Nature Methods

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Nature Methods is a science methodology journal publishing laboratory techniques and methods papers in the life sciences and areas of chemistry relevant to the life sciences.
Updated: 8 years 17 weeks ago

WASP: allele-specific software for robust molecular quantitative trait locus discovery

Sun, 09/13/2015 - 23:00

Nature Methods 12, 1061 (2015). doi:10.1038/nmeth.3582

Authors: Bryce van de Geijn, Graham McVicker, Yoav Gilad & Jonathan K Pritchard

Allele-specific sequencing reads provide a powerful signal for identifying molecular quantitative trait loci (QTLs), but they are challenging to analyze and are prone to technical artifacts. Here we describe WASP, a suite of tools for unbiased allele-specific read mapping and discovery of molecular QTLs. Using simulated reads, RNA-seq reads and chromatin immunoprecipitation sequencing (ChIP-seq) reads, we demonstrate that WASP has a low error rate and is far more powerful than existing QTL-mapping approaches.

Real-time metabolome profiling of the metabolic switch between starvation and growth

Sun, 09/13/2015 - 23:00

Nature Methods 12, 1091 (2015). doi:10.1038/nmeth.3584

Authors: Hannes Link, Tobias Fuhrer, Luca Gerosa, Nicola Zamboni & Uwe Sauer

Bisulfite-free, base-resolution analysis of 5-formylcytosine at the genome scale

Sun, 09/06/2015 - 23:00

Nature Methods 12, 1047 (2015). doi:10.1038/nmeth.3569

Authors: Bo Xia, Dali Han, Xingyu Lu, Zhaozhu Sun, Ankun Zhou, Qiangzong Yin, Hu Zeng, Menghao Liu, Xiang Jiang, Wei Xie, Chuan He & Chengqi Yi

Active DNA demethylation in mammals involves oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). However, genome-wide detection of 5fC at single-base resolution remains challenging. Here we present fC-CET, a bisulfite-free method for whole-genome analysis of 5fC based on selective chemical labeling of 5fC and subsequent C-to-T transition during PCR. Base-resolution 5fC maps showed limited overlap with 5hmC, with 5fC-marked regions more active than 5hmC-marked ones.

Cas9 gRNA engineering for genome editing, activation and repression

Sun, 09/06/2015 - 23:00

Nature Methods 12, 1051 (2015). doi:10.1038/nmeth.3580

Authors: Samira Kiani, Alejandro Chavez, Marcelle Tuttle, Richard N Hall, Raj Chari, Dmitry Ter-Ovanesyan, Jason Qian, Benjamin W Pruitt, Jacob Beal, Suhani Vora, Joanna Buchthal, Emma J K Kowal, Mohammad R Ebrahimkhani, James J Collins, Ron Weiss & George Church

We demonstrate that by altering the length of Cas9-associated guide RNA (gRNA) we were able to control Cas9 nuclease activity and simultaneously perform genome editing and transcriptional regulation with a single Cas9 protein. We exploited these principles to engineer mammalian synthetic circuits with combined transcriptional regulation and kill functions governed by a single multifunctional Cas9 protein.

Interactive analysis and assessment of single-cell copy-number variations

Sun, 09/06/2015 - 23:00

Nature Methods 12, 1058 (2015). doi:10.1038/nmeth.3578

Authors: Tyler Garvin, Robert Aboukhalil, Jude Kendall, Timour Baslan, Gurinder S Atwal, James Hicks, Michael Wigler & Michael C Schatz

We present Ginkgo (http://qb.cshl.edu/ginkgo), a user-friendly, open-source web platform for the analysis of single-cell copy-number variations (CNVs). Ginkgo automatically constructs copy-number profiles of cells from mapped reads and constructs phylogenetic trees of related cells. We validated Ginkgo by reproducing the results of five major studies. After comparing three commonly used single-cell amplification techniques, we concluded that degenerate oligonucleotide-primed PCR is the most consistent for CNV analysis.

SR-Tesseler: a method to segment and quantify localization-based super-resolution microscopy data

Sun, 09/06/2015 - 23:00

Nature Methods 12, 1065 (2015). doi:10.1038/nmeth.3579

Authors: Florian Levet, Eric Hosy, Adel Kechkar, Corey Butler, Anne Beghin, Daniel Choquet & Jean-Baptiste Sibarita

Functional footprinting of regulatory DNA

Sun, 08/30/2015 - 23:00

Nature Methods 12, 927 (2015). doi:10.1038/nmeth.3554

Authors: Jeff Vierstra, Andreas Reik, Kai-Hsin Chang, Sandra Stehling-Sun, Yuanyue Zhou, Sarah J Hinkley, David E Paschon, Lei Zhang, Nikoletta Psatha, Yuri R Bendana, Colleen M O'Neil, Alexander H Song, Andrea K Mich, Pei-Qi Liu, Gary Lee, Daniel E Bauer, Michael C Holmes, Stuart H Orkin, Thalia Papayannopoulou, George Stamatoyannopoulos, Edward J Rebar, Philip D Gregory, Fyodor D Urnov & John A Stamatoyannopoulos

Regulatory regions harbor multiple transcription factor (TF) recognition sites; however, the contribution of individual sites to regulatory function remains challenging to define. We describe an approach that exploits the error-prone nature of genome editing–induced double-strand break repair to map functional elements within regulatory DNA at nucleotide resolution. We demonstrate the approach on a human erythroid enhancer, revealing single TF recognition sites that gate the majority of downstream regulatory function.

Inntags: small self-structured epitopes for innocuous protein tagging

Sun, 08/30/2015 - 23:00

Nature Methods 12, 955 (2015). doi:10.1038/nmeth.3556

Authors: Maya V Georgieva, Galal Yahya, Laia Codó, Raúl Ortiz, Laura Teixidó, José Claros, Ricardo Jara, Mònica Jara, Antoni Iborra, Josep Lluís Gelpí, Carme Gallego, Modesto Orozco & Martí Aldea

Protein tagging is widely used in approaches ranging from affinity purification to fluorescence-based detection in live cells. However, an intrinsic limitation of tagging is that the native function of the protein may be compromised or even abolished by the presence of the tag. Here we describe and characterize a set of small, innocuous protein tags (inntags) that we anticipate will find application in a variety of biological techniques.

Programmed synthesis of three-dimensional tissues

Sun, 08/30/2015 - 23:00

Nature Methods 12, 975 (2015). doi:10.1038/nmeth.3553

Authors: Michael E Todhunter, Noel Y Jee, Alex J Hughes, Maxwell C Coyle, Alec Cerchiari, Justin Farlow, James C Garbe, Mark A LaBarge, Tejal A Desai & Zev J Gartner

CRISPRscan: designing highly efficient sgRNAs for CRISPR-Cas9 targeting in vivo

Sun, 08/30/2015 - 23:00

Nature Methods 12, 982 (2015). doi:10.1038/nmeth.3543

Authors: Miguel A Moreno-Mateos, Charles E Vejnar, Jean-Denis Beaudoin, Juan P Fernandez, Emily K Mis, Mustafa K Khokha & Antonio J Giraldez

Predicting effects of noncoding variants with deep learning–based sequence model

Sun, 08/23/2015 - 23:00

Nature Methods 12, 931 (2015). doi:10.1038/nmeth.3547

Authors: Jian Zhou & Olga G Troyanskaya

Identifying functional effects of noncoding variants is a major challenge in human genetics. To predict the noncoding-variant effects de novo from sequence, we developed a deep learning–based algorithmic framework, DeepSEA (http://deepsea.princeton.edu/), that directly learns a regulatory sequence code from large-scale chromatin-profiling data, enabling prediction of chromatin effects of sequence alterations with single-nucleotide sensitivity. We further used this capability to improve prioritization of functional variants including expression quantitative trait loci (eQTLs) and disease-associated variants.

Oscope identifies oscillatory genes in unsynchronized single-cell RNA-seq experiments

Sun, 08/23/2015 - 23:00

Nature Methods 12, 947 (2015). doi:10.1038/nmeth.3549

Authors: Ning Leng, Li-Fang Chu, Chris Barry, Yuan Li, Jeea Choi, Xiaomao Li, Peng Jiang, Ron M Stewart, James A Thomson & Christina Kendziorski

Oscillatory gene expression is fundamental to development, but technologies for monitoring expression oscillations are limited. We have developed a statistical approach called Oscope to identify and characterize the transcriptional dynamics of oscillating genes in single-cell RNA-seq data from an unsynchronized cell population. Applying Oscope to a number of data sets, we demonstrated its utility and also identified a potential artifact in the Fluidigm C1 platform.

Trajectories of cell-cycle progression from fixed cell populations

Sun, 08/23/2015 - 23:00

Nature Methods 12, 951 (2015). doi:10.1038/nmeth.3545

Authors: Gabriele Gut, Michelle D Tadmor, Dana Pe'er, Lucas Pelkmans & Prisca Liberali

An accurate dissection of sources of cell-to-cell variability is crucial for quantitative biology at the single-cell level but has been challenging for the cell cycle. We present Cycler, a robust method that constructs a continuous trajectory of cell-cycle progression from images of fixed cells. Cycler handles heterogeneous microenvironments and does not require perturbations or genetic markers, making it generally applicable to quantifying multiple sources of cell-to-cell variability in mammalian cells.

Ultrahigh-throughput single-molecule spectroscopy and spectrally resolved super-resolution microscopy

Sun, 08/16/2015 - 23:00

Nature Methods 12, 935 (2015). doi:10.1038/nmeth.3528

Authors: Zhengyang Zhang, Samuel J Kenny, Margaret Hauser, Wan Li & Ke Xu

By developing a wide-field scheme for spectral measurement and implementing photoswitching, we synchronously obtained the fluorescence spectra and positions of ∼106 single molecules in labeled cells in minutes, which consequently enabled spectrally resolved, 'true-color' super-resolution microscopy. The method, called spectrally resolved stochastic optical reconstruction microscopy (SR-STORM), achieved cross-talk–free three-dimensional (3D) imaging for four dyes 10 nm apart in emission spectrum. Excellent resolution was obtained for every channel, and 3D localizations of all molecules were automatically aligned within one imaging path.

EMRinger: side chain–directed model and map validation for 3D cryo-electron microscopy

Sun, 08/16/2015 - 23:00

Nature Methods 12, 943 (2015). doi:10.1038/nmeth.3541

Authors: Benjamin A Barad, Nathaniel Echols, Ray Yu-Ruei Wang, Yifan Cheng, Frank DiMaio, Paul D Adams & James S Fraser

Advances in high-resolution cryo-electron microscopy (cryo-EM) require the development of validation metrics to independently assess map quality and model geometry. We report EMRinger, a tool that assesses the precise fitting of an atomic model into the map during refinement and shows how radiation damage alters scattering from negatively charged amino acids. EMRinger (https://github.com/fraser-lab/EMRinger) will be useful for monitoring progress in resolving and modeling high-resolution features in cryo-EM.

ChIPmentation: fast, robust, low-input ChIP-seq for histones and transcription factors

Sun, 08/16/2015 - 23:00

Nature Methods 12, 963 (2015). doi:10.1038/nmeth.3542

Authors: Christian Schmidl, André F Rendeiro, Nathan C Sheffield & Christoph Bock

Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is widely used to map histone marks and transcription factor binding throughout the genome. Here we present ChIPmentation, a method that combines chromatin immunoprecipitation with sequencing library preparation by Tn5 transposase ('tagmentation'). ChIPmentation introduces sequencing-compatible adaptors in a single-step reaction directly on bead-bound chromatin, which reduces time, cost and input requirements, thus providing a convenient and broadly useful alternative to existing ChIP-seq protocols.

Wirelessly powered, fully internal optogenetics for brain, spinal and peripheral circuits in mice

Sun, 08/16/2015 - 23:00

Nature Methods 12, 969 (2015). doi:10.1038/nmeth.3536

Authors: Kate L Montgomery, Alexander J Yeh, John S Ho, Vivien Tsao, Shrivats Mohan Iyer, Logan Grosenick, Emily A Ferenczi, Yuji Tanabe, Karl Deisseroth, Scott L Delp & Ada S Y Poon