We should also note that the RTC documentation includes instructions on how to properly calibrate the clock for maximum accuracy.
The mutation rate was estimated from the data, using the ages of the sequences to calibrate the clock.
Yet, even in instances where a clock can be assumed or approximated for a particular set of sequences, fossils or clear patterns of vicariance are rarely available to calibrate the clock.
Sequences from 24 patients for which the infection dates were known with certainty were employed to calibrate the clock [ 17].
We calibrated the clock using a mean mutation rate of 3.5%% per lineage per million years, an estimated rate for protein-coding insect mitochondrial DNA [ 55].
However, uncertainty in the absolute age of the evolutionary event used to calibrate the molecular clock, use of different genes or genomic regions, and different methods to estimate divergence times contribute to the variation in divergence time estimates between species (Gaut 2002).
The fact that the virus was in both drills and that the island separated from the mainland 12,000 years ago provided a precise way to calibrate the molecular clock, and comparisons of the SIVs confirmed Marx's suspicion that the jump into chimpanzees must have occurred much earlier than Worobey's previous estimates.
This was used to calibrate the TREx clock using the divergence of pigs and oxen, and pigs and humans.
In the first approach we used fossil data to calibrate the molecular clock with the timing of the Rhinocerontidae and Tapiridae split, and a well know split within Rhinocerontidae.
Using the age of fossils to calibrate the molecular clock, it was estimated that the braconid wasp ancestor of the microgastroid complex lived ≈100 Millions years ago (Mya).
Consequently, their sequences were used to calibrate the molecular clock estimates for the MRCA of each outbreak patient and the PS (Additional file 4).
