However from the electrophoregram the peak height of the shorter allele is way smaller than that of the longer allele whilst it is supposed to be the opposite.Any explanations for this?
Hi. I hope getting your question right. We need to be cautious while doing the scoring. Some peaks could be due to insufficient probe specificity of a marker. You could cross check among all the samples that you have genotyped to identify the consistent peaks. Besides, you also could include a peak height ratio as threshold, such as >or= 1:2 while scoring. Hope this will help.
Some variation in peak heights is not unusual. What is probably a more important factor is the peak spacing. Does the distance between peaks match the repeat motif that you expect for that micros? If you expect repeats of 3 bases then you should find spacing that is roughly a factor of three.
Hi. You are right, the peak height of the shorter allele is way smaller than that of the longer allele, but it is not allways the case. Markes hardly ever show a higher peak in longer allele. You have to check the morphology of your alleles analysing a large number of samples. If you have the possibility, it can help you to analyze this marker in a kown family so you can check the alleles morphology in parents.
The second reason could be that the first peak that you see is a inespecific amplification. Again you can check this options analysing the highest number of samples as possible and detecting the true heterocigocity in individuals that show three alleles.
Good luck!
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