Is sequencing all fragments from the final library also oversampling the population of initially simulated RNA molecules?

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  1. In our simulations, the sequencing depth is obviously limited by the number of fragments in the final library. However, these fragments neither represent all molecules initially expressed in the simulation, nor there is a 1:1 correlation. Fragments in the final library are those ones left after simulated fragmentation, reverse transcription, size selection and PCR. Due to those reasons, many RNA species will not even make it to the final library, thus barring the risk of oversampling in normal setups. Finally, the number of initially simulated molecules as well as the sequencing depth are two distinct parameters which are adjusted to down-sample the final library for producing the required amount of reads.