JP4414823B2 - Gene information display method and display device - Google Patents

Description

  The present invention relates to a display method and display apparatus for gene information used for analysis work for specifying a gene involved in a phenotype such as a disease or an external feature of an individual, and in particular, a DNA containing a gene to be analyzed The present invention relates to a method and an apparatus capable of accurately discriminating and displaying a signal from an analysis object and a noise signal when a fragment is extracted and detected by PCR or electrophoresis.

  After complete decoding of the human genome, gene functional analysis research has been actively conducted. Among them, automation of genotyping, which is the basis for the search for genes involved in phenotypes such as the presence or absence of specific diseases, the degree of drug effects, and the presence or absence of side effects, has attracted particular attention.

Microsatellite Usually, the genomes of the same species have almost similar base sequences, but have different bases in some places. For example, at one locus, one individual may have A and another individual may have T. Such a polymorphism in a single base on the genome between individuals is called SNP (Single Nucleotide Polymorphism).

  On the other hand, there are very many locations (tens of thousands or more) where a short sequence pattern of 2 to 6 bases is repeated several to several tens of times in the genome of an organism. This characteristic arrangement pattern is called a microsatellite. An example of microsatellite appearing on the genome is shown in FIG. The repeating unit in microsatellite is called unit, and the base number of unit is called unit length. For example, in the microsatellite “ATATATAT ...” shown in FIG. 18, the unit is “AT” and the unit length is 2 bases. As shown in FIG. 18, even if the microsatellite has the same unit and unit length among individuals, the number of repetitions may vary depending on the individual.

  As described above, since SNPs and microsatellite can be different among individuals, they are easily distinguishable from other base sequences on the genome, and can be easily detected experimentally. In addition, depending on the species, the approximate position where the SNP and the microsatellite exist on the genome is known, so that it can be used as an index indicating the position on the genome. Because of these properties, SNPs and microsatellite are called DNA markers. In particular, since microsatellite includes a plurality of bases, it has a larger amount of information than SNP and is frequently used as a DNA marker.

  By the way, as shown in FIG. 18, many organism individuals have a set of genomes (homologous chromosomes) derived from a female gamete and a male gamete. Genes present at corresponding sites on a set of genomes are called alleles, and these combinations are called genotypes. As described above, since SNPs and microsatellites on the genome are portions where the nucleotide sequences can be different among individuals, there are generally two or three alleles in SNPs, and several types of microsatellite There are ~ 20 or more alleles. In the example shown in FIG. 18, individual A has a unit “AT” repeated 5 times and 7 times, and individual B has a unit “AT” repeated 6 times. Have two. Here, a state having one different type of allele, such as the individual A, is called heterozygous, and a state having two alleles of the same type, such as the individual B, is called homozygous.

PCR and Electrophoresis Experiments When microsatellite is used as a DNA marker, an experiment such as PCR (Polymerase Chain Reaction) or electrophoresis is performed as an experiment for extracting and detecting a portion where microsatellite appears on the genome. PCR specifies a pair of base sequences called primer sequences at both ends of a microsatellite, and only a microsatellite portion sandwiched between them is replicated as a DNA fragment to obtain a certain amount of sample. Experimental technique. Electrophoresis includes techniques such as gel electrophoresis and capillary electrophoresis, and is an experimental technique for separating amplified DNA fragments on a charged migration path and separating DNA fragments of different lengths. Electrophoresis is a sample separation technique that utilizes the fact that the migration speed in the migration path varies depending on the length of the DNA fragment (the longer the DNA fragment, the smaller the migration speed).

  FIG. 19 is a diagram schematically showing an experimental procedure for extracting and amplifying a DNA fragment of a microsatellite portion by PCR and gel electrophoresis. First, a pair of primer sequences 1900 and 1901 are specified across the target microsatellite, and a genomic region 1902 including the microsatellite and the primer sequence is amplified by a PCR experiment. In the example shown in FIG. 19, two types of PCR amplification products having different lengths from each other are heterozygotes with different numbers of microsatellite repeats on two homologous chromosomes, and the lengths of the microsatellite portions are different. That is, DNA fragments (66 bases and 58 bases) are obtained. When these are electrophoresed on a plate-like gel for a certain period of time, the two kinds of PCR amplification products are separated by the difference in length of their DNA fragments. By attaching a fluorescent dye to each DNA fragment and detecting the intensity and position of the fluorescence signal from each DNA fragment after electrophoresis, as shown in FIG. A graph in which the fluorescence signal intensity (that is, the abundance of DNA fragments) is plotted on the vertical axis. In addition, if a DNA fragment (referred to as a size marker) whose length is known in advance is electrophoresed together with the PCR amplification product and these fluorescent signals are detected, each PCR amplification product can be detected based on the detection position of the size marker. Can know the length of.

  In addition, although the experimental method using gel electrophoresis was described above, the same thing can be performed also by capillary electrophoresis. In capillary electrophoresis, the sample is run through a thin tube packed with gel, and the time taken for each sample to finish moving a fixed distance (usually up to the end of the capillary) is measured. It is a technique to check the thickness. In capillary electrophoresis, it is common to detect a sample with a fluorescence signal detector provided at the end of the capillary rather than scanning the fluorescence signal from the sample in the gel.

Noise generated in PCR and electrophoresis experiments The experimental results shown in FIG. 19 above are obtained when PCR and electrophoresis are performed in an ideal process, and various noises occur in actual experiments. Sometimes. The Stutter peak and + A peak, which are typical noises generated in the PCR and electrophoresis experimental process, will be described below with reference to FIG. For simplicity, FIG. 20 shows only the 66-base DNA fragment (including microsatellite in which “TA” is repeated 12 times) shown in FIG. 19 as an example.

  Stutter peak is noise caused by the phenomenon that the number of repetitions of microsatellite part of DNA fragment to be replicated increases or decreases due to slipped-strand mispairing during PCR reaction. DNA fragments in which the number of repetitions is increased or decreased are observed as noise peaks. As shown in FIG. 20, in addition to a DNA fragment 2000 containing a normal microsatellite in which “TA” is repeated 12 times, a DNA fragment containing an abnormal microsatellite in which “TA” is repeated 11 or 13 times 2001 or 2002 is generated and observed as a Stutter peak in the fluorescence analysis. Since the number of repetitions may increase or decrease, PCR is performed to increase the length of the microsatellite unit length by an integral multiple of the DNA fragment (66 bases) of the same length as the original DNA fragment. DNA fragments with increased or decreased levels may be produced.

  The + A peak is noise caused by the phenomenon that one extra base (usually A) is added to the DNA fragment when replicating the DNA fragment by PCR, and one base is added in the fluorescence analysis. DNA fragments are observed as noise peaks. As shown in FIG. 20, in addition to the DNA fragment 2003 in which one base was added to the normally replicated DNA fragment 2000, abnormal DNA in which the number of microsatellite repeats was increased or decreased due to slipped-strand mispairing. DNA fragments 2004 and 2005 in which one base is added to the fragments 2001 and 2002 may also be generated. These 1-base-added DNA fragments 2003, 2004, and 2005 are observed as different + A peaks in fluorescence analysis.

  In the graph showing the fluorescence analysis results in FIG. 20, a 66-base DNA fragment having the same length as the original DNA fragment is the peak that should be observed (hereinafter referred to as “true peak”), All peaks are noise peaks. It can be seen that a Stutter peak appears at intervals of the microsatellite unit length (at positions of 62 bases, 64 bases, and 68 bases) with respect to this true peak. Furthermore, it can be seen that a + A peak appears at a position (base positions of 63 bases, 65 bases, 67 bases, and 69 bases) that is one base longer than each of the true peak or the Stutter peak. That is, + A peaks appearing at positions of 63 bases, 65 bases, 67 bases, and 69 bases are DNA fragments obtained by adding 1 base to DNA fragments having base lengths of 62 bases, 64 bases, 66 bases, and 68 bases, respectively. It will be supported. In the following, for a certain + A peak, a true peak or a Stutter peak corresponding to a DNA fragment to which the + A peak is generated and from which one base has not been added is referred to as an “original peak”.

In the experimental process of PCR and electrophoresis, it is very important to distinguish a true peak from other noise peaks among a plurality of peaks observed in fluorescence analysis. Among the noise peaks described above, the determination method for the Stutter peak has been widely studied, and accurate determination can be performed by the methods disclosed in Patent Documents 1 to 5, Non-Patent Documents 1 to 4, and the like. Yes. In addition, the software that identifies and removes Stutter peaks includes the software "TrueAllele" from Cybergenetics, the software "SAGA" from LI-COR, the "GenoTyper" from ABI, and the "GeneMapper" from ABI It has been known.
U.S. Pat.No. 5,541,067 U.S. Pat.No. 5,580,728 U.S. Pat.No. 5,876,933 US Patent No. 6,054,268 U.S. Pat.No. 6,274,317 Perlin, MW, et al., “Toward Fully Automated Genotyping: Allele Assignment, Pedigree Construction, Phase Determination, and Recombination Detection in Duchenne Muscular Dystrophy”, Am. J. Hum. Genet. 55, 1994, p777-787 Perlin, MW, et al., “Toward Fully Automated Genotyping: Genotyping Microsatellite Markers by Deconvolution”, Am. J. Hum. Genet. 57, 1995, p1199-1210 Palsson, B., et al., “Using Quality Measures to Facilitate Allele Calling in High-Throughput Genotyping”, Genome Research 9, 1999, p1002-1012 Stoughton, R., et al., “Data-adaptive algorithms for calling alleles in repeat polymorphisms”, Electrophoresis 18, 1997, p1-5 Smith, JR, et al., “Approach to Genotyping Errors Caused by Nontemplated Nucleotide Addition by Taq DNA Polymerase”, Genome Research 5, 1995, p312-317

  On the other hand, it is more difficult to discriminate the + A peak from the graph waveform of the fluorescence signal than to discriminate the Stutter peak. The reason is that the + A peak may be higher or lower than the original peak, and when the same sample (same individual) is used for the same microsatellite multiple times. This is because the appearance of the + A peak (the relative height of the + A peak with respect to the original peak) varies. In Non-Patent Document 5, the experiment and analysis are conducted focusing on the ratio of the height of the true peak to the + A peak, but the above-mentioned difficulty of distinguishing the + A peak can be overcome. Not.

  By the way, there are homozygotes and heterozygotes in one set of microsatellite on the genome, but the graph waveform of the fluorescence signal varies greatly depending on which of the extracted DNA fragments. In the case of a homozygote, only one true peak appears on the graph, and in the case of a heterozygote, two true peaks appear on the graph. However, as is apparent from the graph showing the fluorescence analysis results in FIG. 20, many peaks may appear even in homozygotes. It is impossible to determine whether is a homozygote or a heterozygote.

  Furthermore, in the case of a heterozygote, the peaks overlap each other and the heights of the two true peaks are not necessarily equal, so the waveform becomes very complex. Hereinafter, waveforms appearing in the homozygote and the heterozygote will be described with reference to FIG. First, waveforms appearing in the case of a homozygote are shown in FIGS. (A) is a waveform obtained from a DNA fragment of 66 bases, and (b) is a waveform obtained from a DNA fragment of 68 bases. Next, waveforms appearing in the case of a heterozygote are shown in FIGS. It is assumed that a 66-base DNA fragment and a 68-base DNA fragment are obtained as PCR amplification products from this heterozygote. (C) assumes that the true peak appearing from the 66 base DNA fragment contained in the PCR amplification product and the true peak appearing from the 68 base DNA fragment have the same height (dotted line). Is a waveform obtained by synthesizing the waveform indicated by (2) and the waveform indicated by the broken line. On the other hand, (d) is a synthetic waveform in the case where the true peak appearing from the 66-base DNA fragment contained in the PCR amplification product and the true peak appearing from the 68-base DNA fragment have different heights. In (c), the peaks at 66 bases and 68 bases are relatively higher than the other peaks, so it is easy to guess that these are true peaks. In (d), the peaks at 66 bases are the other peaks. Since it is prominently higher than the peak, there is a possibility of misjudging that it is a homozygote having one true peak at 66 bases. Alternatively, even if the heterozygote is known, it is difficult to determine which two of the relatively high peaks appearing as true peaks.

  Since the waveform data obtained by fluorescence analysis of the PCR amplification product is thus complicated, it is very difficult to correctly determine the number of true peaks and the position of the true peaks. There is no method for accurately making this judgment, and at present, waveform data analysis largely relies on the intuition of skilled researchers, which is a bottleneck in gene function analysis research.

  The present invention has been made in view of such circumstances, and provides a display method and a display device capable of accurately discriminating and displaying a true signal and a noise signal from fluorescence analysis result data of a PCR amplification product. It is something to be offered.

The inventor first discovered that there are the following characteristics regarding the waveform and + A peak appearing in the graph showing the fluorescence analysis result of the PCR amplification product.
-Feature 1 In the waveform obtained in one experiment for one individual, the appearance of the + A peak (the relative height of the + A peak with respect to the original peak) is almost constant. That is, as shown in FIG. 1, if the height of the + A peak is x times that of the true peak, the ratio of the height of the + A peak to other Stutter peaks is also about x times. In heterozygotes, there are two true peaks, but if the ratio of the height of one true peak to the + A peak is x times the other true peak and the + A peak relative to it The height ratio is almost x times.
・Feature 2 + A peak appears differently depending on the microsatellite.
・Characteristic 3 For one microsatellite, the appearance of + A peak varies depending on the individual. Furthermore, the appearance of the + A peak varies even if the same individual is tested multiple times. However, the variation width of the manifestations of the + A peak in between individuals or between experiments, smaller than the variation width of the manifestations of the + A peak between microsatellite.

  Therefore, in consideration of the above characteristics, the present inventor has realized the following functions in order to accurately distinguish and display the true peak and the noise peak in the graph showing the fluorescence analysis result of the PCR amplification product. I came up with the idea.

Function 1- Considering the above feature 1 that groups the waveform appearing in the graph into a pair of + A peak and its original peak, by focusing on the relative height of the + A peak with respect to the original peak, It is possible to determine which is a + A peak and which is an original peak among a pair of peaks different in length by one base. An example of this discrimination method is shown in FIG. In the example of FIG. 2, the original peak of 62 bases and the + A peak corresponding thereto, the original peak of 64 bases and the + A peak corresponding thereto, the original peak of 66 bases and the + A peak corresponding thereto, the original peak of 68 bases The peak and the + A peak corresponding to it are discriminated. By the way, as shown in FIG. 3, when a peak appears every time the length is different by one base, there are two methods for grouping the + A peak and the original peak. Therefore, after distinguishing the original peak and the + A peak with each grouping method, the ratio of the + A peak height and the original peak height of each pair (+ A peak height ÷ Calculate the original peak height). Subsequently, for each grouping method, the variance of the calculated height ratio of each group is calculated, and the grouping method with the smaller variance is selected as the correct grouping method.

  As described above, the result of grouping the waveform appearing in the graph showing the fluorescence analysis result of the PCR amplification product into the pair of the + A peak and the original peak can be output as a display screen as shown in FIG. it can. This display screen includes a graph 400 showing the fluorescence analysis result of the PCR amplification product, a display 401 showing which of the peaks appearing in the graph is a + A peak, and a + A peak and an original of each set. The peak height ratio dispersion value 402 is displayed. In addition, when there are two ways of grouping the + A peak and the original peak, the + A peak and the original peak of each group when a grouping method different from the currently selected grouping method is adopted. The dispersion value of the peak height ratio is also displayed.

  According to this function, not only the microsatellite where the + A peak always appears lower than the original peak, but also the microsatellite where the + A peak appears higher than the original peak, the + A peak and the original peak The discrimination can be performed accurately. The same applies to the microsatellite where the + A peak and the original peak appear at approximately the same height. In the prior art, it is often assumed that the + A peak always appears lower than the original peak, so the original peak is a higher microsatellite, the + A peak appears higher depending on the individual, or the original peak Although it was not possible to make an appropriate judgment for a microsatellite that would appear higher, this problem would be solved by this function.

Furthermore, according to this function, the + A peak and the original peak can be accurately determined regardless of whether the extracted DNA fragment is a homozygote or a heterozygote. The reason will be described with reference to FIG. In the graph shown in FIG. 5, a DNA fragment indicated by a dotted line having a length of 66 bases and a DNA fragment indicated by a broken line having a length of 68 bases appear as a synthesized waveform. Here, a true peak 500 at 66 bases of a DNA fragment indicated by a dotted line having a length of 66 bases (with a height h ₁ ) and a + A peak 501, and a 66 bases of a DNA fragment indicated by a broken line having a length of 68 bases. It is assumed that there is a stutter peak 502 (having a height h ₂ ) and a + A peak 503 corresponding thereto. In this graph, the height ratio between the + A peak and the original peak is substantially constant. Therefore, when the value is approximated to x, the height of the + A peak 501 with respect to the true peak 500 is h ₁ × x, and stutter The height of the + A peak 503 with respect to the peak 502 is h ₂ × x. Then, the peak 504 at 66 bases of the synthesized waveform is h ₁ + h ₂ , and the peak 505 at 67 bases of the synthesized waveform is (h ₁ × x) + (h ₂ × x) = (h ₁ + h ₂ ) × x. In other words, the synthetic waveform from DNA fragments of different base lengths obtained in the case of a heterozygote can also hold the relationship that the height ratio between the + A peak and its original peak is constant (x times). I understand. Therefore, it is not necessary to determine whether the extracted DNA fragment is a homozygote or a heterozygote in order to discriminate the + A peak and the original peak by this function.

Function 2—Function to compare the height ratio value of the original peak and the + A peak with that of another individual Considering the above features 2 and 3, the original peak calculated for a certain microsatellite of an individual and Compare the height ratio of the + A peak with the same height ratio for the same microsatellite of other individuals, and if those values are far apart, the method of grouping the + A peak and the original peak A warning can be displayed to the user for the possibility of a mistake. With this function, the above warning is as shown in FIG. 6 together with the result of grouping the waveform appearing in the graph showing the fluorescence analysis result of the PCR amplification product into a pair of + A peak and its original peak. It can be output as a display screen.

  This display screen includes a graph 600 showing the fluorescence analysis result of the PCR amplification product, a display 601 showing which of the peaks appearing in the graph is a + A peak, and each set of + A peak and original Of the height ratio of the peaks (and dispersion values of the height ratios of the + A peak and the original peak in each group when a grouping method different from the currently selected grouping method is employed) 602 And are displayed. Further, when the data of the height ratio of the original peak and + A peak for the same microsatellite of another individual is held, the histogram 603 thereof is displayed. In addition, in the histogram 603, when the ratio of the height of the original peak of the individual to be analyzed and its + A peak (for example, the average value) is far from that of other individuals, a predetermined warning is displayed. It has come to be.

  According to this function, using the data of the height ratio of the original peak and + A peak accumulated by analysis for various other individuals for the microsatellite to be analyzed, + A in the analysis of new individuals It is possible to prevent the grouping of the peak and the original peak from being set erroneously.

  In particular, as shown in FIG. 7, in this graph, the + A peak does not appear at all in the graph showing the fluorescence analysis result of the PCR amplification product, and only the true peak and the Stutter peak appear. Useful to do. In the graph shown in FIG. 7, stutter peaks b and d and a true peak f appear at intervals of one base, and background noise appears where no such peak appears. Since the peaks a, c, e due to background noise appear next to the stutter peaks b, d and the true peak f, it can be observed as if a + A peak appears. Here, when these peaks are grouped into a set of + A peak and the original peak by the above function 1, when the peak f is correctly interpreted as a true peak (grouping method 1), + A The dispersion value of the peak height ratio is 99.84 (there is no peak on the left of peak a and right of peak f, so it was calculated that there is a peak of height 1 virtually), and peak e is the true peak When it is misinterpreted (grouping method 2), the dispersion value of the height ratio of the original peak and the + A peak is 0. Then, according to the function 1, the grouping method 2 having a smaller variance value is selected as an appropriate grouping method.

  Here, the height ratio between the true peak and + A peak in the grouping method selected by peak discrimination by function 1 is compared with the same height ratio in other individuals of the same microsatellite. If the value is far from the individual, it can be seen that the peak due to background noise was mistakenly interpreted as a + A peak. That is, it is possible to perform more accurate peak discrimination by combining the peak discrimination method using function 1 and the data comparison between individuals using function 2.

Function 3—Displays the discrimination result of each peak appearing in the graph. Based on the processing results of the above functions 1 and 2, each peak appearing in the graph showing the fluorescence analysis result of the PCR amplification product is represented as a true peak and a Stutter peak. , + A peak can be discriminated and the result can be displayed. An example of the display screen is shown in FIG.

  This display screen includes a graph 800 showing the fluorescence analysis result of the PCR amplification product, a display 801 showing whether each peak appearing in the graph is a true peak, a Stutter peak, or a + A peak, and each set + A peak and original peak height ratio variance (and the height of each group + A peak and original peak when using a different grouping method from the currently selected grouping method) Ratio dispersion value) 802, and the height ratio 803 between the true peak and its + A peak is displayed. From this display screen, the user can know the length of the DNA fragment from which the PCR amplification product is replicated.

  In determining which of the original peaks is the true peak, conventional techniques (methods disclosed in Patent Documents 1 to 5, Non-Patent Documents 1 to 4, etc.) are used. And

  As a means for realizing the above functions, the present invention is an apparatus for displaying the result of analyzing the length of a PCR amplification product of a DNA fragment, and displaying the detection signals of PCR amplification products having different lengths in a graph. A processing unit that distinguishes a + A peak corresponding to a detection signal of a PCR amplification product in which one adenine is added to a DNA fragment, and a peak other than the + A peak, and a + A peak and a + A peak The display apparatus which has a process part which displays the result which discriminate | determined from other peaks with a graph is provided.

  In the display device of the present invention, a plurality of peaks appearing in a graph as detection signals of PCR amplification products having different lengths are grouped into two peaks separated by one base in length, and one peak in each pair If the ratio of the height to the other peak is within the specified range, the peak with the longer length is identified as the + A peak, and the peak with the shorter length as the original peak. Features.

  In the display device of the present invention, a plurality of peaks appearing in a graph as detection signals of PCR amplification products having different lengths are grouped into two peaks separated by one base in length, and one peak in each pair When calculating the ratio of the height to the other peak and the variance of the calculated height ratio is within a predetermined range, the peak with the longer length is + A peak and the length is small. One of the peaks is discriminated as the original peak.

  In the display device of the present invention, when there are two grouping methods for grouping a plurality of peaks appearing in the graph as detection signals of PCR amplification products having different lengths, by pairing two peaks separated by one base in length. In the case where the peaks are grouped according to the first grouping method, the distribution of the height ratio between one peak and the other peak in each pair and the peaks are grouped according to the second grouping method. In this case, a comparison is made between the dispersion of the ratio of the height of one peak and the other peak in each pair, and a grouping method in which the dispersion becomes smaller is employed.

  In the display device of the present invention, a plurality of peaks appearing in a graph as detection signals of PCR amplification products having different lengths are grouped into two peaks separated by one base in length, and one peak in each pair The ratio of the height of the other peak is calculated, and when the ratio of the height is not similar to the ratio of the height calculated in the same manner in other individuals, a predetermined warning is displayed. To do.

  In the display device of the present invention, a peak determined to be a peak other than the + A peak is a true peak appearing on the graph as a detection signal of a PCR amplification product having the same length as the amplification source DNA fragment, or amplified. It is characterized by further discriminating whether it is a stutter peak appearing in the graph as a detection signal of a PCR amplification product having a length different from that of the original DNA fragment.

  In the display device of the present invention, when the height ratio between the true peak and the + A peak is not similar to the height ratio between the true peak and the + A peak in other individuals, A warning is displayed.

  The present invention is also a method for displaying the result of analyzing the length of a PCR amplification product of a DNA fragment, the step of displaying a graph of detection signals of PCR amplification products of different lengths, A step of discriminating the + A peak corresponding to the detection signal of the PCR amplification product added with adenine and a peak other than the + A peak, and the result of discriminating the peak other than the + A peak and the + A peak together with the graph A display method including a display step.

  In the display method of the present invention, a plurality of peaks appearing in the graph as detection signals of PCR amplification products having different lengths are grouped as two peaks separated by one base in length, and one peak in each pair If the ratio of the height to the other peak is within the specified range, the peak with the longer length is identified as the + A peak, and the peak with the shorter length as the original peak. Features.

  In the display method of the present invention, a plurality of peaks appearing in the graph as detection signals of PCR amplification products having different lengths are grouped as two peaks separated by one base in length, and one peak in each pair When calculating the ratio of the height to the other peak and the variance of the calculated height ratio is within a predetermined range, the peak with the longer length is + A peak and the length is small. One of the peaks is discriminated as the original peak.

  In the display method of the present invention, when there are two grouping methods in which a plurality of peaks appearing in the graph as detection signals of PCR amplification products having different lengths are grouped as two peaks separated by one base in length. In the case where the peaks are grouped according to the first grouping method, the distribution of the height ratio between one peak and the other peak in each pair and the peaks are grouped according to the second grouping method. In this case, a comparison is made between the dispersion of the ratio of the height of one peak and the other peak in each pair, and a grouping method in which the dispersion becomes smaller is employed.

  In the display method of the present invention, a plurality of peaks appearing in the graph as detection signals of PCR amplification products having different lengths are grouped as two peaks separated by one base in length, and one peak in each pair The ratio of the height of the other peak is calculated, and when the ratio of the height is not similar to the ratio of the height calculated in the same manner in other individuals, a predetermined warning is displayed. To do.

  In the display method of the present invention, a peak determined to be a peak other than the + A peak is a true peak appearing on the graph as a detection signal of a PCR amplification product having the same length as the amplification source DNA fragment, or amplified. It is characterized by further discriminating whether it is a stutter peak appearing in the graph as a detection signal of a PCR amplification product having a length different from that of the original DNA fragment.

  In the display method of the present invention, when the height ratio between the true peak and the + A peak is not similar to the height ratio between the true peak and the + A peak in other individuals, A warning is displayed.

  As described above, according to the gene information display method and display device of the present invention, in the graph showing the fluorescence analysis result of the PCR amplification product, the + A peak and the original peak are automatically discriminated, and the back The possibility of erroneous peak determination due to ground noise is reduced, and further, which peak is a true peak is automatically determined. Thus, the user can accurately know the length of the DNA fragment from which the PCR amplification product is replicated, and can analyze the microsatellite based on the length. In addition, each peak can be accurately identified regardless of whether the DNA fragment of the replication source contains a homozygous or heterozygous microsatellite.

  Hereinafter, the best mode for carrying out the method and apparatus for displaying gene information of the present invention will be described in detail with reference to the accompanying drawings. 9 to 17 are diagrams illustrating the embodiments of the present invention. In these drawings, the same reference numerals denote the same components, and the basic configuration and operation are the same. To do.

  FIG. 9 is a functional block diagram schematically showing the internal configuration of a gene information display system constructed as an embodiment of the present invention. This gene information display system includes a waveform data DB 900 storing waveform data obtained as a result of fluorescence analysis of PCR amplification products after PCR and electrophoresis experiments, a display device 901 for displaying the waveform data and the analysis results in a graph, and a display A keyboard 902 and a pointing device 903 such as a mouse for performing an operation such as selecting an individual or a peak on the displayed graph, a central processing unit 904 for performing necessary arithmetic processing, control processing, and the like. A program memory 905 for storing programs necessary for processing and a data memory 906 for storing data necessary for processing in the central processing unit 904 are provided.

  The program memory 905 includes the above function 1, that is, the + A peak separation processing unit 907 that groups the peaks appearing in the waveform data into pairs of the original peak and the + A peak, and the above function 3, that is, the original peak is true. True peak separation processing unit 908 for determining whether the peak is a peak or a Stutter peak, and the above function 2, ie, the height ratio between the original peak and its + A peak is the same height ratio in other individuals And a warning display processing unit 909 for detecting a large deviation from the above and displaying a warning.

  The data memory 906 includes individual data 910 that holds waveform data for each individual, and waveform data 911 that holds peak data for each waveform data.

  FIG. 10 is a diagram illustrating a data structure of the waveform data DB 900 included in the data memory 906. This data structure WaveFormData [] is an individual ID 1000 for identifying each individual, waveform data 1001 of each individual, and a height ratio 1002 between the true peak and its + A peak in the waveform data. The dispersion of the height ratio between the original peak and its + A peak according to the selected grouping method 1003 and 1003 is the height ratio between the original peak and its + A peak according to a different grouping method from the grouping method in 1003 Data indicating the variance 1004 is included. Note that the data 1002, 1003, and 1004 have NULL values if the calculation has not yet been performed.

  FIG. 11 is a diagram illustrating a data structure of the waveform data 911 included in the data memory 906. This data structure PeakFormData [] is the position where each peak appears (base length) 1100, the height 1101 of each peak, whether the peak is a true peak or a Stutter peak, and + A It includes a label 1102 indicating whether it is a peak, and data indicating a height ratio 1103 between the peak and its + A peak. Note that the data 1102 has a NULL value if analysis has not yet been performed. Further, the data 1103 has a NULL value when the analysis has not been performed yet or when the peak is not the original peak (that is, when it is a + A peak).

  Next, the flow of processing performed in this gene information display system will be described with reference to the flowchart shown in FIG.

  First, the waveform data of each individual is read from the waveform data DB 900 (step 1200). Here, the waveform data of one microsatellite for all individuals stored in the waveform data DB 900 is read and held as individual data 910 and waveform data 911 in the data memory 906. Next, the + A peak and the original peak are grouped for each individual (step 1201). This processing is executed by the + A peak separation processing unit 907 of the program memory 905, and a value indicating that the peak determined to be + A peak is a + A peak in the peak label 1102 of the waveform data 911. In addition, for the peak determined to be the original peak, the height ratio with the + A peak is written in the data 1103 of the waveform data 911. Further, when the grouping is completed for all the waveform data of each individual, the calculated values are written in the data 1002 to 1004 of the individual data 910, respectively. The processing performed by the + A peak separation processing unit 907 will be described in detail later.

  The result of grouping the peaks included in the waveform data of each individual into the + A peak and the original peak in this way is displayed in a graph as shown in FIG. 13 (step 1202). The display screen shown in FIG. 13 shows a result 1300 of grouping each peak into the + A peak and the original peak for the waveform data of a certain individual, the height and height ratio 1301 of each peak set, The variance 1302 of the height ratio of each group according to the grouping method is displayed.

  Further, for each peak determined to be a peak other than the + A peak (original peak) in Step 1201, it is determined whether it is a true peak or a Stutter peak (Step 1203). This processing is executed by the true peak separation processing unit 908 of the program memory 905, and the peak discriminating method uses a conventional technique. The discrimination result of each peak is written in the label 1102 of the waveform data 911. Further, the height ratio between the true peak and the + A peak is calculated for each individual, and the value is written in the data 1002 of the individual data 910.

  The result of discriminating the original peak included in the waveform data of each individual into a true peak and a Stutter peak in this way is displayed in a graph as shown in FIG. 14 (step 1204). The display screen shown in FIG. 14 shows a result 1400 of discriminating each peak into + A peak, true peak, and Stutter peak for the waveform data of a certain individual, the height and height ratio 1401 of each peak set, The dispersion 1402 of the height ratio of both peaks by the two grouping methods of the + A peak and the original peak, and the height ratio 1403 of the true peak and the + A peak are displayed.

  If the height ratio between the true peak and the + A peak greatly deviates from the same height ratio in other individuals as a result of the processing in step 1204, the warning display processing unit 909 in the program memory 905 A predetermined warning is displayed (step 1205). The processing by the warning display processing unit 909 will be described in detail later.

  An example of this warning display screen is shown in FIG. The display screen shown in FIG. 15 shows a result 1500 obtained by discriminating each peak into a + A peak, a true peak, and a Stutter peak for the waveform data of a certain individual, a height and height ratio 1501 of each peak set, The dispersion of the height ratio 1502 of both peaks by the two grouping methods of the + A peak and the original peak, the height ratio 1503 of the true peak and the + A peak, and the true ratio in this individual and other individuals A histogram of the height ratio between this peak and its + A peak and a predetermined warning display 1504 are displayed.

  FIG. 16 is a flowchart showing in detail the processing by the + A peak separation processing unit in step 1201 of FIG. This flowchart shows processing for one individual. First, the highest peak among the peaks included in the individual waveform data is searched (step 1600). Let p be the position of this peak. Next, for the integer i, the ratio “the height of the peak at the position of {p ± (unit length × i) +1} ÷ the height of the peak at the position of {p ± (unit length × i)}” Ask for. The variance of the obtained ratio is set as V1 (step 1601). This corresponds to the ratio and variance assuming that the highest peak is the original peak (assuming it is not a + A peak). If the highest peak is the original peak, the peak at a position that is an integer multiple of the unit length of the microsatellite can be regarded as the original peak. In addition, the peak at a position longer by 1 base than those peaks can be regarded as each + A peak. Then, the ratio “height of the peak at the position of {p ± (unit length × i)} ÷ the height of the peak at the position of {p ± (unit length × i) −1}” with respect to the integer i Ask. The variance of the obtained ratio is set as V2 (step 1602). As a result, contrary to step 1601, the ratio and dispersion when the highest peak is assumed to be the + A peak are obtained.

  Subsequently, V1 and V2 obtained in steps 1601 and 1602 are compared (step 1603) to determine whether the highest peak is the + A peak or the original peak. If V1 ≦ V2, it is determined that the assumption in step 1601 was more appropriate. That is, the highest peak and the peak at an integer multiple of the unit length from that are the original peak (true peak or Stutter peak), and the peak at one base longer than those peaks is + A It is determined that it is a peak (step 1604). Therefore, the peak at the position of {p ± (unit length × i) +1} with respect to the integer i is a + A peak, and each + A peak is a + A peak in the peak label 1102 of the waveform data 911. A value indicating this is written, and for each original peak, the value of the ratio used to obtain V1 is written in the data 1103. Also, the values of V1 and V2 are written in the data 1003 and 1004 of the individual data 910, respectively.

  On the other hand, if V1> V2, it is determined that the assumption in step 1602 was more appropriate. That is, the highest peak and the peak located at an integer multiple of the unit length from the peak are + A peaks, and the peak located at one base shorter than those peaks is determined to be the original peak ( Step 1605). Therefore, it is assumed that the peak at the position of {p ± (unit length × i)} with respect to the integer i is a + A peak, and that each + A peak is a + A peak in the peak label 1102 of the waveform data 911. The value shown is written, and for each original peak, the value of the ratio used to determine V2 is written in data 1103. Also, the values of V2 and V1 are written in the data 1003 and 1004 of the individual data 910, respectively.

  FIG. 17 is a flowchart showing in detail the processing by the warning processing unit in step 1205 of FIG. This flowchart shows processing for one individual. First, for all individuals, the value of the height ratio between the true peak and the + A peak (calculated by the processing in step 1203 in FIG. 12 and stored in the data 1002 of the individual data 910) is totaled, and the average And obtain the standard deviation. Here, the average is E and the standard deviation is σ (step 1700). Next, it is checked whether the height ratio between the true peak calculated for the individual currently processed and the + A peak is E−1.96σ or more and E + 1.96σ or less (step 1701). . Assuming that the height ratio between the true peak and its + A peak follows a normal distribution, if the height ratio between the true peak and its + A peak is outside this range, the lower or higher 2.5% Therefore, it is determined that the value is far from the ratio of other individuals, and a predetermined warning is displayed (step 1702).

  In the above, the threshold for determining whether the height ratio of the true peak and its + A peak is significantly different from other individuals is determined by the standard deviation, but the threshold is determined by other methods. It doesn't matter. For example, several individuals representing a typical waveform may be selected in advance, and the upper and lower limits of the ratio range in that individual may be used as the threshold value, or the threshold value may be entered by the user in advance. Can also be defined. In addition, as a representative value of the ratio of the height of the original peak and its + A peak in one individual, the ratio of the height of the true peak and its + A peak was used, but the original peak and its + A peak were used. The average value, median value, and mode value of the height ratio may be used.

  As mentioned above, although the specific embodiment was shown and demonstrated about the display method and display apparatus of the genetic information of this invention, this invention is not limited to these. A person skilled in the art can make various changes and improvements to the configurations and functions of the invention according to the above-described embodiments or other embodiments without departing from the gist of the present invention.

  In the above-described embodiment, the genotype estimation technique of an individual for the purpose of searching for a gene involved in a phenotype such as a disease using electrophoresis (gel electrophoresis or capillary electrophoresis) is described. The method and apparatus of the present invention can also be applied to the case where the length or molecular weight / mass of a PCR amplification product is examined by another experimental method such as MALDI-TOF / MS. The same applies to genotype estimation technology for individuals aiming to search for genes involved in phenotypes other than diseases, genotype estimation technology for DNA testing, etc., and non-human genotype estimation technologies such as agricultural products and marine products. It is.

  The gene information display method and display apparatus of the present invention can be used by being mounted on, for example, a personal computer used as an experimental data analysis apparatus.

It is a figure which shows the characteristic of how the + A peak appears in the graph which shows the fluorescence analysis result of the PCR amplification product which this inventor discovered. A method for discriminating which is a + A peak and which is an original peak among a pair of peaks different in length by one base by utilizing the feature of the appearance of the + A peak discovered by the present inventors It is a figure which shows the example of. It is a figure which shows the grouping method of + A peak in case a peak appears for every 1 base in length, and the original peak. It is a figure which shows the display screen showing the result of having grouped the waveform which appeared on the graph into the pair of + A peak and its original peak. It is a figure which shows the method of discriminating + A peak and the original peak in the graph obtained from a heterozygote. It is a figure which shows the display screen showing the warning that the grouping method is wrong as needed with the result which grouped the waveform which appeared on the graph into the pair of + A peak and its original peak. It is a figure which shows the method of discriminating a peak in the graph in which the + A peak does not appear at all and only the true peak and the Stutter peak appear. It is a figure which shows the example of the screen which displays the result which discriminate | determined whether each peak which appeared on the graph is a true peak, a Stutter peak, or + A peak. It is a functional block diagram which shows roughly the internal structure of the gene information display system constructed | assembled as one Embodiment of this invention. It is a figure which shows the data structure of the individual data 910 contained in the data memory 906 of the gene information display system shown in FIG. It is a figure which shows the data structure of the waveform data 911 contained in the data memory 906 of the gene information display system shown in FIG. It is a flowchart which shows the flow of the process performed in the gene information display system shown in FIG. It is a figure which shows the screen which carries out the graph display of the result which grouped each peak into + A peak and the original peak about the waveform data of each individual | organism | solid by the + A peak isolation | separation processing part. It is a figure which shows the screen which displays in graph the result of having discriminate | determined the original peak contained in the waveform data of each individual into the true peak and the Stutter peak by the true peak separation processing unit. It is a figure which shows the example of the screen which displays a predetermined | prescribed warning by a warning display process part, when the height ratio of a true peak and the + A peak has remove | deviated significantly from the same height ratio in another individual | organism | solid. 13 is a flowchart showing in detail a process by a + A peak separation processing unit in step 1201 of FIG. 13 is a flowchart showing in detail processing by a warning processing unit in step 1205 of FIG. It is a figure explaining the microsatellite which appears on a genome. It is a figure which shows typically the experimental procedure which extracts and amplifies the DNA fragment of a microsatellite part by PCR and electrophoresis. It is a figure explaining the Stutter peak and + A peak which are the typical noise which arises in the experimental process of PCR and electrophoresis. It is a figure which shows the waveform which appears in the graph of a fluorescence-analysis result about each of a homozygote and a heterozygote.

Explanation of symbols

900 Waveform data DB
901 Display device 902 Keyboard 903 Pointing device 904 Central processing unit 905 Program memory 906 Data memory 907 + A peak separation processing unit 908 True peak separation processing unit 909 Warning display processing unit 910 Individual data 911 Waveform data 1900, 1901 Primer arrangement 1902 Micro Genomic region 2000, 2001, 2003, 2004 DNA fragment containing satellite and primer sequences

Claims (14)

An apparatus for displaying the result of analyzing the length of a PCR amplification product of a DNA fragment,
A processing unit that graphically displays detection signals of PCR amplification products having different lengths, and
A processing unit for discriminating a + A peak corresponding to a detection signal of a PCR amplification product obtained by adding one adenine to a DNA fragment and a peak other than the + A peak;
A display device having a processing unit that displays a result of discriminating a + A peak and a peak other than the + A peak together with a graph.   Plural peaks appearing in the graph as detection signals of PCR amplification products having different lengths are grouped into two peaks separated by one base in length, and the height of one peak and the other peak in each pair When the ratio is within a predetermined range, the peak having the longer length is discriminated as the + A peak, and the peak having the shorter length is discriminated as the original peak. The display device described.   Plural peaks appearing in the graph as detection signals of PCR amplification products having different lengths are grouped into two peaks separated by one base in length, and the height of one peak and the other peak in each pair If the variance of the calculated height ratio is within a predetermined range, the peak with the longer length is + A peak and the peak with the shorter length is its original The display device according to claim 1, wherein the display device is determined as a peak.   When there are two grouping methods for grouping a plurality of peaks appearing in the graph as detection signals of PCR amplification products having different lengths, with two peaks separated by one base in length, the first group When the peaks are grouped according to the dividing method, the dispersion of the ratio of the height of one peak to the other peak in each pair, and one of each pair when the peaks are grouped according to the second grouping method The display device according to claim 2, wherein a grouping method in which the variance is smaller is adopted by comparing the variance of the ratio of the height between the first peak and the other peak. Plural peaks appearing in the graph as detection signals of PCR amplification products having different lengths are grouped into two peaks separated by one base in length, and the height of one peak and the other peak in each pair A processing unit for calculating the ratio of
5. A processing unit that displays a predetermined warning when the calculated height ratio is not similar to the similarly calculated height ratio in other individuals. The display device according to any one of the above. For peaks identified as peaks other than the + A peak, are true peaks appearing in the graph as detection signals of PCR amplification products having the same length as the amplification source DNA fragment, or are different from the amplification source DNA fragment display device according to claim 1, any one of 5, characterized in that to determine the of the PCR amplification product detection signal whether a stutter peak appearing in the graph further as.   When the ratio of the height of the true peak to its + A peak is not similar to the ratio of the height of the true peak to its + A peak in other individuals, a predetermined warning is displayed. The display device according to claim 6. A method for displaying the result of analyzing the length of a PCR amplification product of a DNA fragment,
Displaying a graph of detection signals of PCR amplification products each having a different length;
Discriminating a + A peak corresponding to a detection signal of a PCR amplification product obtained by adding one adenine to a DNA fragment and a peak other than the + A peak;
A display method including a step of displaying a result of discriminating a + A peak and a peak other than the + A peak together with a graph.   Plural peaks appearing in the graph as detection signals of PCR amplification products having different lengths are grouped into two peaks separated by one base in length, and the height of one peak and the other peak in each pair 9. When the ratio is within a predetermined range, a peak having a longer length is discriminated as a + A peak, and a peak having a shorter length is discriminated as its original peak. Display method of description.   Plural peaks appearing in the graph as detection signals of PCR amplification products having different lengths are grouped into two peaks separated by one base in length, and the height of one peak and the other peak in each pair If the variance of the calculated height ratio is within a predetermined range, the peak with the longer length is + A peak and the peak with the shorter length is its original The display method according to claim 8, wherein the display method is determined as a peak.   When there are two grouping methods for grouping a plurality of peaks appearing in the graph as detection signals of PCR amplification products having different lengths, with two peaks separated by one base in length, the first group When the peaks are grouped according to the dividing method, the dispersion of the ratio of the height of one peak to the other peak in each pair, and one of each pair when the peaks are grouped according to the second grouping method 11. The display method according to claim 9, wherein a grouping method in which a variance is smaller is adopted by comparing a variance of a height ratio between the first peak and the other peak. 11.   Plural peaks appearing in the graph as detection signals of PCR amplification products having different lengths are grouped into two peaks separated by one base in length, and the height of one peak and the other peak in each pair The ratio of these is calculated, and when the ratio of the height is not similar to the ratio of the height similarly calculated in other individuals, a predetermined warning is displayed. The display method according to any one of the above items. For peaks identified as peaks other than the + A peak, are true peaks appearing in the graph as detection signals of PCR amplification products having the same length as the amplification source DNA fragment, or are different from the amplification source DNA fragment display method according to claims 8 to any one of 12, characterized in that to determine the of PCR or amplification is stutter peaks appear in the graph as a detection signal of the product further.   When the ratio of the height of the true peak to its + A peak is not similar to the ratio of the height of the true peak to its + A peak in other individuals, a predetermined warning is displayed. The display method according to claim 13.

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