--- title: "Functional Annotation" canonical: "https://help.biobam.com/space/OED0324/3525084067/Functional%20Annotation" format: markdown --- > Macro (toc) # Introduction Functional Annotation of *Severe acute respiratory syndrome coronavirus 2 *(SARS-CoV-2) proteins. ### Dataset description The protein sequences have been downloaded from [NCBI](https://www.ncbi.nlm.nih.gov/genome/?term=sars-cov-2). - Organism: *[Severe acute respiratory syndrome coronavirus 2](https://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=2697049)* - Layout: Fasta ### Original Data - Reference genome: Severe acute respiratory syndrome coronavirus 2 ASM985889v2 - RefSeq: [NC_045512.2](https://www.ncbi.nlm.nih.gov/nuccore/NC_045512.2) - BioProject: [PRJNA485481](https://www.ncbi.nlm.nih.gov/bioproject/PRJNA485481) # Bioinformatic Analysis ## 1- CloudBlast > Macro (anchor) ### Application [Homology search using BLAST](https://biobam.atlassian.net/wiki/spaces/OBD/pages/3335684097) ### Input [SARS-CoV-2 protein Fasta file](https://drive.google.com/file/d/1IQz-gO38AbQzvfHGNMreRnJYtxy7adzc/view?usp=sharing) that has to be [loaded](http://manual.omicsbox.biobam.com/user-manual/omicsbox-modules/module-functional-analysis/load-data/) in OmcisBox. ### Parameters - Blast Program: blastp-fast - Blast DB: Non-redundant protein sequences (nr v5) - Blast Expectation Value (e-Value): 1.0E-3 - Number of Blast Hits: 20 - Blast Description Annotator: True - Word Size: 6 - Low Complexity Filter: True - HSP Length Cutoff: 33 - HSP-Hit Coverage: 0 - Filter By Description: No filter - Save XML results in a folder ### Execution Time 19 min ### Consumed Units Consumed 1174 CloudUnits during this execution. ### Output [Blast XML files](https://drive.google.com/drive/folders/1tPqB8owYaVLZpErx_rdDI_9V-hH-uXo8?usp=sharing) in zip. ## 2- Cloud InterProScan > Macro (anchor) ### Application Retrieve protein family domains with [InterProScan Annotation](https://biobam.atlassian.net/wiki/spaces/OBD/pages/598048967). ### Input Load the [Fasta](https://drive.google.com/file/d/1IQz-gO38AbQzvfHGNMreRnJYtxy7adzc/view?usp=sharing) file or use the saved [Fasta project](https://drive.google.com/file/d/1avjIGWT6uZuAir8R9D6QaeoHQfWKnTKQ/view?usp=sharing). ### Parameters - CDD: True - HAMAP: True - HMMPanther: True - HMMPfam: True - HMMPIR: True - FPrintScan: True - ProfileScan: True - HMMTigr: True - PatternScan: False - Gene3D: True - SFLD: True - SuperFamily: True - Coils: False - MobiDBLite: True - Save InterProScan XML files in a folder ### Execution Time 4 min ### Consumed Units Consumed 194 CloudUnits during this execution. ### Output [InterProScan XML files](https://drive.google.com/file/d/1VEW3rAzwOyrlO6vnhtjlOBTf3QQBPFu4/view?usp=sharing) ## 3- Blast2GO Mapping > Macro (anchor) ### Application Retrieve Gene Ontology terms using [Gene Ontology Mapping](https://biobam.atlassian.net/wiki/spaces/OBD/pages/598114410). ### Input [Fasta](https://drive.google.com/file/d/1IQz-gO38AbQzvfHGNMreRnJYtxy7adzc/view?usp=sharing) and [Blast XML files](https://drive.google.com/drive/folders/1tPqB8owYaVLZpErx_rdDI_9V-hH-uXo8?usp=sharing) from step [1- BLAST](#blast) or the saved [blast and IPS project](https://drive.google.com/file/d/1c-Gop7TXEcQ6EOFLdSdZBXspPyRKOC1g/view?usp=sharing). ### Parameters - Use latest database version: True In this example, it was June 2021 ### Execution Time 2 min ### Output The same [project](https://drive.google.com/drive/folders/1fcP7y78k7HAA8ulUEfzyk9A2vc9pv2gE?usp=sharing) with Gene Ontology terms. ## 4- Blast2GO Annotation > Macro (anchor) ### Application Apply the [Gene Ontology Annotation](https://biobam.atlassian.net/wiki/spaces/OBD/pages/598179910) rule to all GO terms. ### Input Use the [project](https://drive.google.com/drive/folders/1fcP7y78k7HAA8ulUEfzyk9A2vc9pv2gE?usp=sharing) output from step [3- Mapping](#Mapping). ### Parameters - Annotation CutOff: 55 - GO Weight: 5 - Filter GO by Taxonomy: No Filter - E-Value-Hit-Filter: 1.0E-6 - HSP-Hit Coverage CutOff: 0 - Hit Filter: 500 - Only hits with GOs: False - Evidence Code Weights: Default Values ### Execution Time Very Fast ### Output The same [project](https://drive.google.com/drive/folders/1gLW_TddTdxPNFC4_uUGDUpknNVOYIrFk?usp=sharing) with Gene Ontology terms that passed the Annotation rule CutOff. ## 5- EggNOG Annotation > Macro (anchor) ### Application Retrieve additional Gene Ontology terms from orthologs by running [EggNOG](https://biobam.atlassian.net/wiki/spaces/OBD/pages/754450439). ### Input Load the [Fasta](https://drive.google.com/file/d/1IQz-gO38AbQzvfHGNMreRnJYtxy7adzc/view?usp=sharing) file or use the saved [fasta project](https://drive.google.com/file/d/1avjIGWT6uZuAir8R9D6QaeoHQfWKnTKQ/view?usp=sharing). ### Parameters - Target Orthologs: All - GO Evidence: Non-Electronic ### Execution Time 6 min ### Output A new [project](https://drive.google.com/drive/folders/1c2y_Y-ebAUuY9NLqTevfBSyz9rP7Q_fY?usp=sharing) with EggNOG annotations. [Report](https://drive.google.com/file/d/13bHLs8RErdpIMrJUHdKiQCo84mQj-DME/view?usp=sharing) with the ortholog information. ## 6- Merge EggNOG to Annotation ### Application [Merge the Gene Ontology](http://manual.omicsbox.biobam.com/user-manual/omicsbox-modules/module-functional-analysis/eggnog-annotation/#EggNOGAnnotation-MergeEggNOGAnnotations) terms retrieved from EggNog to existing Annotation. ### Input The EggNOG annotation [project](https://drive.google.com/drive/folders/1c2y_Y-ebAUuY9NLqTevfBSyz9rP7Q_fY?usp=sharing) from step [5- EggNOG Annotation](#eggnog) has to be opened in OmcisBox. ### Parameters - Sequence Project: The [project](https://drive.google.com/file/d/1Sccv9M2fQQFkpZ6kUuvHHHTKsgaA955m/view?usp=sharing) with Gene Ontology terms that passed the Annotation rule CutOff from step [4- Blast2GO Annotation](#annotation). - Seed Ortholog E-Value Filter: 1E-3 - Seed Ortholog Bit-Score Filter: 60 ### Execution Time Very fast ### Output The [annotation project](https://drive.google.com/file/d/1ey5qZ0C0AXQKQiRRYY6o8vcrSDjHeYKu/view?usp=sharing) will open with the added Gene Ontology terms. A [chart](https://drive.google.com/file/d/1sIwOd4V7wSzS5hp9CfTR9R18rvtijNVw/view?usp=sharing) with information on the number of GOs that has been merged. ## 7- Merge InterProScan to Annotation ### Application [Merge the Gene Ontology](http://manual.omicsbox.biobam.com/user-manual/omicsbox-modules/module-functional-analysis/interproscan-annotation/#InterProScanAnnotation-MergeInterProScanGOstoAnnotation) terms retrieved from InterProScan to existing Annotation. ### Input Open the [project](https://drive.google.com/file/d/1ey5qZ0C0AXQKQiRRYY6o8vcrSDjHeYKu/view?usp=sharing) with Gene Ontology terms merged from EggNOG and InterProScan results. If no InterProScan results are available in the project, it is possible to run or load the results from step [2- Cloud InterProScan](#IPS) ### Execution Time Very fast ### Output The same [project](https://drive.google.com/file/d/1bFHuEDviEjJ1CV0kp_Mvi0mds9rP-kGM/view?usp=sharing) with the Gene Ontology retrieved from InterProScan. A [chart](https://drive.google.com/file/d/14oKCy79hwAMsXgxtrpz5qfmXV9w7sjdz/view?usp=sharing) with information on the number of GOs that has been merged. ## 8- Functional Enrichment Analysis (Fisher’s Exact Test) ### Application In this case, a [comparative analysis](https://www.biobam.com/run-enrichment-analysis-on-2-groups-of-sequences-with-different-annotation-files/) between SARS-CoV and SARS-CoV-2 will be performed to see if there is a function that is specific to the SARS-CoV-2 strand. ### Input The protein sequences of SARS-CoV have been downloaded from [NCBI](https://www.ncbi.nlm.nih.gov/genome/10320) and analyzed with the above pipeline. Both annotated projects ([SARS-CoV](https://drive.google.com/file/d/1dbFg7dvqOsiR_yIxEFBtRpnibA0SUvCs/view?usp=sharing) and [SARS-CoV-2](https://drive.google.com/file/d/1bFHuEDviEjJ1CV0kp_Mvi0mds9rP-kGM/view?usp=sharing)) have to be merged into a single project and a test set has to be generated. The test set for the Enrichment Analysis will be the identifiers from SARS-CoV-2. #### Merge projects It is possible to [combine 2 projects in OmicsBox](https://www.biobam.com/combine-b2g-projects/) by adding the results to the other. This has to be done in the [file manager](http://manual.omicsbox.biobam.com/user-manual/user-interface/), by selecting both projects, right-clicking on the first project, and selecting Merge. All results have to be added and a new **[Merged project](https://drive.google.com/file/d/15L4w_Ul7duovliZsYtcrtd7SAidehtbf/view?usp=sharing)**[ ](https://drive.google.com/file/d/15L4w_Ul7duovliZsYtcrtd7SAidehtbf/view?usp=sharing)will open. #### Create test set id list It is possible to [create an id list in OmicsBox](https://www.biobam.com/how-to-create-a-gene-list-within-blast2go-to-run-the-functional-enrichment-analysis/) from an annotated project. The [SARS-CoV-2](https://drive.google.com/file/d/1bFHuEDviEjJ1CV0kp_Mvi0mds9rP-kGM/view?usp=sharing) project has to be opened in OmcisBox. First, all sequences have to be marked with Ctrl + A (Windows and Linux) / Cmd + A (Mac) and then right-click on a sequence name to choose “Create ID List of Column: SeqName”. A new tab will open with the sequence identifiers in single columns. This list has to be saved and used as the **[test set](https://drive.google.com/file/d/1WMZW8WIvqBnYOHPkUB8UeYcOUdX9vPLu/view?usp=sharing)** for the Enrichment Analysis. ### Parameters Open the **[Merged project](https://drive.google.com/file/d/15L4w_Ul7duovliZsYtcrtd7SAidehtbf/view?usp=sharing)** in OmicsBox and this will be used as the reference. - Test-Set Files: **[SARS-CoV2_idlist.box](https://drive.google.com/file/d/1WMZW8WIvqBnYOHPkUB8UeYcOUdX9vPLu/view?usp=sharing)** - Reference-Set Files: false - Do Not Filter: false - Filter Value: 0.01 - Filter Mode: P-VALUE - Two Tailed: false - Remove Double IDs: true - Annotations: GO IDs - GO Categories: biological_process,molecular_function,cellular_component ### Execution Time Very fast ### Output [Project](https://drive.google.com/file/d/1atYu292fXFO3hI3ggSY3GiJhPfJ87fLc/view?usp=sharing) containing results of the functional enrichment analysis. In this case, only 1 Gene Ontology term is enriched which is specific for SARS-CoV-2 which is “host cell endosome”. ## Workflow [Example Workflow](https://drive.google.com/file/d/1USEr3zuByEA7IWQ-3VplAjk8aq0FEdcP/view?usp=sharing) ![image](media://5755d1b0-5cd9-4cd5-89c8-e322b353505c)