mutation calling with a reference sequence

[lno]

Hi,
Ive had the following problem and dont know if this can be solved using codoncode. I have a reference sequence for an exon and use this to align traces of 40 samples into a contig. On this contig I perform mutation calling (find mutations). In my version of codoncode (5.0.2), the mutations are not called outside the reference sequence. I would like to have both, know where the reference sequence is (grey or not grey bar on sequences) and have mutation calling. Right now I cant use the reference sequence for fear of losing intronic but potentially important variants. Is there a way that codoncode does call mutations outside the refsequence?
regards,
Alex

[Peter Richterich]

Alex, alignments to a reference sequence are limited to the regions that align to the reference, and only these regions are analyzed when searching for mutations. If your reference sequence includes only the exon, then only the exon region will be analyzed.

If you want to look for mutations in intron sequences that border your exon, you need to make a reference sequence that also includes the intron sequences. Perhaps the easiest way to do this is to start with a Genbank sequence that contains the entire gene and the coding sequence annotation. You can then either edit this reference sequence to include only the parts of the introns you are resequencing, or change the "Alignment" preferences in CodonCode Aligner to clip the alignment to the aligned region or exon.

If you do not have a Genbank sequence with annotations, or need to create it yourself for some reason, you will need to add the proper "codingSequence" tag to the exon region, and probably also a "codonStart" tag. Please check the online help or http://www.codoncode.com/aligner/quicktour/codingregion.htm for more information about adding these tags.