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Recent developments in high-throughput sequencing (HTS) technologies have enabled the reconstruction of complete genome sequence and nucleosome and methylation maps from ancient individuals. A diversity of degradation reactions affect DNA molecules after an organism dies, introducing specific nucleotide misincorporation patterns in HTS data that are typically exploited to distinguish modern contaminants and authentic DNA templates. In this study, we used two different DNA polymerases to amplify Illumina TruSeq DNA libraries built on DNA extracts from ancient equines. The AmpliTaq Gold, widely used in ancient DNA studies, generated amplified libraries showing typical misincorporation patterns. Such patterns were partially lost following AccuPrime Pfx amplification, for which a two-fold reduction of endogenous content was also observed. This is explained by the inability of the AccuPrime Pfx to bypass uracils, which represent the most common post-mortem base modification and derive from the post-mortem deamination of cytosines. Our study, therefore, reveals that amplification of TruSeq DNA libraries with AccuPrime Pfx increases the cost of whole-genome sequencing for samples showing substantial levels of DNA degradation and creates atypical nucleotide misincorporation patterns for data authentication. The method can, however, be exploited to identify ancient methylation marks, and potentially, nucleosome occupancy maps. Statement of significance After an organism dies, DNA molecules are subjected to degradation resulting in fragmentation and bases modifications. After high- throughput sequencing (HTS), it is possible to detect the molecular signature of these damages, demonstrating that the data generated is authentic. DNA extracts from fossil remains indeed contain, besides endogenous ancient molecules, a variety of modern contaminants, but only the former generally exhibits molecular signatures typical of post-mortem damage. Our study reveals that these signatures depend on the protocol used for amplifying DNA samples. It thus helps defining better guidelines for authenticating ancient HTS data. Our results also reveal one molecular approach that limits our ability to exploit the whole complexity of ancient DNA molecules preserved in fossil specimens but can help track ancient methylation marks. Data availability The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are contained within the paper.

Metabolomics is a widely used technology for obtaining direct measures of metabolic activities from diverse biological systems. However, ambiguous metabolite identifications are a common challenge and biochemical interpretation is often limited by incomplete and inaccurate genome-based predictions of enzyme activities (i.e. gene annotations). Metabolite, Annotation, and Gene Integration (MAGI) generates a metabolite-gene association score using a biochemical reaction network. This is calculated by a method that emphasizes consensus between metabolites and genes via biochemical reactions. To demonstrate the potential of this method, we applied MAGI to integrate sequence data and metabolomics data collected from Streptomyces coelicolor A3(2), an extensively characterized bacterium that produces diverse secondary metabolites. Our findings suggest that coupling metabolomics and genomics data by scoring consensus between the two increases the quality of both metabolite identifications and gene annotations in this organism. MAGI also made biochemical predictions for poorly annotated genes that were consistent with the extensive literature on this important organism. This limited analysis suggests that using metabolomics data has the potential to improve annotations in sequenced organisms and also provides testable hypotheses for specific biochemical functions. MAGI is freely available for academic use both as an online tool at https://magi.nersc.gov and with source code available at https://github.com/biorack/magi.

Classically, the estrogen signaling system has two core components: cytochrome P450 aromatase (CYP19), the enzyme complex that catalyzes the rate limiting step in estrogen biosynthesis; and estrogen receptors (ERs), ligand activated transcription factors that interact with the regulatory region of target genes to mediate the biological effects of estrogen. While the importance of estrogens for regulation of reproduction, development and physiology has been well-documented in gnathostome vertebrates, the evolutionary origins of estrogen as a hormone are still unclear. As invertebrates within the phylum Chordata, cephalochordates (e.g., the amphioxus of the genus Branchiostoma) are among the closest invertebrate relatives of the vertebrates and can provide critical insight into the evolution of vertebrate-specific molecules and pathways. To address this question, this paper briefly reviews relevant earlier studies that help to illuminate the history of the aromatase and ER genes, with a particular emphasis on insights from amphioxus and other invertebrates. We then present new analyses of amphioxus aromatase and ER sequence and function, including an in silico model of the amphioxus aromatase protein, and CYP19 gene analysis. CYP19 shares a conserved gene structure with vertebrates (9 coding exons) and moderate sequence conservation (40% amino acid identity with human CYP19). Modeling of the amphioxus aromatase substrate binding site and simulated docking of androstenedione in comparison to the human aromatase shows that the substrate binding site is conserved and predicts that androstenedione could be a substrate for amphioxus CYP19. The amphioxus ER is structurally similar to vertebrate ERs, but differs in sequence and key residues of the ligand binding domain. Consistent with results from other laboratories, amphioxus ER did not bind radiolabeled estradiol, nor did it modulate gene expression on an estrogen-responsive element (ERE) in the presence of estradiol, 4-hydroxytamoxifen, diethylstilbestrol, bisphenol A or genistein. Interestingly, it has been shown that a related gene, the amphioxus "steroid receptor" (SR), can be activated by estrogens and that amphioxus ER can repress this activation. CYP19, ER and SR are all primarily expressed in gonadal tissue, suggesting an ancient paracrine/autocrine signaling role, but it is not yet known how their expression is regulated and, if estrogen is actually synthesized in amphioxus, whether it has a role in mediating any biological effects. Functional studies are clearly needed to link emerging bioinformatics and in vitro molecular biology results with organismal physiology to develop an understanding of the evolution of estrogen signaling. This article is part of a Special Issue entitled 'Marine organisms'.

Although hybridization is a relatively widespread phenomenon in marine ecosystems, the ecological importance of hybrids is poorly understood. As crosses between two distinct genotypes, hybrids might express fitness characteristics similar to either parent species, or they might produce unique phenotypic attributes that make them more or less fit than either parent species. We identified a potential hybrid zone among soft corals in a back-reef community on Guam, where the broadcast-spawning species Sinularia maxima and S. polydactyla co-occur. Morphological and chemical traits confirmed the intermediate and unique status of the putative hybrid. Laboratory cross-fertilization experiments using S. maxima and S. polydactyla gametes demonstrated that barriers to hybridization are absent and that the laboratory-reared hybrids developed specific characteristics identified in adult hybrid field populations. Changes in the populations of each parent species and the hybrid were monitored over a decade. While popula...

The known range of the oyster pathogen Haplosporidium nelsoni Haskin, Stauber, and Mackin (MSX) extends along the North American Atlantic coast from Nova Scotia to Florida. Our study demonstrates that H. nelsoni is also present throughout the Gulf of Mexico. Thirty of 41 oysters (73%) sampled from sites ranging from Florida to as far south as Venezuela were positive for MSX by PCR amplification of the ribosomal rRNA gene complex. DNA sequences cloned from oysters positive for H. nelsoni were $99% identical to H. nelsoni and clearly divergent (

The ubiquitous aquatic bacterium Caulobacter crescentus is highly resistant to uranium (U) and facilitates U biomineralization and thus holds promise as an agent of U bioremediation. To gain an understanding of how C. crescentus tolerates U, we employed transposon (Tn) mutagenesis paired with deep sequencing (Tn-seq) in a global screen for genomic elements required for U resistance. Of the 3,879 annotated genes in the C. crescentus genome, 37 were found to be specifically associated with fitness under U stress, 15 of which were subsequently tested through mutational analysis. Systematic deletion analysis revealed that mutants lacking outer membrane transporters ( rsaF a and rsaF b ), a stress-responsive transcription factor ( cztR ), or a ppGpp synthetase/hydrolase ( spoT ) exhibited a significantly lower survival rate under U stress. RsaF a and RsaF b , which are homologues of TolC in Escherichia coli , have previously been shown to mediate S-layer export. Transcriptional analysis revealed upregulation of rsaF a and rsaF b by 4- and 10-fold, respectively, in the presence of U. We additionally show that rsaF a mutants accumulated higher levels of U than the wild type, with no significant increase in oxidative stress levels. Our results suggest a function for RsaF a and RsaF b in U efflux and/or maintenance of membrane integrity during U stress. In addition, we present data implicating CztR and SpoT in resistance to U stress. Together, our findings reveal novel gene targets that are key to understanding the molecular mechanisms of U resistance in C. crescentus . IMPORTANCE Caulobacter crescentus is an aerobic bacterium that is highly resistant to uranium (U) and has great potential to be used in U bioremediation, but its mechanisms of U resistance are poorly understood. We conducted a Tn-seq screen to identify genes specifically required for U resistance in C. crescentus . The genes that we identified have previously remained elusive using other omics approaches and thus provide significant insight into the mechanisms of U resistance by C. crescentus . In particular, we show that outer membrane transporters RsaF a and RsaF b , previously known as part of the S-layer export machinery, may confer U resistance by U efflux and/or by maintaining membrane integrity during U stress.

Transposon mutagenesis with next-generation sequencing (TnSeq) is a powerful approach to annotate gene function in bacteria, but existing protocols for TnSeq require laborious preparation of every sample before sequencing. Thus, the existing protocols are not amenable to the throughput necessary to identify phenotypes and functions for the majority of genes in diverse bacteria. Here, we present a method, random bar code transposon-site sequencing (RB-TnSeq), which increases the throughput of mutant fitness profiling by incorporating random DNA bar codes into Tn5 and mariner transposons and by using bar code sequencing (BarSeq) to assay mutant fitness. RB-TnSeq can be used with any transposon, and TnSeq is performed once per organism instead of once per sample. Each BarSeq assay requires only a simple PCR, and 48 to 96 samples can be sequenced on one lane of an Illumina HiSeq system. We demonstrate the reproducibility and biological significance of RB-TnSeq with Escherichia coli, Phaeobacter inhibens, Pseudomonas stutzeri, Shewanella amazonensis, and Shewanella oneidensis. To demonstrate the increased throughput of RB-TnSeq, we performed 387 successful genome-wide mutant fitness assays representing 130 different bacterium-carbon source combinations and identified 5,196 genes with significant phenotypes across the five bacteria. In P. inhibens, we used our mutant fitness data to identify genes important for the utilization of diverse carbon substrates, including a putative d-mannose isomerase that is required for mannitol catabolism. RB-TnSeq will enable the cost-effective functional annotation of diverse bacteria using mutant fitness profiling., IMPORTANCE A large challenge in microbiology is the functional assessment of the millions of uncharacterized genes identified by genome sequencing. Transposon mutagenesis coupled to next-generation sequencing (TnSeq) is a powerful approach to assign phenotypes and functions to genes. However, the current strategies for TnSeq are too laborious to be applied to hundreds of experimental conditions across multiple bacteria. Here, we describe an approach, random bar code transposon-site sequencing (RB-TnSeq), which greatly simplifies the measurement of gene fitness by using bar code sequencing (BarSeq) to monitor the abundance of mutants. We performed 387 genome-wide fitness assays across five bacteria and identified phenotypes for over 5,000 genes. RB-TnSeq can be applied to diverse bacteria and is a powerful tool to annotate uncharacterized genes using phenotype data.

Librado, Pablo et al., Yakutia is among the coldest regions in the Northern Hemisphere, showing ∼40% of its territory above the Arctic Circle. Native horses are particularly adapted to this environment, with body sizes and thick winter coats minimizing heat loss. We sequenced complete genomes of two ancient and nine present-day Yakutian horses to elucidate their evolutionary origins. We find that the contemporary population descends from domestic livestock, likely brought by early horse-riders who settled in the region a few centuries ago. The metabolic, anatomical, and physiological adaptations of these horses therefore emerged on very short evolutionary time scales. We show the relative importance of regulatory changes in the adaptive process and identify genes independently selected in cold-adapted human populations and woolly mammoths., This work was supported by the Danish Council for Independent Research, Natural Sciences (Grant 4002-00152B); the Danish National Research Foundation (Grant DNRF94); a Marie-Curie Career Integration grant (Grant FP7 CIG-293845); Initiative d'Excellence Chaires d'attractivité, Université de Toulouse (OURASI); and the International Research Group Program (Grant IRG14-08), Deanship of Scientific Research, King Saud University. P.L. was supported by a Villum Fonden Blokstipendier grant (primary investigator: L.O.); H.J. by a Marie-Curie Initial Training Network grant [EUROTAST (Exploring the History, Archeology, and New Genetics of the Transatlantic Slave Trade); Grant FP7 ITN-290344]; C.G. and L.E. by Marie-Curie Intra-European fellowships (FP7-IEF-328024 and FP7 IEF-302617); M. Schubert by a Lundbeck Foundation grant (Grant R52-A5062); M.A.Y. by a National Science Foundation Graduate Research fellowship; M.F. by a Human Frontier Science Program fellowship (LT000320/2014); A.A. by a Villum Fonden Blokstipendier grant; M. Schubert by an NIH grant (Grant R01-GM40282); and J.K. by the Academy of Finland (Grant 286040). Research work on the ancient Yakut population was supported by the French Archaeological Mission in Oriental Siberia (Ministry of Foreign and European Affairs, France), the North-Eastern Federal University (Yakutsk, Sakha Republic), and the Human Adaptation Program of the French Polar Institute Paul Emile Victor [HUMAD MAFSO (Missions Archéologiques Françaises en Sibérie Orientale) 1038].

Sinularia polydactyla, an abundant Indo-Pacific soft coral species, exhibits biochemical phenotypic plasticity, prompting investigations into differences in mRNA diversity and complexity in response to predation stress. Changes in transcriptome complexity of S. polydactyla cDNA libraries were measured using reannealing rate assays that employ an informatics-based analysis of kinetic profiles. This method allows for quick, high-throughput analysis of sequence complexity and has been used to compare transcriptome-level differences in other marine invertebrates. Here, S. polydactyla colonies were transplanted between two sites exhibiting high and low predation levels. Statistically significant differences between bite scar counts found on different transplant groups suggest site-specific variation in predation. Changes in mRNA pool complexity were quantified to indicate shifts in secondary metabolite concentration between treatment groups. Examining the complexity of the mRNA pool in this soft coral is one of the first steps toward understanding the mechanisms of phenotypic plasticity at a biochemical and molecular level.

The eastern oyster, Crassostrea virginica Gmelin, is a common inhabitant of estuarine and coastal waters from maritime Canada through the Gulf of Mexico. Because mitochondrial DNA haplotypes exhibit a distinct genetic break between Atlantic and Gulf oysters at Cape Canaveral, Florida, the degree of divergence between Atlantic and Gulf oysters in nuclear genes is less well known. We examined patterns of variation in four nuclear loci using restriction fragment analysis of amplified DNA (PCR-RFLP) in oysters (n = 317) from 16 locations spanning the geographic range of C. virginica. Marked differentiation was observed between Atlantic and Gulf populations, with smaller differences detected between North Atlantic and South Atlantic populations. Intermediate populations were observed in both eastern and northwest Florida. Regional population structure was also evident in the Gulf Coast, with Texas oysters highly divergent from all other populations.

Schubert, Michael et al., The domestication of the horse ∼5.5 kya and the emergence of mounted riding, chariotry, and cavalry dramatically transformed human civilization. However, the genetics underlying horse domestication are difficult to reconstruct, given the near extinction of wild horses. We therefore sequenced two ancient horse genomes from Taymyr, Russia (at 7.4- and 24.3-fold coverage), both predating the earliest archeological evidence of domestication. We compared these genomes with genomes of domesticated horses and the wild Przewalski's horse and found genetic structure within Eurasia in the Late Pleistocene, with the ancient population contributing significantly to the genetic variation of domesticated breeds. We furthermore identified a conservative set of 125 potential domestication targets using four complementary scans for genes that have undergone positive selection. One group of genes is involved in muscular and limb development, articular junctions, and the cardiac system, and may represent physiological adaptations to human utilization. A second group consists of genes with cognitive functions, including social behavior, learning capabilities, fear response, and agreeableness, which may have been key for taming horses. We also found that domestication is associated with inbreeding and an excess of deleterious mutations. This genetic load is in line with the "cost of domestication" hypothesis also reported for rice, tomatoes, and dogs, and it is generally attributed to the relaxation of purifying selection resulting from the strong demographic bottlenecks accompanying domestication. Our work demonstrates the power of ancient genomes to reconstruct the complex genetic changes that transformed wild animals into their domesticated forms, and the population context in which this process took place. © 2014, National Academy of Sciences. All rights reserved., This work was supported by the Danish Council for Independent Research, Natural Sciences (FNU); the Danish National Research Foundation (DNFR94); a Marie-Curie Career Integration Grant (FP7 CIG-293845); and the International Research Group Program (IRG14-08), Deanship of Scientific Research (King Saud University, Saudi Arabia). H.J. was supported by a Marie-Curie Initial Training Network Grant (EUROTAST; FP7 ITN-290344); A.G. and L. Ermini were supported by Marie-Curie Intra-European Fellowships (FP7 IEF-299176 and FP7 IEF-302617); M.S. was supported by a Lundbeck Foundation Grant (R52-A5062); I.D., A.F., and L. Excoffier were supported by a Swiss National Science Foundation Grant (31003A-143393); M.H. was supported by a European Research Council (ERC) Consolidator Grant (310763); T.M.-B. was supported by an ERC Starting Grant (260372) and by a Ministerio de Ciencia e Innovación (MICINN) Grant (BFU2011-28549); B.S. was supported by the Packard Foundation; and D.C. was supported by start-up funds to B.S. from the University of California, Santa Cruz.

Epigenetic information is available from contemporary organisms, but is difficult to track back in evolutionary time. Here, we show that genome-wide epigenetic information can be gathered directly from next-generation sequence reads of DNA isolated from ancient remains. Using the genome sequence data generated from hair shafts of a 4000-yr-old Paleo-Eskimo belonging to the Saqqaq culture, we generate the first ancient nucleosome map coupled with a genome-wide survey of cytosine methylation levels. The validity of both nucleosome map and methylation levels were confirmed by the recovery of the expected signals at promoter regions, exon/intron boundaries, and CTCF sites. The top-scoring nucleosome calls revealed distinct DNA positioning biases, attesting to nucleotide-level accuracy. The ancient methylation levels exhibited high conservation over time, clustering closely with modern hair tissues. Using ancient methylation information, we estimated the age at death of the Saqqaq individual and illustrate how epigenetic information can be used to infer ancient gene expression. Similar epigenetic signatures were found in other fossil material, such as 110,000- to 130,000-yr-old bones, supporting the contention that ancient epigenomic information can be reconstructed from a deep past. Our findings lay the foundation for extracting epigenomic information from ancient samples, allowing shifts in epialleles to be tracked through evolutionary time, as well as providing an original window into modern epigenomics.

Sinularia polydactyla, a dioecious, abundant soft coral in the South Pacific, exhibits biochemical pheno- typic plasticity in secondary metabolite production in rela- tion to predation intensity. However, it is unclear to what extent changes in secondary metabolites, such as 11- acetoxypukalide, may result from specific, induced pathway activities at the level of gene expression. To investigate both chemical changes and differences in mRNA diversity in response to predation stress, artificial predation experiments were conducted in situ on colonies of S. polydactyla. Mul- tivariate statistical analyses of coral biochemical metabo- lites and our kinetic transcriptome profiling technique indi- cate that that the induction of 11-acetoxypukalide by predation stress likely results from the upregulation of either one dominant transcript or a very small set of transcripts, indicative of a targeted upregulation rather than a general- ized, genetic stress response. Overall, this work establishes a routine method for integrating high-throughput transcrip- tome and metabolome data sets to allow for the identifica- tion of metabolites whose intracellular concentrations can be readily linked to gene expression events in response to specific treatments in non-model organisms.

A strong foundation of basic and applied research documents that the estuarine fish Fundulus heteroclitus and related species are unique laboratory and field models for understanding how individuals and populations interact with their environment. In this paper we summarize an extensive body of work examining the adaptive responses of Fundulus species to environmental conditions, and describe how this research has contributed importantly to our understanding of physiology, gene regulation, toxicology, and ecological and evolutionary genetics of teleosts and other vertebrates. These explorations have reached a critical juncture at which advancement is hindered by the lack of genomic resources for these species. We suggest that a more complete genomics toolbox for F. heteroclitus and related species will permit researchers to exploit the power of this model organism to rapidly advance our understanding of fundamental biological and pathological mechanisms among vertebrates, as well as ecological strategies and evolutionary processes common to all living organisms.

Secondary metabolites or natural products have been isolated from many marine organisms. These metabolites often have important bioactive functions; however, very little information is available regarding the biosynthesis and regulation of many secondary metabolites. At a time when use of marine-derived metabolites is rapidly expanding in industry and pharmacological fields, a better understanding of the genetic mechanisms controlling secondary metabolite production is necessary. We review the recent development of a novel transcriptome profiling methodology that allows for rapid and high-throughput screening of changes in mRNA sequence pools. The application of genomics-based techniques and the integration of both biochemical and molecular data sets in marine organisms complement ongoing drug discovery efforts.

The soft coral species, Sinularia polydactyla and Sinularia maxima are abundant in shallow coral reef habitats in Guam. Both species are gonochoric and mass spawning events can result in an apparent S. polydactyla–S. maxima hybrid exhibiting morphological characteristics of both parents. These morphological differences prompted our investigation of potential differences in gene expression patterns among these closely related species. In concert with interspecific level differences, intraspecific level differences in transcriptome diversity and complexity were also studied among juvenile, adult male, and adult female S. polydactyla colonies. To uncover these transcriptome-level differences, RNA was extracted from RNAlater®-preserved samples and cDNA libraries constructed using a nano-scale synthesis strategy. Changes in transcriptome complexity (mRNA sequence composition) of these libraries were measured using reannealing rate assays that employ an informatics-based analysis of kinetic profiles. This method allows for quick, high-throughput analysis of sequence complexity and has been used to compare transcriptome-level differences in other marine invertebrates. Comparisons of transcriptome data revealed diagnostic differences in the transcriptome pools. Examination of transcriptome changes in closely related species experiencing similar environmental parameters may enable us to understand developmental, morphological, and sex-specific changes in gene expression patterns.

An automated method for constructing libraries for 454 sequencing significantly reduces the cost and time required., We present an automated, high throughput library construction process for 454 technology. Sample handling errors and cross-contamination are minimized via end-to-end barcoding of plasticware, along with molecular DNA barcoding of constructs. Automation-friendly magnetic bead-based size selection and cleanup steps have been devised, eliminating major bottlenecks and significant sources of error. Using this methodology, one technician can create 96 sequence-ready 454 libraries in 2 days, a dramatic improvement over the standard method.