Answers.Body: <p>Here is a manual (or semi-manual, at best) approach. If you need to do this for very many sequences it won't be practical, but if dealing with one or two genes, it might be enough. The idea is to:</p>
  <ol>
  <li>Get the protein coding transcripts</li>
  <li>Get the proteins they code for</li>
  <li>Align</li>
  </ol>
  <p>In the page you gave, there is a &quot;Show transcript table&quot; button near the top:</p>
  <p><a href="https://i.sstatic.net/FYLSC.png" rel="nofollow noreferrer"><img src="https://i.sstatic.net/FYLSC.png" alt="Screenshot of the EnsEmbl page showing the relevant button" /></a></p>
  <p>If you click on that, you get the list of transcripts and their corresponding proteins:</p>
  <p><a href="https://i.sstatic.net/u25Ur.png" rel="nofollow noreferrer"><img src="https://i.sstatic.net/u25Ur.png" alt="Screenshot of the Ensembl page showing the expanded transcript table" /></a></p>
  <p>You can then click on the &quot;Show/hide columns&quot; and select &quot;Translation ID&quot; so you can get the Ensembl protein corresponding to each transcript:</p>
  <p><a href="https://i.sstatic.net/ktoXx.png" rel="nofollow noreferrer"><img src="https://i.sstatic.net/ktoXx.png" alt="Screenshot of the Ensembl page with &quot;Translation ID&quot; selected" /></a></p>
  <p>If you now deselct everything and only leave &quot;Translation ID&quot; selected:</p>
  <p><a href="https://i.sstatic.net/1UJwv.png" rel="nofollow noreferrer"><img src="https://i.sstatic.net/1UJwv.png" alt="Screenshot of the Ensembl page with only &quot;Translation ID&quot; selected" /></a></p>
  <p>You are left with a nice list of Ensembl proteins. Copy/paste those into a text file:</p>
  <pre><code>ENSP00000369497.3
  ENSP00000499438.2
  ENSP00000505508.1
  ENSP00000439902.1
  ENSP00000514856.2
  ENSP00000434898.2
  ENSP00000506251.1
  ENSP00000499570.2
  ENSP00000433168.2
  ENSP00000499256.2
  ENSP00000514855.1
  </code></pre>
  <p>Now, go to <a href="https://www.ensembl.org/biomart/martview/cc1e3843edd9549a4fb578f900d88d23" rel="nofollow noreferrer">BioMart</a> and retrieve the sequences. Select &quot;Ensembl Genes&quot; as the database and then &quot;Human Genes&quot;:</p>
  <p><a href="https://i.sstatic.net/SgWpW.png" rel="nofollow noreferrer"><img src="https://i.sstatic.net/SgWpW.png" alt="biomart screenshot" /></a></p>
  <p>Then, click on &quot;Filters&quot; (1) and then &quot;Gene&quot; (2), select &quot;Input external references ID list [Max 500 advised]&quot; (3), set the ID to &quot;Protein stable ID(s) with version&quot; (4) and paste the protein list (5):</p>
  <p><a href="https://i.sstatic.net/rDGmI.png" rel="nofollow noreferrer"><img src="https://i.sstatic.net/rDGmI.png" alt="biomart screenshot" /></a></p>
  <p>Next, go to &quot;Attributes&quot; (1) and select &quot;Sequences&quot; (2):</p>
  <p><a href="https://i.sstatic.net/iDwcj.png" rel="nofollow noreferrer"><img src="https://i.sstatic.net/iDwcj.png" alt="biomart screenshot" /></a></p>
  <p>Now, finally, click on &quot;Results&quot; (1) and you will get the sequences in fasta format (2):</p>
  <p><a href="https://i.sstatic.net/26Fx3.png" rel="nofollow noreferrer"><img src="https://i.sstatic.net/26Fx3.png" alt="biomart screenshot" /></a></p>
  <p>I'm afraid they are too long to fit in this answer, but I posted them here: <a href="https://pastebin.com/NZycAmgD" rel="nofollow noreferrer">https://pastebin.com/NZycAmgD</a>.</p>
  <p>You can now paste these sequences into your favorite aligner and get the alignment you were after. Here, I used the online <a href="https://www.ebi.ac.uk/Tools/msa/clustalo/" rel="nofollow noreferrer">clustalΩ</a> (the image only shows the first few lines):</p>
  <p><a href="https://i.sstatic.net/mZap9.png" rel="nofollow noreferrer"><img src="https://i.sstatic.net/mZap9.png" alt="clustalΩ alignment of the BRCA2 proteins" /></a></p>
  <p>You can find the whole alignment in clustal format (text) <a href="https://pastebin.com/GhHeG9CJ" rel="nofollow noreferrer">here</a>.</p>
  
Answers.ContentLicense: CC BY-SA 4.0
Answers.CreationDate: 2024-01-09T21:45:09.077Z
Answers.Text: Thanks @terdon. This is exactly my question. In your clustal omega result screenshot (last figure of your answer) there are a bunch of misalignments for ENST00000700201 compared with others (starting from loc ~105 to the end). I assume the software is forcing different exons aligned together to reduce number of gaps. I mean those gaps should be inserted before loc ~105 on ENST00000700201, not after. But somehow the software prefer before (due to parameters, misalignments outperformed gaps). I can manually adjust it. Easy for one gene, but not for hundreds of genes. That's why I want to downloa
Answers.UserDisplayName: Jen Marylin
Answers.ContentLicense: CC BY-SA 4.0
Answers.CreationDate: 2024-01-09T21:48:50.057Z
Answers.Text: although ENST00000700201 is NMD, not a perfect example. But the question stays for other protein coding transcripts.
Answers.UserDisplayName: Jen Marylin
Answers.ContentLicense: CC BY-SA 4.0
Answers.CreationDate: 2024-01-10T00:16:10.553Z
Answers.Text: @JenMarylin I don't quite understand what you mean. What exons? We're aligning simple protein sequences here, the aligner has no knowledge of the exonic structure of the transcripts that were translated to make these proteins. And this is a multiple sequence alignment, it is positioning the gaps so as to maximize the score of the entire alignment. What is it you want to download? Are you looking for an alignment of the translated _exons_ instead of the actual protein product?
Answers.UserDisplayName: terdon
Answers.ContentLicense: CC BY-SA 4.0
Answers.CreationDate: 2024-01-09T17:05:49.313Z
Answers.LastActivityDate: 2024-01-09T17:05:49.313Z
Answers.UserDisplayName: terdon
Answers.Body: <p>This is a paper that tries to address this problem</p>
  <p>&quot;Splice-Aware Multiple Sequence Alignment of Protein Isoforms&quot;
  <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6508070/" rel="nofollow noreferrer">https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6508070/</a></p>
  <p>I have not evaluated it, but it could be a point of reference</p>
  <p>note that there is also a sequel to this paper from 2023 <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10166558/" rel="nofollow noreferrer">https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10166558/</a></p>
  
Answers.ContentLicense: CC BY-SA 4.0
Answers.CreationDate: 2024-07-08T22:22:19.727Z
Answers.LastActivityDate: 2024-07-08T22:22:19.727Z
Answers.UserDisplayName: Colin D
Body: <p><a href="https://i.sstatic.net/gErzk.png" rel="nofollow noreferrer"><img src="https://i.sstatic.net/gErzk.png" alt="enter image description here" /></a></p>
  <p>[Update] Thanks @terdon. To clarify my question:</p>
  <p>I have a bunch of protein isoforms sequences (produced by the transcripts in the figure) and I want to align protein products (e.g., proteins produced by the first 3 protein-coding transcripts against each other). See there are 3 blocks missing in 5' region in BRCA2-204 compared against BRCA2-206.</p>
  <p>This case might be very easy with any MSA softwares such as clustal omega, but there are complicated cases which may trick software into aligning single amino acids instead of being splicing aware. Instead of manually adjusting the alignments, I want to know whether I can directly download, or &quot;translate&quot;, this figure into alignment results (e.g., fasta format or clustal format)</p>
  <p>This figure should be visible with <a href="https://www.ensembl.org/Homo_sapiens/Gene/Summary?db=core;g=ENSG00000139618;r=13:32315086-32400268" rel="nofollow noreferrer">https://www.ensembl.org/Homo_sapiens/Gene/Summary?db=core;g=ENSG00000139618;r=13:32315086-32400268</a>
  in the gene summary tab</p>
  <p>[Original] Is there a way to directly download the sequence alignment result from Ensembl? If not, what will be the best way to align exons and introns without parameter-tuning</p>
  
Comments.ContentLicense: CC BY-SA 4.0
Comments.CreationDate: 2024-01-09T15:20:16.120Z
Comments.Text: Please [edit] your question and clarify what you need. You mention protein isoforms in the title, but show transcript sequences in the body. Do you want the alignment of the proteins produced by each transcripts against each other? The alignment of each transcript against a reference genome? The alignment of the transcripts against each other? Ideally, give us an example we can look up, a link that we can click on to see what you are using (is that human BRCA2 or something else?) and then explain what you would like to get from that.
Comments.UserDisplayName: terdon
Comments.ContentLicense: CC BY-SA 4.0
Comments.CreationDate: 2024-01-09T15:50:16.573Z
Comments.Text: @terdon Thanks!
Comments.UserDisplayName: Jen Marylin
Comments.ContentLicense: CC BY-SA 4.0
Comments.CreationDate: 2024-01-09T16:20:55.817Z
Comments.Text: Interesting question (upvoted). This might be naive, but if these are transcripts surely they have already been spliced? That aside from eukaryotic genomics its easy, but must have been solved years ago, I suspect a *de novo* pipeline is simply "reinventing the wheel".
Comments.UserDisplayName: M__
Comments.ContentLicense: CC BY-SA 4.0
Comments.CreationDate: 2024-01-09T21:52:42.743Z
Comments.Text: @M__♦ I am not sure I followed. You mean aligning these spliced exons is easy?
Comments.UserDisplayName: Jen Marylin
Comments.ContentLicense: CC BY-SA 4.0
Comments.CreationDate: 2024-01-10T19:40:10.617Z
Comments.Text: I've undeleted the question ... it's a perfectly good question - it has an answer by an expert which is upvoted. My justification is that I had trouble logging in over the last 24 hours - I had to escalate to SE login HQ to get in, hence I couldn't take part in the discussion before the question was deleted. @terdon is an expert on human genetics and I'll defer to their answer.
Comments.UserDisplayName: M__
Comments.ContentLicense: CC BY-SA 4.0
Comments.CreationDate: 2024-01-13T12:44:20.017Z
Comments.Text: Please don't deface your question. Once a question has been posted and answered, you have given SE the right to use the content under [CC-BY-SA](https://stackoverflow.com/help/licensing). Even if your question is no longer useful to you, or if you have your answer, that doesn't mean that other users won't also find it useful so deleting useful content from the site isn't allowed.
Comments.UserDisplayName: terdon
Tags: <phylogenetics><proteins><ensembl><multiple-sequence-alignment>
Title: What is the best way to acquire protein isoform sequence alignment?
ThreadText: # What is the best way to acquire protein isoform sequence alignment?
  
  (Asked by: username_0 on 2024-01-09)
  
  [![enter image description here](https://i.sstatic.net/gErzk.png)](https://i.sstatic.net/gErzk.png)
  
  [Update] Thanks @username_1. To clarify my question:
  
  I have a bunch of protein isoforms sequences (produced by the transcripts in the figure) and I want to align protein products (e.g., proteins produced by the first 3 protein-coding transcripts against each other). See there are 3 blocks missing in 5' region in BRCA2-204 compared against BRCA2-206.
  
  This case might be very easy with any MSA softwares such as clustal omega, but there are complicated cases which may trick software into aligning single amino acids instead of being splicing aware. Instead of manually adjusting the alignments, I want to know whether I can directly download, or "translate", this figure into alignment results (e.g., fasta format or clustal format)
  
  This figure should be visible with <https://www.ensembl.org/Homo_sapiens/Gene/Summary?db=core;g=ENSG00000139618;r=13:32315086-32400268>
  in the gene summary tab
  
  [Original] Is there a way to directly download the sequence alignment result from Ensembl? If not, what will be the best way to align exons and introns without parameter-tuning
  
  **Tags:** phylogenetics, proteins, ensembl, multiple-sequence-alignment
  
  > **Comments:**
  > **username_1**: Please [edit] your question and clarify what you need. You mention protein isoforms in the title, but show transcript sequences in the body. Do you want the alignment of the proteins produced by each transcripts against each other? The alignment of each transcript against a reference genome? The alignment of the transcripts against each other? Ideally, give us an example we can look up, a link that we can click on to see what you are using (is that human BRCA2 or something else?) and then explain what you would like to get from that.  
  > **username_0**: @username_1 Thanks!  
  > **username_2**: Interesting question (upvoted). This might be naive, but if these are transcripts surely they have already been spliced? That aside from eukaryotic genomics its easy, but must have been solved years ago, I suspect a *de novo* pipeline is simply "reinventing the wheel".  
  > **username_0**: @username_2♦ I am not sure I followed. You mean aligning these spliced exons is easy?  
  > **username_2**: I've undeleted the question ... it's a perfectly good question - it has an answer by an expert which is upvoted. My justification is that I had trouble logging in over the last 24 hours - I had to escalate to SE login HQ to get in, hence I couldn't take part in the discussion before the question was deleted. @username_1 is an expert on human genetics and I'll defer to their answer.  
  > **username_1**: Please don't deface your question. Once a question has been posted and answered, you have given SE the right to use the content under [CC-BY-SA](https://stackoverflow.com/help/licensing). Even if your question is no longer useful to you, or if you have your answer, that doesn't mean that other users won't also find it useful so deleting useful content from the site isn't allowed.  
  
  ---
  
  ## 2 answers
  
  ### Answer by username_1 on 2024-01-09. Score: 1
  
  Here is a manual (or semi-manual, at best) approach. If you need to do this for very many sequences it won't be practical, but if dealing with one or two genes, it might be enough. The idea is to:
  
  1. Get the protein coding transcripts
  2. Get the proteins they code for
  3. Align
  
  In the page you gave, there is a "Show transcript table" button near the top:
  
  [![Screenshot of the EnsEmbl page showing the relevant button](https://i.sstatic.net/FYLSC.png)](https://i.sstatic.net/FYLSC.png)
  
  If you click on that, you get the list of transcripts and their corresponding proteins:
  
  [![Screenshot of the Ensembl page showing the expanded transcript table](https://i.sstatic.net/u25Ur.png)](https://i.sstatic.net/u25Ur.png)
  
  You can then click on the "Show/hide columns" and select "Translation ID" so you can get the Ensembl protein corresponding to each transcript:
  
  [![Screenshot of the Ensembl page with "Translation ID" selected](https://i.sstatic.net/ktoXx.png)](https://i.sstatic.net/ktoXx.png)
  
  If you now deselct everything and only leave "Translation ID" selected:
  
  [![Screenshot of the Ensembl page with only "Translation ID" selected](https://i.sstatic.net/1UJwv.png)](https://i.sstatic.net/1UJwv.png)
  
  You are left with a nice list of Ensembl proteins. Copy/paste those into a text file:
  
  ```
  ENSP00000369497.3
  ENSP00000499438.2
  ENSP00000505508.1
  ENSP00000439902.1
  ENSP00000514856.2
  ENSP00000434898.2
  ENSP00000506251.1
  ENSP00000499570.2
  ENSP00000433168.2
  ENSP00000499256.2
  ENSP00000514855.1
  
  ```
  
  Now, go to [BioMart](https://www.ensembl.org/biomart/martview/cc1e3843edd9549a4fb578f900d88d23) and retrieve the sequences. Select "Ensembl Genes" as the database and then "Human Genes":
  
  [![biomart screenshot](https://i.sstatic.net/SgWpW.png)](https://i.sstatic.net/SgWpW.png)
  
  Then, click on "Filters" (1) and then "Gene" (2), select "Input external references ID list [Max 500 advised]" (3), set the ID to "Protein stable ID(s) with version" (4) and paste the protein list (5):
  
  [![biomart screenshot](https://i.sstatic.net/rDGmI.png)](https://i.sstatic.net/rDGmI.png)
  
  Next, go to "Attributes" (1) and select "Sequences" (2):
  
  [![biomart screenshot](https://i.sstatic.net/iDwcj.png)](https://i.sstatic.net/iDwcj.png)
  
  Now, finally, click on "Results" (1) and you will get the sequences in fasta format (2):
  
  [![biomart screenshot](https://i.sstatic.net/26Fx3.png)](https://i.sstatic.net/26Fx3.png)
  
  I'm afraid they are too long to fit in this answer, but I posted them here: <https://pastebin.com/NZycAmgD>.
  
  You can now paste these sequences into your favorite aligner and get the alignment you were after. Here, I used the online [clustalΩ](https://www.ebi.ac.uk/Tools/msa/clustalo/) (the image only shows the first few lines):
  
  [![clustalΩ alignment of the BRCA2 proteins](https://i.sstatic.net/mZap9.png)](https://i.sstatic.net/mZap9.png)
  
  You can find the whole alignment in clustal format (text) [here](https://pastebin.com/GhHeG9CJ).
  
  > **Comments:**
  > **username_0**: Thanks @username_1. This is exactly my question. In your clustal omega result screenshot (last figure of your answer) there are a bunch of misalignments for ENST00000700201 compared with others (starting from loc ~105 to the end). I assume the software is forcing different exons aligned together to reduce number of gaps. I mean those gaps should be inserted before loc ~105 on ENST00000700201, not after. But somehow the software prefer before (due to parameters, misalignments outperformed gaps). I can manually adjust it. Easy for one gene, but not for hundreds of genes. That's why I want to downloa  
  > **username_0**: although ENST00000700201 is NMD, not a perfect example. But the question stays for other protein coding transcripts.  
  > **username_1**: @username_0 I don't quite understand what you mean. What exons? We're aligning simple protein sequences here, the aligner has no knowledge of the exonic structure of the transcripts that were translated to make these proteins. And this is a multiple sequence alignment, it is positioning the gaps so as to maximize the score of the entire alignment. What is it you want to download? Are you looking for an alignment of the translated _exons_ instead of the actual protein product?  
  
  ---
  
  ### Answer by username_3 on 2024-07-08. Score: 0
  
  This is a paper that tries to address this problem
  
  "Splice-Aware Multiple Sequence Alignment of Protein Isoforms"
  <https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6508070/>
  
  I have not evaluated it, but it could be a point of reference
  
  note that there is also a sequel to this paper from 2023 <https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10166558/>
  
  
  ---
  
  