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Image Search Results
Journal: Nature Communications
Article Title: Determining sequencing depth in a single-cell RNA-seq experiment
doi: 10.1038/s41467-020-14482-y
Figure Lengend Snippet: a Description of the sequencing budget allocation problem. Consider estimating the underlying gene distribution (top) from the noisy read counts obtained via sequencing (bottom). With a fixed number of reads to be sequenced, deep sequencing of a few cells accurately estimates each individual cell but lacks coverage of the entire distribution (left), whereas a shallow sequencing of many cells covers the entire population but introduces a lot of noise (right). b Optimal tradeoff. The memory T-cell marker gene S100A4 has 41.7k reads in the pbmc_4k dataset. For estimating the underlying gamma distribution \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${X}_{g} \sim {\rm{Gamma}}({r}_{g},{\theta }_{g})$$\end{document} X g ~ Gamma ( r g , θ g ) , the relative error is plotted as a function of the sequencing depth, where the optimal error is obtained at a depth of one read per cell (orange star) and is two times smaller than that at the current depth of pbmc_4k (red triangle). c Experimental design. To determine the sequencing depth for an experiment, first the relative gene expression level can be obtained via pilot experiments or previous studies (top left). Then the researcher can select a set of genes of interest (i.e., some marker genes highlighted as black dots), of which the smallest relative expression level \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${p}^{* }$$\end{document} p * ( MS4A1 ) defines the reliable detection limit. Finally, the optimal sequencing depth is determined as \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${n}_{{\rm{reads}}}^{* }=1/{p}^{* }$$\end{document} n reads * = 1 ∕ p * (top right). The errors under different tradeoffs are visualized as a function of the genes ordered from the most expressed to the least (bottom). The optimal sequencing budget allocation (orange) minimizes the worst-case error over all the genes of interest (left of the red dashed line), whereas both the deeper sequencing (green) and the shallower sequencing (blue) yield worse results.
Article Snippet: They are publicly available and can be downloaded via the following links:
Techniques: Sequencing, Marker, Expressing
Journal: Nature Communications
Article Title: Determining sequencing depth in a single-cell RNA-seq experiment
doi: 10.1038/s41467-020-14482-y
Figure Lengend Snippet: a Top: for estimating the coefficient of variation (CV), the plug-in estimates become more inflated as the sequencing depth becomes shallower (from right to left along the x axis), whereas the EB estimates are consistent. 3-std confidence intervals are provided for this panel. Middle: both brain_1k and brain_1.3m are from the mouse brain, and hence each gene should have a similar CV value between the two datasets. This is indeed the case for the EB estimator (right), which is adaptive to different sequencing depths. However, as brain_1k is twice deeper than brain_1.3m, the plug-in estimates are biased that most points are above the 45-degree line (red). Bottom: distribution recovery for the gene GZMA from a dataset that is subsampled to be five times shallower (left). The EB estimator provides a reasonable estimation for both the zero proportion and the tail shape, resulting in a small total variation error (right). b Feature selection and PCA. The task is to first select features (genes) based on CV, and then perform PCA on the selected features. The results on the full data (pbmc_4k) and a subsampled (three times shallower) are compared. EB estimates are more consistent between the full data and the subsampled data for both the CV ranks (top) and the PCA plots (bottom).
Article Snippet: They are publicly available and can be downloaded via the following links:
Techniques: Sequencing, Selection
Journal: Nature Communications
Article Title: Determining sequencing depth in a single-cell RNA-seq experiment
doi: 10.1038/s41467-020-14482-y
Figure Lengend Snippet: a Top: the EB-estimated Pearson correlation for some marker genes in pbmc_4k are visualized, ordered by different cell populations (top). The clear block-diagonal structure implies that the EB estimator is capable of capturing the gene functional groups. As a comparison, the plug-in estimator also recovers those modules but with a weaker contrast (bottom left panel, plug-in with 100%). Bottom: a subsample experiment further shows that the EB estimator can recover the module with 5% of the data. For the plug-in estimator, the first block (T cells) is blurred with 25% of the data, and the entire structure vanishes with 10% of the data. b Gene network based on the EB-estimated Pearson correlation using the pbmc_4k dataset. Most gene modules correspond to important cell types or functions, including T cells, B cells, NK-cells, myeloid-derived cells, megakaryocytes/platelets, ribosomal protein genes, and mitochondrially encoded protein-coding genes. c Left: the estimated Pearson correlations between all genes and LCK (1st panel) and CD3D (2nd panel), two known T-cell markers. There are three modes for the EB-estimated values, where the positive mode, the zero mode, and the negative mode correspond to genes in the same module, different modules, and irrelevant genes, respectively. The plug-in estimated values are nonetheless much closer to zero even for the truly correlated ones, indicating an artificial shrinkage of the estimated values. Right: two instances where the EB estimates are significantly different from the plug-in estimates. The axes represent read counts, and the color codes the number of cells. Both gene pairs are biologically validated (see Gene network analysis in Methods). See also Supplementary Figs. – for more examples.
Article Snippet: They are publicly available and can be downloaded via the following links:
Techniques: Marker, Blocking Assay, Functional Assay, Derivative Assay