## 2014 UC Davis Proteomics Workshop

Recently I had the pleasure of teaching data analysis at the 2014 UC Davis Proteomics Workshop. This included a hands on lab for making gene ontology enrichment networks. You can check out my lecture and tutorial below or download all the material.

Introduction

Tutorial

2014 UC Davis Proteomics Workshop Dmitry Grapov is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License.

## Using Repeated Measures to Remove Artifacts from Longitudinal Data

Recently I was tasked with evaluating and most importantly removing analytical variance form a longitudinal metabolomic analysis carried out over a few years and including >2,5000 measurements for >5,000 patients. Even using state-of-the-art analytical instruments and techniques long term biological studies are plagued with unwanted trends which are unrelated to the original experimental design and stem from analytical sources of variance (added noise by the process of measurement). Below is an example of a metabolomic measurement with and without analytical variance.

The noise pattern can be estimated based on replicated measurements of quality control samples embedded at a ratio of 1:10 within the larger experimental design. The process of data normalization is used to remove analytical noise from biological signal on a variable specific basis. At the bottom of this post, you can find an in-depth presentation of how data quality can be estimated and a comparison of many common data normalization approaches. From my analysis I concluded that a relatively simple LOESS normalization is a very powerful method for removal of analytical variance. While LOESS (or LOWESS), locally weighted scatterplot smoothing, is a relatively simple approach to implement; great care has to be taken when optimizing each variable-specific model.

In particular, the span parameter or alpha controls the degree of smoothing and is a major determinant if the model (calculated from repeated measures) is underfit, just right or overfit with regards to correcting analytical noise in samples. Below is a visualization of the effect of the span parameter on the model fit.

One method to estimate the appropriate span parameter is to use cross-validation with quality control samples. Having identified an appropriate span, a LOESS model can be generated from repeated measures data (black points) and is used to remove the analytical noise from all samples (red points).

Having done this we can now evaluate the effect of removing analytical noise from quality control samples (QCs, training data, black points above) and samples (test data, red points) by calculating the relative standard deviation of the measured variable (standard deviation/mean *100). In the case of the single analyte, ornithine, we can see (above) that the LOESS normalization will reduce the overall analytical noise to a large degree. However we can not expect that the performance for the training data (noise only) will converge with that of the test set, which contains both noise and true biological signal.

In addition to evaluating the normalization specific removal of analytical noise on a univariate level we can also use principal components analysis (PCA) to evaluate this for all variables simultaneously. Below is an example of the PCA scores for non-normalized and LOESS normalized data.

We can clearly see that the two largest modes of variance in the raw data explain differences in when the samples were analyzed, which is termed batch effects. Batch effects can mask true biological variability, and one goal of normalizations is to remove them, which we can see is accomplished in the LOESS normalized data (above right).

However be forewarned, proper model validation is critical to avoiding over-fitting and producing complete nonsense.

In case you are interested the full analysis and presentation can be found below as well as the majority of the R code used for the analysis and visualizations.

## Principal Components Analysis Shiny App

I’ve recently started experimenting with making Shiny apps, and today I wanted to make a basic app for calculating and visualizing principal components analysis (PCA). Here is the basic interface I came up with. Test drive the app for yourself or check out the the R code HERE.

library(shiny) runGist("5846650")

Above is an example of the user interface which consists of data upload (from.csv for now), and options for conducting PCA using the pcaMethods package. The various outputs include visualization of the eigenvalues and cross-validated eigenvalues (q2), which are helpful for selecting the optimal number of model components.The PCA scores plot can be used to evaluate extreme (leverage) or moderate (DmodX) outliers. A Hotelling’s T-squared confidence intervals as an ellipse would also be a good addition for this.

The variable loadings can be used to evaluate the effects of data scaling and other pre-treatments.

The next step is to interface the calculation of PCA to a dynamic plot which can be used to map meta data to plotting characteristics.

## Biochemical, Chemical and Mass Spectral Similarity Network

Here is an example of a network leveraging three dominant aspects of metabolomic experiments (biochemical, chemical and mass spectral knowledge) to connect measured variables. This is a network for a blinded data set (sample ids are not known), which I’ve made for a member of my lab presenting their work at the Metabolomics Society Conference in Glasgow, Scotland.

With out knowing the experimental design we can still analyze our data for analytical effects. For example below is a principal components analysis of ~400 samples and 600 variables, where I’ve annotated the sample scores to show data aquisition date (color) and experimental samples or laboratory quality controls (shape). One thing to look for are trends or scores grouping in the PCA scores which are correlated to analytical conditions like, batch, date, technician, etc.

Finally we can take a look at the PCA variable loadings which highlights a major bottleneck in metabolomics experiments, the large amount of structurally unknown molecular features.

Even using feature rich electron impact mass spectra (GC/TOF) only 40% of the unknown variables could be connected to known species based on a cosine correlation >0.75. To give you an idea the cosine correlation or dot product between the mass spectra of two structurally very similar molecules xylose and xylitol is ~ 0.8.