CROSS REFERENCE TO RELATED APPLICATIONS

This application is a National Stage of International Application No. PCT/JP2017/040441 filed Nov. 9, 2017, claiming priority based on Japanese Patent Application No. 2017-004843 filed Jan. 16, 2017.

TECHNICAL FIELD

The present invention relates to a data analyzing device for analytically processing data acquired by an instrumental analysis on a sample, and a computer program for such a device. More specifically, the present invention relates to a data analyzing device and a program for data analysis configured to perform a differential analysis between a plurality of sample groups.

BACKGROUND ART

In recent years, research on an analysis of biomarkers using mass spectrometry has been promoted for early diagnosis of specific diseases or confirmation of therapeutic effects. A portion of those techniques have already been put into practical use. For example, if a substance is discovered which does not exist or barely exists in blood, urine or other types of biological samples collected from healthy individuals, but definitely exists in blood, urine or other types of biological samples collected from patients suffering from certain diseases such as cancer, the substance can be considered to be a promising candidate of the biomarker for the disease in question. A typical approach to searching for such a biomarker is to perform a measurement using a mass spectrometer on a considerable number of samples derived from each of the two or more groups (e.g. a group of healthy individuals and a group of patients), and to perform a differential analysis between the groups based on the data obtained by the measurement.

A conventional and typical procedure for a differential analysis of mass spectrometric data aimed at searching for a biomarker (or any type of generally known marker) is as follows. It is hereinafter assumed that the total number of groups is NG, and that of the samples is NS.

[Step A1] A mass spectrum is acquired for each of a considerable number of samples (NS), and a peak detection is performed in each mass spectrum according to a predetermined algorithm. Then, a peak list which shows the mass-to-charge ratio (m/z) values and signal intensities of the detected peaks, is created for each mass spectrum. The total number of peak lists to be created is the same as that of the samples, NS.

[Step A2] The given samples can be classified into NGgroups based on empirical information (prior information). Accordingly, the peak lists obtained in Step A1 are divided into NGgroups, and a “peak matrix” MPis created in which each peak list is regarded as a column vector, with the signal-intensity values arranged in rows and columns so that signal-intensity values corresponding to the same mass-to-charge ratio are located in the same row. A peak detected in one mass spectrum may not have its counterpart located at the same mass-to-charge ratio in another mass spectrum (i.e. no counterpart in the peak list). In that case, a value of zero can be used as the signal-intensity value of the peak in question in the latter mass spectrum. An example of the empirical information is a piece of information which shows the origin of the sample, such as a piece of information showing whether the sample was collected from a healthy individual or a cancer patient.

FIG. 3Ais a conceptual diagram of the peak matrix. In this peak matrix, the sample information (e.g. sample numbers) is arranged in the horizontal direction (in a row), while the mass-to-charge-ratio values are arranged in the vertical direction (in a column). Each element of the matrix is a signal-intensity value. The total number of columns of this peak matrix MPis equal to the number of samples NS. The total number of rows is equal to the number of all peaks NPdetected across the entire group of samples (without allowing the peaks detected at the same mass-to-charge-ratio value to be counted multiple times).

[Step A3] For each row of the peak matrix MPcreated in Step A2, a univariate analysis (which is typically a t-test, U-test, analysis of variance (ANOVA) or similar statistical hypothesis testing) is performed to determine whether or not there is a significant difference between the groups. Then, the p-value, which shows the statistical reliability of the observed difference, is calculated for each row.

[Step A4] The p-value of each row is compared with a predetermined significance level α (for example, α=0.05 is often used in the t-test) to extract a row, i.e. a peak (or mass-to-charge ratio), at which there is a significant difference between the groups. A substance corresponding to this extracted peak is a marker candidate.

The grouping of the samples into the group of healthy individuals and the group of patients in the previously described example is unambiguous. However, it is often the case that the samples to be analyzed in an actual differential analysis cannot be grouped in a single definite manner. There is also a strong demand for the differential analysis to be performed on the same set of samples using various grouping methods based on different kinds of empirical information, to search for a marker for each grouping method.

For example, in recent years, mass spectrometric techniques for distinguishing or identifying the species or strains of microorganisms have been drawing attention (see Patent Literature or Non-Patent Literature 1). A mass spectrometric process for identifying a microorganism normally includes the steps of assigning a plurality of samples of the same species or strain to one group and performing a differential analysis to search for a mass spectrum peak showing a significant difference between different groups, i.e. between different species or strains. Meanwhile, different species or strains of microorganisms may have some properties or features in common. For example, it is often the case for different species or strains of microorganisms to have common features, such as a specific drug (antibiotic) being particularly effective for different species or strains of microorganisms, or conversely, the different species or strains of microorganisms being resistant to a specific drug. In such cases, it may be necessary to conduct research for each drug to search for a marker which yields a significant difference between a group of samples resistant to one or more drugs and a group of samples having no such resistance, in addition to the search for a marker which yields a significant difference between different species or strains of microorganisms.

In the previously described type of differential analysis, every time the criterion for the grouping (i.e. the kind of empirical information to be used for the grouping) is changed, an operator (user) needs to perform an input operation for each individual sample to specify the group which the sample should belong to according to the new criterion. If there is a considerable number of samples, such a task will be extremely cumbersome and lower the efficiency of the analysis. The same task also constitutes a cause of an inappropriate analysis due to input errors.

CITATION LIST

Patent Literature

Patent Literature 1: JP 2013-85517 A

Non-patent Literature

Non-Patent Literature 1: Y. Hotta and four other authors, “Classification of the GenusBacillusBased on MALDI-TOF MS Analysis of Ribosomal Proteins Coded in S10 and spc Operons”,Journal of Agricultural and Food Chemistry,2011, Vol. 59, No. 10, pp. 5222-5230

SUMMARY OF INVENTION

Technical Problem

The present invention has been developed to solve the previously described problem. Its objective is to provide a data analyzing device and a program for data analysis to be used for a differential analysis based on data acquired for each sample in order to locate a marker for distinguishing between a plurality of groups of samples, where the device or program allows an operator to appropriately change the criterion for the grouping based on the properties, features or other factors which the operator is focused on, and to easily and efficiently perform a differential analysis between the new groups according to the criterion after the change.

Solution to Problem

The data analyzing device according to the present invention developed for solving the previously described problem includes:

a) a data acquiring section configured to acquire data for each of a plurality of samples, the data representing a chromatogram or mass spectrum;

b) an empirical information setting section configured to acquire or set one or more kinds of empirical information for each of the plurality of samples, the empirical information showing a property of each sample;

c) an empirical information selecting section configured to allow an operator to select a condition for grouping based on the one or more kinds of empirical information;

d) a group forming section configured to perform grouping of the plurality of samples into a plurality of groups based on the selected condition for grouping; and

e) a differential analysis section configured to analyze a difference in a peak of the chromatogram or mass spectrum between the plurality of groups formed by the group forming section, using the data acquired by the data acquiring section and grouped into the plurality of groups,

where the data analyzing device is configured to execute the grouping in the group forming section and a differential analysis in the differential analysis section according to a selecting operation by the operator using the condition selecting section.

The program for data analysis according to the present invention developed for solving the previously described problem is a program for data analysis configured to run on a computer to process data for a plurality of samples so as to perform a differential analysis between a plurality of groups into which the plurality of samples are to be classified, based on data representing a chromatogram or mass spectrum acquired for each of the plurality of samples, where the program is configured to make the computer execute a differential analysis including:

a) an empirical information setting step configured to acquire or set one or more kinds of empirical information for each of the plurality of samples, the empirical information showing a property of each sample;

b) a condition selecting step configured to allow an operator to select a condition for grouping based on the one or more kinds of empirical information;

c) a group forming step configured to perform grouping of the plurality of samples into the plurality of groups based on the selected condition for grouping; and

d) a differential analysis step configured to analyze a difference in a peak of the chromatogram or mass spectrum between the plurality of groups formed in the group forming step, using the data grouped into the plurality of groups,

where the computer executes the grouping in the group forming step and a differential analysis in the differential analysis step according to a selecting operation by the operator in the condition selecting step.

In the data analyzing device and the program for data analysis according to the present invention, the data to be analyzed is mass spectrum data collected by a mass spectrometric analysis (including MSnspectrum data where n is an integer equal to or greater than two), or chromatogram data collected by a gas chromatographic or liquid chromatographic analysis.

In the data analyzing device according to the present invention, the data acquiring section acquires chromatogram data or mass spectrum data for each of a plurality of samples by reading the data, for example, from an external storage device. The empirical information setting section acquires, for each of the samples, one or more kinds of empirical information showing a property of the sample by automatically reading the information from an external source, or sets the empirical information according to a manual input by an operator or similar users. The empirical information may include an item of information showing the history or origin of each sample. The condition selecting section allows the operator to select the condition for grouping based on one kind of empirical information which the operator is focused on among the one or more kinds of empirical information which are previously set in the previously described manner. As a specific example, the condition selecting section may display choices of the grouping condition based on one kind of empirical information, or information equivalent to such choices, on a screen of a display unit so that the operator can select and indicate one of the choices with an input unit.

After the grouping condition has been selected by the operator through the condition selecting section, the group forming section responds to the selecting operation and perform the grouping of the plurality of samples to be analyzed into a plurality of groups based on the selected grouping condition. The differential analysis section performs a differential analysis for locating a difference in a peak of the chromatogram or mass spectrum between the groups, using the data corresponding the samples included in each of the plurality of groups formed in the previously described manner. The result of the differential analysis is shown on the screen of the display unit. There is no specific limitation on the method for the differential analysis; for example, statistical hypothesis testing may preferably be used, such as the analysis of variance (ANOVA). A peak which yields a significant difference between the groups can be extracted from the data by the differential analysis. The mass-to-charge ratio, retention time, or substance corresponding to that peak can be used as a marker for distinguishing between the groups.

In a preferable mode of the data analyzing device according to the present invention, the group forming section is configured to perform grouping of the plurality of samples into N groups based on first empirical information concerning each sample, where N is an integer equal to or greater than three, and the data analyzing device further includes:

f) a group rearrangement condition specifying section configured to allow an operator to specify a condition for rearranging the groups using second empirical information concerning each of the N groups formed by the group forming section, or concerning the samples included in each of the N groups; and

g) a group rearranging section configured to form M groups, where N>M, by selecting one or more groups from the N groups formed by the group forming section and merging the selected groups together as needed, based on the condition for rearranging the groups specified through the group rearrangement condition specifying section,

where the differential analysis section is configured to perform a differential analysis between the N groups formed by the group forming section, using the data corresponding to the samples included in each of the N groups, and to perform a differential analysis between the M groups formed by the group rearranging section, using the data corresponding to the samples included in each of the M groups.

According to this mode of configuration, the group forming section perform the grouping of the plurality of samples to be analyzed into N groups based on the first empirical information given to each sample, where N is equal to or greater than three. Normally, the first empirical information is a piece of information for classifying a considerable number of samples into a minimum size of groups.

Typically, the first empirical information is a piece of information showing the history, origin or other aspects of each sample. This information can be included in the sample name or sample identification number given to each sample. This allows the operator to locate the first empirical information in the sample name, sample identification number or similar information of each sample, and manually indicate the group to which the sample should be assigned. In this case, the group forming section can perform the grouping of the samples into N groups according to the indication. Automatic sorting of the samples based on the sample name, sample identification number or similar information is also possible if the first empirical information is included in the sample name, sample identification number or similar information of each sample. In this case, the group forming section can locate the sample name, sample identification number or similar information, and automatically perform the grouping of the samples into N groups based on the located information. The result of the grouping, i.e. the information concerning the samples assigned to each group, may preferably be shown on the screen of the display unit so that the operator can check the result.

Consider the situation in which the operator is going to perform a differential analysis between new groups after merging two or more groups which are similar to each other in a specific property into one group among the groups formed by the group forming section. The operator specifies the group rearrangement condition through the group rearrangement condition specifying section so that the groups labeled with the second empirical information indicating the possession of that specific property should be selected. Based on the specified group rearrangement condition, the group rearranging section rearranges the groups, for example, by selecting two or more groups having that specific property using the second empirical information given to the N groups, and then merging the selected groups into one group as well as merging the other groups into another group. The operator can change the group rearrangement condition specified through the group rearrangement condition specifying section. In that case, the group rearranging section changes the combination of the groups to be selected and merged together. As with the result of the grouping, the result of the group rearrangement, i.e. the information concerning the samples assigned to each group after the rearrangement, may preferably be shown on the screen of the display unit.

In this configuration, the differential analysis section additionally performs the differential analysis between the M rearranged groups formed in the previously described manner, using the data corresponding to the samples included in each of these groups. This differential analysis is based on the M groups which have been formed according to a criterion that is different from the one applied to the differential analysis based on the N groups mentioned earlier. By this analysis, a new peak which yields a significant difference between the groups under the new criterion, i.e. a new marker, can be extracted from the data. Every time the operator changes the group rearrangement condition specified through the group rearrangement condition specifying section in the previously described manner, the differential analysis section performs a new differential analysis based on the rearranged groups. Accordingly, the operator can consecutively specify different properties and search for a marker related to each property.

In the data analyzing device according to the present invention, the group-rearrangement condition specifying section may preferably be configured to allow the operator to cancel the group rearrangement condition by a command which restores the grouping to the original state and allows the operator to newly specify a different group rearrangement condition.

In the data analyzing device according to the present invention, if the data is mass spectrum data, the data analyzing device may further include:

h) a peak detecting section configured to detect peaks from mass spectrum data and create a peak list which shows a signal-intensity value for each of the mass-to-charge ratios of the detected peaks; and

i) a peak matrix creating section configured to create a peak matrix based on the peak lists created for the plurality of samples as well as based on the result of the grouping by the group forming section or the result of the group rearrangement by the group rearranging section, the peak matrix having signal-intensity values as elements arranged in rows and columns in such a manner that the mass-to-charge-ratio values of the peaks are assigned in a row while information which identifies the samples sorted into groups is assigned in a column, and

the differential analysis section is configured to perform a differential analysis on the peak matrix.

The rows and columns in the aforementioned peak matrix are defined for convenience; it is evident that the rows and columns can be transposed. In other words, it is evident that replacing the terms “row” and “column” with each other does not substantially change the previously described configuration.

In the previously described configuration, the peak matrix creating section receives mass spectrum data corresponding to each sample and detects peaks in each mass spectrum according to a predetermined criterion. There is no specific limitation on the predetermined criterion; any commonly known peak-detection method may be used. For example, among the peak-shaped signals observed on a mass spectrum, each signal whose intensity is equal to or higher than a predetermined value may be detected as a true peak, whereas each signal whose intensity is lower than the predetermined value may be considered to be a noise peak. After the peaks have been detected in all mass spectra, the signal-intensity value and mass-to-charge-ratio value of each peak are acquired, and a peak matrix having the signal-intensity values as its elements is created, in which the mass-to-charge-ratio value is assigned in a row, while the information identifying the sample is assigned in a column. The samples assigned in a column should be sorted by the group based on the result of the grouping or that of the group rearrangement. One row in the peak matrix is an array of signal-intensity values for all samples at one specific mass-to-charge ratio.

The differential analysis section performs a differential analysis on the peak matrix by an appropriate method, such as the analysis of variance. In this case, the differential analysis section can extract a peak or mass-to-charge ratio corresponding to a row or column which shows a significant difference in the differential analysis. The substance corresponding to this peak or mass-to-charge ratio serves as a marker for distinguishing between groups.

There is no limitation on the kind of sample to be analyzed in the data analyzing device according to the present invention. As a preferable mode of the present invention, the sample to be analyzed is a microorganism, and the first empirical information is information showing the species and/or strain of the microorganism.

In this case, the second empirical information may be information showing the presence or absence of the resistance to various drugs (antibiotics or antimicrobial agents), or information showing minimum inhibitory concentrations (MIC) or clinically determined thresholds of the drugs.

According to this mode of the invention, it is possible to detect a substance which serves as a marker for distinguishing between species or strains of microorganisms. It is also possible to detect a substance which serves as, or seems to be useful as a marker for distinguishing a microorganism that is characteristically resistant to one specific kind of drug or resistant to a plurality of specific kinds of drugs (i.e. multiple-drug resistant).

Advantageous Effects of Invention

The data analyzing device and the program for data analysis according to the present invention allow an operator to appropriately change the criterion for the grouping based on a property or feature which the operator is focused on, and to easily and efficiently perform a differential analysis between the new groups according to the criterion after the change. Thus, the operator can efficiently detect a substance which serves as a marker that is significantly related to the property or feature of interest.

