The Experts below are selected from a list of 318 Experts worldwide ranked by ideXlab platform
Manuel Reitberger - One of the best experts on this subject based on the ideXlab platform.
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nuclear pores as versatile Reference Standards for quantitative superresolution microscopy
Nature Methods, 2019Co-Authors: Jervis Vermal Thevathasan, Maurice Kahnwald, Konstanty Cieślinski, Philipp Hoess, Sudheer Kumar Peneti, Manuel ReitbergerAbstract:Quantitative fluorescence and superresolution microscopy are often limited by insufficient data quality or artifacts. In this context, it is essential to have biologically relevant control samples to benchmark and optimize the quality of microscopes, labels and imaging conditions. Here, we exploit the stereotypic arrangement of proteins in the nuclear pore complex as in situ Reference structures to characterize the performance of a variety of microscopy modalities. We created four genome edited cell lines in which we endogenously labeled the nucleoporin Nup96 with mEGFP, SNAP-tag, HaloTag or the photoconvertible fluorescent protein mMaple. We demonstrate their use (1) as three-dimensional resolution Standards for calibration and quality control, (2) to quantify absolute labeling efficiencies and (3) as precise Reference Standards for molecular counting. These cell lines will enable the broader community to assess the quality of their microscopes and labels, and to perform quantitative, absolute measurements.
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nuclear pores as versatile Reference Standards for quantitative superresolution microscopy
bioRxiv, 2019Co-Authors: Jervis Vermal Thevathasan, Maurice Kahnwald, Konstanty Cieślinski, Philipp Hoess, Sudheer Kumar Peneti, Manuel Reitberger, Daniel Heid, Krishna Chaitanya KasubaAbstract:Abstract Quantitative fluorescence and superresolution microscopy are often limited by insufficient data quality or artifacts. In this context, it is essential to have biologically relevant control samples to benchmark and optimize the quality of microscopes, labels and imaging conditions. Here we exploit the stereotypic arrangement of proteins in the nuclear pore complex as in situ Reference structures to characterize the performance of a variety of microscopy modalities. We created four genome edited cell lines in which we endogenously labeled the nucleoporin Nup96 with mEGFP, SNAP-tag or HaloTag or the photoconvertible fluorescent protein mMaple. We demonstrate their use a) as 3D resolution Standards for calibration and quality control, b) to quantify absolute labeling efficiencies and c) as precise Reference Standards for molecular counting. These cell lines will enable the broad community to assess the quality of their microscopes and labels, and to perform quantitative, absolute measurements.
Tim R. Mercer - One of the best experts on this subject based on the ideXlab platform.
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chimeric synthetic Reference Standards enable cross validation of positive and negative controls in sars cov 2 molecular tests
Scientific Reports, 2021Co-Authors: Bindu Swapna Madala, Ira W. Deveson, Tim R. Mercer, Andre L M Reis, William D RawlinsonAbstract:DNA synthesis in vitro has enabled the rapid production of Reference Standards. These are used as controls, and allow measurement and improvement of the accuracy and quality of diagnostic tests. Current Reference Standards typically represent target genetic material, and act only as positive controls to assess test sensitivity. However, negative controls are also required to evaluate test specificity. Using a pair of chimeric A/B RNA Standards, this allowed incorporation of positive and negative controls into diagnostic testing for the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2). The chimeric Standards constituted target regions for RT-PCR primer/probe sets that are joined in tandem across two separate synthetic molecules. Accordingly, a target region that is present in standard A provides a positive control, whilst being absent in standard B, thereby providing a negative control. This design enables cross-validation of positive and negative controls between the paired Standards in the same reaction, with identical conditions. This enables control and test failures to be distinguished, increasing confidence in the accuracy of results. The chimeric A/B Standards were assessed using the US Centres for Disease Control real-time RT-PCR protocol, and showed results congruent with other commercial controls in detecting SARS-CoV-2 in patient samples. This chimeric Reference standard design approach offers extensive flexibility, allowing representation of diverse genetic features and distantly related sequences, even from different organisms.
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Reference Standards for next-generation sequencing.
Nature reviews. Genetics, 2017Co-Authors: Simon A. Hardwick, Ira W. Deveson, Tim R. MercerAbstract:Technical errors can hamper the interpretation of next-generation sequencing (NGS) data, which poses a major challenge for the clinical application of this technology. This Review discusses how Reference Standards circumvent this issue by calibrating NGS measurements and evaluating diagnostic performance of NGS-based genetic tests. Next-generation sequencing (NGS) provides a broad investigation of the genome, and it is being readily applied for the diagnosis of disease-associated genetic features. However, the interpretation of NGS data remains challenging owing to the size and complexity of the genome and the technical errors that are introduced during sample preparation, sequencing and analysis. These errors can be understood and mitigated through the use of Reference Standards — well-characterized genetic materials or synthetic spike-in controls that help to calibrate NGS measurements and to evaluate diagnostic performance. The informed use of Reference Standards, and associated statistical principles, ensures rigorous analysis of NGS data and is essential for its future clinical use.
Jervis Vermal Thevathasan - One of the best experts on this subject based on the ideXlab platform.
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nuclear pores as versatile Reference Standards for quantitative superresolution microscopy
Nature Methods, 2019Co-Authors: Jervis Vermal Thevathasan, Maurice Kahnwald, Konstanty Cieślinski, Philipp Hoess, Sudheer Kumar Peneti, Manuel ReitbergerAbstract:Quantitative fluorescence and superresolution microscopy are often limited by insufficient data quality or artifacts. In this context, it is essential to have biologically relevant control samples to benchmark and optimize the quality of microscopes, labels and imaging conditions. Here, we exploit the stereotypic arrangement of proteins in the nuclear pore complex as in situ Reference structures to characterize the performance of a variety of microscopy modalities. We created four genome edited cell lines in which we endogenously labeled the nucleoporin Nup96 with mEGFP, SNAP-tag, HaloTag or the photoconvertible fluorescent protein mMaple. We demonstrate their use (1) as three-dimensional resolution Standards for calibration and quality control, (2) to quantify absolute labeling efficiencies and (3) as precise Reference Standards for molecular counting. These cell lines will enable the broader community to assess the quality of their microscopes and labels, and to perform quantitative, absolute measurements.
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nuclear pores as versatile Reference Standards for quantitative superresolution microscopy
bioRxiv, 2019Co-Authors: Jervis Vermal Thevathasan, Maurice Kahnwald, Konstanty Cieślinski, Philipp Hoess, Sudheer Kumar Peneti, Manuel Reitberger, Daniel Heid, Krishna Chaitanya KasubaAbstract:Abstract Quantitative fluorescence and superresolution microscopy are often limited by insufficient data quality or artifacts. In this context, it is essential to have biologically relevant control samples to benchmark and optimize the quality of microscopes, labels and imaging conditions. Here we exploit the stereotypic arrangement of proteins in the nuclear pore complex as in situ Reference structures to characterize the performance of a variety of microscopy modalities. We created four genome edited cell lines in which we endogenously labeled the nucleoporin Nup96 with mEGFP, SNAP-tag or HaloTag or the photoconvertible fluorescent protein mMaple. We demonstrate their use a) as 3D resolution Standards for calibration and quality control, b) to quantify absolute labeling efficiencies and c) as precise Reference Standards for molecular counting. These cell lines will enable the broad community to assess the quality of their microscopes and labels, and to perform quantitative, absolute measurements.
Ira W. Deveson - One of the best experts on this subject based on the ideXlab platform.
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chimeric synthetic Reference Standards enable cross validation of positive and negative controls in sars cov 2 molecular tests
Scientific Reports, 2021Co-Authors: Bindu Swapna Madala, Ira W. Deveson, Tim R. Mercer, Andre L M Reis, William D RawlinsonAbstract:DNA synthesis in vitro has enabled the rapid production of Reference Standards. These are used as controls, and allow measurement and improvement of the accuracy and quality of diagnostic tests. Current Reference Standards typically represent target genetic material, and act only as positive controls to assess test sensitivity. However, negative controls are also required to evaluate test specificity. Using a pair of chimeric A/B RNA Standards, this allowed incorporation of positive and negative controls into diagnostic testing for the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2). The chimeric Standards constituted target regions for RT-PCR primer/probe sets that are joined in tandem across two separate synthetic molecules. Accordingly, a target region that is present in standard A provides a positive control, whilst being absent in standard B, thereby providing a negative control. This design enables cross-validation of positive and negative controls between the paired Standards in the same reaction, with identical conditions. This enables control and test failures to be distinguished, increasing confidence in the accuracy of results. The chimeric A/B Standards were assessed using the US Centres for Disease Control real-time RT-PCR protocol, and showed results congruent with other commercial controls in detecting SARS-CoV-2 in patient samples. This chimeric Reference standard design approach offers extensive flexibility, allowing representation of diverse genetic features and distantly related sequences, even from different organisms.
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Synthetic microbe communities provide internal Reference Standards for metagenome sequencing and analysis
Nature Communications, 2018Co-Authors: Sheila A. Hardwick, Ted Wong, Bindu S. Kanakamedala, Martin A. Smith, Ira W. Deveson, Sarah E. Ongley, Esteban Marcellin, Wendy Y Chen, Nadia S. Santini, Lars K. NielsenAbstract:The complexity of microbial communities, combined with technical biases in next-generation sequencing, pose a challenge to metagenomic analysis. Here, we develop a set of internal DNA Standards, termed “sequins” (sequencing spike-ins), that together constitute a synthetic community of artificial microbial genomes. Sequins are added to environmental DNA samples prior to library preparation, and undergo concurrent sequencing with the accompanying sample. We validate the performance of sequins by comparison to mock microbial communities, and demonstrate their use in the analysis of real metagenome samples. We show how sequins can be used to measure fold change differences in the size and structure of accompanying microbial communities, and perform quantitative normalization between samples. We further illustrate how sequins can be used to benchmark and optimize new methods, including nanopore long-read sequencing technology. We provide metagenome sequins, along with associated data sets, protocols, and an accompanying software toolkit, as Reference Standards to aid in metagenomic studies.
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Reference Standards for next-generation sequencing.
Nature reviews. Genetics, 2017Co-Authors: Simon A. Hardwick, Ira W. Deveson, Tim R. MercerAbstract:Technical errors can hamper the interpretation of next-generation sequencing (NGS) data, which poses a major challenge for the clinical application of this technology. This Review discusses how Reference Standards circumvent this issue by calibrating NGS measurements and evaluating diagnostic performance of NGS-based genetic tests. Next-generation sequencing (NGS) provides a broad investigation of the genome, and it is being readily applied for the diagnosis of disease-associated genetic features. However, the interpretation of NGS data remains challenging owing to the size and complexity of the genome and the technical errors that are introduced during sample preparation, sequencing and analysis. These errors can be understood and mitigated through the use of Reference Standards — well-characterized genetic materials or synthetic spike-in controls that help to calibrate NGS measurements and to evaluate diagnostic performance. The informed use of Reference Standards, and associated statistical principles, ensures rigorous analysis of NGS data and is essential for its future clinical use.
Philipp Hoess - One of the best experts on this subject based on the ideXlab platform.
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nuclear pores as versatile Reference Standards for quantitative superresolution microscopy
Nature Methods, 2019Co-Authors: Jervis Vermal Thevathasan, Maurice Kahnwald, Konstanty Cieślinski, Philipp Hoess, Sudheer Kumar Peneti, Manuel ReitbergerAbstract:Quantitative fluorescence and superresolution microscopy are often limited by insufficient data quality or artifacts. In this context, it is essential to have biologically relevant control samples to benchmark and optimize the quality of microscopes, labels and imaging conditions. Here, we exploit the stereotypic arrangement of proteins in the nuclear pore complex as in situ Reference structures to characterize the performance of a variety of microscopy modalities. We created four genome edited cell lines in which we endogenously labeled the nucleoporin Nup96 with mEGFP, SNAP-tag, HaloTag or the photoconvertible fluorescent protein mMaple. We demonstrate their use (1) as three-dimensional resolution Standards for calibration and quality control, (2) to quantify absolute labeling efficiencies and (3) as precise Reference Standards for molecular counting. These cell lines will enable the broader community to assess the quality of their microscopes and labels, and to perform quantitative, absolute measurements.
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nuclear pores as versatile Reference Standards for quantitative superresolution microscopy
bioRxiv, 2019Co-Authors: Jervis Vermal Thevathasan, Maurice Kahnwald, Konstanty Cieślinski, Philipp Hoess, Sudheer Kumar Peneti, Manuel Reitberger, Daniel Heid, Krishna Chaitanya KasubaAbstract:Abstract Quantitative fluorescence and superresolution microscopy are often limited by insufficient data quality or artifacts. In this context, it is essential to have biologically relevant control samples to benchmark and optimize the quality of microscopes, labels and imaging conditions. Here we exploit the stereotypic arrangement of proteins in the nuclear pore complex as in situ Reference structures to characterize the performance of a variety of microscopy modalities. We created four genome edited cell lines in which we endogenously labeled the nucleoporin Nup96 with mEGFP, SNAP-tag or HaloTag or the photoconvertible fluorescent protein mMaple. We demonstrate their use a) as 3D resolution Standards for calibration and quality control, b) to quantify absolute labeling efficiencies and c) as precise Reference Standards for molecular counting. These cell lines will enable the broad community to assess the quality of their microscopes and labels, and to perform quantitative, absolute measurements.
