Hi all, I am still a beginner in research. I have recently found an insertion mutation from seqeuncing in a few patient's samples.. But, how do I validate this mutation as true mutation or it could be just sequencing error? Because the occurance rate isnt very high. thank you.
Posted 01 November 2013 - 03:53 AM
the most obvious thing to do would be to repeat the sequencing with the same and fresh samples from the same subjects.
after confirming that there is indeed a mutation you should determine if there is a change in the amino acid sequence.
if so then you should determine if the change affects the protein's conformation, activity, etc.
it may also be a normal variation (eg blue eyes).
Edited by mdfenko, 01 November 2013 - 03:59 AM.
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Posted 01 November 2013 - 04:11 AM
Repeat the PCR and then sequencing. If there was an error, it most likely originated in the PCR reaction. Also to confirm, especially insertions (and especially not 100 %, heterozygous, partial,...) always sequence from both ends.
Insertion makes your sequence shift, on the background of the normal sequence. Sequencing from the other end helps you determine better the start and end of the insertions, and that it is indeed an insertion and not a sequence gone bad near the end.
If the sequencing read can't be done from both sides (too near the oposite end) design new sequencing/PCR primers to check.
Insertions are in general unlikely to be normal variations if they are within coding sequence, as any non triplet insertion will cause a frameshift and seriously affect the protein.
If yoy have low prevalence of the mutation (it is acquired) you can design then sequence specific more sensitive methods, allele specific PCR or so. But that would have it's place after you confirm with repeted sequencing.
Also it should be noted, that insertion that appears near a polynucletide stretch (like seven As in a row, and you get eight one) or near a clear repetitive sequence could be just due to a shift of polymerase on the template, that happens a lot, and I would be really carefull to validate that as an insertion.
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