RNA molecules undergo a vast array of chemical post-transcriptional modifications (PTMs) that can affect their structure and interaction properties. In recent years, a growing number of PTMs have been successfully mapped to the transcriptome using experimental approaches relying on high-throughput sequencing. Oxford Nanopore direct-RNA sequencing has been shown to be sensitive to RNA modifications. We developed and validated Nanocompore, a robust analytical framework that identifies modifications from these data. Our strategy compares an RNA sample of interest against a non-modified control sample, not requiring a training set and allowing the use of replicates. We show that Nanocompore can detect different RNA modifications with position accuracy in vitro, and we apply it to profile m6A in vivo in yeast and human RNAs, as well as in targeted non-coding RNAs. We confirm our results with orthogonal methods and provide novel insights on the co-occurrence of multiple modified residues on individual RNA molecules.

Background: Knowledge of the amount of gene order and synteny conservation between two species gives insights to the extent and mechanisms of divergence. The vertebrateFugu rubripes(pufferfish) has a small genome with little repetitive sequence which makes it attractive as a model genome. Genome compaction and synteny conservation between human andFuguwere studied using data from public databases.Methods: Intron length and map positions of human andFuguorthologues were compared to analyse relative genome compaction and synteny conservation respectivley. The divergence of these two genomes by genome rearrangement was simulated and the results were compared to the real data.Results: Analysis of 199 introns in 22 orthologous genes showed an eight-fold average size reduction inFugu, consistent with the ratio of total genome sizes. There was no consistent pattern relating the size reduction in individual introns or genes to gene base composition in either species. For genes that are neighbours inFugu(genes from the same cosmid or GenBank entry), 40–50% have conserved synteny with a human chromosome. This figure may be underestimated by as much as two-fold, due to problems caused by incomplete human genome sequence data and the existence of dispersed gene families. Some genes that are neighbours inFuguhave human orthologues that are several megabases and tens of genes apart. This is probably caused by small inversions or other intrachromosomal rearrangements.Conclusions: Comparison of observed data to computer simulations suggests that 4000–16 000 chromosomal rearrangements have occured sinceFuguand human shared a common ancestor, implying a faster rate of rearrangement than seen in human/mouse comparisons.

Development of cervical cancer is directly associated with integration of human papillomavirus (HPV) genomes into host chromosomes and subsequent modulation of HPV oncogene expression, which correlates with multi-layered epigenetic changes at the integrated HPV genomes. However, the process of integration itself and dysregulation of host gene expression at sites of integration in our model of HPV16 integrant clone natural selection has remained enigmatic. We now show, using a state-of-the-art ‘HPV integrated site capture’ (HISC) technique, that integration likely occurs through microhomology-mediated repair (MHMR) mechanisms via either a direct process, resulting in host sequence deletion (in our case, partially homozygously) or via a ‘looping’ mechanism by which flanking host regions become amplified. Furthermore, using our ‘HPV16-specific Region Capture Hi-C’ technique, we have determined that chromatin interactions between the integrated virus genome and host chromosomes, both at short- (<500 kbp) and long-range (>500 kbp), appear to drive local host gene dysregulation through the disruption of host:host interactions within (but not exceeding) host structures known as topologically associating domains (TADs). This mechanism of HPV-induced host gene expression modulation indicates that integration of virus genomes near to or within a ‘cancer-causing gene’ is not essential to influence their expression and that these modifications to genome interactions could have a major role in selection of HPV integrants at the early stage of cervical neoplastic progression.