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RNA viruses are distinguished by extensive genetic heterogeneity, which implies classification into subunits, e.g. genotypes, lineages, etc., each with distinct epidemiological or clinical significance. This heterogeneity represents a major challenge in primer and probe design for PCR and DNA microarray assay development. Despite the existence of many software tools it is still common practice to select primer and probe candidates by visually inspecting multiple DNA sequence alignments. Among the main reasons for the inconvenience of primer/probe design software, are the limited capacity to cope both with extensive intra-group sequence variation and with information about sub classifications of variable deep, and the limitations imposes on the number and length homogeneity of the sequences in the target group. Some tools can process only a single sequence at once, while others fail to detect many of the candidates that an expert could identify visually. Defining probes using only one target sequence is not sufficient for the design of robust assays capable of dealing with high target sequence heterogeneity. On the other hand, manual primer/probe selection is both cumbersome and error prone, thus also giving rise to underperforming assays. To help with this selection we developed the Excel worksheet VisualOligoDeg.
VisualOligoDeg facilitate the visualization and selection of appropriate oligonucleotides hybridizing to all the sequences in a multi-alignment, or at least to most of the sequences of a given group.
Open VisualOligoDeg.xlsm in Excel, grant permision to execute macros and save with the name of your project e.g. VisualOliDeg-WNV.xlsm
In general, only red, double yellow unterlined cells are mean to be freely edited.
General information. Include historial of releases.
Assists the import of alignments in FASTA format, with the possibility of selecting a limited region. This sheet can also be used to edit the descriptions of the sequences, to change names, to add up to four levels of classification (e.g. group, species, genotype, subtype, etc.), and to re-export the sequences.
The lower-right quarter of this sheet shows the alignment with nucleotide-specific background colors to facilitate visualization of variations, while the upper row shows an automatically generated consensus sequence constructed using adjustable parameters.
Other rows of the upper-right quarter show various views of nucleotide variability at each alignment position, and hybridization properties of the oligonucleotides beginning at the given position. For example, the workbook determines the minimum and maximum numbers of mismatches between the consensus and all selected target sequences in percent and absolute quantity per position. These calculations are also performed along a running “primer”. The free energy (∆G), enthalpy (∆H), entropy (∆S), a simulated signal intensity (I) and the melting temperature (Tm) using two different methods, including the nearest-neighbor (NN) thermodynamic model (for which different parameter sets can be given) are also shown.
Filters can be applied to restrict the region and the group of sequences to be analyzed. Oligonucleotides can be defined, edited, analyzed and finally, it automates the export of the selected oligonucleotide into the result sheet Oligo. The value of some parameters that influence the calculations can be also modified here.
it make possible to select or modify the set of nearest-neighbor (NN) thermodynamic parameters for DNA hybridization modeling.
Set the function to model Intensity I from ∆G