Dear all,
I have a problem and I do not know why it is happening. When I do my KRAS mutation analysis most of the samples come out as in the image without the predominant red line. However, some samples, as seen in the other image, show that high thymine contamination.
What I don’t understand is, why that high contamination of thymine only shows up on the KRAS codons. The rest of the sequence is clean and does not shows thymine spikes like that.
Thank you for your help.
Kind regards
The broad peak looks like unincorporated dye from the sequencing kit which usually happens when purification of the sequencing reaction is not good. It is worse when there is too little primer or too little pcr product in the sequencing reaction so there is a lot of unused dye to purify away. It usually happens at 2 positions near the start of the sequencing.If you attach one of the .abi sequence files with this problem it would help.
The broad peak looks like unincorporated dye from the sequencing kit which usually happens when purification of the sequencing reaction is not good. It is worse when there is too little primer or too little pcr product in the sequencing reaction so there is a lot of unused dye to purify away. It usually happens at 2 positions near the start of the sequencing.If you attach one of the .abi sequence files with this problem it would help.
You could also try sequencing from the other direction and see if the issue goes away when the region in question isn't at the start of the sequencing run.
I agree with Paul Rutland , looks like a "dye blob" from the kit.
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