Accurate characterization ofEscherichia colitRNA modifications with a simple method of deep-sequencing library preparation

In conventional RNA high-throughput sequencing, modified bases prevent a large fraction of tRNA transcripts to be converted into cDNA libraries. Recent proposals aiming at resolving this issue take advantage of the interference of base modifications with RT enzymes to detect and identify them by establishing signals from aborted cDNA transcripts. Because some modifications, such as methyl groups, do almost not allow RT bypassing, demethylation and highly processive RT enzymes have been used to overcome these obstacles. Working withEscherichia colias a model system, we show that with a conventional (albeit still engineered) RT enzyme and key optimizations in library preparation, all RT-impairing modifications can be highlighted along the entire tRNA length without demethylation procedure. This is achieved by combining deep-sequencing samples, which allows to establish aborted transcription signal of higher accuracy and reproducibility, with the potential for differentiating tiny differences in the state of modification of all cellular tRNAs. In addition, our protocol provides estimates of the relative tRNA abundance.

Automated summary

In RNA high-throughput sequencing, modifications on tRNA transcripts can hinder their conversion into cDNA libraries. By optimizing experimental conditions and utilizing suitable RT enzymes, it is possible to highlight all modifications that affect transcription without the need for demethylation procedures.