Hi! I amplified overlapping PCR products of the same viral genome region from the same sample using two different primer sets. I sequenced both products and there's one nucleotide difference in the result where they overlap. There's no double peak, the result is clear and obvious in both electropherograms. This happens reproducibly and with several samples. What could be causing this?
Thanks for any advice!
In the files I attached there are actually two differences. I know the short product is really short and the differences are near the end, but still, the peaks look nice where the anomaly is.
This is hepatitis A virus. There could be a mixed population but it's not quite as variable as other RNA viruses. I thought I would see double peaks, but maybe I'm wrong.
Thank you for the files Agnes Dencs . The traces are very strong at one end and very weak at the other. This looks like either you are using too much dna template or too much sequencing primer and the chemistry is running out at short sequences. I would not read sample 8.2 until the sequence ggAgA base 34 up to base 359 CTTgA. I would also trim sample 412f at base 19 ggAgA to base 170 gAAgT because the signal is too strong at one end and too weak at the other. Peak spacing is a problem when the signal strength is weak an we see irregular spacing of the peaks with weak signal and they cannot be trusted.. If we include only those bases that are trustworthy then your odd sequenced base is not included. Iwould not trust the sequencing here. I expect if the template to primer ratio was correct that the odd bases would not appear
Thanks! I knew it wasn't well optimized, but I never thought it would cause bad basecalling. I'll try different primer/template ratios and see what happens.
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