mirExTra 2.0 Help
Welcome to the DIANA-mirExtra 2.0 help section!

 1. Differential Expression analysis Module
In the picture below you can explore the interface of Differential Expression analysis Module:


Users can upload miRNA and/or mRNA expression files[1] or select samples from the expression database[2]. miRNAs and mRNAs can be analyzed together or separately. Subsequently, users can select the species annotation, statistical methods, FDR thresholds, file output format and whether they require comparison (volcano, MA, heat map) or homogeneity (PCA, clustering) plots to be created[3]. Following analysis[4], results are presented in a separate pane[5], which is divided into two distinct sections for miRNAs and genes, respectively. It provides information such as transcript name, mean expression in each group, fold change, p-values and FDR level. Entries are colored regarding to the comparison outcome and accompanied by an information button[6], which leads to further details, meta-data, and connects them with internal (DIANA) or external tools and databases. All results can be downloaded in the user selected format[7]. The requested graphs can be viewed and downloaded by pressing the relevant button[8]. Users can forward the results to mirExTra functional analysis modules[9] or to miRPath v3.0 for pathway and GO analyses[10]. A comprehensive help section[11] and an example run[12] support user interaction with the server. Back, reset[13] and home[14] buttons facilitate the navigation between different mirExTra functionalities.

File Setups
The Differential Expression analysis module can import miRNA & Gene Expression files. Users can provide miRNA and gene expression files to miRExtra, in order to perform differential expression analysis using LIMMA, DESeq or EdgeR algorithms. The required file format is presented below.

Gene Expression Files
1st line: Setup and Group names for each sample separated by tabs.
Example:
Setup     Patient     Patient     Patient      Patient     Patient     Healthy      Healthy     Healthy     Healthy

2nd line: ID and Sample names separated by tabs. The number of groups above and sample names have to be equal.
Example:
ID     P1     P2     P3     P4      P5     H1     H2     H3      H4     H5

3rd line till end of the file: Ensemble Gene ID and read count (raw read count / not normalized or RPKM). One number for each sample, separated by tabs.
Example:
ENSG00000000003     15     25     14      12     50     2     1      4     6     10
ENSG00000000005     200     210     250      230     260     30     32      41     56     42
ENSG00000000419     1000     1050     1200      1500     1090     8099     8015      8150     8189     8400
ENSG00000000457     0     1     0     0      0     100     110     310      120     201

Download the 10 first lines of an example file
Download gene expression file used in the example

miRNA Expression Files
1st line: Setup and Group names for each sample separated by tabs.
Example:
Setup     Patient     Patient     Patient      Patient     Patient     Healthy           Healthy     Healthy     Healthy
2nd line: ID and Sample names separated by tabs. The number of groups above and sample names have to be equal
Example:
ID     P1     P2     P3     P4      P5     H1     H2     H3      H4     H5
3rd line till end of the file: miRBase MIMAT or miRNA name (miRBase 18+ nomenclature (-3p / -5p) and read count (raw read count / not normalized or RPKM). One number for each sample, separated by tabs.
Example:
MIMAT0000680     15     25     14      12     50     2     1      4     6     10
MIMAT0000104     200     210     250      230     260     30     32      41     56     42
MIMAT0000253     1000     1050     1200      1500     1090     8099     8015      8150     8189     8400
MIMAT0005824     0     1     0     0      0     100     110     310      120     201

Download the 10 first lines of an example file
Download miRNA expression file used in the example

Selecting Groups from mirExTra Expression DB
Users can select one or even both groups to compare from mirExTra Expression DB. The user interface initially requests a selection of species[1] and then users can select Group A and B directly from the database [2 and 4] or directly upload and import any of the groups [3 and 5]. Any combination of database and imported groups is supported by the web server (both imported or DB groups, imported vs DB and vice versa). Both groups have to belong to the same species. The file format for group import is similar to the main DE module but having only one group per file. The import format is as follows:

Gene Expression Files
1st line: Setup and Group name for each sample separated by tabs (only one group name is allowed)
Example:
Setup     Patient     Patient     Patient      Patient     Patient 

2nd line: ID and Sample names separated by tabs. The number of group mentions above and sample names have to be equal.
Example:
ID     P1     P2     P3     P4      P5

3rd line till end of the file: Ensembl Gene ID and read count (raw read count / not normalized or RPKM). One number for each sample, separated by tabs.
Example:
ENSG00000000003     15     25     14      12     50
ENSG00000000005     200     210     250      230     260
ENSG00000000419     1000     1050     1200      1500     1090
    
miRNA Expression Files
1st line: Setup and Group name for each sample separated by tabs.
Example:
Setup     Patient     Patient     Patient      Patient     Patient  

2nd line: ID and Sample names separated by tabs. The number of group mentions above and sample names have to be equal
Example:
ID     P1     P2     P3     P4 P5

3rd line till end of the file: miRBase MIMAT or miRNA name (miRBase 18+ nomenclature (-3p / -5p) and read count (raw read count / not normalized or RPKM). One number for each sample, separated by tabs.
Example:
MIMAT0000680     15     25     14      12     50 
MIMAT0000104     200     210     250      230     260
MIMAT0000253     1000     1050     1200      1500     1090

 2. Functional Analysis Module
In the picture below you can explore the interface of Functional analysis Module:



Users can directly upload files for analysis[1] or perform the preparatory differential expression analysis directly from mirExTra[2]. Depending on the investigated regulators (miRNAs, TFs), the module offers different setup options[3], including (for TFs) the analyzed species, TF function, assessed promoter region, as well as the relevant thresholds for the regulator and their targets. Following analysis[4], the results are presented in a relevant pane below[5]. Regulators with increased or decreased expression are placed in separate columns. The information link[6] leads to internal and external tools or data. All results can be downloaded[7] and saved for future reference. The module also automatically creates interactive network graphs[8] for the regulators with the lowest p-values. User interaction with the interface is supported by the example[9], help[10] and navigation[11,12] buttons.
Users can also upload their own files of differentially expressed genes and microRNAs, in order to perform the enrichment analysis. The supported file formats are presented below.

Differentially Expressed Genes file
ID, Gene Name and Differential Express outcome separated by tabs. The Gene ID has to be Ensembl Gene ID and differential expression outcome (UP or DOWN in caps). Genes without UP/DOWN notation in the DiffExp column will be used as background (genes present in the analysis but not differentially expressed).
Attention: The column titles must be identical to those provided in the example below.

ID Name DiffExp
ENSG00000122787      AKR1D1      UP     
ENSG00000188112      C6orf132           
ENSG00000228208      C1orf143           
ENSG00000173762      CD7      DOWN     
ENSG00000072657      TRHDE      UP     

Download the 10 first lines of an example file

Differentially Expressed miRNAs file
Identical with the genes file but with MIMAT and miRNA name utilized instead of gene identifiers. miRNAs without UP/DOWN notation in the DiffExp column will be used as background.
Attention: The column titles must be identical to those provided in the example below.

ID Name DiffExp
MIMAT0019734      hsa-miR-4659b-3p      UP     
MIMAT0004551      hsa-miR-30d-3p      UP     
MIMAT0015043      hsa-miR-3168      DOWN     
MIMAT0018188      hsa-miR-3914           
MIMAT0005919      hsa-miR-548o-3p           
MIMAT0022735      hsa-miR-374c-3p      UP     

Download the 10 first lines of an example file

If available, users can add one or more of the following columns: fold change, p-value and/or FDR levels. These extra columns can be utilized to select differentially expressed genes from background. When extra columns are present, the DiffExp column can have UP/DOWN values for all entries, since the added columns will be used to divide entries into differentially expressed genes and background.
Attention: The column titles must be identical to those provided in the example below.

ID DiffExp foldChange PValue FDR
ENSG00000122787      UP      390.6231948      2.23E-31      3.08E-27     
ENSG00000188112      UP      151.6198882      3.80E-26      2.62E-22     
ENSG00000228208      DOWN      116.3257007      5.94E-25      2.74E-21     
ENSG00000173762      DOWN      40.49088751      5.76E-24      1.99E-20     
ENSG00000072657      DOWN      14.02301566      7.55E-20      2.09E-16     

Download the 10 first lines of an example file

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