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compgenomics:modul2 [2025/05/16 09:01] – [Analysis] felixcompgenomics:modul2 [2025/09/23 13:53] (current) – felix
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-Genes in two species that originated from the same ancestral gene in the last common ancestor of the species are good candidates for being 'corresponding' genes (orthologs)+**Assumption 1:** Genes in two species that originated from the same ancestral gene in the last common ancestor of the species are good candidates for being 'corresponding' genes (orthologs)
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-Orthologs are genes in two species that are mutually most similar to each other+**Assumption 2:** Orthologs are genes in two species that are mutually most similar to each other
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-==== Analysis ====+===== Analysis =====
  
 In this course, we will follow the steps of a [[https://www.biorxiv.org/content/10.1101/2024.10.16.618745v2|comparative genomics analysis of plant cell wall degrading enzymes]] (PCDs). Plant cell walls are made up of cellulose and hemi-cellulose, making them two of the most common organic molecules on Earth and key components of the carbon cycle. In this course, we will follow the steps of a [[https://www.biorxiv.org/content/10.1101/2024.10.16.618745v2|comparative genomics analysis of plant cell wall degrading enzymes]] (PCDs). Plant cell walls are made up of cellulose and hemi-cellulose, making them two of the most common organic molecules on Earth and key components of the carbon cycle.
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 To find out how widespread the ability to degrade plant cell wall, really is we trace  the distribution of 235 potential PCDs across all eukaryotic datasets available in the RefSeq database. To find out how widespread the ability to degrade plant cell wall, really is we trace  the distribution of 235 potential PCDs across all eukaryotic datasets available in the RefSeq database.
  
-=== Task 1: Exploration ===+==== Task 1: Finding corresponding genes ====
  
-  - Open a visualizaiton of the results generated using [[https://ebersberger-46-155.biologie.uni-frankfurt.de/phyloppcd/|this interactive web-viewer]]  +The task at hand is to find out which genes "correspond" to each other in different species and should be displayed in the same row.  
-  - <wrap tip>Wait a moment </wrap> for the data to load. Once you were redirected to the "Main profile" page, select a taxonomic rank and click the red **PLOT** button +
-  - Explore the "Main plot" on different taxonomic levels +
-    - Which information is displayed in the rows, which in the columns? +
-    - What patterns can you observe in the plot and how do you interpret them? +
-  - Select the "Dimension reduction" plot from the top menu and explore the plot with "Phylum"-level labels+
  
-=== Task 2: Finding corresponding genes ===+Have another look at the assumptions at the top of the page. Our best bet to find "corresponding" genes between species is to identify orthologs. In practice, this means finding genes in two species that are mutually most similar to each other.
  
-After having explored the results, let's find out how to generate them. The task at hand is to find out which genes "correspond" to each other in different species, and should be displayed together in one row.  +Finding genes with significantly similar sequences is typically done with a BLAST search. To identify orthologs, we will perform a "reciprocal best hit search". As an example, we will use the GH45 type cellulase of //Rhizoctonia solani// ([[https://www.ncbi.nlm.nih.gov/protein/XP_043186466.1?report=genbank&log\$=prottop&blast_rank=1&RID=2A0JJUND016|XP_043186466.1]]).
  
-Have another look at the assumptions at the top of the page. Our best bet to find "corresponding" genes between species is to identify orthologs. In practice, this means finding genes in two species that are mutually most similar to each other.+  - Open [[https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastp&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome|NCBI BLAST]] 
 +  - Start a search with the following parameters: 
 +    - Paste the sequence accession number into the search field: <wrap tip>XP_043186466.1</wrap> 
 +    - Select database: <wrap tip>Reference proteins (refseq_protein)</wrap> 
 +    - Select target species: <wrap tip>//Bradysia coprophila//</wrap> 
 +  - Extract the accession number of the best hit 
 +  - Perform reverse BLAST serach in //Rhizoctonia solani//
  
-Finding genes with significantly similar sequences is typically done with a BLAST search. To identify orthologs, we will perform a "reciprocal best hit serach". As an example, we will use the GH45 type cellulase of //Rhizoctonia solani// ([[https://www.ncbi.nlm.nih.gov/protein/XP_043186466.1?report=genbank&log\$=prottop&blast_rank=1&RID=2A0JJUND016|XP_043186466.1]].+==== Task 2: Exploration ==== 
 + 
 +=== Option 1: Webviewer (if currently online) === 
 + 
 +  - Open the visualizaiton of the results using [[https://ebersberger-46-155.biologie.uni-frankfurt.de/phyloppcd/|this interactive web-viewer]] 
 + 
 +=== Route 2: Local PhyloProfile installation === 
 + 
 +  - Install Miniconda<WRAP> 
 +<code> 
 +mkdir -p ~/miniconda3 
 +wget https://repo.anaconda.com/miniconda/Miniconda3-latest-Linux-x86_64.sh -O ~/miniconda3/miniconda.sh 
 +bash ~/miniconda3/miniconda.sh -b -u -p ~/miniconda3 
 +rm ~/miniconda3/miniconda.sh 
 +</code></WRAP> 
 +  - Install Conplex (our local software management system)<WRAP> 
 +<code> 
 +echo 'CONPLEX_ENVS_FILE=/home/worker/data/conplex/envs.tsv' >> ~/.bashrc 
 +echo 'alias conplex="source /home/worker/bin/conplex.sh"' >> ~/.bashrc 
 +source ~/.bashrc 
 +</code></WRAP> 
 +  -  Download and unpack the input data<WRAP> 
 +<code> 
 +wget https://applbio.biologie.uni-frankfurt.de/download/fDOG_pPCD/pPCD-PhylogeneticProfiles.tar.gz 
 +tar -zxvf pPCD-PhylogeneticProfiles.tar.gz 
 +</code></WRAP> 
 +  - Start Phyloprofile<WRAP> 
 +<code> 
 +conplex load phyloprofile 
 +</code></WRAP> 
 +  - Upload the file you downloaded to PhyloProfile 
 +  - <wrap tip>Wait a moment </wrap> for the data to load. Once you were redirected to the "Main profile" page, select a taxonomic rank and click the red **PLOT** button 
 +  - Explore the "Main plot" on different taxonomic levels 
 +    - You can draw selection boxes on the overview plot and generate a more detailed plot of the selected section 
 +    - Which information is displayed in the rows, which in the columns? 
 +    - What patterns can you observe in the plot and how do you interpret them?
  
-  - +=== Summary and Disussion ===
  
 +  * Record your observations and questions
 +  * Discuss with the group
  
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