The file Latest/Standalone_Annotate_Alignment_V11.py comes with several python3.6.8 functions that can annotate and highlight positions in a Clustal-formatted alignment. The resultfile is a .svg that is fully compatible for further editing in Inkscape/Illustrator or for inspection via your browser.
Please cite
Torsten Schmenger, Gaurav Diwan, Robert Bruce Russell. "PROTEORIZER: A holistic approach to untangle functional consequences of variants of unknown significance", https://doi.org/10.1101/2024.07.16.603688.
and
Gurdeep Singh (1)*+, Torsten Schmenger (1)*+, Juan-Carlos Gonzalez-Sanchez (1), Anastasiia Kutkina (1), Nina Bremec (1), Gaurav Diwan (1), Cristina Lopez (2), Rocio Sotillo (3), Robert B Russell (1)+; "Identify activating, deactivating and resistance variants in protein kinases"; 2023
when using this work.
In general, this script needs only one thing to work:
- an alignment in clustal format mandatory
- optional a python dictionary formatted as {Protein:{Feature:[Residues]}},
- optional a protein feature dictionary, formatted as (and based on protein of interest) {Feature:[Startposition, Endposition]}.
Note: Both dictionaries can either be provided by the user or will be automatically generated by the script. In that case, the script returns both dictionaries for future use.
For example using the files in /Latest:
User prodived dictionaries
python3 Standalone_Annotate_Alignment_V11.py P61586 34 30 RHOA_BlastpExample_ClustalMSA.clustal RHOA_Blastp_info.txt Features_RHOA.txt
Minimal example
python3 Standalone_Annotate_Alignment_V11.py P61586 34 30 RHOA_BlastpExample_ClustalMSA.clustal none none
This applies to users who do not already have an alignment. The following steps can used to create an alignment using blastp and clustal omega.
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Step 1: Download the fasta sequence of your protein of interest. For RHOA you could do this via Uniprot, like this. Note: You can easily build this url using https://rest.uniprot.org/uniprotkb/ + uniprotID +.fasta
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Step 2: Use the downloaded fasta sequence to perform a blast search for similar sequences blastp. For more information on how to use BLAST please see Blast Help. Make sure to select "Swissprot" as a database, or otherwise make sure that the retained accessions will be UniprotIDs.
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Step 3: Select the sequences you prefer and download them.
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| Make sure to download the complete sequences. |
- Step 4: Add the protein of interests fasta manually to the top of the just downloaded file. Change the formatting to roughly mimic the formatting of the remaining entries.
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| This is how your prepared fasta sequences should look like. |
- Step 5: Perform a multiple sequence alignment using Clustal Omega. Make sure to select Protein. Download & save the alignment file for usage with this script. Input the sequences via copy & paste or upload a file.
Make sure you download the complete MSA (including the clustal version, followed by 2 empty lines, followed by the MSA).
- Step 6: Annotate the alignment manually or programmatically with whatever information you want, following the aforementioned format. Recommended to use the Create_Information.py script (see next section).
Python 3.6.8+
- NEW Returns dictionaries of collected information for re-usage by the user in future runs.
- NEW Fused preparation and main script into a single script.
- NEW Upgraded script to python 3 plus added some cosmetic changes.
- New Added tooltips that show up on mouseover events. Works on residues with functional information.
- New Added transparent rectangles to highlight a sequence conservation (= identity) over >= 70 %, based on the sequence of interest. The colors for this are taken from CLUSTAL/Jalview.
- New Change highlighting to circles. Circle radius can later be adjusted based on evidence.
- New Added basic heatmapping above the alignment, showing how many highlights per position & per category we have.
- New Added start and end positions for each displayed sequence.
- Command line functionality.
To use the script we can now execute the following command:
python Standalone_Annotate_Alignment_V11.py P61586 34 30 RHOA_BlastpExample_ClustalMSA.clustal RHOA_Blastp_info.txt Features_RHOA.txt
This command has several fields after calling the script:
| Field | Example | Description |
|---|---|---|
| 0 | P61586 | The uniprot ID of the protein we are interested in |
| 1 | 34 | The position to be highlighted |
| 2 | 30 | The Windowsize, we show +/- the windowsize of residues around the highlighted position |
| 3 | RHOA_BlastpExample_ClustalMSA | The alignment file. |
| 4 | RHOA_Blastp_info.txt | The file containing positional information. IDs must match to those present in the alignment file. Can be left empty by writing "none". The script will then automatically populate the dictionary via Uniprot |
| 5 | Features_RHOA.txt | A file containing structural/domain features, numbering based on protein of interest. IDs must match those present in the alignment file. Can be left empty by writing "none". The script will then automatically populate the dictionary via Interpro |
Note: The script allows for a little hack here. If you want a (large) .svg containing the whole alignment just give a big number in field 2, for example 20000. The script will then produce a complete alignment view. New Giving "none" instead of a position to be highlighted (field 1) works the same + it removed the position specific rectangle.
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Named Output files. The resultfile will already be named depending on the input settings, so one can easily try different settings. The name follows this format:
poi+"_Position"+str(startposition)+"_Windowsize"+str(windowsize)+".svg" -
Conservation: Gives a black rectangle as an indicator of sequence identity (top) for the POI residue at that position.
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Residue Numbering: Gives the residue number (every 10 residues), based on sequence of interest, and highlights the input residue in red.
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Feature Annotation: Gives a colored background based on type of annotation (taken from uniprot) to the respective residue.
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Sorted Sequences: Sequences with fewer uniprot annotations are sorted to the bottom of the alignment.
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GAPs removed: Gaps are printed with white color (i.e. invisible on a white background). Additionally, columns with more than 90 % GAPs are removed from the alignment. Sequences affected by this (i.e. the up to 10 % of sequences that did not have a gap at that position) are kept and not removed.
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Highlighting protein features, here for example p-loop, Switch I and the Effector region of RHOA. We currently support the displaying of up to 9 features (dependent on the given colors in featurecolors on line 2518 of this example script).
The result of executing python3 Standalone_Annotate_Alignment_V11.py P61586 34 30 RHOA_BlastpExample_ClustalMSA.clustal RHOA_Blastp_info.txt Features_RHOA.txt.
tschmenger/Annotate_Alignments is licensed under the GNU General Public License v3.0
Thanks to @gurdeep330 for providing feedback and suggestions.