Significant and pervasive effects of RNA degradation on Nanopore direct RNA sequencing

Oxford Nanopore direct RNA sequencing (DRS) is capable of sequencing complete RNA molecules and accurately measuring both gene and isoform expression. However, as DRS is designed to profile intact native RNA the precision of gene and isoform quantification may be more heavily dependent upon RNA integrity, (as measured by RIN), than other RNA-seq methodologies. It is currently unclear how RNA integrity impacts DRS or if RNA degradation can be corrected for. RNA from in-vivo, post-mortem and clinical samples have often undergone some degradation, therefore, to enable its application to the widest range of samples, it is essential to determine the appropriate RNA integrity range to generate high quality data from DRS. To determine the impact of RNA degradation on DRS we performed a degradation time-course using SH-SY5Y neuroblastoma cells. Our results demonstrate that degradation is a significant and pervasive factor that can bias downstream analysis. This includes a reduction in library complexity resulting in an overrepresentation of short genes and isoforms. Increased mRNA length was closely correlated with rates of degradation, while DNA binding proteins and transcription factors showing the highest susceptibility to degradation. Degradation also biases differential expression analyses; however, we find explicit correction can almost fully recover meaningful biological signal. Overall, we find samples with RIN > 9.5 can be treated as undegraded and samples with RIN > 7 can be utilised for DRS with appropriate correction. These results establish the suitability of DRS for a wide range of samples whilst limiting the confounding effect of degradation on downstream analysis