Dear respected researchers and experts,

I seek your valuable opinions and suggestions. I am calculating NUMT insertion time. I have filtered the NUMT IDs (sequences) using the following criteria: Identity percentage less than 96 and alignment length greater than 7000 bp.

I have used two methods for the calculation:

Method 1:

I ran a script in Python (Pycharm) to calculate Divergence and Divergence Proportion. The formula used for divergence is: Divergence = 100 - Identity percentage.

I already know the substitution rate for the order Orthoptera, which is 13.554 times 10 to the power of -9 substitutions per site per year (subs/s/y). Then, I calculated the insertion time using the following formula: NUMT Insertion Time = Divergence / 2 times Nuclear Substitution Rate.

Method 2:

In this method, I extracted NUMT sequences and aligned them with the mtDNA reference genome using ClustalW in MEGA software. I calculated the P-distance (pairwise difference) and then used the following formula to calculate insertion time: NUMT Insertion Time = P-distance / 2 times Nuclear Substitution Rate.

Both methods give different NUMT insertion times, and I am confused about which one to rely on. Could you provide guidance on this or if I have made any mistakes?

Thank you for your help!