duper is a null mutation of Cryptochrome 1 in Syrian hamsters.

The duper mutation is a recessive mutation that shortens the period length of the circadian rhythm in Syrian hamsters. These animals show a large phase shift when responding to light pulses. Limited genetic resources for the Syrian hamster (Mesocricetus auratus) presented a major obstacle to cloning duper. This caused the duper mutation to remain unknown for over a decade. In this study, we did a de novo genome assembly of Syrian hamsters with long-read sequencing data from two different platforms, Pacific Biosciences and Oxford Nanopore Technologies. Using two distinct ecotypes and a fast homozygosity mapping strategy, we identified duper as an early nonsense allele of Cryptochrome 1 (Cry1) leading to a short, unstable protein. CRY1 is known as a highly conserved component of the repressive limb of the core circadian clock. The genome assembly and other genomic datasets generated in this study will facilitate the use of the Syrian hamster in biomedical research.

Cross-Site Evaluation of Commercial Sanger Sequencing Chemistries.

Sanger sequencing remains an essential tool utilized by researchers. Despite competition from commercial sequencing providers, many academic sequencing core facilities continue to offer these services based on a model of competitive pricing, knowledgeable technical support, and rapid turnaround time. In-house Sanger sequencing remains a viable core service and, until recently, Applied Biosystems BigDye Terminator chemistry was the only commercially available solution for Sanger DNA sequencing on Applied Biosystems (ABI) instruments; however, several new products employing novel dye chemistries and reaction configurations have entered the market. As a result, there is a need to benchmark the performance of these new chemistries on various DNA templates, including difficult-to-sequence templates, and their amenability to commonly employed cost-saving measures, such as dye dilution and reaction miniaturization. To evaluate these new reagents, a study was designed to compare the quality of Sanger sequencing data produced by ABI BigDye and commercially available kits from 2 other vendors using both control and difficult-to-sequence DNA templates under various reaction conditions. This study will serve as a valuable resource to core facilities conducting Sanger sequencing that wish to evaluate the use of an alternative chemistry in their sequencing core.

FLNC Gene Splice Mutations Cause Dilated Cardiomyopathy.

OBJECTIVE: To identify novel dilated cardiomyopathy (DCM) causing genes, and to elucidate the pathological mechanism leading to DCM by utilizing zebrafish as a model organism. BACKGROUND: DCM, a major cause of heart failure, is frequently familial and caused by a genetic defect. However, only 50% of DCM cases can be attributed to a known DCM gene variant, motivating the ongoing search for novel disease genes. METHODS: We performed whole exome sequencing (WES) in two multigenerational Italian families and one US family with arrhythmogenic DCM without skeletal muscle defects, in whom prior genetic testing had been unrevealing. Pathogenic variants were sought by a combination of bioinformatic filtering and cosegregation testing among affected individuals within the families. We performed function assays and generated a zebrafish morpholino knockdown model. RESULTS: A novel filamin C gene splicing variant (FLNC c.7251+1 G>A) was identified by WES in all affected family members in the two Italian families. A separate novel splicing mutation (FLNC c.5669-1delG) was identified in the US family. Western blot analysis of cardiac heart tissue from an affected individual showed decreased FLNC protein, supporting a haploinsufficiency model of pathogenesis. To further analyze this model, a morpholino knockdown of the ortholog filamin Cb in zebrafish was created which resulted in abnormal cardiac function and ultrastructure. CONCLUSIONS: Using WES, we identified two novel FLNC splicing variants as the likely cause of DCM in three families. We provided protein expression and in vivo zebrafish data supporting haploinsufficiency as the pathogenic mechanism leading to DCM.