I found some bases with disagreement in the assembly of the contig. I read that because it is a nuclear marker, this disagreement may not be due to a failure in sequencing but due to heterozygosity, and therefore I should not change the base in the contig by the highest-quality criterion, but set it as an IUPAC ambiguity code.
On the other hand, I was told that when it is heterozygous, the peaks are usually the same size. But it is not always easy to determine how equal it is.
So I would like to know if there is a cut-off point or defined protocol to determine if a mismatch is due to heterozygosity or an error in sequencing on one of the strands?
Look for read coverage over the sites in question and observe allelic frequencies before deciding how to proceed. With coverage you can gain confidence and establish a PValue which you then use to determine if the polymorphic site you are observing is likely to be the result of some random process - most likely sequencing errors. Assuming your PValue is
Dear Gabriel Novaes-e-Fagundes ,
Are you performing Sanger or whole-genome sequencing? What is the organism you are dealing with and what is the gene?
Look for read coverage over the sites in question and observe allelic frequencies before deciding how to proceed. With coverage you can gain confidence and establish a PValue which you then use to determine if the polymorphic site you are observing is likely to be the result of some random process - most likely sequencing errors. Assuming your PValue is
Dear Gabriel,
Please read our article "Sequence motifs capable of forming DNA stem-loop structures act as a replication diode". FEBS Open Bio. 4:7(7):944-952. Shirak A., Seroussi U., Gootwine E., Seroussi E. (2017).
Andrey Shirak - what is the relevancy of the paper you referenced to the question asked by Gabriel Novaes-e-Fagundes ?
Dear Dr Stuart Stephen,
In our article, we have proven that secondary structures of DNA lead to inequality in heights of picks (distortion from 1:1 ratio) in Sanger sequencing up to a situation where one of two alleles turn out to be silent completely. I assume that this technical disturbance affects also on genome assembly.
Sincerely
Andrey
you should be particularly careful if the changes are either
1 Close to the sequencing primer where sequencing is usually poor or
2 Close to the end of a long read where the signal strength is falling and becoming comparable to background signal noise.
Also lookat the sequence generally and if ,for instance, you find a lot of one base eg C is found under other strong bases then distrust Cs which are a bit stronger and sequence in the reverse direction also.
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