ORIOGEN (O rder R estricted I nference for O rdered G ene E xpressioN )

Inputs Screen:

Input file name

Output file name

Ontology file name

Total number of dose groups or time points

Vector of sample sizes per dose group or time point

Number of initial bootstrap samples

Number of final bootstrap samples

FDR level for performing the actual test

Percentile to use for s0 adjustment

Bootstrap random seed

Take log of signal

Longitudinal sampling

Transpose input file

 

Input file name:

This field specifies the path and name of the file to be analyzed.

Users can directly enter the file path and name, or use the browse button for file selection.

All fields in the input file must be tab-delimited.

ORIOGEN does not normalize the input data. It is recommended that the user pre-process the data by applying a suitable normalization method before submitting the data to ORIOGEN. ORIOGEN selects and clusters genes on the basis of the mean of the expression values provided to ORIOGEN.

The format of the input file should be as follows:

Header row (optional): Row 1 can be a header row (if present it is ignored) or data row.

Column 1: Contains the gene ID. The gene ID is an alpha-numeric character string used to identify the gene and can also be used as the key when performing a gene look-up on selected genes. The format of the gene ID used for gene look-up can be any of the following:

Probe ID (example: A_42_P453131)

Systemic name (example: 216994_Rn)

GenBank Accession Number (example: BE109018)

UniGene ID (example: Rn.19577)

Column 2 (optional): May contain a gene description string. If present, this string will be saved into the output file if the gene gets selected and will appear on the popup window if the user clicks on this gene in the Results graph.

All Remaining Columns: Contain tab-delimited numeric gene expression data. Missing values in the input data should be represented by a single period(i.e. ".").

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Output file name (Fitted means/Raw means):

This field specifies the path and name of the file containing the genes selected by the ORIOGEN software.

Users can directly enter the file path and name, or use the browse button for file selection.

The fields in the output file are tab-delimited.

The format of the file is as follows:

Column 1: Contains the counter number of the gene selected, starting at number one.

Column 2: Contains the row number from the input file of the selected gene.

Column 3: Contains the gene ID.

Column 4: Contains the user provided gene description from the input file if present, blank otherwise.

Column 5: Contains the profile number of the selected gene.

Column 6: Contains the computed P-value.

Column 7: Contains the computed Q-value.

Columns 8 and Higher: Contains the following:

Last 3 Columns: The last three columns of the output file may contain the following fields depending on the results of the ontology look-up procedure. If the ontology file is not specified, or the look-up procedure finds no data, these fields will not be present.

NOTE: In addition to the output file specified above, ORIOGEN will also create two additional files. The first is a raw output file that contains the input data for the genes that were selected. This file will have the same name as the primary output file, with "(Raw)" appended to the end of the filename. The second additional file will have a similar structure to the output file, but will contain the rejected genes and their corresponding results. This file will have the same name as the primary output file, with "(RejectedGenes)" appended to the end of the filename.

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Ontology file name:

This field specifies the path and name of the file used for the ontology look-up for a selected gene. Ontology files are available for download from ftp://ftp.tigr.org/pub/data/tgi/Resourcerer/.

ORIOGEN uses the following procedure to find the ontology data for a particular gene:

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Total number of dose groups or time points:

This field specifies the total number of dose groups or time points present in the input file. The maximum number of dose groups or time points supported in this release is 30.

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Vector of sample sizes per dose/time point:

This field specifies the sample sizes that are associated with each dose/time point.

Sample sizes can be individually entered using the "Enter Sample Sizes" button, or as a string with values separated by commas.

The ORIOGEN software performs a check to ensure that the input data is correct with respect to dose/time points. For example, if a user specifies 4 dose/time points with each sample size being 4, then the input file must contain 16 data values for each gene (including a period "." for missing values). If the sum of the sample sizes for each dose/time point does not equal the number of data values, an error message is displayed.

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Number of initial bootstrap samples:

This field specifies the number of initial bootstrap samples to be used in the analysis. This is the starting point for the adaptive bootstrapping routine. The program will evaluate each gene using this number of bootstraps and will accept or reject some of them based on a confidence interval around the calculated p-value. Genes that are not accepted or rejected are evaluated again after the number of bootstraps has been doubled.

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Number of final bootstrap samples:

This field specifies the number of final bootstrap samples to be used in the analysis. As described above, the number of bootstraps is doubled on each round of the adaptive routine to evaluate genes that have not yet been accepted or rejected. When doubling the number of bootstraps will cause it to exceed the final number specified, each undecided gene is accepted or rejected based on its latest calculated p-value.

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FDR value for performing the test for profiles:

This field specifies the false discovery rate level to be used for selecting significant genes. Genes that meet this criteria are selected and written to the output files specified above.

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Percentile to use for s0 adjustment:

This field specifies the percentile to use for the s0 adjustment used in the calculation of the goodness-of-fit statistic. A value of 0 disables this adjustment. Higher values reduce the effects of low sample variances in the data. Recommended values are 0 for sampling with replacement and 10 for longitudinal sampling.

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Bootstrap random seed:

Automatic random seed uses a constantly changing seed value for the random number generator. Manual random seed uses a user provided seed value for the random number generator.

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Take log of signal:

If this option is checked, ORIOGEN will take the log of all signal values in the Input file before calculating means or performing any processing.

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Longitudinal sampling:

If this option is checked, ORIOGEN will assume that the data was derived from a longitudinal sampling (repeated measures) study and will adjust the statistic accordingly. For example, for a time-course study with 3 replicates, the data file should be set up so that each time point is showing data from the same list of subjects. See the "About ORIOGEN" screen for a detailed description of how ORIOGEN handles longitudinal sampling.

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Transpose Input File:

If this option is checked, ORIOGEN will assume that the input file is arranged with each gene represented by a single column and each replicate represented by a single row.

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