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Recent RNA-sequencing technology and associated bioinformatics have led to identification of tens of thousands of putative human chimeric RNAs, i.e. RNAs containing sequences from two different genes, most of which are derived from neighboring genes on the same chromosome. In this essay, we redefine "two neighboring genes" as those producing individual transcripts, and point out two known mechanisms for chimeric RNA formation, i.e. transcription from a fusion gene or trans-splicing of two RNAs. By our definition, most putative RNA chimeras derived from canonically-defined neighboring genes may either be technical artifacts or be cis-splicing products of 5'- or 3'-extended RNA of either partner that is redefined herein as an unannotated gene, whereas trans-splicing events are rare in human cells. Therefore, most authentic chimeric RNAs result from fusion genes, about 1,000 of which have been identified hitherto. We propose a hypothesis of "consecutive reverse transcriptions (RTs)", i.e. another RT reaction following the previous one, for how most spurious chimeric RNAs, especially those containing a short homologous sequence, may be generated during RT, especially in RNA-sequencing wherein RNAs are fragmented. We also point out that RNA samples contain numerous RNA and DNA shreds that can serve as endogenous random primers for RT and ensuing polymerase chain reactions (PCR), creating artifacts in RT-PCR.

journal of cancer

Hypothesis: Artifacts, Including Spurious Chimeric RNAs with a Short Homologous Sequence, Caused by Consecutive Reverse Transcriptions and Endogenous Random Primers