Showing papers in "Nature Methods in 2015"
TL;DR: Tests showed that HISAT is the fastest system currently available, with equal or better accuracy than any other method, and requires only 4.3 gigabytes of memory.
Abstract: HISAT (hierarchical indexing for spliced alignment of transcripts) is a highly efficient system for aligning reads from RNA sequencing experiments. HISAT uses an indexing scheme based on the Burrows-Wheeler transform and the Ferragina-Manzini (FM) index, employing two types of indexes for alignment: a whole-genome FM index to anchor each alignment and numerous local FM indexes for very rapid extensions of these alignments. HISAT's hierarchical index for the human genome contains 48,000 local FM indexes, each representing a genomic region of ∼64,000 bp. Tests on real and simulated data sets showed that HISAT is the fastest system currently available, with equal or better accuracy than any other method. Despite its large number of indexes, HISAT requires only 4.3 gigabytes of memory. HISAT supports genomes of any size, including those larger than 4 billion bases.
13,192 citations
TL;DR: DIAMOND is introduced, an open-source algorithm based on double indexing that is 20,000 times faster than BLASTX on short reads and has a similar degree of sensitivity.
Abstract: The alignment of sequencing reads against a protein reference database is a major computational bottleneck in metagenomics and data-intensive evolutionary projects. Although recent tools offer improved performance over the gold standard BLASTX, they exhibit only a modest speedup or low sensitivity. We introduce DIAMOND, an open-source algorithm based on double indexing that is 20,000 times faster than BLASTX on short reads and has a similar degree of sensitivity.
7,164 citations
TL;DR: CIBERSORT outperformed other methods with respect to noise, unknown mixture content and closely related cell types when applied to enumeration of hematopoietic subsets in RNA mixtures from fresh, frozen and fixed tissues, including solid tumors.
Abstract: We introduce CIBERSORT, a method for characterizing cell composition of complex tissues from their gene expression profiles When applied to enumeration of hematopoietic subsets in RNA mixtures from fresh, frozen and fixed tissues, including solid tumors, CIBERSORT outperformed other methods with respect to noise, unknown mixture content and closely related cell types CIBERSORT should enable large-scale analysis of RNA mixtures for cellular biomarkers and therapeutic targets (http://cibersortstanfordedu/)
6,967 citations
TL;DR: A stand-alone I-TASSER Suite that can be used for off-line protein structure and function prediction and three complementary algorithms to enhance function inferences are developed, the consensus of which is derived by COACH4 using support vector machines.
Abstract: The lowest free-energy conformations are identified by structure clustering. A second round of assembly simulation is conducted, starting from the centroid models, to remove steric clashes and refine global topology. Final atomic structure models are constructed from the low-energy conformations by a two-step atomic-level energy minimization approach. The correctness of the global model is assessed by the confidence score, which is based on the significance of threading alignments and the density of structure clustering; the residue-level local quality of the structural models and B factor of the target protein are evaluated by a newly developed method, ResQ, built on the variation of modeling simulations and the uncertainty of homologous alignments through support vector regression training. For function annotation, the structure models with the highest confidence scores are matched against the BioLiP5 database of ligand-protein interactions to detect homologous function templates. Functional insights on ligand-binding site (LBS), Enzyme Commission (EC) and Gene Ontology (GO) are deduced from the functional templates. We developed three complementary algorithms (COFACTOR, TM-SITE and S-SITE) to enhance function inferences, the consensus of which is derived by COACH4 using support vector machines. Detailed instructions for installation, implementation and result interpretation of the Suite can be found in the Supplementary Methods and Supplementary Tables 1 and 2. The I-TASSER Suite pipeline was tested in recent communitywide structure and function prediction experiments, including CASP10 (ref. 1) and CAMEO2. Overall, I-TASSER generated the correct fold with a template modeling score (TM-score) >0.5 for 10 out of 36 “New Fold” (NF) targets in the CASP10, which have no homologous templates in the Protein Data Bank (PDB). Of the 110 template-based modeling targets, 92 had a TM-score >0.5, and 89 had the templates drawn closer to the native with an average r.m.s. deviation improvement of 1.05 Å in the same threadingaligned regions6. In CAMEO, COACH generated LBS predictions for 4,271 targets with an average accuracy 0.86, which was 20% higher than that of the second-best method in the experiment. Here we illustrate I-TASSER Suite–based structure and function modeling using six examples (Fig. 1b–g) from the communitywide blind tests1,2. R0006 and R0007 are two NF targets from CASP10, and I-TASSER constructed models of correct fold with a TM-score of 0.62 for both targets (Fig. 1b,c). An illustration of local quality estimation by ResQ is shown for T0652, which has an average error 0.75 Å compared to the actual deviation of the model from the native (Fig. 1h). The four LBS prediction examples (Fig. 1d–g) are from CASP10 (ref. 1) and CAMEO2; COACH generated ligand models all with a ligand r.m.s. deviation below 2 Å. COACH also correctly assigned the threeand fourdigit EC numbers to the enzyme targets C0050 and C0046 (Supplementary Table 3). In summary, we developed a stand-alone I-TASSER Suite that can be used for off-line protein structure and function prediction. The I-TASSER Suite: protein structure and function prediction
4,693 citations
Harvard University1, Genentech2, Fred Hutchinson Cancer Research Center3, State University of Campinas4, University of Maryland, College Park5, National Institutes of Health6, University of Cambridge7, University of California, Riverside8, Novartis9, Johns Hopkins University10, University of Washington11, Walter and Eliza Hall Institute of Medical Research12, City University of New York13
TL;DR: An overview of Bioconductor, an open-source, open-development software project for the analysis and comprehension of high-throughput data in genomics and molecular biology, which comprises 934 interoperable packages contributed by a large, diverse community of scientists.
Abstract: Bioconductor is an open-source, open-development software project for the analysis and comprehension of high-throughput data in genomics and molecular biology. The project aims to enable interdisciplinary research, collaboration and rapid development of scientific software. Based on the statistical programming language R, Bioconductor comprises 934 interoperable packages contributed by a large, diverse community of scientists. Packages cover a range of bioinformatic and statistical applications. They undergo formal initial review and continuous automated testing. We present an overview for prospective users and contributors.
2,818 citations
TL;DR: A deep learning–based algorithmic framework, DeepSEA, is developed that directly learns a regulatory sequence code from large-scale chromatin-profiling data, enabling prediction of chromatin effects of sequence alterations with single-nucleotide sensitivity and improving prioritization of functional variants.
Abstract: Identifying functional effects of noncoding variants is a major challenge in human genetics. To predict the noncoding-variant effects de novo from sequence, we developed a deep learning-based algorithmic framework, DeepSEA (http://deepsea.princeton.edu/), that directly learns a regulatory sequence code from large-scale chromatin-profiling data, enabling prediction of chromatin effects of sequence alterations with single-nucleotide sensitivity. We further used this capability to improve prioritization of functional variants including expression quantitative trait loci (eQTLs) and disease-associated variants.
1,744 citations
TL;DR: Improvements in the underlying pipeline for identifying marker genes and the profiling procedure resulted in much improved quantitative performances (higher correlation with true abundances, lower false positive and false negative rates).
Abstract: Profiling of all domains of life. Marker and quasi-marker genes are now identified not only for microbes (Bacteria and Archaea), but also for viruses and Eukaryotic microbes (Fungi, Protozoa) that are crucial components of microbial communities. A 6-fold increase in the number of considered species. Markers are now identified from >16,000 reference genomes and >7,000 unique species, dramatically expanding the comprehensiveness of the method. The new pipeline for identifying marker genes is also scalable to the quickly increasing number of reference genomes. See Supplementary Tables 1-3. Introduction of the concept of quasi-markers, allowing more comprehensive and accurate profiling. For species with less than 200 markers, MetaPhlAn2 adopts additional quasi-marker sequences (Supplementary Note 2) that are occasionally present in other genomes (because of vertical conservation or horizontal transfer). At profiling time, if no other markers of the potentially confounding species are detected, the corresponding quasi-local markers are used to improve the quality and accuracy of the profiling. Addition of strain-specific barcoding for microbial strain tracking. MetaPhlAn2 includes a completely new feature that exploits marker combinations to perform species-specific and genus-specific “barcoding” for strains in metagenomic samples (Supplementary Note 7). This feature can be used for culture-free pathogen tracking in epidemiology studies and strain tracking across microbiome samples. See Supplementary Figs. 12-20. Strain-level identification for organisms with sequenced genomes. For the case in which a microbiome includes strains that are very close to one of those already sequenced, MetaPhlAn2 is now able to identify such strains and readily reports their abundances. See Supplementary Note 7, Supplementary Table 13, and Supplementary Fig. 21. Improvement of false positive and false negative rates. Improvements in the underlying pipeline for identifying marker genes (including the increment of the adopted genomes and the use of quasi-markers) and the profiling procedure resulted in much improved quantitative performances (higher correlation with true abundances, lower false positive and false negative rates). See the validation on synthetic metagenomes in Supplementary Note 4. Estimation of the percentage of reads mapped against known reference genomes. MetaPhlAn2 is now able to estimate the number of reads that would map against genomes of each clade detected as present and for which an estimation of its relative abundance is provided by the default output. See Supplementary Note 3 for details. Integration of MetaPhlAn with post-processing and visualization tools. The MetaPhlAn2 package now includes a set of post-processing and visualization tools (“utils” subfolder of the MetaPhlAn2 repository). Multiple MetaPhlAn profiles can in fact be merged in an abundance table (“merge_metaphlan_tables.py”), exported as BIOM files, visualized as heatmap (“metaphlan_hclust_heatmap.py” or the integrated “hclust2” package), GraPhlAn plots (“export2graphlan.py” and the GraPhlAn package1), Krona2 plots (“metaphlan2krona.py”), and single microbe barplot across samples and conditions (“plot_bug.py”).
1,618 citations
TL;DR: For a reversed-phase LC-MS/MS analysis of nine algal strains, MS-DIAL using an enriched LipidBlast library identified 1,023 lipid compounds, highlighting the chemotaxonomic relationships between theAlgal strains.
Abstract: Data-independent acquisition (DIA) in liquid chromatography (LC) coupled to tandem mass spectrometry (MS/MS) provides comprehensive untargeted acquisition of molecular data. We provide an open-source software pipeline, which we call MS-DIAL, for DIA-based identification and quantification of small molecules by mass spectral deconvolution. For a reversed-phase LC-MS/MS analysis of nine algal strains, MS-DIAL using an enriched LipidBlast library identified 1,023 lipid compounds, highlighting the chemotaxonomic relationships between the algal strains.
1,609 citations
TL;DR: An improved transcriptional regulator obtained through the rational design of a tripartite activator, VP64-p65-Rta (VPR), fused to nuclease-null Cas9 is described and demonstrated in activating endogenous coding and noncoding genes and stimulating neuronal differentiation of human induced pluripotent stem cells (iPSCs).
Abstract: The RNA-guided nuclease Cas9 can be reengineered as a programmable transcription factor. However, modest levels of gene activation have limited potential applications. We describe an improved transcriptional regulator obtained through the rational design of a tripartite activator, VP64-p65-Rta (VPR), fused to nuclease-null Cas9. We demonstrate its utility in activating endogenous coding and noncoding genes, targeting several genes simultaneously and stimulating neuronal differentiation of human induced pluripotent stem cells (iPSCs).
1,147 citations
TL;DR: Inspired by molecular modeling, the N,N-dimethylamino substituents in tetramethylrhodamine are replaced with four-membered azetidine rings, which doubles the quantum efficiency and improves the photon yield of the dye in applications ranging from in vitro single-molecule measurements to super-resolution imaging.
Abstract: Specific labeling of biomolecules with bright fluorophores is the keystone of fluorescence microscopy. Genetically encoded self-labeling tag proteins can be coupled to synthetic dyes inside living cells, resulting in brighter reporters than fluorescent proteins. Intracellular labeling using these techniques requires cell-permeable fluorescent ligands, however, limiting utility to a small number of classic fluorophores. Here we describe a simple structural modification that improves the brightness and photostability of dyes while preserving spectral properties and cell permeability. Inspired by molecular modeling, we replaced the N,N-dimethylamino substituents in tetramethylrhodamine with four-membered azetidine rings. This addition of two carbon atoms doubles the quantum efficiency and improves the photon yield of the dye in applications ranging from in vitro single-molecule measurements to super-resolution imaging. The novel substitution is generalizable, yielding a palette of chemical dyes with improved quantum efficiencies that spans the UV and visible range.
1,140 citations
TL;DR: This document describes the development and use of MiXCR, a software for comprehensive adaptive immunity profiling, and some of the techniques used in its development.
Abstract: MiXCR: software for comprehensive adaptive immunity profiling. MiXCR: software for comprehensive adaptive immunity profiling. MiXCR: software for comprehensive adaptive immunity profiling. MiXCR: software for comprehensive adaptive immunity profiling.MiXCR: software for comprehensive adaptive immunity profiling.
TL;DR: This work has assembled de novo the Escherichia coli K-12 MG1655 chromosome in a single 4.6-Mb contig using only nanopore data and reconstructs gene order and has 99.5% nucleotide identity.
Abstract: We have assembled de novo the Escherichia coli K-12 MG1655 chromosome in a single 4.6-Mb contig using only nanopore data. Our method has three stages: (i) overlaps are detected between reads and then corrected by a multiple-alignment process; (ii) corrected reads are assembled using the Celera Assembler; and (iii) the assembly is polished using a probabilistic model of the signal-level data. The assembly reconstructs gene order and has 99.5% nucleotide identity.
TL;DR: A simple and reproducible 3D culture approach for generating a laminated cerebral cortex–like structure, named human cortical spheroids (hCSs), from pluripotent stem cells, which demonstrate that cortical neurons participate in network activity and produce complex synaptic events.
Abstract: The human cerebral cortex develops through an elaborate succession of cellular events that, when disrupted, can lead to neuropsychiatric disease. The ability to reprogram somatic cells into pluripotent cells that can be differentiated in vitro provides a unique opportunity to study normal and abnormal corticogenesis. Here, we present a simple and reproducible 3D culture approach for generating a laminated cerebral cortex-like structure, named human cortical spheroids (hCSs), from pluripotent stem cells. hCSs contain neurons from both deep and superficial cortical layers and map transcriptionally to in vivo fetal development. These neurons are electrophysiologically mature, display spontaneous activity, are surrounded by nonreactive astrocytes and form functional synapses. Experiments in acute hCS slices demonstrate that cortical neurons participate in network activity and produce complex synaptic events. These 3D cultures should allow a detailed interrogation of human cortical development, function and disease, and may prove a versatile platform for generating other neuronal and glial subtypes in vitro.
TL;DR: m6A individual-nucleotide-resolution cross-linking and immunoprecipitation (miCLIP) is developed and used to demonstrate that antibodies to m6A can induce specific mutational signatures at m 6A residues after ultraviolet light–induced antibody-RNA cross- linking and reverse transcription.
Abstract: N(6)-methyladenosine (m6A) is the most abundant modified base in eukaryotic mRNA and has been linked to diverse effects on mRNA fate. Current mapping approaches localize m6A residues to transcript regions 100-200 nt long but cannot identify precise m6A positions on a transcriptome-wide level. Here we developed m6A individual-nucleotide-resolution cross-linking and immunoprecipitation (miCLIP) and used it to demonstrate that antibodies to m6A can induce specific mutational signatures at m6A residues after ultraviolet light-induced antibody-RNA cross-linking and reverse transcription. We found that these antibodies similarly induced mutational signatures at N(6),2'-O-dimethyladenosine (m6Am), a modification found at the first nucleotide of certain mRNAs. Using these signatures, we mapped m6A and m6Am at single-nucleotide resolution in human and mouse mRNA and identified small nucleolar RNAs (snoRNAs) as a new class of m6A-containing non-coding RNAs (ncRNAs).
TL;DR: It is shown that targeting Cas9 to the germ line using a Cas9-nanos 3′ UTR led to the generation of maternal-zygotic mutants, as well as increased viability and decreased somatic mutations.
Abstract: CRISPR-Cas9 technology provides a powerful system for genome engineering. However, variable activity across different single guide RNAs (sgRNAs) remains a significant limitation. We analyzed the molecular features that influence sgRNA stability, activity and loading into Cas9 in vivo. We observed that guanine enrichment and adenine depletion increased sgRNA stability and activity, whereas differential sgRNA loading, nucleosome positioning and Cas9 off-target binding were not major determinants. We also identified sgRNAs truncated by one or two nucleotides and containing 5' mismatches as efficient alternatives to canonical sgRNAs. On the basis of these results, we created a predictive sgRNA-scoring algorithm, CRISPRscan, that effectively captures the sequence features affecting the activity of CRISPR-Cas9 in vivo. Finally, we show that targeting Cas9 to the germ line using a Cas9-nanos 3' UTR led to the generation of maternal-zygotic mutants, as well as increased viability and decreased somatic mutations. These results identify determinants that influence Cas9 activity and provide a framework for the design of highly efficient sgRNAs for genome targeting in vivo.
TL;DR: Yeast-display evolution is used to improve the catalytic efficiency of APEX, enabling the use of electron microscopy to resolve the submitochondrial localization of calcium uptake regulatory protein MICU1 and superior enrichment of endogenous mitochondrial and endoplasmic reticulum membrane proteins.
Abstract: APEX is an engineered peroxidase that functions as an electron microscopy tag and a promiscuous labeling enzyme for live-cell proteomics. Because limited sensitivity precludes applications requiring low APEX expression, we used yeast-display evolution to improve its catalytic efficiency. APEX2 is far more active in cells, enabling the use of electron microscopy to resolve the submitochondrial localization of calcium uptake regulatory protein MICU1. APEX2 also permits superior enrichment of endogenous mitochondrial and endoplasmic reticulum membrane proteins.
TL;DR: Digenome-seq is a robust, sensitive, unbiased and cost-effective method for profiling genome-wide off-target effects of programmable nucleases including Cas9, and shows that Cas9 off- target effects can be avoided by replacing 'promiscuous' single guide RNAs (sgRNAs) with modified sgRNAs.
Abstract: Although RNA-guided genome editing via the CRISPR-Cas9 system is now widely used in biomedical research, genome-wide target specificities of Cas9 nucleases remain controversial. Here we present Digenome-seq, in vitro Cas9-digested whole-genome sequencing, to profile genome-wide Cas9 off-target effects in human cells. This in vitro digest yields sequence reads with the same 5' ends at cleavage sites that can be computationally identified. We validated off-target sites at which insertions or deletions were induced with frequencies below 0.1%, near the detection limit of targeted deep sequencing. We also showed that Cas9 nucleases can be highly specific, inducing off-target mutations at merely several, rather than thousands of, sites in the entire genome and that Cas9 off-target effects can be avoided by replacing 'promiscuous' single guide RNAs (sgRNAs) with modified sgRNAs. Digenome-seq is a robust, sensitive, unbiased and cost-effective method for profiling genome-wide off-target effects of programmable nucleases including Cas9.
TL;DR: These results demonstrate that repression mediated by dCas9-KRAB is sufficiently specific to disrupt the activity of individual enhancers via local modification of the epigenome.
Abstract: Epigenome editing with the CRISPR (clustered, regularly interspaced, short palindromic repeats)-Cas9 platform is a promising technology for modulating gene expression to direct cell phenotype and to dissect the causal epigenetic mechanisms of gene regulation. Fusions of nuclease-inactive dCas9 to the Kruppel-associated box (KRAB) repressor (dCas9-KRAB) can silence target gene expression, but the genome-wide specificity and the extent of heterochromatin formation catalyzed by dCas9-KRAB are not known. We targeted dCas9-KRAB to the HS2 enhancer, a distal regulatory element that orchestrates the expression of multiple globin genes, and observed highly specific induction of H3K9 trimethylation (H3K9me3) at the enhancer and decreased chromatin accessibility of both the enhancer and its promoter targets. Targeted epigenetic modification of HS2 silenced the expression of multiple globin genes, with minimal off-target changes in global gene expression. These results demonstrate that repression mediated by dCas9-KRAB is sufficiently specific to disrupt the activity of individual enhancers via local modification of the epigenome.
TL;DR: EMRinger, a tool that assesses the precise fitting of an atomic model into the map during refinement and shows how radiation damage alters scattering from negatively charged amino acids is reported.
Abstract: Advances in high-resolution cryo-electron microscopy (cryo-EM) require the development of validation metrics to independently assess map quality and model geometry. We report EMRinger, a tool that assesses the precise fitting of an atomic model into the map during refinement and shows how radiation damage alters scattering from negatively charged amino acids. EMRinger (https://github.com/fraser-lab/EMRinger) will be useful for monitoring progress in resolving and modeling high-resolution features in cryo-EM.
TL;DR: This work introduces network-based stratification (NBS), a method to integrate somatic tumor genomes with gene networks that allows for stratification of cancer into informative subtypes by clustering together patients with mutations in similar network regions.
Abstract: The embodiments provide a method of for stratification of cancer into one or more informative subtypes of a subject in need thereof The embodiments, further provide assigning a subject in need into one or more informative subtypes, including assigning a subject in need into an informative subtype wherein the subtype is an ovarian cancer subtype
TL;DR: G&T-seq, a method for separating and sequencing genomic DNA and full-length mRNA from single cells, is described and cellular properties that could not be inferred from DNA or RNA sequencing alone are discovered.
Abstract: G&T-seq offers robust full-length transcript and whole-genome sequencing simultaneously from a single cell.
TL;DR: A microchip technology (the Cluster-Chip) is developed to capture CTC clusters independently of tumor-specific markers from unprocessed blood to enable the detailed characterization of their biological properties and role in metastasis.
Abstract: Cancer cells metastasize through the bloodstream either as single migratory circulating tumor cells (CTCs) or as multicellular groupings (CTC clusters). Existing technologies for CTC enrichment are designed to isolate single CTCs, and although CTC clusters are detectable in some cases, their true prevalence and significance remain to be determined. Here we developed a microchip technology (the Cluster-Chip) to capture CTC clusters independently of tumor-specific markers from unprocessed blood. CTC clusters are isolated through specialized bifurcating traps under low-shear stress conditions that preserve their integrity, and even two-cell clusters are captured efficiently. Using the Cluster-Chip, we identified CTC clusters in 30-40% of patients with metastatic breast or prostate cancer or with melanoma. RNA sequencing of CTC clusters confirmed their tumor origin and identified tissue-derived macrophages within the clusters. Efficient capture of CTC clusters will enable the detailed characterization of their biological properties and role in metastasis.
TL;DR: It is explained why P is fickle to discourage the ill-informed practice of interpreting analyses based predominantly on this statistic.
Abstract: The reliability and reproducibility of science are under scrutiny. However, a major cause of this lack of repeatability is not being considered: the wide sample-to-sample variability in the P value. We explain why P is fickle to discourage the ill-informed practice of interpreting analyses based predominantly on this statistic.
TL;DR: This work presents a single-nucleotide-variant detection tool that uses maximum-likelihood parameter estimates and marginalization over many possible read alignments to achieve precision and recall of up to 99% by pairing the authors' high-confidence alignment strategy with long MinION reads.
Abstract: Speed, single-base sensitivity and long read lengths make nanopores a promising technology for high-throughput sequencing. We evaluated and optimized the performance of the MinION nanopore sequencer using M13 genomic DNA and used expectation maximization to obtain robust maximum-likelihood estimates for insertion, deletion and substitution error rates (4.9%, 7.8% and 5.1%, respectively). Over 99% of high-quality 2D MinION reads mapped to the reference at a mean identity of 85%. We present a single-nucleotide-variant detection tool that uses maximum-likelihood parameter estimates and marginalization over many possible read alignments to achieve precision and recall of up to 99%. By pairing our high-confidence alignment strategy with long MinION reads, we resolved the copy number for a cancer-testis gene family (CT47) within an unresolved region of human chromosome Xq24.
TL;DR: A nuclease-deficient Cas9 (dCas9)–histone demethylase fusion is used to functionally characterize previously described and new enhancer elements for their roles in the embryonic stem cell state and distinguishes the mechanism of action of dCas9-LSD1 at enhancers from previous d Cas9-effectors.
Abstract: Targeting a histone demethylase to specific enhancers with Cas9 allows modification of epigenetic marks at the enhancers.
TL;DR: This work implemented a single-wavelength pulse-width-based method with a one-dimensional imaging rate of 100 kHz to image blood oxygenation with capillary-level resolution for three-dimensional high-resolution, high-speed imaging of the mouse brain.
Abstract: We present fast functional photoacoustic microscopy (PAM) for three-dimensional high-resolution, high-speed imaging of the mouse brain, complementary to other imaging modalities. We implemented a single-wavelength pulse-width-based method with a one-dimensional imaging rate of 100 kHz to image blood oxygenation with capillary-level resolution. We applied PAM to image the vascular morphology, blood oxygenation, blood flow and oxygen metabolism in both resting and stimulated states in the mouse brain.
TL;DR: DIA-Umpire enables targeted extraction of quantitative information based on peptides initially identified in only a subset of the samples, resulting in more consistent quantification across multiple samples.
Abstract: As a result of recent improvements in mass spectrometry (MS), there is increased interest in data-independent acquisition (DIA) strategies in which all peptides are systematically fragmented using wide mass-isolation windows ('multiplex fragmentation'). DIA-Umpire (http://diaumpire.sourceforge.net/), a comprehensive computational workflow and open-source software for DIA data, detects precursor and fragment chromatographic features and assembles them into pseudo-tandem MS spectra. These spectra can be identified with conventional database-searching and protein-inference tools, allowing sensitive, untargeted analysis of DIA data without the need for a spectral library. Quantification is done with both precursor- and fragment-ion intensities. Furthermore, DIA-Umpire enables targeted extraction of quantitative information based on peptides initially identified in only a subset of the samples, resulting in more consistent quantification across multiple samples. We demonstrated the performance of the method with control samples of varying complexity and publicly available glycoproteomics and affinity purification-MS data.
TL;DR: Real-time deformability cytometry (RT-DC) is introduced for continuous cell mechanical characterization of large populations with analysis rates greater than 100 cells/s and adds a new marker-free dimension to flow cytometry with diverse applications in biology, biotechnology and medicine.
Abstract: Real-time deformability cytometry allows the continuous mechanical characterization of cells with high throughput and is applied to distinguish cell-cycle phases, track differentiated cells and profile cell populations in whole blood.
TL;DR: This approach extends the optogenetic toolkit beyond the specificity obtained with genetic or viral approaches, enabling high-throughput, flexible and long-term optical interrogation of functionally defined neural circuits with single-cell and single-spike resolution in the mouse brain in vivo.
Abstract: We describe an all-optical strategy for simultaneously manipulating and recording the activity of multiple neurons with cellular resolution in vivo. We performed simultaneous two-photon optogenetic activation and calcium imaging by coexpression of a red-shifted opsin and a genetically encoded calcium indicator. A spatial light modulator allows tens of user-selected neurons to be targeted for spatiotemporally precise concurrent optogenetic activation, while simultaneous fast calcium imaging provides high-resolution network-wide readout of the manipulation with negligible optical cross-talk. Proof-of-principle experiments in mouse barrel cortex demonstrate interrogation of the same neuronal population during different behavioral states and targeting of neuronal ensembles based on their functional signature. This approach extends the optogenetic toolkit beyond the specificity obtained with genetic or viral approaches, enabling high-throughput, flexible and long-term optical interrogation of functionally defined neural circuits with single-cell and single-spike resolution in the mouse brain in vivo.