JP2007327743A - Method and system for analyzing gene copy - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for correcting the amount of the genome DNA of a chromosome measured, using a probe complementary to genome DNA. <P>SOLUTION: The amount of genome DNA derived from an experiment is corrected, by using the correction tendency value related to a plurality of experimental processes in the measurement of the amount of the genome DNA of the chromosome and the correction factor calculated from the tendency value. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a gene copy analysis method and apparatus, and in particular, correction of measured values of genomic DNA amount and genomic DNA amount ratio of chromosomes of cells, identification of gene copy number of chromosomes, identification of haplotypes by the correction and detection method, In addition, the present invention relates to a technique for displaying the measurement results relating to chromosomal gene defects and abnormal amplification in an easily understandable manner.

In recent years, DNA microarray technology has been widely used to analyze the amount of genomic DNA at an arbitrary site of a chromosome mainly for the purpose of cancer diagnosis. For example, Non-Patent Document 1, Non-Patent Document 2, Non-Patent Document 3, Non-Patent Document 4 and the like.
Here, the amount of genomic DNA of a chromosome refers to the amount of genomic DNA at a specific site in a chromosomal gene and depends on the number of gene copies. In normal cells, the genome DNA usually has two copies of alleles derived from the parents, for a total of 2 copies. In diseases such as cancer, the number of gene copies is increased or decreased in the genome.

Various methods using microarrays have been developed to detect the genomic DNA content and gene copy number of normal and diseased cells, including the CGA (Comparative genome hybridization) microarray method using BAC (Bacterial Artificial Chromosome). (Non-patent document 1, Non-patent document 2, Non-patent document 3, Non-patent document 4). In order to analyze mutations to chromosomal genes in diseases such as cancer in more detail, changes in the amount of genomic DNA for each allelic chromosome must be detected.
In particular, in the analysis of LOH (loss of heterozygosity), it is possible to understand which allele of the chromosome, that is, maternal or paternal origin (Non-patent Documents 5 and 6).
Furthermore, in the analysis of the amount of genomic DNA of a chromosome, a method using a genome typing microarray using an oligonucleotide has been reported (Non-patent Document 7).

However, in the conventional method, the process of analyzing a part of the whole genome amplified by PCR (Representational analysis) has the disadvantage that it contains noise data that cannot be ignored depending on many experimental conditions and individual experimental conditions. is there.
Therefore, in the method for detecting the amount of genomic DNA of a chromosome and the number of gene copies, it is necessary to correct these noise data in order to obtain more accurate data. In addition, there has been no report of analyzing the genomic DNA amount for each allele of the chromosome in the methods for detecting the genomic DNA amount of the chromosome so far, and more accurate by detecting the genomic DNA amount and the gene copy number for each allele. Data is obtained and haplotypes can be detected from the data.

Ishkanian, A.S. et al Nat Genet, 36, 299-303 (2004) Pinkel, D. et al. Nature Genetic 20, 207-211 (1998) Pollack, J.R. et al Nature Genetic 23, 41-46 (1999) Lucito, R. Et al. Genome Research 13, 2291-2305 (2003) Robinson, W.P.Bioessays. 22, 452-9 (2000) Murthy, S.K.Mod Pathol. 15, 1241-50 (2002) Bignell, G.R. et al. Genome Resesrch 14, 287-295 (2004) Kennedy, G.C.Nature Biotechnology 21, 1233-7 (2003)

  The present invention was created in view of the above-mentioned background art, and aims to correct the measurement value related to the genomic DNA amount of the chromosome and the genomic DNA amount ratio by a suitable method, and to improve the accuracy thereof, and thus Provide technologies that contribute to the analysis of gene copy number, such as identification of genomic DNA amount and gene copy number for each allele, and identification of haplotypes.

As a result of diligent research to solve the above problems, the present inventor amplifies a chromosomal gene as a factor that makes the measurement result unstable in a method for measuring the amount of genomic DNA of a chromosome using a microarray or the like. It was found that there were specific experimental conditions such as a step, a step of hybridizing the amplified PCR product to a probe on the array, and a step of scanning a fluorescent signal.
By correcting for these experimental conditions, a correction method for measuring the amount of genomic DNA of a chromosome was completed. Moreover, it discovered that the said measurement result can be converted into the gene copy number of a chromosome by correct | amending the amount of genomic DNA of a chromosome. The present invention has been completed based on this finding. That is, the present invention is as follows.

  The invention according to claim 1 is the gene copy analysis method, wherein the genomic DNA amount of the chromosome (hereinafter referred to as the genomic DNA amount) or the ratio of the genomic DNA amount of the chromosomes of two different or identical cells (hereinafter referred to as the genomic DNA amount). ), And a method for correcting any of the values (hereinafter referred to as measured values). In the method, following the measurement value measurement step of measuring a predetermined genomic DNA amount or genomic DNA amount ratio, a correction tendency value calculation means calculates a measurement value of the genomic amount or genome amount ratio for each correction parameter value in a coordinate system. A correction tendency value calculation step for calculating a correction tendency value related to a predetermined parameter relating to the measured value by smoothing the plot value and a correction factor calculation means for calculating the correction factor from the correction tendency value The method includes a factor calculation step and a measurement value correction calculation step of correcting the measurement value with the correction factor by the measurement value correction calculation means.

  According to the second aspect of the present invention, the measured value is a genomic DNA amount or a genomic DNA amount ratio measured using a probe complementary to genomic DNA (hereinafter referred to as a probe), and a correction factor is calculated. In the process, a correction factor for each correction reference value is calculated from the correction tendency value. The correction reference value means each correction parameter value corresponding to an arbitrary probe among the correction parameter values, and the correction factor can be calculated from the correction tendency value at the correction reference value. The correction factor is a value for correcting the measured genomic DNA amount data.

  According to the third aspect of the present invention, in the correction tendency value calculating step in the gene copy analysis method, the measurement value for the length of each gene fragment obtained by cleaving a chromosomal gene with a specific restriction enzyme is used for each correction in the coordinate system. The correction tendency value related to the length of the gene fragment is calculated by plotting the parameter value and smoothing the plotted value.

  The invention according to claim 4 is a method for measuring the ratio of G and C bases contained in each gene fragment obtained by cleaving a chromosomal gene with a specific restriction enzyme in the correction tendency value calculating step in the gene copy analysis method. The correction tendency value related to the GC base content is calculated by plotting the value with respect to each correction parameter value in the coordinate system and smoothing the plotted value.

  In the invention according to claim 5, in the correction tendency value calculation step in the gene copy analysis method, 20 to 220 bases continuous for each genetic fragment obtained by cleaving a chromosomal gene with a specific restriction enzyme With respect to the maximum value of the ratio of G and C bases contained in the gene fragment corresponding to the fixed frame, obtained by moving a fixed frame having a specific length selected from the range of the gene by one base from the end of the gene. By plotting the measured value against each correction parameter value in the coordinate system and smoothing the plotted value, the correction tendency value related to the GC base content of the fixed frame is calculated. .

  According to the sixth aspect of the present invention, in the correction tendency value calculating step in the gene copy analysis method, the measurement value for the hybridization free energy of the probe base sequence is plotted against each correction parameter value in the coordinate system. Then, the correction tendency value related to hybridization is calculated by smoothing the plot value.

  The invention according to claim 7 is characterized in that, in the gene copy analysis method, the signal intensity of a probe set complementary to a completely complementary genomic DNA for detecting a specific site of each chromosome in the correction tendency value calculation step. The correction tendency value related to the signal intensity of the control sample is calculated by plotting the measured value with respect to the intermediate value of each of the correction parameter values in the coordinate system with respect to each correction parameter value and smoothing the plotted value.

  The invention according to claim 8 is characterized in that, in the gene copy analysis method, the correction tendency value calculating step obtains an average curve of each plot using a least mean square method, and the average curve is approximated using a polynomial. Thus, the method includes a step of calculating each correction tendency value.

In the invention according to claim 9, the correction factor calculation step in the gene copy analysis method calculates a correction factor for each probe corresponding to each correction tendency value based on the following equation: Including a process.
(Formula) Correction factor = A + Σ (B _k X _k + C _k X _k ² + D _k X _k ³ ...)
Here, although the polynomial is described up to the third-order term, any number of terms equal to or higher than the first-order term (B _k X _k ) can be used. For example, the correction factor = A + ΣB _k X _k may be used. In the equations, A to D... Are average curve coefficient (correction tendency) values, and X _k is a correction parameter value according to claims 3 to 7 where k = 1 to 5, respectively.

  The invention according to claim 10 is characterized in that, in the gene copy analysis method, the method of identifying the gene copy number of the chromosome uses the correction calculation result value of the measurement value according to claims 1 to 9, and the plot means When the correction calculation result values for the chromosome positions arranged in the correct order are plotted on the coordinate system, the step of extracting the data shown in a step shape, the gene copy number identification means has the same correction calculation result value A step of setting a region having the lowest average value of the correction calculation result values at the positions of two or more consecutive chromosomes belonging to the rank as a region having a gene copy number of 0; The present invention provides a method for analyzing gene copies, comprising the step of setting a low region as a region having a gene copy number of 1, and identifying the gene copy number of a chromosome.

  The invention according to claim 11 is the method of displaying the result of identifying the gene copy number of the chromosome in the gene copy analysis method, wherein the image processing means uses the chromosome 1 to chromosome 22, X chromosome and Y chromosome. A reproduction image of the chromosome shape is displayed on the image display means for all or a part thereof, and the gene copy number is visually associated with the region where the gene copy number of the chromosome is increased or decreased on the reproduction image. It is characterized by displaying that the area is increasing or decreasing.

  According to the twelfth aspect of the present invention, in the gene copy analysis method, the haplotype identifying method uses the correction calculation result value of the measurement value according to any one of the first to ninth aspects, and in a physical order by a plotting means. The step of extracting the data shown in a staircase pattern when the correction calculation result values for the positions of the arranged chromosomes are plotted on the coordinate system, the haplotype identification means, the correction calculation result values belong to the same rank 2 The method includes the step of identifying the position of a plurality of consecutive chromosomes as a haplotype, and identifying the haplotype.

  In the invention according to claim 13, the method for displaying the result of identifying the haplotype in the gene copy analysis method is that the image processing means uses the first to the 22nd chromosomes, the X chromosome and the Y chromosome all or one of them. Displaying a chromosome shape-like reproduction image on the image display means, and visually displaying that the specific haplotype exists in the region by associating the region where the specific haplotype exists on the reproduction image. It is a feature.

  The invention according to claim 14 is a genome typing microarray in which the probe can identify SNPs in the measurement value measurement step in the gene copy analysis method, and measuring the amount of genomic DNA of a chromosome for each chromosome allele It is characterized by.

  The invention according to claim 15 is the gene copy analysis method, wherein the measurement value is an amount of genomic DNA measured by amplifying the gene, and the correction factor calculation step includes corrections related to a plurality of parameters. If there are trend values, the correction factors are calculated by adding them together.

    In the invention according to claim 16, the result of measuring either the value of the genomic DNA amount of a chromosome or the ratio of the genomic DNA amounts of chromosomes of two different or identical cells (hereinafter referred to as a measured value). An input means for inputting; a correction tendency value calculating means for calculating a correction tendency value relating to a predetermined parameter by plotting the measured value in a coordinate system with respect to each correction parameter value and smoothing the plotted value; The present invention provides a gene copy analyzing apparatus that corrects a measured value by including a correction factor calculating means for calculating a correction factor from the correction tendency value and a measured value correction calculating means for correcting the measured value with the correction factor. .

  The present invention can also provide an apparatus for analyzing gene copies. That is, the invention according to claim 16 is a gene copy analyzing apparatus, which is one of the values of the genomic DNA amount of a chromosome or the ratio of the genomic DNA amounts of chromosomes of two different or identical cells (hereinafter referred to as the following). A correction tendency for calculating a correction tendency value according to a predetermined parameter by plotting the measurement value in a coordinate system and smoothing the plot value. A correction factor calculation unit that calculates a correction factor from a correction tendency value, and a measurement value correction calculation unit that corrects the measurement value with the correction factor, thereby correcting the measurement value; To do.

According to the present invention, a method for correcting a measurement result in a method for analyzing a gene copy, particularly a method for measuring the genomic DNA amount of a chromosome using a microarray, a method for detecting a gene copy number of a chromosome from the measurement result after the correction process, and A method is provided for displaying chromosomal gene defects or abnormal amplification in a visually comprehensible manner.
The correction method according to the present invention shows that the bias of the measurement result of the genomic DNA amount of the chromosome using a microarray or the like is remarkably reduced, and the stability and reliability of the measurement result is increased.
It was also shown that the absolute value of the chromosome gene copy number can be detected for the first time by converting the genomic DNA amount of the chromosome by the correction method into the gene copy number. From the result of the correction method, a new method for identifying a haplotype can also be provided.
Furthermore, by providing the display method of the present invention, it is possible to convey the deficiency and abnormal amplification of a specific gene on each chromosome so that it can be easily understood visually.

  Hereinafter, the present invention will be described in detail. FIG. 1 is a processing diagram of a method for correcting the genomic DNA amount of a chromosome or the ratio of genomic DNA amounts of chromosomes of two different or identical cells in the gene copy analysis method according to the present invention. FIG. 2 is a block diagram of the gene copy analysis apparatus (1) according to the present invention. The device (1) is realized by a general-purpose personal computer or the like, and includes a memory (3), a hard disk (4), and a display screen that operate in conjunction with a central processing unit (CPU) (2). (5) etc.

1. Measurement of genomic DNA content of chromosomes using microarray (measurement value measurement process)
First, the amount of genomic DNA to be input to the device (1) is measured. The measurement of the genomic DNA amount of a chromosome using a microarray will be described. First, a sample adjustment method will be described, but the following methods, procedures, materials, capacities, and the like are not particularly limited.
First, a chromosomal gene is extracted from an arbitrary cell or tissue. The chromosomal gene extraction method involves treating the cell extract with a proteolytic enzyme, decomposing the proteins in the cells and the chromosome, and then treating them with phenol / chloroform to separate the proteins into the aqueous layer. Take out a certain part (part containing DNA). Next, the extracted chromosomal gene is cleaved with an arbitrary restriction enzyme. The method for treating the restriction enzyme is not particularly limited, but here, the chromosomal gene was cleaved with the restriction enzyme XbaI.

  Next, an adapter molecule is bound to each gene fragment (fragment) cleaved with the restriction enzyme XbaI. The adapter molecule is an oligonucleotide containing a restriction enzyme cleavage site and further having an arbitrary gene sequence. Ligation reaction between the adapter molecule and the gene fragment is performed, and the adapter molecule is bound to the gene fragment. The gene is then amplified by PCR using specific primers corresponding to the adapter molecule. Gene fragments amplified by the PCR reaction correspond to about 2% of the total genomic DNA. The amount of gene fragment amplified by the PCR method varies depending on the number of gene copies of genomic DNA. If the number of gene copies of genomic DNA grown by the PCR method is large, the amount of gene fragments amplified accordingly increases.

Next, a fluorescent label is applied to the gene fragment amplified by PCR. The fluorescent substance used for the fluorescent label is not particularly limited, and examples include labeling with a radioactive substance instead of the fluorescent label.

  Second, hybridization is performed with an array of fluorescently labeled PCR products and oligonucleotide probes. The type of the oligonucleotide microarray is not particularly limited. When an array capable of genome typing is used, chromosomal gene defects and abnormal amplification can be detected separately for each chromosome allele. Furthermore, by considering information on whether a specific allele is derived from a paternal or maternal, it is possible to detect from which origin a chromosomal gene defect or the like originates. By quantitatively measuring the signal intensity for each probe, the amount of genomic DNA in the gene region corresponding to the specific probe can be detected.

  In addition, when measuring the amount of genomic DNA of only a specific gene, it may be amplified by PCR or the like using a specific primer gene for the gene to be measured instead of the adapter molecule, such as RT-PCR or There is also a method of measuring the amount of genomic DNA of a chromosome by amplifying a specific gene such as LAMP method and quantitatively measuring the amount of this gene product. In this case, the step of hybridization with the probe described below is not required.

  The measured value may be the amount of genomic DNA or the ratio of the genomic DNA amounts of two or different cells in the same cell (genomic DNA amount ratio). The genomic DNA content ratio is also measured by the same method as described above. Hereinafter, the description will focus on the genomic DNA amount ratio, but the present invention can be similarly applied to the genomic DNA amount.

2. Method to correct the measured value (correction tendency value calculation process)
The present invention provides a correction method for reducing the bias in the raw data (primary data) of the obtained measurement value by using a correction factor obtained by quantifying the bias tendency generated in the measurement process for determining the amount of chromosomal genomic DNA. . That is, the correction method uses various measurement conditions or measurement results in the measurement process for determining the genomic DNA amount or genomic DNA amount ratio of a chromosome. Correction is performed with the data value for each probe for each chromosome position.

  The primary data consists of a component that truly reflects the amount of genomic DNA of the chromosome and a noise component that is affected by the measurement conditions such as the PCR process. This correction method is characterized by removing this noise component. The removal of the noise component includes dividing the primary data by the “correction factor” calculated from the “correction reference value” and the “correction tendency value”, or subtracting the “noise component” from the primary data.

  In addition, the correction factor and the noise component may be calculated using all or a part of “correction reference value” and “correction tendency value” related to five correction parameters described below. “Correction tendency value” refers to a correction tendency value relating to a predetermined parameter relating to a measurement value of the genomic DNA amount, plots the measurement value in each correction parameter in a coordinate system, and smoothes the average value from the plotted value. Obtaining from the coefficient of the average curve. It is desirable to obtain the “correction tendency value” from a plurality of probes that can measure as many chromosomal sites as possible (see FIG. 3 and the like).

The “correction reference value” means each correction parameter value corresponding to an arbitrary probe among the correction parameter values, and the correction factor can be calculated from the correction tendency value at the correction reference value. The “correction factor” is a value for correcting the data of the measured genomic DNA amount, and is a value corresponding to each probe that can be calculated from one or more correction parameters.

  Here, the correction trend value is obtained by smoothing the first-order data into the same curve using a least square method or other known method for obtaining an average curve, and converting the curve into an arbitrary function (polynomial approximation). At the time of smoothing to the average curve, it may be determined by excluding abnormal values of primary data (particularly far from the average curve compared to other primary data).

  The “correction parameters” causing the bias are the following five. Here, the bias means that the measurement value becomes unstable by causing “variation” or “fluctuation” in the measurement value. The first parameter is the length of the gene fragment cut with XbaI. In this implementation step, the chromosome of the cell is first extracted and purified, and then the chromosome is cleaved with the restriction enzyme XbaI. The length of the gene fragment cleaved at that time is referred to. Here, although XbaI was mentioned as a typical restriction enzyme, the types of restriction enzymes such as BamI and XhoI are not limited. This parameter is expected to cause a bias in the process of amplifying genes by PCR.

  As an example showing this, FIG. 3 or FIG. 4 is shown. These are graphs showing the ratio of signal intensity to the length of the gene fragment. The gene fragment is a gene fragment obtained by cleaving a chromosomal gene with a restriction enzyme XbaI. The vertical axis in FIG. 3 represents the signal intensity ratio, which is the ratio of the gene amplification amount of the Down syndrome chromosome / the gene amplification amount of the healthy human chromosome. The horizontal axis indicates the length of the gene fragment. FIG. 4 uses those obtained from the same H1437 cells. The measured value compared the data which experimented the same cell separately. The signal intensity ratio is the amount of gene amplification of H1437 cells / the amount of gene amplification of H1437 cells. The horizontal axis indicates the length of the gene fragment cut with the restriction enzyme, and the vertical axis indicates the signal intensity ratio.

For example, smoothing may be performed from each plot value in the coordinate system of FIG. 3 or FIG. 4 to an average curve, and the coefficient obtained from the average curve related to the length of the gene fragment. The “correction reference value” herein refers to the length of each gene fragment on the horizontal axis, and correction factor data relating to an arbitrary gene fragment can be derived from the correction reference value and the correction tendency value.

  The bias due to the length of the gene fragment may be a fixed bias value, the correction factor may be a fixed value, or the bias value may vary from experiment to experiment. You may decide. This is because the bias does not completely depend on the length of the gene fragment, and it is considered that a bias due to other reasons is also included.

  The second parameter is the ratio (%) of the GC base pair content contained in the gene fragment cleaved with the restriction enzyme. The number of bases of the gene fragment cleaved with the restriction enzyme referred to in the first parameter is divided by the number of GC base pair pairs contained in the gene fragment and multiplied by 100. Since the gene fragment is double-stranded, paying attention to either gene chain, the number of base G (guanine) and base C (cytosine) may be calculated, or conversely, base A (adenine) Alternatively, the number of bases T (thymine) may be calculated, and the number of bases of AC may be subtracted from the number of bases of the entire gene fragment.

  As an example showing this, FIG. 5 and FIG. 6 are shown. FIG. 5 is a graph showing the relationship between the GC content and the signal intensity ratio in a gene fragment cleaved with the restriction enzyme XbaI. The signal intensity ratio is the amount of gene amplification of the chromosome of Down syndrome / the amount of gene amplification of the chromosome of a healthy person. The horizontal axis represents the GC base content in the gene fragment, and the vertical axis represents the signal intensity ratio. FIG. 6 is a graph showing the genomic DNA amount of the chromosome of the same H1437 cell. The measured value was obtained by comparing data obtained by experimenting the same cell separately. The signal intensity ratio is the amount of gene amplification of H1437 cells / the amount of gene amplification of H1437 cells. The horizontal axis represents a part of the probe with respect to the chromosome position, and the vertical axis represents the signal intensity ratio.

It can be obtained by smoothing an averaged curve from each plot value in the coordinate system of FIGS. 5 and 6 and obtaining the coefficient from the averaged curve related to the GC content. Here, the “correction reference value” refers to the GC content of the gene fragment, and correction factor data with an arbitrary GC content can be derived from the correction reference value and the correction tendency value.

  The first and second parameters are expected to cause a bias in the process of amplifying genes by PCR. The bias based on the ratio of the GC base pair content contained in the gene fragment is assumed to have a fixed bias value, the correction factor may be a fixed value, and the bias value varies from measurement to measurement. The bias value may be measured and determined. This is because the bias does not depend on the ratio of the GC base pair content, and a bias due to other reasons is considered to be included. Here, all gene sequence information is obtained from Human Genome Build 34 (from July 2003 UCSC genome build) (described in Non-Patent Document 9), but other gene sequence databases are used for gene sequence information. Alternatively, the genomic gene sequence may be determined independently.

HYPERLINK "http://genome.ucsc.edu/" http://genome.ucsc.edu/

  The third parameter is the ratio (%) of GC base pair content in the specific region. Here, the specific region refers to a continuous region of 100 bp (base pair) having the highest GC base pair content ratio among gene fragments cut with a restriction enzyme. GC base pair content ratio (%) in a specific region means that a 100 bp fixed frame is shifted by one base from the end of the same gene fragment with respect to any restriction enzyme fragment gene, and is included in that Each GC content is obtained, and the GC base pair content (%) in a specific region having the highest GC base pair content is calculated.

  The calculation method is obtained by the method described in the second parameter. In addition, this specific region is not necessarily limited to 100 bp, and may be the number of bases in the range of 20 bp to 220 bp. This factor is expected to cause a bias in the process of amplifying genes by PCR (FIGS. 7 and 8).

  As an example showing this, FIG. 7 and FIG. 8 are shown. FIG. 7 is a graph showing a signal intensity ratio with respect to the CG content in a specific region of a gene fragment. The vertical axis represents the signal intensity ratio, which is the amount of gene amplification of chromosomes of Down syndrome / the amount of gene amplification of chromosomes of healthy individuals. The horizontal axis indicates the maximum GC content within 100 bp in a specific region as a percentage for the same fragment. Random data can be seen in the region where the GC content is higher than the region indicated by the arrow. Such a region showing the random signal intensity ratio may be excluded in the data acquisition process. Further, FIG. 8 was obtained by comparing data obtained by separately experimenting with the same cell (H1437 cell). The vertical axis indicates the signal intensity ratio as in FIG. 7, and is the gene amplification amount of H1437 cells / gene amplification amount of H1437 cells. The horizontal axis indicates the GC content in a specific region as a percentage as in FIG.

Examples include smoothing an averaged curve from each plot value in the coordinate system of FIG. 7 or 8 and obtaining the coefficient from the averaged curve related to the GC content in a specific region. The “correction reference value” here refers to the GC content in a specific region, and correction factor data with an arbitrary GC content can be derived from the correction reference value and the correction tendency value.

  The bias based on the ratio of the GC base pair content contained in a specific region may be a fixed bias value, the correction factor may be a fixed value, or the bias value varies from experiment to experiment, and the bias value is measured individually. May be determined. Here, the bias does not depend on the ratio of the GC base pair content, and it is considered that a bias due to other reasons is also included. The portion (6) having a high GC base pair content ratio (%) may be excluded because the measured value greatly deviates from the approximate equation curve (FIG. 7).

The fourth parameter is the hybridization free energy (Kcal / mol) of the probe base sequence itself. The method for calculating the hybridization free energy is not particularly limited, but here, it was calculated using OligoScreen ^™ . Alternatively, hybridization free energy may be calculated using a calculation method using the GC base content as an index. Hybridization free energy is expected to cause a bias in the hybridization and washing process of a gene complementary to the probe.

  As an example showing this, FIG. 9 and FIG. 10 are given. FIG. 9 is a graph showing the ratio of signal intensity to the hybridization free energy (Kcal / mol) of the probe gene sequence itself. The signal intensity ratio is the amount of gene amplification of the chromosome of Down syndrome / the amount of gene amplification of the chromosome of a healthy person. The horizontal axis represents the hybridization free energy (Kcal / mol) of the probe gene sequence itself, and the vertical axis represents the signal intensity ratio. In addition, FIG. 10 was obtained by comparing data obtained by separately experimenting with the same cells (H1437 cells). The vertical axis represents the signal intensity ratio, which is gene amplification amount of H1437 cells / gene amplification amount of H1437 cells. The horizontal axis indicates the hybridization free energy (Kcal / mol) of the probe gene sequence itself.

For example, smoothing is performed from each plot value in the coordinate system of FIG. 9 or 10 to an averaged curve, and the coefficient is obtained from an averaged curve related to hybridization free energy. The “correction reference value” herein refers to each hybridization free energy value, and correction factor data at an arbitrary hybridization free energy value can be derived from the correction reference value and the correction tendency value.

  The fifth parameter is an intermediate value (log scaled) of the signal intensity of the probe set of Prefect Match (PM). It means the signal intensity of the control sample, and means the intermediate value of the signal intensity of a plurality of probes that detect a specific chromosomal site (specific SNP site). The intermediate value may be obtained after eliminating the highest value and the lowest value.

  An example of this is shown in FIG. FIG. 11 is a graph showing the signal intensity with respect to the intermediate value of the signal intensity of the probe set which is a Perfect Match (PM). This intermediate value refers to an intermediate value of signal intensities of a plurality of probes that detect a specific chromosomal site (specific SNP site). The vertical axis represents the signal intensity ratio, which is the amount of gene amplification of chromosomes of Down syndrome / the amount of gene amplification of chromosomes of healthy individuals. The horizontal axis indicates the intermediate value of the signal intensity of the probe set that is Prefect Match (PM).

For example, smoothing is performed from each plot value of the coordinate system of FIG. 11 to an average curve, and the coefficient is obtained from an average curve coefficient related to an intermediate value of the signal intensity of the probe set. The “correction reference value” here refers to the intermediate value of the signal intensity of each probe set, and the correction factor data at the intermediate value of the signal intensity of any probe set is derived from the correction reference value and the correction tendency value. Can do.

3. Method to correct the measurement result of the amount of genomic DNA of a chromosome (calculation process of correction factor)
In the measurement value correction in the gene copy number analysis method according to the present invention, all or part of the “correction reference value” and “correction tendency value” related to the above five correction parameters is used. "Correction factor" is calculated. As will be described later, the “correction factor” for the probe can be derived from the “correction tendency value” and the “correction reference value” for the plurality of correction parameters for each probe.

  Next, the primary data in a probe can be corrected using the correction factor in the probe, whereby the primary data can be corrected. Correction of primary data using this correction factor includes a method of dividing primary data by a correction factor and a method of subtracting primary data by a correction factor, as will be described below.

  Hereinafter, a method for obtaining a comprehensive correction factor from correction tendency values and correction reference values in a plurality of measurement steps will be described. The plurality of measurement steps refers to all or part of the above five correction parameters. Here, by way of example, an overall correction factor (predicted bias value / expected data relating to five correction requirements) is shown using the above five correction factors as a polynomial of a cubic function. Here, the polynomial may be obtained by a function of another order such as a linear function. Further, although it is desirable to use all of the correction parameters X1 to X5, only an arbitrary correction parameter may be used.

(Number 1) Expected data = A + Σ (B k X k + C k X k 2 + D k X k 3)
In Equation 1, K takes the following values for correction requirements 1 to 5, respectively. B, C, and D represent coefficients of a cubic function, and A represents an intercept of the function.
X1 is the length (bp) of the gene fragment cleaved with XbaI,
X2 is the ratio (%) of the GC base pair content contained in the gene fragment cleaved with the restriction enzyme, and the region showing a high GC content is excluded from the data and smoothed into a curve. Good.
X3 is the ratio of GC base pair content in a specific region (%)
X4 is the hybridization free energy (Kcal / mol) of the probe gene sequence itself.
X5 is an intermediate value (log scaled) of the signal intensity of the probe set of Prefect Match (PM). It means the signal intensity of the control sample, and means the intermediate value of the signal intensity of a plurality of probes that detect a specific chromosomal site (specific SNP site). The intermediate value may be obtained after eliminating the highest value and the lowest value.

  Depending on the microarray used, X1, X2, X3, and X4 take fixed values, but X5, A, B, C, and D vary from measurement to measurement. It is desirable to calculate each measurement individually. However, values other than X5 may be fixed to each probe.

  The figure which shows the measured value and correction result which concern on this invention is shown in FIG.12 and FIG.13. FIG. 12 is a graph showing a signal intensity ratio before correction of the present application with respect to a chromosome of a healthy person having a chromosome of Down syndrome. Here, the signal intensity ratio is the amount of gene amplification of the chromosome of Down syndrome / the amount of gene amplification of the chromosome of a healthy person. An array of probe sets capable of 10K mapping was used in order of each physical gene position centered on chromosome 21. The vertical axis represents the signal intensity ratio before correction, and the horizontal axis represents the physical gene position around chromosome 21.

  FIG. 13 is a graph obtained by processing the graph shown in FIG. 12 using the correction method of the present application. It has been shown that the signal intensity ratio is clearly increased in the region of chromosome 21 of the patient with Down's syndrome by the correction process, in which the variation and fluctuations in the signal intensity ratio data are significantly reduced (fluctuations) ( Area indicated by an arrow). The signal intensity ratio is the amount of gene amplification of the chromosome of Down syndrome / the amount of gene amplification of the chromosome of a healthy person. The vertical axis shows the signal intensity ratio, and the horizontal axis shows the physical gene position around chromosome 21.

4). A method for identifying the gene copy number of a chromosome, and a method for obtaining an absolute value of the gene copy number of a chromosome by adding a specific step to the correction method of the present invention are provided. It is shown that when the intensity ratio of the amount of genomic DNA is corrected by the correction method of the present invention, the bias of the measurement value is reduced, and the measurement value is clearly stepped. By setting the average value of the minimum signal intensity ratios among the stepwise continuous measurement values as “0”, the signal intensity ratio and the absolute value of the gene copy number in each chromosomal region can be converted.

  FIG. 14 is a graph showing the results of genome DNA amount and gene copy number analysis (Allelic dosage analysis) by allele. The figure shows the amount of gene amplification of chromosomes of cancer patients / the amount of gene amplification of chromosomes of healthy individuals for each probe. By defining the minimum signal intensity ratio in any allele as “0” as the number of gene copies per cell, the absolute number of gene copies in each chromosomal region can be calculated. The vertical axis (left side) represents the signal intensity ratio, and the vertical axis (right side) represents the absolute value of the gene copy number per cell defined in the present invention. Alleles with a high gene copy number are indicated by bold lines (7), and alleles with a low gene copy number are indicated by thin lines (8).

  FIG. 15 is a graph showing the results of analyzing the amount of genomic DNA for each allele in the whole genome under the same conditions as in FIG. This graph shows the signal intensity ratio for each allele. By defining the minimum signal intensity ratio in any allele as “0” as the number of gene copies per cell, the absolute number of gene copies in each chromosomal region can be calculated. The vertical axis (left side) represents the signal intensity ratio, and the vertical axis (right side) represents the absolute value of the gene copy number per cell defined in the present invention. The horizontal axis shows the physical gene position of the whole genome in order. Alleles with a high gene copy number are indicated by bold lines (7), and alleles with a low gene copy number are indicated by thin lines (8). LOH on the X chromosome is clearly shown in one allele (arrow).

This process is summarized as follows. That is, among the stepwise continuous measurement values, the average value of the minimum signal intensity ratio is set to “0”, and the average value of the next highest continuous signal intensity compared to “0” is set to “1”. Next, “2” and “3” are requested. Thereby, the absolute value of the gene copy number can be detected from the value of the signal intensity ratio.

In order to obtain the minimum signal intensity ratio on the chromosome, it is desirable that one of the comparison controls is a healthy person. When the signal intensity ratio was determined for almost all of the chromosomes, it was shown that the gene copy number of most chromosomes was 0, 1, 2, or 3.
In addition, when detecting the gene copy number of a chromosome, it is necessary to use a genome typing microarray that can measure the amount of genomic DNA for each allele in addition to the correction method according to the present invention.

5). The method for identifying a haplotype on a chromosome can also be used for a method for detecting a haplotype on a chromosome according to the present invention. Here, the haplotype, when inherited from the parent, the chromosomal gene is inherited in an appropriate block unit, and a continuous gene block at that time is called a haplotype. This haplotype generally contains a plurality of SNPs.

When measuring the amount of genomic DNA of a chromosome, the haplotype of the chromosome can be detected by making the microarray a genome typing microarray that enables measurement for each allele. This is because in the microarray, a probe having one SNP is usually arranged on the array and has the minimum haplotype component. Moreover, highly reliable detection can be performed by processing with a measured value by the correction method of the present invention.

By correcting the genomic DNA amount data of the chromosome with the correction method of the present invention, a clear stepwise chromosomal genomic DNA amount or gene copy number can be obtained as described above, and consecutive chromosomes having the same signal intensity ratio. The upper segment can be found, and the haplotype can be detected more reliably. A segment (block) on consecutive chromosomes (alleles) with the same signal intensity ratio is defined as a single haplotype (FIGS. 14 and 15). For example, a continuous region (9) in which the two alleles have different signal intensity ratios shown in FIG. 14 is predicted to indicate the haplotype of the gene. The horizontal axis indicates the physical gene position of the chromosomal gene. The vertical axis represents the signal intensity ratio.

6). Method for Displaying Chromosomal Gene Deletion and Abnormal Amplification According to the present invention, the number of chromosome gene copies for each chromosome position can be determined. As shown in FIG. 14 and FIG. 15, the display method is such that the vertical axis indicates the genomic DNA amount (signal intensity ratio) or gene copy number, and the horizontal axis indicates the probe corresponding to the chromosome position (Locus). It was common to express in the physical order of chromosomes.

  In the present invention, all or part of chromosomes 1 to 22, chromosomes X and Y are displayed on a screen or the like according to the shape of the chromosome, such as its length, and the number of gene copies of the chromosome is A deficient region is associated and deleted, and an abnormal amplification of the gene copy number is associated with the region, and a display method indicating that there is amplification according to the gene copy number (FIG. 16). . In addition, depending on the number of gene copies, modulation such as increasing the color of the corresponding region of the chromosome may be mentioned. The long chromosome shown in FIG. 16 indicates the 4th chromosome, and the short chromosome indicates the 18th chromosome.

7). Method for displaying haplotypes on chromosomes As described above, haplotypes on chromosomes can also be detected according to the present invention. This haplotype display can also be shown by a graph of gene copy number, but the chromosomes are all chromosomes 1 to 23, X and Y depending on the shape of the chromosome such as its length. Alternatively, a method may be used in which a part of the information is displayed on a screen or the like, the regions are associated with each haplotype of the chromosome, and the region is colored differently to display the information as a spatial information to a third party.

Experimental example 1

Hereinafter, the present invention will be described more specifically with reference to the following experimental example 1. However, the scope of the present invention is not limited to the following experimental example.
In this experimental example, an experiment was performed using chromosome 21 which is a gene responsible for Down's syndrome. It is known that chromosome 21 of Down syndrome patients has an increased gene copy number compared to that of healthy individuals.

  We compared chromosome 21 of a patient with Down's syndrome with that of a healthy person. FIG. 12 shows the ratio of the signal intensity for each probe in the Down syndrome patient divided by the signal intensity in the healthy person by obtaining the signal intensity for each probe on the array by the method described above for the genomes of healthy persons and Down syndrome patients. It is calculated. The graph shown in FIG. 12 shows the signal intensity ratio depending on the number of gene copies of the chromosome for each specific part of the chromosome. However, since there is a lot of signal noise, the signal intensity ratio is not necessarily the number of gene copies of the chromosome. It is shown that it is not dependent data.

When the primary data is processed by the correction method according to the present invention, it is shown that “fluctuation” and “variation” of the data included in the primary data are remarkably reduced as shown in FIG. Further, it was shown that there is almost no difference between the average value data represented by the primary data before correction and the average value data represented by the secondary data corrected by the present correction method (FIGS. 12 and 13). The median and SD of the signal intensity ratio of primary data in chromosome 21 and other chromosomes were 1.30 ± 0.39 and 1.00 ± 0.28, respectively. On the other hand, secondary data on chromosome 21 and other chromosomes after the correction processing by the correction method of the present application were 1.29 ± 0.18 and 0.99 ± 0.09, respectively.

In another experimental example, using the same H1437 cell, an experiment was performed to measure the genomic DNA content ratio of chromosomes from data obtained by experimenting separately. Measurement data before correction (FIG. 17) and measurement data after correction (FIG. 18) are shown. In these figures, the vertical axis represents the signal intensity ratio, which is the gene amplification amount of H1437 cells / the gene amplification amount of H1437 cells, and the horizontal axis represents a part of the probe for the chromosome position. FIG. 17 and FIG. 18 show that the intermediate value and its SD value are 1.00 ± 0.08 and 1.00 ± 0.06, and the signal intensity value is not changed, and only the variation of the data is reduced.

Experimental example 2

  Next, the result of the correction method (GIM; Genome Imbalance Map) in this application and the result of the CGH (Comparative genomic hybridization) array (Genosensor array 300, Vysis) spotted from the BAC (Bacterial artificial chromosome) used in the past Compared. It is shown below as Experimental Example 2. The two methods showed good correlation from high copy range to low copy range, but uncorrelated sites were also found in some chromosomal sites (FIG. 19). This is because the BAC probe is about 200 Kb long compared to the SNP typing array used in the correction method of the present application, and even within this distance, the gene copy number is averaged even if the gene copy number is different. It is considered that a different gene copy number was calculated.

  In addition, we conducted an experiment to compare the gene copy number of the chromosomes of male and female. Regarding the difference in the number of single gene copies in the X chromosome, the data by the present correction method (GIM) was compared with the CGH array that spotted BAC. It was shown that it can be more clearly distinguished. The signal intensity ratio (Asian ± SD) between the X chromosome and the other autosomes is 0.99 ± 0.09 / 0.63 ± 0.08 in the GIM method, while 1.00 ± 0.09 in the BAC array measurement. /0.71±0.14. The Asian gene copy number of the X chromosome is 0.71, which is only 0.29 different from that of the autosome (in the case of the GIM method, there is a difference of 0.36), and the SD value is 0.14, which is 0.08 of the GIM method. Larger than Therefore, the GIM method of the present application can distinguish the gene copy number more clearly in the X chromosome than the CGH method spotted with BAX (FIG. 20).

Experimental example 3

  Experimental example 3 includes detection of gene copy number in cancer cells. That is, the correction method of the present invention was applied to obtain the amount of genomic DNA and gene copy number of cancer cells. Cells were measured for the amount of genomic DNA of all chromosomal genes using chromosomes obtained from H1395 lung cancer cell line and normal cells. FIG. 21 shows the signal intensity ratio between the chromosomal genes of cancer cells and normal cells on chromosomes 8 and 9p. The signal intensity ratio is the gene amplification amount of chromosomes of cancer patients / gene amplification amount of chromosomes of healthy individuals. That is, it represents the ratio of genomic DNA content. In the figure, the left arrow 8q24 indicates a site where amplification of the c-myc oncogene is observed, and the right arrow indicates an unknown genome growth region. The scale on the right side of the vertical axis shows the result of calculating the gene copy number. (See Figure 15)

  The number of gene copies in which a gene is deleted or abnormally amplified in a specific chromosomal region by determining the ratio of the genomic DNA amount of chromosomes obtained from normal cells and specific cancer cells and correcting this by the correction method of the present invention Can be shown. Thereby, it can be applied to a method for diagnosing cancer and other genetic diseases, and a disease-related gene can be identified by specifying the number of gene copies of disease cells. Further, by determining the amount of genomic DNA of a chromosome for each allele, it is possible to identify whether the abnormal gene copy number of the chromosome is derived from either the paternal or the maternal (FIGS. 3 and 7).

It is a processing figure of correction of the measured value in the analysis method of gene copy by the present invention. It is a block diagram of the gene copy analysis apparatus by this invention. It is the graph which showed signal intensity ratio with respect to the length of a gene fragment (the amount of chromosome genomic DNA of Down's syndrome / normal amount of chromosome genomic DNA). It is the graph which showed the signal intensity ratio (the genomic DNA amount of the chromosome of the same H1437 cell) with respect to the length of a gene fragment. It is the graph which showed the relationship between GC content contained in a gene fragment, and signal intensity ratio (the amount of chromosome genomic DNA of Down's syndrome / normal amount of chromosome genomic DNA). It is the graph which showed the relationship between GC content contained in a gene fragment, and signal intensity ratio (genomic DNA amount of the chromosome of the same H1437 cell). It is the graph which showed signal intensity ratio (the amount of chromosomal genomic DNA of Down's syndrome / normal amount of chromosomal genomic DNA) with respect to CG content in the specific region of a gene fragment. It is the graph which showed signal intensity ratio (genomic DNA amount of the chromosome of the same H1437 cell) with respect to CG content in the specific area | region of a gene fragment. It is a graph which shows signal intensity ratio (chromosomal genomic DNA amount of Down syndrome / normal chromosomal genomic DNA amount) with respect to hybridization free energy (Kcal / mol) which the gene sequence of the probe itself has. It is a graph which shows the signal intensity ratio (the genomic DNA amount of the chromosome of the same H1437 cell) with respect to the hybridization free energy (Kcal / mol) which the gene sequence itself has. It is a graph which shows signal intensity ratio (the amount of chromosomal genomic DNA of Down's syndrome / normal amount of chromosomal genomic DNA) with respect to the intermediate value (log scaled) of the signal intensity of the probe set of Prefect Match (PM). It is a graph which shows the signal intensity ratio (the amount of chromosomal genomic DNA of Down's syndrome / the amount of normal chromosomal genomic DNA) for each chromosome position before correction of the present application. It is the graph which processed the graph shown by FIG. 12 with the correction method of this application. It is a graph of the result of having analyzed genomic DNA amount and gene copy number analysis (Allelic dosage analysis) according to the allele of a chromosome. It is a graph which shows the result of having analyzed the genomic DNA amount and gene copy number for every allele in the whole genome. It is a figure which shows the display of the defect | deletion of chromosome gene and abnormal amplification by the display method of this invention. It is a graph which shows the signal intensity ratio (the amount of genome DNA of the chromosome of the same H1437 cell) for every chromosome position before correction of this application. It is the graph which processed the graph shown by FIG. 17 with the correction method of this application. It is a graph which shows the comparison of the result by this application correction method, and the result of the CGH array which spotted BAC. It is a graph which shows the comparison with the result by this application correction method, and the result of the CGH array which spotted BAC for every order of the position of a chromosome. It is a graph which shows the signal intensity ratio of the chromosomal gene of the cancer cell and normal cell in the 8th chromosome and the 9th chromosome.

Explanation of symbols

1 Gene copy analyzer 2 Central processing unit (CPU)
3 Memory 4 Hard disk 5 Display device 6 High ratio of GC base pair content 7 Allele with high gene copy number 8 Allele with low gene copy number 9 Region indicating haplotype

Claims (16)

In the gene copy analysis method,
One of the values of the genomic DNA amount of chromosomes (hereinafter referred to as genomic DNA amount), or the ratio of the genomic DNA amounts of chromosomes of two different or identical cells (hereinafter referred to as genomic DNA amount ratio) ( Hereinafter, the method of correcting the measured value)
Following the measurement value measurement step for measuring a predetermined genomic DNA amount or genomic DNA amount ratio,
The correction tendency value calculation means plots the measurement value of the genome amount or the genome amount ratio with respect to each correction parameter value in the coordinate system, and smoothes the plot value to obtain the correction tendency value related to the predetermined correction parameter related to the measurement value. A correction tendency value calculation step to calculate,
A correction factor calculation step of calculating a correction factor from the correction tendency value by a correction factor calculation means;
And a measurement value correction calculation step of correcting the measurement value with the correction factor by a measurement value correction calculation means. The measured value is a genomic DNA amount measured using a plurality of probes complementary to genomic DNA (hereinafter referred to as probes), or a genomic DNA amount ratio,
2. The gene copy analysis method according to claim 1, wherein, in the correction factor calculation step, a correction factor at each correction reference value is calculated from the correction tendency value. In the correction tendency value calculation step in the gene copy analysis method,
By plotting the measured values for the length of each gene fragment obtained by cleaving a chromosomal gene with a specific restriction enzyme in the coordinate system and smoothing the plotted value, the correction tendency value related to the length of the gene fragment is calculated. The method for analyzing gene copies according to claim 1 or 2, wherein: In the correction tendency value calculation step in the gene copy analysis method,
By plotting the measured values for the ratio of G and C bases contained in each gene fragment obtained by cleaving a chromosomal gene with a specific restriction enzyme in the coordinate system, and smoothing the plotted values, it is related to the GC base content. The method of analyzing gene copies according to any one of claims 1 to 3, wherein a correction tendency value is calculated. In the correction tendency value calculation step in the gene copy analysis method,
For each genetic fragment obtained by cleaving a chromosomal gene with a specific restriction enzyme, a fixed frame having a specific length selected from a continuous range of 20 to 220 bases is moved from the end of the gene one base at a time. The measured value for the maximum value of the ratio of G and C bases contained in the gene fragment corresponding to the fixed frame obtained on the coordinate system is plotted in the coordinate system, and the plotted value is smoothed to obtain the GC of the fixed frame. The method of analyzing gene copies according to any one of claims 1 to 4, wherein a correction tendency value related to the base content is calculated. In the correction tendency value calculation step in the gene copy analysis method,
Plotting the measured values for the ratio of G and C bases or hybridization free energy in the base sequence of the probe complementary to the genomic DNA in the coordinate system, and smoothing the plotted value, the correction tendency related to hybridization 6. The gene copy analysis method according to claim 1, wherein a value is calculated. In the gene copy analysis method,
In the correction tendency value calculation step,
Control by plotting the measured value against the intermediate value of the signal intensity of the probe set perfectly complementary to the genomic DNA in the probe set that detects a specific site of each chromosome and smoothing the plot value 7. The gene copy analysis method according to claim 1, wherein a correction tendency value related to the signal intensity of the sample is calculated. In the gene copy analysis method,
The correction tendency value calculation step includes a step of calculating each correction tendency value by obtaining an average curve of each plot using at least the least mean square method and approximating the average curve using a polynomial. The gene copy analysis method according to any one of claims 1 to 7, The correction factor calculation step in the gene copy analysis method comprises:
A method for calculating a correction factor in each corresponding probe from each correction tendency value,
(Formula) Correction factor = A + Σ (B _k X _k + C _k X _k ² + D _k X _k ³ ...)
(In the above polynomial, any number of terms equal to or higher than the first order term can be used. A to D in the equation are coefficients of an average curve, and X _k is a correction parameter value according to claims 3 to 7 where k = 1 to 5, respectively. )
The gene copy analysis method according to claim 8, further comprising a step of being calculated based on: In the measurement value measurement step in the gene copy analysis method,
The gene copy analysis method according to any one of claims 1 to 9, wherein the probe is a genome typing microarray capable of identifying SNPs, and the amount of genomic DNA of a chromosome is measured for each chromosome allele. In the gene copy analysis method,
A method for identifying the gene copy number of a chromosome uses a correction calculation result value of a measurement value according to claims 1 to 9,
A step in which the extraction means extracts the data shown in a staircase when the correction calculation result values for the positions of the chromosomes arranged in physical order are plotted in the coordinate system by the plotting means;
A step in which the gene copy number identification means sets the region where the average value of the correction calculation result values at the position of two or more consecutive chromosomes having the correction calculation result values belonging to the same rank as the lowest is the gene copy number 0 region; ,
A method for analyzing gene copies, wherein the gene copy number identification means includes a step of setting a region having the next lowest average value as a region having a gene copy number 1, and identifying the gene copy number of a chromosome. A method of displaying the result of identifying the gene copy number of the chromosome in the gene copy analysis method,
The image processing means displays chromosome-like reproduction images on the image display means for all or part of the first to 22nd chromosomes, the X chromosome and the Y chromosome,
11. The reproduction image is displayed by associating a region where the gene copy number of the chromosome is increasing or decreasing and visually indicating that the gene copy number is increasing or decreasing in the region. Analysis method of gene copy. In the gene copy analysis method,
A method for identifying a haplotype uses a result of correction calculation of measured values according to claims 1 to 13,
A step in which the extraction means extracts the data shown in a staircase when the correction calculation result values for the positions of the chromosomes arranged in physical order are plotted in the coordinate system by the plotting means;
A gene copy analysis method comprising: identifying a haplotype, wherein the haplotype identifying means includes a step of identifying a position of a plurality of two or more consecutive chromosomes whose correction calculation result values belong to the same rank as a haplotype. A method of displaying a result of identifying a haplotype in the gene copy analysis method,
The image processing means displays chromosome-like reproduction images on the image display means for all or part of the first to 22nd chromosomes, the X chromosome and the Y chromosome,
13. The gene copy analysis method according to claim 12, wherein a region in which the specific haplotype is present is associated with the reproduced image and the fact that the specific haplotype exists in the region is visually displayed. In the gene copy analysis method,
The measured value is the amount of genomic DNA measured by amplifying the gene,
The gene copy analysis according to any one of claims 1, 3, 4, 5 and 8, wherein, in the correction factor calculation step, when there are correction tendency values related to a plurality of parameters, the correction factors are calculated by adding them together. Method. An input means for inputting the result of measuring either the value of the genomic DNA amount of the chromosome or the ratio of the genomic DNA amount of the chromosomes of two different or identical cells (hereinafter referred to as a measured value);
A correction tendency value calculating means for calculating a correction tendency value according to a predetermined parameter by plotting the measurement value in a coordinate system and smoothing the plot value;
Correction factor calculating means for calculating a correction factor from the correction tendency value;
A gene copy analysis apparatus, comprising: a measurement value correction calculation unit that corrects and calculates a measurement value with the correction factor.

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