ABSTRACT
Motivation: Single-cell RNA-sequencing (scRNA-seq) technology enables researchers to investigate a genome at the cellular level with unprecedented resolution. An organism consists of a heterogeneous collection of cell types, each of which plays a distinct role in various biological processes. Hence, the first step of scRNA-seq data analysis is often to distinguish cell types so they can be investigated separately. Researchers have recently developed several automated cell-type annotation tools, requiring neither biological knowledge nor subjective human decisions. Dropout is a crucial characteristic of scRNA-seq data widely used in differential expression analysis. However, no current cell annotation method explicitly utilizes dropout information. Fully utilizing dropout information motivated this work. Results: We present scAnnotate, a cell annotation tool that fully utilizes dropout information. We model every gene's marginal distribution using a mixture model, which describes both the dropout proportion and the distribution of the non-dropout expression levels. Then, using an ensemble machine learning approach, we combine the mixture models of all genes into a single model for cell-type annotation. This combining approach can avoid estimating numerous parameters in the high-dimensional joint distribution of all genes. Using 14 real scRNA-seq datasets, we demonstrate that scAnnotate is competitive against nine existing annotation methods. Furthermore, because of its distinct modelling strategy, scAnnotate's misclassified cells differ greatly from competitor methods. This suggests using scAnnotate together with other methods could further improve annotation accuracy. Availability and implementation: We implemented scAnnotate as an R package and made it publicly available from CRAN: https://cran.r-project.org/package=scAnnotate. Supplementary information: Supplementary data are available at Bioinformatics Advances online.
ABSTRACT
Sorting rare cells from heterogeneous mixtures makes a significant contribution to biological research and medical treatment. However, the performances of traditional methods are limited due to the time-consuming preparation, poor purity, and recovery rate. In this paper, we proposed a cell screening method based on the automated microrobotic aspirate-and-place strategy under fluorescence microscopy. A fast autofocusing visual feedback (FAVF) method is introduced for precise and real-time three-dimensional (3D) location. In the context of this method, the scalable correlation coefficient (SCC) matching is presented for planar locating cells with regions of interest (ROI) created for autofocusing. When the overlap occurs, target cells are separated by a segmentation algorithm. To meet the shallow depth of field (DOF) limitation of the microscope, the improved multiple depth from defocus (MDFD) algorithm is used for depth detection, taking 850 ms a time with an accuracy rate of 96.79%. The neighborhood search based algorithm is applied for the tracking of the micropipette. Finally, experiments of screening NIH/3T3 (mouse embryonic fibroblast) cells verifies the feasibility and validity of this method with an average speed of 5 cells/min, 95% purity, and 80% recovery rate. Moreover, such versatile functions as cell counting and injection, for example, could be achieved by this expandable system.
