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Methylation of cytosines is the prototypic epigenetic modification of the DNA. It has been implicated in various regulatory mechanisms throughout the animal kingdom and particularly in vertebrates. We mapped DNA methylation in 580 animal species (535 vertebrates, 45 invertebrates), resulting in 2443 genome-scale, base-resolution DNA methylation profiles of primary tissue samples from various organs. Reference-genome independent analysis of this comprehensive dataset quantified the association of DNA methylation with the underlying genomic DNA sequence throughout vertebrate evolution. We observed a broadly conserved link with two major transitions – once in the first vertebrates and again with the emergence of reptiles. Cross-species comparisons focusing on individual organs supported a deeply conserved association of DNA methylation with tissue type, and cross-mapping analysis of DNA methylation at gene promoters revealed evolutionary changes for orthologous genes with conserved DNA methylation patterns. In summary, this study establishes a large resource of vertebrate and invertebrate DNA methylomes, it showcases the power of reference-free epigenome analysis in species for which no reference genomes are available, and it contributes an epigenetic perspective to the study of vertebrate evolution.

Autoimmune disease has both a predisposing and a protective effect toward malignancy. Though studies have investigated the risk of malignancy in patients with autoimmune disease, there is limited research on how autoimmunity affects survival.This study compared survival in patients with lung cancer with and without autoimmune disease. Patients with lung cancer were culled from the Surveillance, Epidemiology, and End Results Medicare databases (2007-2014), and autoimmune diseases were identified using diagnosis codes.The overall prevalence of investigated autoimmune diseases among the 112,445 patients was 22.7%. Overall survival (OS) (The prevalence of rheumatoid arthritis, inflammatory bowel disease, and systemic lupus erythematous was highly enriched compared with the general population. The improvement in OS and CSM was larger in NSCLC than in SCLC, suggesting a larger role for the immune system in NSCLC. Alternate explanations for the improved survival include lead time bias, better access to health care, and a survival or autoimmunity-inducing genetic factor.

Significance The axolotl is an important model organism because it is a tetrapod with a similar body plan to humans. Unlike humans, the axolotl regenerates limbs and other complex tissues. Therefore, the axolotl contributes to understanding evolution, development, and regeneration. With sophisticated tools for gene modification and tissue labeling, a fully assembled genome sequence was a sorely missing resource. Assembly was difficult because the genome size is 10× that of humans. Here, we use a cross-linking strategy called Hi-C to link together fragmented genome sequences to chromosome scale. We show that gene regulation occurs over very large genomic distances and that mitotic chromosomes are packaged efficiently., Vertebrates harbor recognizably orthologous gene complements but vary 100-fold in genome size. How chromosomal organization scales with genome expansion is unclear, and how acute changes in gene regulation, as during axolotl limb regeneration, occur in the context of a vast genome has remained a riddle. Here, we describe the chromosome-scale assembly of the giant, 32 Gb axolotl genome. Hi-C contact data revealed the scaling properties of interphase and mitotic chromosome organization. Analysis of the assembly yielded understanding of the evolution of large, syntenic multigene clusters, including the Major Histocompatibility Complex (MHC) and the functional regulatory landscape of the Fibroblast Growth Factor 8 (Axfgf8) region. The axolotl serves as a primary model for studying successful regeneration.

The axolotl (Ambystoma mexicanum) provides critical models for studying regeneration, evolution, and development. However, its large genome (∼32 Gb) presents a formidable barrier to genetic analyses. Recent efforts have yielded genome assemblies consisting of thousands of unordered scaffolds that resolve gene structures, but do not yet permit large-scale analyses of genome structure and function. We adapted an established mapping approach to leverage dense SNP typing information and for the first time assemble the axolotl genome into 14 chromosomes. Moreover, we used fluorescence in situ hybridization to verify the structure of these 14 scaffolds and assign each to its corresponding physical chromosome. This new assembly covers 27.3 Gb and encompasses 94% of annotated gene models on chromosomal scaffolds. We show the assembly's utility by resolving genome-wide orthologies between the axolotl and other vertebrates, identifying the footprints of historical introgression events that occurred during the development of axolotl genetic stocks, and precisely mapping several phenotypes including a large deletion underlying the cardiac mutant. This chromosome-scale assembly will greatly facilitate studies of the axolotl in biological research.

e17001 Background: Prostate cancer is thought of as an immunologically cold tumor, however a small proportion of patients treated with immunotherapy have impressive responses. Studies into the effect of autoimmune disease on risk of prostate cancer have yielded contradictory results and little is known about the survival of patients with concurrent autoimmune disease and prostate cancer. This study compared outcomes in patients with prostate cancer with and without autoimmune disease. Methods: This study was a retrospective analysis of patients from the SEER-Medicare databases from 2007-2014 with prostate cancer. Patients with a history of autoimmune disease were identified using ICD-9 codes. The effects of autoimmune disease on overall survival (OS) and cancer-specific survival (CSS) were estimated using multivariable Cox regression and Gray’s method respectively controlling the effects of age, race and chronic kidney disease (CKD). The cumulative CSS was estimated taking death as a competing risk. Results: The overall prevalence of investigated autoimmune diseases among the 172,061 patients with prostate cancer was 23.74%. The most common autoimmune diseases identified were rheumatoid arthritis (RA) (20.93%), psoriasis (2.43%) and ulcerative colitis (UC) (0.91%). In stage IV prostate cancer, OS (p = 0.018) and CSS (p < 0.001) were significantly higher in patients with autoimmune disease, with a median OS of 55 months compared to 48 months in patients without autoimmune disease. After adjusting for the effects of age, race, and CKD, autoimmune disease was still predictive of higher OS (HR: 1.41, 95% CI: 1.33 – 1.5, p < 0.0001) and CSS (HR: 1.30, 95% CI: 1.21 – 1.39, p < 0.0001). Patients with autoimmune disease and stage II and III prostate cancer had lower OS (p values < 0.0001) compared to patients without autoimmune disease. Conclusions: The study showed higher prevalence of RA, Crohn disease, UC, and systemic lupus erythematosus in patients with prostate cancer compared to cohorts of similar age ranges in the general population. History of autoimmune disease predicted significantly higher OS and CSS in patients with stage IV prostate cancer even when age, race and CKD were controlled for. It is possible that an improvement in OS with a history of autoimmune disease was evident in stage IV but not in stages II and III because anti-tumor autoimmune activity plays a larger role when prostate cancer effects regional lymph nodes or has distant metastases.

The lampbrush chromosomes (LBCs) in oocytes of the Mexican axolotl (Ambystoma mexicanum) were identified some time ago by their relative lengths and predicted centromeres, but they have never been associated completely with the mitotic karyotype, linkage maps or genome assembly. We identified 9 of the axolotl LBCs using RNAseq to identify actively transcribed genes and 13 BAC (bacterial artificial clone) probes containing pieces of active genes. Using read coverage analysis to find candidate centromere sequences, we developed a centromere probe that localizes to all 14 centromeres. Measurements of relative LBC arm lengths and polymerase III localization patterns enabled us to identify all LBCs. This study presents a relatively simple and reliable way to identify each axolotl LBC cytologically and to anchor chromosome-length sequences (from the axolotl genome assembly) to the physical LBCs by immunostaining and fluorescence in situ hybridization. Our data will facilitate a more detailed transcription analysis of individual LBC loops.

In the Mexican axolotl (Ambystoma mexicanum) sex is known to be determined by a single Mendelian factor, yet the sex chromosomes of this model salamander do not exhibit morphological differentiation that is typical of many vertebrate taxa that possess a single sex-determining locus. Differentiated sex chromosomes are thought to evolve rapidly in the context of a Mendelian sex-determining gene and, therefore, undifferentiated chromosomes provide an exceptional opportunity to reconstruct early events in sex chromosome evolution. Whole chromosome sequencing, whole genome resequencing (48 individuals from a backcross of axolotl and tiger salamander) andin situhybridization were used to identify a homomorphic chromosome that carries anA. mexicanumsex determining factor and identify sequences that are present only on the W chromosome. Altogether, these sequences cover ~300 kb, or roughly 1/100,000thof the ~32 Gb genome. Notably, these W-specific sequences also contain a recently duplicated copy of the ATRX gene: a known component of mammalian sex-determining pathways. This gene (designatedATRW) is one of the few functional (non-repetitive) genes in the chromosomal segment and maps to the tip of chromosome 9 near the markerE24C3, which was previously found to be linked to the sex-determining locus. These analyses provide highly predictive markers for diagnosing sex inA. mexicanumand identifyATRWas a strong candidate for the primary sex determining locus or alternately a strong candidate for a recently acquired, sexually antagonistic gene.AUTHOR SUMMARYSex chromosomes are thought to follow fairly stereotypical evolutionary trajectories that result in differentiation of sex-specific chromosomes. In the salamander A. mexicanum (the axolotl), sex is determined by a single Mendelian locus, yet the sex chromosomes are essentially undifferentiated, suggesting that these sex chromosomes have recently acquired a sex locus and are in the early stages of differentiating. Although Mendelian sex determination was first reported for the axolotl more than 70 years ago, no sex-specific sequences have been identified for this important model species. Here, we apply new technologies and approaches to identify and validate a tiny region of female-specific DNA within the gigantic genome of the axolotl (1/100,000th of the genome). This region contains a limited number of genes, including a duplicate copy of the ATRX gene which, has been previously shown to contribute to mammalian sex determination. Our analyses suggest that this gene, which we refer to as ATRW, evolved from a recent duplication and presents a strong candidate for the primary sex determining factor of the axolotl, or alternately a recently evolved sexually antagonistic gene.

The sea lamprey (Petromyzon marinus) serves as a comparative model for reconstructing vertebrate evolution. To enable more informed analyses, we developed a new assembly of the lamprey germline genome that integrates several complementary data sets. Analysis of this highly contiguous (chromosome-scale) assembly shows that both chromosomal and whole-genome duplications have played significant roles in the evolution of ancestral vertebrate and lamprey genomes, including chromosomes that carry the six lamprey HOX clusters. The assembly also contains several hundred genes that are reproducibly eliminated from somatic cells during early development in lamprey. Comparative analyses show that gnathostome (mouse) homologs of these genes are frequently marked by polycomb repressive complexes (PRCs) in embryonic stem cells, suggesting overlaps in the regulatory logic of somatic DNA elimination and bivalent states that are regulated by early embryonic PRCs. This new assembly will enhance diverse studies that are informed by lampreys’ unique biology and evolutionary/comparative perspective.

It is generally accepted that many genes present in vertebrate genomes owe their origin to two whole-genome duplications that occurred deep in the ancestry of the vertebrate lineage. However, details regarding the timing and outcome of these duplications are not well resolved. We present high-density meiotic and comparative genomic maps for the sea lamprey (Petromyzon marinus), a representative of an ancient lineage that diverged from all other vertebrates ∼550 million years ago. Linkage analyses yielded a total of 95 linkage groups, similar to the estimated number of germline chromosomes (1n ∼ 99), spanning a total of 5570.25 cM. Comparative mapping data yield strong support for the hypothesis that a single whole-genome duplication occurred in the basal vertebrate lineage, but do not strongly support a hypothetical second event. Rather, these comparative maps reveal several evolutionarily independent segmental duplications occurring over the last 600+ million years of chordate evolution. This refined history of vertebrate genome duplication should permit more precise investigations of vertebrate evolution.

Genetic linkage maps are fundamental resources that enable diverse genetic and genomic approaches, including quantitative trait locus (QTL) analyses and comparative studies of genome evolution. It is straightforward to build linkage maps for species that are amenable to laboratory culture and genetic crossing designs, and that have relatively small genomes and few chromosomes. It is more difficult to generate linkage maps for species that do not meet these criteria. Here, we introduce a method to rapidly build linkage maps for salamanders, which are known for their enormous genome sizes. As proof of principle, we developed a linkage map with thousands of molecular markers (N=2349) for the Eastern newt ( Notophthalmus viridescens ). The map contains 12 linkage groups (152.3–934.7 cM), only one more than the number of chromosome pairs. Importantly, this map was generated using RNA isolated from a single wild caught female and her 28 offspring. We used the map to reveal chromosome-scale conservation of synteny among N. viridescens, A. mexicanum (Urodela), and chicken (Amniota), and to identify large conserved segments between N. viridescens and Xenopus tropicalis (Anura). We also show that met1, a major effect QTL that regulates the expression of alternate metamorphic and paedomorphic modes of development in Ambystoma, associates with a chromosomal fusion that is not found in the N. viridescens map. Our results shed new light on the ancestral amphibian karyotype and reveal specific fusion and translocation events that shaped the genomes of three amphibian model taxa. The ability to rapidly build linkage maps for large salamander genomes will enable genetic and genomic analyses within this important vertebrate group, and more generally, empower comparative studies of vertebrate biology and evolution.

Vertebrates exhibit substantial diversity in genome size and some of the largest genomes exist in species that uniquely inform diverse areas of basic and biomedical research. For example, the salamander Ambystoma mexicanum (the Mexican axolotl) is a model organism for studies of regeneration, development and genome evolution, yet its genome is ~10× larger than the human genome. As part of a hierarchical approach toward improving genome resources for the species, we generated 600 Gb of shotgun sequence data and developed methods for sequencing individual laser-captured chromosomes. Based on these data, we estimate that the A. mexicanum genome is ~32 Gb. Notably, as much as 19 Gb of the A. mexicanum genome can potentially be considered single copy, which presumably reflects the evolutionary diversification of mobile elements that accumulated during an ancient episode of genome expansion. Chromosome-targeted sequencing permitted the development of assemblies within the constraints of modern computational platforms, allowed us to place 2062 genes on the two smallest A. mexicanum chromosomes and resolves key events in the history of vertebrate genome evolution. Our analyses show that the capture and sequencing of individual chromosomes is likely to provide valuable information for the systematic sequencing, assembly and scaffolding of large genomes.

In the version of this article initially published, the present addresses for authors Dorit Hockman and Chris Amemiya were switched. The error has been corrected in the HTML and PDF versions of the article.

The sea lamprey (Petromyzon marinus) serves as a comparative model for reconstructing vertebrate evolution. To enable more informed analyses, we developed a new assembly of the lamprey germline genome that integrates several complementary datasets. Analysis of this highly contiguous (chromosome-scale) assembly reveals that both chromosomal and whole-genome duplications have played significant roles in the evolution of ancestral vertebrate and lamprey genomes, including chromosomes that carry the six lamprey HOX clusters. The assembly also contains several hundred genes that are reproducibly eliminated from somatic cells during early development in lamprey. Comparative analyses show that gnathostome (mouse) homologs of these genes are frequently marked by Polycomb Repressive Complexes (PRCs) in embryonic stem cells, suggesting overlaps in the regulatory logic of somatic DNA elimination and repressive/bivalent states that are regulated by early embryonic PRCs. This new assembly will enhance diverse studies that are informed by lampreys’ unique biology and evolutionary/comparative perspective.