Predicting priming sites within transcripts
Compute the probability of internal priming along individual transcripts, given the transcript sequences, the primer sequence and a cutoff in the energy of interaction beyond which interactions should be reported. The following software https://github.com/fukunagatsu/RIblast could be used to predict interactions. Given the energy of interaction, the probability of hybridization at a given position i is given by pi = exp(-E/kT), where k is the Bolzmann constant (1.380649×10−23 J⋅K−1) and T is the temperature in degrees Kelvin (we can take that to be 298 (or 25 degrees Celsius). The probability of a position to be chosen as priming site should be computed as pi_norm = pi/sum_i pi.
Input:
- fasta-formatted file of transcript sequences
- primer sequence
- cutoff for the energy of interaction (energy of interaction should be < cutoff)
Output: gff-formatted file potential priming sites within each transcript, for each priming site reporting their coordinate within the corresponding transcript and the associated probability.