Design Signatures - Inputs

1. Experimental Application:

   Sets the type of experiment that will be used to differentiate PCR amplicons:

• Sequencing:

  The amplicon signatures will distinguished by DNA sequencing. This method will maximize variability in the 5-mer profile of each group. The PCR product size (#5 below) should be carefully considered to match the sequencing method being used (e.g., Sanger = 600-1500, Next-Gen = 300-700 base pairs), and the length of input sequences.

• High Resolution Melting (HRM) analysis:

  Amplicon signatures will be differentiated by the distance between melt curves, typically using a "saturating" dye such as EvaGreen. This method will maximize the area between different melt curves. Setting the primer length (#4 below) and PCR product size (#5 below) to be short might improve the results (e.g., primers 15-21 nucleotides and PCR Product 45-300 nucleotides). Also, consider selecting potential restriction enzymes (#7 below) to further diversify the amplicon signatures of longer amplicons.

• Fragment Length Polymorphism (FLP) analysis:

  Amplicons will be differentiated by their length using a gel run with moderate to high resolution. For FLP, the PCR product size sets the minimum and maximum lengths that can be detected (e.g., 150 to 900 base pairs). Therefore, it is important to specify a wide range of PCR product sizes (#5 below) in the inputs. Furthermore, it is likely that using a restriction enzyme (#7 below) will greatly diversify the length signatures after amplification.

2. Primers:

   Set constraints on the designed primers by limiting their lengths and maximum number of different permutations. Primers are designed to be equal length, and include ambiguity codes to generate additional permutations. For example, an "S" in the primer requires two permutations because it refers to either a "C" or "G" in that position.

3. PCR Product Amplicon:

   Specify the desired amplicon size, which determines the distance separation between the start of the forward and reverse primers on the target template.

4. Reaction Conditions:

   These inputs are used to determine the optimal length for each primer that will maximize sensitivity of the primer pair. The default values are sufficient for most experiments. Concentrations should be provided as they are in the final PCR reaction. For example, if you add 1µL of a 10µM stock of each primer to a 25µL total reaction volume then the final primer concentration is 1µL*10µM/25µL = 400nM. The program will calculate a Sodium Equivalent concentration based on the formula [Na⁺] + 3.33*([Mg⁺⁺] - [dNTP])^0.5. To use an alternative sodium equivalent concentration simply enter it as the sodium concentration and enter zero for the magnesium and dNTP concentrations.

5. Restriction Enzymes (optional):

   When performing an HRM or FLP experiment, a restriction enzyme can be used to further diversify amplicon signatures. The list of all standard restriction enzymes sold by New England BioLabs is provided for selection. Up to 10 enzymes may be chosen, although they will be considered independently (not used in combination). It may be useful to use an online "cutting" program to find potential enzymes that will digest your input sequences.

6. FASTA File:

   Choose a text file containing the sequence records to use for primer design. Here are some remarks on the input file:

• Sequences must be in FASTA format where each new sequence record begins with a ">" symbol on a single line containing the name of the group where it belongs, and subsequent lines contain the actual sequence. There are no restrictions on the number of nucleotides per line or the length of sequences.
• The size of the uploaded file is restricted to be less than 50 MB. This limit corresponds to roughly 50,000 sequences of length 1,000 nucleotides. To analyze more than 50MB of sequences, please download DECIPHER for use on your computer.
• Ideally, groups should differentiate the different organisms (i.e., strains) that you hope to distinguish. Most commonly, there will be a distinct group name for each input sequence. In cases where there are multiple different alleles per organism (e.g., duplicate genes), the groups should be defined such that all of the alleles belonging to an organism have the same group name.
• An example input FASTA file can be downloaded here. It contains four groups: the Human IDH2 common allele, and three less common alleles with a single nucleotide polymorphism (SNP). Note that the four groups all have distinct names.
• Each FASTA record must have an identifier listing only the group to which it belongs. Up to 1,000 groups are permitted. For example, a correctly formatted input file containing the groups "ABC" and "XYZ" might look like:
  >XYZ
  ACTGACTGT...
  >XYZ
  ACTGGACTG...
  >ABC
  GGCACTGAA...
  >Etc.
  ...