Q: Is your automatic alignment result seriously faulty?
A: Your alignment or some of your sequences are fundamentally incorrect if a large proportion of the
resulting alignment table contains red positions and numerous insertions. Tips:
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Check whether any sequence is in reverse complement orientation. E.g. Use the "Load / FASTA (auto align auto reverse)" option.
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For circular molecules (mtDNA and plasmids), check whether the last
nucleotides of your entered sequences transgress the first nucleotide of
the reference sequence. Remedy: Rearrange your reference sequence (or
alternatively, your entered sequences) before starting DNA Alignment.
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Check for sequencing errors in those sequences which after alignment contain numerous insertions or red positions.
Q: Does your automatic alignment result contain insertions instead of mismatches?
A: Try increasing the values of the
alignment parameters.
Q: Are you using DNA Alignment to format data including an outgroup for Fluxus' Network software?
A: The alignment algorithm is designed for closely related sequences without large-scale re-arrangements or duplications.
If the outgroup is very different from the rest of the data set and causes alignment problems:
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Omit the outgroup from alignment and from initial work analysis, and produce an unrooted network.
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Then, try aligning the outgroup against some or all of your data to find out which nucleotide positions correspond to which nps in your main data set.
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With this knowledge of nucleotide numbering, inspect your unrooted network manually for internal nodes which are most similar to the outgroup.
