Formalin-fixed paraffin-embedded (FFPE) samples are the only remaining biological archive for many toxicological and clinical studies, yet their use in genomics has been limited due to nucleic acid damage from formalin fixation. Older FFPE samples with highly degraded RNA pose a particularly difficult technical challenge. Probe-based targeted sequencing technologies show promise in addressing this issue but have not been directly compared to standard whole-genome RNA-Sequencing (RNA-Seq) methods. In this study, we evaluated dose-dependent transcriptional changes from paired frozen (FROZ) and FFPE liver samples stored for over 20 years using targeted resequencing (TempO-Seq) and whole-genome RNA-Seq methods. Samples were originally collected from male mice exposed to a reference chemical (dichloroacetic acid, DCA) at 0, 198, 313, and 427 mg/kg-day (n = 6/dose) by drinking water for 6 days. TempO-Seq showed high overlap in differentially expressed genes (DEGs) between matched FFPE and FROZ samples and high concordance in fold-change values across the two highest dose levels of DCA vs. control (R2 ≥ 0.94). Similarly, high concordance in fold-change values was observed between TempO-Seq FFPE and RNA-Seq FROZ results (R2 ≥ 0.92). In contrast, RNA-Seq FFPE samples showed few overlapping DEGs compared to FROZ RNA-Seq (≤5 for all dose groups). Modeling of DCA-dependent changes in gene sets identified benchmark doses from TempO-Seq FROZ and FFPE samples within 1.4-fold of RNA-Seq FROZ samples (93.9 mg/kg-d), whereas RNA-Seq FFPE samples were 3.3-fold higher (310.3 mg/kg-d). This work demonstrates that targeted sequencing may provide a more robust method for quantifying gene expression profiles from aged archival FFPE samples.

The rise of high-throughput RNA sequencing (RNA-seq) and de novo transcriptome assembly has had a transformative impact on how we identify and study genes in the phototransduction cascade of non-model organisms. But the advantage provided by the nearly automated annotation of RNA-seq transcriptomes may at the same time hinder the possibility for gene discovery and the discovery of new gene functions. For example, standard functional annotation based on domain homology to known protein families can only confirm group membership, not identify the emergence of new biochemical function. In this study, we show the importance of developing a strategy that circumvents the limitations of semiautomated annotation and apply this workflow to photosensitivity as a means to discover non-opsin photoreceptors. We hypothesize that non-opsin G-protein-coupled receptor (GPCR) proteins may have chromophore-binding lysines in locations that differ from opsin. Here, we provide the first case study describing non-opsin light-sensitive GPCRs based on tissue-specific RNA-seq data of the common bay scallop Argopecten irradians (Lamarck, 1819). Using a combination of sequence analysis and three-dimensional protein modeling, we identified two candidate proteins. We tested their photochemical properties and provide evidence showing that these two proteins incorporate 11-cis and/or all-trans retinal and react to light photochemically. Based on this case study, we demonstrate that there is potential for the discovery of new light-sensitive GPCRs, and we have developed a workflow that starts from RNA-seq assemblies to the discovery of new non-opsin, GPCR-based photopigments.