Single cell transcriptome analysis using next generation sequencing.

The heterogeneity of tissues, especially in cancer research, is a central issue in transcriptome analysis. In recent years, research has primarily focused on the development of methods for single cell analysis. Single cell analysis aims at gaining (novel) insights into biological processes of healthy and diseased cells. Some of the challenges in transcriptome analysis concern low abundance of sample starting material, necessary sample amplification steps and subsequent analysis. In this study, two fundamentally different approaches to amplification were compared using next-generation sequencing analysis: I. exponential amplification using polymerase-chain-reaction (PCR) and II. linear amplification. For both approaches, protocols for single cell extraction, cell lysis, cDNA synthesis, cDNA amplification and preparation of next-generation sequencing libraries were developed. We could successfully show that transcriptome analysis of low numbers of cells is feasible with both exponential and linear amplification. Using exponential amplification, the highest amplification rates up to 106 were possible. The reproducibility of results is a strength of the linear amplification method. The analysis of next generation sequencing data in single cell samples showed detectable expression in at least 16.000 genes. The variance between samples results in a need to work with a greater amount of biological replicates. In summary it can be said that single cell transcriptome analysis with next generation sequencing is possible but improvements leading to a higher yield of transcriptome reads is required. In the near future by comparing single cancer cells with healthy ones for example, a basis for improved prognosis and diagnosis can be realised

DART-ID increases single-cell proteome coverage.

Analysis by liquid chromatography and tandem mass spectrometry (LC-MS/MS) can identify and quantify thousands of proteins in microgram-level samples, such as those comprised of thousands of cells. This process, however, remains challenging for smaller samples, such as the proteomes of single mammalian cells, because reduced protein levels reduce the number of confidently sequenced peptides. To alleviate this reduction, we developed Data-driven Alignment of Retention Times for IDentification (DART-ID). DART-ID implements principled Bayesian frameworks for global retention time (RT) alignment and for incorporating RT estimates towards improved confidence estimates of peptide-spectrum-matches. When applied to bulk or to single-cell samples, DART-ID increased the number of data points by 30-50% at 1% FDR, and thus decreased missing data. Benchmarks indicate excellent quantification of peptides upgraded by DART-ID and support their utility for quantitative analysis, such as identifying cell types and cell-type specific proteins. The additional datapoints provided by DART-ID boost the statistical power and double the number of proteins identified as differentially abundant in monocytes and T-cells. DART-ID can be applied to diverse experimental designs and is freely available at http://dart-id.slavovlab.net

Technical note: Bias and the quantification of stability

Research on bias in machine learning algorithms has generally been concerned with the impact of bias on predictive accuracy. We believe that there are other factors that should also play a role in the evaluation of bias. One such factor is the stability of the algorithm; in other words, the repeatability of the results. If we obtain two sets of data from the same phenomenon, with the same underlying probability distribution, then we would like our learning algorithm to induce approximately the same concepts from both sets of data. This paper introduces a method for quantifying stability, based on a measure of the agreement between concepts. We also discuss the relationships among stability, predictive accuracy, and bias

X-ray microtomographic characterization and quantification of the strain rate dependent failure mechanism in cenosphere epoxy syntactic foams

This work investigates the failure mechanism in cenosphere epoxy syntactic foams at the quasi-static and dynamic strain rates. Split-Hopkinson pressure bar experiments are controlled to stop dynamic deformation of the foams at various strain stages. The internal microstructure at each strain is characterized in the x-ray microtomography and compared to the microstructure in the foams deformed quasi-statically. The microscopic observations reveal that the failure process in syntactic foams at the low and high rates is dominated by the crushing of cenospheres and the cracking of the epoxy matrix. However, the mechanism of failure in the foam is significantly affected by the strain rate. Compared to quasi-static compression, macro-cracks form earlier in the matrix at dynamic rates and can propagate to split cenospheres. The volume of the damage as defined by the failure of both cenospheres and the matrix is calculated from the x-ray microtomographic images. It is found that the damage can be quantitatively related to the strain and the strain rate using an empirical equation