Genome-Wide Mapping of DNA Methylation 5mC by Methylated DNA Immunoprecipitation (MeDIP)-Sequencing Methods and Protocols

Methylated DNA immunoprecipitation is a large scale purification technique. It enables the isolation of methylated DNA fragments for subsequent locus-specific or genome-wide analysis. Here we describe an immunoprecipitation protocol using a monoclonal mouse anti 5-methyl-cytidine antibody followed by next-generation sequencing (MeDIP-Seq)

Middle Pleistocene vertebrate fossils from the Nefud Desert, Saudi Arabia: Implications for biogeography and palaeoecology

The current paucity of Pleistocene vertebrate records from the Arabian Peninsula - a landmass of over 3 million km2 - is a significant gap in our knowledge of the Quaternary. Such data are critical lines of contextual evidence for considering animal and hominin dispersals between Africa and Eurasia generally, and hominin palaeoecology in the Pleistocene landscapes of the Arabian interior specifically. Here, we describe an important contribution to the record and report stratigraphically-constrained fossils of mammals, birds and reptiles from recent excavations at Ti’s al Ghadah in the southwestern Nefud Desert. Combined U-series and ESR analyses of Oryx sp. teeth indicate that the assemblage is Middle Pleistocene in age and dates to ca. 500 ka. The identified fauna is a biogeographical admixture that consists of likely endemics and taxa of African and Eurasian affinity and includes extinct and extant (or related Pleistocene forms of) mammals (Palaeoloxodon cf. recki, Panthera cf. gombaszogenis, Equus hemionus, cf. Crocuta crocuta, Vulpes sp., Canis anthus, Oryx sp.), the first Pleistocene records of birds from the Arabian Peninsula (Struthio sp., Neophron percnopterus, Milvus cf. migrans, Tachybaptus sp. Anas sp., Pterocles orientalis, Motacilla cf. alba) and reptiles (Varanidae/Uromastyx sp.). We infer that the assemblage reflects mortality in populations of herbivorous animals and their predators and scavengers that were attracted to freshwater and plant resources in the inter-dune basin. At present, there is no evidence to suggest hominin agency in the accumulation of the bone assemblages. The inferred ecological characteristics of the taxa recovered indicate the presence, at least periodically, of substantial water-bodies and open grassland habitats

Plant-RRBS, a bisulfite and next-generation sequencing-based methylome profiling method enriching for coverage of cytosine positions

Background: Cytosine methylation in plant genomes is important for the regulation of gene transcription and transposon activity. Genome-wide methylomes are studied upon mutation of the DNA methyltransferases, adaptation to environmental stresses or during development. However, from basic biology to breeding programs, there is a need to monitor multiple samples to determine transgenerational methylation inheritance or differential cytosine methylation. Methylome data obtained by sodium hydrogen sulfite (bisulfite)-conversion and next-generation sequencing (NGS) provide genome- wide information on cytosine methylation. However, a profiling method that detects cytosine methylation state dispersed over the genome would allow high-throughput analysis of multiple plant samples with distinct epigenetic signatures. We use specific restriction endonucleases to enrich for cytosine coverage in a bisulfite and NGS-based profiling method, which was compared to whole-genome bisulfite sequencing of the same plant material. Methods: We established an effective methylome profiling method in plants, termed plant-reduced representation bisulfite sequencing (plant-RRBS), using optimized double restriction endonuclease digestion, fragment end repair, adapter ligation, followed by bisulfite conversion, PCR amplification and NGS. We report a performant laboratory protocol and a straightforward bioinformatics data analysis pipeline for plant-RRBS, applicable for any reference-sequenced plant species. Results: As a proof of concept, methylome profiling was performed using an Oryza sativa ssp. indica pure breeding line and a derived epigenetically altered line (epiline). Plant-RRBS detects methylation levels at tens of millions of cytosine positions deduced from bisulfite conversion in multiple samples. To evaluate the method, the coverage of cytosine positions, the intra-line similarity and the differential cytosine methylation levels between the pure breeding line and the epiline were determined. Plant-RRBS reproducibly covers commonly up to one fourth of the cytosine positions in the rice genome when using MspI-DpnII within a group of five biological replicates of a line. The method predominantly detects cytosine methylation in putative promoter regions and not-annotated regions in rice. Conclusions: Plant-RRBS offers high-throughput and broad, genome- dispersed methylation detection by effective read number generation obtained from reproducibly covered genome fractions using optimized endonuclease combinations, facilitating comparative analyses of multi-sample studies for cytosine methylation and transgenerational stability in experimental material and plant breeding populations

Calibration of the charge and energy loss per unit length of the MicroBooNE liquid argon time projection chamber using muons and protons

We describe a method used to calibrate the position- and time-dependent response of the MicroBooNE liquid argon time projection chamber anode wires to ionization particle energy loss. The method makes use of crossing cosmic-ray muons to partially correct anode wire signals for multiple effects as a function of time and position, including cross-connected TPC wires, space charge effects, electron attachment to impurities, diffusion, and recombination. The overall energy scale is then determined using fully-contained beam-induced muons originating and stopping in the active region of the detector. Using this method, we obtain an absolute energy scale uncertainty of 2% in data. We use stopping protons to further refine the relation between the measured charge and the energy loss for highly-ionizing particles. This data-driven detector calibration improves both the measurement of total deposited energy and particle identification based on energy loss per unit length as a function of residual range. As an example, the proton selection efficiency is increased by 2% after detector calibration

Reconstruction and measurement of (100) MeV energy electromagnetic activity from π0 arrow γγ decays in the MicroBooNE LArTPC

We present results on the reconstruction of electromagnetic (EM) activity from photons produced in charged current νμ interactions with final state π0s. We employ a fully-automated reconstruction chain capable of identifying EM showers of (100) MeV energy, relying on a combination of traditional reconstruction techniques together with novel machine-learning approaches. These studies demonstrate good energy resolution, and good agreement between data and simulation, relying on the reconstructed invariant π0 mass and other photon distributions for validation. The reconstruction techniques developed are applied to a selection of νμ + Ar → μ + π0 + X candidate events to demonstrate the potential for calorimetric separation of photons from electrons and reconstruction of π0 kinematics

ReadDepth: A Parallel R Package for Detecting Copy Number Alterations from Short Sequencing Reads

Copy number alterations are important contributors to many genetic diseases, including cancer. We present the readDepth package for R, which can detect these aberrations by measuring the depth of coverage obtained by massively parallel sequencing of the genome. In addition to achieving higher accuracy than existing packages, our tool runs much faster by utilizing multi-core architectures to parallelize the processing of these large data sets. In contrast to other published methods, readDepth does not require the sequencing of a reference sample, and uses a robust statistical model that accounts for overdispersed data. It includes a method for effectively increasing the resolution obtained from low-coverage experiments by utilizing breakpoint information from paired end sequencing to do positional refinement. We also demonstrate a method for inferring copy number using reads generated by whole-genome bisulfite sequencing, thus enabling integrative study of epigenomic and copy number alterations. Finally, we apply this tool to two genomes, showing that it performs well on genomes sequenced to both low and high coverage. The readDepth package runs on Linux and MacOSX, is released under the Apache 2.0 license, and is available at http://code.google.com/p/readdepth/

Analytic philosophy for biomedical research: the imperative of applying yesterday's timeless messages to today's impasses

The mantra that "the best way to predict the future is to invent it" (attributed to the computer scientist Alan Kay) exemplifies some of the expectations from the technical and innovative sides of biomedical research at present. However, for technical advancements to make real impacts both on patient health and genuine scientific understanding, quite a number of lingering challenges facing the entire spectrum from protein biology all the way to randomized controlled trials should start to be overcome. The proposal in this chapter is that philosophy is essential in this process. By reviewing select examples from the history of science and philosophy, disciplines which were indistinguishable until the mid-nineteenth century, I argue that progress toward the many impasses in biomedicine can be achieved by emphasizing theoretical work (in the true sense of the word 'theory') as a vital foundation for experimental biology. Furthermore, a philosophical biology program that could provide a framework for theoretical investigations is outlined

Measurements of neutrino oscillation in appearance and disappearance channels by the T2K experiment with 6.6 x 10(20) protons on target

111 pages, 45 figures, submitted to Physical Review D. Minor revisions to text following referee comments111 pages, 45 figures, submitted to Physical Review D. Minor revisions to text following referee comments111 pages, 45 figures, submitted to Physical Review D. Minor revisions to text following referee commentsWe thank the J-PARC staff for superb accelerator performance and the CERN NA61/SHINE Collaboration for providing valuable particle production data. We acknowledge the support of MEXT, Japan; NSERC, NRC, and CFI, Canada; CEA and CNRS/IN2P3, France; DFG, Germany; INFN, Italy; National Science Centre (NCN), Poland; RSF, RFBR and MES, Russia; MINECO and ERDF funds, Spain; SNSF and SER, Switzerland; STFC, UK; and the U. S. Deparment of Energy, USA. We also thank CERN for the UA1/NOMAD magnet, DESY for the HERA-B magnet mover system, NII for SINET4, the WestGrid and SciNet consortia in Compute Canada, GridPP, UK, and the Emerald High Performance Computing facility in the Centre for Innovation, UK. In addition, participation of individual researchers and institutions has been further supported by funds from ERC (FP7), EU; JSPS, Japan; Royal Society, UK; and DOE Early Career program, USA

Measurement of the electron neutrino charged-current interaction rate on water with the T2K ND280 pi(0) detector

10 pages, 6 figures, Submitted to PRDhttp://journals.aps.org/prd/abstract/10.1103/PhysRevD.91.112010© 2015 American Physical Society11 pages, 6 figures, as accepted to PRD11 pages, 6 figures, as accepted to PRD11 pages, 6 figures, as accepted to PR

A Powerful Method for Transcriptional Profiling of Specific Cell Types in Eukaryotes: Laser-Assisted Microdissection and RNA Sequencing

The acquisition of distinct cell fates is central to the development of multicellular organisms and is largely mediated by gene expression patterns specific to individual cells and tissues. A spatially and temporally resolved analysis of gene expression facilitates the elucidation of transcriptional networks linked to cellular identity and function. We present an approach that allows cell type-specific transcriptional profiling of distinct target cells, which are rare and difficult to access, with unprecedented sensitivity and resolution. We combined laser-assisted microdissection (LAM), linear amplification starting from <1 ng of total RNA, and RNA-sequencing (RNA-Seq). As a model we used the central cell of the Arabidopsis thaliana female gametophyte, one of the female gametes harbored in the reproductive organs of the flower. We estimated the number of expressed genes to be more than twice the number reported previously in a study using LAM and ATH1 microarrays, and identified several classes of genes that were systematically underrepresented in the transcriptome measured with the ATH1 microarray. Among them are many genes that are likely to be important for developmental processes and specific cellular functions. In addition, we identified several intergenic regions, which are likely to be transcribed, and describe a considerable fraction of reads mapping to introns and regions flanking annotated loci, which may represent alternative transcript isoforms. Finally, we performed a de novo assembly of the transcriptome and show that the method is suitable for studying individual cell types of organisms lacking reference sequence information, demonstrating that this approach can be applied to most eukaryotic organisms