ExPairwiseAlignment - Revision history

Henni: /* Global alignment */

2026-09-14T13:32:56Z

Global alignment

← Older revision		Revision as of 15:32, 14 September 2026
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	[[Image:Office-notes-line_drawing.png\|30px\|left]]		[[Image:Office-notes-line_drawing.png\|30px\|left]]
	:'''QUESTION 2.2''':		:'''QUESTION 2.2''':
	:Now, perform the same alignment without applying gap penalties. Which values changed?		:Now, perform the same alignment without applying end gap penalties. Which values changed?

	=== Local Alignment ===		=== Local Alignment ===

Henni: /* Epilogue — mostly for fun */

2026-09-10T14:19:54Z

Epilogue — mostly for fun

← Older revision		Revision as of 16:19, 10 September 2026
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	==Epilogue — mostly for fun==		==Epilogue — mostly for fun==
	[[Image:Cogs_brain.png\|50px\|right]]		[[Image:Cogs_brain.png\|50px\|right]]
	Go to UniProt and find the sequences with the IDs GLBE_CHITH and GLB7A_CHITH. These are two globins from a midge, a very small and annoying insect. If you have ever been to Scotland in the summer, you will know about midges. Make a global alignment of GLBE_CHITH and GLB7A_CHITH.		Go to UniProt and find the sequences with the IDs GLBE_CHITH and GLB7A_CHITH. These are two globins from a midge, a very small and annoying insect. If you have ever been to Scotland in the summer, you will know about midges. Make a global alignment of GLBE_CHITH and GLB7A_CHITH. '''Note:''' remember to set the gap penalties back to their default values: 10 for opening and 0.5 for elongation.

	[[Image:Office-notes-line_drawing.png\|30px\|left]]		[[Image:Office-notes-line_drawing.png\|30px\|left]]
	:'''QUESTION 14''':		:'''QUESTION 14''':
	Note that there is a gap of 6 positions in GLBE_CHITH. What is the corresponding 6 amino acid long sequence of GLB7A_CHITH? This is an authentic example! Nature truly is fascinating...		Note that there is a gap of 6 positions in GLBE_CHITH. What is the corresponding 6 amino acid long sequence of GLB7A_CHITH? This is an authentic example! Nature truly is fascinating...

Henni: /* Release the Gaps! */

2026-09-10T14:12:41Z

Release the Gaps!

← Older revision		Revision as of 16:12, 10 September 2026
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	* Still, use local protein alignment.		* Still, use local protein alignment.
	* Set the matrix back to "BLOSUM62"		* Set the matrix back to "BLOSUM62"
	* Set the Gap opening penalty to 1.0 ~~(smallest possible value).~~		* Set the Gap opening penalty to 1.0
	* Set the Gap extend penalty to 0.~~0005 (smallest possible value).~~		* Set the Gap extend penalty to 0.01
	* Align Savinase and Tripeptidyl Peptidase		* Align Savinase and Tripeptidyl Peptidase

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	:'''QUESTION 13''':		:'''QUESTION 13''':
	:* How do the quality parameters look this time (Length, score, gaps, identity, similarity)?		:* How do the quality parameters look this time (Length, score, gaps, identity, similarity)?
	:* Is this ~~alignment~~ biologically meaningful at all?		:* Look at the alignment. Is this biologically meaningful at all?

	==Epilogue — mostly for fun==		==Epilogue — mostly for fun==

Henni: /* Release the Gaps! */

2026-09-10T14:09:30Z

Release the Gaps!

← Older revision		Revision as of 16:09, 10 September 2026
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	As the last part of the comparison of sequences — let's allow as many gaps as possible, by making it almost "free" for the algorithm to insert gaps.		As the last part of the comparison of sequences — let's allow as many gaps as possible, by making it almost "free" for the algorithm to insert gaps.

	* ~~Go the the page for~~ local protein alignment.		* Still, use local protein alignment.
	* Click "More Options"
	* Set the matrix back to "BLOSUM62"		* Set the matrix back to "BLOSUM62"
	* Set the Gap opening penalty to 1.0 (smallest possible value).		* Set the Gap opening penalty to 1.0 (smallest possible value).

Henni: /* Choose the Matrix */

2026-09-10T14:04:40Z

Choose the Matrix

← Older revision		Revision as of 16:04, 10 September 2026
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	First, let's see how the alignment depends on the choice of substitution matrix.		First, let's see how the alignment depends on the choice of substitution matrix.

	* ~~Go the the page for~~ local protein alignment.		* Again, use local protein alignment.
	* Click "More Options"
	* Choose the substitution matrix "BLOSUM90" instead of the default (BLOSUM62)		* Choose the substitution matrix "BLOSUM90" instead of the default (BLOSUM62)
	* Align Savinase and Tripeptidyl Peptidase		* Align Savinase and Tripeptidyl Peptidase (NOT shuffled)
	* Redo the alignment, this time with the matrix "BLOSUM30"		* Redo the alignment once again, this time with the matrix "BLOSUM30"

	~~You can see the BLOSUM matrices on this page: [[BLOSUM matrix examples]]~~

			You can see the three BLOSUM matrices on this page: [[BLOSUM matrix examples]]

	[[Image:Office-notes-line_drawing.png\|30px\|left]]		[[Image:Office-notes-line_drawing.png\|30px\|left]]
	:'''QUESTION 12''':		:'''QUESTION 12''':
	:* What are the alignment results (Length, score, gaps, identity, similarity)?		:* What are the alignment results (Length, score, gaps, identity, similarity)?
	:* How do alignment length and % identity depend on the BLOSUM number (compare also to your answer to question 8)?		:* How do alignment length and % identity depend on the BLOSUM number (compare also to your answer to question 8, using BLOSUM62)?

	=== Release the Gaps! ===		=== Release the Gaps! ===

Henni: /* Part 2 — about gaps and dubious alignments */

2026-09-10T13:59:47Z

Part 2 — about gaps and dubious alignments

← Older revision		Revision as of 15:59, 10 September 2026
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	[[Image:Office-notes-line_drawing.png\|30px\|left]]		[[Image:Office-notes-line_drawing.png\|30px\|left]]
	:'''QUESTION 6''':		:'''QUESTION 6''':
	: Compare Savinase to the human peptidase by global alignment ~~(Needle)~~ — remember again to ~~set~~ <u>~~End Gap Penalty~~</u> ~~to "true"~~ — and report the following:		: Compare Savinase to the human peptidase by global alignment — remember again to tick <u>Apply end gap penalties</u> — and report the following:
	:* Alignment score		:* Alignment score
	:* Alignment length		:* Alignment length
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	[[Image:Office-notes-line_drawing.png\|30px\|left]]		[[Image:Office-notes-line_drawing.png\|30px\|left]]
	:'''QUESTION 7''':		:'''QUESTION 7''':
	:Repeat the alignment with <u>~~End Gap Penalty~~</u> ~~set to "false"~~ and report the same results as above.		:Repeat the alignment with <u>Apply end gap penalties</u> unticked and report the same results as above. Does the alignment look different this time?


	[[Image:Office-notes-line_drawing.png\|30px\|left]]		[[Image:Office-notes-line_drawing.png\|30px\|left]]
	:'''QUESTION 8''':		:'''QUESTION 8''':
	:Repeat the alignment again — this time using the local alignment algorithm ~~(Water)~~ — and report the same results as above.		:Repeat the alignment again — this time using the local alignment algorithm — and report the same results as above.


	[[Image:Office-notes-line_drawing.png\|30px\|left]]		[[Image:Office-notes-line_drawing.png\|30px\|left]]
	:'''QUESTION 9''':		:'''QUESTION 9''':
	:Do you think local or global alignment is best for finding ~~similar parts of~~ distantly related proteins? Why?		:Do you think local or global alignment is best for finding similarities in distantly related proteins? Why?
	:*Hint: Distantly related proteins typically share a core, that relates to the function of the protein		:*Hint: Distantly related proteins typically share a core, that relates to the function of the protein

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	:'''When is an alignment "true"?'''		:'''When is an alignment "true"?'''

	:When we perform an alignment of two (or more) sequences, it's actually '''a statement of evolutionary relationship''': We ~~believe~~ that the sequences share a '''common ancestor''' - and that the sequences thus have diverged from an identical gene/protein throughout the eons.		:When we perform an alignment of two (or more) sequences, it's actually '''a statement of evolutionary relationship''': We hypothesize that the sequences share a '''common ancestor''' - and that the sequences thus have diverged from an identical gene/protein throughout the eons.

	:The problem that we face right now in this part of the exercise, is that we're actually '''NOT sure''' if the two peptidases are related at all. As we discussed in the lecture, it's often possible to get at least some kind of alignment from unrelated sequences, but then the alignment score will be low. But in order to investigate if the alignment score we got from the alignment above indicates that the alignment is trustworthy, we need to get an idea of what '''range of alignment scores''' we can expect by aligning '''truly unrelated sequences'''.		:The problem that we face right now in this part of the exercise, is that we're actually '''NOT sure''' if the two peptidases are related at all. As we discussed in the lecture, it's often possible to get at least some kind of alignment from unrelated sequences, but then the alignment score will be low. But in order to investigate if the alignment score we got from the alignment above indicates that the alignment is trustworthy, we need to get an idea of what '''range of alignment scores''' we can expect by aligning '''truly unrelated sequences'''.
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	* Open the <!-- SeqShuffle Server (http://www.cbs.dtu.dk/biotools/SeqShuffle-1.0/) --> "Shuffle Protein" page (http://www.bioinformatics.org/sms2/shuffle_protein.html) in a new window/tab, paste in the tripeptidyl peptidase sequence and shuffle it.		* Open the <!-- SeqShuffle Server (http://www.cbs.dtu.dk/biotools/SeqShuffle-1.0/) --> "Shuffle Protein" page (http://www.bioinformatics.org/sms2/shuffle_protein.html) in a new window/tab, paste in the tripeptidyl peptidase sequence and shuffle it.
	* Then align Savinase and the shuffled tripeptidyl peptidase sequence using local alignment.		* Then align Savinase and the shuffled tripeptidyl peptidase sequence using local alignment.
	* Repeat the above procedure ~~two~~ more times, so that you align Savinase with ''~~three~~ different'' shuffled versions of tripeptidyl peptidase.		* Repeat the above procedure four more times, so that you align Savinase with ''five different'' shuffled versions of tripeptidyl peptidase.

Henni: /* Local Alignment */

2026-09-10T13:42:41Z

Local Alignment

← Older revision		Revision as of 15:42, 10 September 2026
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	=== Local Alignment ===		=== Local Alignment ===
	Now, perform the alignment once again (preferably in a new browser tab/window, so it's easy to compare), but this time ~~use~~ the ~~Water~~ (~~short for~~ Smith-Waterman) algorithm~~. This can (again) be found on http://www.ebi.ac.uk/Tools/psa/ . Again use protein alignment~~.		Now, perform the alignment once again (preferably in a new browser tab/window, so it's easy to compare), but this time choose the <u>Local (Smith-Waterman)</u> algorithm.

	[[Image:Office-notes-line_drawing.png\|30px\|left]]		[[Image:Office-notes-line_drawing.png\|30px\|left]]

Henni: /* Global alignment */

2026-09-10T13:37:22Z

Global alignment

← Older revision		Revision as of 15:37, 10 September 2026
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	* Below the sequence boxes you find the alignment options. Ensure that <u>Matrix</u> is set to "BLOSUM62"		* Below the sequence boxes you find the alignment options. Ensure that <u>Matrix</u> is set to "BLOSUM62"
	* Also, tick the box <U>Apply end gap penalties</u> (otherwise, the algorithm ignores gaps at the very ends of the alignment, ''i.e.'' gaps positioned at the end of one of the sequences would not cost anything).		* Also, tick the box <U>Apply end gap penalties</u> (otherwise, the algorithm ignores gaps at the very ends of the alignment, ''i.e.'' gaps positioned at the end of one of the sequences would not cost anything).
	* Finally, perform the alignment by clicking the appropriate button.		* Finally, perform the alignment by clicking the appropriate button and scroll down to see the result.

	*Have a look at the result. Notice that the degree of similarity between aligned amino acids is indicated with the following symbols: "<tt>\|</tt>" indicates a perfect match, "<tt>:</tt>" indicates a "close" mismatch (positive value on the BLOSUM matrix, also known as a ''conservative substitution'' — the amino acids share some physico-chemical properties), and "<tt>.</tt>" indicates a "far" mismatch (zero or negative value on the BLOSUM matrix).		Have a look at the result. Notice that the degree of similarity between aligned amino acids is indicated with the following symbols: "<tt>\|</tt>" indicates a perfect match, "<tt>:</tt>" indicates a "close" mismatch (positive value on the BLOSUM matrix, also known as a ''conservative substitution'' — the amino acids share some physico-chemical properties), and "<tt>.</tt>" indicates a "far" mismatch (zero or negative value on the BLOSUM matrix).

	[[Image:Office-notes-line_drawing.png\|30px\|left]]		[[Image:Office-notes-line_drawing.png\|30px\|left]]
	:'''QUESTION 2''':		:'''QUESTION 2.1''':
	: Report the following values / observations from the alignment.		:Report the following values / observations from the alignment.
	:* Alignment score		:* Alignment score
	:* Alignment length		:* Alignment length
	:* % and fraction Identity (The value reported for "Identity" includes perfect matches only)		:* % and fraction Identity (The value reported for "Identity" includes perfect matches only)
	:* % and fraction Similarity (The value reported for "Similarity" includes perfect matches + "close" mismatches)		:* % and fraction Similarity (The value reported for "Similarity" includes perfect matches + "close" mismatches)

			[[Image:Office-notes-line_drawing.png\|30px\|left]]
			:'''QUESTION 2.2''':
			:Now, perform the same alignment without applying gap penalties. Which values changed?

	=== Local Alignment ===		=== Local Alignment ===

Henni: /* Global alignment */

2026-09-10T13:27:40Z

Global alignment

← Older revision		Revision as of 15:27, 10 September 2026
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	=== Global alignment ===		=== Global alignment ===
	Your first task is to make a '''global alignment'''.		Your first task is to make a '''global alignment'''.
	* ~~In the Needle section~~ (~~Short for~~ Needleman & Wunsch) at the ~~site http://www~~.~~ebi.ac.uk/Tools/psa/, Click the button labeled "Protein" and paste~~ in the sequences ~~- including~~ the header line - one sequence in each search box.		* Choose <u>Protein Sequences</u> and <u>Global (Needleman-Wunsch)</u> at the top of the page. Paste in the sequences (it doesn't matter whether you include the header line) one sequence in each search box.
	* ~~Click~~ the ~~button labeled "More~~ options~~" (under "STEP 2") and ensure~~ that <u>Matrix</u> is set to "BLOSUM62"		* Below the sequence boxes you find the alignment options. Ensure that <u>Matrix</u> is set to "BLOSUM62"
	* Also ~~under "options" ("STEP 2") set~~ <u>~~End Gap Penalty~~</u> ~~to "true"~~ (otherwise, the algorithm ignores gaps at the very ends of the ~~alignments~~, ''i.e.'' gaps positioned at the end of one of the sequences would not cost anything).		* Also, tick the box <U>Apply end gap penalties</u> (otherwise, the algorithm ignores gaps at the very ends of the alignment, ''i.e.'' gaps positioned at the end of one of the sequences would not cost anything).
			* Finally, perform the alignment by clicking the appropriate button.
	* Finally, ~~start~~ the alignment by clicking the button ~~labeled "Submit"~~

	*Have a look at the result. Notice that the degree of similarity between aligned amino acids is indicated with the following symbols: "<tt>\|</tt>" indicates a perfect match, "<tt>:</tt>" indicates a "close" mismatch (positive value on the BLOSUM matrix, also known as a ''conservative substitution'' — the amino acids share some physico-chemical properties), and "<tt>.</tt>" indicates a "far" mismatch (zero or negative value on the BLOSUM matrix).		*Have a look at the result. Notice that the degree of similarity between aligned amino acids is indicated with the following symbols: "<tt>\|</tt>" indicates a perfect match, "<tt>:</tt>" indicates a "close" mismatch (positive value on the BLOSUM matrix, also known as a ''conservative substitution'' — the amino acids share some physico-chemical properties), and "<tt>.</tt>" indicates a "far" mismatch (zero or negative value on the BLOSUM matrix).


	[[Image:Office-notes-line_drawing.png\|30px\|left]]		[[Image:Office-notes-line_drawing.png\|30px\|left]]

Henni: /* Part 1 — basic use */

2026-09-10T13:13:51Z

Part 1 — basic use

← Older revision		Revision as of 15:13, 10 September 2026
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	Go back to https://teaching.healthtech.dtu.dk/alignment/ and open the "'''Pairwise Sequence Alignment Tool'''". This tool does not display the alignment matrix, but it can work with long sequences and allows for affine gap penalties (different values for gap opening and gap elongation).		Go back to https://teaching.healthtech.dtu.dk/alignment/ and open the "'''Pairwise Sequence Alignment Tool'''". This tool does not display the alignment matrix, but it can work with long sequences and allows for affine gap penalties (different values for gap opening and gap elongation).

	'''Note:''' Previously, we used the alignment webpage at EBI: https://www.ebi.ac.uk/jdispatcher/psa (you are welcome to try if you want), but that is a server-based application which becomes slow when 150+ students connect at the same time. The new local solution, which was vibe-coded in 2025 by a bachelor student as part of a special course, works much faster since all calculations are carried out on your own computers via JavaScript.		<div style="background-color: lavender; border: solid thin grey;">
			:'''Note:''' Previously, we used the alignment webpage at EBI: https://www.ebi.ac.uk/jdispatcher/psa (you are welcome to try if you want), but that is a server-based application which becomes slow when 150+ students connect at the same time. The new local solution, which was vibe-coded in 2025 by a bachelor student as part of a special course, works much faster since all calculations are carried out on your own computers via JavaScript.
			</div>

	<!--		<!--
	[[File:PairwiseAlignment.png\|thumb\|210px\|right\|The different options at EBI (local and global alignment)]]		[[File:PairwiseAlignment.png\|thumb\|210px\|right\|The different options at EBI (local and global alignment)]]