Background: RNA sequencing methods have already altered our view of the extent and complexity of bacterial and eukaryotic transcriptomes, revealing rare transcript isoforms (circular RNAs, RNA chimeras) that could play an important role in their biology./nResults: We performed an analysis of chimera formation by four different computational approaches, including a custom designed pipeline, to study the transcriptomes of M. pneumoniae and P. aeruginosa, as well as mixtures of both. We found that rare transcript isoforms detected by conventional pipelines of analysis could be artifacts of the experimental procedure used in the library preparation, and that they are protocol-dependent. Conclusion: By using a customized pipeline we show that optimal library preparation protocol and the pipeline to analyze the results are crucial to identify real chimeric RNAs. Keywords: Chimeric RNAs, Fusion transcripts, RNA-seq, Library preparation protocols

The research leading to these results has received funding from the European Union Seventh Framework Programme (FP7/2007-2013), through the European Research Council, under grant agreement Nr. 232913, the Fundación Botín, the Spanish Ministry of Economy and Competitiveness (BIO2007-61762), the National Plan of R + D + i, the ISCIII -Subdirección General de Evaluación y/nFomento de la Investigación- (PI10/01702), and the European Regional Development Fund (ERDF) to the ICREA Research Professor LS. We acknowledge support from the Spanish Ministry of Economy and Competitiveness, ‘Centro de Excelencia Severo Ochoa 2013-2017’ (SEV-2012-0208)