STEP 1 P-value specifics

---

Results generated through the Functional Analysis are filtered according to the user-specified P-value:

• Type, which represents the type of the P-value that must be taken into account in the filtering. A user can choose to filter on: (Raw P-value)-based filter or (Adjusted P-value)-based filter (Benjamini and Hochberg).
• Threshold, which represents the upper bound limit value for the set of P-values the user wishes to display.

The figure below shows the module which allows to set the P-value type and threshold.

STEP 2 Resources selection

---

The Functional Analysis can be performed using different resources:

• Link Gene Ontology (GO terms)
• Link KEGG (Pathways)
• Link Reactome (Pathways)

Select the resources in the Functional Analysis to be considered, as shown in the following figure:

STEP 3 CPU cores selection

---

It is possible to perform all the analyses using a single core CPU or taking advantage of a multiple CPU cores. In this latter case, tools and the analysis are executed in parallel in order to optimize execution time, hiding the underlying complexity to the user.
The following figure shows a hypothetical system equipped with 8 CPU cores, as well as a dropdown menu reporting all selectable CPU cores. In this example, it is possible to select from 1 to 8 cores.

---

Analysis execution

---

Click on the Submit button to start the Functional Analysis. isoTar will show you a box reporting the analysis status, as well as the stopping button to shut down its execution (both shown below).

After the conclusion of the analysis (based on predicted targets), results are grouped according to each selected resource (i.e., GO term, KEGG, Reactome), and details are displayed by variation type (i.e., Substitution, isomiR).
For each resource, the Functional Analysis is performed considering wild type, variation, and wild type vs. variation (comparison). Depending on which resource is selected, isoTar provides different details. Below, we report results for the three main resources here available.

GO term

The GO term-based Functional Analysis is performed for each pair wild type-variation, specified as input for the Prediction Analysis. Then, results are grouped per Ontology category:

• Biological Process (BP);
• Cellular Component (CC);
• Molecular Function (MF).

A typical analysis provides information according to:

• Wild Type
  A GO term list for BP, CC, and MF, respectively, is supplied with additional details. Such GO terms are identified considering all the consensus predicted targets associated to the wild type miRNA;

• Variation
  A GO term list for BP, CC, and MF, respectively, is supplied with additional details. Such GO terms are identified considering all the consensus predicted targets associated to the variation miRNA;

• Wild Type vs. Variation
  

  • A GO term list for each of the GO category (BP, CC, and MF) is supplied with additional details. GO terms are identified considering those consensus predicted targets that are associated with each of the following:

    • Wild type only
    • Variation only
    • Wild type-variation intersection

Results are displayed adopting the same format employed when displaying prediction results. Results are grouped per variation type and ontology category (i.e., BP, CC, MF). Each specified variation is associated with:

• Variation details, such as type (i.e., Substitution or isomiR), position (for Substitution), and so on;
• The number of identified GO terms (filtered per P-value type and threshold) for each ontology category, supplied with GO term trees and histograms;
• The number of GO terms (per ontology category) considering only those predicted targets associated to the wild type, variation, and their intersection, respectively. GO term trees and histograms are provided as well;

Each number (colored in blue) represents the number of GO terms for a specific wild type or variation (i.e., Substitution or isomiR) and ontology category (i.e., BP, CC, MF). By clicking on it, the system will show you a modal box (figure below) reporting the GO terms list supplied with additional information. Results can be downloaded in different data formats, such as Excel, PDF, TSV, and so forth.

Histograms are available for each ontology category. They report:

---

• Comparison between wild type and variation. As an example, we report (figure below) a histogram associated with the wild type and the variation at position 6 according to the ontology category CC (wild type in blue and variation in red);
  
  
• Comparison between wild type only, variation only, and their intersection. As an example, we report (figure below) a histogram associated with the comparison between the wild type and the variation (wild type only, variation only, and their intersection) at position 6 according to the ontology category CC (wild type in blue, variation in red, and intersection in green);
  
  

Histograms can be downloaded in different formats, such as PDF, PNG, JPEG, and SVG.

---

GO term trees are available for each ontology type. They are associated with:

• Each wild type and their variation(s). As an example, we report (figure below) two GO term trees associated with the wild type and the variation at position 6, respectively, according to the ontology type CC (in green, the GO term nodes for which the P-value is also reported);
  
  Wild Type
  Variation

• Comparison between wild type only, variation only, and their intersection. As an example, we report (figure below) the GO term tree associated with the comparison between the wild type and the variation (wild type only, variation only, and their intersection) at position 6 according to the ontology type CC;
  
  Wild Type vs. Variation

  Colors are used to highlight GO term nodes, according to all possible combinations among all possible intersection subsets. A table reporting the adopted colors is provided as well (figure below).

  

GO term trees can be downloaded in PDF format.

KEGG & Reactome

The Pathway-based Functional Analysis is performed for each pair wild type-variation, specified as input for the Prediction Analysis. A typical analysis provides information according to:

• Wild Type
  A pathways list is supplied with additional details. Pathways are identified considering all consensus predicted targets associated to the wild type;

• Variation
  A pathways list is supplied with additional details. Pathways are identified considering all consensus predicted targets associated to the variation;

• Wild Type vs. Variation
  

  • A pathways list is supplied with additional details. Pathways are identified considering those consensus predicted targets that are respectively associated only to:

    • Wild type
    • Variation
    • Wild type-variation intersection

Results are displayed adopting the same format employed when displaying prediction results (grouped per variation type). Each specified variation is associated with:

• Variation details, such as type (i.e., Substitution or isomiR), position (Substitution only), and so on;
• The number of identified pathways (filtered by P-value type and threshold), supplied with histograms;
• The number of pathways considering only those predicted targets associated with the wild type, variation, and their intersection, respectively. Histograms are provided as well;

Each number (colored in blue) represents the number of pathways for a specific variation (i.e., Substitution or isomiR). By clicking on it, the system will show you a modal box (figure below) reporting the pathways list supplied with additional information. Results can be downloaded in different data formats, such as Excel, PDF, TSV, and so forth.

---

Histograms report:

• Comparison between wild type and variation. As an example, we report (figure below) a histogram associated with the wild type and the variation at position 6 according to KEGG (wild type in blue and variation in red);
  
  
• Comparison between wild type only, variation only, and their intersection. As an example, we report (figure below) a histogram associated with the comparison between the wild type and the variation (wild type only, variation only, and their intersection) at position 6 according to KEGG (wild type in blue, variation in red, and intersection in green);
  
  

Histograms can be downloaded in different formats, such as PDF, PNG, JPEG, and SVG.

What is described above is valid for Reactome results al well.