Developing an automated workflow to analyze differential gene expression in cells through long-read Nanopore RNA sequencing
Introduction
RNA-sequencing
RNA sequencing (RNA-Seq) technology has, since its inception shortly after next-generation sequencing (NGS), made large advancements in our ability to study the transcriptome (Weber 2015). Long-read RNA-Sequencing technology, such as the Nanopore MinION, enables us to analyze full-length transcripts over short read RNA-Seq data. While very potent, short-read RNA-Seq has its drawbacks due to its short nature, such as when high amounts of repeating sequences are involved and the challenges in overlapping regions (van Dijk et al. 2018). As full-length isoforms are lost due to the fragmenting nature of short-read RNA-Seq, we can instead turn to long-read RNA-Seq. While this technology can suffer from lower throughput and accuracy, it does allow us to directly sequence native RNA sequences. (Ament et al. 2024). With this versatile tool we can analyze the effects of the exposure of human cells to environmental contaminants, such as microplastics (MPs).
Microplastics
Over the past decade we have gone from detecting and identifying MPs in marine habitats and oceans worldwide (Thompson et al. 2004) to analyzing human samples and finding them in large groups of people everywhere such as in testicles (Zhao et al. 2023), breastmilk (Saraluck et al. 2024), placenta (Ragusa et al. 2021), liver (Barceló, Picó, and Alfarhan 2023) and respiratory systems (Amato-Lourenço et al. 2021). Since then there has been increasingly more research regarding the topic as evidenced by numerous publications exploring the many routes of exposure and their pathways of MP toxicity. And while it is observed that high concentrations of MPs can cause inflammatory lesions, and may be a factor in the increasing incidence of neurodegenerative diseases, immune disorders and cancers (Prata et al. 2020), the interactions and long-term effects require more studies as they are not yet fully understood. One such pathway is the gene expression, Lei et al. (2018) found that the exposing zebrafish Danio Rerio and nematode Caenorhabditis elegans to MPs caused the intestine to have reduced calcium levels and increased expression of gluthathione S-transferase which indicated intestinal damage and oxidative stress.
Aim of this project
In order to better understand the biological effect of MPs we aim to utilize long-read RNA-Seq analyses, facilitated by the Oxford Nanopore MinION, to explore the impact on human gene expression to analyze potential downstream health issues. By developing an automated data science workflow which makes use of minimap2, bambu and DESeq2 for discovery, quantification and differential gene expression we aim to provide a workflow that serves as a foundation for our research and a potential foundation for more reproducible data analyses in the future.