Sequence Alignments Hemochromatosis

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Protocol

Sorry for the inconvenience (not beeing able to read something), we're rerunning some data...

Protocol

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. Only 6 of these were PDB-IDs.

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.

PSI-Blast

<figtable id="psiblastdist">

	Table 2: 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. 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.

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 because of 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 3: Runtime analysis of PSI-BLAST. </figtable>

HHblits

<figtable id="hhblitsdist">

	E-Value and identity distributions of the HHblits search against Uniprot20.

</figtable>

Comparison

<figtable id="psiblastoverlap">

	Nothing to see here....

</figtable>

<figtable id="alloverlap">

	Nothing to see here....

</figtable>

Multiple Sequence Alignments

Dataset

<figtable id="msagroup60">

Uniprot AC (Group 60-99)	Identity	Comment
G3QU39	99.14
H2PI54	97.41
Q6B0J5	95.98
G7P2L8	94.54
F7GRH8	90.52
F7DKE9	86.18
Q9GL42	79.89
F6RUG7	78.16
G3THV5	75.21
G5BQE5	67.66

</figtable>

<figtable id="msagroup40">

Uniprot AC (Group 00-40)	Identity	Comment
P16391	36.03
P05534	35.13
Q30597	33.72
P01900	32.11
Q31093	31.74
P14432	29.14
Q860W6	27.74
Q31615	26.38
Q31206	25.87
P01921	21.10

</figtable>

<figtable id="msagroup00">

Uniprot AC (Group 00-99)	Identity	Comment
Q6B0J5	95.98
F7GRH8	90.52
F7DKE9	86.18
Q9GL42	79.89
G5BQE5	67.66
G1PHG2	57.43
F7C3B3	40.11
Q30597	33.72
Q860W6	27.74
P01921	21.10

</figtable>