Hi colleagues;
Can any one help with attached image of Sanger sequencing, where Alignment result shows SNP cant be confirmed by the other primer ( 2 nucleotides difference). One primer shows SNP and other confirmatory primer shows wild type exon. I am confused with possibility of getting such weird result? Have any one ever come across situation like that?
Many thanks
Can you attach the original .abi sequencing files for this result please?
thanks very much
ps you have highlighted the wrong base in the picture
Dear Paul and Abhijeet
Thank you for your reply and apologies for the confusion. Yes The correct nucleotide that should be highlighted is the 6th nucleotide (230F) not as indicated. To further clarify, I performed sequencing for RHD exon 3 gene. Forward primer (230F) showed there are 2 mutations ,whereas reverse primer (230R) showed normal gene sequencing with no mutation. Alignment result showed different nucleotides ( at mutation sites ) between both sequencing though they were sequenced from the same PCR product (hope I am clear enough now). Paul, please find attached the chromatogram files as requested.
Thank you Mujtaba for the files it does help to have all of the data.
The sequence qualities are both good with signal strengths of 1500 to 4600 and backgrounds of 5-7 so very good signal to noise ratio so it looks like the sequencing reaction is very good and also the template is very clean.
The results were from runs about a month apart so sample mix up is possible but I think there is another explanation. Sanger sequencing is normally poor quality close to the sequencing primer and improves after about 30 bases from the primer. If we align the 2 sequences we see that in the F sequence ACCCT is right in the middle of the sequence so is likely to be correct but the AAcct in the R sequence is near the end of the sequence where peaks are broad and background is high and this sequence is less trustworthy.
Similarly GGTGC in F is right at the end of the sequence where the quality of the electropherogram is very poor and should not be trusted while GGCGC in R is right in the middle of an excellent region of sequencing and should be trusted.
If your amplimer was bigger then I think that both sequences would be the same. Similarly if the amplimer was cloned then sequenced with plasid primers then they would look the same I think.
Base 42 in F CA(AG)CT looks like a possible snp but again not seen in F so not totally convincing.
Hope this makes sense. P
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