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Mutation Annotator

Purpose of this program

This program is designed to help annotate sequence variants in a cDNA sequence. The program was written to help me create/test another more complex program that automatically finds and annotates mutations in trace files.

insilicase icon This program can be downloaded here.

To use the program follow these steps:

  • Press the 'Sequence' button and import the file containing the cDNA sequence. The sequence will then appear in the panel, the upper line is the original sequence and the lower sequence is the edited sequence.
  • Select the start of the open reading frame from the list box at the bottom left of the form or enter it into the text box next to the 'Start codon' label. The ORFs amino acid sequence should now be visible. (Use the scroll bar to navigate along the sequence.)
  • Choose the nucleotide you wish to mutate by clicking on the nucleotide. A box will appear around the selected residue.
    1. To change a single base press the 'A', 'C', 'G' or 'T' button.
    2. To delete nucleotides press the 'Del' button and enter the number of bases to remove.
    3. To insert nucleotides press the 'In' button and enter the sequence to insert.
    4. To create an InDel press then 'InDel' button and first enter the number of bases to delete and then enter the sequence to insert
  • When a mutation is entered, the mutation is 'Described' in the label above the row of buttons and the positions where the original and variant sequences differ are coloured in red. If the sequence change affects the amino acid sequence, click on the amino acid residue and the protein variant will be annotated. An indication of the affect of the AA change is given as the PAM value score for the wildtype and variant sequence (See Important notes for explanation). To see the whole label, you my have to elongate the window.
  • To reset the mutant sequence press the 'Reset' button.

Important note

If a number of sequence variants are close together (i.e aCgTg > aAgAg) it should not be written as g.2C>A and g.4T>A but combined to be g.2_4delCGTinsAGA.

Also the program will not scan around a mutant to combine variations therefore if you create an Insert, deletion or InDel and then select a different residue and then move back, the variant will not be annotated correctly. To get the correct nomenclature press the 'Reset' button and then re-enter the variant.

Pam scores

These values are used to create sequence alignments and are generated by comparing evolutionary linked sequences. For example
AA = p.75W>G Pam score = 17 > -7
The wild type W residue is usually highly conserved and so scores 17. A change to G is rarely seen and so scores -7. This means that a change from 17 to -7 is very significant where as
AA = p.91E>D Pam score = 4 > 3
E is not a highly conserved AA (in evolution) and so only scores 4, similarly a change from E to D is common and so scores 3. This may underestimate a changes true importance if it is part of a motif or conserved structure.

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