Color markings among felid species display both a remarkable diversity and a common underlying periodicity. A similar range of patterns in domestic cats suggests a conserved mechanism whose appearance can be altered by selection. We identified the gene responsible for tabby pattern variation in domestic cats as Transmembrane aminopeptidase Q (Taqpep), which encodes a membrane-bound metalloprotease. Analyzing 31 other felid species, we identified Taqpep as the cause of the rare king cheetah phenotype, in which spots coalesce into blotches and stripes. Histologic, genomic expression, and transgenic mouse studies indicate that paracrine expression of Endothelin3 (Edn3) coordinates localized color differences. We propose a two-stage model in which Taqpep helps to establish a periodic pre-pattern during skin development that is later implemented by differential expression of Edn3.

Studies of natural variation have revealed that the winter-annual habit of many accessions of Arabidopsis is conferred by two genes, FRIGIDA (FRI) and FLOWERING LOCUS C (FLC),whose activities impose a vernalization requirement. To better understand the mechanism underlying the winter-annual habit, a genetic screen was performed to identify mutants that suppress the late-flowering behavior of a non-vernalized winter-annual strain. We have identified a locus, FRIGIDA-ESSENTIAL 1 (FES1), which, like FRI, is specifically required for the upregulation of FLC expression. FES1 is predicted to encode a protein with a CCCH zinc finger, but the predicted sequence does not otherwise share significant similarity with other known proteins. fes1 is a complete suppressor of FRI-mediated delayed flowering, but has little effect on the late-flowering phenotype of autonomous-pathway mutants. Thus, FES1activity is required for the FRI-mediated winter-annual habit, but not for the similar phenotype resulting from autonomous-pathway mutations. Epistasis analysis between FES1, FRI and another specific suppressor of FRI-containing lines, FRIGIDA-LIKE 1(FRL1), indicates that these genes do not function in a linear pathway, but instead act cooperatively to promote the expression of FLC.

In cancer drug discovery, it is important to investigate the genetic determinants of response or resistance to cancer therapy as well as factors that contribute to adverse events in the course of clinical trials. Despite the emergence of new technologies and the ability to measure more diverse analytes (e.g., circulating tumor cell (CTC), circulating tumor DNA (ctDNA), etc.), tumor tissue is still the most common and reliable source for biomarker investigation. Because of its worldwide use and ability to preserve samples for many decades at ambient temperature, formalin-fixed, paraffin-embedded tumor tissue (FFPE) is likely to be the preferred choice for tissue preservation in clinical practice for the foreseeable future. Multiple analyses are routinely performed on the same FFPE samples (such as Immunohistochemistry (IHC), in situ hybridization, RNAseq, DNAseq, TILseq, Methyl-Seq, etc.). Thus, specimen prioritization and optimization of the isolation of analytes is critical to ensure successful completion of each assay. FFPE is notorious for producing suboptimal DNA quality and low DNA yield. However, commercial vendors tend to request higher DNA sample mass than what is actually required for downstream assays, which restricts the breadth of biomarker work that can be performed. We evaluated multiple genomics service laboratories to assess the current state of NGS pre-analytical processing of FFPE. Significant differences in pre-analytical capabilities were observed. Key aspects are highlighted and recommendations are made to improve the current practice in translational research.

Next-generation sequencing technologies offer new approaches for global measurements of gene expression but are mostly limited to organisms for which a high-quality assembled reference genome sequence is available. We present a method for gene expression profiling called EDGE, or EcoP15I-tagged Digital Gene Expression, based on ultra-highthroughput sequencing of 27-bp cDNA fragments that uniquely tag the corresponding gene, thereby allowing direct quantification of transcript abundance. We show that EDGE is capable of assaying for expression in >99% of genes in the genome and achieves saturation after 6-8 million reads. EDGE exhibits very little technical noise, reveals a large (10 6 ) dynamic range of gene expression, and is particularly suited for quantification of transcript abundance in non-model organisms where a high-quality annotated genome is not available. In a direct comparison with RNA-seq, both methods provide similar assessments of relative transcript abundance, but EDGE does better at detecting gene expression differences for poorly expressed genes and does not exhibit transcript length bias. Applying EDGE to laboratory mice, we show that a loss-of-function mutation in the melanocortin 1 receptor (Mc1r), recognized as a Mendelian determinant of yellow hair color in many different mammals, also causes reduced expression of genes involved in the interferon response. To illustrate the application of EDGE to a non-model organism, we examine skin biopsy samples from a cheetah (Acinonyx jubatus) and identify genes likely to control differences in the color of spotted versus non-spotted regions.

In Arabidopsis thaliana, DICER-LIKE 1 and DICER-LIKE 3 are involved in the generation of small RNAs. Double mutants between dicer-like 1 and dicer-like 3 exhibit a delay in flowering that is caused by increased expression of the floral repressor FLOWERING LOCUS C. This delayed-flowering phenotype is similar to that of autonomous-pathway mutants, and the flowering delay can be overcome by vernalization.