Double Digest RADseq: An Inexpensive Method for De Novo SNP Discovery and Genotyping in Model and Non-Model Species

The ability to efficiently and accurately determine genotypes is a keystone technology in modern genetics, crucial to studies ranging from clinical diagnostics, to genotype-phenotype association, to reconstruction of ancestry and the detection of selection. To date, high capacity, low cost genotyping has been largely achieved via “SNP chip” microarray-based platforms which require substantial prior knowledge of both genome sequence and variability, and once designed are suitable only for those targeted variable nucleotide sites. This method introduces substantial ascertainment bias and inherently precludes detection of rare or population-specific variants, a major source of information for both population history and genotype-phenotype association. Recent developments in reduced-representation genome sequencing experiments on massively parallel sequencers (commonly referred to as RAD-tag or RADseq) have brought direct sequencing to the problem of population genotyping, but increased cost and procedural and analytical complexity have limited their widespread adoption. Here, we describe a complete laboratory protocol, including a custom combinatorial indexing method, and accompanying software tools to facilitate genotyping across large numbers (hundreds or more) of individuals for a range of markers (hundreds to hundreds of thousands). Our method requires no prior genomic knowledge and achieves per-site and per-individual costs below that of current SNP chip technology, while requiring similar hands-on time investment, comparable amounts of input DNA, and downstream analysis times on the order of hours. Finally, we provide empirical results from the application of this method to both genotyping in a laboratory cross and in wild populations. Because of its flexibility, this modified RADseq approach promises to be applicable to a diversity of biological questions in a wide range of organisms

Transcriptomics of Tasmanian devil (Sarcophilus harrisii) ear tissue reveals homogeneous gene expression patterns across a heterogeneous landscape

In an era of unprecedented global change, exploring patterns of gene expression among wild populations across their geographic range is crucial for characterizing adaptive potential. RNA-sequencing studies have successfully characterized gene expression differences among populations experiencing divergent environmental conditions in a wide variety of taxa. However, few of these studies have identified transcriptomic signatures to multivariate, environmental stimuli among populations in their natural environments. Herein, we aim to identify environmental and sex-driven patterns of gene expression in the Tasmanian devil (Sarcophilus harrisii), a critically endangered species that occupies a heterogeneous environment. We performed RNA-sequencing on ear tissue biopsies from adult male and female devils from three populations at the extremes of their geographic range. There were no transcriptome-wide patterns of differential gene expression that would be suggestive of significant, environmentally-driven transcriptomic responses. The general lack of transcriptome-wide variation in gene expression levels across the devil’s geographic range is consistent with previous studies that documented low levels of genetic variation in the species. However, genes previously implicated in local adaptation to abiotic environment in devils were enriched for differentially expressed genes. Additionally, three modules of co-expressed genes were significantly associated with either population of origin or sex

Genomics of Divergence along a Continuum of Parapatric Population Differentiation

MM received funding from the Max Planck innovation funds for this project. PGDF was supported by a Marie Curie European Reintegration Grant (proposal nr 270891). CE was supported by German Science Foundation grants (DFG, EI 841/4-1 and EI 841/6-1)

The devil is in the details: Genomics of transmissible cancers in Tasmanian devils

Cancer poses one of the greatest human health threats of our time. Fortunately, aside from a few rare cases of cancer transmission in immune-suppressed organ transplant recipients or a small number of transmission events from mother to fetus, cancers are not spread from human to human. However, transmissible cancers have been detected in vertebrate and invertebrate animals, sometimes with devastating effects. Four examples of transmissible cancers are now known: 1) canine transmissible venereal tumor (CTVT) in dogs, 2) a tumor in a laboratory population of Syrian hamsters that is no longer cultured, 3) infectious neoplasias in at least four species of bivalve mollusks, and 4) two independently derived transmissible cancers (devil facial tumor disease [DFTD]) in Tasmanian devils (Fig 1A and 1B). The etiologic agents of CTVT, the bivalve cancers, and DFTDare the transplants (allografts) of the neoplastic cells themselves, but the etiologic agent is unknown for the hamster tumor.The effects of these transmissible cancers on their respective host populations vary. CTVT is spread in dogs through sexual contact and is at least 11,000 years old, placing the timing of its origin close to that of the domestication of dogs. Although genomic analyses of the tumor suggest evasion of multiple components of the dog immune system, dogs most commonly survive and often show evidence of spontaneous tumor regression within a year of initial diagnosis. For the infectious bivalve neoplasias, which have existed for at least 40 years, population effects vary from enzootic infections with no noticeable effects on population sizes to evidence of a catastrophic population decline. In Tasmanian devils (Fig 1A), the first infectious tumor discovered (DFT1; Fig 1B) has spread across approximately 95% of the geographic range of Tasmanian devils since 1996 (Fig 1C). DFTD is almost always fatal (Fig 1B), with >90% declines in infected localities and an overall species-wide decline exceeding 80%. Transmission dynamics appear consistent with frequency dependence, with DFTD spread by biting during social interactions, resulting in predictions of extinction from standard epidemiological models. Despite these predictions, long-infected devil populations persist at reduced densities, suggesting that individual-level variability in fecundity and tumor growth rate in infected individuals are key for understanding epidemiological dynamics. Additionally, the origin of the second, independent lineage of DFTD (i.e., DFT2) within 20 years of the discovery of DFT1 suggests that transmissible cancers may be a recurring part of the Tasmanian devils' evolutionary history, without causing extinction

The genomic basis of tumor regression in Tasmanian devils (Sarcophilus harrisii)

Understanding the genetic basis of disease-related phenotypes, such as cancer susceptibility, is crucial for the advancement of personalized medicine. Although most cancers are somatic in origin, a small number of transmissible cancers have been documented. Two such cancers have emerged in the Tasmanian devil (Sarcophilus harrisii) and now threaten the species with extinction. Recently, cases of natural tumor regression in Tasmanian devils infected with the clonally contagious cancer have been detected. We used whole-genome sequencing and FST-based approaches to identify the genetic basis of tumor regression by comparing the genomes of seven individuals that underwent tumor regression with those of three infected individuals that did not. We found three highly differentiated candidate genomic regions containing several genes related to immune response and/or cancer risk, indicating that the genomic basis of tumor regression was polygenic. Within these genomic regions, we identified putative regulatory variation in candidate genes but no nonsynonymous variation, suggesting that natural tumor regression may be driven, at least in part, by differential host expression of key loci. Comparative oncology can provide insight into the genetic basis of cancer risk, tumor development, and the pathogenicity of cancer, particularly due to our limited ability to monitor natural, untreated tumor progression in human patients. Our results support the hypothesis that host immune response is necessary for triggering tumor regression, providing candidate genes that may translate to novel treatments in human and nonhuman cancers