Hello,
my question is more directed to those who have experience in treating sequencing data: alignment and phylogenetic tree construction. I have ratovirus sequences coming for NGS (clean sequences), i added other sequences from ncbi for alignment (fig.1). In fig.1 is the 5'end of the VP1 gene of rotavirus. So i would like to know if the aligned sequences are comparable as they are already, even with the presence of gaps (dashes in the figure)-nucleotide 1 does not begin at the same position for different rota genotypes. Or should the sequences align perfectly for them to be compared by removing all the selected nucleotides in fig.2. If yes, should i consider not touching any ORF region?
In my view option 2 is better. I will suggest that make phylogeny with both alignment and if you have information about there correct phylogeny then whichever gives good result just take that. If you simply do option2 then you might loose some important information about phylogeny. So its better to perform both analysis and select only that which gives relevant and meaningful information.
Suggestions:
1. Make sure not to include primer regions (this especially applies to the NCBI sequences, e.g. see the primers used in Article Interaction of Rotavirus Polymerase VP1 with Nonstructural P...
). This could create a lot of artifacts.2. Alignment from Fig. 1 will add a lot of ambiguities if the gaps are interpreted as N's. Bootstrapping could then point into a wrong direction depending on the type of analysis carried out.
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