Difference between revisions of "Sequence Alignments Hemochromatosis"
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The distributions for the e-Values and identities of these hits are shown in <xr id="blastdist"/>. Most of the e-Values are between 1e-50 and 2e-3 (cutoff). Only few hits have a better e-Value. The identities are piled between 20% and 40% with two peaks at around 27% and 34% respectively. |
The distributions for the e-Values and identities of these hits are shown in <xr id="blastdist"/>. Most of the e-Values are between 1e-50 and 2e-3 (cutoff). Only few hits have a better e-Value. The identities are piled between 20% and 40% with two peaks at around 27% and 34% respectively. |
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− | For the evaluation of the results we compared the hits' GO terms |
+ | For the evaluation of the results we compared the hits' GO terms. <xr id="blastgo"/> shows that the majority of the hits share almost all of their GO terms with the HFE protein. In contrast, only about 10% to 15% of HFE's GO terms are shared by most of the hits. This might be caused by the fact that most of the hit proteins didn't have as much GO terms as HFE (27 GO terms). |
<figtable id="blastgo"> |
<figtable id="blastgo"> |
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− | <figure id="blastcops"> |
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− | [[File:hemo_cops_blast.png|thumb|200px|left|<font size=1>'''Figure 1:''' COPS classification of BLAST hits.]] |
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− | </figure> |
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Revision as of 15:57, 7 May 2012
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Contents
Task Description
Protocol
Sorry for the inconvenience (not beeing able to read something), we're rerunning some data...
Reference Sequence
Sequence from Uniprot: Q30201
>sp|Q30201|HFE_HUMAN Hereditary hemochromatosis protein OS=Homo sapiens GN=HFE PE=1 SV=1 MGPRARPALLLLMLLQTAVLQGRLLRSHSLHYLFMGASEQDLGLSLFEALGYVDDQLFVF YDHESRRVEPRTPWVSSRISSQMWLQLSQSLKGWDHMFTVDFWTIMENHNHSKESHTLQV ILGCEMQEDNSTEGYWKYGYDGQDHLEFCPDTLDWRAAEPRAWPTKLEWERHKIRARQNR AYLERDCPAQLQQLLELGRGVLDQQVPPLVKVTHHVTSSVTTLRCRALNYYPQNITMKWL KDKQPMDAKEFEPKDVLPNGDGTYQGWITLAVPPGEEQRYTCQVEHPGLDQPLIVIWEPS PSGTLVIGVISGIAVFVVILFIGILFIILRKRQGSRGAMGHYVLAERE
Sequence Searches
BLAST
<figtable id="blastdist">
Table 1: E-Value and identity distributions of the Blast search against Big80. |
</figtable>
The first BLAST search against the Big80 database reached the hit limit of 250 sequences with an e-Value of e-30 for the worst hit. So we did it again with a new limit of 1500 reported hits. These hits were then filtered for unique IDs and an e-Value cutoff of 2e-3. After the filtering 1159 hits were left.
The distributions for the e-Values and identities of these hits are shown in <xr id="blastdist"/>. Most of the e-Values are between 1e-50 and 2e-3 (cutoff). Only few hits have a better e-Value. The identities are piled between 20% and 40% with two peaks at around 27% and 34% respectively.
For the evaluation of the results we compared the hits' GO terms. <xr id="blastgo"/> shows that the majority of the hits share almost all of their GO terms with the HFE protein. In contrast, only about 10% to 15% of HFE's GO terms are shared by most of the hits. This might be caused by the fact that most of the hit proteins didn't have as much GO terms as HFE (27 GO terms).
<figtable id="blastgo">
Table 2: Common GO terms within BLAST hits. |
</figtable>
PSI-BLAST
<figtable id="psiblastdist">
Table 3: E-Value and identity distributions of the PSI-BLast search against Big80. |
</figtable>
After the BLAST search, we also performed several searches with PSI-BLAST. This time we increased the number of reported hits and used a variety of parameter combinations to test their impact on the search results. The parameters to be changed were 'h' and 'j'. The first one, 'h', sets the e-Value cutoff for the inclusion of sequences into the PSI-BLAST-profile. The second one, 'j', is for the number of iterations for the PSI-BLAST search. For each of these parameters we used two different values: 2e-3 and 1e-10 for 'h', 2 and 10 for 'j'. This resulted in a total of 4 combinations.
The first 4 searches were against Big80 with a maximum of 10000 reported hits. We also saved the PSI-BLAST profiles for later (see bellow). The hits for each individual parameter combination were again filtered for unique IDs and an e-Value cutoff of 2e-3. This resulted in the following number of hits:
- h=2e-3, j=2: 1892 (2786 prefiltered)
- h=2e-3, j=10: 1704 (2734 prefiltered)
- h=1e-10, j=2: 2058 (3574 prefiltered)
- h=1e-10, j=10: 2035 (3458 prefiltered)
<xr id="psiblastdist"/> shows the e-Value and identity distributions for the Big80 results. In contrast to the previous BLAST search, we have more significant e-Values, but the identity shifts a bit to the left (lower identity). The differences between the parameter combinations are quite easy to spot. The lower e-Value cutoff (1-e10) also produces more significant hits (lower e-Values). This might be caused by the inclusion of fewer sequences into the profile and therefore a higher specificity for more closely related sequences (i.e. low e-Values). An increased number of iterations on the other hand reduces the number of significant hits and seems to slightly reduce the average identity.
After the searches against Big80 we also ran PSI-BLAST against the Big database. We reused the profiles from the Big80 runs and also increased the maximum of reported hits to 100000.
- h=2e-3, j=2: 23840 (25934 prefiltered)
- h=2e-3, j=10: 25756 (30616 prefiltered)
- h=1e-10, j=2: 26483 (28766 prefiltered)
- h=1e-10, j=10: 27535 (29609 prefiltered)
The two combinations with 10 iterations threw multiple error messages (but finished the process nevertheless). These errors were due to an internal code failure of PSI-BLAST and caused by too many possible hits.
The performances of the different PSI-BLAST runs (see <xr id="psiblastruntime"/>) show that the cutoff for the profiles ('h') doesn't really affect the runtime. The number of iteration on the other hand has a big impact on the runtime. The size of the database, of course, also affects the runtime. The exceptionally high runtime for the 10-iteration runs against Big might also be caused by the errors mentioned above.
<figtable id="psiblastruntime">
Iterations | 2 | 2 | 10 | 10 |
---|---|---|---|---|
E-Value | 0.002 | 10E-10 | 0.002 | 10E-10 |
Big80 | 3m21 | 3m6 | 16m39 | 16m41 |
Big | 28m17 | 26m43 | 367m15 | 64m4 |
Table 4: Runtime analysis of PSI-BLAST. </figtable>
The GO term and COPS classification analysis yields similar results to BLAST. The different parameter values have almost no effect on the GO terms. The only visible effect is that a higher iteration count seems to have a small negative effect on the GO term analysis. In the COPS analysis (see <xr id="psiblastcops"/>) the parameters seem to make almost no difference, but a more strict e-Value cutoff and higher iteration count both have a negative effect (see Comparison section, <xr id="allcops"/>).
<figtable id="psiblastgo">
Table 5: Common GO terms within PSI-BLAST hits. |
</figtable>
<figtable id="psiblastcops">
Table 6: COPS classification of PSI-BLAST hits. |
</figtable>
HHblits
<figtable id="hhblitsdist">
Table 7: E-Value and identity distributions of the HHblits search against Uniprot20. |
</figtable>
The final sequence search algorithm we apllied was HHblits. This time we searched against another database, Uniprot20. We set the number of reported hits (clusters) to 600 which corresponds to a worst e-Value of 0.0021. After filtering for unique clusters, we had 585 clusters left. Within these clusters we had 27588 unique Uniprot ACs. The most significant cluster with an e-Value of 2e-131 and an 40% identities accounted for 2771 (about 10% total) of these Uniprot ACs.
In <xr id="hhblitsdist"/> you can see the distribution of the clusters' e-Values and identities. Like in the BLAST results, most e-Values are between 1e-50 and 2e-3. The majority of the identities are again between 20% and 40%, with a peak at around 26%.
The runtime was 13m 13s. This means that HHblits' is more or less between BLAST's and PSI-BLAST's runtime.
HHblits shows about the same results for the GO term analysis (xr id="hhblitsgo"/>) regarding the percentage of HFE's GO terms. In contrast to the previous results, HHblits also has a high peak for the hit's GO terms at around 65% to 70%. This means that HHblits finds more proteins with a more distant related function than the other algorithms. The COPS classification is similar to PSI-BLAST (database: Big) in the L99 and L80 clusters, but HHblits has a better performance to find proteins in the L30, L40, and L60 clusters than PSI-BLAST (database: Big).
<figtable id="hhblitsgo">
Table 8: Common GO terms within HHblits hits. |
</figtable>
<figure id="hhblitscops">
</figure>
Comparison
<figtable id="psiblastoverlap">
Overlap between the different PSI-BLAST runs. |
</figtable>
<figtable id="alloverlap">
Overlap between the different search algorithms. In the first figure (only) PSI-BLAST includes the sum of all unique hits from all PSI-BLAST iterations (against Big80). |
</figtable>
<figure id="allcops">
</figure>
Multiple Sequence Alignments
Dataset
<figtable id="msagroup60">
Uniprot AC (Group 60-99) | Identity | Comment |
---|---|---|
G3QU39 | 99.14 | Uncharacterized protein OS=Gorilla gorilla gorilla |
H2PI54 | 97.41 | Uncharacterized protein OS=Pongo abelii |
Q6B0J5 | 95.98 | HFE protein OS=Homo sapiens |
G7P2L8 | 94.54 | Putative uncharacterized protein OS=Macaca fascicularis |
F7GRH8 | 90.52 | Uncharacterized protein OS=Callithrix jacchus |
F7DKE9 | 86.18 | Uncharacterized protein OS=Callithrix jacchus |
Q9GL42 | 79.89 | Hereditary hemochromatosis protein homolog OS=Dicerorhinus sumatrensis |
F6RUG7 | 78.16 | Uncharacterized protein OS=Equus caballus |
G3THV5 | 75.21 | Uncharacterized protein (Fragment) OS=Loxodonta africana |
G5BQE5 | 67.66 | Hereditary hemochromatosis protein-like protein OS=Heterocephalus glaber |
</figtable>
<figtable id="msagroup40">
Uniprot AC (Group 00-40) | Identity | Comment |
---|---|---|
P16391 | 36.03 | RT1 class I histocompatibility antigen, AA alpha chain OS=Rattus norvegicus |
P05534 | 35.13 | HLA class I histocompatibility antigen, A-24 alpha chain OS=Homo sapiens |
Q30597 | 33.72 | MHC class I Mamu-A*02 (Fragment) OS=Macaca mulatta |
P01900 | 32.11 | H-2 class I histocompatibility antigen, D-D alpha chain OS=Mus musculus |
Q31093 | 31.74 | Histocompatibility 2, M region locus 3 OS=Mus musculus |
P14432 | 29.14 | H-2 class I histocompatibility antigen, TLA(B) alpha chain OS=Mus musculus |
Q860W6 | 27.74 | Major histocompatibility complex class Ib M10.5 (Fragment) OS=Mus musculus |
Q31615 | 26.38 | MHC class I H2-TL-27-129 mRNA (b haplotype), complete cds OS=Mus musculus |
Q31206 | 25.87 | MHC class I H2-TL-T10-129 mRNA (b haplotype), complete cds OS=Mus musculus |
P01921 | 21.10 | H-2 class II histocompatibility antigen, A-D beta chain OS=Mus musculus |
</figtable>
<figtable id="msagroup00">
Uniprot AC (Group 00-99) | Identity | Comment |
---|---|---|
Q6B0J5 | 95.98 | HFE protein OS=Homo sapiens |
F7GRH8 | 90.52 | Uncharacterized protein OS=Callithrix jacchus |
F7DKE9 | 86.18 | Uncharacterized protein OS=Callithrix jacchus |
Q9GL42 | 79.89 | Hereditary hemochromatosis protein homolog OS=Dicerorhinus sumatrensis |
G5BQE5 | 67.66 | Hereditary hemochromatosis protein-like protein OS=Heterocephalus glaber |
G1PHG2 | 57.43 | Uncharacterized protein (Fragment) OS=Myotis lucifugus |
F7C3B3 | 40.11 | Uncharacterized protein OS=Macaca mulatta |
Q30597 | 33.72 | MHC class I Mamu-A*02 (Fragment) OS=Macaca mulatta |
Q860W6 | 27.74 | Major histocompatibility complex class Ib M10.5 (Fragment) OS=Mus musculus |
P01921 | 21.10 | H-2 class II histocompatibility antigen, A-D beta chain OS=Mus musculus |
</figtable>
CLustalW
ClustalW 0-40
Sequence | Gap |
---|---|
Q30597 | 79 |
P01900 | 71 |
Q860W6 | 109 |
Q31093 | 100 |
Q31615 | 57 |
P05534 | 71 |
P01921 | 171 |
Q30201 | 88 |
P16391 | 65 |
P14432 | 52 |
Q31206 | 30 |
Conserved |
---|
23 |
ClustalW 0-99
Sequence | Gap |
---|---|
Q30597 | 42 |
Q860W6 | 72 |
G5BQE5 | 32 |
Q9GL42 | 51 |
G1PHG2 | 61 |
Q6B0J5 | 54 |
P01921 | 134 |
Q30201 | 51 |
F7C3B3 | 146 |
F7DKE9 | 62 |
F7GRH8 | 51 |
Conserved |
---|
21 |
ClustalW 60-99
Sequence | Gap |
---|---|
G3THV5 | 12 |
H2PI54 | 25 |
F6RUG7 | 25 |
G5BQE5 | 6 |
Q9GL42 | 25 |
Q6B0J5 | 28 |
Q30201 | 25 |
G3QU39 | 25 |
F7GRH8 | 25 |
F7DKE9 | 36 |
G7P2L8 | 25 |
Conserved |
---|
175 |
Muscle
Muscle Group 0-40
Sequence | Gap |
---|---|
P01900 | 82 |
Q30597 | 90 |
Q31093 | 111 |
Q860W6 | 120 |
Q31615 | 68 |
P05534 | 82 |
Q30201 | 99 |
P01921 | 182 |
P16391 | 76 |
P14432 | 63 |
Q31206 | 41 |
Conserved |
---|
22 |
Muscle Group 0-99
Sequence | Gap |
---|---|
Q30597 | 44 |
Q860W6 | 74 |
G5BQE5 | 34 |
Q9GL42 | 53 |
Q6B0J5 | 56 |
G1PHG2 | 63 |
Q30201 | 53 |
P01921 | 136 |
F7C3B3 | 148 |
F7DKE9 | 64 |
F7GRH8 | 53 |
Conserved |
---|
22 |
Muscle Group 60-99
Sequence | Gap |
---|---|
G3THV5 | 12 |
H2PI54 | 25 |
F6RUG7 | 25 |
G5BQE5 | 6 |
Q9GL42 | 25 |
Q6B0J5 | 28 |
Q30201 | 25 |
G3QU39 | 25 |
F7GRH8 | 25 |
F7DKE9 | 36 |
G7P2L8 | 25 |
Conserved |
---|
175 |
T-Coffee
ClustalW 0-40
Sequence | Gap | ||
---|---|---|---|
Q30597 | 92|- | P01900 | 84 |
Q31093 | 113 | ||
Q860W6 | 122 | ||
Q31615 | 70 | ||
P05534 | 84|- | P01921 | 184 |
Q30201 | 101 | ||
P14432 | 65 | ||
P16391 | 78 | ||
Q31206 | 43 |
Conserved |
---|
2 |
ClustalW 0-99
Sequence | Gap |
---|---|
Q30597 | 51 |
Q860W6 | 81 |
G5BQE5 | 41 |
Q9GL42 | 60 |
Q6B0J5 | 63 |
G1PHG2 | 70 |
P01921 | 143 |
Q30201 | 60 |
F7C3B3 | 155 |
F7DKE9 | 71 |
F7GRH8 | 60 |
Conserved |
---|
22 |
ClustalW 60-99
Sequence | Gap |
---|---|
G3THV5 | 12 |
H2PI54 | 25 |
F6RUG7 | 25 |
G5BQE5 | 6 |
Q9GL42 | 25 |
Q6B0J5 | 28 |
Q30201 | 25 |
G3QU39 | 25 |
G7P2L8 | 25 |
F7GRH8 | 25 |
F7DKE9 | 25 |
Conserved |
---|
175 |
3D-Coffee