Next, an optional fragmentation process (nebulization, hydrolysis, adaptive focusing acoustics, …) may be comprised in the simulation. In general the simulation distinguishes 2 different mechanisms of RNA degradation, a mechanical/physical breaking process (PHYSICAL) and cleavage that is less dependant on physical properties (CHEMICAL). Choice of the fragmentation nature will influence the distribution of fragment lengths after the simulated fragmentation. Furthermore, also dependant on the adopted method, you should provide a realistic estimation of the maximum molecule length (FRAG_LAMBDA) that is not broken in the applied protocol. For instance, ~500nt cDNA molecules are known to be problematic to break with usual nebulization strategies. Naturally, this will mark about the upper limit of the fragment distribution yielded.
Simulation of fragmentation is an iterative process, where in each round a fragment is assigned a certain breaking probability, for PHYSICAL fragmentation
(5)
P b =1−exp −length(cDNA) FRAG_LAMBDA
and for CHEMICAL fragmentation
(6)
P b =1−(length(cDNA)−λ) −2
On the occurrences of breaks is decided in Bernoulli trials, the location of the respective breakpoint is normally distributed around the middle of the molecule (PHYSICAL), respectively uniformly distributed along the molecule (CHEMICAL). Finally, you specify whether the fragmentation step is carried out after or before the reverse transcription from RNA to DNA. Finally, in some protocols there is a step after RT and fragmentation that filters the generated cDNA fragments by size (FILTERING). If so, provide the minimum , (FILT_MIN) and the maximum (FILT_MAX) length of the fragments you want to retain for the sequencing.
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