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Pre Normalization

Critical to any processing pipeline is the ability to summarize and visualize data, both pre and post processing. Tidyproteomics covers this well with both a summary() function and several plot_() functions. The summary function (described in more detail vignette("summarizing")) utilizes the same syntax inherent to subset() to generate summary statistics on any variable set, including all annotated and accounting terms.

Post Normalization

Visualizing data post processing is an important aspect of data analysis and great care is taken to explore the data post normalization with a variety of plot functions. Each of these are intended to display graphs that should lend insights such as the quantitative dynamic ranges pre and post normalizations plot_normalization(), the sample specific CVs, dynamic grange plot_variation_cv() and principal component variation plot_variation_pca() for each normalization.

library("dplyr")
library("tidyproteomics")

rdata <- hela_proteins %>% 
  normalize(.method = c("scaled", "median", "linear", "loess", "randomforest"))

rdata %>% plot_normalization()
#> Warning: Removed 73038 rows containing non-finite outside the scale range
#> (`stat_boxplot()`).

Variation

Coefficient of Variation and Dynamic Range

The statistical assessment often referred to as CVs (Coefficient of Variation) or RSD (Relative Standard Deviation) attempts to measure the dispersion in a measurement. CVs in proteomics is plural because we often measure hundreds or thousands of proteins simultaneously. Understanding that variability and the effects of normalization will help improve the accuracy of your experiments.

#> TableGrob (2 x 2) "arrange": 3 grobs
#>   z     cells    name                grob
#> 1 1 (2-2,1-1) arrange      gtable[layout]
#> 2 2 (2-2,2-2) arrange      gtable[layout]
#> 3 3 (1-1,1-2) arrange text[GRID.text.741]

Principal Component Analysis

This is a plot of the accumulative variation explained by each of the principal components. Ideally, normalization show improve the first few principal components, removing the measurement and instrument variability, exposing the underlying biological variability. This plot show help visuallize that.

Dynamic Range

Perhaps more intriguing is the plot in plot_dynamic_range() which shows a density heat map of sample specific CVs in relation to quantitative abundance. This plot highlights how CVs increase at the lower quantitative range and, more importantly, how each normalization method can address these large variances. Again, note how random forest normalization is best able to minimize the CVs at the lower quantitative range.

rdata %>% plot_dynamic_range()
#> Warning in ggplot2::geom_point(ggplot2::aes(x = range_x, y = range_y), color = "lightblue"): All aesthetics have length 1, but the data has 60912 rows.
#>  Please consider using `annotate()` or provide this layer with data containing
#>   a single row.

Clustering

Once normalization and imputation methods have been implemented and selected it is often desired to visualize the unbiased clustering of samples. This can be accomplished with the plot_heatmap() and plot_pca() functions to generate plots.

Heatmap

PCA

rdata %>% plot_pca()

Plot alternative variables, in this case the 3rd and 4th prinicpal components.

rdata %>% plot_pca(variables = c('PC3', 'PC4'))