Nanopore Sequencing in the Regenerating Axolotl Limb

In the landscape of modern genomics, RNA sequencing (RNA-seq) has become a cornerstone for deciphering intricate molecular compositions. However, the field's progress has been challenged by the costs and limitations inherent in traditional short-read sequencing methods. Oxford Nanopore Technologies (ONT) has rapidly emerged as a transformative force in the field of RNA-seq by utilizing nanopores - nanometer-scale channel proteins - as artificial transport mechanisms. This innovative approach presents an exciting long-read sequencing alternative that counters the limitations of conventional short-read sequencing methodologies, such as the occurrence of multi-mapping reads and difficulty of generating de novo assemblies. In addition, ONT provides a series of distinct library preparation alternatives allowing for additional epigenetic and transcriptomic analyses.

Here we present an optimized, cost-effective, and streamlined protocol for ONT RNA-seq that maximizes the potential of this technology in diverse research settings. We use the axolotl salamander (Ambystoma mexicanum) to test ONT technology in a regenerative biology setting, developing a pipeline which can be applied to tissues to characterize differential gene expression in varied contexts. To validate our characterization pipeline, we compared our axolotl intact limb and blastema expression data to established short-read RNA-seq datasets and observed consistent differential expression of well-established markers. Additionally, we characterized a dataset derived from mutant axolotls to identify misregulated genes by immunohistochemistry in tissue samples.

Our data indicate that ONT technology represents a convenient and low-cost approach which can generate in-house agnostic differential gene expression data for various tissue sets. We hope to further broaden our understanding of long-read sequencing by including a wider range of epigenetic and transcriptomic analyses that are currently unavailable in traditional short-read RNA-seq alternatives.