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Showing papers in "Nature Methods in 2013"


Journal ArticleDOI
TL;DR: The UPARSE pipeline reports operational taxonomic unit (OTU) sequences with ≤1% incorrect bases in artificial microbial community tests, compared with >3% correct bases commonly reported by other methods.
Abstract: Amplified marker-gene sequences can be used to understand microbial community structure, but they suffer from a high level of sequencing and amplification artifacts. The UPARSE pipeline reports operational taxonomic unit (OTU) sequences with ≤1% incorrect bases in artificial microbial community tests, compared with >3% incorrect bases commonly reported by other methods. The improved accuracy results in far fewer OTUs, consistently closer to the expected number of species in a community.

11,329 citations


Journal ArticleDOI
TL;DR: The feasibility of analyzing an individual's epigenome on a timescale compatible with clinical decision-making is demonstrated and classes of DNA-binding factors that strictly avoided, could tolerate or tended to overlap with nucleosomes are discovered.
Abstract: We describe an assay for transposase-accessible chromatin using sequencing (ATAC-seq), based on direct in vitro transposition of sequencing adaptors into native chromatin, as a rapid and sensitive method for integrative epigenomic analysis. ATAC-seq captures open chromatin sites using a simple two-step protocol with 500-50,000 cells and reveals the interplay between genomic locations of open chromatin, DNA-binding proteins, individual nucleosomes and chromatin compaction at nucleotide resolution. We discovered classes of DNA-binding factors that strictly avoided, could tolerate or tended to overlap with nucleosomes. Using ATAC-seq maps of human CD4(+) T cells from a proband obtained on consecutive days, we demonstrated the feasibility of analyzing an individual's epigenome on a timescale compatible with clinical decision-making.

4,984 citations


Journal ArticleDOI
TL;DR: This work presents a hierarchical genome-assembly process (HGAP) for high-quality de novo microbial genome assemblies using only a single, long-insert shotgun DNA library in conjunction with Single Molecule, Real-Time (SMRT) DNA sequencing.
Abstract: We present a hierarchical genome-assembly process (HGAP) for high-quality de novo microbial genome assemblies using only a single, long-insert shotgun DNA library in conjunction with Single Molecule, Real-Time (SMRT) DNA sequencing. Our method uses the longest reads as seeds to recruit all other reads for construction of highly accurate preassembled reads through a directed acyclic graph-based consensus procedure, which we follow with assembly using off-the-shelf long-read assemblers. In contrast to hybrid approaches, HGAP does not require highly accurate raw reads for error correction. We demonstrate efficient genome assembly for several microorganisms using as few as three SMRT Cell zero-mode waveguide arrays of sequencing and for BACs using just one SMRT Cell. Long repeat regions can be successfully resolved with this workflow. We also describe a consensus algorithm that incorporates SMRT sequencing primary quality values to produce de novo genome sequence exceeding 99.999% accuracy.

3,647 citations


Journal ArticleDOI
TL;DR: It is demonstrated that high-quality read length and abundance are the primary factors differentiating correct from erroneous reads produced by Illumina GAIIx, HiSeq and MiSeq instruments.
Abstract: High-throughput sequencing has revolutionized microbial ecology, but read quality remains a considerable barrier to accurate taxonomy assignment and α-diversity assessment for microbial communities. We demonstrate that high-quality read length and abundance are the primary factors differentiating correct from erroneous reads produced by Illumina GAIIx, HiSeq and MiSeq instruments. We present guidelines for user-defined quality-filtering strategies, enabling efficient extraction of high-quality data and facilitating interpretation of Illumina sequencing results.

2,931 citations


Journal ArticleDOI
TL;DR: Smart-seq2 with improved reverse transcription, template switching and preamplification to increase both yield and length of cDNA libraries generated from individual cells to improve detection, coverage, bias and accuracy.
Abstract: Single-cell gene expression analyses hold promise for characterizing cellular heterogeneity, but current methods compromise on either the coverage, the sensitivity or the throughput. Here, we introduce Smart-seq2 with improved reverse transcription, template switching and preamplification to increase both yield and length of cDNA libraries generated from individual cells. Smart-seq2 transcriptome libraries have improved detection, coverage, bias and accuracy compared to Smart-seq libraries and are generated with off-the-shelf reagents at lower cost.

1,834 citations


Journal ArticleDOI
TL;DR: This approach determined a 3.3-Å-resolution structure of an ∼700-kDa protein with D7 symmetry, the Thermoplasma acidophilum 20S proteasome, showing clear side-chain density and greatly enhances image quality and data acquisition efficiency.
Abstract: In recent work with large high-symmetry viruses, single-particle electron cryomicroscopy (cryo-EM) has achieved the determination of near-atomic-resolution structures by allowing direct fitting of atomic models into experimental density maps. However, achieving this goal with smaller particles of lower symmetry remains challenging. Using a newly developed single electron-counting detector, we confirmed that electron beam-induced motion substantially degrades resolution, and we showed that the combination of rapid readout and nearly noiseless electron counting allow image blurring to be corrected to subpixel accuracy, restoring intrinsic image information to high resolution (Thon rings visible to ∼3 A). Using this approach, we determined a 3.3-A-resolution structure of an ∼700-kDa protein with D7 symmetry, the Thermoplasma acidophilum 20S proteasome, showing clear side-chain density. Our method greatly enhances image quality and data acquisition efficiency-key bottlenecks in applying near-atomic-resolution cryo-EM to a broad range of protein samples.

1,726 citations


Journal ArticleDOI
TL;DR: It is shown that metagenomeSeq outperforms the tools currently used in this field and relies on a novel normalization technique and a statistical model that accounts for undersampling in large-scale marker-gene studies.
Abstract: We introduce a methodology to assess differential abundance in sparse high-throughput microbial marker-gene survey data. Our approach, implemented in the metagenomeSeq Bioconductor package, relies on a novel normalization technique and a statistical model that accounts for undersampling-a common feature of large-scale marker-gene studies. Using simulated data and several published microbiota data sets, we show that metagenomeSeq outperforms the tools currently used in this field.

1,664 citations


Journal ArticleDOI
TL;DR: The contaminant repository for affinity purification (the CRAPome) is presented and its use for scoring protein-protein interactions is described and aggregating negative controls from multiple AP-MS studies can increase coverage and improve the characterization of background associated with a given experimental protocol.
Abstract: Affinity purification coupled with mass spectrometry (AP-MS) is a widely used approach for the identification of protein-protein interactions. However, for any given protein of interest, determining which of the identified polypeptides represent bona fide interactors versus those that are background contaminants (for example, proteins that interact with the solid-phase support, affinity reagent or epitope tag) is a challenging task. The standard approach is to identify nonspecific interactions using one or more negative-control purifications, but many small-scale AP-MS studies do not capture a complete, accurate background protein set when available controls are limited. Fortunately, negative controls are largely bait independent. Hence, aggregating negative controls from multiple AP-MS studies can increase coverage and improve the characterization of background associated with a given experimental protocol. Here we present the contaminant repository for affinity purification (the CRAPome) and describe its use for scoring protein-protein interactions. The repository (currently available for Homo sapiens and Saccharomyces cerevisiae) and computational tools are freely accessible at http://www.crapome.org/.

1,355 citations


Journal ArticleDOI
TL;DR: A Cas9-based transactivator that is targeted to DNA sequences by guide RNA molecules is created, demonstrating a simple and versatile approach for RNA-guided gene activation.
Abstract: Technologies for engineering synthetic transcription factors have enabled many advances in medical and scientific research. In contrast to existing methods based on engineering of DNA-binding proteins, we created a Cas9-based transactivator that is targeted to DNA sequences by guide RNA molecules. Coexpression of this transactivator and combinations of guide RNAs in human cells induced specific expression of endogenous target genes, demonstrating a simple and versatile approach for RNA-guided gene activation.

1,247 citations


Journal ArticleDOI
TL;DR: The computational performance of SHAPEIT2 is competitive compared to other methods and had the property that SER decreases as sample size increases, illustrating that the SHAPEit2 model can adapt to data sets with very high SNP density.
Abstract: SHAPEIT2 uses multithreading so that multiple cores can be used to phase whole chromosomes, allowing users to make the best use of their computational resources. We tested SHAPEIT2 on several large-sample, whole-chromosome data sets from a range of SNP genotyping chips (Supplementary Note 1). SHAPEIT2 outperforms other methods (Fig. 1a–c) in terms of switch error rate (SER) and the mean distance between switch errors (Supplementary Figs. 1 and 2). As compared to SHAPEIT1, SHAPEIT2 reduced SER by as much as 45% on these data sets. For example, on 1,229 Vietnamese samples assayed on the Illumina 660K chip on chromosome 22, the SERs of SHAPEIT2 (K = 100, W = 2 Mb), SHAPEIT1 (K = 100) (ref. 2), HAPI-UR (v1.01) (ref. 4), Beagle (v3.3) (ref. 5), Impute2 v2.1.2 (K = 100) (ref. 3), MaCH v1.0.18 (K = 100) (ref. 6) and fastPHASE (v1.4) (ref. 7) were 2.87%, 4.64%, 4.75%, 5.14%, 5.57%, 6.05% and 6.34%, respectively. In general, SHAPEIT2 with low values of K outperformed SHAPEIT1 with high values of K (Fig. 1a–c). As the number of samples increased (up to ~9,000 samples in our tests), we found that SHAPEIT2 outperformed other methods and had the property that SER decreases as sample size increases (Fig. 1d). We assessed accuracy on sequence data by phasing 381 European samples from the 1000 Genomes Project (TGP) together with genotypes from two trio parents sequenced at high coverage. We found that SHAPEIT2 (K = 100, W = 0.3 Mb) reduced SER by 38% compared to Beagle (Supplementary Table 1 and Supplementary Note 2), illustrating that the SHAPEIT2 model can adapt to data sets with very high SNP density. The computational performance of SHAPEIT2 is competitive compared to other methods. Figure 1e shows the computational Improved whole-chromosome phasing for disease and population genetic studies

1,242 citations


Journal ArticleDOI
TL;DR: Light-sheet microscopy is used to record activity from the entire volume of the brain of the larval zebrafish in vivo at 0.8 Hz, capturing more than 80% of all neurons at single-cell resolution, demonstrating how this technique can be used to reveal functionally defined circuits across the brain.
Abstract: Brain function relies on communication between large populations of neurons across multiple brain areas, a full understanding of which would require knowledge of the time-varying activity of all neurons in the central nervous system. Here we use light-sheet microscopy to record activity, reported through the genetically encoded calcium indicator GCaMP5G, from the entire volume of the brain of the larval zebrafish in vivo at 0.8 Hz, capturing more than 80% of all neurons at single-cell resolution. Demonstrating how this technique can be used to reveal functionally defined circuits across the brain, we identify two populations of neurons with correlated activity patterns. One circuit consists of hindbrain neurons functionally coupled to spinal cord neuropil. The other consists of an anatomically symmetric population in the anterior hindbrain, with activity in the left and right halves oscillating in antiphase, on a timescale of 20 s, and coupled to equally slow oscillations in the inferior olive.

Journal ArticleDOI
TL;DR: The Cas9 targeting methodology is described, current and prospective engineering advances are detail and potential applications ranging from basic science to the clinic are suggested.
Abstract: RNA-guided Cas9 nucleases derived from clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems have dramatically transformed our ability to edit the genomes of diverse organisms. We believe tools and techniques based on Cas9, a single unifying factor capable of colocalizing RNA, DNA and protein, will grant unprecedented control over cellular organization, regulation and behavior. Here we describe the Cas9 targeting methodology, detail current and prospective engineering advances and suggest potential applications ranging from basic science to the clinic.

Journal ArticleDOI
TL;DR: The term ‘proteoform’ is proposed to be used to designate all of the different molecular forms in which the protein product of a single gene can be found, including changes due to genetic variations, alternatively spliced RNA transcripts and posttranslational modifications.
Abstract: genetic differences and not to variation at the protein level2. The term “protein species” was proposed in 2009 (ref. 2) but does not distinguish between proteins originating from different genes and those originating from a single gene, and thus we find it confusing. A similar issue arises with the term “protein variants.” The UniProt Knowledgebase (a definitive, gene-centric protein database)5 uses the term “isoform” in yet a different manner, one that denotes related forms of protein molecules arising from the same gene by alternative splicing or variable promoter usage (Fig. 1). Such events create a variable set of protein sequences that significantly change the numbering of amino acids for the protein as compared to the canonical sequence. These changes to the base primary sequence are referred to by some as “isoforms” and are denoted in UniProt by a –1, –2 and so on following the accession number (Fig. 1). However, genetic changes (for example, mutations and polymorphisms) are not covered by this terminology and create a conflict with the IUPAC definition of isoform2. Differences in IUPAC and UniProt definitions notwithstanding, the terms “variants” and “isoforms” were intended to describe proteins derived from distinct DNA or RNA; their use to describe modified proteins is confusing. Accordingly, we propose that the term ‘proteoform’ be used to designate all of the different molecular forms in which the protein product of a single gene can be found, including changes due to genetic variations, alternatively spliced RNA transcripts and posttranslational modifications (Fig. 1). Any gene or protein processing events such as those using inteins or RNA-editing mechanisms are now covered cleanly by the term ‘proteoform’. The term should include all post-translational modifications in the PSI-MOD ontology except those classified as reagent-derivatized or isotope-labeled residues (see the Supplementary Note for a precise definition). Products of multigene families should continue to be categorized on the basis of sequence identity (for example, >90%, >99% and so on). The term is compatible with a gene-centric approach for referring to proteins, which we support, because grouping related forms of proteins together even though they are the products of different genes leads to imprecision in protein identification5. We have begun to use the term ‘proteoform’ in our own writing and presentations, and we find it to be intuitive and readily grasped Proteoform: a single term describing protein complexity

Journal ArticleDOI
TL;DR: A comparison of microRNA quantification by ddPCR and real-time PCR revealed greater precision and improved day-to-day reproducibility of dd PCR but with comparable sensitivity, which translated to superior diagnostic performance for identifying individuals with cancer.
Abstract: Nanoliter-sized droplet technology paired with digital PCR (ddPCR) holds promise for highly precise, absolute nucleic acid quantification. Our comparison of microRNA quantification by ddPCR and real-time PCR revealed greater precision (coefficients of variation decreased 37-86%) and improved day-to-day reproducibility (by a factor of seven) of ddPCR but with comparable sensitivity. When we applied ddPCR to serum microRNA biomarker analysis, this translated to superior diagnostic performance for identifying individuals with cancer.

Journal ArticleDOI
TL;DR: This proof-of-principle work shows that clustered regularly interspaced short palindromic repeat (CRISPR)-Cas systems can target heterologous effector domains to endogenous sites in human cells.
Abstract: Short guide RNAs (gRNAs) can direct catalytically inactive CRISPR-associated 9 nuclease (dCas9) to repress endogenous genes in bacteria and human cells. Here we show that single or multiple gRNAs can direct dCas9 fused to a VP64 transcriptional activation domain to increase expression of endogenous human genes. This proof-of-principle work shows that clustered regularly interspaced short palindromic repeat (CRISPR)-Cas systems can target heterologous effector domains to endogenous sites in human cells.

Journal ArticleDOI
TL;DR: In this article, a monomeric yellow green fluorescent protein, mNeonGreen, derived from a tetrameric fluorescent protein from the cephalochordate Branchiostoma lanceolatum, was described.
Abstract: We report a monomeric yellow-green fluorescent protein, mNeonGreen, derived from a tetrameric fluorescent protein from the cephalochordate Branchiostoma lanceolatum. mNeonGreen is the brightest monomeric green or yellow fluorescent protein yet described to our knowledge, performs exceptionally well as a fusion tag for traditional imaging as well as stochastic single-molecule superresolution imaging and is an excellent fluorescence resonance energy transfer (FRET) acceptor for the newest cyan fluorescent proteins.

Journal ArticleDOI
TL;DR: A quantitative statistical method is developed to distinguish true biological variability from the high levels of technical noise in single-cell experiments and quantifies the statistical significance of observed cell-to-cell variability in expression strength on a gene-by-gene basis.
Abstract: A statistical method that uses spike-ins to model the dependence of technical noise on transcript abundance in single-cell RNA-seq experiments allows identification of genes wherein observed variability in read counts can be reliably interpreted as a signal of biological variability as opposed to the effect of technical noise. Single-cell RNA-seq can yield valuable insights about the variability within a population of seemingly homogeneous cells. We developed a quantitative statistical method to distinguish true biological variability from the high levels of technical noise in single-cell experiments. Our approach quantifies the statistical significance of observed cell-to-cell variability in expression strength on a gene-by-gene basis. We validate our approach using two independent data sets from Arabidopsis thaliana and Mus musculus.

Journal ArticleDOI
TL;DR: eXpress is a software package for efficient probabilistic assignment of ambiguously mapping sequenced fragments that can determine abundances of sequenced molecules in real time and can be applied to ChIP-seq, metagenomics and other large-scale sequencing data.
Abstract: We present eXpress, a software package for efficient probabilistic assignment of ambiguously mapping sequenced fragments. eXpress uses a streaming algorithm with linear run time and constant memory use. It can determine abundances of sequenced molecules in real time and can be applied to ChIP-seq, metagenomics and other large-scale sequencing data. We demonstrate its use on RNA-seq data and show that eXpress achieves greater efficiency than other quantification methods.

Journal ArticleDOI
TL;DR: The use of clustered, regularly interspaced, short palindromic repeats (CRISPR)-associated endonuclease Cas9 to target genomic sequences in the Caenorhabditis elegans germ line using single-guide RNAs that are expressed from a U6 small nuclear RNA promoter is reported.
Abstract: CRISPR-Cas systems have been used with single-guide RNAs for accurate gene disruption and conversion in multiple biological systems. Here we report the use of the endonuclease Cas9 to target genomic sequences in the C. elegans germline, utilizing single-guide RNAs that are expressed from a U6 small nuclear RNA promoter. Our results demonstrate that targeted, heritable genetic alterations can be achieved in C. elegans, providing a convenient and effective approach for generating loss-of-function mutants.

Journal ArticleDOI
TL;DR: A method to edit the C. elegans genome using the clustered, regularly interspersed, short palindromic repeats (CRISPR) RNA-guided Cas9 nuclease and homologous recombination and it is demonstrated that Cas9 is able to induce DNA double-strand breaks with specificity for targeted sites.
Abstract: Study of the nematode Caenorhabditis elegans has provided important insights in a wide range of fields in biology. The ability to precisely modify genomes is critical to fully realize the utility of model organisms. Here we report a method to edit the C. elegans genome using the clustered, regularly interspersed, short palindromic repeats (CRISPR) RNA-guided Cas9 nuclease and homologous recombination. We demonstrate that Cas9 is able to induce DNA double-strand breaks with specificity for targeted sites and that these breaks can be repaired efficiently by homologous recombination. By supplying engineered homologous repair templates, we generated gfp knock-ins and targeted mutations. Together our results outline a flexible methodology to produce essentially any desired modification in the C. elegans genome quickly and at low cost. This technology is an important addition to the array of genetic techniques already available in this experimentally tractable model organism.

Journal ArticleDOI
Predrag Radivojac1, Wyatt T. Clark1, Tal Ronnen Oron2, Alexandra M. Schnoes3, Tobias Wittkop2, Artem Sokolov4, Artem Sokolov5, Kiley Graim5, Christopher S. Funk6, Karin Verspoor6, Asa Ben-Hur5, Gaurav Pandey7, Gaurav Pandey8, Jeffrey M. Yunes7, Ameet Talwalkar7, Susanna Repo7, Susanna Repo9, Michael L Souza7, Damiano Piovesan10, Rita Casadio10, Zheng Wang11, Jianlin Cheng11, Hai Fang, Julian Gough12, Patrik Koskinen13, Petri Törönen13, Jussi Nokso-Koivisto13, Liisa Holm13, Domenico Cozzetto14, Daniel W. A. Buchan14, Kevin Bryson14, David T. Jones14, Bhakti Limaye15, Harshal Inamdar15, Avik Datta15, Sunitha K Manjari15, Rajendra Joshi15, Meghana Chitale16, Daisuke Kihara16, Andreas Martin Lisewski17, Serkan Erdin17, Eric Venner17, Olivier Lichtarge17, Robert Rentzsch14, Haixuan Yang18, Alfonso E. Romero18, Prajwal Bhat18, Alberto Paccanaro18, Tobias Hamp19, Rebecca Kaßner19, Stefan Seemayer19, Esmeralda Vicedo19, Christian Schaefer19, Dominik Achten19, Florian Auer19, Ariane Boehm19, Tatjana Braun19, Maximilian Hecht19, Mark Heron19, Peter Hönigschmid19, Thomas A. Hopf19, Stefanie Kaufmann19, Michael Kiening19, Denis Krompass19, Cedric Landerer19, Yannick Mahlich19, Manfred Roos19, Jari Björne20, Tapio Salakoski20, Andrew Wong21, Hagit Shatkay21, Hagit Shatkay22, Fanny Gatzmann23, Ingolf Sommer23, Mark N. Wass24, Michael J.E. Sternberg24, Nives Škunca, Fran Supek, Matko Bošnjak, Panče Panov, Sašo Džeroski, Tomislav Šmuc, Yiannis A. I. Kourmpetis25, Yiannis A. I. Kourmpetis26, Aalt D. J. van Dijk25, Cajo J. F. ter Braak25, Yuanpeng Zhou27, Qingtian Gong27, Xinran Dong27, Weidong Tian27, Marco Falda28, Paolo Fontana, Enrico Lavezzo28, Barbara Di Camillo28, Stefano Toppo28, Liang Lan29, Nemanja Djuric29, Yuhong Guo29, Slobodan Vucetic29, Amos Marc Bairoch30, Amos Marc Bairoch31, Michal Linial32, Patricia C. Babbitt3, Steven E. Brenner7, Christine A. Orengo14, Burkhard Rost19, Sean D. Mooney2, Iddo Friedberg33 
TL;DR: Today's best protein function prediction algorithms substantially outperform widely used first-generation methods, with large gains on all types of targets, and there is considerable need for improvement of currently available tools.
Abstract: Automated annotation of protein function is challenging. As the number of sequenced genomes rapidly grows, the overwhelming majority of protein products can only be annotated computationally. If computational predictions are to be relied upon, it is crucial that the accuracy of these methods be high. Here we report the results from the first large-scale community-based critical assessment of protein function annotation (CAFA) experiment. Fifty-four methods representing the state of the art for protein function prediction were evaluated on a target set of 866 proteins from 11 organisms. Two findings stand out: (i) today's best protein function prediction algorithms substantially outperform widely used first-generation methods, with large gains on all types of targets; and (ii) although the top methods perform well enough to guide experiments, there is considerable need for improvement of currently available tools.

Journal ArticleDOI
TL;DR: An intensity-based glutamate-sensing fluorescent reporter with signal-to-noise ratio and kinetics appropriate for in vivo imaging is described and its utility for visualizing glutamate release by neurons and astrocytes in increasingly intact neurological systems is validated.
Abstract: We describe an intensity-based glutamate-sensing fluorescent reporter (iGluSnFR) with signal-to-noise ratio and kinetics appropriate for in vivo imaging. We engineered iGluSnFR in vitro to maximize its fluorescence change, and we validated its utility for visualizing glutamate release by neurons and astrocytes in increasingly intact neurological systems. In hippocampal culture, iGluSnFR detected single field stimulus-evoked glutamate release events. In pyramidal neurons in acute brain slices, glutamate uncaging at single spines showed that iGluSnFR responds robustly and specifically to glutamate in situ, and responses correlate with voltage changes. In mouse retina, iGluSnFR-expressing neurons showed intact light-evoked excitatory currents, and the sensor revealed tonic glutamate signaling in response to light stimuli. In worms, glutamate signals preceded and predicted postsynaptic calcium transients. In zebrafish, iGluSnFR revealed spatial organization of direction-selective synaptic activity in the optic tectum. Finally, in mouse forelimb motor cortex, iGluSnFR expression in layer V pyramidal neurons revealed task-dependent single-spine activity during running.

Journal ArticleDOI
TL;DR: It is demonstrated that the engineered platform allows for the generation of three-dimensional, aligned cardiac tissues (biowires) with frequent striations and that the responses of immature human myocardium to electrical stimulation and pacing are in agreement with cardiomyocyte maturation.
Abstract: Directed differentiation protocols enable derivation of cardiomyocytes from human pluripotent stem cells (hPSCs) and permit engineering of human myocardium in vitro However, hPSC-derived cardiomyocytes are reflective of very early human development, limiting their utility in the generation of in vitro models of mature myocardium Here we describe a platform that combines three-dimensional cell cultivation with electrical stimulation to mature hPSC-derived cardiac tissues We used quantitative structural, molecular and electrophysiological analyses to explain the responses of immature human myocardium to electrical stimulation and pacing We demonstrated that the engineered platform allows for the generation of three-dimensional, aligned cardiac tissues (biowires) with frequent striations Biowires submitted to electrical stimulation had markedly increased myofibril ultrastructural organization, elevated conduction velocity and improved both electrophysiological and Ca(2+) handling properties compared to nonstimulated controls These changes were in agreement with cardiomyocyte maturation and were dependent on the stimulation rate

Journal ArticleDOI
TL;DR: This work provides a freely available computer-generated tandem mass spectral library of 212,516 spectra covering 119,200 compounds from 26 lipid compound classes, including phospholipids, glycerolipid, bacterial lipoglycans and plant glycolipids.
Abstract: Current tandem mass spectral libraries for lipid annotations in metabolomics are limited in size and diversity. We provide a freely available computer-generated tandem mass spectral library of 212,516 spectra covering 119,200 compounds from 26 lipid compound classes, including phospholipids, glycerolipids, bacterial lipoglycans and plant glycolipids. We show platform independence by using tandem mass spectra from 40 different mass spectrometer types including low-resolution and high-resolution instruments.

Journal ArticleDOI
TL;DR: An expression cassette architecture for genetic elements controlling transcription and translation initiation in Escherichia coli is developed, demonstrating that arbitrary genes are reliably expressed to within twofold relative target expression windows with ∼93% reliability.
Abstract: An inability to reliably predict quantitative behaviors for novel combinations of genetic elements limits the rational engineering of biological systems. We developed an expression cassette architecture for genetic elements controlling transcription and translation initiation in Escherichia coli: transcription elements encode a common mRNA start, and translation elements use an overlapping genetic motif found in many natural systems. We engineered libraries of constitutive and repressor-regulated promoters along with translation initiation elements following these definitions. We measured activity distributions for each library and selected elements that collectively resulted in expression across a 1,000-fold observed dynamic range. We studied all combinations of curated elements, demonstrating that arbitrary genes are reliably expressed to within twofold relative target expression windows with ∼93% reliability. We expect the genetic element definitions validated here can be collectively expanded to create collections of public-domain standard biological parts that support reliable forward engineering of gene expression at genome scales.

Journal ArticleDOI
TL;DR: Hydrogel-based structures can be built from within biological tissue to allow subsequent removal of lipids without mechanical disassembly of the tissue, creating a tissue-hydrogel hybrid that is physically stable, that preserves fine structure, proteins and nucleic acids, and that is permeable to both visible-spectrum photons and exogenous macromolecules.
Abstract: With potential relevance for brain-mapping work, hydrogel-based structures can now be built from within biological tissue to allow subsequent removal of lipids without mechanical disassembly of the tissue. This process creates a tissue-hydrogel hybrid that is physically stable, that preserves fine structure, proteins and nucleic acids, and that is permeable to both visible-spectrum photons and exogenous macromolecules. Here we highlight relevant challenges and opportunities of this approach, especially with regard to integration with complementary methodologies for brain-mapping studies.

Journal ArticleDOI
TL;DR: Improvements for sequencing 16S ribosomal RNA (rRNA) amplicons, a cornerstone technique in metagenomics, are described through unique tagging of template molecules before PCR, which substantially enriching microbial sequences without introducing bias.
Abstract: We describe improvements for sequencing 16S ribosomal RNA (rRNA) amplicons, a cornerstone technique in metagenomics. Through unique tagging of template molecules before PCR, amplicon sequences can be mapped to their original templates to correct amplification bias and sequencing error with software we provide. PCR clamps block amplification of contaminating sequences from a eukaryotic host, thereby substantially enriching microbial sequences without introducing bias.

Journal ArticleDOI
TL;DR: This work introduces a measure based on Fourier ring correlation (FRC) that can be computed directly from an image and demonstrates its validity and benefits on two-dimensional (2D) and 3D localization microscopy images of tubulin and actin filaments.
Abstract: Resolution in optical nanoscopy (or super-resolution microscopy) depends on the localization uncertainty and density of single fluorescent labels and on the sample's spatial structure. Currently there is no integral, practical resolution measure that accounts for all factors. We introduce a measure based on Fourier ring correlation (FRC) that can be computed directly from an image. We demonstrate its validity and benefits on two-dimensional (2D) and 3D localization microscopy images of tubulin and actin filaments. Our FRC resolution method makes it possible to compare achieved resolutions in images taken with different nanoscopy methods, to optimize and rank different emitter localization and labeling strategies, to define a stopping criterion for data acquisition, to describe image anisotropy and heterogeneity, and even to estimate the average number of localizations per emitter. Our findings challenge the current focus on obtaining the best localization precision, showing instead how the best image resolution can be achieved as fast as possible.

Journal ArticleDOI
TL;DR: The results show that most algorithms are able to identify discrete transcript components with high success rates but that assembly of complete isoform structures poses a major challenge even when all constituent elements are identified.
Abstract: We evaluated 25 protocol variants of 14 independent computational methods for exon identification, transcript reconstruction and expression-level quantification from RNA-seq data. Our results show that most algorithms are able to identify discrete transcript components with high success rates but that assembly of complete isoform structures poses a major challenge even when all constituent elements are identified. Expression-level estimates also varied widely across methods, even when based on similar transcript models. Consequently, the complexity of higher eukaryotic genomes imposes severe limitations on transcript recall and splice product discrimination that are likely to remain limiting factors for the analysis of current-generation RNA-seq data.

Journal ArticleDOI
TL;DR: In situ sequencing of point mutations and multiplexed gene expression profiling in human breast cancer tissue sections is demonstrated and the method for parallel targeted analysis of short RNA fragments in morphologically preserved cells and tissue is developed.
Abstract: Tissue gene expression profiling is performed on homogenates or on populations of isolated single cells to resolve molecular states of different cell types. In both approaches, histological context is lost. We have developed an in situ sequencing method for parallel targeted analysis of short RNA fragments in morphologically preserved cells and tissue. We demonstrate in situ sequencing of point mutations and multiplexed gene expression profiling in human breast cancer tissue sections.