Three million images and morphological profiles of cells treated with matched chemical and genetic perturbations.

The identification of genetic and chemical perturbations with similar impacts on cell morphology can elucidate compounds' mechanisms of action or novel regulators of genetic pathways. Research on methods for identifying such similarities has lagged due to a lack of carefully designed and well-annotated image sets of cells treated with chemical and genetic perturbations. Here we create such a Resource dataset, CPJUMP1, in which each perturbed gene's product is a known target of at least two chemical compounds in the dataset. We systematically explore the directionality of correlations among perturbations that target the same protein encoded by a given gene, and we find that identifying matches between chemical and genetic perturbations is a challenging task. Our dataset and baseline analyses provide a benchmark for evaluating methods that measure perturbation similarities and impact, and more generally, learn effective representations of cellular state from microscopy images. Such advancements would accelerate the applications of image-based profiling of cellular states, such as uncovering drug mode of action or probing functional genomics.

High-dimensional phenotyping to define the genetic basis of cellular morphology.

The morphology of cells is dynamic and mediated by genetic and environmental factors. Characterizing how genetic variation impacts cell morphology can provide an important link between disease association and cellular function. Here, we combine genomic sequencing and high-content imaging approaches on iPSCs from 297 unique donors to investigate the relationship between genetic variants and cellular morphology to map what we term cell morphological quantitative trait loci (cmQTLs). We identify novel associations between rare protein altering variants in WASF2, TSPAN15, and PRLR with several morphological traits related to cell shape, nucleic granularity, and mitochondrial distribution. Knockdown of these genes by CRISPRi confirms their role in cell morphology. Analysis of common variants yields one significant association and nominate over 300 variants with suggestive evidence (P < 10-6) of association with one or more morphology traits. We then use these data to make predictions about sample size requirements for increasing discovery in cellular genetic studies. We conclude that, similar to molecular phenotypes, morphological profiling can yield insight about the function of genes and variants.

Pervasive mislocalization of pathogenic coding variants underlying human disorders.

Widespread sequencing has yielded thousands of missense variants predicted or confirmed as disease-causing. This creates a new bottleneck: determining the functional impact of each variant - largely a painstaking, customized process undertaken one or a few genes or variants at a time. Here, we established a high-throughput imaging platform to assay the impact of coding variation on protein localization, evaluating 3,547 missense variants of over 1,000 genes and phenotypes. We discovered that mislocalization is a common consequence of coding variation, affecting about one-sixth of all pathogenic missense variants, all cellular compartments, and recessive and dominant disorders alike. Mislocalization is primarily driven by effects on protein stability and membrane insertion rather than disruptions of trafficking signals or specific interactions. Furthermore, mislocalization patterns help explain pleiotropy and disease severity and provide insights on variants of unknown significance. Our publicly available resource will likely accelerate the understanding of coding variation in human diseases.

A genome-wide atlas of human cell morphology.

A key challenge of the modern genomics era is developing data-driven representations of gene function. Here, we present the first unbiased morphology-based genome-wide perturbation atlas in human cells, containing three genome-scale genotype-phenotype maps comprising >20,000 single-gene CRISPR-Cas9-based knockout experiments in >30 million cells. Our optical pooled cell profiling approach (PERISCOPE) combines a de-stainable high-dimensional phenotyping panel (based on Cell Painting1,2) with optical sequencing of molecular barcodes and a scalable open-source analysis pipeline to facilitate massively parallel screening of pooled perturbation libraries. This approach provides high-dimensional phenotypic profiles of individual cells, while simultaneously enabling interrogation of subcellular processes. Our atlas reconstructs known pathways and protein-protein interaction networks, identifies culture media-specific responses to gene knockout, and clusters thousands of human genes by phenotypic similarity. Using this atlas, we identify the poorly-characterized disease-associated transmembrane protein TMEM251/LYSET as a Golgi-resident protein essential for mannose-6-phosphate-dependent trafficking of lysosomal enzymes, showing the power of these representations. In sum, our atlas and screening technology represent a rich and accessible resource for connecting genes to cellular functions at scale.

Interpreting Image-based Profiles using Similarity Clustering and Single-Cell Visualization.

Image-based profiling quantitatively assesses the effects of perturbations on cells by capturing a breadth of changes via microscopy. Here, we provide two complementary protocols to help explore and interpret data from image-based profiling experiments. In the first protocol, we examine the similarity among perturbed cell samples using data from compounds that cluster by their mechanisms of action. The protocol includes steps to examine feature-driving differences between samples and to visualize correlations between features and treatments to create interpretable heatmaps using the open-source web tool Morpheus. In the second protocol, we show how to interactively explore images together with the numerical data, and we provide scripts to create visualizations of representative single cells and image sites to understand how changes in features are reflected in the images. Together, these two tutorials help researchers interpret image-based data to speed up research. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Exploratory analysis of profile similarities and driving features Basic Protocol 2: Image and single-cell visualization following profile interpretation.

Morphology and gene expression profiling provide complementary information for mapping cell state.

Morphological and gene expression profiling can cost-effectively capture thousands of features in thousands of samples across perturbations by disease, mutation, or drug treatments, but it is unclear to what extent the two modalities capture overlapping versus complementary information. Here, using both the L1000 and Cell Painting assays to profile gene expression and cell morphology, respectively, we perturb human A549 lung cancer cells with 1,327 small molecules from the Drug Repurposing Hub across six doses, providing a data resource including dose-response data from both assays. The two assays capture both shared and complementary information for mapping cell state. Cell Painting profiles from compound perturbations are more reproducible and show more diversity but measure fewer distinct groups of features. Applying unsupervised and supervised methods to predict compound mechanisms of action (MOAs) and gene targets, we find that the two assays not only provide a partially shared but also a complementary view of drug mechanisms. Given the numerous applications of profiling in biology, our analyses provide guidance for planning experiments that profile cells for detecting distinct cell types, disease phenotypes, and response to chemical or genetic perturbations.

High-dimensional gene expression and morphology profiles of cells across 28,000 genetic and chemical perturbations.

Cells can be perturbed by various chemical and genetic treatments and the impact on gene expression and morphology can be measured via transcriptomic profiling and image-based assays, respectively. The patterns observed in these high-dimensional profile data can power a dozen applications in drug discovery and basic biology research, but both types of profiles are rarely available for large-scale experiments. Here, we provide a collection of four datasets with both gene expression and morphological profile data useful for developing and testing multimodal methodologies. Roughly a thousand features are measured for each of the two data types, across more than 28,000 chemical and genetic perturbations. We define biological problems that use the shared and complementary information in these two data modalities, provide baseline analysis and evaluation metrics for multi-omic applications, and make the data resource publicly available ( https://broad.io/rosetta/ ).

Publisher Correction: Nucleus segmentation across imaging experiments: the 2018 Data Science Bowl.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Nucleus segmentation across imaging experiments: the 2018 Data Science Bowl.

Segmenting the nuclei of cells in microscopy images is often the first step in the quantitative analysis of imaging data for biological and biomedical applications. Many bioimage analysis tools can segment nuclei in images but need to be selected and configured for every experiment. The 2018 Data Science Bowl attracted 3,891 teams worldwide to make the first attempt to build a segmentation method that could be applied to any two-dimensional light microscopy image of stained nuclei across experiments, with no human interaction. Top participants in the challenge succeeded in this task, developing deep-learning-based models that identified cell nuclei across many image types and experimental conditions without the need to manually adjust segmentation parameters. This represents an important step toward configuration-free bioimage analysis software tools.

AUTOMATIC RENAL SEGMENTATION IN DCE-MRI USING CONVOLUTIONAL NEURAL NETWORKS.

Kidney function evaluation using dynamic contrast-enhanced MRI (DCE-MRI) images could help in diagnosis and treatment of kidney diseases of children. Automatic segmentation of renal parenchyma is an important step in this process. In this paper, we propose a time and memory efficient fully automated segmentation method which achieves high segmentation accuracy with running time in the order of seconds in both normal kidneys and kidneys with hydronephrosis. The proposed method is based on a cascaded application of two 3D convolutional neural networks that employs spatial and temporal information at the same time in order to learn the tasks of localization and segmentation of kidneys, respectively. Segmentation performance is evaluated on both normal and abnormal kidneys with varying levels of hydronephrosis. We achieved a mean dice coefficient of 91.4 and 83.6 for normal and abnormal kidneys of pediatric patients, respectively.

EEG-assisted Modulation of Sound Sources in the Auditory Scene.

Noninvasive EEG (electroencephalography) based auditory attention detection could be useful for improved hearing aids in the future. This work is a novel attempt to investigate the feasibility of online modulation of sound sources by probabilistic detection of auditory attention, using a noninvasive EEG-based brain computer interface. Proposed online system modulates the upcoming sound sources through gain adaptation which employs probabilistic decisions (soft decisions) from a classifier trained on offline calibration data. In this work, calibration EEG data were collected in sessions where the participants listened to two sound sources (one attended and one unattended). Cross-correlation coefficients between the EEG measurements and the attended and unattended sound source envelope (estimates) are used to show differences in sharpness and delays of neural responses for attended versus unattended sound source. Salient features to distinguish attended sources from the unattended ones in the correlation patterns have been identified, and later they have been used to train an auditory attention classifier. Using this classifier, we have shown high offline detection performance with single channel EEG measurements compared to the existing approaches in the literature which employ large number of channels. In addition, using the classifier trained offline in the calibration session, we have shown the performance of the online sound source modulation system. We observe that online sound source modulation system is able to keep the level of attended sound source higher than the unattended source.

Context-aware recursive bayesian graph traversal in BCIs.

Noninvasive brain computer interfaces (BCI), and more specifically Electroencephalography (EEG) based systems for intent detection need to compensate for the low signal to noise ratio of EEG signals. In many applications, the temporal dependency information from consecutive decisions and contextual data can be used to provide a prior probability for the upcoming decision. In this study we proposed two probabilistic graphical models (PGMs), using context information and previously observed EEG evidences to estimate a probability distribution over the decision space in graph based decision-making mechanism. In this approach, user moves a pointer to the desired vertex in the graph in which each vertex represents an action. To select a vertex, a "Select" command, or a proposed probabilistic Selection criterion (PSC) can be used to automatically detect the user intended vertex. Performance of different PGMs and Selection criteria combinations are compared over a keyboard based on a graph layout. Based on the simulation results, probabilistic Selection criterion along with the probabilistic graphical model provides the highest performance boost for individuals with pour calibration performance and achieving the same performance for individuals with high calibration performance.

A Graphical Model for Online Auditory Scene Modulation Using EEG Evidence for Attention.

Recent findings indicate that brain interfaces have the potential to enable attention-guided auditory scene analysis and manipulation in applications, such as hearing aids and augmented/virtual environments. Specifically, noninvasively acquired electroencephalography (EEG) signals have been demonstrated to carry some evidence regarding, which of multiple synchronous speech waveforms the subject attends to. In this paper, we demonstrate that: 1) using data- and model-driven cross-correlation features yield competitive binary auditory attention classification results with at most 20 s of EEG from 16 channels or even a single well-positioned channel; 2) a model calibrated using equal-energy speech waveforms competing for attention could perform well on estimating attention in closed-loop unbalanced-energy speech waveform situations, where the speech amplitudes are modulated by the estimated attention posterior probability distribution; 3) such a model would perform even better if it is corrected (linearly, in this instance) based on EEG evidence dependence on speech weights in the mixture; and 4) calibrating a model based on population EEG could result in acceptable performance for new individuals/users; therefore, EEG-based auditory attention classifiers may generalize across individuals, leading to reduced or eliminated calibration time and effort.