Parameters
Parameter Name | Default Value | Parameter Range | Description |
|---|---|---|---|
| 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) |
Algorithm
Input: RNA polymers annotated by their start end end coordinates on the transcript sequence they originate from (LIB_FILE) and parameters for reverse transcription.
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 or poly-dT primers (RT_PRIMER). According to the nature of primers and the template fidelity of the reverse transcriptase enzyme (RT_MIN and RT_MAX), the algorithm determines start point and extension separately for first and for second strand synthesis.
- During first strand synthesis, poly-dT primers induce priming events in the poly-A tail, whereas random primers provoke successful initiation events along the entire molecule, and anchored primers trigger exactly one priming event at the 3-end of the respective fragment. In sequencing protocols without sequence-specific biases, each priming event is assigned a random location uniformly sampled along the corresponding stretch. Optionally, start points of first strand synthesis are determined by importance sampling according to weights of an optional PWM capturing sequence-biases.
- The point where second strand synthesis initiates is simulated by the length of the first DNA strand, which can be between RT_MIN and RT_MAX nucleotides, but maximally the distance of the first strand synthesis priming event from the 5’-end of the RNA template. The point of priming second strand synthesis in the presence of sequence biases is drawn from a distribution according to the PWM capturing the bias, or from a uniform distribution otherwise.
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.
Output: Reversely transcribed DNA molecules annotated by their start end end coordinates on the transcript sequence they originate from (LIB_FILE).
Overview
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