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.

Tissue- and development-stage-specific mRNA and heterogeneous CNV signatures of human ribosomal proteins in normal and cancer samples.

We give results from a detailed analysis of human Ribosomal Protein (RP) levels in normal and cancer samples and cell lines from large mRNA, copy number variation and ribosome profiling datasets. After normalizing total RP mRNA levels per sample, we find highly consistent tissue specific RP mRNA signatures in normal and tumor samples. Multiple RP mRNA-subtypes exist in several cancers, with significant survival and genomic differences. Some RP mRNA variations among subtypes correlate with copy number loss of RP genes. In kidney cancer, RP subtypes map to molecular subtypes related to cell-of-origin. Pan-cancer analysis of TCGA data showed widespread single/double copy loss of RP genes, without significantly affecting survival. In several cancer cell lines, CRISPR-Cas9 knockout of RP genes did not affect cell viability. Matched RP ribosome profiling and mRNA data in humans and rodents stratified by tissue and development stage and were strongly correlated, showing that RP translation rates were proportional to mRNA levels. In a small dataset of human adult and fetal tissues, RP protein levels showed development stage and tissue specific heterogeneity of RP levels. Our results suggest that heterogeneous RP levels play a significant functional role in cellular physiology, in both normal and disease states.

flowLearn: fast and precise identification and quality checking of cell populations in flow cytometry.

Motivation: Identification of cell populations in flow cytometry is a critical part of the analysis and lays the groundwork for many applications and research discovery. The current paradigm of manual analysis is time consuming and subjective. A common goal of users is to replace manual analysis with automated methods that replicate their results. Supervised tools provide the best performance in such a use case, however they require fine parameterization to obtain the best results. Hence, there is a strong need for methods that are fast to setup, accurate and interpretable. Results: flowLearn is a semi-supervised approach for the quality-checked identification of cell populations. Using a very small number of manually gated samples, through density alignments it is able to predict gates on other samples with high accuracy and speed. On two state-of-the-art datasets, our tool achieves median(F1)-measures exceeding 0.99 for 31%, and 0.90 for 80% of all analyzed populations. Furthermore, users can directly interpret and adjust automated gates on new sample files to iteratively improve the initial training. Availability and implementation: FlowLearn is available as an R package on https://github.com/mlux86/flowLearn. Evaluation data is publicly available online. Details can be found in the Supplementary Material. Supplementary information: Supplementary data are available at Bioinformatics online.

acdc - Automated Contamination Detection and Confidence estimation for single-cell genome data.

BACKGROUND: A major obstacle in single-cell sequencing is sample contamination with foreign DNA. To guarantee clean genome assemblies and to prevent the introduction of contamination into public databases, considerable quality control efforts are put into post-sequencing analysis. Contamination screening generally relies on reference-based methods such as database alignment or marker gene search, which limits the set of detectable contaminants to organisms with closely related reference species. As genomic coverage in the tree of life is highly fragmented, there is an urgent need for a reference-free methodology for contaminant identification in sequence data. RESULTS: We present acdc, a tool specifically developed to aid the quality control process of genomic sequence data. By combining supervised and unsupervised methods, it reliably detects both known and de novo contaminants. First, 16S rRNA gene prediction and the inclusion of ultrafast exact alignment techniques allow sequence classification using existing knowledge from databases. Second, reference-free inspection is enabled by the use of state-of-the-art machine learning techniques that include fast, non-linear dimensionality reduction of oligonucleotide signatures and subsequent clustering algorithms that automatically estimate the number of clusters. The latter also enables the removal of any contaminant, yielding a clean sample. Furthermore, given the data complexity and the ill-posedness of clustering, acdc employs bootstrapping techniques to provide statistically profound confidence values. Tested on a large number of samples from diverse sequencing projects, our software is able to quickly and accurately identify contamination. Results are displayed in an interactive user interface. Acdc can be run from the web as well as a dedicated command line application, which allows easy integration into large sequencing project analysis workflows. CONCLUSIONS: Acdc can reliably detect contamination in single-cell genome data. In addition to database-driven detection, it complements existing tools by its unsupervised techniques, which allow for the detection of de novo contaminants. Our contribution has the potential to drastically reduce the amount of resources put into these processes, particularly in the context of limited availability of reference species. As single-cell genome data continues to grow rapidly, acdc adds to the toolkit of crucial quality assurance tools.