Hello,
It is my understanding that you can detect heterozygosity by looking for double peaks (see attached image) in sanger sequencing chromatograms.
I have obtained sanger sequencing chromatograms from two genomic regions from the same individual (grasshoppers in this case) using two different sets of primers.
In one chromatogram (lets say genomic region A), I see several double-peaks which I take as signs of heterozygosity in this female since this region is known to contain X-linked polymorphism and this individual happens to have an X chromosome from each of the two populations.
In the other chromatogram (lets say genomic region B), there are no double peaks. This genomic region does contain a SNP.
What does this mean?
Wouldn't it be correct to expect double peaks in the genomic region B too?
Thank you very much.
All of your peaks look very small (scale is missing in pic), if I would be you, I would have check the overall quality of the sequence before going into any interpretation of the chromatograms.
Hi Sachin,
peaks like these ones are referred to heterozygous SNP, indeed. the problem is the quality of the sequence, peaks are very small as Abhijeet Singh, and the background is very high. I would review your protocols, see if primers are specific (use primer bast at NCBI or in silica PCR at UCSC), obtain a really fine band on agarose gel without smears, and follow the manufacturer's protocol for sanger purification.
fred
Hi Sachin,
This shouldn't happen normally, except for the beginning and end of your sequencing chromatogram. This background noise is probably due to the poor quality of the sequencing. Insufficient amount of DNA template or primer and and presence of inhibitory contaminants such as phenol and EDTA in your samples could lead to low peaks.
Regards,
Parham
Hi Pathan;
as you can see noise is all long of the sequence. no way to say if it's noise or a real SNP (or there is an other one at G169).
fred
Frederic Lepretre
Parham Habibzadeh
Abhijeet Singh
Thank you very much for your help.
Hi. I would like to know which software you have used to detect heterozygosity in sanger sequencing chromatograms.
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