Hi,
Can anyone please guide me about this chromatogram? I can not say it is a sequencing error as sequence chromatogram is quite clear. And same type of duplication exist in 5 samples. is it a mutation? if it is so, then how can I discuss it, as its not like an insertion mutation.
Thank You.
Below is the attached file of sequence
AA, GG
A copy of the .abi sequence file might be more useful Tayyaba Saleem but what I think is happening here is that your data does not exactly fit the basecaller applied to the sequencing run. A basecaller identifies peaks of fluorescence and assigns a base (cagt) to that peak based on colour. One simple way of basecalling is equal spacing where the spacing between peaks is used to calculate where the next peak will be and this can cause calling errors (additional base) when a peak is very slightly wider than the previous called peaks and the trailing edge of the single peak is identified as a significant signal and basecalled. Sequencers have a choice of different basecallers in their software and I am sure that if you re analysed your data using a different basecaller it would correctly call these bases. It might introduce different errors at the ends of the sequence but these odd bases should be correctly called. It is possible that desalting was not perfect and that the spacing between early peaks is larger than later in the sequence maybe all of the bases are closer together and this is causing the miscall. It is good sequencing imperfectly called
A copy of the .abi sequence file might be more useful Tayyaba Saleem but what I think is happening here is that your data does not exactly fit the basecaller applied to the sequencing run. A basecaller identifies peaks of fluorescence and assigns a base (cagt) to that peak based on colour. One simple way of basecalling is equal spacing where the spacing between peaks is used to calculate where the next peak will be and this can cause calling errors (additional base) when a peak is very slightly wider than the previous called peaks and the trailing edge of the single peak is identified as a significant signal and basecalled. Sequencers have a choice of different basecallers in their software and I am sure that if you re analysed your data using a different basecaller it would correctly call these bases. It might introduce different errors at the ends of the sequence but these odd bases should be correctly called. It is possible that desalting was not perfect and that the spacing between early peaks is larger than later in the sequence maybe all of the bases are closer together and this is causing the miscall. It is good sequencing imperfectly called
Tayyaba, ignore the letters at the top and trust on the sequence peaks. I suggest you to align the electropherogram with a reference sequence. here some software to do it:
https://dna-analysis.yale.edu/dna-sequencing-services/contig-assembly
https://www.monash.edu/__data/assets/pdf_file/0020/2015534/electropherogram-software.pdf
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