Nature Methods Contents: March 2015 Volume 12 pp 161 - 272

Advertisement Understanding Cancer MetabolismSeahorse Bioscience presents: Understanding the tumor micro environment, and its effect on tumor metabolism, progression and therapy. Webinar presented by Robert Gillies, Ph.D., Moffitt Cancer Center. Watch this webinar at: www.seahorsebio.com/nature TABLE OF CONTENTS March 2015 Volume 12, Issue 3 In This Issue Editorial This Month Correspondence Research Highlights Commentary Technology Feature News and Views Brief Communications Articles Advertisement Tried. True. New. New products.More products. Click link to receive your free copy of the New 2015-16 Catalog and Product Guide. Worthington develops and produces high quality purified enzymes, proteins, nucleic acids and cell isolation kits: www.worthington-biochem.com Subscribe   Facebook   RSS   Recommend to library   Twitter   Advertisement Multicolor flow cytometry How have the advances of modern multicolor flow cytometry driven research forward, especially in the field of immunology? Access this new Collection from Nature Immunology free online. Produced with support from: BD Biosciences    IN THIS ISSUE In This Issue    EDITORIAL Mapping the epigenome   p161 doi:10.1038/nmeth.3315 The Roadmap Epigenomics Project generates valuable resources and tools and also highlights questions that still need addressing. THIS MONTH The Author File: Robert E. Campbell   p163 Vivien Marx doi:10.1038/nmeth.3294 Developing biosensors takes patience and running through the snow. Points of Significance: Split plot design   pp165 - 166 Naomi Altman and Martin Krzywinski doi:10.1038/nmeth.3293 When some factors are harder to vary than others, a split plot design can be efficient. CORRESPONDENCE dSysMap: exploring the edgetic role of disease mutations   pp167 - 168 Roberto Mosca, Jofre Tenorio-Laranga, Roger Olivella, Victor Alcalde, Arnaud Céol et al. doi:10.1038/nmeth.3289 RESEARCH HIGHLIGHTS Bigger is better for super-resolution Expansion microscopy uses enlarged samples for high-resolution imaging with conventional microscopes. Fast volumetric imaging in live samples Swept, confocally aligned planar excitation imaging is a fast light-sheet microscopy technique that can be applied to live samples such as behaving animals. Kindred cells among the crowd Modeling cell-cycle state from the transcriptional profiles of single cells can improve the ability to group cells by function. Complex regulatory control with CRISPR Guide RNAs can serve as scaffolds to flexibly recruit different effector modules to specific genomic loci. A visual aid for cellular imaging analysis A new software tool plots quantitative cell traits as cell-like glyphs. Diamonds for MRI Three groups report key steps toward nanometer-scale magnetic resonance imaging using nitrogen-vacancy centers in diamond. Methods in Brief Optogenetic positioning of organelles | Probing molecular stoichiometry at super-resolution | The genome and transcriptome of a single cell |Closing in on video-rate STED nanoscopy Tools in Brief CRISPR off-target detection | A nonblinking quantum dot |Targeted electrophysiology at any depth | Cell positioning with acoustic waves Methods  JOBS of the week PhD position - Proteogenomics Universities of Rio de Janeiro and Groningen Postdoctoral Fellow Stanford University School of Medicine Bioinformatician in the Pompeu Fabra University Universitat Pompeu Fabra (UPF) Postdoc University of California - Davis Post-doctoral Position in Computational Biology The University of Texas at San Antonio More Science jobs from Methods EVENT EMBO Practical Course: Current Methods in Cell Biology 14th Sept - 22nd Sept 2015 Heidelberg, Germany More science events from COMMENTARY The fickle P value generates irreproducible results   pp179 - 185 Lewis G Halsey, Douglas Curran-Everett, Sarah L Vowler and Gordon B Drummond doi:10.1038/nmeth.3288 The reliability and reproducibility of science are under scrutiny. However, a major cause of this lack of repeatability is not being considered: the wide sample-to-sample variability in the Pvalue. We explain why P is fickle to discourage the ill-informed practice of interpreting analyses based predominantly on this statistic. TECHNOLOGY FEATURE Probes: paths to photostability   pp187 - 190 Vivien Marx doi:10.1038/nmeth.3295 Blinking and photobleaching of fluorophores cause challenges in a whole range of imaging experiments. Here are some ways researchers are approaching fluorophore photostability. NEWS AND VIEWS Determining the epigenome using DNA alone   pp191 - 192 Coby Viner and Michael M Hoffman doi:10.1038/nmeth.3291 A computational method predicts epigenomic marks and DNA methylation patterns from DNA sequence. See also: Article by Whitaker et al. BRIEF COMMUNICATIONS Ratiometric biosensors based on dimerization-dependent fluorescent protein exchange   pp195 - 198 Yidan Ding, Jiao Li, Jhon Ralph Enterina, Yi Shen, Issan Zhang et al. doi:10.1038/nmeth.3261 This paper reports a new strategy for the rapid and efficient generation of ratiometric fluorescent sensors for a variety of cellular processes. Real-time deformability cytometry: on-the-fly cell mechanical phenotyping   pp199 - 202 Oliver Otto, Philipp Rosendahl, Alexander Mietke, Stefan Golfier, Christoph Herold et al. doi:10.1038/nmeth.3281 Real-time deformability cytometry allows the continuous mechanical characterization of cells with high throughput and is applied to distinguish cell-cycle phases, track differentiated cells and profile cell populations in whole blood. Massively parallel single-amino-acid mutagenesis   pp203 - 206 Jacob O Kitzman, Lea M Starita, Russell S Lo, Stanley Fields and Jay Shendure doi:10.1038/nmeth.3223 A comprehensive allelic series for a protein of interest with a single mutation per cDNA template can be generated using a combination of microarray-based DNA synthesis and overlap-extension mutagenesis. Reporters for sensitive and quantitative measurement of auxin response   pp207 - 210 Che-Yang Liao, Wouter Smet, Geraldine Brunoud, Saiko Yoshida, Teva Vernoux et al. doi:10.1038/nmeth.3279 Two fluorescent reporters allow high-resolution visualization of auxin response; DR5v2 is more sensitive than existing auxin response reporters, and a ratiometric version of DII-Venus named R2D2 allows for accurate quantification of auxin input. Targeted exploration and analysis of large cross-platform human transcriptomic compendia   pp211 - 214 Qian Zhu, Aaron K Wong, Arjun Krishnan, Miriam R Aure, Alicja Tadych et al. doi:10.1038/nmeth.3249 The search engine SEEK allows multigene query across a large number of human expression data sets from array and sequencing platforms. Fixation-resistant photoactivatable fluorescent proteins for CLEM   pp215 - 218 Maria G Paez-Segala, Mei G Sun, Gleb Shtengel, Sarada Viswanathan, Michelle A Baird et al. doi:10.1038/nmeth.3225 The fluorescent proteins mEos4a and mEos4b maintain their fluorescence and photoconversion after fixation with osmium. This property enables applications such as correlative super-resolution and electron microscopy. Improved and expanded Q-system reagents for genetic manipulations   pp219 - 222 Olena Riabinina, David Luginbuhl, Elizabeth Marr, Sha Liu, Mark N Wu et al. doi:10.1038/nmeth.3250 Binary expression systems such as the Gal4-UAS or Q-systems are useful tools for genetic manipulation in Drosophila. Here, improved reagents for the Q-system are described. Sequence tag-based analysis of microbial population dynamics   pp223 - 226 Sören Abel, Pia Abel zur Wiesch, Hsiao-Han Chang, Brigid M Davis, Marc Lipsitch et al. doi:10.1038/nmeth.3253 The combination of molecularly tagging bacteria prior to infection and high-throughput sequencing of infected sites allows the quantitative assessment of the founding population size and analysis of bacterial migration patterns. A sensor for quantification of macromolecular crowding in living cells   pp227 - 229 Arnold J Boersma, Inge S Zuhorn and Bert Poolman doi:10.1038/nmeth.3257 A genetically encodable, FRET-based sensor enables quantitative measurements of macromolecular crowding in living bacterial and mammalian cells. Coverage recommendations for methylation analysis by whole-genome bisulfite sequencing   pp230 - 232 Michael J Ziller, Kasper D Hansen, Alexander Meissner and Martin J Aryee doi:10.1038/nmeth.3152 Results show the minimum sequencing depth required for the discovery of differentially methylated regions at desired sensitivity and specificity and the trade-off between adding more replicates versus increasing sequencing depth. Inferring biological tasks using Pareto analysis of high-dimensional data   pp233 - 235 Yuval Hart, Hila Sheftel, Jean Hausser, Pablo Szekely, Noa Bossel Ben-Moshe et al. doi:10.1038/nmeth.3254 Pareto task inference (ParTI) computes a polytype that encloses a data set and determines the enrichment of features around the vertices (archetypes) of the polytype, which allows the identification of the task the archetype represents. Advertisement Get in Touch with Superior Gel/Blot Imaging PerformanceChemiDoc-ItTS2 Imagers capture vivid gel/blot images with a touch of the screen! Systems feature large touch interface, ultra-sensitive gel/blot detection plus new One-Touch image capture templates. UVP offers life science workflow solutions including nucleic acid isolation kits. UVP LLC (800) 452-6788, (909) 946-3197   ARTICLES Digenome-seq: genome-wide profiling of CRISPR-Cas9 off-target effects in human cells   pp237 - 243 Daesik Kim, Sangsu Bae, Jeongbin Park, Eunji Kim, Seokjoong Kim et al. doi:10.1038/nmeth.3284 In vitro digestion of genomic DNA with Cas9 and single guide RNAs (sgRNAs) yields genome-wide off-target sites at frequencies below 0.1%. Off-target sites can be further reduced with modified sgRNAs. A general method to improve fluorophores for live-cell and single-molecule microscopy   pp244 - 250 Jonathan B Grimm, Brian P English, Jiji Chen, Joel P Slaughter, Zhengjian Zhang et al. doi:10.1038/nmeth.3256 A simple and general chemical structure change to a panel of cell-permeable small-molecule fluorophores increases their brightness and photostability, which will enable improved single-molecule studies and super-resolution imaging. Ribose-seq: global mapping of ribonucleotides embedded in genomic DNA   pp251 - 257 Kyung Duk Koh, Sathya Balachander, Jay R Hesselberth and Francesca Storici doi:10.1038/nmeth.3259 Ribose-seq allows ribose nucleotide (rNMP) incorporation to be detected genome-wide in DNA at single-base resolution and is demonstrated on budding yeast. DIA-Umpire: comprehensive computational framework for data-independent acquisition proteomics   pp258 - 264 Chih-Chiang Tsou, Dmitry Avtonomov, Brett Larsen, Monika Tucholska, Hyungwon Choi et al. doi:10.1038/nmeth.3255 The computational workflow of DIA-Umpire allows untargeted peptide identificationdirectly from DIA (data-independent acquisition) proteomics data without dependence on a spectral library for data extraction Predicting the human epigenome from DNA motifs   pp265 - 272 John W Whitaker, Zhao Chen and Wei Wang doi:10.1038/nmeth.3065 The analysis pipeline Epigram finds DNA motifs predictive of histone modifications and regions that lack DNA methylation. See also: News and Views by Viner & Hoffman Advertisement Droplet Digital™ PCR (ddPCR™) is leading the wayLast year more than 150 peer-reviewed publications took advantage of Droplet Digital PCR's unmatched ability to detect rare nucleic acid species. From tracking cancer biomarkers to quantifying rare mutations, gene expression, and miniscule viral loads, Droplet Digital PCR led to more discoveries than any other digital PCR platform. Visit our ddPCR page for application guides, publication lists, and webinars that explore the world of ddPCR.   Natureevents is a fully searchable, multi-disciplinary database designed to maximise exposure for events organisers. The contents of the Natureevents Directory are now live. The digital version is available here. Find the latest scientific conferences, courses, meetings and symposia on natureevents.com. For event advertising opportunities across the Nature Publishing Group portfolio please contact natureevents@nature.com  More Nature Events