Difference between revisions of "Lab journal task 8"

From Bioinformatikpedia
(→‎MutationTaster)
(→‎Mutation analysis)
 
(10 intermediate revisions by the same user not shown)
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282 C -6 -6 -8 -9 12 -8 -9 -8 -8 -7 -7 -8 -7 -5 -8 -5 -6 -8 <span style="color:OrangeRed">'''-5'''</span> -6 0 1 0 0 94 0 0 0 0 0 0 0 0 1 0 1 0 0 <span style="color:OrangeRed">'''1'''</span> 0 4.31 inf
  
282 C -6 -6 -8 -9 12 -8 -9 -8 -8 -7 -7 -8 -7 -5 -8 -5 -6 -8 <span style="color:OrangeRed">'''-5'''</span> -6 0 1 0 0 94 0 0 0 0 0 0 0 0 1 0 1 0 0 <span style="color:OrangeRed">'''1'''</span> 0 4.31 inf
 
330 R -5 5 -4 -6 1 -1 -5 -6 2 -5 -5 3 <span style="color:OrangeRed">'''-3'''</span> 0 -5 -4 -5 8 4 -6 1 28 1 0 3 2 0 0 4 1 1 18 <span style="color:OrangeRed">'''0'''</span> 3 1 1 0 23 14 0 1.41 inf
  
330 R -5 5 -4 -6 1 -1 -5 -6 2 -5 -5 3 <span style="color:OrangeRed">'''-3'''</span> 0 -5 -4 -5 8 4 -6 1 28 1 0 3 2 0 0 4 1 1 18 <span style="color:OrangeRed">'''0'''</span> 3 1 1 0 23 14 0 1.41 inf
−
The mutations are marked in purple.
 +
The mutations are marked in red.
   
−
For the creation of the MSA, we first searched for homologous mammalian sequences with the NCBI BlastP online tool in the UniprotKB/SwissProt and restricted the organisms to Mammalia. The E-value cutoff was set to 0.1 and Psi-Blast was used as Algorithm. The maximum target sequences threshold was set to 100, bacause we only wanted to get the close homologs, but we did a second run with a threshold of 20000 to get also remote homologous seuences. All other parameters were left as default. We performed two iterations and then downloaded all matched sequences in fasta format.
 +
For the creation of the MSA, we first searched for homologous mammalian sequences with the NCBI BlastP online tool in the UniprotKB/SwissProt and restricted the organisms to Mammalia. The E-value cutoff was set to 0.1 and Psi-Blast was used as Algorithm. The maximum target sequences threshold was set to 100, because we only wanted to get the close homologs, but we did a second run with a threshold of 20000 to get also remote homologous seuqences. All other parameters were left as default. We performed two iterations and then downloaded all matched sequences in fasta format.
−
Those sequences were then used as input for [http://www.ebi.ac.uk/Tools/services/web_clustalw2/toolform.ebi ClustalW]. The MSA was gerenerated with default parameters. Jalview was then used to save the alignment in fasta format.
 +
Those sequences were then used as input for [http://www.ebi.ac.uk/Tools/services/web_clustalw2/toolform.ebi ClustalW]. The MSA was generated with default parameters. Jalview was then used to save the alignment in fasta format.
 
The commandline version of Blast 2.2.25+ was then used to generate a PSSM for the HFE protein Q30201 from the MSA:
  
The commandline version of Blast 2.2.25+ was then used to generate a PSSM for the HFE protein Q30201 from the MSA:
 
psiblast -subject Q30201.fasta -in_msa alignment.fa -out_ascii_pssm pssm.txt
  
psiblast -subject Q30201.fasta -in_msa alignment.fa -out_ascii_pssm pssm.txt
Line 178: Line 178:
   
 
== MutationTaster ==
 
== MutationTaster ==
−
MutationTaster was also executed from the web interface.
+
MutationTaster was also executed from the web interface. <br>
−
Gene: HFE
 +
Gene: HFE<br>
 
Transcript: ENST00000357618 (protein_coding, 5286 bases) NM_000410
 
Transcript: ENST00000357618 (protein_coding, 5286 bases) NM_000410
   
Line 223: Line 223:
 
Prediction
 
Prediction
 
polymorphism
  
polymorphism
−
Model: simple_aae, prob: 0.999999996637944 (explain)
 +
Model: simple_aae, prob: 0.999999996637944 (explain)
 
Summary
 
Summary
 
amino acid sequence changed
  
amino acid sequence changed
Line 233: Line 233:
 
Prediction
  
Prediction
 
polymorphism
  
polymorphism
−
Model: simple_aae, prob: 0.707489599782817 (explain)
 +
Model: simple_aae, prob: 0.707489599782817 (explain)
−
Summary
+
Summary
 
amino acid sequence changed
  
amino acid sequence changed
 
known disease mutation at this position (HGMD CM004106)
  
known disease mutation at this position (HGMD CM004106)
Line 244: Line 244:
 
Prediction
  
Prediction
 
disease causing
  
disease causing
−
Model: simple_aae, prob: 0.999999979100498 (explain)
 +
Model: simple_aae, prob: 0.999999979100498 (explain)
−
Summary
+
Summary
 
amino acid sequence changed
  
amino acid sequence changed
 
known disease mutation at this position (HGMD CM081301)
  
known disease mutation at this position (HGMD CM081301)
Line 253: Line 253:
 
Prediction
  
Prediction
 
polymorphism
  
polymorphism
−
Model: simple_aae, prob: 0.999999999993365 (explain)
 +
Model: simple_aae, prob: 0.999999999993365 (explain)
 
Summary
 
Summary
 
amino acid sequence changed
  
amino acid sequence changed
Line 263: Line 263:
 
Prediction
  
Prediction
 
disease causing
  
disease causing
−
Model: simple_aae, prob: 0.999999999736277 (classification due to ClinVar, real probability is shown anyway) (explain)
 +
Model: simple_aae, prob: 0.999999999736277 (classification due to ClinVar, real probability is shown anyway) (explain)
 
Summary
 
Summary
 
amino acid sequence changed
  
amino acid sequence changed
Line 275: Line 275:
 
Prediction
  
Prediction
 
disease causing
  
disease causing
−
Model: simple_aae, prob: 8.23173030693237e-06 (classification due to ClinVar, real probability is shown anyway) (explain)
 +
Model: simple_aae, prob: 8.23173030693237e-06 (classification due to ClinVar, real probability is shown anyway) (explain)
 
Summary
 
Summary
 
amino acid sequence changed
  
amino acid sequence changed
Line 283: Line 283:
 
splice site changes
  
splice site changes
   
−
== SNAP ==
 +
== SNAP2 ==
  +
  +
SNAP2 was executed from the command line on the biolab computers. first, we created a ~/.snap2rc file that contains the paths to several databases that are needed for snap2:
  +
<source lang="bash">
  +
[snap2]
  +
# snapfun_utildir=path - path to package utilities, default: /usr/share/snap2
  +
snap2dir=/usr/share/snap2
  +
#use snap cache [0|1], default: 0
  +
use_snap_cache=0
  +
#snap cache fetch executable
  +
snapc_fetch=/usr/bin/snapc_fetch
  +
#snap cache store executable
  +
snapc_store=/usr/bin/snapc_store
  +
#snap cache root - overrides snap-cache-mgr configuration
  +
snap_cache_root=
  +
# blastpgp_processors
  +
blastpgp_processors=1
  +
#use predictprotein cache, default: 0
  +
use_pp_cache=0
  +
#predictprotein executable
  +
pp_exe=/usr/bin/predictprotein
  +
#sift executable
  +
sift_exe=/usr/bin/sift_for_submitting_fasta_seq.csh
  +
#reprof executable
  +
reprof_exe=/usr/bin/reprof
  +
#blast executable
  +
blast_exe=/usr/bin/blastpgp
  +
  +
[data]
  +
# swiss_dat=path - location of UniProt/Swiss-Prot dat file
  +
swiss_dat=/mnt/project/pracstrucfunc13/data/swissprot/20120501/uniprot_sprot.dat
  +
# db_swiss=path - path to ID index of Swiss-Prot dat file (generated by /usr/share/librg-utils-perl/dbSwiss.pl)
  +
db_swiss=/mnt/project/pracstrucfunc13/data/swissprot/20120501/dbswiss
  +
# PHAT substitution matrix
  +
phat_matrix=/usr/share/snap2/phat.txt
  +
  +
[blast]
  +
# big80=path - path to redundancy reduced database (UniProtKB 80 or equivalent)
  +
big80=/mnt/project/pracstrucfunc13/data/big/big_80
  +
# swiss=path - path to SwissProt database
  +
swiss=/mnt/project/pracstrucfunc13/data/swissprot/uniprot_sprot
  +
</source>
  +
SNAP2 was then excuted with the following command:
  +
snapfun -i Q30201.fasta -m mutations.txt -o results.txt
  +
  +
where mutations.txt is a file that contains the mutations in the one letter code.
  +
The results are the following:
  +
nsSNP Prediction Reliability Expected Accuracy
  +
V53M Neutral 0 53%
  +
H63D Non-neutral 2 63%
  +
R67H Non-neutral 4 71%
  +
M97I Neutral 7 87%
  +
N130S Neutral 3 66%
  +
E168Q Neutral 7 87%
  +
L183P Non-neutral 8 91%
  +
T217I Neutral 0 53%
  +
C282Y Non-neutral 7 85%
  +
R330M Non-neutral 7 85%

Latest revision as of 14:43, 26 September 2013

Mutation selection

10 mutations were randomly selected from HGMD and dbSNP.

Mutation analysis

The description of the physicochemical properties is based on the entry for amino acids in wikipedia.

The mutations were visualized with Pymol. Because the pdb structure 1A6Z starts at position 22 in the reference structure, we subtracted 22 from the codon position to get the position of the mutation in the structure. The mutatins were done following the description in Use PyMOL for this. We did mutations for the first 9 SNPs but the last one (Arg330Met) could not be visualized, because the pdb structure is shorter than the reference sequence and only contains the residues 22 to 297. The rotamer for each mutated residue was selected based on the orientation and the size and color of the discs. We selected the rotamers with the least and smallest red discs if there was none without. For residues that are located on the border of the protein, we also tried to find rotamers that are not pointed into the solvent.

The secondary structure of the location of the mutation was taken from the DSSP assignment of the 1A6Z_A structure.

The BLOSUM62 matrix was taken from BLOSUM62 and the PAM250 matrix from PAM250.

PSSM fom PsiBlast with 5 iterations and default parameters using the /mnt/project/pracstrucfunc13/data/big/big_80 database: 

blastpgp -i /mnt/home/student/betza/data/hfe.fasta -d /mnt/project/pracstrucfunc13/data/big/big_80 -j 5 -o /mnt/home/student/betza/task8/psiblast/iter5_big80.results
 -Q /mnt/home/student/betza/task8/psiblast/iter5_big80.pssm -C /mnt/home/student/betza/task8/psiblast/iter5_big80.chk

The resulting PSSM is the following (only the 10 mutation positions are shown): 

Last position-specific scoring matrix computed, weighted observed percentages rounded down, information per position, and relative weight of gapless real matches to pseudocounts
          A  R  N  D  C  Q  E  G  H  I  L  K  M  F  P  S  T  W  Y  V    A   R   N   D   C   Q   E   G   H   I   L   K   M   F   P   S   T   W   Y   V	
  53 V   -3 -6 -7 -7 -3 -6 -6 -7 -2  0  4 -6  1 -1 -6 -4 -5 -6 -5  6    2   0   0   0   1   0   0   0   1   4  37   0   3   2   0   1   0   0   0  47  1.32 inf
  63 H   -3 -1  2 -3  1 -5 -4 -1  0 -4 -5  0 -3 -7 -3  6 -1 -2 -7 -5    1   3   8   2   3   0   1   5   2   1   1   5   1   0   1  62   4   1   0   1  1.26 inf
  67 R   -3  5  0 -3 -3  2  0 -3 -1 -2 -3  3 -2 -6 -2  0  2 -1 -5 -4    2  33   4   1   1   7   5   2   1   2   2  16   1   0   2   5  13   1   0   1  0.69 inf
  97 M   -1  0  0  0  2  0  0 -1  2  0 -1  0  2  1  0 -1  0  4  1 -1    5   5   5   5   5   5   5   5   5   5   5   5   5   5   5   5   5   5   5   5  0.09 inf
 130 N   -4  0  2  0 -2 -2 -1  5 -2 -2 -4  0 -1 -4 -4 -1  2 -1 -6 -3    1   5  10   4   1   2   4  40   1   3   2   5   1   1   1   5  11   1   0   2  0.67 inf
 168 E   -3  1 -1 -2 -3 -1  0 -3 -2  0  1  4  0  0 -3 -3 -3  0  2  0    2   6   4   2   1   2   7   2   1   6  14  22   2   4   2   3   1   1   7   6  0.29 inf
 183 L   -4 -3 -3 -4 -2 -4 -4 -5 -1  1  5 -2  2 -1 -4 -3 -3 -1  1  2    2   2   1   1   1   1   1   1   1   6  45   3   4   3   1   2   2   1   5  13  0.68 inf
 217 T    0 -3 -2  0 -5 -1 -1  1 -1 -3  1 -2 -1 -3  3  3  0 -6 -1 -1    8   1   1   4   0   3   3  10   1   1  12   2   1   1  15  22   6   0   2   5  0.30 inf
 282 C   -6 -6 -8 -9 12 -8 -9 -8 -8 -7 -7 -8 -7 -5 -8 -5 -6 -8 -5 -6    0   1   0   0  94   0   0   0   0   0   0   0   0   1   0   1   0   0   1   0  4.31 inf
 330 R   -5  5 -4 -6  1 -1 -5 -6  2 -5 -5  3 -3  0 -5 -4 -5  8  4 -6    1  28   1   0   3   2   0   0   4   1   1  18   0   3   1   1   0  23  14   0  1.41 inf

The mutations are marked in red.

For the creation of the MSA, we first searched for homologous mammalian sequences with the NCBI BlastP online tool in the UniprotKB/SwissProt and restricted the organisms to Mammalia. The E-value cutoff was set to 0.1 and Psi-Blast was used as Algorithm. The maximum target sequences threshold was set to 100, because we only wanted to get the close homologs, but we did a second run with a threshold of 20000 to get also remote homologous seuqences. All other parameters were left as default. We performed two iterations and then downloaded all matched sequences in fasta format. Those sequences were then used as input for ClustalW. The MSA was generated with default parameters. Jalview was then used to save the alignment in fasta format. The commandline version of Blast 2.2.25+ was then used to generate a PSSM for the HFE protein Q30201 from the MSA: 

psiblast -subject Q30201.fasta -in_msa alignment.fa -out_ascii_pssm pssm.txt

The resulting PSSM for the first 100 sequences is the following: 

Last position-specific scoring matrix computed, weighted observed percentages rounded down, information per position, and relative weight of gapless real matches to pseudocounts

          A  R  N  D  C  Q  E  G  H  I  L  K  M  F  P  S  T  W  Y  V   A   R   N   D   C   Q   E   G   H   I   L   K   M   F   P   S   T   W   Y   V
  53 V    0 -3 -3 -4 -1 -3 -3 -4 -3  2  1 -3  1 -1 -3 -2  0 -3 -1  5    0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0 100  0.47 inf
  63 H    0 -1  0 -1 -2  0  0 -1  5 -3 -3 -1 -2 -2 -1  4  1 -3 -1 -2    0   0   0   0   0   0   0   0  26   0   0   0   0   0   0  74   0   0   0   0  0.47 inf
  67 R   -2  6 -1 -2 -4  1  0 -3 -1 -3 -3  2 -2 -3 -2 -1 -1 -3 -2 -3    0 100   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0  0.91 inf
  97 M    0 -2 -1 -2 -1 -1 -1 -2 -2  0  0 -1  3 -2 -2  1  4  1 -2  0    1   0   0   0   0   0   0   0   0   4   0   0  23   0   0   7  62   2   0   0  0.37 inf
 130 N   -2  6  3 -1 -4  0 -1 -2  0 -4 -3  1 -2 -3 -1 -1 -1 -3 -2 -3    0  75  23   0   0   0   0   0   0   0   0   0   0   0   2   0   0   0   0   0  0.73 inf
 168 E   -1  1 -1  0 -4  1  3 -2 -1 -3 -3  5 -2 -4 -1 -1 -1 -3 -2 -3    0   0   0   0   0   0  23   0   0   0   0  77   0   0   0   0   0   0   0   0  0.62 inf
 183 L   -2 -2 -4 -4 -1 -2 -3 -4 -3  1  4 -3  2  0 -3 -3 -1 -2 -1  1    0   0   0   0   0   0   0   0   0   0  96   0   4   0   0   0   0   0   0   0  0.51 inf
 217 T    1  2 -2 -2 -1 -1 -1 -2 -2  2  0  0  0 -2 -2  0  1 -3 -2  2   15  25   0   0   0   0   0   0   0  12   0   0   0   0   0   0  12   0   0  35  0.15 inf
 282 C   -1 -4 -3 -4  9 -3 -4 -3 -3 -2 -2 -3 -2 -3 -3 -1 -1 -3 -3 -1    0   0   0   0 100   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0  1.41 inf
 330 R   -1  3 -2 -3 -2  0 -2 -3 -2  1  1  0  5 -1 -3 -2 -1 -2 -2  1    0  30   0   0   0   0   0   0   0  14   6   0  47   0   0   0   0   0   0   3  0.31 inf

This is the PSSM for all homologous sequences: 

Last position-specific scoring matrix computed, weighted observed percentages rounded down, information per position, and relative weight of gapless real matches to pseudocounts
          A  R  N  D  C  Q  E  G  H  I  L  K  M  F  P  S  T  W  Y  V   A   R   N   D   C   Q   E   G   H   I   L   K   M   F   P   S   T   W   Y   V
  53 V    0 -2  1 -2 -3  1  0  0  2  0  0  0 -1  1 -3  0  0  4  1  0    7   0   7   0   0   7   7   7   7   7   7   7   0   7   0   7   7   7   7   7  0.11 inf
  63 H    0  1  0 -1  0  1  0  1  0 -2  0  1 -2 -1  0  1  0 -3 -2 -1    6   7   5   3   3   6   5  10   3   0  10  14   0   3   3  15   4   0   0   4  0.09 inf
  67 R   -2  3 -1 -1 -4  1 -1 -4  1  1 -1  0  1 -3 -1  0  0  4  0  0    2  20   3   3   0   6   2   0   4   9   6   5   4   0   3   7   8   8   4   6  0.26 inf
  97 M   -1 -1 -1 -1  1 -1 -1 -1 -3  1  0 -1  2  0 -1  0  1  2  1  2    4   3   3   4   3   3   3   3   0   7  10   3   7   3   3   5  12   3   5  16  0.10 inf
 130 N   -1  2  2  2 -4  0  1  2  1 -3 -1  0  0 -3  0 -1 -1  0 -3 -2    3  12  12  13   0   2   9  17   4   0   7   5   2   0   5   2   3   2   0   2  0.23 inf
 168 E   -1  0  0  1  2  1  0 -2 -3  1  2  2 -1  1 -3 -2 -1 -3 -2 -2    3   3   5   7   5   5   4   2   0   9  25  19   0   8   0   0   3   0   0   0  0.18 inf
 183 L   -1 -4 -1 -4  3 -1 -4  0 -4  1  4 -4  2 -3  0  0 -3 -4 -4 -1    4   0   3   0   6   3   0   9   0   7  47   0   6   0   4   9   0   0   0   2  0.50 inf
 217 T    1  0  0  0 -2  0  0  0 -2  0  0 -1  1  0  0  0  0 -3 -2  0   15   7   4   5   0   5   5   9   0   6  12   0   6   5   5   4   5   0   0   7  0.04 inf
 282 C    0 -4  1 -4  8  1 -4 -4 -4 -3 -1 -4 -3 -3 -4  1 -3 -4  1  1    7   0   7   0  46   8   0   0   0   0   7   0   0   0   0  10   0   0   6  10  1.16 inf
 330 R    0 -1 -2 -3  2 -1 -3  3 -1 -1  0 -1  0 -1  1 -1  0  4  0  0    6   4   1   0   6   2   0  25   1   2   8   5   3   2   6   2   6   7   3   9  0.22 inf

SIFT

SIFT was executed from the web interface with default parameters

Val53Met 

Substitution at pos 53 from V to M is predicted to AFFECT PROTEIN FUNCTION with a score of 0.00.
   Median sequence conservation: 3.03
   Sequences represented at this position:396

His63Asp 

Substitution at pos 63 from H to D is predicted to AFFECT PROTEIN FUNCTION with a score of 0.00.
   Median sequence conservation: 3.03
   Sequences represented at this position:396

Arg67His 

Substitution at pos 67 from R to H is predicted to AFFECT PROTEIN FUNCTION with a score of 0.00.
   Median sequence conservation: 3.03
   Sequences represented at this position:396

Met97Ile 

Substitution at pos 97 from M to I is predicted to be TOLERATED with a score of 0.53.
   Median sequence conservation: 3.03
   Sequences represented at this position:395

Asn130Ser 

Substitution at pos 130 from N to S is predicted to AFFECT PROTEIN FUNCTION with a score of 0.02.
   Median sequence conservation: 3.03
   Sequences represented at this position:396

Glu168Gln 

Substitution at pos 168 from E to Q is predicted to AFFECT PROTEIN FUNCTION with a score of 0.01.
   Median sequence conservation: 3.03
   Sequences represented at this position:396

Leu183Pro 

Substitution at pos 183 from L to P is predicted to AFFECT PROTEIN FUNCTION with a score of 0.00.
   Median sequence conservation: 3.03
   Sequences represented at this position:396

Thr217Ile 

Substitution at pos 217 from T to I is predicted to be TOLERATED with a score of 0.92.
   Median sequence conservation: 3.03
   Sequences represented at this position:396

Cys282Tyr 

Substitution at pos 282 from C to Y is predicted to AFFECT PROTEIN FUNCTION with a score of 0.00.
   Median sequence conservation: 3.03
   Sequences represented at this position:396

Arg330Met 

Substitution at pos 330 from R to M is predicted to be TOLERATED with a score of 0.11.
   Median sequence conservation: 3.01
   Sequences represented at this position:255

Polyphen2

Polyphen was executed from the web interface using default parameters

Val53Met 

HumDiv
This mutation is predicted to be probably damaging with a score of 0.998 (sensitivity: 0.27; specificity: 0.99)
HumVar
This mutation is predicted to be possibly damaging with a score of 0.841 (sensitivity: 0.73; specificity: 0.88)

His63Asp 

HumDiv
This mutation is predicted to be benign with a score of 0.142 (sensitivity: 0.92; specificity: 0.86)
HumVar
This mutation is predicted to be benign with a score of 0.161 (sensitivity: 0.89; specificity: 0.72)

Arg67His 

HumDiv
This mutation is predicted to be benign with a score of 0.145 (sensitivity: 0.92; specificity: 0.86)
HumVar
This mutation is predicted to be benign with a score of 0.031 (sensitivity: 0.94; specificity: 0.59)

Met97Ile 

HumDiv
This mutation is predicted to be possibly damaging with a score of 0.575 (sensitivity: 0.88; specificity: 0.91)
HumVar
This mutation is predicted to be benign with a score of 0.114 (sensitivity: 0.90; specificity: 0.69)

Asn130Ser 

HumDiv
This mutation is predicted to be possibly damaging with a score of 0.883 (sensitivity: 0.82; specificity: 0.94)
HumVar
This mutation is predicted to be benign with a score of 0.282 (sensitivity: 0.87; specificity: 0.76)

Glu168Gln 

HumDiv
This mutation is predicted to be probably damaging with a score of 1.000 (sensitivity: 0.00; specificity: 1.00)
HumVar
This mutation is predicted to be probably damaging with a score of 0.980 (sensitivity: 0.57; specificity: 0.94)

Leu183Pro 

HumDiv
This mutation is predicted to be probably damaging with a score of 1.000 (sensitivity: 0.00; specificity: 1.00)
HumVar
This mutation is predicted to be probably damaging with a score of 1.000 (sensitivity: 0.00; specificity: 1.00)

Thr217Ile 

HumDiv
This mutation is predicted to be benign with a score of 0.118 (sensitivity: 0.93; specificity: 0.86)
HumVar
This mutation is predicted to be benign with a score of 0.097 (sensitivity: 0.91; specificity: 0.68)

Cys282Tyr 

HumDiv
This mutation is predicted to be probably damaging with a score of 0.961 (sensitivity: 0.78; specificity: 0.95)
HumVar
This mutation is predicted to be possibly damaging with a score of 0.667 (sensitivity: 0.79; specificity: 0.84)

Arg330Met 

HumDiv
This mutation is predicted to be probably damaging with a score of 0.997 (sensitivity: 0.41; specificity: 0.98)
HumVar
This mutation is predicted to be possibly damaging with a score of 0.781 (sensitivity: 0.76; specificity: 0.87)

MutationTaster

MutationTaster was also executed from the web interface.
Gene: HFE
Transcript: ENST00000357618 (protein_coding, 5286 bases) NM_000410

Val53Met 

Prediction 
disease causing
Model: simple_aae, prob: 0.974610381496817      (explain)
Summary 	
   amino acid sequence changed
   known disease mutation at this position (HGMD CM994469)
   protein features (might be) affected
   splice site changes

His63Asp 

Prediction	
disease causing 
Model: simple_aae, prob: 0.974610381496817      (explain)
Summary 	
   amino acid sequence changed
   known disease mutation at this position (HGMD CM994469)
   protein features (might be) affected
   splice site changes

Arg67His 

Prediction
polymorphism
Model: simple_aae, prob: 0.999999997930159      (explain)
Summary 	
   amino acid sequence changed
   listed as SNP
   protein features (might be) affected
   splice site changes

Met97Ile 

Prediction	
disease causing
Model: simple_aae, prob: 0.943409356836766      (explain)
Summary 	
   amino acid sequence changed
   protein features (might be) affected

Asn130Ser 

Prediction	
polymorphism
Model: simple_aae, prob: 0.999999996637944      (explain)
Summary 	
   amino acid sequence changed
   listed as SNP
   protein features (might be) affected
   splice site changes

Glu168Gln 

Prediction
polymorphism
Model: simple_aae, prob: 0.707489599782817      (explain)
Summary 	
   amino acid sequence changed
   known disease mutation at this position (HGMD CM004106)
   known disease mutation at this position (HGMD CM004810)
   protein features (might be) affected
   splice site changes

Leu183Pro 

Prediction
disease causing
Model: simple_aae, prob: 0.999999979100498      (explain)
Summary 	
   amino acid sequence changed
   known disease mutation at this position (HGMD CM081301)
   protein features (might be) affected

Thr217Ile 

Prediction
polymorphism
Model: simple_aae, prob: 0.999999999993365      (explain)
Summary 	
   amino acid sequence changed
   listed as SNP
   protein features (might be) affected
   splice site changes

Cys282Tyr 

Prediction
disease causing
Model: simple_aae, prob: 0.999999999736277 (classification due to ClinVar, real probability is shown anyway)      (explain)
Summary 	
   amino acid sequence changed
   heterozygous in TGP
   known disease mutation at this position (HGMD CM004391)
   known disease mutation at this position (HGMD CM960828)
   known disease mutation: rs1800562 (pathogenic)
   protein features (might be) affected

Arg330Met 

Prediction
disease causing
Model: simple_aae, prob: 8.23173030693237e-06 (classification due to ClinVar, real probability is shown anyway)      (explain)
Summary 	
   amino acid sequence changed
   known disease mutation at this position (HGMD CM990722)
   known disease mutation: rs111033558 (pathogenic)
   protein features (might be) affected
   splice site changes

SNAP2

SNAP2 was executed from the command line on the biolab computers. first, we created a ~/.snap2rc file that contains the paths to several databases that are needed for snap2: <source lang="bash"> [snap2]

  1. snapfun_utildir=path - path to package utilities, default: /usr/share/snap2

snap2dir=/usr/share/snap2

  1. use snap cache [0|1], default: 0

use_snap_cache=0

  1. snap cache fetch executable

snapc_fetch=/usr/bin/snapc_fetch

  1. snap cache store executable

snapc_store=/usr/bin/snapc_store

  1. snap cache root - overrides snap-cache-mgr configuration

snap_cache_root=

  1. blastpgp_processors

blastpgp_processors=1

  1. use predictprotein cache, default: 0

use_pp_cache=0

  1. predictprotein executable

pp_exe=/usr/bin/predictprotein

  1. sift executable

sift_exe=/usr/bin/sift_for_submitting_fasta_seq.csh

  1. reprof executable

reprof_exe=/usr/bin/reprof

  1. blast executable

blast_exe=/usr/bin/blastpgp

[data]

  1. swiss_dat=path - location of UniProt/Swiss-Prot dat file

swiss_dat=/mnt/project/pracstrucfunc13/data/swissprot/20120501/uniprot_sprot.dat

  1. db_swiss=path - path to ID index of Swiss-Prot dat file (generated by /usr/share/librg-utils-perl/dbSwiss.pl)

db_swiss=/mnt/project/pracstrucfunc13/data/swissprot/20120501/dbswiss

  1. PHAT substitution matrix

phat_matrix=/usr/share/snap2/phat.txt

[blast]

  1. big80=path - path to redundancy reduced database (UniProtKB 80 or equivalent)

big80=/mnt/project/pracstrucfunc13/data/big/big_80

  1. swiss=path - path to SwissProt database

swiss=/mnt/project/pracstrucfunc13/data/swissprot/uniprot_sprot </source> SNAP2 was then excuted with the following command: 

snapfun -i Q30201.fasta -m mutations.txt -o results.txt

where mutations.txt is a file that contains the mutations in the one letter code. The results are the following: 

nsSNP  Prediction  Reliability Expected Accuracy 
V53M    Neutral         0       53%
H63D    Non-neutral     2       63%
R67H    Non-neutral     4       71%
M97I    Neutral         7       87%
N130S   Neutral         3       66%
E168Q   Neutral         7       87%
L183P   Non-neutral     8       91%
T217I   Neutral         0       53%
C282Y   Non-neutral     7       85%
R330M   Non-neutral     7       85%
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