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. 2019 Dec 20;9:19477. doi: 10.1038/s41598-019-55573-1

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© The Author(s) 2019

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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The hidden break and how to diagnose it. (A) After post-transcriptional processing, the eukaryotic pre-rRNA molecule is cleaved to produce the 18S, 5.8S and 28S subunits. The RIN works normally in species that follow this rule. (B) In some species, the 28S subunit gets cleaved into 28Sα and 28Sβ, a phenomenon known as the hidden break. This phenomenon can be detected via electrophorograms where, in species with a break, only one peak, instead of two, is observed. (C) Our method allows the computational discovery of hidden breaks, by mapping RNA-Seq reads onto the 28S rRNA sequence and measuring (i) the read coverage and (ii) the log₂ of the ratio of forward/reverse reads mapped in each position of the 28S. In species with a break, e.g. Bombyx mori, a drop in coverage is observed in the middle of the break region. Additionally, the log₂ of the forward/reverse reads ratio drops before the hidden break site and increases immediately after it. These features are not observed in Aporometra wilsoni, an animal that does not possess the break. The vertical lines represent the conserved regions flanking the hidden break site.