Hello everyone
I conducted sequencing prosedure for RNA genome segment (about 700nt) , for this reason I employed the random hexamer primer for cDNA synthesis .
after sequencing (by sanger sequencer ) the result show very good quality chromatogram but their is a significant High number of mutations .
so, I wondered, if there is an evidence that random hexamer priming can produce sequencing error .
I appreciate any idea .
If most of the mutations are close to the sequencing primers ( within about 30bases) or at the ends of the sequence where signal intensity is weak then these may be sequencing errors rather than production errors. can we see an original .abi file please? Could they also be polymorphisms normally found in the sequence?
thank you Paul Rutland
yes we know the technical issues of the chromatogram, the chromatogram is clean and have good quality .
In that case I would expect that the low annealing temperature of hexamers would mean that the enzyme is extending at well below its optimum working temperature so would be more liable to making errors but unfortunately I have no evidence to back this guess up. It may be relevant that often random 9mers are used in cdna kits which would anneal at higher temperatures.
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