In unsupervised learning (UML), no labels are provided, and the learning algorithm focuses solely on detecting structure in unlabelled input data. One generally differentiates between
There is however one important caveat with clustering: clustering algorithms will always find clusters, even when none exists. It is thus essential to critically assess and validate (for example using existing domain knowledge) the results of any clustering algorithm.
UML presents specific challenges and benefits:
This chapter has as objectives to
In this chapter, we are going to learn about dimensionality reduction, also called ordination. The goal of dimensionality reduction is to transform a high-dimensional data into data of lesser dimensions while minimising the loss of information.
Dimensionality reduction is used as a data transformation technique for input to other machine learning methods such as classifications, or as a very efficient visualisation technique, which is the use case we will focus on here. We are going to focus on a widerly used method called Principal Component Analysis (PCA).
We are going to use the following dataset to illustrate some important concepts related to PCA. The small dataset show below represents the measurement of genes x and y in 20 samples. We will be using the scaled and centred version of this dataset.
The goal of dimensionality reduction is to reduce the number of dimensions in a way that the new data remains useful. One way to reduce a 2-dimensional data is by projecting the data onto a lines. Below, we project our data on the x and y axes. These are called linear projections.
In general, and in particular in the projections above, we lose information when reducing the number of dimensions (above, from 2 (plane) to 1 (line)). In the first example above (left), we lose all the measurements of gene y. In the second example (right), we lose all the measurements of gene x.
The goal of dimensionality reduction is to limit this loss.
We know already about linear regression. Below, we use the lm
function to regress y onto x (left) and x onto y
(right). These regression lines give us an approximate linear
relationship between the expression of genes x and y. The
relationship differs depending on which gene we choose to be the
predictor and the response.
We now want a line that minimises distances in both directions, as shown below. This line, called principal component, is also the ones that maximises the variance of the projections along itself.
The second principal component is then chosen to be orthogonal to the first one. In our case above, there is only one possibility.
In the example above the variance, the variance along the PCs are 1.77 and 0.23 respectively. The first one explains 88.6% or that variance, and the second one merely 11.4%. This is also reflected in the different scales along the x and y axis.
To account for these differences in variation along the different PCs, it is better to represent a PCA plot as a rectangle, using an aspect ratio that is illustrative of the respective variances.
Principal components are linear combinations of the variables that were originally measured, they provide a new coordinate system. The PC in the previous example is a linear combination of gene_x and gene_y:
\[ PC = c_{1} ~ gene_{x} + c_{2} ~ gene_{y} \]
It has coefficients \((c_1, c_2)\), also called loading.
PCA will find linear combinations of the original variables. These new linear combinations will maximise the variance of the data.
Principal Component Analysis (PCA) is a technique that transforms the original n-dimensional data into a new space.
These new dimensions are linear combinations of the original data, i.e. they are composed of proportions of the original variables.
Along these new dimensions, called principal components, the data expresses most of its variability along the first PC, then second, …
Principal components are orthogonal to each other, i.e. non-correlated.
In R, we can use the prcomp
function. A summary of the prcomp
output shows that along PC1, we are able to retain close to 92%
of the total variability in the data.
## Importance of components:
## PC1 PC2
## Standard deviation 1.3308 0.4784
## Proportion of Variance 0.8856 0.1144
## Cumulative Proportion 0.8856 1.0000
This pca_xy
variable is an object of class prcomp
. To learn what
it contains, we can look at its structure with str
and read the
?prcomp
manual page.
## List of 5
## $ sdev : num [1:2] 1.331 0.478
## $ rotation: num [1:2, 1:2] 0.707 0.707 -0.707 0.707
## ..- attr(*, "dimnames")=List of 2
## .. ..$ : chr [1:2] "gene_x" "gene_y"
## .. ..$ : chr [1:2] "PC1" "PC2"
## $ center : Named num [1:2] 4.05e-16 1.53e-17
## ..- attr(*, "names")= chr [1:2] "gene_x" "gene_y"
## $ scale : logi FALSE
## $ x : num [1:20, 1:2] -2.728 -2.4 -2.257 -0.945 -0.14 ...
## ..- attr(*, "dimnames")=List of 2
## .. ..$ : NULL
## .. ..$ : chr [1:2] "PC1" "PC2"
## - attr(*, "class")= chr "prcomp"
We are going to focus on two elements:
## [1] 1.7711407 0.2288593
## [1] 0.8855704 0.1144296
## [1] 0.8855704 1.0000000
## PC1 PC2
## [1,] -2.72848772 0.17019003
## [2,] -2.40018291 0.11371158
## [3,] -2.25654153 -0.17987834
## [4,] -0.94491834 0.14224604
## [5,] -0.14044618 0.86760695
## [6,] -0.85917747 0.06485259
## [7,] -1.26714805 -0.38570171
## [8,] -0.13426336 0.01962124
## [9,] 0.14678704 0.14527416
## [10,] 1.08677349 0.67252884
## [11,] 0.93793985 0.43069778
## [12,] 0.62416628 0.08630825
## [13,] 0.05477564 -0.56143151
## [14,] 1.20838962 0.46257985
## [15,] 0.58230063 -0.44200381
## [16,] 1.48091889 0.42011071
## [17,] 1.13374563 -0.12764440
## [18,] 1.28218805 -0.32631519
## [19,] 1.46938699 -0.32768480
## [20,] 0.72379344 -1.24506826
A biplot features all original points re-mapped (rotated) along the first two PCs as well as the original features as vectors along the same PCs. Feature vectors that are in the same direction in PC space are also correlated in the original data space.
One important piece of information when using PCA is the proportion of variance explained along the PCs (see above), in particular when dealing with high dimensional data, as PC1 and PC2 (that are generally used for visualisation), might only account for an insufficient proportion of variance to be relevant on their own. This can be visualised on a screeplot, that can be produced with
► Question
Repeat the analysis above with the xy0
data, that you can load
from the rWSBIM1322
data. First scale it, then repeat the PCA
analysis as shown above.
► Solution
► Question
Load the cptac_se_prot
data, scale the data (see below for
why scaling is important), then perform the PCA analysis and interpret
it. Also produce a PCA for PCs 2 and 3.
► Solution
► Question
In the exercise above, the PCA was performed on the features (proteins). Transpose the data and produce a PCA of the samples. Interprete the figures.
► Solution
We haven’t looked at other prcomp
parameters, other that the first
one, x
. There are two other ones that are or importance, in
particular in the light of the section on pre-processing above, which
are center
and scale.
. The former is set to TRUE
by default,
while the second one is set the FALSE
.
► Question
Perform a PCA analysis on the mtcars
dataset with and without
scaling. Compare and interpret the results.
► Solution
Real datasets often come with missing values. In R, these should be
encoded using NA
. Unfortunately, prcomp()
cannot deal with missing
values, and observations containing NA
values will be dropped
automatically. This is a viable solution only when the proportion of
missing values is low. Otherwise, it is possible to impute missing
values (which often requires great care) or use an implementation of
PCA such as non-linear iterative partial least squares (NIPALS),
that support missing values.
Finally, we should be careful when using categorical data in any of
the unsupervised methods. Categories are generally represented as
factors, which are encoded as integer levels, and might give the
impression that a distance between levels is a relevant measure (which
it is not, unless the factors are ordered). In such situations,
categorical data can be dropped, or it is possible to encode
categories as binary dummy variables. For example, if we have 3
categories, say A
, B
and C
, we would create two dummy variables
to encode the categories as:
## x y
## A 1 0
## B 0 1
## C 0 0
so that the distance between each category are approximately equal to 1.
In this section, we will describe the detailed PCA analysis and
interpretation of two real-life proteomics datasets. Both of these
datasets are available in the pRolocdata
package.
We are first going to focus on the data from Mulvey et al. (2015), where they present the Dynamic proteomic profiling of extra-embryonic endoderm differentiation in mouse embryonic stem cells (Mulvey et al. 2015Mulvey, C M, C Schröter, L Gatto, D Dikicioglu, I Baris Fidaner, A Christoforou, M J Deery, et al. 2015. “Dynamic Proteomic Profiling of Extra-Embryonic Endoderm Differentiation in Mouse Embryonic Stem Cells.” Stem Cells, June. https://doi.org/10.1002/stem.2067.).
During mammalian preimplantation development, the cells of the blastocyst’s inner cell mass differentiate into the epiblast and primitive endoderm lineages, which give rise to the fetus and extra-embryonic tissues, respectively. Extra-embryonic endoderm (XEN) differentiation can be modeled in vitro by induced expression of GATA transcription factors in mouse embryonic stem cells. Here, we use this GATA-inducible system to quantitatively monitor the dynamics of global proteomic changes during the early stages of this differentiation event and also investigate the fully differentiated phenotype, as represented by embryo-derived XEN cells. Using mass spectrometry-based quantitative proteomic profiling with multivariate data analysis tools, we reproducibly quantified 2,336 proteins across three biological replicates and have identified clusters of proteins characterized by distinct, dynamic temporal abundance profiles. We first used this approach to highlight novel marker candidates of the pluripotent state and XEN differentiation. Through functional annotation enrichment analysis, we have shown that the downregulation of chromatin-modifying enzymes, the reorganization of membrane trafficking machinery, and the breakdown of cell-cell adhesion are successive steps of the extra-embryonic differentiation process. Thus, applying a range of sophisticated clustering approaches to a time-resolved proteomic dataset has allowed the elucidation of complex biological processes which characterize stem cell differentiation and could establish a general paradigm for the investigation of these processes.
► Question
Load the mulvey2015_se
data from the pRolocdata
package. The data
comes as an object of class SummarizedExperiment
. Familiarise
yourself with its experimental design stored in the colData slot.
► Solution
Here, we will want to do a PCA analysis on the samples. We want to remap the
18 samples from a 2337-dimensional space into 2, or
possibly 3 dimensions. This will require to transpose the data matrix
before passing it to the prcomp
function.
► Question
mulvey2015_se
data and
display a summary of the results.
► Solution
► Question
Assuming we are happy with a reduced space explaining 90% of the variance of the data, how many PCs do we need?
Visualise the variance captured by the 18 PCs. To do so, you can use
the fviz_screeplot
function from the factoextra
package.
► Solution
► Question
Visualise your PCA analysis on a biplot. You can use the fviz_pca
function from the factoextra
package, or the fviz_pca_ind
function to focus on the individuals (the rows of the original
input).
Use the habillage
or col.ind
arguments10 The habillage
only uses (or converts to) factors, and is
this appropriate for categories. If the variable to be
highlighted on the PCA plot is continuous, then col.ind
should
be used. See ?fviz_pca_ind
for details. of the
fviz_pca_ind()
function to highlight the time experimental
variable. Interpret the figures in the light of the experimental
design.
► Solution
► Question
To visualise the data in 3 dimension, you can use the plot3d
function from the rgl
package, that provides means to rotate the
cube along the three first PCs.
► Solution
In this section, we are going to use PCA to explore spatial map of mouse pluripotent stem cell by Christoforou et al. (Christoforou et al. 2016Christoforou, A, C M Mulvey, L M Breckels, A Geladaki, T Hurrell, P C Hayward, T Naake, et al. 2016. “A Draft Map of the Mouse Pluripotent Stem Cell Spatial Proteome.” Nat Commun 7: 8992. https://doi.org/10.1038/ncomms9992.).
Knowledge of the subcellular distribution of proteins is vital for understanding cellular mechanisms. Capturing the subcellular proteome in a single experiment has proven challenging, with studies focusing on specific compartments or assigning proteins to subcellular niches with low resolution and/or accuracy. Here we introduce hyperLOPIT, a method that couples extensive fractionation, quantitative high-resolution accurate mass spectrometry with multivariate data analysis. We apply hyperLOPIT to a pluripotent stem cell population whose subcellular proteome has not been extensively studied. We provide localization data on over 5,000 proteins with unprecedented spatial resolution to reveal the organization of organelles, sub-organellar compartments, protein complexes, functional networks and steady-state dynamics of proteins and unexpected subcellular locations. The method paves the way for characterizing the impact of post-transcriptional and post-translational modification on protein location and studies involving proteome-level locational changes on cellular perturbation. An interactive open-source resource is presented that enables exploration of these data.
► Question
Load the hyperLOPIT2015_se
data from the pRolocdata
package. Familiarise yourself with its experimental design and its
feature annotation stored in the rowData slot.
► Solution
Here, we will want to do a PCA analysis on the features. We want to
remap the 5032 proteins from a
20-dimensional space into 2, or
possibly 3 dimensions. Here, we can use the data as it when
passing it to the prcomp
function.
► Question
hyperLOPIT2015_se
data and
display a summary of the results.
► Solution
► Question
Assuming we are happy with a reduced space explaining 75% of the variance of the data, how many PCs do we need?
Visualise the variance captured by the 18 PCs. To do so, you can use
the fviz_screeplot
function from the factoextra
package.
► Solution
► Question
Visualise your PCA analysis on a biplot. You can use the fviz_pca
function from the factoextra
package, or the fviz_pca_var
function to focus on the variables (the columns of the original
input).
Interpret the variables on the biplot. Which ones are correlated? Is this to be expected in the light of the experimental design.
Use the fviz_pca_ind
function to focus on the individuals (the
rows of the orgininal input). Use the habillage
argument of the
fviz_pca_ind
function to highlight sub-cellular locations of the
proteins (use the "markers"
feature variable) and set the geom
argument to "point"
to drop the feature names from the plot (which
correspond to geom = "text"
). Interpret the figures in the light of
the spatial proteomics experiment.
Repeat the figure above setting the "final.assignment"
variable to
annotate the plot. Compare and interpret the two plots.
► Solution
A short video (in French, with subtitles in multiple languages) summarises the solution for this spatial proteomics PCA application.
► Question
Professor Simpson and her team wanted to test the effect of drug A and drug B on fibroblast. They measured gene expression using RNA-Seq in three conditions: a control without any drug, cells in presence of drug A, and cells in presence of drug B. Each of these were measured in triplicates in a single batch by the same operator.
The figure below shows the PCA plot of the 9 samples, coloured by condition. Interpreted the figure in the light of the experimentally design and suggest how to best analyse these data.
► Question
Consider the same experimental design as above, but with the PCA plot below. Interpret the figure in the light of the experimental design and suggest how to best analyse these data.
► Question
Researchers of the famous FLVIT (Fosse-La-Ville Institute of Technology) wanted to test the effect of a newly identified anti-cancer drug C121 on KEK293 cells. They measured proteins expression using quantitative mass spectrometry in these two conditions and 4 replicates. Each of these were measured in triplicates in a single batch.
Interpret the PCA plot in the light of the experimental design. Do you think the analysis is likely to provide significant results?
► Question
In the following experimental design, clinicians have used quantitative PCR to measure the expression of 96 micro RNAs in the blood of three patients and three healthy donor, with the goal of identifying novel biomarkers.
In the light of the experimental design, interpret the PCA plot. What are the chances to find good biomarkers?
► Question
The team of Professor Von der Brille have quantified the gene expression of Jurkat cells using two RNA-Seq protocols. 25 samples were tested with kit 1 by operator A and B (12 and 13 samples respectively), and 27 samples were processed with kit 2 by operators A and C (20 and 7 samples respectively).
In the light of the experimental design, interpret the PCA plot. What would you suggest to do before testing whether using kit 1 and 2 have an impact on the data?
► Question
Load the tdata1
dataset from the rWSBIM1322
package and run a PCA
analysis on the samples. Interpret the results, knowing that these
data are random.
► Question
Using the function kem2.tsv
from the rWSBIM1207
package, import
the RNA-seq data and perform a PCA analysis on the samples.
► Question
Load the metab1
data from the rWSBIM1322
package (version >=
0.1.10
) and perform a PCA analysis on the samples. These data
represent mass spectrometry-based metabolomics data. Do the samples
seem to be randomly placed? Do you notice any structure explained by
the labels?
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