Showing papers in "Nature Methods in 2007"
TL;DR: This work clarifies the preferred methodology by addressing four issues based on observed decoy hit frequencies: the major assumptions made with this database search strategy are reasonable, concatenated target-decoy database searches are preferable to separate target and decoydatabase searches, and the theoretical error associated with target-Decoy false positive (FP) rate measurements can be estimated.
Abstract: Liquid chromatography and tandem mass spectrometry (LC-MS/MS) has become the preferred method for conducting large-scale surveys of proteomes. Automated interpretation of tandem mass spectrometry (MS/MS) spectra can be problematic, however, for a variety of reasons. As most sequence search engines return results even for 'unmatchable' spectra, proteome researchers must devise ways to distinguish correct from incorrect peptide identifications. The target-decoy search strategy represents a straightforward and effective way to manage this effort. Despite the apparent simplicity of this method, some controversy surrounds its successful application. Here we clarify our preferred methodology by addressing four issues based on observed decoy hit frequencies: (i) the major assumptions made with this database search strategy are reasonable; (ii) concatenated target-decoy database searches are preferable to separate target and decoy database searches; (iii) the theoretical error associated with target-decoy false positive (FP) rate measurements can be estimated; and (iv) alternate methods for constructing decoy databases are similarly effective once certain considerations are taken into account.
3,602 citations
TL;DR: These competitive inhibitors are transcripts expressed from strong promoters, containing multiple, tandem binding sites to a microRNA of interest that specifically inhibit microRNAs with a complementary heptameric seed, such that a single sponge can be used to block an entire microRNA seed family.
Abstract: MicroRNAs are predicted to regulate thousands of mammalian genes, but relatively few targets have been experimentally validated and few microRNA loss-of-function phenotypes have been assigned. As an alternative to chemically modified antisense oligonucleotides, we developed microRNA inhibitors that can be expressed in cells, as RNAs produced from transgenes. Termed 'microRNA sponges', these competitive inhibitors are transcripts expressed from strong promoters, containing multiple, tandem binding sites to a microRNA of interest. When vectors encoding these sponges are transiently transfected into cultured cells, sponges derepress microRNA targets at least as strongly as chemically modified antisense oligonucleotides. They specifically inhibit microRNAs with a complementary heptameric seed, such that a single sponge can be used to block an entire microRNA seed family. RNA polymerase II promoter (Pol II)-driven sponges contain a fluorescence reporter gene for identification and sorting of sponge-treated cells. We envision the use of stably expressed sponges in animal models of disease and development.
2,054 citations
TL;DR: An algorithm, called Percolator, is described, for improving the rate of confident peptide identifications from a collection of tandem mass spectra, using semi-supervised machine learning to discriminate between correct and decoy spectrum identifications.
Abstract: Shotgun proteomics uses liquid chromatography-tandem mass spectrometry to identify proteins in complex biological samples We describe an algorithm, called Percolator, for improving the rate of confident peptide identifications from a collection of tandem mass spectra Percolator uses semi-supervised machine learning to discriminate between correct and decoy spectrum identifications, correctly assigning peptides to 17% more spectra from a tryptic Saccharomyces cerevisiae dataset, and up to 77% more spectra from non-tryptic digests, relative to a fully supervised approach
1,836 citations
TL;DR: ChIP-seq identified 41,582 and 11,004 putative STAT1-binding regions in stimulated and unstimulated cells, respectively, and found 24 loci known to contain STAT1 interferon-responsive binding sites, including 24 that were enriched in sequences similar to known STAT1 binding motifs.
Abstract: We developed a method, ChIP-sequencing (ChIP-seq), combining chromatin immunoprecipitation (ChIP) and massively parallel sequencing to identify mammalian DNA sequences bound by transcription factors in vivo. We used ChIP-seq to map STAT1 targets in interferon-γ (IFN-γ)–stimulated and unstimulated human HeLa S3 cells, and compared the method's performance to ChIP-PCR and to ChIP-chip for four chromosomes. By ChIP-seq, using 15.1 and 12.9 million uniquely mapped sequence reads, and an estimated false discovery rate of less than 0.001, we identified 41,582 and 11,004 putative STAT1-binding regions in stimulated and unstimulated cells, respectively. Of the 34 loci known to contain STAT1 interferon-responsive binding sites, ChIP-seq found 24 (71%). ChIP-seq targets were enriched in sequences similar to known STAT1 binding motifs. Comparisons with two ChIP-PCR data sets suggested that ChIP-seq sensitivity was between 70% and 92% and specificity was at least 95%.
1,444 citations
TL;DR: A robust and generalized method for the culturing of various human breast cell lines in three dimensions is described and the preparation of cellular extracts from these cultures for molecular analyses are described.
Abstract: Extracellular matrix is a key regulator of normal homeostasis and tissue phenotype. Important signals are lost when cells are cultured ex vivo on two-dimensional plastic substrata. Many of these crucial microenvironmental cues may be restored using three-dimensional (3D) cultures of laminin-rich extracellular matrix (lrECM). These 3D culture assays allow phenotypic discrimination between nonmalignant and malignant mammary cells, as the former grown in a 3D context form polarized, growth-arrested acinus-like colonies whereas the latter form disorganized, proliferative and nonpolar colonies. Signaling pathways that function in parallel in cells cultured on plastic become reciprocally integrated when the cells are exposed to basement membrane-like gels. Appropriate 3D culture thus provides a more physiologically relevant approach to the analysis of gene function and cell phenotype ex vivo. We describe here a robust and generalized method for the culturing of various human breast cell lines in three dimensions and describe the preparation of cellular extracts from these cultures for molecular analyses. The procedure below describes the 3D 'embedded' assay, in which cells are cultured embedded in an lrECM gel (Fig. 1). By lrECM, we refer to the solubilized extract derived from the Engelbreth-Holm-Swarm mouse sarcoma cells. For a discussion of user options regarding 3D matrices, see Box 1. Alternatively, the 3D 'on-top' assay, in which cells are cultured on top of a thin lrECM gel overlaid with a dilute solution of lrECM, may be used as described in Box 2 (Fig. 1 and Fig. 2).
1,214 citations
TL;DR: This new technique allows optical sectioning of fixed mouse brains with cellular resolution and can be used to detect single GFP-labeled neurons in excised mouse hippocampi and is ideally suited for high-throughput phenotype screening of transgenic mice and thus will benefit the investigation of disease models.
Abstract: Visualizing entire neuronal networks for analysis in the intact brain has been impossible up to now. Techniques like computer tomography or magnetic resonance imaging (MRI) do not yield cellular resolution, and mechanical slicing procedures are insufficient to achieve high-resolution reconstructions in three dimensions. Here we present an approach that allows imaging of whole fixed mouse brains. We modified 'ultramicroscopy' by combining it with a special procedure to clear tissue. We show that this new technique allows optical sectioning of fixed mouse brains with cellular resolution and can be used to detect single GFP-labeled neurons in excised mouse hippocampi. We obtained three-dimensional (3D) images of dendritic trees and spines of populations of CA1 neurons in isolated hippocampi. Also in fruit flies and in mouse embryos, we were able to visualize details of the anatomy by imaging autofluorescence. Our method is ideally suited for high-throughput phenotype screening of transgenic mice and thus will benefit the investigation of disease models.
1,140 citations
TL;DR: A new cloning method, sequence and ligation–independent cloning (SLIC), which allows the assembly of multiple DNA fragments in a single reaction using in vitro homologous recombination and single-strand annealing, which allows much greater versatility in the generation of recombinant DNA for the purposes of synthetic biology.
Abstract: We describe a new cloning method, sequence and ligation–independent cloning (SLIC), which allows the assembly of multiple DNA fragments in a single reaction using in vitro homologous recombination and single-strand annealing. SLIC mimics in vivo homologous recombination by relying on exonuclease-generated ssDNA overhangs in insert and vector fragments, and the assembly of these fragments by recombination in vitro. SLIC inserts can also be prepared by incomplete PCR (iPCR) or mixed PCR. SLIC allows efficient and reproducible assembly of recombinant DNA with as many as 5 and 10 fragments simultaneously. SLIC circumvents the sequence requirements of traditional methods and functions much more efficiently at very low DNA concentrations when combined with RecA to catalyze homologous recombination. This flexibility allows much greater versatility in the generation of recombinant DNA for the purposes of synthetic biology.
992 citations
TL;DR: Immonium ions generated via HCD pinpoint modifications such as phosphotyrosine with very high confidence are generated via higher-energy C-trap dissociation (HCD) and this work shows that an added octopole collision cell facilitates de novo sequencing.
Abstract: Peptide sequencing is the basis of mass spectrometry–driven proteomics. Here we show that in the linear ion trap–orbitrap mass spectrometer (LTQ Orbitrap) peptide ions can be efficiently fragmented by high-accuracy and full-mass-range tandem mass spectrometry (MS/MS) via higher-energy C-trap dissociation (HCD). Immonium ions generated via HCD pinpoint modifications such as phosphotyrosine with very high confidence. Additionally we show that an added octopole collision cell facilitates de novo sequencing.
905 citations
TL;DR: Important examples of widely used caged compounds, their design features and synthesis, as well as practical details of how to use them with living cells are described.
Abstract: Caged compounds are light-sensitive probes that functionally encapsulate biomolecules in an inactive form. Irradiation liberates the trapped molecule, permitting targeted perturbation of a biological process. Uncaging technology and fluorescence microscopy are 'optically orthogonal': the former allows control, and the latter, observation of cellular function. Used in conjunction with other technologies (for example, patch clamp and/or genetics), the light beam becomes a uniquely powerful tool to stimulate a selected biological target in space or time. Here I describe important examples of widely used caged compounds, their design features and synthesis, as well as practical details of how to use them with living cells.
828 citations
TL;DR: This new biomimetic model may provide a broadly applicable 3D culture system to study the effect of microenvironmental conditions on tumor malignancy in vitro and in vivo.
Abstract: Microenvironmental conditions control tumorigenesis and biomimetic culture systems that allow for in vitro and in vivo tumor modeling may greatly aid studies of cancer cells' dependency on these conditions. We engineered three-dimensional (3D) human tumor models using carcinoma cells in polymeric scaffolds that recreated microenvironmental characteristics representative of tumors in vivo. Strikingly, the angiogenic characteristics of tumor cells were dramatically altered upon 3D culture within this system, and corresponded much more closely to tumors formed in vivo. Cells in this model were also less sensitive to chemotherapy and yielded tumors with enhanced malignant potential. We assessed the broad relevance of these findings with 3D culture of other tumor cell lines in this same model, comparison with standard 3D Matrigel culture and in vivo experiments. This new biomimetic model may provide a broadly applicable 3D culture system to study the effect of microenvironmental conditions on tumor malignancy in vitro and in vivo.
809 citations
TL;DR: The current state of the art of MALDI-IMS is discussed along with some recent applications and technological developments that illustrate not only its current capabilities but also the future potential of the technique to provide a better understanding of the underlying molecular mechanisms of biological processes.
Abstract: Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) is emerging as a powerful tool for investigating the distribution of molecules within biological systems through the direct analysis of thin tissue sections. Unique among imaging methods, MALDI-IMS can determine the distribution of hundreds of unknown compounds in a single measurement. We discuss the current state of the art of MALDI-IMS along with some recent applications and technological developments that illustrate not only its current capabilities but also the future potential of the technique to provide a better understanding of the underlying molecular mechanisms of biological processes.
TL;DR: The aim of this review is introduce biologists to current strategies in mass spectrometry–based proteomics that are used to characterize protein post-translational modifications, noting strengths and shortcomings of various approaches.
Abstract: Post-translational modifications of proteins control many biological processes, and examining their diversity is critical for understanding mechanisms of cell regulation. Mass spectrometry is a fundamental tool for detecting and mapping covalent modifications and quantifying their changes. Modern approaches have made large-scale experiments possible, screening complex mixtures of proteins for alterations in chemical modifications. By profiling protein chemistries, biologists can gain deeper insight into biological control. The aim of this review is introduce biologists to current strategies in mass spectrometry–based proteomics that are used to characterize protein post-translational modifications, noting strengths and shortcomings of various approaches.
TL;DR: High-density microarrays were applied to the enrichment of specific sequences from the human genome for high-throughput sequencing to supersede multiplex PCR for the large-scale analysis of genomic regions.
Abstract: We applied high-density microarrays to the enrichment of specific sequences from the human genome for high-throughput sequencing. After capture of 6,726 approximately 500-base 'exon' segments, and of 'locus-specific' regions ranging in size from 200 kb to 5 Mb, followed by sequencing on a 454 Life Sciences FLX sequencer, most sequence reads represented selection targets. These direct selection methods supersede multiplex PCR for the large-scale analysis of genomic regions.
TL;DR: This review discusses critical issues related to data processing and analysis in proteomics and describes available methods and tools and places special emphasis on the elaboration of results that are supported by sound statistical arguments.
Abstract: The analysis of the large amount of data generated in mass spectrometry-based proteomics experiments represents a significant challenge and is currently a bottleneck in many proteomics projects. In this review we discuss critical issues related to data processing and analysis in proteomics and describe available methods and tools. We place special emphasis on the elaboration of results that are supported by sound statistical arguments.
TL;DR: A single-molecule technique for counting subunits of proteins in live cell membranes by observing bleaching steps of GFP fused to a protein of interest is described.
Abstract: The subunit number and stoichiometry of membrane-bound proteins are difficult to determine without disrupting their membrane environment. Here we describe a single-molecule technique for counting subunits of proteins in live cell membranes by observing bleaching steps of GFP fused to a protein of interest. After testing the method with proteins of known stoichiometry expressed in Xenopus laevis oocytes, we resolved the composition of NMDA receptors composed of NR1 and NR3 subunits.
TL;DR: A far-red fluorescent protein, named Katushka, is reported, which is seven- to tenfold brighter compared to the spectrally close HcRed or mPlum, and is characterized by fast maturation as well as a high pH-stability and photostability, which makes it the protein of choice for visualization in living tissues.
Abstract: For deep imaging of animal tissues, the optical window favorable for light penetration is in near-infrared wavelengths, which requires proteins with emission spectra in the far-red wavelengths. Here we report a far-red fluorescent protein, named Katushka, which is seven- to tenfold brighter compared to the spectrally close HcRed or mPlum, and is characterized by fast maturation as well as a high pH-stability and photostability. These unique characteristics make Katushka the protein of choice for visualization in living tissues. We demonstrate superiority of Katushka for whole-body imaging by direct comparison with other red and far-red fluorescent proteins. We also describe a monomeric version of Katushka, named mKate, which is characterized by high brightness and photostability, and should be an excellent fluorescent label for protein tagging in the far-red part of the spectrum.
TL;DR: TagRFP is reported, a monomeric red fluorescent protein, which is characterized by high brightness, complete chromophore maturation, prolonged fluorescence lifetime and high pH-stability, which makes it an excellent tag for protein localization studies and fluorescence resonance energy transfer (FRET) applications.
Abstract: Fluorescent proteins have become extremely popular tools for in vivo imaging and especially for the study of localization, motility and interaction of proteins in living cells. Here we report TagRFP, a monomeric red fluorescent protein, which is characterized by high brightness, complete chromophore maturation, prolonged fluorescence lifetime and high pH-stability. These properties make TagRFP an excellent tag for protein localization studies and fluorescence resonance energy transfer (FRET) applications.
TL;DR: A deletion-mutant rabies virus encoding EGFP is constructed and found to be an excellent tool for studying detailed morphology and physiology of neurons projecting to injection sites within the mammalian brain.
Abstract: We have constructed a deletion-mutant rabies virus encoding EGFP and find it to be an excellent tool for studying detailed morphology and physiology of neurons projecting to injection sites within the mammalian brain. The virus cannot spread beyond initially infected cells yet, unlike other viral vectors, replicates its core within them. The cells therefore fluoresce intensely, revealing fine dendritic and axonal structure with no background from partially or faintly labeled cells.
TL;DR: The results suggest that the three methods detect different, partially overlapping segments of the phosphoproteome and that, at present, no single method is sufficient for a comprehensive phosphopeptides analysis.
Abstract: The ability to routinely analyze and quantitatively measure changes in protein phosphorylation on a proteome-wide scale is essential for biological and clinical research. We assessed the ability of three common phosphopeptide isolation methods (phosphoramidate chemistry (PAC), immobilized metal affinity chromatography (IMAC) and titanium dioxide) to reproducibly, specifically and comprehensively isolate phosphopeptides from complex mixtures. Phosphopeptides were isolated from aliquots of a tryptic digest of the cytosolic fraction of Drosophila melanogaster Kc167 cells and analyzed by liquid chromatography-electrospray ionization tandem mass spectrometry. Each method reproducibly isolated phosphopeptides. The methods, however, differed in their specificity of isolation and, notably, in the set of phosphopeptides isolated. The results suggest that the three methods detect different, partially overlapping segments of the phosphoproteome and that, at present, no single method is sufficient for a comprehensive phosphoproteome analysis.
TL;DR: Two microfluidic chips, the 'behavior' chip and the 'olfactory' chip, are developed for imaging of neuronal and behavioral responses in C. elegans and revealed previously unknown properties of AVA and ASH neurons.
Abstract: The nematode C. elegans is an excellent model organism for studying behavior at the neuronal level. Because of the organism's small size, it is challenging to deliver stimuli to C. elegans and monitor neuronal activity in a controlled environment. To address this problem, we developed two microfluidic chips, the 'behavior' chip and the 'olfactory' chip for imaging of neuronal and behavioral responses in C. elegans. We used the behavior chip to correlate the activity of AVA command interneurons with the worm locomotion pattern. We used the olfactory chip to record responses from ASH sensory neurons exposed to high-osmotic-strength stimulus. Observation of neuronal responses in these devices revealed previously unknown properties of AVA and ASH neurons. The use of these chips can be extended to correlate the activity of sensory neurons, interneurons and motor neurons with the worm's behavior.
TL;DR: PhyloPythia is presented, a composition-based classifier that combines higher-level generic clades from a set of 340 completed genomes with sample-derived population models and allows the accurate classification of most sequence fragments across all considered taxonomic ranks, even for unknown organisms.
Abstract: Metagenome studies have retrieved vast amounts of sequence data from a variety of environments leading to new discoveries and insights into the uncultured microbial world. Except for very simple communities, the encountered diversity has made fragment assembly and the subsequent analysis a challenging problem. A taxonomic characterization of metagenomic fragments is required for a deeper understanding of shotgun-sequenced microbial communities, but success has mostly been limited to sequences containing phylogenetic marker genes. Here we present PhyloPythia, a composition-based classifier that combines higher-level generic clades from a set of 340 completed genomes with sample-derived population models. Extensive analyses on synthetic and real metagenome data sets showed that PhyloPythia allows the accurate classification of most sequence fragments across all considered taxonomic ranks, even for unknown organisms. The method requires no more than 100 kb of training sequence for the creation of accurate models of sample-specific populations and can assign fragments ≥1 kb with high specificity.
TL;DR: It is shown that targeting oligonucleotides released from programmable microarrays can be used to capture and amplify ∼10,000 human exons in a single multiplex reaction, and it is anticipated that highly multiplexed methods for targeted amplification will enable the comprehensive resequencing ofhuman exons at a fraction of the cost of whole-genome resequenced.
Abstract: A new generation of technologies is poised to reduce DNA sequencing costs by several orders of magnitude. But our ability to fully leverage the power of these technologies is crippled by the absence of suitable 'front-end' methods for isolating complex subsets of a mammalian genome at a scale that matches the throughput at which these platforms will routinely operate. We show that targeting oligonucleotides released from programmable microarrays can be used to capture and amplify approximately 10,000 human exons in a single multiplex reaction. Additionally, we show integration of this protocol with ultra-high-throughput sequencing for targeted variation discovery. Although the multiplex capture reaction is highly specific, we found that nonuniform capture is a key issue that will need to be resolved by additional optimization. We anticipate that highly multiplexed methods for targeted amplification will enable the comprehensive resequencing of human exons at a fraction of the cost of whole-genome resequencing.
TL;DR: In this article, the authors reported stimulated emission depletion (STED) fluorescence microscopy with continuous wave (CW) laser beams, achieving a resolution of 29-60 nm in the focal plane.
Abstract: We report stimulated emission depletion (STED) fluorescence microscopy with continuous wave (CW) laser beams. Lateral fluorescence confinement from the scanning focal spot delivered a resolution of 29-60 nm in the focal plane, corresponding to a 5-8-fold improvement over the diffraction barrier. Axial spot confinement increased the axial resolution by 3.5-fold. We observed three-dimensional (3D) subdiffraction resolution in 3D image stacks. Viable for fluorophores with low triplet yield, the use of CW light sources greatly simplifies the implementation of this concept of far-field fluorescence nanoscopy.
TL;DR: In this paper, a facile allelic exchange system was proposed by identifying and expression of mycobacteriophage-encoded recombination proteins, adapting a strategy developed previously for recombineering in Escherichia coli.
Abstract: Genetic dissection of M. tuberculosis is complicated by its slow growth and its high rate of illegitimate recombination relative to homologous DNA exchange. We report here the development of a facile allelic exchange system by identification and expression of mycobacteriophage-encoded recombination proteins, adapting a strategy developed previously for recombineering in Escherichia coli. Identifiable recombination proteins are rare in mycobacteriophages, and only 1 of 30 genomically characterized mycobacteriophages (Che9c) encodes homologs of both RecE and RecT. Expression and biochemical characterization show that Che9c gp60 and gp61 encode exonuclease and DNA-binding activities, respectively, and expression of these proteins substantially elevates recombination facilitating allelic exchange in both M. smegmatis and M. tuberculosis. Mycobacterial recombineering thus provides a simple approach for the construction of gene replacement mutants in both slow- and fast-growing mycobacteria.
TL;DR: Recent approaches and applications of top-down mass spectrometry are outlined as well as an outlook for its future.
Abstract: Top-down mass spectrometry is an emerging technology which strives to preserve the post-translationally modified forms of proteins present in vivo by measuring them intact, rather than measuring peptides produced from them by proteolysis. The top-down technology is beginning to capture the interest of biologists and mass spectrometrists alike, with a main goal of deciphering interaction networks operative in cellular pathways. Here we outline recent approaches and applications of top-down mass spectrometry as well as an outlook for its future.
TL;DR: To bioengineer ectodermal organs such as teeth and whisker follicles, a three-dimensional organ-germ culture method is developed, which generated a structurally correct tooth, after both in vitro organ culture and transplantation under a tooth cavity in vivo.
Abstract: To bioengineer ectodermal organs such as teeth and whisker follicles, we developed a three-dimensional organ-germ culture method. The bioengineered tooth germ generated a structurally correct tooth, after both in vitro organ culture as well as transplantation under a tooth cavity in vivo, showing penetration of blood vessels and nerve fibers. Our method provides a substantial advance in the development of bioengineered organ replacement strategies and regenerative therapies.
TL;DR: It is demonstrated, by cryo-electron tomography of Escherichia coli cells, that a focused ion beam (FIB) can be used to thin whole frozen-hydrated cells in a convenient and essentially artifact-free way.
Abstract: Cryo-electron microscopy can provide high-resolution structural information about cells and organelles in the nearly native, frozen-hydrated state. Applicability, however, is limited by difficulties encountered in preparing suitably thin, vitreously frozen biological specimens. We demonstrate, by cryo-electron tomography of Escherichia coli cells, that a focused ion beam (FIB) can be used to thin whole frozen-hydrated cells in a convenient and essentially artifact-free way.
TL;DR: A step-by-step guide to the application of FCS to cellular systems, including methods for minimizing artifacts, optimizing measurement conditions and obtaining parameter values in the face of diverse and complex conditions of the living cell.
Abstract: In fluorescence correlation spectroscopy (FCS), information about molecular dynamics is extracted by recording the fluctuating signal of a pico- to micromolar concentration of fluorescent molecules in solution. As primary parameters, FCS provides time constants of the fluorescence emission, as well as numbers and dwell times of the observed particles diffusing through the open volume. A biochemical reaction or macromolecular rearrangement causing changes in these parameters, when linked to fluorescence readout, can be quantified by FCS. Since the measurements are now routinely performed in a laser-illuminated confocal spot, making measurements in living cells is straightforward. Different cellular compartments, such as the nucleus, the cytoplasm, or the plasma membrane, can be easily distinguished and addressed. In addition to biochemical reactions, the local environment of macromolecules, for example, ion concentrations, pH, or viscosity, can be probed. Thus, FCS is a versatile and attractive technique for researchers striving for a quantitative understanding of interactions and dynamics of biological and in particular cellular systems.
TL;DR: It is demonstrated that large human genomic regions, on the order of hundreds of kilobases, can be enriched and resequenced with resequencing arrays.
Abstract: We developed a general method, microarray-based genomic selection (MGS), capable of selecting and enriching targeted sequences from complex eukaryotic genomes without the repeat blocking steps necessary for bacterial artificial chromosome (BAC)-based genomic selection. We demonstrate that large human genomic regions, on the order of hundreds of kilobases, can be enriched and resequenced with resequencing arrays. MGS, when combined with a next-generation resequencing technology, can enable large-scale resequencing in single-investigator laboratories.
TL;DR: This work presents a maximum likelihood–based classification method that does not depend on prior knowledge about the structural variability, and demonstrates its effectiveness for two macromolecular assemblies with different types of conformational variability: the Escherichia coli ribosome and Simian virus 40 large T-antigen.
Abstract: Although three-dimensional electron microscopy (3D-EM) permits structural characterization of macromolecular assemblies in distinct functional states, the inability to classify projections from structurally heterogeneous samples has severely limited its application. We present a maximum likelihood–based classification method that does not depend on prior knowledge about the structural variability, and demonstrate its effectiveness for two macromolecular assemblies with different types of conformational variability: the Escherichia coli ribosome and Simian virus 40 (SV40) large T-antigen.