Exercise:AntigenProcessing ans - Revision history

Carol: /* GibbsCluster */

2026-09-22T20:40:27Z

GibbsCluster

← Older revision		Revision as of 22:40, 22 September 2026
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	*'''Q10: Does the number of clusters found by GibbsCluster correspond to the number of HLA alleles in your sample?'''		*'''Q10: Does the number of clusters found by GibbsCluster correspond to the number of HLA alleles in your sample?'''

	'''Answer:''' ~~Not necessarily~~. ~~The number of sequence~~ clusters ~~identified by GibbsCluster does~~ '''~~not have to equal the number of HLA molecules expressed by the sample~~'''.		'''Answer:''' No. When all five RA patients were combined, GibbsCluster tested solutions containing 1–5 clusters but selected a solution containing only '''one sequence motif'''.

	~~In this experiment, after taking DRB4 into account, there are three relevant~~ HLA-DR ~~specificities~~:		This does not correspond to the number of HLA-DR molecules present in the samples. Across the five patients there are already '''six different DRB1 alleles''':

	~~DRB1*01:01~~		DRB1_0101
	~~DRB1*04:01~~		DRB1_0401
	~~DRB4*01:01~~		DRB1_0402
			DRB1_0801
			DRB1_1104
			DRB1_1501

	~~However~~, ~~an unsupervised clustering analysis may recover only the strongest distinguishable motifs and may fail to resolve a smaller peptide population such as~~ the ~~DRB4 repertoire as an independent cluster.~~		If the secondary HLA-DR molecules identified in the previous exercise are also considered, the dataset additionally contains:

	~~'''Explanation:''' GibbsCluster does not know the patient's HLA genotype. It only examines patterns in the peptide sequences and asks how the sequences can best be partitioned into clusters.~~		DRB3_0202
			DRB4_0101
			DRB5_0101

	~~Therefore:~~		Thus, several different HLA-DR specificities are present in the combined dataset, but GibbsCluster finds only '''one dominant motif'''.

	number of sequence clusters ≠ necessarily number of HLA alleles		This illustrates an important limitation of unsupervised motif deconvolution: the number of sequence clusters does not necessarily correspond directly to the number of HLA alleles. Different HLA molecules can have overlapping binding preferences, and smaller motifs may be hidden by larger, dominant peptide populations.

	~~Two alleles with similar motifs may be merged into~~ one ~~cluster, a small allele~~-~~specific repertoire may not form a sufficiently strong independent~~ cluster~~, or noisy~~ peptides ~~may affect the clustering solution.~~		In the selected one-cluster solution:

			Cluster 1 948 peptides
			Outliers 94 peptides

	~~This contrasts with MHCMotifDecon, where the known HLA molecules are explicitly supplied to the algorithm.~~

	*'''Q11: Can you identify other differences between the GibbsCluster solution and the MHCMotifDecon solution?'''		*'''Q11: Can you identify other differences between the GibbsCluster solution and the MHCMotifDecon solution?'''

	'''Answer:''' Yes. The ~~most important difference is that~~ '''MHCMotifDecon ~~is supervised by~~ HLA-binding predictions ~~and known~~ HLA ~~typing~~, ~~whereas~~ GibbsCluster is an ~~unsupervised sequence~~-~~clustering method~~'''.		'''Answer:''' Yes. The two methods produce quite different interpretations of the same immunopeptidomics data.

			'''MHCMotifDecon''' uses the known HLA genotype of each patient together with peptide–HLA binding predictions. It therefore separates the peptides into motifs corresponding to specific HLA molecules, for example:

			RA2 DRB1_0101,DRB1_0401,DRB4_0101
			RA5 DRB1_0801,DRB1_1501,DRB5_0101

			In contrast, '''GibbsCluster does not know which HLA alleles are present'''. It groups peptides only according to similarities in their amino-acid sequences. Consequently, when peptides from all five patients are combined, the different HLA-specific motifs are largely merged into a single dominant motif rather than being separated according to HLA allele.

			GibbsCluster also identifies '''outliers''' rather than an allele-specific Trash group. In the selected solution, 94 peptides were classified as outliers.

			Another important difference is that GibbsCluster first collapses the input to '''unique peptide sequences'''. In this analysis, 1072 unique sequences were read from the combined dataset. In contrast, MHCMotifDecon analysed the peptides separately according to their patient labels, allowing the same peptide to contribute independently to different patient-specific analyses.

	~~'''Explanation:'''~~		Overall, MHCMotifDecon is better able to separate overlapping HLA motifs when the HLA genotype is known, whereas GibbsCluster attempts to discover motifs directly from the peptide sequences without prior HLA information.

	With '''MHCMotifDecon''':		With '''MHCMotifDecon''':

Carol: /* MHCMotifDecon */

2026-09-22T20:18:59Z

MHCMotifDecon

← Older revision		Revision as of 22:18, 22 September 2026
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	'''Answer:''' After including the secondary HLA-DR molecules, '''97 peptides''' were assigned to DRB3, DRB4 or DRB5.		'''Answer:''' After including the secondary HLA-DR molecules, '''97 peptides''' were assigned to DRB3, DRB4 or DRB5.

			[[Image:MHCMotifDecon_rank20_extendedHLA.png\|center\|800px]]

	The assignments for each patient were:		The assignments for each patient were:

Carol: /* MHCMotifDecon */

2026-09-22T20:18:01Z

MHCMotifDecon

← Older revision		Revision as of 22:18, 22 September 2026
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	*'''Q6: How many peptides were assigned to each of the alleles, and how many were assigned to the trash cluster?'''		*'''Q6: How many peptides were assigned to each of the alleles, and how many were assigned to the trash cluster?'''

	[[Image:MHCMotifDecon_rank20.png\|center\|800px]]:		[[Image:MHCMotifDecon_rank20.png\|center\|800px]]



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	'''Answer:''' Lowering the %Rank threshold from '''20% to 2%''' makes the peptide assignment considerably more stringent. Peptides with a best predicted binding rank between 2% and 20% are no longer accepted as binders and are instead assigned to the '''Trash''' cluster.		'''Answer:''' Lowering the %Rank threshold from '''20% to 2%''' makes the peptide assignment considerably more stringent. Peptides with a best predicted binding rank between 2% and 20% are no longer accepted as binders and are instead assigned to the '''Trash''' cluster.

			[[Image:MHCMotifDecon_rank2.png\|center\|800px]]

	The effect can be seen directly by comparing the two runs:		The effect can be seen directly by comparing the two runs:

Carol: /* MHCMotifDecon */

2026-09-22T20:17:17Z

MHCMotifDecon

← Older revision		Revision as of 22:17, 22 September 2026
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	*'''Q6: How many peptides were assigned to each of the alleles, and how many were assigned to the trash cluster?'''		*'''Q6: How many peptides were assigned to each of the alleles, and how many were assigned to the trash cluster?'''

	[[Image:MHCMotifDecon_rank20.png\|~~left~~\|800px]]:		[[Image:MHCMotifDecon_rank20.png\|center\|800px]]:

Carol: /* MHCMotifDecon */

2026-09-22T20:17:07Z

MHCMotifDecon

← Older revision		Revision as of 22:17, 22 September 2026
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	*'''Q6: How many peptides were assigned to each of the alleles, and how many were assigned to the trash cluster?'''		*'''Q6: How many peptides were assigned to each of the alleles, and how many were assigned to the trash cluster?'''

	[[Image:MHCMotifDecon_rank20.png\|left\|800px]]		[[Image:MHCMotifDecon_rank20.png\|left\|800px]]:

Carol: /* MHCMotifDecon */

2026-09-22T20:16:53Z

MHCMotifDecon

← Older revision		Revision as of 22:16, 22 September 2026
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	*'''Q6: How many peptides were assigned to each of the alleles, and how many were assigned to the trash cluster?'''		*'''Q6: How many peptides were assigned to each of the alleles, and how many were assigned to the trash cluster?'''

	[[Image:MHCMotifDecon_rank20.png\|~~center~~\|~~500px~~]]		[[Image:MHCMotifDecon_rank20.png\|left\|800px]]

Carol: /* MHCMotifDecon */

2026-09-22T20:16:33Z

MHCMotifDecon

← Older revision		Revision as of 22:16, 22 September 2026
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	*'''Q6: How many peptides were assigned to each of the alleles, and how many were assigned to the trash cluster?'''		*'''Q6: How many peptides were assigned to each of the alleles, and how many were assigned to the trash cluster?'''

	[[Image:MHCMotifDecon_rank20.png\|center\|~~100px~~]]		[[Image:MHCMotifDecon_rank20.png\|center\|500px]]

Carol: /* MHCMotifDecon */

2026-09-22T20:16:21Z

MHCMotifDecon

← Older revision		Revision as of 22:16, 22 September 2026
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	*'''Q6: How many peptides were assigned to each of the alleles, and how many were assigned to the trash cluster?'''		*'''Q6: How many peptides were assigned to each of the alleles, and how many were assigned to the trash cluster?'''

	[[~~image~~:MHCMotifDecon_rank20.png\|center~~\|frame~~\|100px ~~MHC Motif Decon rank20~~]]		[[Image:MHCMotifDecon_rank20.png\|center\|100px]]

Carol: /* MHCMotifDecon */

2026-09-22T20:15:42Z

MHCMotifDecon

← Older revision		Revision as of 22:15, 22 September 2026
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	*'''Q6: How many peptides were assigned to each of the alleles, and how many were assigned to the trash cluster?'''		*'''Q6: How many peptides were assigned to each of the alleles, and how many were assigned to the trash cluster?'''

	[[image:MHCMotifDecon_rank20.png\|center\|frame\|100px\| MHC Motif Decon rank20]]		[[image:MHCMotifDecon_rank20.png\|center\|frame\|100px MHC Motif Decon rank20]]

Carol: /* MHCMotifDecon */

2026-09-22T20:15:26Z

MHCMotifDecon

← Older revision		Revision as of 22:15, 22 September 2026
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	*'''Q6: How many peptides were assigned to each of the alleles, and how many were assigned to the trash cluster?'''		*'''Q6: How many peptides were assigned to each of the alleles, and how many were assigned to the trash cluster?'''

	[[image:MHCMotifDecon_rank20.png\|center\|frame\|~~800px~~ MHC Motif Decon rank20]]		[[image:MHCMotifDecon_rank20.png\|center\|frame\|100px\| MHC Motif Decon rank20]]