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Nature Methods

Ibrahim Numanagić, James K Bonfield, Faraz Hach, Jan Voges, Jörn Ostermann, Claudio Alberti, Marco Mattavelli, S Cenk Sahinalp
High-throughput sequencing (HTS) data are commonly stored as raw sequencing reads in FASTQ format or as reads mapped to a reference, in SAM format, both with large memory footprints. Worldwide growth of HTS data has prompted the development of compression methods that aim to significantly reduce HTS data size. Here we report on a benchmarking study of available compression methods on a comprehensive set of HTS data using an automated framework.
October 24, 2016: Nature Methods
Jonathan B Grimm, Brian P English, Heejun Choi, Anand K Muthusamy, Brian P Mehl, Peng Dong, Timothy A Brown, Jennifer Lippincott-Schwartz, Zhe Liu, Timothée Lionnet, Luke D Lavis
Small-molecule fluorophores are important tools for advanced imaging experiments. We previously reported a general method to improve small, cell-permeable fluorophores which resulted in the azetidine-containing 'Janelia Fluor' (JF) dyes. Here, we refine and extend the utility of these dyes by synthesizing photoactivatable derivatives that are compatible with live-cell labeling strategies. Once activated, these derived compounds retain the superior brightness and photostability of the JF dyes, enabling improved single-particle tracking and facile localization microscopy experiments...
October 24, 2016: Nature Methods
Yuchen Gao, Xin Xiong, Spencer Wong, Emeric J Charles, Wendell A Lim, Lei S Qi
The ability to dynamically manipulate the transcriptome is important for studying how gene networks direct cellular functions and how network perturbations cause disease. Nuclease-dead CRISPR-dCas9 transcriptional regulators, while offering an approach for controlling individual gene expression, remain incapable of dynamically coordinating complex transcriptional events. Here, we describe a flexible dCas9-based platform for chemical-inducible complex gene regulation. From a screen of chemical- and light-inducible dimerization systems, we identified two potent chemical inducers that mediate efficient gene activation and repression in mammalian cells...
October 24, 2016: Nature Methods
Christine Koehler, Paul F Sauter, Mirella Wawryszyn, Gemma Estrada Girona, Kapil Gupta, Jonathan J M Landry, Markus Hsi-Yang Fritz, Ksenija Radic, Jan-Erik Hoffmann, Zhuo A Chen, Juan Zou, Piau Siong Tan, Bence Galik, Sini Junttila, Peggy Stolt-Bergner, Giancarlo Pruneri, Attila Gyenesei, Carsten Schultz, Moritz Bosse Biskup, Hueseyin Besir, Vladimir Benes, Juri Rappsilber, Martin Jechlinger, Jan O Korbel, Imre Berger, Stefan Braese, Edward A Lemke
We present a baculovirus-based protein engineering method that enables site-specific introduction of unique functionalities in a eukaryotic protein complex recombinantly produced in insect cells. We demonstrate the versatility of this efficient and robust protein production platform, 'MultiBacTAG', (i) for the fluorescent labeling of target proteins and biologics using click chemistries, (ii) for glycoengineering of antibodies, and (iii) for structure-function studies of novel eukaryotic complexes using single-molecule Förster resonance energy transfer as well as site-specific crosslinking strategies...
October 17, 2016: Nature Methods
Chen-Shan Chin, Paul Peluso, Fritz J Sedlazeck, Maria Nattestad, Gregory T Concepcion, Alicia Clum, Christopher Dunn, Ronan O'Malley, Rosa Figueroa-Balderas, Abraham Morales-Cruz, Grant R Cramer, Massimo Delledonne, Chongyuan Luo, Joseph R Ecker, Dario Cantu, David R Rank, Michael C Schatz
While genome assembly projects have been successful in many haploid and inbred species, the assembly of noninbred or rearranged heterozygous genomes remains a major challenge. To address this challenge, we introduce the open-source FALCON and FALCON-Unzip algorithms ( to assemble long-read sequencing data into highly accurate, contiguous, and correctly phased diploid genomes. We generate new reference sequences for heterozygous samples including an F1 hybrid of Arabidopsis thaliana, the widely cultivated Vitis vinifera cv...
October 17, 2016: Nature Methods
Xingqi Chen, Ying Shen, Will Draper, Jason D Buenrostro, Ulrike Litzenburger, Seung Woo Cho, Ansuman T Satpathy, Ava C Carter, Rajarshi P Ghosh, Alexandra East-Seletsky, Jennifer A Doudna, William J Greenleaf, Jan T Liphardt, Howard Y Chang
Spatial organization of the genome plays a central role in gene expression, DNA replication, and repair. But current epigenomic approaches largely map DNA regulatory elements outside of the native context of the nucleus. Here we report assay of transposase-accessible chromatin with visualization (ATAC-see), a transposase-mediated imaging technology that employs direct imaging of the accessible genome in situ, cell sorting, and deep sequencing to reveal the identity of the imaged elements. ATAC-see revealed the cell-type-specific spatial organization of the accessible genome and the coordinated process of neutrophil chromatin extrusion, termed NETosis...
October 17, 2016: Nature Methods
K M Naga Srinivas Nadella, Hana Roš, Chiara Baragli, Victoria A Griffiths, George Konstantinou, Theo Koimtzis, Geoffrey J Evans, Paul A Kirkby, R Angus Silver
Understanding how neural circuits process information requires rapid measurements of activity from identified neurons distributed in 3D space. Here we describe an acousto-optic lens two-photon microscope that performs high-speed focusing and line scanning within a volume spanning hundreds of micrometers. We demonstrate its random-access functionality by selectively imaging cerebellar interneurons sparsely distributed in 3D space and by simultaneously recording from the soma, proximal and distal dendrites of neocortical pyramidal cells in awake behaving mice...
October 17, 2016: Nature Methods
Jason A Reuter, Damek V Spacek, Reetesh K Pai, Michael P Snyder
Paired DNA and RNA profiling is increasingly employed in genomics research to uncover molecular mechanisms of disease and to explore personal genotype and phenotype correlations. Here, we introduce Simul-seq, a technique for the production of high-quality whole-genome and transcriptome sequencing libraries from small quantities of cells or tissues. We apply the method to laser-capture-microdissected esophageal adenocarcinoma tissue, revealing a highly aneuploid tumor genome with extensive blocks of increased homozygosity and corresponding increases in allele-specific expression...
October 10, 2016: Nature Methods
Yunqing Ma, Jiayuan Zhang, Weijie Yin, Zhenchao Zhang, Yan Song, Xing Chang
A large number of genetic variants have been associated with human diseases. However, the lack of a genetic diversification approach has impeded our ability to interrogate functions of genetic variants in mammalian cells. Current screening methods can only be used to disrupt a gene or alter its expression. Here we report the fusion of activation-induced cytidine deaminase (AID) with nuclease-inactive clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (dCas9) for efficient genetic diversification, which enabled high-throughput screening of functional variants...
October 10, 2016: Nature Methods
Tsung-Han S Hsieh, Geoffrey Fudenberg, Anton Goloborodko, Oliver J Rando
We present Micro-C XL, an improved method for analysis of chromosome folding at mononucleosome resolution. Using long crosslinkers and isolation of insoluble chromatin, Micro-C XL increases signal-to-noise ratio. Micro-C XL maps of budding and fission yeast genomes capture both short-range chromosome fiber features such as chromosomally interacting domains and higher order features such as centromere clustering. Micro-C XL provides a single assay to interrogate chromosome folding at length scales from the nucleosome to the full genome...
October 10, 2016: Nature Methods
Emily E Wrenbeck, Justin R Klesmith, James A Stapleton, Adebola Adeniran, Keith E J Tyo, Timothy A Whitehead
Deep mutational scanning is a foundational tool for addressing the functional consequences of large numbers of mutants, but a more efficient and accessible method for construction of user-defined mutagenesis libraries is needed. Here we present nicking mutagenesis, a robust, single-day, one-pot saturation mutagenesis method performed on routinely prepped plasmid dsDNA. The method can be used to produce comprehensive or single- or multi-site saturation mutagenesis libraries.
October 10, 2016: Nature Methods
Olga Levin-Kravets, Neta Tanner, Noa Shohat, Ilan Attali, Tal Keren-Kaplan, Anna Shusterman, Shay Artzi, Alexander Varvak, Yael Reshef, Xiaojing Shi, Ori Zucker, Tamir Baram, Corine Katina, Inbar Pilzer, Shay Ben-Aroya, Gali Prag
About one-third of the eukaryotic proteome undergoes ubiquitylation, but the enzymatic cascades leading to substrate modification are largely unknown. We present a genetic selection tool that utilizes Escherichia coli, which lack deubiquitylases, to identify interactions along ubiquitylation cascades. Coexpression of split antibiotic resistance protein tethered to ubiquitin and ubiquitylation target together with a functional ubiquitylation apparatus results in a covalent assembly of the resistance protein, giving rise to bacterial growth on selective media...
October 3, 2016: Nature Methods
Gabe Haller, David Alvarado, Kevin McCall, Robi D Mitra, Matthew B Dobbs, Christina A Gurnett
Large-scale mutagenesis of target DNA sequences allows researchers to comprehensively assess the effects of single-nucleotide changes. Here we demonstrate the construction of a systematic allelic series (SAS) using massively parallel single-nucleotide mutagenesis with reversibly terminated deoxyinosine triphosphates (rtITP). We created a mutational library containing every possible single-nucleotide mutation surrounding the active site of the TEM-1 β-lactamase gene. When combined with high-throughput functional assays, SAS mutational libraries can expedite the functional assessment of genetic variation...
October 3, 2016: Nature Methods
Samuel H Henager, Nam Chu, Zan Chen, David Bolduc, Daniel R Dempsey, Yousang Hwang, James Wells, Philip A Cole
Expressed protein ligation is a valuable method for protein semisynthesis that involves the reaction of recombinant protein C-terminal thioesters with N-terminal cysteine (N-Cys)-containing peptides, but the requirement of a Cys residue at the ligation junction can limit the utility of this method. Here we employ subtiligase variants to efficiently ligate Cys-free peptides to protein thioesters. Using this method, we have more accurately determined the effect of C-terminal phosphorylation on the tumor suppressor protein PTEN...
September 26, 2016: Nature Methods
Maxwell R Mumbach, Adam J Rubin, Ryan A Flynn, Chao Dai, Paul A Khavari, William J Greenleaf, Howard Y Chang
Genome conformation is central to gene control but challenging to interrogate. Here we present HiChIP, a protein-centric chromatin conformation method. HiChIP improves the yield of conformation-informative reads by over 10-fold and lowers the input requirement over 100-fold relative to that of ChIA-PET. HiChIP of cohesin reveals multiscale genome architecture with greater signal-to-background ratios than those of in situ Hi-C.
September 19, 2016: Nature Methods
Takayuki Miki, Masashi Awa, Yuki Nishikawa, Shigeki Kiyonaka, Masaki Wakabayashi, Yasushi Ishihama, Itaru Hamachi
Zinc signaling and dynamics play significant roles in many physiological responses and diseases. To understand the physiological roles of zinc in detail, comprehensive identification of proteins under high concentration of mobile zinc ion is crucial. We developed a 'conditional proteomics' approach to identify proteins involved in zinc homeostasis based on a chemical proteomic strategy that utilizes designer zinc-responsive labeling reagents to tag such proteins and quantitative mass spectrometry for their identification...
September 12, 2016: Nature Methods
Yuchen Liu, Yonghao Zhan, Zhicong Chen, Anbang He, Jianfa Li, Hanwei Wu, Li Liu, Chengle Zhuang, Junhao Lin, Xiaoqiang Guo, Qiaoxia Zhang, Weiren Huang, Zhiming Cai
The complex phenotypes of eukaryotic cells are controlled by decision-making circuits and signaling pathways. A key obstacle to implementing artificial connections in signaling networks has been the lack of synthetic devices for efficient sensing, processing and control of biological signals. By extending sgRNAs to include modified riboswitches that recognize specific signals, we can create CRISPR-Cas9-based 'signal conductors' that regulate transcription of endogenous genes in response to external or internal signals of interest...
September 5, 2016: Nature Methods
Barbara M Grüner, Christopher J Schulze, Dian Yang, Daisuke Ogasawara, Melissa M Dix, Zoë N Rogers, Chen-Hua Chuang, Christopher D McFarland, Shin-Heng Chiou, J Mark Brown, Benjamin F Cravatt, Matthew Bogyo, Monte M Winslow
Phenotype-based small-molecule screening is a powerful method to identify molecules that regulate cellular functions. However, such screens are generally performed in vitro under conditions that do not necessarily model complex physiological conditions or disease states. Here, we use molecular cell barcoding to enable direct in vivo phenotypic screening of small-molecule libraries. The multiplexed nature of this approach allows rapid in vivo analysis of hundreds to thousands of compounds. Using this platform, we screened >700 covalent inhibitors directed toward hydrolases for their effect on pancreatic cancer metastatic seeding...
October 2016: Nature Methods
Wei Leong Chew, Mohammadsharif Tabebordbar, Jason K W Cheng, Prashant Mali, Elizabeth Y Wu, Alex H M Ng, Kexian Zhu, Amy J Wagers, George M Church
CRISPR-Cas9 delivery by adeno-associated virus (AAV) holds promise for gene therapy but faces critical barriers on account of its potential immunogenicity and limited payload capacity. Here, we demonstrate genome engineering in postnatal mice using AAV-split-Cas9, a multifunctional platform customizable for genome editing, transcriptional regulation, and other previously impracticable applications of AAV-CRISPR-Cas9. We identify crucial parameters that impact efficacy and clinical translation of our platform, including viral biodistribution, editing efficiencies in various organs, antigenicity, immunological reactions, and physiological outcomes...
October 2016: Nature Methods
Mathias Uhlen, Anita Bandrowski, Steven Carr, Aled Edwards, Jan Ellenberg, Emma Lundberg, David L Rimm, Henry Rodriguez, Tara Hiltke, Michael Snyder, Tadashi Yamamoto
No abstract text is available yet for this article.
October 2016: Nature Methods
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