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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.    Results  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.    Electronic supplementary material  The online version of this article (doi:10.1186/1471-2164-15-633) contains supplementary material, which is available to authorized users.

Assessing the hodgepodge of non-mapped reads in bacterial transcriptomes: real or artifactual RNA chimeras?