Comparison of triploid and diploid rainbow trout (Oncorhynchus mykiss) fine-scale movement, migration and catchability in lowland lakes of western Washington.

Fisheries managers stock triploid (i.e., infertile, artificially produced) rainbow trout Oncorhynchus mykiss in North American lakes to support sport fisheries while minimizing the risk of genetic introgression between hatchery and wild trout. In Washington State, the Washington Department of Fish and Wildlife (WDFW) allocates approximately US $3 million annually to stock hatchery-origin rainbow trout in > 600 lakes, yet only about 10% of them are triploids. Many lakes in Washington State drain into waters that support wild anadromous steelhead O. mykiss that are listed as threatened under the U.S. Endangered Species Act. As a result, there is a strong interest in understanding the costs and benefits associated with stocking sterile, triploid rainbow trout as an alternative to traditional diploids. The objectives of this study were to compare triploid and diploid rainbow trout in terms of: (1) contribution to the sport fishery catch, (2) fine-scale movements within the study lakes, (3) rate of emigration from the lake, and (4) natural mortality. Our results demonstrated that triploid and diploid trout had similar day-night distribution patterns, but triploid trout exhibited a lower emigration rate from the lake and lower catch rates in some lakes. Overall, triploid rainbow trout represent a viable alternative to stocking of diploids, especially in lakes draining to rivers, because they are sterile, have comparable home ranges, and less often migrate.

A robust targeted sequencing approach for low input and variable quality DNA from clinical samples.

Next-generation deep sequencing of gene panels is being adopted as a diagnostic test to identify actionable mutations in cancer patient samples. However, clinical samples, such as formalin-fixed, paraffin-embedded specimens, frequently provide low quantities of degraded, poor quality DNA. To overcome these issues, many sequencing assays rely on extensive PCR amplification leading to an accumulation of bias and artifacts. Thus, there is a need for a targeted sequencing assay that performs well with DNA of low quality and quantity without relying on extensive PCR amplification. We evaluate the performance of a targeted sequencing assay based on Oligonucleotide Selective Sequencing, which permits the enrichment of genes and regions of interest and the identification of sequence variants from low amounts of damaged DNA. This assay utilizes a repair process adapted to clinical FFPE samples, followed by adaptor ligation to single stranded DNA and a primer-based capture technique. Our approach generates sequence libraries of high fidelity with reduced reliance on extensive PCR amplification-this facilitates the accurate assessment of copy number alterations in addition to delivering accurate single nucleotide variant and insertion/deletion detection. We apply this method to capture and sequence the exons of a panel of 130 cancer-related genes, from which we obtain high read coverage uniformity across the targeted regions at starting input DNA amounts as low as 10 ng per sample. We demonstrate the performance using a series of reference DNA samples, and by identifying sequence variants in DNA from matched clinical samples originating from different tissue types.

High-throughput droplet digital PCR system for absolute quantitation of DNA copy number.

Digital PCR enables the absolute quantitation of nucleic acids in a sample. The lack of scalable and practical technologies for digital PCR implementation has hampered the widespread adoption of this inherently powerful technique. Here we describe a high-throughput droplet digital PCR (ddPCR) system that enables processing of ~2 million PCR reactions using conventional TaqMan assays with a 96-well plate workflow. Three applications demonstrate that the massive partitioning afforded by our ddPCR system provides orders of magnitude more precision and sensitivity than real-time PCR. First, we show the accurate measurement of germline copy number variation. Second, for rare alleles, we show sensitive detection of mutant DNA in a 100,000-fold excess of wildtype background. Third, we demonstrate absolute quantitation of circulating fetal and maternal DNA from cell-free plasma. We anticipate this ddPCR system will allow researchers to explore complex genetic landscapes, discover and validate new disease associations, and define a new era of molecular diagnostics.

Genetic dissection of a major mouse obesity QTL (Carfhg2): integration of gene expression and causality modeling.

HG.CAST-(D9Mit249-D9Mit133) (HG9) congenic mice are homozygous for CAST/EiJ chromosome (Chr) 9 alleles from approximately 9 to 84 Mbp on a C57BL6/J-hg/hg (HG) background. This region contains the carcass fat in high growth mice (Carfhg2) quantitative trait locus (QTL), and while its obesity-promoting effects have been confirmed in HG9 mice, its underlying genetic basis remains elusive. To refine the location of Carfhg2, we preformed a linkage analysis in two congenic F2 intercrosses and progeny-tested a recombinant F2 male. These analyses narrowed Carfhg2 to between 33.0 and 40.8 Mbp on Chr 9. To identify candidate genes we measured the expression of 44 transcripts surrounding the Carfhg2 peak in adipose, brain, liver, and muscle tissues from F2 mice using Biomark 48.48 Dynamic Arrays. In total, 68% (30 of the 44) of genes were regulated by a significant expression QTL (eQTL) in at least one tissue. To prioritize genes with eQTL we used Network Edge Orienting, a causality modeling tool. These analyses advance our goal of identifying the molecular basis of Carfhg2.