New Recommendations for Optimal Sequencing Depth in Alternative Splicing Studies

As part of an international research team led by Olga Tsoy from the University of Hamburg, the Division of Data Science in Biomedicine contributed to the development of new guidelines for determining the optimal sequencing depth in RNA sequencing (RNA-Seq) studies focusing on alternative splicing (AS). By analyzing extensive RNA-Seq datasets from human tissues such as the heart, adipose tissue, and hypothalamus, the researchers found that commonly used sequencing depths of 50 to 150 million reads may not be sufficient to capture the full range of AS events, especially in lowly expressed genes.
The study recommends a sequencing depth of 150 to 200 million reads for reliably detecting AS events in lowly expressed genes and 100 to 150 million reads for highly expressed genes. This deeper sequencing allows for the identification of additional biologically relevant AS events that are important for understanding disease mechanisms and tissue-specific gene regulation.
The findings highlight the importance of careful experimental design in RNA-Seq studies and offer valuable guidelines for researchers aiming to comprehensively assess alternative splicing. The study also notes that large RNA-Seq projects like GTEx and TCGA may not have sufficient sequencing depth for a complete AS analysis.
This work provides a solid foundation for future studies seeking a more thorough understanding of complex biological processes and disease pathways.