flowCut: An R package for automated removal of outlier events and flagging of files based on time versus fluorescence analysis.

Technical artifacts such as clogging that occur during the data acquisition process of flow cytometry data can cause spurious events and fluorescence intensity shifting that impact the quality of the data and its analysis results. These events should be identified and potentially removed before being passed to the next stage of analysis. flowCut, an R package, automatically detects anomaly events in flow cytometry experiments and flags files for potential review. Its results are on par with manual analysis and it outperforms existing automated approaches.

High-throughput phenotyping reveals expansive genetic and structural underpinnings of immune variation.

By developing a high-density murine immunophenotyping platform compatible with high-throughput genetic screening, we have established profound contributions of genetics and structure to immune variation (http://www.immunophenotype.org). Specifically, high-throughput phenotyping of 530 unique mouse gene knockouts identified 140 monogenic 'hits', of which most had no previous immunologic association. Furthermore, hits were collectively enriched in genes for which humans show poor tolerance to loss of function. The immunophenotyping platform also exposed dense correlation networks linking immune parameters with each other and with specific physiologic traits. Such linkages limit freedom of movement for individual immune parameters, thereby imposing genetically regulated 'immunologic structures', the integrity of which was associated with immunocompetence. Hence, we provide an expanded genetic resource and structural perspective for understanding and monitoring immune variation in health and disease.

Single Cell Phenotypic Profiling of 27 DLBCL Cases Reveals Marked Intertumoral and Intratumoral Heterogeneity.

Diffuse large B-cell lymphoma (DLBCL) is the most common histologic subtype of non-Hodgkin lymphoma and is notorious for its clinical heterogeneity. Patient outcomes can be predicted by cell-of-origin (COO) classification, demonstrating that the underlying transcriptional signature of malignant B-cells informs biological behavior in the context of standard combination chemotherapy regimens. In the current study, we used mass cytometry (CyTOF) to examine tumor phenotypes at the protein level with single cell resolution in a collection of 27 diagnostic DLBCL biopsy specimens from treatment naïve patients. We found that malignant B-cells from each patient occupied unique regions in 37-dimensional phenotypic space with no apparent clustering of samples into discrete subtypes. Interestingly, variable MHC class II expression was found to be the greatest contributor to phenotypic diversity. Within individual tumors, a subset of cases showed multiple phenotypic subpopulations, and in one case, we were able to demonstrate direct correspondence between protein-level phenotypic subsets and DNA mutation-defined subclones. In summary, CyTOF analysis can resolve both intertumoral and intratumoral heterogeneity among primary samples and reveals that each case of DLBCL is unique and may be comprised of multiple, genetically distinct subclones. © 2019 International Society for Advancement of Cytometry.

ddPCRclust: an R package and Shiny app for automated analysis of multiplexed ddPCR data.

Motivation: Droplet digital PCR (ddPCR) is an emerging technology for quantifying DNA. By partitioning the target DNA into ∼20 000 droplets, each serving as its own PCR reaction compartment, a very high sensitivity of DNA quantification can be achieved. However, manual analysis of the data is time consuming and algorithms for automated analysis of non-orthogonal, multiplexed ddPCR data are unavailable, presenting a major bottleneck for the advancement of ddPCR transitioning from low-throughput to high-throughput. Results: ddPCRclust is an R package for automated analysis of data from Bio-Rad's droplet digital PCR systems (QX100 and QX200). It can automatically analyze and visualize multiplexed ddPCR experiments with up to four targets per reaction. Results are on par with manual analysis, but only take minutes to compute instead of hours. The accompanying Shiny app ddPCRvis provides easy access to the functionalities of ddPCRclust through a web-browser based GUI. Availability and implementation: R package: https://github.com/bgbrink/ddPCRclust; Interface: https://github.com/bgbrink/ddPCRvis/; Web: https://bibiserv.cebitec.uni-bielefeld.de/ddPCRvis/. Supplementary information: Supplementary data are available at Bioinformatics online.

High throughput automated analysis of big flow cytometry data.

The rapid expansion of flow cytometry applications has outpaced the functionality of traditional manual analysis tools used to interpret flow cytometry data. Scientists are faced with the daunting prospect of manually identifying interesting cell populations in 50-dimensional datasets, equalling the complexity previously only reached in mass cytometry. Data can no longer be analyzed or interpreted fully by manual approaches. While automated gating has been the focus of intense efforts, there are many significant additional steps to the analytical pipeline (e.g., cleaning the raw files, event outlier detection, extracting immunophenotypes). We review the components of a customized automated analysis pipeline that can be generally applied to large scale flow cytometry data. We demonstrate these methodologies on data collected by the International Mouse Phenotyping Consortium (IMPC).

flowCL: ontology-based cell population labelling in flow cytometry.

MOTIVATION: Finding one or more cell populations of interest, such as those correlating to a specific disease, is critical when analysing flow cytometry data. However, labelling of cell populations is not well defined, making it difficult to integrate the output of algorithms to external knowledge sources. RESULTS: We developed flowCL, a software package that performs semantic labelling of cell populations based on their surface markers and applied it to labelling of the Federation of Clinical Immunology Societies Human Immunology Project Consortium lyoplate populations as a use case. CONCLUSION: By providing automated labelling of cell populations based on their immunophenotype, flowCL allows for unambiguous and reproducible identification of standardized cell types. AVAILABILITY AND IMPLEMENTATION: Code, R script and documentation are available under the Artistic 2.0 license through Bioconductor (http://www.bioconductor.org/packages/devel/bioc/html/flowCL.html). CONTACT: rbrinkman@bccrc.ca SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.