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| 2. |
- Beal, Jacob, et al.
(författare)
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Robust estimation of bacterial cell count from optical density
- 2020
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Ingår i: Communications Biology. - : Springer Science and Business Media LLC. - 2399-3642. ; 3:1
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Tidskriftsartikel (refereegranskat)abstract
- Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data.
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| 3. |
- Klionsky, Daniel J., et al.
(författare)
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Guidelines for the use and interpretation of assays for monitoring autophagy
- 2012
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Ingår i: Autophagy. - : Informa UK Limited. - 1554-8635 .- 1554-8627. ; 8:4, s. 445-544
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Forskningsöversikt (refereegranskat)abstract
- In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.
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| 4. |
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- 2019
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Tidskriftsartikel (refereegranskat)
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| 5. |
- Callaway, EM, et al.
(författare)
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A multimodal cell census and atlas of the mammalian primary motor cortex
- 2021
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Ingår i: Nature. - : Springer Science and Business Media LLC. - 1476-4687 .- 0028-0836. ; 598:7879, s. 86-102
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Tidskriftsartikel (refereegranskat)abstract
- Here we report the generation of a multimodal cell census and atlas of the mammalian primary motor cortex as the initial product of the BRAIN Initiative Cell Census Network (BICCN). This was achieved by coordinated large-scale analyses of single-cell transcriptomes, chromatin accessibility, DNA methylomes, spatially resolved single-cell transcriptomes, morphological and electrophysiological properties and cellular resolution input–output mapping, integrated through cross-modal computational analysis. Our results advance the collective knowledge and understanding of brain cell-type organization1–5. First, our study reveals a unified molecular genetic landscape of cortical cell types that integrates their transcriptome, open chromatin and DNA methylation maps. Second, cross-species analysis achieves a consensus taxonomy of transcriptomic types and their hierarchical organization that is conserved from mouse to marmoset and human. Third, in situ single-cell transcriptomics provides a spatially resolved cell-type atlas of the motor cortex. Fourth, cross-modal analysis provides compelling evidence for the transcriptomic, epigenomic and gene regulatory basis of neuronal phenotypes such as their physiological and anatomical properties, demonstrating the biological validity and genomic underpinning of neuron types. We further present an extensive genetic toolset for targeting glutamatergic neuron types towards linking their molecular and developmental identity to their circuit function. Together, our results establish a unifying and mechanistic framework of neuronal cell-type organization that integrates multi-layered molecular genetic and spatial information with multi-faceted phenotypic properties.
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| 6. |
- Chapman, Lesley M, et al.
(författare)
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A crowdsourced set of curated structural variants for the human genome
- 2020
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Ingår i: PLoS Computational Biology. - : Public Library of Science (PLoS). - 1553-7358. ; 16:6
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Tidskriftsartikel (refereegranskat)abstract
- A high quality benchmark for small variants encompassing 88 to 90% of the reference genome has been developed for seven Genome in a Bottle (GIAB) reference samples. However a reliable benchmark for large indels and structural variants (SVs) is more challenging. In this study, we manually curated 1235 SVs, which can ultimately be used to evaluate SV callers or train machine learning models. We developed a crowdsourcing app - SVCurator - to help GIAB curators manually review large indels and SVs within the human genome, and report their genotype and size accuracy. SVCurator displays images from short, long, and linked read sequencing data from the GIAB Ashkenazi Jewish Trio son [NIST RM 8391/HG002]. We asked curators to assign labels describing SV type (deletion or insertion), size accuracy, and genotype for 1235 putative insertions and deletions sampled from different size bins between 20 and 892,149 bp. 'Expert' curators were 93% concordant with each other, and 37 of the 61 curators had at least 78% concordance with a set of 'expert' curators. The curators were least concordant for complex SVs and SVs that had inaccurate breakpoints or size predictions. After filtering events with low concordance among curators, we produced high confidence labels for 935 events. The SVCurator crowdsourced labels were 94.5% concordant with the heuristic-based draft benchmark SV callset from GIAB. We found that curators can successfully evaluate putative SVs when given evidence from multiple sequencing technologies.
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| 7. |
- Chapman, Lesley M, et al.
(författare)
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SVCurator: A Crowdsourcing app to visualize evidence of structural variants for the human genome
- 2019
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- A high quality benchmark for small variants encompassing 88 to 90% of the reference genome has been developed for seven Genome in a Bottle (GIAB) reference samples. However a reliable benchmark for large indels and structural variants (SVs) is yet to be defined. In this study, we manually curated 1235 SVs which can ultimately be used to evaluate SV callers ortrain machine learning models. We developed a crowdsourcing app - SVCurator - to help curators manually review large indels and SVs within the human genome, and report their genotype and size accuracy.SVCurator is a Python Flask-based web platform that displays images from short, long, and linked read sequencing data from the GIAB Ashkenazi Jewish Trio son [NIST RM 8391/HG002]. We asked curators to assign labels describing SV type (deletion or insertion), size accuracy, and genotype for 1235 putative insertions and deletions sampled from different size bins between 20 and 892,149 bp. The crowdsourced results were highly concordant with 37 out ofthe 61 curators having at least 78% concordance with a set of ‘expert’ curators, where there was 93% concordance amongst ‘expert’ curators. This produced high confidence labels for 935 events. When compared to the heuristic-based draft benchmark SV callset from GIAB, the SVCurator crowdsourced labels were 94.5% concordant with the benchmark set. We found that curators can successfully evaluate putative SVs when given evidence from multiple sequencing technologies.
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| 8. |
- Wang, Anqi, et al.
(författare)
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Characterizing prostate cancer risk through multi-ancestry genome-wide discovery of 187 novel risk variants
- 2023
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Ingår i: Nature Genetics. - : Springer Nature. - 1061-4036 .- 1546-1718. ; 55:12, s. 2065-2074
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Tidskriftsartikel (refereegranskat)abstract
- The transferability and clinical value of genetic risk scores (GRSs) across populations remain limited due to an imbalance in genetic studies across ancestrally diverse populations. Here we conducted a multi-ancestry genome-wide association study of 156,319 prostate cancer cases and 788,443 controls of European, African, Asian and Hispanic men, reflecting a 57% increase in the number of non-European cases over previous prostate cancer genome-wide association studies. We identified 187 novel risk variants for prostate cancer, increasing the total number of risk variants to 451. An externally replicated multi-ancestry GRS was associated with risk that ranged from 1.8 (per standard deviation) in African ancestry men to 2.2 in European ancestry men. The GRS was associated with a greater risk of aggressive versus non-aggressive disease in men of African ancestry (P = 0.03). Our study presents novel prostate cancer susceptibility loci and a GRS with effective risk stratification across ancestry groups.
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