PCR reaction was performed using phusion high-fidelity PCR Mastermix (New England Biolabs LTD., Beijing, China) with the following condition: 94 °C for 3 min (1 cycle), 94 °C for 45 °C50 °C for 60 °C72 °C for 90 s (35 cycles), and a last step of 72 °C for 10 min.
PCR conditions were as follows: 95°C for 2 min followed by 35 cycles of 95°C for 20 s, 55°C for 10 s and 70°C for 15 s, ending with a last step of 72°C for 10 min to ensure complete amplification of the target region.
The following thermal cycling conditions were used: 3 min of initial denaturation at 94 °C; 35 cycles of denaturation at 94 °C for 45 s, annealing at 50 °C for 60 s, and elongation at 72 °C for 90 s; and a last step at 72 °C for 10 min.
We applied PMM on the sampled data and we reported the rank of gene g within pathogen p. This was repeated 1000 times for each combination of n s and n p. As a last step we calculated for each combination the mean and variance of the rank for gene g within pathogen p. For the resampling we omitted genes that have less than 6 siRNA sets, in order to have a good resampling basis.
In a last step, Ka/Ks values for each pair-wise alignment were calculated.
Amplification was performed using Go Tag DNA Polymerase (Promega) with the following thermal conditions: 95°C for 2 min followed by 40 cycles of 95°C for 30 sec, 60°C for 30 sec and 72°C for 30 sec and as a last step, final extension at 72°C for 10 min was run.
In a last step, we again tested for invariance of factor means.
▪ As a last step chose N t th column (for which we have only one choice).
In a last step, the testing system is adjusted for the industrial environment and integrated into an industrial production line.
In a last step, we performed group-mean centering for the two variables in order to focus on the effect at the individual level while controlling for the effect of the program.
▪ j th Column is chosen such that it contains a power of ϕ at i th row (i, j = 2,..., N t -1) ▪ As a last step chose N t th column (for which we have only one choice).
