library(knitr) opts_chunk$set(fig.width = 6, fig.height = 4)
Suppose we have collected measurements about bacterial abundances from a number of samples, and those samples fall into one of several groups. We want to know if there is a statistically significant difference between the groups, that is, whether it looks like the microbiome samples from the different groups look like they could all have come from all come from the same distribution.
One good non-parametric family of tests for this problem is based on the Friedman-Rafsky[^1] test. The idea is to compute distances between the samples, create a graph based on those distances, and use the number of edges between samples of the same type (the number of "pure edges") as a test statistic. We can then compute a $p$-value by comparing the observed test statistic to the distribution of the test statistic under the permutation distribution.
[^1]: Friedman, J.H. and Rafsky, L.C. "Multivariate generalizations of the Wald-Wolfowitz and Smirnov two-sample tests." The Annals of Statistics (1979):697-717.
From the description above, we see that we have some choices to
make. We need to define a distance between the samples and choose a
method for creating a graph from those distances. These choices are
responsible for most of the arguments to graph_perm_test
, the
primary function in this package.
The distance
argument in graph_perm_test
allows you to specify a
distance. This can be any distance implemented in phyloseq
, and it
should be taken from the following list:
library(phyloseq) unlist(distanceMethodList)
You can see the help page on distances for more information. The distance should be chosen carefully and should reflect the type of differences between samples you are interested in.
graph_perm_test
allows you to specify one of four options for a type
of graph: a minimum spanning tree, a $k$-nearest neighbors graph, and
two types of thresholded graphs. These are passed to the type
argument.
type = "mst"
creates a
minimum spanning tree. The
minimum spanning tree places edges between the samples so that all
of the samples are connected and the sum of the distances between
samples connected by an edge is minimized.type = "knn"
creates a $k$-nearest neighbors graph. For each
sample, we place an edge between it and its $k$ nearest
neighbors. This of course requires you to specify $k$ with the
argument knn
. A small number, on the order of 1 to 3 is likely a
good choice.type = "threshold.distance"
creates a distance threshold graph,
and requires you to specify max.dist
. The graph will be created by
placing an edge between any pair of points where the distance
between them is less than max.dist
.type = "threshold.nedges"
creates a distance threshold graph, and
requires you to specify nedges
. The graph will be created by
computing distances between every pair of samples, and placing an
edge between the nedges
pairs of samples with the smallest
distances between them.Note that the knn
argument is only used with type = "knn"
, the
max.dist
argument is only used if type = "threshold.distance"
,
and the nedges
argument is only used if type =
"threshold.nedges"
. type = "mst"
requires no additional
arguments.
In some simulations we saw that the minimum spanning tree and k-nearest neighbors had the most power. The minimum spanning tree is the simplest choice since it doesnâ€™t require specifying any further parameters, but if you have reason to believe that other types of graphs would be more appropriate in your application they are also available. The $k$-nearest neighbors graph might be desirable because it gives an interpretable test statistic: the number of nearest neighbors that are of the same type.
Suppose that we have collected the data in the enterotype
dataset,
which is available in the phyloseq package as a phyloseq
object. We
can load the data and look at it with the following commands:
library(ggplot2) # not necessary, but I like the white background with ggplot theme_set(theme_bw()) library(phyloseqGraphTest) data(enterotype) enterotype
Suppose we want to test for differences between sequencing platforms
(the SeqTech
column in the sample data). We have also decided we
want to use the Jaccard dissimilarity and a $k$-nearest neighbors
graph with $k$ = 1 to perform our test. Then we would use the
following commands to run the test and view the output:
gt = graph_perm_test(enterotype, sampletype = "SeqTech", distance = "jaccard", type = "knn", knn = 1) gt
We see that the difference between sequencenig platforms is statistically significant, with a $p$-value of .002. The effect is also quite substantial: we see from the observed test statistic that out of the 221 total edges in the 1-nearest neighbors graph, 197 of them connect samples of the same type.
The output from graph_perm_test
is a psgraphtest
object, which is
a list containing information about the test. The elements of the list
are:
observed
: The observed test statistic, the number of pure edges.
perm
: A vector containing the value of the test statistic (the
number of pure edges) in each of the permuted datasets.
pval
: The p-value for the permutation test. This is the fraction
of times the number of pure edges in the permuted dataset exceeded
the number of pure edges in the observed dataset.
net
: The graph used for testing.
sampletype
: A vector containing the group label for each sample.
type
: The type of graph used.
These can be inspected by hand, but the package also contains some functions for plotting the results.
The function plot_test_network
plots the graph we created on the
samples, the sample identities, and the edge types (pure or mixed,
i.e. edges between samples of the same type or edges between samples
of different types). Here we see that the nearest neighbor graph
connects largely samples of the same type.
plot_test_network(gt)
The function plot_permutations
will plot a histogram of the number
of pure edges in each of the permuted datasets along with the number
of pure edges in the observed dataset. For this dataset, we see that
the number of pure edges in the observed dataset is well outside of
the permutation distribution.
plot_permutations(gt)
There are a couple of other arguments to the graph_perm_test
function. nperm
is the number of permutations to use for the
test. The default is 499, and it can be increased or decreased
depending on how much computational time you have and how closely you
want to approximate the full permutation distribution.
You can also specify a stratifying variable using the grouping
argument. This is necessary in repeated measures designs. Suppose for
instance that we have mice in two different litters, and we would like
to test for equality of the distributions from the two litters. If we
have more than one sample taken from each of the mice, permuting the
litter label over all the samples independently will not give a valid
test because of the dependence between samples taken from the same
mouse. We can fix this by considering the mice the independent units
and permuting the litter label over mouse instead of over sample to
obtain a valid test.
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