of windows consisting of several overlapping or non-overlapping windows that can cover all or part of a class of chromosomes.
  • a simulation window (simulation window combination) that has a strong correlation with the window of the first type of chromosome refers to a specific simulation window present on the chromosome other than the first type of chromosome currently studied in the study genome, the specific There is a strong correlation between the simulation window and the first type of chromosome window under the current study. It is expressed as follows: For each of the normal biological samples, there is always a relationship that is derived from this sample. If the amount of a nucleic acid molecule of a type of chromosome window changes, the molecular weight of the nucleic acid derived from the analog window (simulation window combination) which is highly correlated with the first type of chromosome window also changes correspondingly, and this amount changes. Correlation in which another quantity also changes can be approximated by a correlation function expression. See Method 100 for the selection of the simulation window (simulation window combination).
  • Unbalanced here means that the biological sample detected at this time is stored in the first type of chromosome or the first type of chromosome region studied by comparing the true value in the confidence interval calculated according to the simulation window and the function relation.
  • Chr in the present invention is an abbreviation for "chromosome", for example, "Chr 18" represents ⁇ chromosome 18.
  • the dye double helix chain includes a positive strand, a reverse strand, and the forward and reverse strands are complementary, that is, one strand base sequence is determined, and the base sequence of the other complementary strand is also determined. Thus, each of these positions appears in pairs in pairs, and the base pairs are complementary: A is complementary to T and C is complementary to G.
  • any position refers to a pair of base pairs, including both bases in the positive strand and complementary bases in the reverse strand.
  • the positioning positions in the present invention are all in units of bp, that is, abbreviations of base pa ir (base pairs).
  • base pa ir base pairs
  • chr21: 150 - 450kbp represents a window of 150000 bases to 450000 base pairs on chromosome 21.
  • the positive chain refers to the chain of 5, -3
  • the reverse chain is the one complementary to it, and the direction should be 3, -5.
  • the base of chr21: 150 bp refers to the base from the positive strand, the direction 5, -3, the first base, and the base at 150. Right; from the negative chain, direction 3, -5, the number of the first base starts, the base pair at 150.
  • a method of finding a combination of analog windows or windows that is highly correlated with a first type of chromosome window combination is provided.
  • the essence of the method of "looking for a simulated window or window combination” is to find a simulation window or simulation that has a strong correlation with the first type of chromosome window or window combination through an innovative correlation analysis of behavior values between windows.
  • a window combination for example, a behavioral value correlation coefficient between a window in a simulated window combination that can be referred to as a first type of chromosome window combination and a corresponding class of chromosome window is close to 1, and can be obtained universally
  • the function relation is used to express the correlation between the behavior values of the two windows.
  • the function relation is a unary function relationship between the two. If multiple strong correlation windows are found, the function relationship The formula is to fit the optimized multivariate function relation.
  • a diagnostic method for diagnosing aneuploid chromosomes by judging whether the amount of a nucleic acid molecule combined with a steroid-like chromosome window derived from the same sample is within a confidence interval, and the confidence interval is derived from The behavior value of the above-mentioned search for the determined simulation window combination and the correlation between the respective windows are determined together.
  • Embodiment 1 - v ⁇ ⁇ ⁇ Step 1 Obtain the sequencing data required for modeling
  • a DNA library is prepared, and the nucleic acid fragment molecule is ligated to a So lexa sequencing linker, and then the nucleic acid molecule having a purified nucleic acid fragment of 150 to 300 base pairs in length is isolated.
  • the nucleic acid molecule linked to the adaptor can hybridize with the surface complement of the f low ce ll. Under certain conditions, the nucleic acid molecules grow in clusters and then pass through 36 cycles of sequencing on the Illumina Genome Analyzer, which is equivalent to each nucleic acid molecule. A 35 base pair nucleic acid fragment was detected.
  • the sequence information measured is subjected to a P i pel ine process, and finally the ELAND alignment result using the human genome sequence in NCBI as a reference sequence can be obtained.
  • the inventors knocked out fragments of nucleic acid molecules that are in tandem repeats and transpositions of the genome, and only counted those nucleic acid fragment molecules that can be located at unique positions in the genome. In this way, only about one-quarter to one-third of all nucleic acid molecules detected can be located in a unique location in the genome.
  • these statistical nucleic acid molecules are called Unique nucleic acid molecules, and the amount of this unique nucleic acid molecule still represents the amount of nucleic acid molecules distributed in specific regions of the genome.
  • This step corresponds to steps 110, 120 of Fig. 1.
  • Step 2 Establish a chromosome 21 detection model
  • the process of establishing the mathematical model is optimized by computer program.
  • the computer program completes the correlation window search through different parameter selections, finds the optimal parameters, and establishes a functional relationship between the first type of chromosome window and the second type of chromosome window.
  • each of the measured nucleic acid fragment molecules can be mapped to the exact position of the genome by means of bioinformatics analysis.
  • a cutting window is performed for each sample.
  • Each chromosome constituting the genome is segmented into several window sequences. Cutting the entire genome into windows of appropriate size is equivalent to treating each chromosome as a combination of windows with overlapping or non-overlapping regions.
  • chromosome 21 has a total of about 47 Mega base pairs. If the size of l OMega is a window, and the position of the beginning of each window slides back to the size of lMega, then chromosome 21 corresponds to 47 overlapping OMega sizes. Window.
  • the length of the desired window and the length of the window overlap region can be artificially defined, and also the window number, which produces a mapping of the pair of window numbers to the true coordinates of the window on each chromosome on the genome.
  • 300k is the window size
  • 150k is the over lap splitting window (that is, the starting end of each window is the front The beginning of a window slides back 150k).
  • Table 1 shows the 40 windows after cutting chromosome 21. If the window corresponding to a window is labeled chr21, 2 , its corresponding position on the genome is chr21 : 150 - 45 O
  • Table 1 shows the labels of the windows obtained by artificially cutting chromosome 21 and the position coordinates of the window on the genome. Only 40 windows are shown in the table. For example, if the window is labeled (chr21, 180), it means that it is the 180th window after cutting chromosome 21, and the specific position on chromosome 21 is the coordinates (chr21: 26850 - 27150kbp), which is 2,658,500 on chromosome 21. Base pair and between 2,725,500 base pairs. Thus, each window label represents the mapped position of the corresponding chromosome, that is, an artificially cut chromosomal region.
  • the chromosome 4 is a non-first type chromosome, and if the size of the 300k base pair is a window, and the position of each window starts to slide backward by 150k base pairs, the same manner can be cut into several windows.
  • Table 2 lists - twenty window labels and positional information on the chromosome, ie window coordinates. If the window corresponding to a window is labeled chr4, 2, its corresponding position on the genome is chr4: 150 - 450kbp.
  • the window is labeled (chr4, 643), it means that it is the 180th window on which chromosome 4 is cut, and the specific position on chromosome 4 is the coordinates (chr4: 96300 - 96600kbp), that is, on chromosome 4. Between 963,300 base pairs and 966,000 base pairs.
  • step 150 based on the information obtained by sequencing, the samples prepared and sequenced under different conditions were counted for the amount of nucleic acid fragment molecules derived from each window sequence.
  • the window of chromosome 21 refers to the window combination of chromosomes with clinical research value.
  • the coordinates of a window in this window combination are (chr21: 89000 - 89200bp), indicating that it is on chromosome 21, and this window region ends from the 8890th base pair to the ⁇ 89200 base pairs.
  • the measured starting coordinate of a certain nucleic acid fragment molecule is chr21: 89100, and the measured molecular fragment length is 35 base pairs, then the sequence position of the nucleic acid fragment molecule is just at the window coordinate Inside, it indicates that the nucleic acid fragment molecule is derived from this window region.
  • the number of nucleic acid molecules in which all sequence positions of a sample are located in this window can be counted, and the number of nucleic acids can be called the nucleic acid molecule derived from this window.
  • the amount is added, by combining the quantities of the nucleic acid fragment molecules constituting the window of the chromosome-like window combination, the molecular weight of the nucleic acid derived from the first type of chromosome window combination can be obtained.
  • the first type of chromosome window combination can be based on The condition partially or collectively covers the first type of chromosome one or more times.
  • step 160 for each window of chromosome 21, the window analysis on the entire genome (except chromosome 21) is related to this window, select the window group retention with strong correlation, and determine the functional relationship between them. formula, At this time, a single window of chromosome 21 may correspond to multiple related windows to form a related window combination.
  • the statistically derived nucleic acid derived from the second type of chromosome window have a molecular weight of Xi
  • the statistically derived nucleic acid derived from the first type of chromosome window has a molecular weight of Y i
  • i represents a sample number
  • the observed sample is (XI, Yl), ( X2, Y2), ...( ⁇ , ⁇ )
  • the square of the correlation coefficient of the two sets of data can be calculated. The closer the square of the correlation coefficient is to 1, the intercept is close to 0, indicating that the correlation is higher.
  • Each chromosome 21 and its individual correlation window can determine a functional relationship.
  • 89 biological samples for sample preparation and sequencing under different experimental conditions and sequencing conditions were prepared, and the amount of nucleic acid fragments derived from each window region sequence of each sample was separately counted, that is, for each sample, Each window cut on the genome corresponds to a behavior value. For 89 samples, there are 89 behavior values in each window.
  • a window with a very high correlation with this window is searched for on the genome (except chromosome 21).
  • chromosome 21 For convenience of explanation, only three windows are used here for illustration. See Table 5, a window derived from a combination of chromosomes of the first class, numbered (chr21, 180), and two windows derived from non-first chromosomes. , the numbers are window (chr l, 256), window (chr4, 642).
  • the fourth row shows the magnitude of the nucleic acid molecule from the genomic window labeled (chr21, 180) window for each of the 89 samples corresponding to the sample number, which is also 89 behavioral values, denoted as yi, i value from 1 Get 89,
  • ⁇ can be taken to 89, and the correlation coefficient of the two columns of values is studied. The closer the square of the correlation coefficient is to 1, the correlation is The higher the sex, that is, when the behavior value of one window changes, the other window will change accordingly.
  • the correlation coefficient is close to 1, it can be studied according to the observation sample whether the behavior of the two windows obeys the linear relationship, as shown in Table 3.
  • the behavior values of the window (chr4, 642) and the window (chr21, 180) obey the linear relationship, and the expression of the linear equation and the square of the correlation coefficient R can be obtained. See Fig.
  • the window in the first type of chromosome window combination can be used to find one or more highly correlated ones.
  • the simulation window In the future, only one X value needs to be known, that is, only the amount of the analog window derived from the window corresponding to the embodiment window is calculated, and an analog value can be calculated by the relation or Analog value confidence interval.
  • Table 4 discloses a combination of simulation windows scattered at other locations in the genome that are strongly correlated with the first type of window combination selected by the method 100 process steps, and only a combination of 20 windows is shown in the table.
  • the first column shows the window label on the first type of chromosome, the second column shows its window coordinates; the third column shows the corresponding first The chromosomes of the simulation window with extremely strong correlations, the four columns display the corresponding simulation window coordinates; the fifth column shows the relational expression of the window and the related simulation window; the sixth column shows the square of the correlation coefficient, ie R
  • the square value the closer the R squared value is to 1, indicates that the higher the correlation between the two windows, that is, the molecular weight of the nucleic acid derived from one window changes, the molecular weight of the nucleic acid derived from another window will change accordingly.
  • simulation windows with strong correlation with the chromosome 21 window combination can be combined to form a simulation window combination, and a modified confidence interval is calculated according to the amount of the nucleic acid molecules derived from the combination of the simulation window or the simulation window.
  • step 170 since each window on chromosome 21 may have multiple windows associated with it, in order to fully use the sequencing data, the detection accuracy is improved, and the correlation function relationships of these windows are fitted, and according to the actual data. Correction is performed to finally determine the functional relationship of each window in chromosome 21 that can find a strong correlation on the genome.
  • Step 3 Sequencing the test sample
  • a biological sample is obtained from a pregnant woman, which may be plasma or other suitable sample. This biological sample contains nucleic acid molecules derived from pregnant women as well as fetuses.
  • the nucleic acid molecules in the sample are randomly sequenced.
  • the sequenced nucleic acid fragment molecule represents a part of the entire genome and can be localized on each chromosome.
  • the nucleic acid molecule in the sample can be measured only at one end (such as measuring 35 base pairs at one end) or both ends, or the entire nucleic acid The molecules are all measured.
  • Sequencing can be sequenced by large-scale, high-throughput parallel sequencers such as the 454, So lexa, SOL iD systems, and single-molecule sequencing or nanopore sequencing.
  • sequencers such as the 454, So lexa, SOL iD systems, and single-molecule sequencing or nanopore sequencing.
  • sample 1 sample 2 is a set of experimental conditions (group A); sample 3, sample 4, sample 7, sample 8 is another set of experimental conditions (group B).
  • group A 5 ml of maternal plasma was extracted, free DNA was extracted using QIAamp DNA Mi cro kit (QIAGEN, 56304), SE library was constructed using NEB reagent according to the Illumina/Solexa library procedure, SE36 was sequenced, and group B was used to extract 600 ul of maternal plasma.
  • TIANamp Mi cro DNA Ki t (Tiangen DP316) was used to extract free DNA, and the PEI library was constructed using Enzymatics reagent according to the Illumina/Solexa library procedure, and sequenced on SE36+7.
  • This step is shown in steps 210 and 220 of Figure 2.
  • Step 4 Detect the sample, according to the flow chart of Figure 2.
  • each of the measured nucleic acid fragment molecules can be localized to the exact location of the genome by means of bioinformatics analysis. As shown in Table 1, the positional coordinates of the nucleic acid fragment molecules which can be localized to each chromosome as determined by sequencing on the chromosome are shown.
  • Table 5 shows the positional information of some nucleic acid molecules obtained after sequencing of biological samples 1 and 2 to the first ten starting sites on chromosome 4 and chromosome 21.
  • the starting point of the first nucleic acid fragment molecule that can be mapped to chromosome 21 is 997943, that is, the starting point of the nucleic acid fragment molecule is located on chromosome 21 9797443 base pair position.
  • the starting point of the first nucleic acid fragment molecule that can be mapped to chromosome 4 is 19219, that is, the starting point of the nucleic acid fragment molecule is located at the 19th pair of base pairs of chromosome 4.
  • step 240 as in the model building, 300k is the window size, and 150k is the overlap to cut the window for all the chromosomes of the 8 samples to be detected.
  • the statistics are from the 21st. The amount of nucleic acid fragment molecules in each window of the chromosome.
  • step 260 based on the core Ml! MJMd Mil acid three three three three... Fragment position information, statistics body body
  • step 270 by determining the 250th tire of each sample, 2 222
  • J is the source of II II 1 l 3 3 3 3 3 ⁇ 33! Whether the amount of the nucleic acid fragment molecule in the chromosome 21 window is ⁇ 4*_ ⁇ - obtained in step 260 of the same sample. .. often often believe that the fetus is at high risk of aneuploidy on chromosome 21.
  • Table 6 Results obtained by detecting the eight samples in the examples by the method of the present invention Sample Real value Y Analog value 95% confidence interval HI
  • the true value Y column in Table 6 is the statistically derived molecular weight of the nucleic acid derived from the chromosome 21 window combination of each sample; the nucleic acid molecular weight and phase of the simulated window combination with strong correlation with the chromosome 21 window combination in the sample are counted.
  • a relational expression (as shown in Table 4), followed by a confidence interval (confidence level: 95%) obtained from statistical knowledge. For a sample of a study, when its true value exists in the calculated confidence In the interval, it can be considered that the sample under study is normal. On the contrary, if the true value exists outside the confidence interval of the analog value, it is unlikely that the sample is normal.
  • sample 1 sample 2, sample 3, and sample 4 whose true values are derived from the molecular weight of the chromosome 21 combination are outside the confidence interval, the conclusion obtained by this method is that they are 21 trisomy
  • the result is proved by the results of the karyotype analysis.
  • the true values of samples 1 and 2 are above the confidence interval of their respective simulated values, which can be determined as chromosome 21 aneuploidy; while sample 5 and sample 6 are true. The values are within the confidence interval of the respective analog values and the sample can be considered normal.
  • Figure 8 shows the relationship between the true values of normal and abnormal samples and their respective analog value confidence intervals. Four nucleic acid samples from pregnant women's peripheral blood are displayed. The true values of samples 3 and 4 are above the confidence interval of their respective simulated values, which can be determined as chromosome 21 aneuploidy; and the true values of samples 7 and 8 are in their respective Within the confidence interval of the analog value, the sample can be considered normal.
  • Step 1 Get the sequencing data needed for modeling
  • This step corresponds to steps 110, 120 of Fig. 1.
  • Step 2 Establish a chromosome 21 detection model
  • the process of establishing the mathematical model is optimized by computer program.
  • the computer program completes the correlation window search through different parameter selections, finds the optimal parameters, and establishes a functional relationship between the chromosome window of the first class and the chromosome window of the second type.
  • each of the measured nucleic acid fragment molecules can be mapped to the exact location of the genome by means of bioinformatics analysis.
  • a cutting window is performed for each sample.
  • Each chromosome constituting the genome is divided into a number of window sequences.
  • 300k is the window size
  • 150k is the over l ap segmentation window (that is, the start of each window). The end slides 150k backwards at the beginning of the previous window.
  • step 150 based on the information obtained by sequencing, the samples prepared and sequenced under different conditions were counted for the amount of nucleic acid fragment molecules derived from each window sequence.
  • step 160 for each window of chromosome 18, the window analysis on the entire genome (except chromosome 21) is related to the Yiyi window, select the window group retention with strong correlation, and determine the functional relationship between them. In this case, if a single window of chromosome 18 corresponds to multiple related windows, a related window combination is formed.
  • the sample number is displayed, which is the number of the 53 samples studied in this example; the second row shows that the sample from the 53 samples corresponding to the sample number is labeled (chrl, 8)
  • the magnitude of the nucleic acid molecule on the window, ie 53 behavioral values, is recorded as 4, and the value of 1 is taken from 1 to 53.
  • the third row shows the amount of nucleic acid molecules (53 behavior values) from the genomic window labeled (chrl 8, 259) for each of the 53 samples corresponding to the sample number, denoted xi, and the value of i is taken from 1 53
  • the fourth row shows the amount of nucleic acid molecules (53 behavior values) from the genomic window labeled (chrl 8, 259) for each of the 53 samples corresponding to the sample number, denoted as yi, and the value of i is taken from 1. To 53.
  • the correlation between the window (chr8, 710) and the window (chrl 8, 259) is studied.
  • the observed samples are (XI, YD, (X2. Y2), ... ( ⁇ , ⁇ ), in this embodiment.
  • ⁇ can be taken to 53, to study the relationship between the two columns of values
  • only one X value needs to be known, that is, only the amount of the analog window derived from the window corresponding to the embodiment window is calculated, and a confidence interval can be calculated by the function relation.
  • Table 8 discloses the combination of windows scattered at other locations in the genome that are strongly correlated with chromosome 18 selected by the above steps selected by the method 100, and only 20 of them are shown.
  • the first column shows the window label on chromosome 18, the second column shows its window coordinates, the third column shows the label of the window with strong correlation with the corresponding chromosome 18, and the fourth column shows the corresponding window coordinates.
  • a window with a strong correlation with the chromosome 18 window combination can be combined, and a modified confidence interval is calculated according to the amount of the nucleic acid molecules derived from each window, and the method 200 is used to judge the chromosome aneuploidy. .
  • Step 3 Sequencing the test sample
  • a biological sample is obtained from a pregnant woman.
  • the biological sample can be plasma or other suitable sample containing nucleic acid molecules derived from pregnant women and fetuses.
  • the nucleic acid molecules in the sample are randomly sequenced.
  • the sequenced nucleic acid fragment molecules represent a portion of the entire genome and can be localized on each chromosome.
  • sample 1 sample 2
  • sample 4 is a set of experimental conditions (group A); sample 3, sample 5, sample 6 is another set of experimental conditions (group B), group A is to extract 5ml maternal plasma
  • group A is to extract 5ml maternal plasma
  • QIAamp DNA Mi cro kit QIAGEN, 56304
  • SE36 was sequenced and the sputum group was used to extract 600 ul maternal plasma using TIANamp Mi cro DNA K it ( T iangen DP316)
  • TIANamp Mi cro DNA K it T iangen DP316
  • the free DNA was extracted and the PEI library was constructed using Enzymatics reagent according to the II lumina/Solexa library procedure, and sequenced on SE36+7.
  • This step is shown in steps 210 and 220 of Figure 2.
  • each of the measured nucleic acid fragment molecules can be localized to the exact location of the genome by means of bioinformatics analysis. As shown in Table 1, the coordinates of the starting position of the nucleic acid fragment molecule which can be localized to each of the nemeric chromosomes measured by sequencing on the chromosome are shown.
  • step 240 as in the model building, 300k is the window size, and 150k is over l ap to cut the window for all the chromosomes of the six samples to be tested.
  • step 250 based on the nucleic acid fragment position information, the nucleic acid fragment derived from each window of chromosome 18 is counted. The amount of children.
  • step 260 based on the nucleic acid fragment position information, the amount of nucleic acid molecules derived from the window of each sample having a strong correlation with the chromosome 18 window is counted, and the confidence interval is calculated according to the functional relationship.
  • step 270 by determining whether the amount of the nucleic acid fragment molecule derived from the chromosome 21 window obtained in step 250 of each sample is within the confidence interval obtained in step 260 of the same sample, it can be determined whether the fetus is Chromosome 18 has a high risk of aneuploidy.
  • the true value in Table 9 is the statistically derived molecular weight of the nucleic acid derived from the chromosome 18 window combination of each sample; the nucleic acid molecular weight of the simulated window combination with strong correlation with the chromosome 18 window combination in the sample is counted. And related function expressions (as shown in Table 8), and then a confidence interval (confidence level: 95%) obtained through statistical knowledge. For a sample of a study, when its true value exists within the calculated confidence interval, it can be considered that the sample under study is normal, and conversely, if the true value exists outside the confidence interval of the analog value , the risk that the sample is aneuploid is very high.
  • sample 4 sample 5 and sample 6 whose true values are derived from the molecular weight of the chromosome 21 combination are outside the confidence interval, the conclusion obtained by this method is that they are chromosome 18 aneuploidy. Fetal samples, the results were confirmed by the results of the karyotype analysis.

Abstract

L'invention concerne un procédé de détection de l'aneuploïdie chromosomique fœtale, dans lequel le diagnostic prénatal de l'aneuploïdie chromosomique fœtale est réalisé à l'aide des valeurs de quantités de molécules d'acide nucléique dans les fenêtres du premier type de chromosome et des valeurs des quantités de molécules d'acide nucléique dans les fenêtres correspondantes du second type de chromosome et de la relation fonctionnelle entre elles.
PCT/CN2010/000568 2010-04-23 2010-04-23 Procédé de détection de l'aneuploïdie chromosomique fœtale WO2011130880A1 (fr)

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CN104156631B (zh) * 2014-07-14 2017-07-18 天津华大基因科技有限公司 染色体三倍体检验方法
CN104789686A (zh) * 2015-05-06 2015-07-22 安诺优达基因科技(北京)有限公司 检测染色体非整倍性的试剂盒和装置
CN104789466A (zh) * 2015-05-06 2015-07-22 安诺优达基因科技(北京)有限公司 检测染色体非整倍性的试剂盒和装置
CN105316420A (zh) * 2015-12-01 2016-02-10 钦州市妇幼保健院 一种单管四色双重相对荧光定量pcr技术快速检测人13号和18号染色体数目的试剂盒
CN111226281A (zh) * 2019-12-31 2020-06-02 深圳华大临床检验中心 确定染色体非整倍性、构建分类模型的方法和装置
WO2021134513A1 (fr) * 2019-12-31 2021-07-08 深圳华大医学检验实验室 Procédés pour déterminer une aneuploïdie chromosomique et construire un modèle de classification et dispositif
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