Parameter Name | Default Value | Parameter Range | Description |
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RTRANSCRIPTION | true | {true,false} | switch reverse transcription on/off |
RT_PRIMER | RH | {PDT,RH} | chooses random (RH) or poly-dT primers for first strand synthesis |
RT_MOTIF | sequence motif that | ||
RT_MIN | 500 | >0 | the minimum stretch that is polymerized by the reverse transcriptase enzyme |
RT_MAX | 5,500 | >0 | the maximum stretch that is polymerized by the reverse transcriptase enzyme (template-fidelity) |
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Input: RNA polymers annotated by their start end end coordinates on the transcript sequence they originate from (LIB_FILE) and parameters for reverse transcription. |
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Current sequencing technologies have to transform RNA into double-stranded DNA molecules before sequencing, either before or after fragmentation. For the first strand synthesis the Flux Simulator provides random priming |
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or poly-dT primers (RT_PRIMER) |
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. According to the nature of primers and the template fidelity of the reverse transcriptase enzyme (RT_MIN |
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and RT_MAX) |
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, the algorithm determines start point and extension separately for first and for second strand synthesis.
In the case of multiple priming events with random primers, several enzymes concurrently transcribe parts of the RNA molecule, and collisions with downstream DNA-RNA hybrids are resolved by displacement according to a Bernoulli trial. |
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Output: Reversely transcribed DNA molecules annotated by their start end end coordinates on the transcript sequence they originate from (LIB_FILE). |
(2)
Eq.(2) compares length() of the considered molecule RNA x to the average length of the RNA molecules in the reaction. To prevent the loss, especially of shorter transcripts, that may incur due to molecule numbers that are lower than in real experiments, at least one successful priming event is enforced on every RNA molecule. The length of the generated RNA molecule then is determined by an uniform randomly distributed variable U=[0;1[ .
(3)
In the case of multiple priming events are extended along the same RNA molecule, upstream primed first strand cDNA synthesis can displace further downstream bound primers. The chance of for a DNA-RNA hybrid to be displaced is most likely a function of its distance dist to the closest upstream priming event. The FLUX SIMULATOR currently decides on the displacement of overlapping RT extensions in a Bernoulli trial. To be specific, downstream primers get displaced if for a uniformly drawn random variable U=[0;1[ holds
(4)
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