Modelling and simulating generic RNA-Seq experiments with the flux simulator.

Abstract : High-throughput sequencing of cDNA libraries constructed from cellular RNA complements (RNA-Seq) naturally provides a digital quantitative measurement for every expressed RNA molecule. Nature, impact and mutual interference of biases in different experimental setups are, however, still poorly understood-mostly due to the lack of data from intermediate protocol steps. We analysed multiple RNA-Seq experiments, involving different sample preparation protocols and sequencing platforms: we broke them down into their common-and currently indispensable-technical components (reverse transcription, fragmentation, adapter ligation, PCR amplification, gel segregation and sequencing), investigating how such different steps influence abundance and distribution of the sequenced reads. For each of those steps, we developed universally applicable models, which can be parameterised by empirical attributes of any experimental protocol. Our models are implemented in a computer simulation pipeline called the Flux Simulator, and we show that read distributions generated by different combinations of these models reproduce well corresponding evidence obtained from the corresponding experimental setups. We further demonstrate that our in silico RNA-Seq provides insights about hidden precursors that determine the final configuration of reads along gene bodies; enhancing or compensatory effects that explain apparently controversial observations can be observed. Moreover, our simulations identify hitherto unreported sources of systematic bias from RNA hydrolysis, a fragmentation technique currently employed by most RNA-Seq protocols.
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https://hal.inria.fr/hal-00744755
Contributeur : Marie-France Sagot <>
Soumis le : mardi 23 octobre 2012 - 17:53:48
Dernière modification le : jeudi 28 juin 2018 - 14:37:52

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Thasso Griebel, Benedikt Zacher, Paolo Ribeca, Emanuele Raineri, Vincent Lacroix, et al.. Modelling and simulating generic RNA-Seq experiments with the flux simulator.. Nucleic Acids Research, Oxford University Press, 2012, 40 (20), pp.10073-10083. 〈10.1093/nar/gks666〉. 〈hal-00744755〉

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