Using single-cell genomics to understand developmental processes and cell fate decisions.

High-throughput -omics techniques have revolutionised biology, allowing for thorough and unbiased characterisation of the molecular states of biological systems. However, cellular decision-making is inherently a unicellular process to which "bulk" -omics techniques are poorly suited, as they capture ensemble averages of cell states. Recently developed single-cell methods bridge this gap, allowing high-throughput molecular surveys of individual cells. In this review, we cover core concepts of analysis of single-cell gene expression data and highlight areas of developmental biology where single-cell techniques have made important contributions. These include understanding of cell-to-cell heterogeneity, the tracing of differentiation pathways, quantification of gene expression from specific alleles, and the future directions of cell lineage tracing and spatial gene expression analysis.J.A.G. was supported by Wellcome Trust Grant “Systematic Identification of Lineage Specification in Murine Gastrulation” (109081/Z/15/A). A.S. was supported by Wellcome Trust Grant “Tracing early mammalian lineage decisions by single cell genomics” (105031/B/14/Z). J.C.M. was supported by core funding from Cancer Research UK (award no. A17197) and EMBL

Mosaic autosomal aneuploidies are detectable from single-cell RNAseq data.

BACKGROUND: Aneuploidies are copy number variants that affect entire chromosomes. They are seen commonly in cancer, embryonic stem cells, human embryos, and in various trisomic diseases. Aneuploidies frequently affect only a subset of cells in a sample; this is known as "mosaic" aneuploidy. A cell that harbours an aneuploidy exhibits disrupted gene expression patterns which can alter its behaviour. However, detection of aneuploidies using conventional single-cell DNA-sequencing protocols is slow and expensive. METHODS: We have developed a method that uses chromosome-wide expression imbalances to identify aneuploidies from single-cell RNA-seq data. The method provides quantitative aneuploidy calls, and is integrated into an R software package available on GitHub and as an Additional file of this manuscript. RESULTS: We validate our approach using data with known copy number, identifying the vast majority of aneuploidies with a low rate of false discovery. We show further support for the method's efficacy by exploiting allele-specific gene expression levels, and differential expression analyses. CONCLUSIONS: The method is quick and easy to apply, straightforward to interpret, and represents a substantial cost saving compared to single-cell genome sequencing techniques. However, the method is less well suited to data where gene expression is highly variable. The results obtained from the method can be used to investigate the consequences of aneuploidy itself, or to exclude aneuploidy-affected expression values from conventional scRNA-seq data analysis

Why do patients decline surgical trials? Findings from a qualitative interview study embedded in the Cancer Research UK BOLERO trial (Bladder cancer: Open versus Lapararoscopic or RObotic cystectomy)

Background Surgical trials have typically experienced recruitment difficulties when compared with other types of oncology trials. Qualitative studies have an important role to play in exploring reasons for low recruitment, although to date few such studies have been carried out that are embedded in surgical trials. The BOLERO trial (Bladder cancer: Open versus Lapararoscopic or RObotic cystectomy) is a study to determine the feasibility of randomisation to open versus laparoscopic access/robotic cystectomy in patients with bladder cancer. We describe the results of a qualitative study embedded within the clinical trial that explored why patients decline randomisation. Methods Ten semi-structured interviews with patients who declined randomisation to the clinical trial, and two interviews with recruiting research nurses were conducted. Data were analysed for key themes. Results The majority of patients declined the trial because they had preferences for a particular treatment arm, and in usual practice could choose which surgical method they would be given. In most cases the robotic option was preferred. Patients described an intuitive ‘sense’ that favoured the new technology and had carried out their own inquiries, including Internet research and talking with previous patients and friends and family with medical backgrounds. Medical histories and lifestyle considerations also shaped these personalised choices. Of importance too, however, were the messages patients perceived from their clinical encounters. Whilst some patients felt their surgeon favoured the robotic option, others interpreted ‘indirect’ cues such as the ‘established’ reputation of the surgeon and surgical method and comments made during clinical assessments. Many patients expressed a wish for greater direction from their surgeon when making these decisions. Conclusion For trials where the ‘new technology’ is available to patients, there will likely be difficulties with recruitment. Greater attention could be paid to how messages about treatment options and the trial are conveyed across the whole clinical setting. However, if it is too difficult to challenge such messages, then questions should be asked about whether genuine and convincing equipoise can be presented and perceived in such trials. This calls for consideration of whether alternative methods of generating evidence could be used when evaluating surgical techniques which are established and routinely available

Bioactive nutrients - Time for tolerable upper intake levels to address safety.

There is increasing interest by consumers, researchers, and regulators into the roles that certain bioactive compounds, derived from plants and other natural sources, can play in health maintenance and promotion, and even prolonging a productive quality of life. Research has rapidly emerged suggesting that a wide range of compounds and mixtures in and from plants (such as fruits and vegetables, tea and cocoa) and animals (such as fish and probiotics) may exert substantial health benefits. There is interest in exploring the possibility of establishing recommended intakes or dietary guidance for certain bioactive substances to help educate consumers. A key aspect of establishing dietary guidance is the assessment of safety/toxicity of these substances. Toxicologists need to be involved in both the development of the safety framework and in the evaluation of the science to establish maximum intake/upper limits

Detection and removal of barcode swapping in single-cell RNA-seq data.

Barcode swapping results in the mislabelling of sequencing reads between multiplexed samples on patterned flow-cell Illumina sequencing machines. This may compromise the validity of numerous genomic assays; however, the severity and consequences of barcode swapping remain poorly understood. We have used two statistical approaches to robustly quantify the fraction of swapped reads in two plate-based single-cell RNA-sequencing datasets. We found that approximately 2.5% of reads were mislabelled between samples on the HiSeq 4000, which is lower than previous reports. We observed no correlation between the swapped fraction of reads and the concentration of free barcode across plates. Furthermore, we have demonstrated that barcode swapping may generate complex but artefactual cell libraries in droplet-based single-cell RNA-sequencing studies. To eliminate these artefacts, we have developed an algorithm to exclude individual molecules that have swapped between samples in 10x Genomics experiments, allowing the continued use of cutting-edge sequencing machines for these assays

J.S. Bell's Concept of Local Causality

John Stewart Bell's famous 1964 theorem is widely regarded as one of the most important developments in the foundations of physics. It has even been described as "the most profound discovery of science." Yet even as we approach the 50th anniversary of Bell's discovery, its meaning and implications remain controversial. Many textbooks and commentators report that Bell's theorem refutes the possibility (suggested especially by Einstein, Podolsky, and Rosen in 1935) of supplementing ordinary quantum theory with additional ("hidden") variables that might restore determinism and/or some notion of an observer-independent reality. On this view, Bell's theorem supports the orthodox Copenhagen interpretation. Bell's own view of his theorem, however, was quite different. He instead took the theorem as establishing an "essential conflict" between the now well-tested empirical predictions of quantum theory and relativistic \emph{local causality}. The goal of the present paper is, in general, to make Bell's own views more widely known and, in particular, to explain in detail Bell's little-known mathematical formulation of the concept of relativistic local causality on which his theorem rests. We thus collect and organize many of Bell's crucial statements on these topics, which are scattered throughout his writings, into a self-contained, pedagogical discussion including elaborations of the concepts "beable", "completeness", and "causality" which figure in the formulation. We also show how local causality (as formulated by Bell) can be used to derive an empirically testable Bell-type inequality, and how it can be used to recapitulate the EPR argument.Comment: 19 pages, 4 figure

H I ABSORPTION TOWARD H II REGIONS AT SMALL GALACTIC LONGITUDES

We make a comprehensive study of H I absorption toward H II regions located within |l| < 10°. Structures in the extreme inner Galaxy are traced using the longitude-velocity space distribution of this absorption. We find significant H I absorption associated with the Near and Far 3 kpc Arms, the Connecting Arm, Bania's Clump 1, and the H I Tilted Disk. We also constrain the line-of-sight distances to H II regions, by using H I absorption spectra together with the H II region velocities measured by radio recombination lines