Genefisher2 is a tool supporting biologists finding
primers for degenerated sequences. Design of degenerated Primers is a feasable task that
hardly can be handled manually.
For this it incorporates BatCons2, a simple tool to create ambiguous consensus sequences from alignments containing amino acid or DNA sequences.
1. a) AA - Consensus Backtranslation
Calculates the backtranslation of an amino acid alignment by using the consensus codon table and afterwards the ambiguous consensus sequence of that alignment.
When using this function, please note that the degeneracy of the primer and the 3' end is likely to be very high. Please change your parameters accordingly.
1. b) AA - Codon Table Backtranslation
Calculates the backtranslation of an amino acid alignment, by using a custom codon table, and afterwards the ambiguous consensus sequence of that alignment.
When using this function, please note that the degeneracy of the primer and the 3' end is likely to be very high. Please change your parameters accordingly.
1. c) DNA
Calculates the ambiguous consensus sequence of the alignment.
2.) Primer Calculation
Primers are calculated based on the consensus sequence that was generated in the last steps.
In-/Output values
INPUT :: Codon Usage Table
A codon usage table for backtranslation from amino acids. Must be a file of CGC type (like CodonFrequency ouput in Wisconsin Package, see http://www.kazusa.or.jp/codon). Please note that all fractions will be recalculated from 'count' from the codon usage table, as most tables lack proper 'fraction' values.
INPUT :: AA Alignment
An alignment made from amino acid sequences.
INPUT :: DNA Alignment
An alignment made from possibly ambiguous DNA sequences.
INPUT :: Input Sequence
The (consensus)-sequence will be the basis for primer calculation.
OUTPUT :: Consensus Sequence
The consensus sequence of the given alignment.
OUTPUT :: GeneFisher OutputGeneFisher Output has a special text-format.
Parameter
| Noise Threshold |
If the percentage of any given nucleotide is lower than the noise threshold, this nucleotide is not part of the consensus, except all nucleotides are below the noise threshold. Integer between 0 ans 100. Default: 20. Set to 0 to disable.
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| Conservation Threshold |
If the percentage of any given nucleotide is higher than the conservation threshold, we assume this position to be perfectly conserved and only nucleotides greater than the threshold appear in the consensus. Integer between 0 ans 100. Default: 80. Set to 100 to disable.
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| Cutoff Threshold |
During backtranslation only codons with a fraction above this value will be considered to avoid high degeneracy. Please note that all fractions will be recalculated from 'count' from the codon usage table, as most tables lack proper 'fraction' values. Float between 0 and 1. Default: 0.
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| Maximal Difference of Melting Temperature |
Specifies the maximal temperature difference allowed between two primers when computing primer pairs. |
| Primer Degeneracy |
The maximal allowed degree of degeneracy for the generated Primer. Primer degeneracy allows a certain ambiguity in the resulting primer-sequence. If the degeneracy values reach this threshold, the priming site is rejected.
The degeneracy is calculated as follows:
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| Display only Unique Primer |
Display only unique primers. |
| Salt Concentration |
The Na+ concentration for the PCR for temperature calculation. |
| Minimal Melting Temperature |
Minimal allowed melting temperature of the primer calculated from basepair composition. |
| Minimal 3' GC-Content |
Minimal allowed content of Guanin and Cytosin of the primer's 3'-End. Lower values are rejected. |
| Maximal GC-Content |
Maximal allowed content of Guanin and Cytosin in the designed Primer. |
| Maximal 3' GC-Content |
Maximal allowed content of Guanin and Cytosin of the primer's 3'-End. Higher values are rejected. |
| Minimal Primer Length |
Minimal length of the generated Primer |
| Oligonucleotide Concentration |
The oligonucleotide concentration for the PCR for temperature calculation. |
| Minimal GC-Content |
Minimal allowed content of Guanin and Cytosin in the designed Primer. |
| Maximal Product Size |
Maximal size of the product that will be produced by the PCR. |
| Maxmimal Primer Length |
Maximal length of the generated Primer |
| Maximal Melting Temperature |
Maximal allowed melting temperature of the primer calculated from basepair composition. |
| Primer 3' Length |
Defines the length of the primer's 3'-end for 3' degeneracy and GC-Content calculation. |
| Minimal Product Size |
Minimal size of the product that will be produced by the PCR. |
| 3' degeneracy |
Maximal degeneracy of the primer's 3'-end. If the degeneracy values reach this threshold, the priming site is rejected.
The degeneracy is calculated as follows:
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| Nucleotide at 3'-End |
Nucleotide that can occure at the 3'-end of the primer. This nucleotide is expected to have a strong influence on amplification success. Multiple selections are allowed. |
Genefisher is a Toolchain. At the end of the chain degenerated Primers are calculated.
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