When you want to get to know and love your data

Posts tagged “imDEV

Tutorials Covering Biological Data Analysis Strategies

I’ve posted two new tutorials focused on intermediate and advanced strategies for biological, and specifically metabolomic data analysis (click titles for pdfs).




PCA to PLS modeling analysis strategy for WIDE DATA

Working with wide data is already hard enough, add to this row outliers and things can get murky fast.

Here is an example of an anlysis of a wide data set, 24 rows  x 84 columns.

Using imDEV, written in R, to calculate and visualize a principal components analysis (PCA) on this data set. We find that 7 components capture >80% of the variance in the data or X. We can  also clearly see that the first dimension, capturing 35% of the variance in X, is skewed towards one outlier,  larger black point in the plots in the lower left triangle of the figure below, representing PCA scores.

pca scatter plot matrix

In this plot representing the results from a PCA:

  • Bottom left triangle  = PCA SCORES,  red and cyan ellipses display 2 groups, outlier is marked by a larger black point
  • Diagonal = PCA Eigen values or variance in X explained by the component
  • Top right triangle = PCA Loadings, representing linear combinations variable weights  to reconstruct Sample scores

In actuality the large variance in the outlier is due to a single value imputation used to back fill missing values in 40% of the columns (variable) for this row (sample).

Removing this sample (and another more moderate outlier) and using partial least squares projection to latent structures discriminant analysis or PLS-DA to generate a projection of  X which maximizes its covariance with Y which here is sample membership among the two groups noted by point  and ellipse color  (red = group 1, cyan = group 2).

PLS scores with outlier removed

The PLS scores separation for the two groups is largely captured in the first latent variable (LV1, x-axis). However we can’t be sure that this separation is better than random chance. To test this we can generate permuted NULL models by using our original X data to discriminate between randomly permuted sample group assignments (Y). When doing the random assignments we can optionally conserve the proportion of cases in each group or maintain this at 1:1.

PLS permuted models

Comparing our models (vertical hashed line) cross-validated fit to the training data , Q^2, to 100 randomly permuted models (TOP PANEL above, cyan distribution), we see that generally our “one” model is better fit than that achieved for the random data. Another interesting parameter is the comparison of our model’s root mean error of prediction, RMSEP, or out of sample error to the  permuted models’ (BOTTOM PANEL above, dark green distribution).

To have more confidence in the assessment of our model we can conduct training and testing validations. We can do this by randomly splitting our original X data into 2/3 training and 1/3 test sets. By using the training data to fit the model, and then using this to predict group memberships for the test data we can get an idea of the model’s out of sample classification error, visualized below using an receiver operator characteristic curve (ROC, RIGHT PANEL).

1 model performanec

Wow this one model looks “perfect”, based on its assessment using one training/testing evaluation (ROC curve above). However it is important to repeat this procedure to evaluate its performance for other random splits of the data into training and test sets.

After permuting the samples training/test assignments 100 times; now we see that our original “one” model (cyan line in ROC curve in the TOP PANEL) was overly optimistic compared to the average performance of 100 models (green lines and distribution above).

100 model performance

Now that we have more confidence in our models performance we can compare the distributions for its performance metrics to those we calculated for the permuted NULL models above. In the case of the “one” model we can use a single sample t-Test or for the “100” model a two-sample t-Test to determine the probability of achieving a similar performance to our model by random chance.

comparison of permuted to robust model metrics

Now by looking at our models RMSEP compared to random chance (BOTTOM LEFT PANEL, out of sample error for our model, green, compared to random chance, yellow) we can be confident that our model is worth exploring further.

For example, we can now investigate the variables’ weights and loadings in the  PLS model to understand key differences in these parameters which are driving the above models discrimination  performance of our two groups of interest. 

Visualization of Multivariate Biological Models (PLS-DA and O-PLS-DA)

Its not uncommon to be faced by multiple questions at the same time. For instance imagine the following experimental design. You have one MAIN question: what is different between groups A  and B, but among groups A and B are subgroups 1 and 2. This complicates things because now the answer to the MAIN question (what is different between A and B) may be slightly different for the two sub groups A|1, A|2 and B|1, B|2.



In statistics we can account for these types of experimental designs by choosing different tests. For instance in the case outlined above we could use a two-way analysis of variance (2-way ANOVA) to identify differences between A|B which are independent of differences between 1|2 (and interaction between A|B and 1|2). In the case of multivariate modeling we can achieve a similar effect by using covariate adjustments. For example we can use the residuals from a simple linear model for  differences between 1|2 as the 1|2-effect adjusted data to be used to test for differences between A|B. Here is a visual example of this approach using:

2) 1|2–adjusted PLS-DA model for A|B
1) PCA to evaluate the data variance between A and B (GREEN and RED) and 1 and 2 (SMALL or LARGE)

3) 1|2–adjusted O-PLS-DA model for A|B

Based on the PCA we see that the differences between A|B are also affected by 1|2. This is evident in distribution of scores based on LARGE|SMALL among A ( A|1 (GREEN|SMALL) is more different (further right) from all B than A|2 (GREEN|LARGE). The same can be said for B, and in particular the greatest differences between all groups is between those which have the greatest separation in the X-axis (1st principal component) which are RED|LARGE and GREEN|SMALL. 

To identify the greatest difference between RED|GREEN which is independent of differences due to SMALL|LARGE, we can use a SMALL|LARGE -adjusted data to create a PLS-DA model to discriminate between RED|GREEN.

This projection of the differences between A|B is the same for SMALL|LARGE groups. Ideally we want the two groups scores to be maximally separated in the X-axis or 1st LV. We see that this is not the case above, and instead the explanation of how the variables  contribute to differences between  GREEN|RED needs to be answered by explaining scores variance in X and Y axes or  two dimensions.

Next we try the O-PLS-DA algorithm, which aims to rotate the projection of the data to maximize the separation between GREEN|RED on the X-axis and capture unrelated or orthogonal variance on the Y-axis.
The O-PLS-DA model loadings for the 1st LV provide information regarding differences in variable magnitudes between the two groups (GREEN|RED).

We can use network mapping to visualize these weights within a domain specific context. In the case of metabolomics data this is best achieved using biochemical/chemical similarity networks.

We can create these networks by assigning edges between vertices (representing metabolites) based on biochemical relationships (KEGG RPAIRs ) or chemical similarities (Tanimoto coefficient >0.7). We can then map the O-PLS-DA model loadings to this network’s visual properties (vertex: size, color, border, and inset graphic).


For example we can map vertex size to the matabolite’s importance in the explained discrimination between groups (loading on O-PLS-DA LV 1) and color the direction of change (blue, decrease; red, increase). Metabolites displaying significant differences between RED and GREEN groups (two-way ANOVA, p < 0.05 adjusting for 1|2) are shown at maximum size, with a black border and contain a box-plot  visualization.

Here is  network mapping the O-PLS-DA model loadings into a biological context and displaying graphs for import parameters means among groups stratified by A|B and 1|2 (left to right: A|1, A|2,B|1,B|2).


Here is  another network with the same edge and vertex properties as above, except the inset graphs show differences between groups A|B adjusted for the effect of 1|2.


Data analysis approaches to modeling changes in primary metabolism

Modeling Short-term Glucose Effects on Primary Metabolism

Comparison of Serum vs Urine metabolites +

Primary metabolites in human serum or urine.

serum urine idOh oh, there seem to be some outliers: serum samples  looking like urine and vice versa. Fix these and evaluate using PCA and hierarchical clustering on rank correlations.

fix assignments

Now things look more believable. Next let us test the effects of data pre-treatment on PLS-DA model scores for a 3 group comparison in serum. Ideally group scores would be maximally resolved in the dimension of the first latent variable (x) and inter-group variance would be orthogonal or in the y-axis.

scaling vs normalization

Compared to raw data (TOP) where ~ 3 top variables (glucose, urea and mannitol) dominate the variance structure, the autoscaled model, due to variable-wise  mean subtraction and division by the standard deviation, displays a more balanced contribution to scores variance by variables. The larger separation between  WHITE  and RED class scores  along the x-axis suggest  improved classifier performance over raw data model and overview of samples with scores outside their respective group’s Hotelling’s T ellipse (95%) might point to  a sample outlier to further investigate or potentially exclude from the current test.

Discriminating Between Iris Species

The Iris data set is a famous for its use to compare unsupervised classifiers.

The goal is to use information about flower characteristics to accurately classify the 3 species of Iris. We can look at scatter plots of the 4 variables in the data set and see that no single variable nor bivariate combination can achieve this.

One approach to improve the separation between the two closely related Iris species, I.versicolor (blue) and I.virginica (green), is to use a combination of all 4 measurements, by constructing principal components (PCs).

Using the singular value decomposition to calculate PCs we see that the sample scores above are not resolved for the two species of interest.

Another approach is to use a supervised projection method like partial least squares (PLS), to identify Latent Variables (LVs) which are data projections similar to those of PCA, but  which are also correlated with the species label. Interestingly this approach leads to a projection which changes the relative orientation of  I. versicolor and I. verginica to I. setaosa. However,  this supervised approach is not enough to identify a hyperplane of separation between all three species.

Non-linear PCA via neural networks can be used to identify the hypersurface of separation, shown above. Looking at the scores we can see that  this  approach is the most success for resolving the  two closely related species. However, the loadings from this method, which help relate how the variables are combined achieve the classification, are impossible to interpret. In the case of the function used above(nlPca, pcaMethods R package)  the loadings are literally NA.