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Nature Methods

Zoë N Rogers, Christopher D McFarland, Ian P Winters, Santiago Naranjo, Chen-Hua Chuang, Dmitri Petrov, Monte M Winslow
Cancer growth is a multistage, stochastic evolutionary process. While cancer genome sequencing has been instrumental in identifying the genomic alterations that occur in human tumors, the consequences of these alterations on tumor growth remain largely unexplored. Conventional genetically engineered mouse models enable the study of tumor growth in vivo, but they are neither readily scalable nor sufficiently quantitative to unravel the magnitude and mode of action of many tumor-suppressor genes. Here, we present a method that integrates tumor barcoding with ultradeep barcode sequencing (Tuba-seq) to interrogate tumor-suppressor function in mouse models of human cancer...
May 22, 2017: Nature Methods
David A Knowles, Joe R Davis, Hilary Edgington, Anil Raj, Marie-Julie Favé, Xiaowei Zhu, James B Potash, Myrna M Weissman, Jianxin Shi, Douglas F Levinson, Philip Awadalla, Sara Mostafavi, Stephen B Montgomery, Alexis Battle
Identifying interactions between genetics and the environment (GxE) remains challenging. We have developed EAGLE, a hierarchical Bayesian model for identifying GxE interactions based on associations between environmental variables and allele-specific expression. Combining whole-blood RNA-seq with extensive environmental annotations collected from 922 human individuals, we identified 35 GxE interactions, compared with only four using standard GxE interaction testing. EAGLE provides new opportunities for researchers to identify GxE interactions using functional genomic data...
May 22, 2017: Nature Methods
Catalina A Vallejos, Davide Risso, Antonio Scialdone, Sandrine Dudoit, John C Marioni
Single-cell transcriptomics is becoming an important component of the molecular biologist's toolkit. A critical step when analyzing data generated using this technology is normalization. However, normalization is typically performed using methods developed for bulk RNA sequencing or even microarray data, and the suitability of these methods for single-cell transcriptomics has not been assessed. We here discuss commonly used normalization approaches and illustrate how these can produce misleading results. Finally, we present alternative approaches and provide recommendations for single-cell RNA sequencing users...
May 15, 2017: Nature Methods
Aaron T L Lun, Arianne C Richard, John C Marioni
When comparing biological conditions using mass cytometry data, a key challenge is to identify cellular populations that change in abundance. Here, we present a computational strategy for detecting 'differentially abundant' populations by assigning cells to hyperspheres, testing for significant differences between conditions and controlling the spatial false discovery rate. Our method ( outperforms other approaches in simulations and finds novel patterns of differential abundance in real data...
May 15, 2017: Nature Methods
Qing Dai, Sharon Moshitch-Moshkovitz, Dali Han, Nitzan Kol, Ninette Amariglio, Gideon Rechavi, Dan Dominissini, Chuan He
The ribose of RNA nucleotides can be 2'-O-methylated (Nm). Despite advances in high-throughput detection, the inert chemical nature of Nm still limits sensitivity and precludes mapping in mRNA. We leveraged the differential reactivity of 2'-O-methylated and 2'-hydroxylated nucleosides to periodate oxidation to develop Nm-seq, a sensitive method for transcriptome-wide mapping of Nm with base precision. Nm-seq uncovered thousands of Nm sites in human mRNA with features suggesting functional roles.
May 15, 2017: Nature Methods
Nan Yang, Soham Chanda, Samuele Marro, Yi-Han Ng, Justyna A Janas, Daniel Haag, Cheen Euong Ang, Yunshuo Tang, Quetzal Flores, Moritz Mall, Orly Wapinski, Mavis Li, Henrik Ahlenius, John L Rubenstein, Howard Y Chang, Arturo Alvarez Buylla, Thomas C Südhof, Marius Wernig
Approaches to differentiating pluripotent stem cells (PSCs) into neurons currently face two major challenges-(i) generated cells are immature, with limited functional properties; and (ii) cultures exhibit heterogeneous neuronal subtypes and maturation stages. Using lineage-determining transcription factors, we previously developed a single-step method to generate glutamatergic neurons from human PSCs. Here, we show that transient expression of the transcription factors Ascl1 and Dlx2 (AD) induces the generation of exclusively GABAergic neurons from human PSCs with a high degree of synaptic maturation...
May 15, 2017: Nature Methods
Joshua A Bagley, Daniel Reumann, Shan Bian, Julie Lévi-Strauss, Juergen A Knoblich
Human brain development involves complex interactions between different regions, including long-distance neuronal migration or formation of major axonal tracts. Different brain regions can be cultured in vitro within 3D cerebral organoids, but the random arrangement of regional identities limits the reliable analysis of complex phenotypes. Here, we describe a coculture method combining brain regions of choice within one organoid tissue. By fusing organoids of dorsal and ventral forebrain identities, we generate a dorsal-ventral axis...
May 10, 2017: Nature Methods
Jung-Hoon Park, Lingjie Kong, Yifeng Zhou, Meng Cui
Adaptive optics can correct for optical aberrations. We developed multi-pupil adaptive optics (MPAO), which enables simultaneous wavefront correction over a field of view of 450 × 450 μm(2) and expands the correction area to nine times that of conventional methods. MPAO's ability to perform spatially independent wavefront control further enables 3D nonplanar imaging. We applied MPAO to in vivo structural and functional imaging in the mouse brain.
May 8, 2017: Nature Methods
Subha Kalyaanamoorthy, Bui Quang Minh, Thomas K F Wong, Arndt von Haeseler, Lars S Jermiin
Model-based molecular phylogenetics plays an important role in comparisons of genomic data, and model selection is a key step in all such analyses. We present ModelFinder, a fast model-selection method that greatly improves the accuracy of phylogenetic estimates by incorporating a model of rate heterogeneity across sites not previously considered in this context and by allowing concurrent searches of model space and tree space.
May 8, 2017: Nature Methods
Dan Du, Assen Roguev, David E Gordon, Meng Chen, Si-Han Chen, Michael Shales, John Paul Shen, Trey Ideker, Prashant Mali, Lei S Qi, Nevan J Krogan
We describe a combinatorial CRISPR interference (CRISPRi) screening platform for mapping genetic interactions in mammalian cells. We targeted 107 chromatin-regulation factors in human cells with pools of either single or double single guide RNAs (sgRNAs) to downregulate individual genes or gene pairs, respectively. Relative enrichment analysis of individual sgRNAs or sgRNA pairs allowed for quantitative characterization of genetic interactions, and comparison with protein-protein-interaction data revealed a functional map of chromatin regulation...
May 8, 2017: Nature Methods
Peter Cameron, Chris K Fuller, Paul D Donohoue, Brittnee N Jones, Matthew S Thompson, Matthew M Carter, Scott Gradia, Bastien Vidal, Elizabeth Garner, Euan M Slorach, Elaine Lau, Lynda M Banh, Alexandra M Lied, Leslie S Edwards, Alexander H Settle, Daniel Capurso, Victor Llaca, Stéphane Deschamps, Mark Cigan, Joshua K Young, Andrew P May
RNA-guided CRISPR-Cas9 endonucleases are widely used for genome engineering, but our understanding of Cas9 specificity remains incomplete. Here, we developed a biochemical method (SITE-Seq), using Cas9 programmed with single-guide RNAs (sgRNAs), to identify the sequence of cut sites within genomic DNA. Cells edited with the same Cas9-sgRNA complexes are then assayed for mutations at each cut site using amplicon sequencing. We used SITE-Seq to examine Cas9 specificity with sgRNAs targeting the human genome. The number of sites identified depended on sgRNA sequence and nuclease concentration...
May 1, 2017: Nature Methods
Shengdar Q Tsai, Nhu T Nguyen, Jose Malagon-Lopez, Ved V Topkar, Martin J Aryee, J Keith Joung
Sensitive detection of off-target effects is important for translating CRISPR-Cas9 nucleases into human therapeutics. In vitro biochemical methods for finding off-targets offer the potential advantages of greater reproducibility and scalability while avoiding limitations associated with strategies that require the culture and manipulation of living cells. Here we describe circularization for in vitro reporting of cleavage effects by sequencing (CIRCLE-seq), a highly sensitive, sequencing-efficient in vitro screening strategy that outperforms existing cell-based or biochemical approaches for identifying CRISPR-Cas9 genome-wide off-target mutations...
May 1, 2017: Nature Methods
Thomas Willems, Dina Zielinski, Jie Yuan, Assaf Gordon, Melissa Gymrek, Yaniv Erlich
Short tandem repeats (STRs) are highly variable elements that play a pivotal role in multiple genetic diseases, population genetics applications, and forensic casework. However, it has proven problematic to genotype STRs from high-throughput sequencing data. Here, we describe HipSTR, a novel haplotype-based method for robustly genotyping and phasing STRs from Illumina sequencing data, and we report a genome-wide analysis and validation of de novo STR mutations. HipSTR is freely available at https://hipstr-tool...
April 24, 2017: Nature Methods
Rhonda Bacher, Li-Fang Chu, Ning Leng, Audrey P Gasch, James A Thomson, Ron M Stewart, Michael Newton, Christina Kendziorski
The normalization of RNA-seq data is essential for accurate downstream inference, but the assumptions upon which most normalization methods are based are not applicable in the single-cell setting. Consequently, applying existing normalization methods to single-cell RNA-seq data introduces artifacts that bias downstream analyses. To address this, we introduce SCnorm for accurate and efficient normalization of single-cell RNA-seq data.
April 17, 2017: Nature Methods
Yarui Diao, Rongxin Fang, Bin Li, Zhipeng Meng, Juntao Yu, Yunjiang Qiu, Kimberly C Lin, Hui Huang, Tristin Liu, Ryan J Marina, Inkyung Jung, Yin Shen, Kun-Liang Guan, Bing Ren
Millions of cis-regulatory elements are predicted to be present in the human genome, but direct evidence for their biological function is scarce. Here we report a high-throughput method, cis-regulatory element scan by tiling-deletion and sequencing (CREST-seq), for the unbiased discovery and functional assessment of cis-regulatory sequences in the genome. We used it to interrogate the 2-Mb POU5F1 locus in human embryonic stem cells, and identified 45 cis-regulatory elements. A majority of these elements have active chromatin marks, DNase hypersensitivity, and occupancy by multiple transcription factors, which confirms the utility of chromatin signatures in cis-element mapping...
April 17, 2017: Nature Methods
Daniel Agudelo, Alexis Duringer, Lusiné Bozoyan, Caroline C Huard, Sophie Carter, Jeremy Loehr, Dafni Synodinou, Mathieu Drouin, Jayme Salsman, Graham Dellaire, Josée Laganière, Yannick Doyon
Targeted genome editing enables the creation of bona fide cellular models for biological research and may be applied to human cell-based therapies. Therefore, broadly applicable and versatile methods for increasing its efficacy in cell populations are highly desirable. We designed a simple and robust coselection strategy for enrichment of cells with either nuclease-driven nonhomologous end joining (NHEJ) or homology-directed repair (HDR) events by harnessing the multiplexing capabilities of CRISPR-Cas9 and Cpf1 systems...
April 17, 2017: Nature Methods
Jae-Byum Chang, Fei Chen, Young-Gyu Yoon, Erica E Jung, Hazen Babcock, Jeong Seuk Kang, Shoh Asano, Ho-Jun Suk, Nikita Pak, Paul W Tillberg, Asmamaw T Wassie, Dawen Cai, Edward S Boyden
We recently developed a method called expansion microscopy, in which preserved biological specimens are physically magnified by embedding them in a densely crosslinked polyelectrolyte gel, anchoring key labels or biomolecules to the gel, mechanically homogenizing the specimen, and then swelling the gel-specimen composite by ∼4.5× in linear dimension. Here we describe iterative expansion microscopy (iExM), in which a sample is expanded ∼20×. After preliminary expansion a second swellable polymer mesh is formed in the space newly opened up by the first expansion, and the sample is expanded again...
April 17, 2017: Nature Methods
John Paul Shen, Dongxin Zhao, Roman Sasik, Jens Luebeck, Amanda Birmingham, Ana Bojorquez-Gomez, Katherine Licon, Kristin Klepper, Daniel Pekin, Alex N Beckett, Kyle Salinas Sanchez, Alex Thomas, Chih-Chung Kuo, Dan Du, Assen Roguev, Nathan E Lewis, Aaron N Chang, Jason F Kreisberg, Nevan Krogan, Lei Qi, Trey Ideker, Prashant Mali
We developed a systematic approach to map human genetic networks by combinatorial CRISPR-Cas9 perturbations coupled to robust analysis of growth kinetics. We targeted all pairs of 73 cancer genes with dual guide RNAs in three cell lines, comprising 141,912 tests of interaction. Numerous therapeutically relevant interactions were identified, and these patterns replicated with combinatorial drugs at 75% precision. From these results, we anticipate that cellular context will be critical to synthetic-lethal therapies...
March 20, 2017: Nature Methods
Alexander Song, Adam S Charles, Sue Ann Koay, Jeff L Gauthier, Stephan Y Thiberge, Jonathan W Pillow, David W Tank
Two-photon laser scanning microscopy of calcium dynamics using fluorescent indicators is a widely used imaging method for large-scale recording of neural activity in vivo. Here, we introduce volumetric two-photon imaging of neurons using stereoscopy (vTwINS), a volumetric calcium imaging method that uses an elongated, V-shaped point spread function to image a 3D brain volume. Single neurons project to spatially displaced 'image pairs' in the resulting 2D image, and the separation distance between projections is proportional to depth in the volume...
March 20, 2017: Nature Methods
Wei Zhang, Alexander W Lohman, Yevgeniya Zhuravlova, Xiaocen Lu, Matthew D Wiens, Hiofan Hoi, Sine Yaganoglu, Manuel A Mohr, Elena N Kitova, John S Klassen, Periklis Pantazis, Roger J Thompson, Robert E Campbell
To expand the range of experiments that are accessible with optogenetics, we developed a photocleavable protein (PhoCl) that spontaneously dissociates into two fragments after violet-light-induced cleavage of a specific bond in the protein backbone. We demonstrated that PhoCl can be used to engineer light-activatable Cre recombinase, Gal4 transcription factor, and a viral protease that in turn was used to activate opening of the large-pore ion channel Pannexin-1.
March 13, 2017: Nature Methods
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