cfi-toolkit 0.1.0__py3-none-any.whl

cfi_toolkit-0.1.0.dist-info/METADATA

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+Metadata-Version: 2.4
+Name: cfi-toolkit
+Version: 0.1.0
+Summary: CFI: Cell Functionality and Interaction Analysis Tool
+License: GPL-3
+License-File: LICENSE
+Keywords: scRNA_seq,SEQ,GO,pathways,interactions,gene ontology,diseases,enrichment,OMIC
+Author: Jakub Kubis - JBS
+Author-email: jbiosystem@gmail.com
+Requires-Python: >=3.12,<3.13
+Classifier: License :: Other/Proprietary License
+Classifier: Programming Language :: Python :: 3
+Classifier: Programming Language :: Python :: 3.12
+Requires-Dist: gedspy (>=2.1.9,<3.0.0)
+Requires-Dist: jdti (>=0.2.2,<0.3.0)
+Requires-Dist: jvectorgraph (>=1.0.2,<2.0.0)
+Requires-Dist: llvmlite (>=0.46.0,<0.47.0)
+Requires-Dist: pandas (<3.0.0)
+Project-URL: Homepage, https://github.com/jkubis96/CFI
+Project-URL: Repository, https://github.com/jkubis96/CFI
+Description-Content-Type: text/markdown
+
+## CFI: Cell Functionality and Interaction Analysis Tool
+
+![Python version](https://img.shields.io/badge/python-%E2%89%A53.12-blue?logo=python&logoColor=white.png) ![License](https://img.shields.io/badge/license-GPLv3-blue) ![Docs](https://img.shields.io/badge/docs-available-blueviolet)
+
+
+
+
+<p align="right">
+<img  src="https://github.com/jkubis96/Logos/blob/main/logos/jbs_current.png?raw=true" alt="drawing" width="200" />
+</p>
+
+
+### Author: Jakub Kubiś 
+
+<div align="left">
+ Institute of Bioorganic Chemistry<br />
+ Polish Academy of Sciences<br />
+</div>
+
+
+## Description
+
+
+
+
+</br>
+
+
+CFI (Cell Functionality and Interaction Analysis Tool) is an analytical platform designed for the investigation of cellular functions, intra-cellular gene interactions, and intercellular communication, including ligand–receptor interactions and adhesion junctions.
+
+The tool integrates the advanced enrichment and interaction analysis capabilities of [GEDSpy](https://github.com/jkubis96/GEDSpy) with the single-cell data processing functionalities provided by [JDtI](https://github.com/jkubis96/JDtI).
+
+CFI extends these capabilities by enabling the identification of direct cell–cell interactions, dominant biological processes, and gene–gene interaction networks within individual cells, along with comprehensive visualization of these relationships.
+
+
+<p align="center">
+<img  src="fig/log.png" alt="drawing" width="500" />
+</p>
+
+
+CFI is designed to streamline the interpretation of biological data, enabling researchers to perform in-depth analyses of cellular functions and interactions for drug target discovery.
+
+
+</br>
+
+Included data bases:
+
+* [Gene Ontology (GO-TERM)](http://geneontology.org/)
+* [KEGG (Kyoto Encyclopedia of Genes and Genomes)](https://www.genome.jp/kegg/)
+* [Reactome](https://reactome.org/)
+* [HPA (Human Protein Atlas)](https://www.proteinatlas.org/)
+* [NCBI](https://www.ncbi.nlm.nih.gov/)
+* [STRING](https://string-db.org/)
+* [IntAct](https://www.ebi.ac.uk/intact/home)
+* [CellTalk](https://tcm.zju.edu.cn/celltalkdb/)
+* [CellPhone](https://www.cellphonedb.org/)
+
+
+*If you use CFI, please remember to cite both CFI and the original sources of the data you utilized in your work.*
+
+In the case of interactions network analyses, it is recommended to use the  [JVectorGraph](https://github.com/jkubis96/JVectorGraph) library to easily adjust and customise graph and network visualisations from the Python side.
+
+
+<br />
+
+## 📚 Table of Contents
+
+- [Installation](#installation)
+- [Documentation](#doc)
+- [Example usage](#example)
+  - [1. Cell functionality](#bf)
+    - [1.1. Create project](#bf1)
+    - [1.2. Calculate cell marker genes](#bf2)
+    - [1.3. Marker gene enrichment analysis](#bf3)
+    - [1.4. Cell inside gene interactions](#bf4)
+    - [1.5. Cell-cell interactions](#bf5)
+    - [1.6. Saving & loading project](#bf6)
+  - [2. Comparison of cell interaction sets](#br)
+    - [2.1. Create projects](#br1)
+    - [2.2. Calculate interactions](#br2)
+    - [2.3. Comparison analysis](#br3)
+
+
+<br />
+
+
+# Installation <a id="installation"></a>
+
+#### In command line write:
+
+```
+pip install cfi
+```
+
+
+
+
+## Documentation <a id="doc"></a>
+
+
+Documentation for classes and functions is available here 👉 [Documentation 📄](https://jkubis96.github.io/CFI/cfi.html)
+
+
+<br />
+
+
+## Example usage <a id="example"></a>
+
+### 1. Cell functionality <a id="bf"></a>
+
+##### 1.1. Create project <a id="bf1"></a>
+
+```
+# ------------------------------------------------------------
+# Import required standard and project-specific libraries
+# ------------------------------------------------------------
+
+import os
+from jdti import COMPsc          # JDtI module for handling single-cell projects
+from cfi import CellFunCon       # Cell functional connectivity / enrichment analysis
+
+
+# ------------------------------------------------------------
+# Load single-cell sequencing data using the JDtI framework
+# ------------------------------------------------------------
+
+# Define the project directory containing input data
+# and specify the sample identifiers to be loaded
+
+jseq_object = COMPsc.project_dir(
+    os.path.join(os.getcwd(), "data"),   # path to data directory
+    ["s1"]                               # list of project/sample IDs
+)
+
+# Load sparse expression matrices from the project structure
+# normalized_data=True ensures that pre-normalized counts are used
+
+jseq_object.load_sparse_from_projects(normalized_data=True)
+
+
+# ------------------------------------------------------------
+# Initialize CellFunCon analysis object
+# ------------------------------------------------------------
+
+# Create a CellFunCon instance using the loaded single-cell dataset.
+# This object enables downstream functional enrichment,
+# interaction analysis, and pathway inference.
+
+instance = CellFunCon(jseq_object)
+```
+
+<br />
+
+
+##### 1.2. Calculate cell marker genes <a id="bf2"></a>
+
+```
+# ------------------------------------------------------------
+# Required step before functional enrichment analysis
+# ------------------------------------------------------------
+# Identify cell-type–specific marker genes based on:
+# - minimum expression threshold (min_exp)
+# - minimum fraction of expressing cells (min_pct)
+# - parallel computation across multiple processes (n_proc)
+#
+# The resulting marker set is used as input for downstream
+# functional enrichment and pathway analysis.
+
+instance.calculate_cells_markers(
+    min_exp=0,
+    min_pct=0.05,
+    n_proc=10
+)
+```
+
+<br />
+
+
+##### 1.3. Marker gene enrichment analysis <a id="bf3"></a>
+
+```
+# ------------------------------------------------------------
+# Perform functional enrichment analysis for each cell population
+# ------------------------------------------------------------
+# This step evaluates overrepresentation of biological functions,
+# pathways, and gene sets using previously identified marker genes.
+#
+# Parameters:
+# - p_value : significance threshold for enrichment results
+# - log_fc  : minimum log fold-change required for marker inclusion
+# - top_max : maximum number of top-ranked markers used per cell type
+#
+# The output provides functionally annotated cell states
+# for downstream biological interpretation.
+
+instance.enrich_cells_fucntionality(
+    p_value=0.05,
+    log_fc=0.25,
+    top_max=500
+)
+```
+
+<br />
+
+
+* GO-TERM
+
+```
+data = instance.get_enrichment_data( 
+                        data_type = 'GO-TERM', 
+                        p_value = 0.05, 
+                        test = 'FISH', 
+                        adj = 'BH', 
+                        parent_inc = False, 
+                        top_n = 50)
+```
+
+| parent     | parent_genes                                   | parent_pval_FISH | parent_pval_BIN | parent_n | parent_pct | child     | child_genes                              | child_pval_FISH | child_pval_BIN | child_n | child_pct | parent_name                         | child_name                                              | cell             | source  |
+|------------|-------------------------------------------------|------------------|-----------------|----------|------------|-----------|-------------------------------------------|------------------|----------------|---------|-----------|-------------------------------------|----------------------------------------------------------|------------------|---------|
+| GO:0060090 | ['LETMD1', 'TNRC6A']                            | 0.01397          | 0.01402         | 2        | 0.04348    | GO:0030674 | ['TNRC6A', 'SRRT', 'CRADD', 'SLMAP']      | 3.22e-05         | 3.30e-05       | 4       | 0.08696   | MF : molecular adaptor activity     | MF : protein-macromolecule adaptor activity              | STRIATUM_1 # s1 | GO-TERM |
+| GO:0032991 | ['CLU','SRRT','HSP90AA1','SNX4','CHEK1','PPP3CB'] | 9.38e-05         | 9.49e-05        | 6        | 0.13043    | GO:0005955 | ['PPP3CB']                                | 0.00459          | 0.00458        | 1       | 0.02174   | CC : protein-containing complex     | CC : calcineurin complex                                | STRIATUM_1 # s1 | GO-TERM |
+| GO:0032991 | ['CLU','SRRT','HSP90AA1','SNX4','CHEK1','PPP3CB'] | 9.38e-05         | 9.49e-05        | 6        | 0.13043    | GO:0031428 | ['FBL']                                   | 0.00535          | 0.00535        | 1       | 0.02174   | CC : protein-containing complex     | CC : box C/D methylation guide snoRNP complex           | STRIATUM_1 # s1 | GO-TERM |
+| GO:0032991 | ['CLU','SRRT','HSP90AA1','SNX4','CHEK1','PPP3CB'] | 9.38e-05         | 9.49e-05        | 6        | 0.13043    | GO:0005664 | ['ORC6']                                  | 0.00611          | 0.00611        | 1       | 0.02174   | CC : protein-containing complex     | CC : nuclear origin of replication recognition complex  | STRIATUM_1 # s1 | GO-TERM |
+| GO:0032991 | ['CLU','SRRT','HSP90AA1','SNX4','CHEK1','PPP3CB'] | 9.38e-05         | 9.49e-05        | 6        | 0.13043    | GO:0008287 | ['PPP3CB']                                | 0.00611          | 0.00611        | 1       | 0.02174   | CC : protein-containing complex     | CC : protein serine/threonine phosphatase complex       | STRIATUM_1 # s1 | GO-TERM |
+
+<br />
+
+
+ Visualization
+
+```
+from cfi import encrichment_cell_heatmap
+
+fig = encrichment_cell_heatmap(data = data,
+                             fig_size = (3,3), 
+                             sets = None,
+                             top_n = 3,
+                             test = 'FISH', 
+                             adj = 'BH', 
+                             parent_inc = False,
+                             font_size = 16,
+                             clustering = 'ward',
+                             scale = True)
+```
+
+
+<p align="center">
+<img  src="fig/heatmap.svg" alt="drawing" width="450" />
+</p>
+
+
+<br />
+
+* KEGG
+
+```
+data = instance.get_enrichment_data( 
+                        data_type = 'KEGG', 
+                        test = 'FISH', 
+                        adj = 'BH', 
+                        parent_inc = False, 
+                        top_n = 50)
+```
+| 2nd                     | 2nd_genes                                                                 | 2nd_pval | 2nd_n | 2nd_pct | 3rd                     | 3rd_genes                                   | 3rd_pval | 3rd_n | 3rd_pct | cell             | source |
+|-------------------------|---------------------------------------------------------------------------|----------|-------|---------|--------------------------|-----------------------------------------------|----------|-------|---------|------------------|--------|
+| Neurodegenerative disease | ['ATXN10','VDAC2','PSMA3','APC','SETX','CREBBP','PSMD8','PSMD7','NDUFA4'] | 1.10e-05 | 9     | 0.093   | Spinocerebellar ataxia   | ['ATXN10','VDAC2','PSMA3','PSMD8','PSMD7']    | 7.51e-06 | 5     | 0.052   | STRIATUM_2 # s1 | KEGG   |
+| Neurodegenerative disease | ['ATXN10','VDAC2','PSMA3','APC','SETX','CREBBP','PSMD8','PSMD7','NDUFA4'] | 1.10e-05 | 9     | 0.093   | Huntington disease       | ['VDAC2','PSMA3','CREBBP','PSMD8','PSMD7','NDUFA4'] | 2.08e-05 | 6 | 0.062 | STRIATUM_2 # s1 | KEGG |
+| Neurodegenerative disease | ['ATXN10','VDAC2','PSMA3','APC','SETX','CREBBP','PSMD8','PSMD7','NDUFA4'] | 1.10e-05 | 9     | 0.093   | Alzheimer disease        | ['VDAC2','PSMA3','APC','PSMD8','PSMD7','NDUFA4'] | 8.20e-05 | 6 | 0.062 | STRIATUM_2 # s1 | KEGG |
+| Neurodegenerative disease | ['ATXN10','VDAC2','PSMA3','APC','SETX','CREBBP','PSMD8','PSMD7','NDUFA4'] | 1.10e-05 | 9     | 0.093   | Prion disease            | ['VDAC2','PSMA3','PSMD8','PSMD7','NDUFA4']    | 1.23e-04 | 5     | 0.052   | STRIATUM_2 # s1 | KEGG   |
+| Neurodegenerative disease | ['ATXN10','VDAC2','PSMA3','APC','SETX','CREBBP','PSMD8','PSMD7','NDUFA4'] | 1.10e-05 | 9     | 0.093   | Parkinson disease        | ['VDAC2','PSMA3','PSMD8','PSMD7','NDUFA4']    | 1.77e-04 | 5     | 0.052   | STRIATUM_2 # s1 | KEGG   |
+
+
+<br />
+
+Visualization
+
+```
+from cfi import encrichment_cell_heatmap
+
+fig = encrichment_cell_heatmap(data = enr,
+                             fig_size = (3,3), 
+                             sets = None,
+                             top_n = 3,
+                             test = 'FISH', 
+                             adj = 'BH', 
+                             parent_inc = False,
+                             font_size = 16,
+                             clustering = 'ward',
+                             scale = True)
+```
+
+
+<p align="center">
+<img  src="fig/heatmap2.svg" alt="drawing" width="450" />
+</p>
+
+
+> [!NOTE]
+> In this case, **no significant KEGG terms** were detected for the second cell type.  
+> To indicate the absence of specific terms, use  
+> `instance1.get_included_cells()` to display both cells at the top.
+
+<br />
+
+Visualization
+
+```
+from cfi import encrichment_cell_heatmap
+
+fig2 = encrichment_cell_heatmap(data = enr,
+                             fig_size = (3,3), 
+                             sets = instance1.get_included_cells(),
+                             top_n = 3,
+                             test = 'FISH', 
+                             adj = 'BH', 
+                             parent_inc = False,
+                             font_size = 16,
+                             clustering = 'ward',
+                             scale = True)
+```
+
+<br />
+
+ Visualization
+
+
+
+<p align="center">
+<img  src="fig/heatmap3.svg" alt="drawing" width="450" />
+</p>
+
+
+<br />
+
+
+* REACTOME
+
+```
+data = instance.get_enrichment_data( 
+                        data_type = 'REACTOME', 
+                        p_value = 0.05, 
+                        test = 'FISH', 
+                        adj = 'BH', 
+                        parent_inc = False, 
+                        top_n = 50)
+```
+
+| pathway                                      | genes        | p-value | n | pct  | top level pathway        | top genes            | cell             | source   |
+|----------------------------------------------|-------------|--------|---|------|---------------------------|----------------------|------------------|----------|
+| Resistance of ERBB2 KD mutants to tesevatinib | [HSP90AA1]  | 0.00306 | 1 | 0.0217 | Disease                   | [RNGTT,HSP90AA1,HMGB1] | STRIATUM_1 # s1 | REACTOME |
+| Resistance of ERBB2 KD mutants to osimertinib | [HSP90AA1]  | 0.00306 | 1 | 0.0217 | Disease                   | [RNGTT,HSP90AA1,HMGB1] | STRIATUM_1 # s1 | REACTOME |
+| Resistance of ERBB2 KD mutants to AEE788      | [HSP90AA1]  | 0.00306 | 1 | 0.0217 | Disease                   | [RNGTT,HSP90AA1,HMGB1] | STRIATUM_1 # s1 | REACTOME |
+| Apoptosis induced DNA fragmentation           | [HMGB1]     | 0.00991 | 1 | 0.0217 | Programmed Cell Death     | [HSP90AA1,HMGB1]       | STRIATUM_1 # s1 | REACTOME |
+| Regulation of CDH11 mRNA translation by miRNAs| [TNRC6A]    | 0.00839 | 1 | 0.0217 | Cell-Cell communication   | [TNRC6A]               | STRIATUM_1 # s1 | REACTOME |
+
+<br />
+
+Visualization
+
+```
+from cfi import encrichment_cell_heatmap
+
+fig = encrichment_cell_heatmap(data = enr,
+                             fig_size = (3,3), 
+                             sets = instance1.get_included_cells(),
+                             top_n = 3,
+                             test = 'FISH', 
+                             adj = 'BH', 
+                             parent_inc = False,
+                             font_size = 16,
+                             clustering = 'ward',
+                             scale = True)
+```
+
+
+<p align="center">
+<img  src="fig/heatmap4.svg" alt="drawing" width="450" />
+</p>
+
+
+<br />
+
+* Specificity (HPA)
+
+```
+data = instance.get_enrichment_data( 
+                        data_type = 'specificity', 
+                        p_value = 1, 
+                        test = 'FISH', 
+                        adj = 'BH', 
+                        parent_inc = False, 
+                        top_n = 50)
+```
+| specificity            | genes                | p-value | n | pct  | cell             | source      |
+|------------------------|----------------------|--------|---|------|------------------|-------------|
+| Cardiomyocytes         | [SLMAP]              | 0.116  | 1 | 0.0217 | STRIATUM_1 # s1 | specificity |
+| Proximal tubular cells | [ALDH6A1]            | 0.098  | 1 | 0.0217 | STRIATUM_1 # s1 | specificity |
+| granulocytes           | [CLU, ALDH6A1, TIMP2]| 0.132  | 3 | 0.0652 | STRIATUM_1 # s1 | specificity |
+| liver                  | [ALDH6A1]            | 0.288  | 1 | 0.0217 | STRIATUM_1 # s1 | specificity |
+| kidney                 | [ALDH6A1]            | 0.146  | 1 | 0.0217 | STRIATUM_1 # s1 | specificity |
+| Schwann cells          | [GRIK3]              | 0.030  | 1 | 0.0217 | STRIATUM_1 # s1 | specificity |
+
+<br />
+
+Visualization
+
+```
+from cfi import encrichment_cell_heatmap
+
+fig = encrichment_cell_heatmap(data = enr,
+                             fig_size = (3,3), 
+                             sets = instance1.get_included_cells(),
+                             top_n = 3,
+                             test = 'FISH', 
+                             adj = 'BH', 
+                             parent_inc = False,
+                             font_size = 16,
+                             clustering = 'ward',
+                             scale = True)
+```
+
+
+<p align="center">
+<img  src="fig/heatmap5.svg" alt="drawing" width="450" />
+</p>
+
+<br />
+
+##### 1.4. Cell inside gene interactions <a id="bf4"></a>
+
+View available cell names
+
+```
+# ------------------------------------------------------------
+# List all cell populations included in the dataset
+# ------------------------------------------------------------
+# Useful for verifying dataset structure before selecting
+# specific cell populations for further investigation.
+
+instance.get_included_cells()
+```
+
+<p align="center">
+<img  src="fig/out_cell.bmp" alt="drawing" width="450" />
+</p>
+
+
+
+Retrieve interaction data for the selected cell
+
+```
+cell_int = instance.get_gene_interactions('STRIATUM_1 # s1')
+```
+
+| A    | B       | interaction_type     | connection_type     | source      |
+|------|---------|----------------------|---------------------|-------------|
+| CLU  | CLU     | physical association | protein -> protein  | Alzheimers  |
+| CLU  | HSP90AA1| physical association | protein -> protein  | Alzheimers  |
+| FBL  | KRR1    |                      | gene -> gene        | STRING      |
+| FBL  | KRR1    |                      | protein -> protein  | STRING      |
+| KRR1 | FBL     |                      | gene -> gene        | STRING      |
+| KRR1 | FBL     |                      | protein -> protein  | STRING      |
+
+
+<br />
+
+ Visualization
+
+```
+from cfi import gene_interaction_network
+
+fig5 = gene_interaction_network(idata = cell_int, min_con = 2)
+
+from JVG import JVG
+
+nt = JVG.NxEditor(fig5)
+nt.edit()
+```
+
+
+<p align="center">
+<img  src="fig/gin.bmp" alt="drawing" width="300" />
+</p>
+
+<br />
+
+
+##### 1.5. Cell-cell interactions <a id="bf5"></a>
+
+
+Calculate cells interactions
+
+```
+# ------------------------------------------------------------
+# Infer functional and molecular connections between cell populations
+# ------------------------------------------------------------
+# This step identifies potential interactions based on
+# inferred molecular communication signals
+#
+# The resulting network describes relationships between cells
+# and can be used for downstream visualization, pathway analysis,
+# or biological interpretation of tissue organization.
+
+instance.calculate_cell_connections()
+```
+
+Get data
+
+```
+cell_con = instance.get_cell_connections()
+```
+
+| interaction              | directionality     | classification                 | modulatory_effect | interactor1 | interactor2 | cell1       | cell2       |
+|--------------------------|--------------------|---------------------------------|-------------------|-------------|-------------|-------------|-------------|
+| CDH2 -> CDH2             | Adhesion-Adhesion  | Adhesion by Cadherin           |                   | CDH2        | CDH2        | STRIATUM_1  | STRIATUM_2  |
+| CDH6 -> CDH6             | Adhesion-Adhesion  | Adhesion by Cadherin           |                   | CDH6        | CDH6        | STRIATUM_1  | STRIATUM_2  |
+| CDH7 -> CDH7             | Adhesion-Adhesion  | Adhesion by Cadherin           |                   | CDH7        | CDH7        | STRIATUM_1  | STRIATUM_2  |
+| COL11A1 -> ITGA1+ITGB1   | Adhesion-Adhesion  | Adhesion by Collagen/Integrin  |                   | COL11A1     | ITGA1       | STRIATUM_1  | STRIATUM_2  |
+| COL11A1 -> ITGA1+ITGB1   | Adhesion-Adhesion  | Adhesion by Collagen/Integrin  |                   | COL11A1     | ITGB1       | STRIATUM_1  | STRIATUM_2  |
+
+<br />
+
+Visualization
+
+```
+from cfi import draw_cell_conections
+
+fig = draw_cell_conections(cell_con)
+
+from JVG import JVG
+
+nt = JVG.NxEditor(fig)
+nt.edit()
+```
+
+<p align="center">
+<img  src="fig/cell_con.bmp" alt="drawing" width="300" />
+</p>
+
+<br />
+
+
+
+##### 1.6. Saving & loading project <a id="bf6"></a>
+
+
+Saving current project
+
+```
+instance.save_project('project')
+```
+
+
+Loading previously saved project
+
+```
+from cfi import CellFunCon   
+
+instance = CellFunCon.load_project('project.psc')
+```
+
+
+<br />
+
+
+### 2. Comparison of cell interaction sets <a id="br"></a>
+
+##### 2.1. Create projects <a id="br1"></a>
+
+```
+# ------------------------------------------------------------
+# Import required libraries
+# ------------------------------------------------------------
+import os
+from jdti import COMPsc          # JDtI module for single-cell project handling
+from cfi import CellFunCon       # Functional analysis and cell interaction inference
+
+
+# ------------------------------------------------------------
+# Load single-cell datasets for two experimental conditions
+# ------------------------------------------------------------
+# Each COMPsc object represents an independent project/sample
+# loaded from the same data directory but identified by
+# different sample IDs (e.g., control vs. case).
+
+jseq_object1 = COMPsc.project_dir(
+    os.path.join(os.getcwd(), "data"),
+    ["s1"]   # first dataset / condition
+)
+jseq_object1.load_sparse_from_projects(normalized_data=True)
+
+
+jseq_object2 = COMPsc.project_dir(
+    os.path.join(os.getcwd(), "data"),
+    ["s2"]   # second dataset / condition
+)
+jseq_object2.load_sparse_from_projects(normalized_data=True)
+
+
+# ------------------------------------------------------------
+# Initialize CellFunCon analysis objects for comparative study
+# ------------------------------------------------------------
+# Separate instances enable:
+# - independent marker detection
+# - condition-specific functional enrichment
+# - downstream comparison of cell functionality and interactions
+
+instance1 = CellFunCon(jseq_object1)
+instance2 = CellFunCon(jseq_object2)
+```
+<br />
+
+
+##### 2.2. Calculate interactions <a id="br2"></a>
+
+```
+# ------------------------------------------------------------
+# Calculate cell–cell connection networks for each dataset
+# ------------------------------------------------------------
+# This step reconstructs potential functional and molecular
+# interactions between cell populations independently for:
+# - dataset 1 (e.g., control)
+# - dataset 2 (e.g., disease / treated condition)
+
+instance1.calculate_cell_connections()
+instance2.calculate_cell_connections()
+```
+
+<br />
+
+
+##### 2.3. Comparison analysis <a id="br3"></a>
+
+```
+# ------------------------------------------------------------
+# Compare cell–cell interaction networks between conditions
+# ------------------------------------------------------------
+# This step performs a comparative analysis of inferred
+# cell–cell connections across multiple biological conditions.
+#
+# Each entry in `instances_dict` represents a fully processed
+# CellFunCon object with reconstructed interaction networks.
+# Here, we contrast:
+# - healthy condition
+# - disease condition
+
+from cfi import compare_connections
+
+instances_dict = {
+    "healthy": instance2,
+    "disease": instance1
+}
+
+comparison = compare_connections(instances_dict=instances_dict, 
+                                 cells_compartment = None, 
+                                 connection_type  = ['Adhesion-Adhesion',
+                                                     'Gap-Gap',
+                                                     'Ligand-Ligand',
+                                                     'Ligand-Receptor',
+                                                     'Receptor-Receptor',
+                                                     'Undefined'])
+```
+
+| feature | p-value  | pct_valid | pct_ctrl | FC    | log(FC) | norm_diff | group   |
+|---------|----------|-----------|----------|-------|---------|-----------|---------|
+| HSP90B1 | 4.75e-14 | 0.164     | 0.935    | 0.160 | -2.65   | -6.58     | healthy |
+| CANX    | 5.90e-14 | 0.091     | 0.848    | 0.102 | -3.30   | -5.53     | healthy |
+| ARPC5   | 1.34e-11 | 0.055     | 0.739    | 0.080 | -3.64   | -4.93     | healthy |
+| CALR    | 5.47e-09 | 0.182     | 0.761    | 0.226 | -2.14   | -4.41     | healthy |
+| APLP2   | 1.16e-08 | 0.073     | 0.609    | 0.115 | -3.12   | -3.73     | healthy |
+| ITGB1   | 1.44e-08 | 0.055     | 0.587    | 0.098 | -3.36   | -3.61     | healthy |
+
+<br />
+
+Visualization
+
+```
+from cfi import volcano_plot_conections
+
+fig = volcano_plot_conections(
+    deg_data = comparison,
+    p_adj = True,
+    top = 25,
+    top_rank = "p_value",
+    p_val = 0.05,
+    lfc = 0.25,
+    rescale_adj = True,
+    image_width = 12,
+    image_high = 12,
+)
+```
+
+
+<p align="center">
+<img  src="fig/volcano.svg" alt="drawing" width="450" />
+</p>
+
+
+<br />
+
+An example analysis pipeline is available here → [Example file](example.py)
+
+<br />
+
+
+#### Have fun JBS©
+
+
+
+
+
+
+
+