I have found homozygous mutation in a certain gene using NGS (c.436G>C) and when I tried sanger confirmation and segregation within the family. I got these two chromatograms; one for the patient and the other for both father and mother and also normal sibling. I wonder how I can interpret this chromatogram as it seems that there is an insertion of C in the parents and sibling chromatogram while the patient is clearly substitution of G with C.
If the first chromatogram is representative of the parents and the sibling it would appears they are all heterozygous for this position, while the patient is homozygous G>C. The blue (i.e. C) peak in the first chromatogram is roughly half the size of the peak in the second, while also being accompanied by a second black (i.e. G) peak. Sanger sequencing can have problems with the resolution, which would explain why these two peaks, though on the same genetic position, aren't overlapping exactly.
There is quality problem in chromatogram-1. G(black) at 3rd position have residual peak, which create noise and interpreted as extra G at 5th position in chromatogram.
I think both the chromatogram have same sequence.
The first chromatogram has a quality problem... The insertion that you are seeing is most likely background noise of the sequencing. You need to resubmit the sample for Sanger sequencing and be very sure that the sequence has been cleaned well... I assume that you are suing exoSAP or other enzymatic/physical method to clean up your sequences.
Dear colleagues, As Patrick said this is the chromatogram of expected heterozygous individuals and that's why as I think the peaks of G and C in this chromatogram is half the peak of homozygous C in chromatogram 2. I don't think it is a quality problem. I do use ExoSAP to clean up PCR products. what do you think about this interpretation?
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