Showing papers in "Nature Methods in 2011"
TL;DR: SignalP 4.0 was the best signal-peptide predictor for all three organism types but was not in all cases as good as SignalP 3.0 according to cleavage-site sensitivity or signal- peptide correlation when there are no transmembrane proteins present.
Abstract: We benchmarked SignalP 4.0 against SignalP 3.0 and ten other signal peptide prediction algorithms (Fig. 1). We compared prediction performance using the Matthews correlation coefficient16, for which each sequence was counted as a true or false positive or negative. To test SignalP 4.0 performance, we did not use data that had been used in training the networks or selecting the optimal architecture, and the test data did not contain homologs to the training and optimization data (Supplementary Methods). The test set for SignalP 3.0 was also independent of the training set because we removed sequences used to construct SignalP 3.0 and their homologs from the benchmark data. For other algorithms more recent than SignalP 3.0, the benchmark data may include data used to train the methods, possibly leading to slight overestimations of their performance. Our results show that SignalP 4.0 was the best signal-peptide predictor for all three organism types (Fig. 1). This comes at a price, however, because SignalP 4.0 was not in all cases as good as SignalP 3.0 according to cleavage-site sensitivity or signal-peptide correlation when there are no transmembrane proteins present (Supplementary Results). An ideal method would have the best SignalP 4.0: discriminating signal peptides from transmembrane regions
8,370 citations
TL;DR: An automated brain-mapping framework that uses text-mining, meta-analysis and machine-learning techniques to generate a large database of mappings between neural and cognitive states is described and validated.
Abstract: The rapid growth of the literature on neuroimaging in humans has led to major advances in our understanding of human brain function but has also made it increasingly difficult to aggregate and synthesize neuroimaging findings. Here we describe and validate an automated brain-mapping framework that uses text-mining, meta-analysis and machine-learning techniques to generate a large database of mappings between neural and cognitive states. We show that our approach can be used to automatically conduct large-scale, high-quality neuroimaging meta-analyses, address long-standing inferential problems in the neuroimaging literature and support accurate 'decoding' of broad cognitive states from brain activity in both entire studies and individual human subjects. Collectively, our results have validated a powerful and generative framework for synthesizing human neuroimaging data on an unprecedented scale.
2,853 citations
TL;DR: A simple method is reported, using p53 suppression and nontransforming L-Myc, to generate human induced pluripotent stem cells (iPSCs) with episomal plasmid vectors, which may provide iPSCs suitable for autologous and allologous stem-cell therapy in the future.
Abstract: Human induced pluripotent stem cells are generated with episomal plasmid vectors at increased efficiency using non-transforming L-Myc and knockdown of p53 Also in this issue, Chen et al report defined conditions for human cell reprogramming and culture
1,712 citations
TL;DR: A cell culture system in which all protein reagents for liquid media, attachment surfaces and splitting are chemically defined is formed, and improved derivation efficiencies of vector-free human iPSCs with an episomal approach are demonstrated.
Abstract: We re-examine the individual components for human embryonic stem cell (ESC) and induced pluripotent stem cell (iPSC) culture and formulate a cell culture system in which all protein reagents for liquid media, attachment surfaces and splitting are chemically defined. A major improvement is the lack of a serum albumin component, as variations in either animal- or human-sourced albumin batches have previously plagued human ESC and iPSC culture with inconsistencies. Using this new medium (E8) and vitronectin-coated surfaces, we demonstrate improved derivation efficiencies of vector-free human iPSCs with an episomal approach. This simplified E8 medium should facilitate both the research use and clinical applications of human ESCs and iPSCs and their derivatives, and should be applicable to other reprogramming methods.
1,330 citations
TL;DR: This analysis quantitatively characterized the switching properties of 26 organic dyes and directly related these properties to the quality of super-resolution images, providing guidelines for characterization ofsuper-resolution probes and a resource for selecting probes based on performance.
Abstract: One approach to super-resolution fluorescence imaging uses sequential activation and localization of individual fluorophores to achieve high spatial resolution. Essential to this technique is the choice of fluorescent probes; the properties of the probes, including photons per switching event, on-off duty cycle, photostability and number of switching cycles, largely dictate the quality of super-resolution images. Although many probes have been reported, a systematic characterization of the properties of these probes and their impact on super-resolution image quality has been described in only a few cases. Here we quantitatively characterized the switching properties of 26 organic dyes and directly related these properties to the quality of super-resolution images. This analysis provides guidelines for characterization of super-resolution probes and a resource for selecting probes based on performance. Our evaluation identified several photoswitchable dyes with good to excellent performance in four independent spectral ranges, with which we demonstrated low-cross-talk, four-color super-resolution imaging.
1,188 citations
TL;DR: SourceTracker, a Bayesian approach to estimate the proportion of contaminants in a given community that come from possible source environments, is presented, and microbial surveys from neonatal intensive care units, offices and molecular biology laboratories are applied.
Abstract: Contamination is a critical issue in high-throughput metagenomic studies, yet progress toward a comprehensive solution has been limited. We present SourceTracker, a Bayesian approach to estimate the proportion of contaminants in a given community that come from possible source environments. We applied SourceTracker to microbial surveys from neonatal intensive care units (NICUs), offices and molecular biology laboratories, and provide a database of known contaminants for future testing.
1,131 citations
TL;DR: This work describes factored spectrally transformed linear mixed models (FaST-LMM), an algorithm for genome-wide association studies (GWAS) that scales linearly with cohort size in both run time and memory use.
Abstract: We describe factored spectrally transformed linear mixed models (FaST-LMM), an algorithm for genome-wide association studies (GWAS) that scales linearly with cohort size in both run time and memory use. On Wellcome Trust data for 15,000 individuals, FaST-LMM ran an order of magnitude faster than current efficient algorithms. Our algorithm can analyze data for 120,000 individuals in just a few hours, whereas current algorithms fail on data for even 20,000 individuals (http://mscompbio.codeplex.com/).
1,106 citations
TL;DR: The major conceptual and practical challenges of high-throughput RNA sequencing, the general classes of solutions for each category, and the interdependence between these categories are highlighted and discussed.
Abstract: High-throughput RNA sequencing (RNA-seq) promises a comprehensive picture of the transcriptome, allowing for the complete annotation and quantification of all genes and their isoforms across samples. Realizing this promise requires increasingly complex computational methods. These computational challenges fall into three main categories: (i) read mapping, (ii) transcriptome reconstruction and (iii) expression quantification. Here we explain the major conceptual and practical challenges, and the general classes of solutions for each category. Finally, we highlight the interdependence between these categories and discuss the benefits for different biological applications.
1,045 citations
TL;DR: Scanned Bessel beams are used in conjunction with structured illumination and/or two-photon excitation to create thinner light sheets better suited to three-dimensional (3D) subcellular imaging.
Abstract: A key challenge when imaging living cells is how to noninvasively extract the most spatiotemporal information possible. Unlike popular wide-field and confocal methods, plane-illumination microscopy limits excitation to the information-rich vicinity of the focal plane, providing effective optical sectioning and high speed while minimizing out-of-focus background and premature photobleaching. Here we used scanned Bessel beams in conjunction with structured illumination and/or two-photon excitation to create thinner light sheets (<0.5 μm) better suited to three-dimensional (3D) subcellular imaging. As demonstrated by imaging the dynamics of mitochondria, filopodia, membrane ruffles, intracellular vesicles and mitotic chromosomes in live cells, the microscope currently offers 3D isotropic resolution down to ∼0.3 μm, speeds up to nearly 200 image planes per second and the ability to noninvasively acquire hundreds of 3D data volumes from single living cells encompassing tens of thousands of image frames.
1,007 citations
TL;DR: It is reported that applying triple-stage mass spectrometry (MS3) almost completely eliminates interference in the two-proteome model.
Abstract: Quantitative mass spectrometry-based proteomics is highly versatile but not easily multiplexed. Isobaric labeling strategies allow mass spectrometry-based multiplexed proteome quantification; however, ratio distortion owing to protein quantification interference is a common effect. We present a two-proteome model (mixture of human and yeast proteins) in a sixplex isobaric labeling system to fully document the interference effect, and we report that applying triple-stage mass spectrometry (MS3) almost completely eliminates interference.
870 citations
TL;DR: A miniature (1.9 g) integrated fluorescence microscope made from mass-producible parts, including a semiconductor light source and sensor enables high-speed cellular imaging across ∼0.5 mm2 areas in active mice and allows concurrent tracking of Ca2+ spiking in >200 Purkinje neurons across nine cerebellar microzones.
Abstract: The light microscope is traditionally an instrument of substantial size and expense. Its miniaturized integration would enable many new applications based on mass-producible, tiny microscopes. Key prospective usages include brain imaging in behaving animals for relating cellular dynamics to animal behavior. Here we introduce a miniature (1.9 g) integrated fluorescence microscope made from mass-producible parts, including a semiconductor light source and sensor. This device enables high-speed cellular imaging across ∼0.5 mm2 areas in active mice. This capability allowed concurrent tracking of Ca2+ spiking in >200 Purkinje neurons across nine cerebellar microzones. During mouse locomotion, individual microzones exhibited large-scale, synchronized Ca2+ spiking. This is a mesoscopic neural dynamic missed by prior techniques for studying the brain at other length scales. Overall, the integrated microscope is a potentially transformative technology that permits distribution to many animals and enables diverse usages, such as portable diagnostics or microscope arrays for large-scale screens.
TL;DR: A computational tool for predicting the structure of DNA Origami objects is introduced and information is provided on the conditions under which DNA origami objects can be expected to maintain their structure.
Abstract: Molecular self-assembly with scaffolded DNA origami enables building custom-shaped nanometer-scale objects with molecular weights in the megadalton regime. Here we provide a practical guide for design and assembly of scaffolded DNA origami objects. We also introduce a computational tool for predicting the structure of DNA origami objects and provide information on the conditions under which DNA origami objects can be expected to maintain their structure.
TL;DR: It is shown that shRNAs, modeled on an endogenous microRNA, are extremely effective at silencing gene expression during oogenesis and the progress toward building a genome-wide shRNA resource is described.
Abstract: Short hairpin RNAs, expressed from microRNA scaffold–containing vectors, efficiently silence gene expression in female germ cells as well as somatic cells in the fly. A genome-wide resource is being developed.
TL;DR: It is concluded that high-quality sequencing approaches must be considered in conjunction with high-throughput sequencing for comparative genomics analyses and studies of genome evolution.
Abstract: High-throughput sequencing technologies promise to transform the fields of genetics and comparative biology by delivering tens of thousands of genomes in the near future. Although it is feasible to construct de novo genome assemblies in a few months, there has been relatively little attention to what is lost by sole application of short sequence reads. We compared the recent de novo assemblies using the short oligonucleotide analysis package (SOAP), generated from the genomes of a Han Chinese individual and a Yoruban individual, to experimentally validated genomic features. We found that de novo assemblies were 16.2% shorter than the reference genome and that 420.2 megabase pairs of common repeats and 99.1% of validated duplicated sequences were missing from the genome. Consequently, over 2,377 coding exons were completely missing. We conclude that high-quality sequencing approaches must be considered in conjunction with high-throughput sequencing for comparative genomics analyses and studies of genome evolution.
TL;DR: Super-resolution fluorescence imaging of live cells with high spatiotemporal resolution using stochastic optical reconstruction microscopy (STORM) opens a new window for characterizing cellular structures in living cells at the ultrastructural level.
Abstract: We report super-resolution fluorescence imaging of live cells with high spatiotemporal resolution using stochastic optical reconstruction microscopy (STORM). By labeling proteins either directly or via SNAP tags with photoswitchable dyes, we obtained two-dimensional (2D) and 3D super-resolution images of living cells, using clathrin-coated pits and the transferrin cargo as model systems. Bright, fast-switching probes enabled us to achieve 2D imaging at spatial resolutions of ∼25 nm and temporal resolutions as fast as 0.5 s. We also demonstrated live-cell 3D super-resolution imaging. We obtained 3D spatial resolution of ∼30 nm in the lateral direction and ∼50 nm in the axial direction at time resolutions as fast as 1-2 s with several independent snapshots. Using photoswitchable dyes with distinct emission wavelengths, we also demonstrated two-color 3D super-resolution imaging in live cells. These imaging capabilities open a new window for characterizing cellular structures in living cells at the ultrastructural level.
TL;DR: 'Significance analysis of interactome' (SAINT), a computational tool that assigns confidence scores to protein-protein interaction data generated using affinity purification–mass spectrometry (AP-MS), is presented.
Abstract: We present 'significance analysis of interactome' (SAINT), a computational tool that assigns confidence scores to protein-protein interaction data generated using affinity purification-mass spectrometry (AP-MS). The method uses label-free quantitative data and constructs separate distributions for true and false interactions to derive the probability of a bona fide protein-protein interaction. We show that SAINT is applicable to data of different scales and protein connectivity and allows transparent analysis of AP-MS data.
TL;DR: It is demonstrated the versatility of a collection of insertions of the transposon Minos-mediated integration cassette (MiMIC), in Drosophila melanogaster, which can revert insertions that function as gene traps and cause mutant phenotypes to revert to wild type by RMCE and modify insertions to control GAL4 or QF overexpression systems or perform lineage analysis using the Flp recombinase system.
Abstract: We demonstrate the versatility of a collection of insertions of the transposon Minos-mediated integration cassette (MiMIC), in Drosophila melanogaster. MiMIC contains a gene-trap cassette and the yellow+ marker flanked by two inverted bacteriophage ΦC31 integrase attP sites. MiMIC integrates almost at random in the genome to create sites for DNAmanipulation. The attP sites allow the replacement of the intervening sequence of the transposon with any other sequence through recombinase-mediated cassette exchange (RMCE). We can revert insertions that function as gene traps and cause mutant phenotypes to revert to wild type by RMCE and modify insertions to control GAL4 or QF overexpression systems or perform lineage analysis using the Flp recombinase system. Insertions in coding introns can be exchanged with protein-tag cassettes to create fusion proteins to follow protein expression and perform biochemical experiments. The applications of MiMIC vastly extend the D. melanogaster toolkit.
TL;DR: The two-photon absorption properties of a wide variety of fluorescent proteins, including new far-red variants, are reviewed to produce a comprehensive guide to choosing the right fluorescent protein and excitation wavelength for two-Photon applications.
Abstract: Two-photon excitation of fluorescent proteins is an attractive approach for imaging living systems. Today researchers are eager to know which proteins are the brightest and what the best excitation wavelengths are. Here we review the two-photon absorption properties of a wide variety of fluorescent proteins, including new far-red variants, to produce a comprehensive guide to choosing the right fluorescent protein and excitation wavelength for two-photon applications.
TL;DR: In this paper, the authors used a bacterial artificial chromosome (BAC) transgenic strategy to express the H134R variant of channelrhodopsin-2, ChR2(H134R), under the control of cell type-specific promoter elements.
Abstract: Optogenetic methods have emerged as powerful tools for dissecting neural circuit connectivity, function and dysfunction. We used a bacterial artificial chromosome (BAC) transgenic strategy to express the H134R variant of channelrhodopsin-2, ChR2(H134R), under the control of cell type–specific promoter elements. We performed an extensive functional characterization of the newly established VGAT-ChR2(H134R)-EYFP, ChAT-ChR2(H134R)-EYFP, Tph2-ChR2(H134R)-EYFP and Pvalb(H134R)-ChR2-EYFP BAC transgenic mouse lines and demonstrate the utility of these lines for precisely controlling action-potential firing of GABAergic, cholinergic, serotonergic and parvalbumin-expressing neuron subsets using blue light. This resource of cell type–specific ChR2(H134R) mouse lines will facilitate the precise mapping of neuronal connectivity and the dissection of the neural basis of behavior.
TL;DR: Context-dependent assembly (CoDA) is described, a platform for engineering ZFNs using only standard cloning techniques or custom DNA synthesis and rapidly altered 20 genes in Danio rerio, Arabidopsis thaliana and Glycine max.
Abstract: Context-dependent assembly (CoDA) of zinc finger nucleases is reported. Starting from an archive of zinc finger modules known to function well together, effective multifinger arrays can be constructed using standard techniques. Also in this issue, Doyon et al. report rational design of nucleases with improved cleavage activity.
TL;DR: PC-PALM is an effective tool with broad applicability for analysis of protein heterogeneity and function, adaptable to other single-molecule strategies, and shows dramatic changes in glycosylphosphatidylinositol (GPI)-anchored protein arrangement under varying perturbations.
Abstract: Photoactivated localization microscopy (PALM) is a powerful approach for investigating protein organization, yet tools for quantitative, spatial analysis of PALM datasets are largely missing. Combining pair-correlation analysis with PALM (PC-PALM), we provide a method to analyze complex patterns of protein organization across the plasma membrane without determination of absolute protein numbers. The approach uses an algorithm to distinguish a single protein with multiple appearances from clusters of proteins. This enables quantification of different parameters of spatial organization, including the presence of protein clusters, their size, density and abundance in the plasma membrane. Using this method, we demonstrate distinct nanoscale organization of plasma-membrane proteins with different membrane anchoring and lipid partitioning characteristics in COS-7 cells, and show dramatic changes in glycosylphosphatidylinositol (GPI)-anchored protein arrangement under varying perturbations. PC-PALM is thus an effective tool with broad applicability for analysis of protein heterogeneity and function, adaptable to other single-molecule strategies.
TL;DR: A diverse array of small molecule–based fluorescent probes is available for many different types of biological experiments and some of the most interesting applications are discussed.
Abstract: A diverse array of small molecule–based fluorescent probes is available for many different types of biological experiments. Here we examine the history of these probes and discuss some of the most interesting applications.
TL;DR: This work describes the production of a sequence-confirmed, clonal collection of over 16,100 human open-reading frames encoded in a versatile Gateway vector system, thereby enabling both targeted experiments and high-throughput screens in diverse cell types.
Abstract: Functional characterization of the human genome requires tools for systematically modulating gene expression in both loss-of-function and gain-of-function experiments. We describe the production of a sequence-confirmed, clonal collection of over 16,100 human open-reading frames (ORFs) encoded in a versatile Gateway vector system. Using this ORFeome resource, we created a genome-scale expression collection in a lentiviral vector, thereby enabling both targeted experiments and high-throughput screens in diverse cell types.
TL;DR: FUS identifies regions of brain activation and was used to image whisker-evoked cortical and thalamic responses and the propagation of epileptiform seizures in the rat brain.
Abstract: We present functional ultrasound (fUS), a method for imaging transient changes in blood volume in the whole brain at better spatiotemporal resolution than with other functional brain imaging modalities. fUS uses plane-wave illumination at high frame rate and can measure blood volumes in smaller vessels than previous ultrasound methods. fUS identifies regions of brain activation and was used to image whisker-evoked cortical and thalamic responses and the propagation of epileptiform seizures in the rat brain.
TL;DR: Methods for using simple ssDNA oligonucleotides in tandem with ZFNs to efficiently produce human cell lines with three distinct genetic outcomes: targeted point mutation, targeted genomic deletion of up to 100 kb and targeted insertion of small genetic elements concomitant with large genomic deletions.
Abstract: Zinc-finger nucleases (ZFNs) have enabled highly efficient gene targeting in multiple cell types and organisms. Here we describe methods for using simple ssDNA oligonucleotides in tandem with ZFNs to efficiently produce human cell lines with three distinct genetic outcomes: (i) targeted point mutation, (ii) targeted genomic deletion of up to 100 kb and (iii) targeted insertion of small genetic elements concomitant with large genomic deletions.
TL;DR: In this article, the authors present mProphet, a fully automated system that computes accurate error rates for the identification of targeted peptides in SRM data sets and maximizes specificity and sensitivity by combining relevant features in the data into a statistical model.
Abstract: Selected reaction monitoring (SRM) is a targeted mass spectrometric method that is increasingly used in proteomics for the detection and quantification of sets of preselected proteins at high sensitivity, reproducibility and accuracy. Currently, data from SRM measurements are mostly evaluated subjectively by manual inspection on the basis of ad hoc criteria, precluding the consistent analysis of different data sets and an objective assessment of their error rates. Here we present mProphet, a fully automated system that computes accurate error rates for the identification of targeted peptides in SRM data sets and maximizes specificity and sensitivity by combining relevant features in the data into a statistical model.
TL;DR: Application of CREST to whole-genome sequencing data from five pediatric T-lineage acute lymphoblastic leukemias and a human melanoma cell line, COLO-829, identified 160 somatic structural variations.
Abstract: We developed 'clipping reveals structure' (CREST), an algorithm that uses next-generation sequencing reads with partial alignments to a reference genome to directly map structural variations at the nucleotide level of resolution. Application of CREST to whole-genome sequencing data from five pediatric T-lineage acute lymphoblastic leukemias (T-ALLs) and a human melanoma cell line, COLO-829, identified 160 somatic structural variations. Experimental validation exceeded 80%, demonstrating that CREST had a high predictive accuracy.
TL;DR: An energy compensation model of ZFN specificity in which excess binding energy contributes to off-target ZFN cleavage is established and strategies for the improvement of future ZFN design are suggested.
Abstract: An in vitro method for examining cleavage patterns of zinc-finger nucleases (ZFNs) identifies previously unknown off-target cleavage sites. Some of the sites are present in the human genome and show evidence for ZFN-induced cleavage in cultured human cell lines.
TL;DR: 3D SIM is applied to living samples and recorded whole cells at up to 5 s per volume for >50 time points with 120-nm lateral and 360-nm axial resolution, demonstrating the technique by imaging microtubules in S2 cells and mitochondria in HeLa cells.
Abstract: Three-dimensional (3D) structured-illumination microscopy (SIM) can double the lateral and axial resolution of a wide-field fluorescence microscope but has been too slow for live imaging. Here we apply 3D SIM to living samples and record whole cells at up to 5 s per volume for >50 time points with 120-nm lateral and 360-nm axial resolution. We demonstrate the technique by imaging microtubules in S2 cells and mitochondria in HeLa cells.
TL;DR: The results confirm the expectation from original CLIP publications that RNA-binding proteins do not protect their binding sites sufficiently under the denaturing conditions used during the CLIP procedure, and show that extensive digestion with sequence-specific RNases strongly biases the recovered binding sites.
Abstract: Cross-linking and immunoprecipitation (CLIP) is increasingly used to map transcriptome-wide binding sites of RNA-binding proteins. We developed a method for CLIP data analysis, and applied it to compare CLIP with photoactivatable ribonucleoside-enhanced CLIP (PAR-CLIP) and to uncover how differences in cross-linking and ribonuclease digestion affect the identified sites. We found only small differences in accuracies of these methods in identifying binding sites of HuR, which binds low-complexity sequences, and Argonaute 2, which has a complex binding specificity. We found that cross-link-induced mutations led to single-nucleotide resolution for both PAR-CLIP and CLIP. Our results confirm the expectation from original CLIP publications that RNA-binding proteins do not protect their binding sites sufficiently under the denaturing conditions used during the CLIP procedure, and we show that extensive digestion with sequence-specific RNases strongly biases the recovered binding sites. This bias can be substantially reduced by milder nuclease digestion conditions.