fdr thresholding
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FDR ThresholdingFDR Thresholding
Caleb J. Emmons
Slide: 1
What is FDR?What is FDR?
Slide: 2
If decoy proteins are present
Protein FDR = # decoy proteins identified
# target proteins identified
Peptide FDR = # spectra from decoy proteins
# spectra from target proteins
The FDR BrowserThe FDR Browser
How does FDR Thresholding work?How does FDR Thresholding work?
Slide: 4
1
54
32
The “FDR Landscape”
Minimum number o
f peptid
es
How does FDR Thresholding work?How does FDR Thresholding work?
Slide: 5The “FDR Landscape”
Some Fine PointsSome Fine Points
Slide: 6
Confusing!
Protein ClusteringProtein Clustering
Poster 509, Tuesday 10:30-1:00
Informatics: Quantification/Validation
Caleb J. Emmons
Slide: 7
What is a Cluster?What is a Cluster?
Slide: 8
Total Peptide EvidenceTotal Peptide Evidence
Slide: 9
PEtot(A) = sum of peptide probabilitiesover all peptides matching A
Protein PEtot
K1C10 1481%
K1C14 1061%
K1C16 852%
K1C17 503%
Joint Peptide EvidenceJoint Peptide Evidence
Slide: 10
PEjoint(A, B) = sum of peptide probabilitiesover all peptides matching A and B
PEjoint K1C10 K1C14 K1C16
K1C14 184%
K1C16 184% 661%
K1C17 84% 375% 175%
Proteins A and B are in the same cluster if they are directly similar, or if they can be connected with a sequence of proteins that are directly similar.
Cluster FormationCluster Formation
Slide: 11
A ≈ B if 1) their joint evidence is at least 95%, and2) their joint evidence is at least half of the total evidence for A or B
Directly similar
Clusters
Cluster FormationCluster Formation
Slide: 12
Protein PEtot
K1C10 1481%
K1C14 1061%
K1C16 852%
K1C17 503%
PEjoint K1C10 K1C14 K1C16
K1C14 184%
K1C16 184% 661%
K1C17 84% 375% 175%
A ≈ B ?K1C10 K1C14 K1C16
K1C14 no
K1C16 no yes
K1C17 no yes no
Peptide-Protein WeightsPeptide-Protein Weights
Slide: 13
A B C
PEexcl(C) = sum of peptide probabilitiesover all peptides exclusively matching C
W(p, C) =
Spectrum CountingSpectrum Counting
Slide: 14
SEQ1, +3
SEQ1, +2
SEQ2, +2
SEQ3, +2
SEQ7, +3
SEQ7, +3
SEQ4,+2
SEQ5, +2A
B
C
Protein A 3 2 3 1
Protein B 4 2 4 2
Protein C 4 2 3 1
Cluster of B&C
6 5 5 4
Exclusive peptide/spectrum: associated only with this single cluster/protein
Unique peptides: only consider amino acid sequences
Unique spectra: only consider amino acid sequence, modifications, & charge state
Quantitative ValuesQuantitative Values
Slide: 15
Total and Weighted Spectrum Counts run over all spectra in the cluster
Total Ion Current (TIC) and Precursor Intensity may be computed, treating the cluster as a collection of spectra.
Normalized Spectral Abundance Factor (NSAF) roughly consists of a ratio of an exclusive spectrum count and protein length, so does not make direct sense on the level of cluster (as clusters do not have a ‘length’). However, the average NSAF over the member proteins gives an interpretable value. Similarly, we compute the Exponentially Modified Protein Abundance Index (emPAI) as an average over the member proteins in the cluster.
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