Sequence Alignments Hemochromatosis

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Protocol

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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.

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.

<figtable id="blastgo">

	Overlap of hit's GO terms.	Overlap of HFE's GO terms.
	Table 2: Common GO terms within BLAST hits.

</figtable>

<figure id="blastcops">

Figure 1: COPS classification of BLAST hits.

</figure>

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>

<figtable id="psiblastgo">

	Overlap of hit's GO terms (Big80).	Overlap of HFE's GO terms (Big80).
	Overlap of hit's GO terms (Big).	Overlap of HFE's GO terms (Big).
	Table 5: Common GO terms within PSI-BLAST hits.

</figtable>

<figtable id="psiblastcops">

	Database: Big80
	Database: Big
	Table 6: COPS classification of PSI-BLAST hits.

</figtable>

HHblits

<figtable id="hhblitsdist">

	Table 4: 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.

<figtable id="hhblitsgo">

	Overlap of hit's GO terms.	Overlap of HFE's GO terms.
	Table X: Common GO terms within HHblits hits.

</figtable>

COPS classification of HHblits hits.

Comparison

<figtable id="psiblastoverlap">

	Database: Big80.	Database: Big.
	Overlap between the different PSI-BLAST runs.

</figtable>

<figtable id="alloverlap">

	Databases: Big80, Uniprot20.	Databases: Big, Uniprot20.	Databases: Big, Uniprot20.	Database: Big80.
	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>

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

Muscle

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