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  • For each gene, the percentage of the variance of the normalized log-expression values that is explained by the (log-transformed) total spike-in counts, the sex or age of the mouse, or the tissue of origin is calculated. Each curve corresponds to one factor and represents the distribution of percentages across all genes.

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    Nonetheless, we demonstrate how to account for uninteresting technical factors by using sex as an example. We set up a design matrix with the sex of the animal as the explanatory factor for each cell. This ensures that any sex-specific changes in expression will be modelled in our downstream analyses. We do not block on the tissue of origin, despite the fact that it explains more of the variance than sex in Figure 20 . This is because the tissue factor is likely to be associated with genuine differences between cell types, so including it in the model might regress out interesting biological effects.

    Other relevant factors include the chip or plate on which the cells were processed and the batch in which the libraries were sequenced. Blocking on these factors may be necessary to account for batch effects that are often observed in scRNA-seq data ( Hicks et al. , 2015 ; Tung et al. , 2016 ).

    We identify HVGs that may be involved in driving population heterogeneity. This is done by fitting a trend to the technical variances for the spike-in transcripts. We then compute the biological component of the variance for each endogenous gene by subtracting the fitted value of the trend from the total variance.

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    suggests that the trend is fitted accurately to the technical variances. Errors in fitting are negligible due to the precision of the variance estimates in a large dataset containing thousands of cells. The technical and total variances are also much smaller than those in the HSC dataset. This is due to the use of UMIs which reduces the noise caused by variable PCR amplification. Furthermore, the spike-in trend is consistently lower than the variances of the endogenous genes. This reflects the heterogeneity in gene expression across cells of different types. It also means the previous strategy of fitting a trend to the endogenous variances would not be appropriate here (or necessary, given the quality of the spike-in trend).

    The red line represents the mean-dependent trend in the technical variance of the spike-in transcripts (also highlighted as red points).

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    HVGs are identified as genes with large positive biological components. These are saved to file for future reference. Note that some of the p-values are reported as zero due to numerical imprecision.

    If you run a dev script again, you will see that hash number has been updated in both files.

    This issue is known and there is even a stack overflow question about it:

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    After trying a lot of plugins that claim they solve this problem I have finally came to two types of solution:

    Replace [chukhash] with just [hash] in .css extract plugin. This was one of the solutions to the issue . This appears to be a conflict with webpack 4.3 which introduced a [contenthash] variable of its . In conjunction, use this plugin: webpack-md5-hash

    [chukhash] [hash] webpack-md5-hash

    Now if you make changes to your main.scss file and run dev script, only a new style.css should be generated with a new hash.

    const WebpackMd5Hash = require('webpack-md5-hash'); style.[hash].css new WebpackMd5Hash()

    Now lets test our .js files. Now both files change hash.

    There might also be some conflicts still, so now lets try mini-css-extract plugin .

    now lets try mini-css-extract plugin

    The Mini CSS plugin is meant to replace extract-text plugin and provide you with better future compatibility.

    I have restructured my webpack file to compile style.css with /mini-css-extract-plugin and it works for me.

    /mini-css-extract-plugin and it works for me. // const ExtractTextPlugin = require('extract-text-webpack-plugin'); const MiniCssExtractPlugin = require("mini-css-extract-plugin"); { test: /\.scss$/, use: [ 'style-loader', MiniCssExtractPlugin.loader, 'css-loader', 'sass-loader'] } // new ExtractTextPlugin( // {filename: 'style.[hash].css', disable: false, allChunks: true } // ), new MiniCssExtractPlugin({ filename: 'style.[contenthash].css', }),

    As Nikolay Volkov pointed out, ‘style-loader’ might not be necessary anymore since MiniCssExtractPlugin.loader does the same. Though it might be true I would still recommend to leave it for the fallback.

    MiniCssExtractPlugin.loader does the same.

    To have out output .css polished, we can add PostCSS on top.

    PostCSS provides you with autoprefixer, cssnano and other nice and handy stuff. I will show what I am using on a daily basis. We will need postcss-loader. We will also install autoprefixer as we will need it later.

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