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  • Strelka2: fast and accurate calling of germline and somatic variants
    Nat. Methods (IF 26.919) Pub Date : 2018-07-16
    Sangtae Kim, Konrad Scheffler, Aaron L. Halpern, Mitchell A. Bekritsky, Eunho Noh, Morten Källberg, Xiaoyu Chen, Yeonbin Kim, Doruk Beyter, Peter Krusche, Christopher T. Saunders

    We describe Strelka2 (https://github.com/Illumina/strelka), an open-source small-variant-calling method for research and clinical germline and somatic sequencing applications. Strelka2 introduces a novel mixture-model-based estimation of insertion/deletion error parameters from each sample, an efficient tiered haplotype-modeling strategy, and a normal sample contamination model to improve liquid tumor analysis. For both germline and somatic calling, Strelka2 substantially outperformed the current leading tools in terms of both variant-calling accuracy and computing cost.

    更新日期:2018-07-18
  • An enhanced CRISPR repressor for targeted mammalian gene regulation
    Nat. Methods (IF 26.919) Pub Date : 2018-07-16
    Nan Cher Yeo, Alejandro Chavez, Alissa Lance-Byrne, Yingleong Chan, David Menn, Denitsa Milanova, Chih-Chung Kuo, Xiaoge Guo, Sumana Sharma, Angela Tung, Ryan J. Cecchi, Marcelle Tuttle, Swechchha Pradhan, Elaine T. Lim, Noah Davidsohn, Mo R. Ebrahimkhani, James J. Collins, Nathan E. Lewis, Samira Kiani, George M. Church

    The RNA-guided endonuclease Cas9 can be converted into a programmable transcriptional repressor, but inefficiencies in target-gene silencing have limited its utility. Here we describe an improved Cas9 repressor based on the C-terminal fusion of a rationally designed bipartite repressor domain, KRAB–MeCP2, to nuclease-dead Cas9. We demonstrate the system’s superiority in silencing coding and noncoding genes, simultaneously repressing a series of target genes, improving the results of single and dual guide RNA library screens, and enabling new architectures of synthetic genetic circuits.

    更新日期:2018-07-18
  • Active PSF shaping and adaptive optics enable volumetric localization microscopy through brain sections
    Nat. Methods (IF 26.919) Pub Date : 2018-07-16
    Michael J. Mlodzianoski, Paul J. Cheng-Hathaway, Shane M. Bemiller, Tyler J. McCray, Sheng Liu, David A. Miller, Bruce T. Lamb, Gary E. Landreth, Fang Huang

    Application of single-molecule switching nanoscopy (SMSN) beyond the coverslip surface poses substantial challenges due to sample-induced aberrations that distort and blur single-molecule emission patterns. We combined active shaping of point spread functions and efficient adaptive optics to enable robust 3D-SMSN imaging within tissues. This development allowed us to image through 30-μm-thick brain sections to visualize and reconstruct the morphology and the nanoscale details of amyloid-β filaments in a mouse model of Alzheimer’s disease.

    更新日期:2018-07-18
  • High-precision automated reconstruction of neurons with flood-filling networks
    Nat. Methods (IF 26.919) Pub Date : 2018-07-16
    Michał Januszewski, Jörgen Kornfeld, Peter H. Li, Art Pope, Tim Blakely, Larry Lindsey, Jeremy Maitin-Shepard, Mike Tyka, Winfried Denk, Viren Jain

    Reconstruction of neural circuits from volume electron microscopy data requires the tracing of cells in their entirety, including all their neurites. Automated approaches have been developed for tracing, but their error rates are too high to generate reliable circuit diagrams without extensive human proofreading. We present flood-filling networks, a method for automated segmentation that, similar to most previous efforts, uses convolutional neural networks, but contains in addition a recurrent pathway that allows the iterative optimization and extension of individual neuronal processes. We used flood-filling networks to trace neurons in a dataset obtained by serial block-face electron microscopy of a zebra finch brain. Using our method, we achieved a mean error-free neurite path length of 1.1 mm, and we observed only four mergers in a test set with a path length of 97 mm. The performance of flood-filling networks was an order of magnitude better than that of previous approaches applied to this dataset, although with substantially increased computational costs.

    更新日期:2018-07-18
  • A synthetic-diploid benchmark for accurate variant-calling evaluation
    Nat. Methods (IF 26.919) Pub Date : 2018-07-16
    Heng Li, Jonathan M. Bloom, Yossi Farjoun, Mark Fleharty, Laura Gauthier, Benjamin Neale, Daniel MacArthur

    Existing benchmark datasets for use in evaluating variant-calling accuracy are constructed from a consensus of known short-variant callers, and they are thus biased toward easy regions that are accessible by these algorithms. We derived a new benchmark dataset from the de novo PacBio assemblies of two fully homozygous human cell lines, which provides a relatively more accurate and less biased estimate of small-variant-calling error rates in a realistic context.

    更新日期:2018-07-18
  • Genome-wide C-SWAT library for high-throughput yeast genome tagging
    Nat. Methods (IF 26.919) Pub Date : 2018-07-09
    Matthias Meurer, Yuanqiang Duan, Ehud Sass, Ilia Kats, Konrad Herbst, Benjamin C. Buchmuller, Verena Dederer, Florian Huber, Daniel Kirrmaier, Martin Štefl, Koen Van Laer, Tobias P. Dick, Marius K. Lemberg, Anton Khmelinskii, Emmanuel D. Levy, Michael Knop

    Here we describe a C-SWAT library for high-throughput tagging of Saccharomyces cerevisiae open reading frames (ORFs). In 5,661 strains, we inserted an acceptor module after each ORF that can be efficiently replaced with tags or regulatory elements. We validated the library with targeted sequencing and tagged the proteome with bright fluorescent proteins to quantify the effect of heterologous transcription terminators on protein expression and to localize previously undetected proteins.

    更新日期:2018-07-10
  • Fast reversibly photoswitching red fluorescent proteins for live-cell RESOLFT nanoscopy
    Nat. Methods (IF 26.919) Pub Date : 2018-07-09
    Francesca Pennacchietti, Ekaterina O. Serebrovskaya, Aline R. Faro, Irina I. Shemyakina, Nina G. Bozhanova, Alexey A. Kotlobay, Nadya G. Gurskaya, Andreas Bodén, Jes Dreier, Dmitry M. Chudakov, Konstantin A. Lukyanov, Vladislav V. Verkhusha, Alexander S. Mishin, Ilaria Testa

    Reversibly photoswitchable fluorescent proteins (rsFPs) are gaining popularity as tags for optical nanoscopy because they make it possible to image with lower light doses. However, green rsFPs need violet-blue light for photoswitching, which is potentially phototoxic and highly scattering. We developed new rsFPs based on FusionRed that are reversibly photoswitchable with green-orange light. The rsFusionReds are bright and exhibit rapid photoswitching, thereby enabling nanoscale imaging of living cells.

    更新日期:2018-07-10
  • Genome-wide SWAp-Tag yeast libraries for proteome exploration
    Nat. Methods (IF 26.919) Pub Date : 2018-07-09
    Uri Weill, Ido Yofe, Ehud Sass, Bram Stynen, Dan Davidi, Janani Natarajan, Reut Ben-Menachem, Zohar Avihou, Omer Goldman, Nofar Harpaz, Silvia Chuartzman, Kiril Kniazev, Barbara Knoblach, Janina Laborenz, Felix Boos, Jacqueline Kowarzyk, Shifra Ben-Dor, Einat Zalckvar, Johannes M. Herrmann, Richard A. Rachubinski, Ophry Pines, Doron Rapaport, Stephen W. Michnick, Emmanuel D. Levy, Maya Schuldiner

    Yeast libraries revolutionized the systematic study of cell biology. To extensively increase the number of such libraries, we used our previously devised SWAp-Tag (SWAT) approach to construct a genome-wide library of ~5,500 strains carrying the SWAT NOP1promoter-GFP module at the N terminus of proteins. In addition, we created six diverse libraries that restored the native regulation, created an overexpression library with a Cherry tag, or enabled protein complementation assays from two fragments of an enzyme or fluorophore. We developed methods utilizing these SWAT collections to systematically characterize the yeast proteome for protein abundance, localization, topology, and interactions.

    更新日期:2018-07-10
  • Targeting Cas9
    Nat. Methods (IF 26.919) Pub Date : 2018-07-02
    Nicole Rusk

    Targeting Cas9Targeting Cas9, Published online: 02 July 2018; doi:10.1038/s41592-018-0061-8Targeting Cas9

    更新日期:2018-07-05
  • Inferring neuronal activity from gene expression
    Nat. Methods (IF 26.919) Pub Date : 2018-07-02
    Nina Vogt

    Inferring neuronal activity from gene expressionInferring neuronal activity from gene expression, Published online: 02 July 2018; doi:10.1038/s41592-018-0064-5Inferring neuronal activity from gene expression

    更新日期:2018-07-05
  • Metastasis in a dish
    Nat. Methods (IF 26.919) Pub Date : 2018-07-02
    Tal Nawy

    Metastasis in a dishMetastasis in a dish, Published online: 02 July 2018; doi:10.1038/s41592-018-0067-2Metastasis in a dish

    更新日期:2018-07-05
  • Weighing single proteins with light
    Nat. Methods (IF 26.919) Pub Date : 2018-07-02
    Rita Strack

    Weighing single proteins with lightWeighing single proteins with light, Published online: 02 July 2018; doi:10.1038/s41592-018-0055-6Interferometric scattering microscopy enables quantitative mass imaging and biophysical characterization of single biomolecules in solution.

    更新日期:2018-07-05
  • Sensing cellular metabolites
    Nat. Methods (IF 26.919) Pub Date : 2018-07-02
    Rita Strack

    Sensing cellular metabolitesSensing cellular metabolites, Published online: 02 July 2018; doi:10.1038/s41592-018-0062-7Sensing cellular metabolites

    更新日期:2018-07-05
  • StaPLs: versatile genetically encoded modules for engineering drug-inducible proteins
    Nat. Methods (IF 26.919) Pub Date : 2018-07-02
    Conor L. Jacobs, Ryan K. Badiee, Michael Z. Lin

    Robust approaches for chemogenetic control of protein function would have many biological applications. We developed stabilizable polypeptide linkages (StaPLs) based on hepatitis C virus protease. StaPLs undergo autoproteolysis to cleave proteins by default, whereas protease inhibitors prevent cleavage and preserve protein function. We created StaPLs responsive to different clinically approved drugs to bidirectionally control transcription with zinc-finger-based effectors, and used StaPLs to create single-chain, drug-stabilizable variants of CRISPR–Cas9 and caspase-9.

    更新日期:2018-07-02
  • Correlating behavior and neural activity at high resolution
    Nat. Methods (IF 26.919) Pub Date : 2018-07-02
    Nina Vogt

    Correlating behavior and neural activity at high resolutionCorrelating behavior and neural activity at high resolution, Published online: 02 July 2018; doi:10.1038/s41592-018-0057-4A combination of behavioral analysis and neural activity recording provides a glimpse into how the brain orchestrates complex behaviors.

    更新日期:2018-07-02
  • Chemogenetic control of gene expression and cell signaling with antiviral drugs
    Nat. Methods (IF 26.919) Pub Date : 2018-07-02
    Elliot P. Tague, Hannah L. Dotson, Shannon N. Tunney, D. Christopher Sloas, John T. Ngo

    We developed a method in which the NS3 cis-protease from hepatitis C virus can be used as a ligand-inducible connection to control the function and localization of engineered proteins in mammalian cells. To demonstrate the versatility of this approach, we designed drug-sensitive transcription factors and transmembrane signaling proteins, the activities of which can be tightly and reversibly controlled through the use of clinically tested antiviral protease inhibitors.

    更新日期:2018-07-02
  • Transcripts from a spliceosome
    Nat. Methods (IF 26.919) Pub Date : 2018-07-02
    Tal Nawy

    Transcripts from a spliceosomeTranscripts from a spliceosome, Published online: 02 July 2018; doi:10.1038/s41592-018-0058-3Two new methods provide high-resolution profiles of splicing events in yeast.

    更新日期:2018-07-02
  • Bioconda: sustainable and comprehensive software distribution for the life sciences
    Nat. Methods (IF 26.919) Pub Date : 2018-07-02
    Björn Grüning, Ryan Dale, Andreas Sjödin, Brad A. Chapman, Jillian Rowe, Christopher H. Tomkins-Tinch, Renan Valieris, Johannes Köster

    Bioconda: sustainable and comprehensive software distribution for the life sciencesBioconda: sustainable and comprehensive software distribution for the life sciences, Published online: 02 July 2018; doi:10.1038/s41592-018-0046-7Bioconda: sustainable and comprehensive software distribution for the life sciences

    更新日期:2018-07-02
  • US universities go East
    Nat. Methods (IF 26.919) Pub Date : 2018-07-02

    US universities go EastUS universities go East, Published online: 02 July 2018; doi:10.1038/s41592-018-0060-9The global expansion of Western universities is not without challenges but can provide manifold opportunities, particularly in the Middle East.

    更新日期:2018-07-02
  • Efficient and precise editing of endogenous transcripts with SNAP-tagged ADARs
    Nat. Methods (IF 26.919) Pub Date : 2018-07-02
    Paul Vogel, Matin Moschref, Qin Li, Tobias Merkle, Karthika D. Selvasaravanan, Jin Billy Li, Thorsten Stafforst

    Molecular tools that target RNA at specific sites allow recoding of RNA information and processing. SNAP-tagged deaminases guided by a chemically stabilized guide RNA can edit targeted adenosine to inosine in several endogenous transcripts simultaneously, with high efficiency (up to 90%), high potency, sufficient editing duration, and high precision. We used adenosine deaminases acting on RNA (ADARs) fused to SNAP-tag for the efficient and concurrent editing of two disease-relevant signaling transcripts, KRAS and STAT1. We also demonstrate improved performance compared with that of the recently described Cas13b–ADAR.

    更新日期:2018-07-02
  • Controlling protein function with HCV protease
    Nat. Methods (IF 26.919) Pub Date : 2018-07-02
    Bryan C. Dickinson

    Controlling protein function with HCV proteaseControlling protein function with HCV protease, Published online: 02 July 2018; doi:10.1038/s41592-018-0043-xTwo methods for controlling protein activities with small molecules provide a general solution to a long-standing challenge in mammalian synthetic biology.

    更新日期:2018-07-02
  • A reassessment of DNA-immunoprecipitation-based genomic profiling
    Nat. Methods (IF 26.919) Pub Date : 2018-06-25
    Antonio Lentini, Cathrine Lagerwall, Svante Vikingsson, Heidi K. Mjoseng, Karolos Douvlataniotis, Hartmut Vogt, Henrik Green, Richard R. Meehan, Mikael Benson, Colm E. Nestor

    DNA immunoprecipitation followed by sequencing (DIP-seq) is a common enrichment method for profiling DNA modifications in mammalian genomes. However, the results of independent DIP-seq studies often show considerable variation between profiles of the same genome and between profiles obtained by alternative methods. Here we show that these differences are primarily due to the intrinsic affinity of IgG for short unmodified DNA repeats. This pervasive experimental error accounts for 50–99% of regions identified as ‘enriched’ for DNA modifications in DIP-seq data. Correction of this error profoundly altered DNA-modification profiles for numerous cell types, including mouse embryonic stem cells, and subsequently revealed novel associations among DNA modifications, chromatin modifications and biological processes. We conclude that both matched input and IgG controls are essential in order for the results of DIP-based assays to be interpreted correctly, and that complementary, non-antibody-based techniques should be used to validate DIP-based findings to avoid further misinterpretation of genome-wide profiling data.

    更新日期:2018-06-27
  • DeTiN: overcoming tumor-in-normal contamination
    Nat. Methods (IF 26.919) Pub Date : 2018-06-25
    Amaro Taylor-Weiner, Chip Stewart, Thomas Giordano, Mendy Miller, Mara Rosenberg, Alyssa Macbeth, Niall Lennon, Esther Rheinbay, Dan-Avi Landau, Catherine J. Wu, Gad Getz

    Comparison of sequencing data from a tumor sample with data from a matched germline control is a key step for accurate detection of somatic mutations. Detection sensitivity for somatic variants is greatly reduced when the matched normal sample is contaminated with tumor cells. To overcome this limitation, we developed deTiN, a method that estimates the tumor-in-normal (TiN) contamination level and, in cases affected by contamination, improves sensitivity by reclassifying initially discarded variants as somatic.

    更新日期:2018-06-27
  • SAVER: gene expression recovery for single-cell RNA sequencing
    Nat. Methods (IF 26.919) Pub Date : 2018-06-25
    Mo Huang, Jingshu Wang, Eduardo Torre, Hannah Dueck, Sydney Shaffer, Roberto Bonasio, John I. Murray, Arjun Raj, Mingyao Li, Nancy R. Zhang

    In single-cell RNA sequencing (scRNA-seq) studies, only a small fraction of the transcripts present in each cell are sequenced. This leads to unreliable quantification of genes with low or moderate expression, which hinders downstream analysis. To address this challenge, we developed SAVER (single-cell analysis via expression recovery), an expression recovery method for unique molecule index (UMI)-based scRNA-seq data that borrows information across genes and cells to provide accurate expression estimates for all genes.

    更新日期:2018-06-27
  • GeNets: a unified web platform for network-based genomic analyses
    Nat. Methods (IF 26.919) Pub Date : 2018-06-18
    Taibo Li, April Kim, Joseph Rosenbluh, Heiko Horn, Liraz Greenfeld, David An, Andrew Zimmer, Arthur Liberzon, Jon Bistline, Ted Natoli, Yang Li, Aviad Tsherniak, Rajiv Narayan, Aravind Subramanian, Ted Liefeld, Bang Wong, Dawn Thompson, Sarah Calvo, Steve Carr, Jesse Boehm, Jake Jaffe, Jill Mesirov, Nir Hacohen, Aviv Regev, Kasper Lage

    Functional genomics networks are widely used to identify unexpected pathway relationships in large genomic datasets. However, it is challenging to compare the signal-to-noise ratios of different networks and to identify the optimal network with which to interpret a particular genetic dataset. We present GeNets, a platform in which users can train a machine-learning model (Quack) to carry out these comparisons and execute, store, and share analyses of genetic and RNA-sequencing datasets.

    更新日期:2018-06-18
  • EASI-tag enables accurate multiplexed and interference-free MS2-based proteome quantification
    Nat. Methods (IF 26.919) Pub Date : 2018-06-18
    Sebastian Virreira Winter, Florian Meier, Christoph Wichmann, Juergen Cox, Matthias Mann, Felix Meissner

    We developed EASI-tag (easily abstractable sulfoxide-based isobaric-tag), a new type of amine-derivatizing and sulfoxide-containing isobaric labeling reagents for highly accurate quantitative proteomics analysis using mass spectrometry. We observed that EASI-tag labels dissociate at low collision energy and generate peptide-coupled, interference-free reporter ions with high yield. Efficient isolation of 12C precursors and quantification at the MS2 level allowed accurate determination of quantitative differences between up to six multiplexed samples.

    更新日期:2018-06-18
  • A comparison of methods to assess cell mechanical properties
    Nat. Methods (IF 26.919) Pub Date : 2018-06-18
    Pei-Hsun Wu, Dikla Raz-Ben Aroush, Atef Asnacios, Wei-Chiang Chen, Maxim E. Dokukin, Bryant L. Doss, Pauline Durand-Smet, Andrew Ekpenyong, Jochen Guck, Nataliia V. Guz, Paul A. Janmey, Jerry S. H. Lee, Nicole M. Moore, Albrecht Ott, Yeh-Chuin Poh, Robert Ros, Mathias Sander, Igor Sokolov, Jack R. Staunton, Ning Wang, Graeme Whyte, Denis Wirtz

    The mechanical properties of cells influence their cellular and subcellular functions, including cell adhesion, migration, polarization, and differentiation, as well as organelle organization and trafficking inside the cytoplasm. Yet reported values of cell stiffness and viscosity vary substantially, which suggests differences in how the results of different methods are obtained or analyzed by different groups. To address this issue and illustrate the complementarity of certain approaches, here we present, analyze, and critically compare measurements obtained by means of some of the most widely used methods for cell mechanics: atomic force microscopy, magnetic twisting cytometry, particle-tracking microrheology, parallel-plate rheometry, cell monolayer rheology, and optical stretching. These measurements highlight how elastic and viscous moduli of MCF-7 breast cancer cells can vary 1,000-fold and 100-fold, respectively. We discuss the sources of these variations, including the level of applied mechanical stress, the rate of deformation, the geometry of the probe, the location probed in the cell, and the extracellular microenvironment.

    更新日期:2018-06-18
  • Author Correction: Informed-Proteomics: open-source software package for top-down proteomics
    Nat. Methods (IF 26.919) Pub Date : 2018-06-13
    Jungkap Park, Paul D Piehowski, Christopher Wilkins, Mowei Zhou, Joshua Mendoza, Grant M Fujimoto, Bryson C Gibbons, Jared B Shaw, Yufeng Shen, Anil K Shukla, Ronald J Moore, Tao Liu, Vladislav A Petyuk, Nikola Tolić, Ljiljana Paša-Tolić, Richard D Smith, Samuel H Payne, Sangtae Kim

    Author Correction: Informed-Proteomics: open-source software package for top-down proteomics Author Correction: Informed-Proteomics: open-source software package for top-down proteomics, Published online: 13 June 2018; doi:10.1038/s41592-018-0040-0 Author Correction: Informed-Proteomics: open-source software package for top-down proteomics

    更新日期:2018-06-14
  • Comprehensive comparative analysis of 5′-end RNA-sequencing methods
    Nat. Methods (IF 26.919) Pub Date : 2018-06-04
    Xian Adiconis, Adam L. Haber, Sean K. Simmons, Ami Levy Moonshine, Zhe Ji, Michele A. Busby, Xi Shi, Justin Jacques, Madeline A. Lancaster, Jen Q. Pan, Aviv Regev, Joshua Z. Levin

    Specialized RNA-seq methods are required to identify the 5′ ends of transcripts, which are critical for studies of gene regulation, but these methods have not been systematically benchmarked. We directly compared six such methods, including the performance of five methods on a single human cellular RNA sample and a new spike-in RNA assay that helps circumvent challenges resulting from uncertainties in annotation and RNA processing. We found that the ‘cap analysis of gene expression’ (CAGE) method performed best for mRNA and that most of its unannotated peaks were supported by evidence from other genomic methods. We applied CAGE to eight brain-related samples and determined sample-specific transcription start site (TSS) usage, as well as a transcriptome-wide shift in TSS usage between fetal and adult brain.

    更新日期:2018-06-05
  • A home for integrative structural models
    Nat. Methods (IF 26.919) Pub Date : 2018-05-31
    Allison Doerr

    A home for integrative structural models A home for integrative structural models, Published online: 31 May 2018; doi:10.1038/s41592-018-0032-0 PDB-Dev hosts integrative structural models of biomolecular assemblies solved by hybrid methods.

    更新日期:2018-06-01
  • MaxQuant goes Linux
    Nat. Methods (IF 26.919) Pub Date : 2018-05-31
    Pavel Sinitcyn, Shivani Tiwary, Jan Rudolph, Petra Gutenbrunner, Christoph Wichmann, Şule Yılmaz, Hamid Hamzeiy, Favio Salinas, Jürgen Cox

    MaxQuant goes Linux MaxQuant goes Linux, Published online: 31 May 2018; doi:10.1038/s41592-018-0018-y MaxQuant goes Linux

    更新日期:2018-06-01
  • A pan-cancer atlas
    Nat. Methods (IF 26.919) Pub Date : 2018-05-31
    Tal Nawy

    A pan-cancer atlas A pan-cancer atlas, Published online: 31 May 2018; doi:10.1038/s41592-018-0020-4 A pan-cancer atlas

    更新日期:2018-06-01
  • RBP census
    Nat. Methods (IF 26.919) Pub Date : 2018-05-31
    Nicole Rusk

    RBP census RBP census, Published online: 31 May 2018; doi:10.1038/s41592-018-0025-z RBP census

    更新日期:2018-06-01
  • Stem cells: lineage tracing lets single cells talk about their past
    Nat. Methods (IF 26.919) Pub Date : 2018-05-31
    Vivien Marx

    Stem cells: lineage tracing lets single cells talk about their past Stem cells: lineage tracing lets single cells talk about their past, Published online: 31 May 2018; doi:10.1038/s41592-018-0016-0 Multipotent stem cells can become a variety of cell types. A flurry of new approaches enable lineage tracing at single-cell resolution.

    更新日期:2018-06-01
  • Hessian structured illumination microscopy
    Nat. Methods (IF 26.919) Pub Date : 2018-05-31
    Rita Strack

    Hessian structured illumination microscopy Hessian structured illumination microscopy, Published online: 31 May 2018; doi:10.1038/s41592-018-0023-1 Hessian structured illumination microscopy

    更新日期:2018-06-01
  • A home for brain organoids
    Nat. Methods (IF 26.919) Pub Date : 2018-05-31
    Tal Nawy

    A home for brain organoids A home for brain organoids, Published online: 31 May 2018; doi:10.1038/s41592-018-0029-8 Inside a mouse brain, human cerebral organoids can show their potential.

    更新日期:2018-06-01
  • RNAs that shape the genome
    Nat. Methods (IF 26.919) Pub Date : 2018-05-31
    Nicole Rusk

    RNAs that shape the genome RNAs that shape the genome, Published online: 31 May 2018; doi:10.1038/s41592-018-0030-2 Chromatin-associated RNA sequencing elucidates the role of RNAs in genome regulation.

    更新日期:2018-06-01
  • Nanoparticles for super-resolution microscopy and single-molecule tracking
    Nat. Methods (IF 26.919) Pub Date : 2018-05-28
    Dayong Jin, Peng Xi, Baoming Wang, Le Zhang, Jörg Enderlein, Antoine M. van Oijen

    We review the use of luminescent nanoparticles in super-resolution imaging and single-molecule tracking, and showcase novel approaches to super-resolution imaging that leverage the brightness, stability, and unique optical-switching properties of these nanoparticles. We also discuss the challenges associated with their use in biological systems, including intracellular delivery and molecular targeting. In doing so, we hope to provide practical guidance for biologists and continue to bridge the fields of super-resolution imaging and nanoparticle engineering to support their mutual advancement.

    更新日期:2018-05-29
  • Mapping the physical network of cellular interactions
    Nat. Methods (IF 26.919) Pub Date : 2018-05-21
    Jean-Charles Boisset, Judith Vivié, Dominic Grün, Mauro J. Muraro, Anna Lyubimova, Alexander van Oudenaarden

    A cell’s function is influenced by the environment, or niche, in which it resides. Studies of niches usually require assumptions about the cell types present, which impedes the discovery of new cell types or interactions. Here we describe ProximID, an approach for building a cellular network based on physical cell interaction and single-cell mRNA sequencing, and show that it can be used to discover new preferential cellular interactions without prior knowledge of component cell types. ProximID found specific interactions between megakaryocytes and mature neutrophils and between plasma cells and myeloblasts and/or promyelocytes (precursors of neutrophils) in mouse bone marrow, and it identified a Tac1+ enteroendocrine cell–Lgr5+ stem cell interaction in small intestine crypts. This strategy can be used to discover new niches or preferential interactions in a variety of organs.

    更新日期:2018-05-22
  • CRISPR off-target analysis in genetically engineered rats and mice
    Nat. Methods (IF 26.919) Pub Date : 2018-05-21
    Keith R. Anderson, Maximilian Haeussler, Colin Watanabe, Vasantharajan Janakiraman, Jessica Lund, Zora Modrusan, Jeremy Stinson, Qixin Bei, Andrew Buechler, Charles Yu, Sobha R. Thamminana, Lucinda Tam, Michael-Anne Sowick, Tuija Alcantar, Natasha O’Neil, Jinjie Li, Linda Ta, Lisa Lima, Merone Roose-Girma, Xin Rairdan, Steffen Durinck, Søren Warming

    Despite widespread use of CRISPR, comprehensive data on the frequency and impact of Cas9-mediated off-targets in modified rodents are limited. Here we present deep-sequencing data from 81 genome-editing projects on mouse and rat genomes at 1,423 predicted off-target sites, 32 of which were confirmed, and show that high-fidelity Cas9 versions reduced off-target mutation rates in vivo. Using whole-genome sequencing data from ten mouse embryos, treated with a single guide RNA (sgRNA), and from their genetic parents, we found 43 off-targets, 30 of which were predicted by an adapted version of GUIDE-seq.

    更新日期:2018-05-22
  • Automated, parallel mass spectrometry imaging and structural identification of lipids
    Nat. Methods (IF 26.919) Pub Date : 2018-05-21
    Shane R. Ellis, Martin R. L. Paine, Gert B. Eijkel, Josch K. Pauling, Peter Husen, Mark W. Jervelund, Martin Hermansson, Christer S. Ejsing, Ron M. A. Heeren

    We report a method that enables automated data-dependent acquisition of lipid tandem mass spectrometry data in parallel with a high-resolution mass spectrometry imaging experiment. The method does not increase the total image acquisition time and is combined with automatic structural assignments. This lipidome-per-pixel approach automatically identified and validated 104 unique molecular lipids and their spatial locations from rat cerebellar tissue.

    更新日期:2018-05-22
  • Author Correction: High-speed volumetric imaging of neuronal activity in freely moving rodents
    Nat. Methods (IF 26.919) Pub Date : 2018-05-21
    Oliver Skocek, Tobias Nöbauer, Lukas Weilguny, Francisca Martínez Traub, Chuying Naomi Xia, Maxim I. Molodtsov, Abhinav Grama, Masahito Yamagata, Daniel Aharoni, David D. Cox, Peyman Golshani, Alipasha Vaziri

    Author Correction: High-speed volumetric imaging of neuronal activity in freely moving rodents Author Correction: High-speed volumetric imaging of neuronal activity in freely moving rodents, Published online: 21 May 2018; doi:10.1038/s41592-018-0034-y Author Correction: High-speed volumetric imaging of neuronal activity in freely moving rodents

    更新日期:2018-05-21
  • Single-shot super-resolution total internal reflection fluorescence microscopy
    Nat. Methods (IF 26.919) Pub Date : 2018-05-07
    Min Guo, Panagiotis Chandris, John Paul Giannini, Adam J. Trexler, Robert Fischer, Jiji Chen, Harshad D. Vishwasrao, Ivan Rey-Suarez, Yicong Wu, Xufeng Wu, Clare M. Waterman, George H. Patterson, Arpita Upadhyaya, Justin W. Taraska, Hari Shroff

    We combined instant structured illumination microscopy (iSIM) with total internal reflection fluorescence microscopy (TIRFM) in an approach referred to as instant TIRF-SIM, thereby improving the lateral spatial resolution of TIRFM to 115 ± 13 nm without compromising speed, and enabling imaging frame rates up to 100 Hz over hundreds of time points. We applied instant TIRF-SIM to multiple live samples and achieved rapid, high-contrast super-resolution imaging close to the coverslip surface.

    更新日期:2018-05-08
  • Discovery of proteins associated with a predefined genomic locus via dCas9–APEX-mediated proximity labeling
    Nat. Methods (IF 26.919) Pub Date : 2018-05-07
    Samuel A. Myers, Jason Wright, Ryan Peckner, Brian T. Kalish, Feng Zhang, Steven A. Carr

    Regulation of gene expression is primarily controlled by changes in the proteins that occupy genes’ regulatory elements. We developed genomic locus proteomics (GLoPro), in which we combine CRISPR-based genome targeting, proximity labeling, and quantitative proteomics to discover proteins associated with a specific genomic locus in native cellular contexts.

    更新日期:2018-05-08
  • High-speed volumetric imaging of neuronal activity in freely moving rodents
    Nat. Methods (IF 26.919) Pub Date : 2018-05-07
    Oliver Skocek, Tobias Nöbauer, Lukas Weilguny, Francisca Martínez Traub, Chuying Naomi Xia, Maxim I. Molodtsov, Abhinav Grama, Masahito Yamagata, Daniel Aharoni, David D. Cox, Peyman Golshani, Alipasha Vaziri

    Thus far, optical recording of neuronal activity in freely behaving animals has been limited to a thin axial range. We present a head-mounted miniaturized light-field microscope (MiniLFM) capable of capturing neuronal network activity within a volume of 700 × 600 × 360 µm3 at 16 Hz in the hippocampus of freely moving mice. We demonstrate that neurons separated by as little as ~15 µm and at depths up to 360 µm can be discriminated.

    更新日期:2018-05-08
  • BoxCar acquisition method enables single-shot proteomics at a depth of 10,000 proteins in 100 minutes
    Nat. Methods (IF 26.919) Pub Date : 2018-05-07
    Florian Meier, Philipp E. Geyer, Sebastian Virreira Winter, Juergen Cox, Matthias Mann

    Great advances have been made in sensitivity and acquisition speed on the Orbitrap mass analyzer, enabling increasingly deep proteome coverage. However, these advances have been mainly limited to the MS2 level, whereas ion beam sampling for the MS1 scans remains extremely inefficient. Here we report a data-acquisition method, termed BoxCar, in which filling multiple narrow mass-to-charge segments increases the mean ion injection time more than tenfold as compared to that of a standard full scan. In 1-h analyses, the method provided MS1-level evidence for more than 90% of the proteome of a human cancer cell line that had previously been identified in 24 fractions, and it quantified more than 6,200 proteins in ten of ten replicates. In mouse brain tissue, we detected more than 10,000 proteins in only 100 min, and sensitivity extended into the low-attomolar range.

    更新日期:2018-05-08
  • C-BERST: defining subnuclear proteomic landscapes at genomic elements with dCas9–APEX2
    Nat. Methods (IF 26.919) Pub Date : 2018-05-07
    Xin D. Gao, Li-Chun Tu, Aamir Mir, Tomás Rodriguez, Yuehe Ding, John Leszyk, Job Dekker, Scott A. Shaffer, Lihua Julie Zhu, Scot A. Wolfe, Erik J. Sontheimer

    Mapping proteomic composition at distinct genomic loci in living cells has been a long-standing challenge. Here we report that dCas9–APEX2 biotinylation at genomic elements by restricted spatial tagging (C-BERST) allows the rapid, unbiased mapping of proteomes near defined genomic loci, as demonstrated for telomeres and centromeres. C-BERST enables the high-throughput identification of proteins associated with specific sequences, thereby facilitating annotation of these factors and their roles.

    更新日期:2018-05-08
  • Accurate detection of complex structural variations using single-molecule sequencing
    Nat. Methods (IF 26.919) Pub Date : 2018-04-30
    Fritz J. Sedlazeck, Philipp Rescheneder, Moritz Smolka, Han Fang, Maria Nattestad, Arndt von Haeseler, Michael C. Schatz

    Structural variations are the greatest source of genetic variation, but they remain poorly understood because of technological limitations. Single-molecule long-read sequencing has the potential to dramatically advance the field, although high error rates are a challenge with existing methods. Addressing this need, we introduce open-source methods for long-read alignment (NGMLR; https://github.com/philres/ngmlr) and structural variant identification (Sniffles; https://github.com/fritzsedlazeck/Sniffles) that provide unprecedented sensitivity and precision for variant detection, even in repeat-rich regions and for complex nested events that can have substantial effects on human health. In several long-read datasets, including healthy and cancerous human genomes, we discovered thousands of novel variants and categorized systematic errors in short-read approaches. NGMLR and Sniffles can automatically filter false events and operate on low-coverage data, thereby reducing the high costs that have hindered the application of long reads in clinical and research settings.

    更新日期:2018-04-30
  • Picky comprehensively detects high-resolution structural variants in nanopore long reads
    Nat. Methods (IF 26.919) Pub Date : 2018-04-30
    Liang Gong, Chee-Hong Wong, Wei-Chung Cheng, Harianto Tjong, Francesca Menghi, Chew Yee Ngan, Edison T. Liu, Chia-Lin Wei

    Acquired genomic structural variants (SVs) are major hallmarks of cancer genomes, but they are challenging to reconstruct from short-read sequencing data. Here we exploited the long reads of the nanopore platform using our customized pipeline, Picky (https://github.com/TheJacksonLaboratory/Picky), to reveal SVs of diverse architecture in a breast cancer model. We identified the full spectrum of SVs with superior specificity and sensitivity relative to short-read analyses, and uncovered repetitive DNA as the major source of variation. Examination of genome-wide breakpoints at nucleotide resolution uncovered micro-insertions as the common structural features associated with SVs. Breakpoint density across the genome is associated with the propensity for interchromosomal connectivity and was found to be enriched in promoters and transcribed regions of the genome. Furthermore, we observed an over-representation of reciprocal translocations from chromosomal double-crossovers through phased SVs. We demonstrate that Picky analysis is an effective tool for comprehensive detection of SVs in cancer genomes from long-read data.

    更新日期:2018-04-30
  • Self-interference 3D super-resolution microscopy for deep tissue investigations
    Nat. Methods (IF 26.919) Pub Date : 2018-04-30
    Pierre Bon, Jeanne Linarès-Loyez, Maxime Feyeux, Kevin Alessandri, Brahim Lounis, Pierre Nassoy, Laurent Cognet

    Fluorescence localization microscopy has achieved near-molecular resolution capable of revealing ultra-structures, with a broad range of applications, especially in cellular biology. However, it remains challenging to attain such resolution in three dimensions and inside biological tissues beyond the first cell layer. Here we introduce SELFI, a framework for 3D single-molecule localization within multicellular specimens and tissues. The approach relies on self-interference generated within the microscope's point spread function (PSF) to simultaneously encode equiphase and intensity fluorescence signals, which together provide the 3D position of an emitter. We combined SELFI with conventional localization microscopy to visualize F-actin 3D filament networks and reveal the spatial distribution of the transcription factor OCT4 in human induced pluripotent stem cells at depths up to 50 µm inside uncleared tissue spheroids. SELFI paves the way to nanoscale investigations of native cellular processes in intact tissues.

    更新日期:2018-04-30
  • Computational correction of index switching in multiplexed sequencing libraries
    Nat. Methods (IF 26.919) Pub Date : 2018-04-27
    Anton J M Larsson, Geoff Stanley, Rahul Sinha, Irving L Weissman, Rickard Sandberg

    Computational correction of index switching in multiplexed sequencing libraries Computational correction of index switching in multiplexed sequencing libraries, Published online: 27 April 2018; doi:10.1038/nmeth.4666 Computational correction of index switching in multiplexed sequencing libraries

    更新日期:2018-04-28
  • Neuroscience: Extracting neuronal activity from microendoscopy videos
    Nat. Methods (IF 26.919) Pub Date : 2018-04-27

    Neuroscience: Extracting neuronal activity from microendoscopy videos Neuroscience: Extracting neuronal activity from microendoscopy videos, Published online: 27 April 2018; doi:10.1038/nmeth.4670 Neuroscience: Extracting neuronal activity from microendoscopy videos

    更新日期:2018-04-28
  • Neuroscience: Chronic imaging of the fruit fly brain
    Nat. Methods (IF 26.919) Pub Date : 2018-04-27

    Neuroscience: Chronic imaging of the fruit fly brain Neuroscience: Chronic imaging of the fruit fly brain, Published online: 27 April 2018; doi:10.1038/nmeth.4668 Neuroscience: Chronic imaging of the fruit fly brain

    更新日期:2018-04-28
  • Structural biology: cisTEM software for cryo-EM
    Nat. Methods (IF 26.919) Pub Date : 2018-04-27

    Structural biology: cisTEM software for cryo-EM Structural biology: cisTEM software for cryo-EM, Published online: 27 April 2018; doi:10.1038/nmeth.4672 Structural biology: cisTEM software for cryo-EM

    更新日期:2018-04-28
  • Microbiology: Metabolomes from metagenomics and modeling
    Nat. Methods (IF 26.919) Pub Date : 2018-04-27

    Microbiology: Metabolomes from metagenomics and modeling Microbiology: Metabolomes from metagenomics and modeling, Published online: 27 April 2018; doi:10.1038/nmeth.4674 Microbiology: Metabolomes from metagenomics and modeling

    更新日期:2018-04-28
  • Retraction: Unexpected mutations after CRISPR–Cas9 editing in vivo
    Nat. Methods (IF 26.919) Pub Date : 2018-04-27
    Kellie A Schaefer, Wen-Hsuan Wu, Diana F Colgan, Stephen H Tsang, Alexander G Bassuk, Vinit B Mahajan

    Retraction: Unexpected mutations after CRISPR–Cas9 editing in vivo Retraction: Unexpected mutations after CRISPR–Cas9 editing in vivo, Published online: 27 April 2018; doi:10.1038/nmeth0518-394a Retraction: Unexpected mutations after CRISPR–Cas9 editing in vivo

    更新日期:2018-04-28
  • Harris Wang
    Nat. Methods (IF 26.919) Pub Date : 2018-04-27
    Vivien Marx

    Harris Wang Harris Wang, Published online: 27 April 2018; doi:10.1038/nmeth.4676 A regulatory vocabulary for synthetic biology and why baby diapers matter.

    更新日期:2018-04-28
  • Imaging: Correlation analysis in single-molecule localization microscopy
    Nat. Methods (IF 26.919) Pub Date : 2018-04-27
    Christian Schnell

    Imaging: Correlation analysis in single-molecule localization microscopy Imaging: Correlation analysis in single-molecule localization microscopy, Published online: 27 April 2018; doi:10.1038/nmeth.4680 A coordinate-based framework quantifies the correlation and interaction of biomolecules in images acquired with super-resolution microscopy techniques.

    更新日期:2018-04-28
  • Real-time 3D single-molecule localization using experimental point spread functions
    Nat. Methods (IF 26.919) Pub Date : 2018-04-09
    Yiming Li, Markus Mund, Philipp Hoess, Joran Deschamps, Ulf Matti, Bianca Nijmeijer, Vilma Jimenez Sabinina, Jan Ellenberg, Ingmar Schoen, Jonas Ries

    We present a real-time fitter for 3D single-molecule localization microscopy using experimental point spread functions (PSFs) that achieves minimal uncertainty in 3D on any microscope and is compatible with any PSF engineering approach. We used this method to image cellular structures and attained unprecedented image quality for astigmatic PSFs. The fitter compensates for most optical aberrations and makes accurate 3D super-resolution microscopy broadly accessible, even on standard microscopes without dedicated 3D optics.

    更新日期:2018-04-09
  • FateID infers cell fate bias in multipotent progenitors from single-cell RNA-seq data
    Nat. Methods (IF 26.919) Pub Date : 2018-04-09
    Josip S Herman, Sagar, Dominic Grün

    To understand stem cell differentiation along multiple lineages, it is necessary to resolve heterogeneous cellular states and the ancestral relationships between them. We developed a robotic miniaturized CEL-Seq2 implementation to carry out deep single-cell RNA-seq of ∼2,000 mouse hematopoietic progenitors enriched for lymphoid lineages, and used an improved clustering algorithm, RaceID3, to identify cell types. To resolve subtle transcriptome differences indicative of lineage biases, we developed FateID, an iterative supervised learning algorithm for the probabilistic quantification of cell fate bias in progenitor populations. Here we used FateID to delineate domains of fate bias and enable the derivation of high-resolution differentiation trajectories, thereby revealing a common progenitor population of B cells and plasmacytoid dendritic cells, which we validated by in vitro differentiation assays. We expect that FateID will improve understanding of the process of cell fate choice in complex multi-lineage differentiation systems.

    更新日期:2018-04-09
Some contents have been Reproduced with permission of the American Chemical Society.
Some contents have been Reproduced by permission of The Royal Society of Chemistry.
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