The application of RNA-sequencing has led to numerous breakthroughs related to investigating gene expression levels in complex biological systems. Among these are knowledge of how organisms, such as the vertebrate model organism zebrafish (Danio rerio), respond to toxicant exposure. Recently, the development of 3′ RNA-seq has allowed for the determination of gene expression levels with a fraction of the required reads compared to standard RNA-seq. While 3′ RNA-seq has many advantages, a comparison to standard RNA-seq has not been performed in the context of whole organism toxicity and sparse data.
Oregon State University researchers examined samples from zebrafish exposed to perfluorobutane sulfonamide (FBSA) with either 3′ or standard RNA-seq to determine the advantages of each with regards to the identification of functionally enriched pathways. They found that 3′ and standard RNA-seq showed specific advantages when focusing on annotated or unannotated regions of the genome. They also found that standard RNA-seq identified more differentially expressed genes (DEGs), but that this advantage disappeared under conditions of sparse data. The researchers also found that standard RNA-seq had a significant advantage in identifying functionally enriched pathways via analysis of DEG lists but that this advantage was minimal when identifying pathways via gene set enrichment analysis of all genes.
Statistical significance and intersection of 3′ and standard RNA-seq enriched functions. For all functions found by both standard and 3′ RNA-seq DEG functional enrichment the −log10 of the q-value is shown. Red bars represent q-values of functions enriched from the 3′ RNA-seq DEG list and blue bars represent functions enriched from the standard RNA-seq DEG list.
These results show that each approach has experimental conditions where they may be advantageous. These observations can help guide others in the choice of 3′ RNA-seq vs standard RNA sequencing to query gene expression levels in a range of biological systems.