This vignette provides an overview of the Bioconductor package ASSIGN (Adaptive Signature Selection and InteGratioN) for signature-based profiling of heterogeneous biological pathways. ASSIGN is a computational tool used to evaluate the pathway deregulation/activation status in individual patient samples. ASSIGN employs a flexible Bayesian factor analysis approach that adapts predetermined pathway signatures derived either from a literature search or from perturbation experiments to create cell-/tissue-specific pathway signatures. The deregulation/activation level of each context-specific pathway is quantified to a score, which represents the extent to which a patient sample matches thepathway deregulation/activation signature.
Some distinctive features of ASSIGN are:
In the following examples, we will illustrate how to run ASSIGN
using either the easy to use
assign.wrapper function for simple
analysis or each individual ASSIGN step for more detailed
For either analysis, we will first load ASSIGN and create a
'tempdir' under the user’s current working directory. All
output generated in this vignette will be saved in
library(ASSIGN) dir.create("tempdir") tempdir <- "tempdir"
Next, load the training data, test data, training labels, and test labels. The
training dataset is a G (number of genomic measurements) x N (number of samples
in pathway perturbation experiments) matrix, including five oncogenic pathways:
B-Catenin, E2F3, MYC, RAS, and SRC pathways in this example. The training data
labels denote the column indices of control and experimental samples for each
perturbation experiment. For example, we specify the column indices of the 10
RAS control samples to be 1:10, and column indices of 10 RAS activated samples
to be 39:48. The test dataset is a G (number of genomic measurements) x N
(number of patient samples) matrix. The test data labels denote the classes of
the N test samples. In our example, test samples 1-53 are adenocarcinoma and
samples 54-111 are squamous cell carcinoma. We specify
'Squamous' in the vector of test data labels. Note that the test data labels
are optional. ASSIGN outputs additional validation plots to
evaluate classification accuracy when test data labels are provided.
data(trainingData1) data(testData1) data(geneList1) trainingLabel1 <- list(control = list(bcat=1:10, e2f3=1:10, myc=1:10, ras=1:10, src=1:10), bcat = 11:19, e2f3 = 20:28, myc= 29:38, ras = 39:48, src = 49:55) testLabel1 <- rep(c("Adeno", "Squamous"), c(53,58))
We developed an all-in-one
assign.wrapper function to run
ASSIGN with one command. For most users,
assign.wrapper will be
assign.wrapper function outputs the following files:
If training data is provided,
assign.wrapper also outputs the
Adaptive_S is TRUE,
assign.wrapper also outputs the following files:
Finally, if the
testLabel argument is not NULL,
assign.wrapper also outputs the following files:
Here we illustrate how to run
assign.wrapper function with three
examples. To start, create a temporary directory
'tempdir' and load training
and test datasets. The individual parameters are described in detail in the
sections below and the ASSIGN reference manual.
dir.create(file.path(tempdir,"wrapper_example1")) assign.wrapper(trainingData=trainingData1, testData=testData1, trainingLabel=trainingLabel1, testLabel=testLabel1, geneList=NULL, n_sigGene=rep(200,5), adaptive_B=TRUE, adaptive_S=FALSE, mixture_beta=TRUE, outputDir=file.path(tempdir,"wrapper_example1"), iter=2000, burn_in=1000)
dir.create(file.path(tempdir,"wrapper_example2")) assign.wrapper(trainingData=trainingData1, testData=testData1, trainingLabel=trainingLabel1, testLabel=NULL, geneList=geneList1, n_sigGene=NULL, adaptive_B=TRUE, adaptive_S=FALSE, mixture_beta=TRUE, outputDir=file.path(tempdir,"wrapper_example2"), iter=2000, burn_in=1000)
dir.create(file.path(tempdir,"wrapper_example3")) assign.wrapper(trainingData=NULL, testData=testData1, trainingLabel=NULL, testLabel=NULL, geneList=geneList1, n_sigGene=NULL, adaptive_B=TRUE, adaptive_S=TRUE, mixture_beta=TRUE, outputDir=file.path(tempdir,"wrapper_example3"), iter=2000, burn_in=1000)
We developed a series of functions:
that work in concert to produce detailed results.
We first run the
assign.preprocess function on the input datasets.
When the genomic measurements (e.g., gene expression profiles) of training
samples are provided, but predetermined pathway signature gene lists are not
assign.preprocess function utilizes a Bayesian
univariate regression module to select a gene set (usually 50-200 genes, but
this can be specified by the user) based on the absolute value of the regression
coefficient (fold change) and the posterior probability of the variable to be
selected (statistical significance). Since we have no predetermined gene lists
to provide, we leave the
geneList option as default NULL. Here we
specify 200 signature genes for each of the five pathways.
# training dataset is available; # the gene list of pathway signature is NOT available processed.data <- assign.preprocess(trainingData=trainingData1, testData=testData1, trainingLabel=trainingLabel1, geneList=NULL, n_sigGene=rep(200,5))
Alternatively, the users can have both the training data and the
curated/predetermined pathway signatures. Some genes in the curated pathway
signatures, although not significantly differentially expressed, need to be
included for the purpose of prediction. In this case, we specify the
geneList parameters when both the training
dataset and predetermined signature gene list are available.
# training dataset is available; # the gene list of pathway signature is available processed.data <- assign.preprocess(trainingData=trainingData1, testData=testData1, trainingLabel=trainingLabel1, geneList=geneList1)
In some cases, the expression profiles (training dataset) is unavailable. Only
the knowledge-based gene list or gene list from the joint knowledge of some
prior profiling experiments is available. In this case, we specify
geneList and leave the
trainingLabel as default NULL.
# training dataset is NOT available; # the gene list of pathway signature is available processed.data <- assign.preprocess(trainingData=NULL, testData=testData1, trainingLabel=NULL, geneList=geneList1)
assign.preprocess function returns the processed training
trainingData_sub) and test dataset (
testData_sub) as well as the
prior parameters for the background vector (
B_vector), signature matrix
S_matrix) and the probability signature matrix (
differentially expressed gene lists of each pathway (
details of the
assign.preprocess output are described in the
section of the manual page of
assign.preprocess function. The output data of
assign.preprocess function are used as the input data of the
assign.mcmc function, Y, Bg, and X are specified as the
output of the
assign.preprocess function. The
Adaptive_S (adaptive signature) and
mixture_beta (regularization of signature strength) can be specified
TRUE or FALSE based on the analysis context. When training and test samples are
from the different cell or tissue types, we recommend the adaptive background
option to be TRUE. Notice that when the training dataset is not available, the
adaptive signature option must be set TRUE, meaning that the magnitude of the
signature should be estimated from the test dataset. The default
(iteration) is 2000. Particularly, when training datasets are unavailable, it is
better to specify the
X option in the
assign.mcmc using a
more informative X (specify up- or down- regulated genes) to initiate the model.
mcmc.chain <- assign.mcmc(Y=processed.data$testData_sub, Bg = processed.data$B_vector, X=processed.data$S_matrix, Delta_prior_p = processed.data$Pi_matrix, iter = 2000, adaptive_B=TRUE, adaptive_S=FALSE, mixture_beta=TRUE)
assign.mcmc function returns the MCMC chain recording default
2000 iterations for each parameter. The details of
output are described in the
'value' section of the manual page of
We can make a trace plot to check the convergence of the model parameters
burn_in default is 0, so
that the trace plot starts from the first iteration. Additional iterations
can be specified if the MCMC chain does not converge in 2000 iterations.
trace.plot <- assign.convergence(test=mcmc.chain, burn_in=0, iter=2000, parameter="B", whichGene=1, whichSample=NA, whichPath=NA)
assign.convergence function returns a vector of the estimated values from
each Gibbs sampling iteration of the model parameter to be checked and a trace
plot of the parameter.
We then apply the
assign.summary function to compute the posterior
mean of each parameter. Typically we use the second half of the MCMC chain to
compute the posterior mean. We specify the default burn-in period to be the
first 1000 iteration and the default total iteration to be 2000. The 1000
burn-in iterations are discarded when we compute the posterior mean. The
have to set the same as those in the
mcmc.pos.mean <- assign.summary(test=mcmc.chain, burn_in=1000, iter=2000, adaptive_B=TRUE, adaptive_S=FALSE, mixture_beta=TRUE)
assign.summary function returns the posterior mean of each
parameter. The details of the
assign.summary output are described in
'value' section of the manual page of
assign.cv.output generates the cross-validation results in the
training samples. Output files from
# For cross-validation, Y in the assign.mcmc function # should be specified as processed.data$trainingData_sub. assign.cv.output(processed.data=processed.data, mcmc.pos.mean.trainingData=mcmc.pos.mean, trainingData=trainingData1, trainingLabel=trainingLabel1, adaptive_B=FALSE, adaptive_S=FALSE, mixture_beta=TRUE, outputDir=tempdir)
assign.output generates the prediction results in the test
samples. Output files from
Adaptive_Sis specified TRUE.
testLabelargument is not NULL.
The user needs to specify the output directory in the
outputDir option, when
assign.output(processed.data=processed.data, mcmc.pos.mean.testData=mcmc.pos.mean, trainingData=trainingData1, testData=testData1, trainingLabel=trainingLabel1, testLabel=testLabel1, geneList=NULL, adaptive_B=TRUE, adaptive_S=FALSE, mixture_beta=TRUE, outputDir=tempdir)
The ASSIGN package allows a signature to be adapted to fit other biological contexts, reducing the contribution of specific genes in the signature to better match heterogeneity observed in the test dataset. Occasionally, adapting a signature may reduce the importance of key signature genes. For example, if a signature is created by overexpressing an oncogenic gene in a cell line, but during the adaptation step, ASSIGN reduces the importance of that key gene, the quality of the ASSIGN predictions may be reduced. Alternatively, if a gene in the signature is associated with some other heterogeneity in the data, such as smoking status, ASSIGN may adapt to differences in that gene, rather than the actual desired signature activity predictions. To this end, we have added the ability to provide a list of key genes to anchor in the signature, and genes to exclude from the signature. ASSIGN accomplishes this by setting the probability of inclusion into the signature to one for anchor genes, and zero for exclude genes. The change in expression values can still adapt, increasing or reducing the fold change associated with each gene in the signature, but the anchor genes will always contribute to the final signature, and the exclude genes will not.
dir.create(file.path(tempdir, "anchor_exclude_example")) anchorList = list(bcat="224321_at", e2f3="202589_at", myc="221891_x_at", ras="201820_at", src="224567_x_at") excludeList = list(bcat="1555340_x_at", e2f3="1555340_x_at", myc="1555340_x_at", ras="204748_at", src="1555339_at") assign.wrapper(trainingData=trainingData1, testData=testData1, trainingLabel=trainingLabel1, testLabel=NULL, geneList=geneList1, n_sigGene=NULL, adaptive_B=TRUE, adaptive_S=TRUE, mixture_beta=TRUE, outputDir=file.path(tempdir, "anchor_exclude_example"), anchorGenes=anchorList, excludeGenes=excludeList, iter=2000, burn_in=1000)
By default, ASSIGN Bayesian gene selection chooses the signature genes with an equal fraction of genes that increase with pathway activity and genes that decrease with pathway activity. Use the pctUp parameter to modify this fraction. Set pctUP to NULL to select the most significant genes, regardless of direction.
When running ASSIGN, the number of genes in the gene list can affect the predictions that ASSIGN produces, but it is not always clear how long the gene list should be. Included within ASSIGN is the optimization procedure used in the publication Activity of distinct growth factor receptor network components in breast tumors uncovers two biologically relevant subtypes. The function allows you to optimize the gene list lengths for the pathways included in the paper using your own correlation data and gene list lengths. This function runs ASSIGN pathway prediction on various gene list lengths to find the optimum gene list length for the GFRN pathways by correlating the ASSIGN predictions to a matrix of correlation data that you provide. This function takes a long time to run because you are running ASSIGN many times on many pathways, so I recommend parallelizing by pathway or running the ASSIGN predictions first (long and parallelizable) and then running the correlation step (quick) separately.
The following example optimizes the pathway length for the AKT pathway based on correlating ASSIGN predictions with proteomics data. First, read in the test data that you want to predict using ASSIGN and the data (e.g. proteomics data) that will be used for correlation:
dir.create(file.path(tempdir, "optimization_example")) setwd(file.path(tempdir, "optimization_example")) testData <- read.table("https://drive.google.com/uc?authuser=0&id=1mJICN4z_aCeh4JuPzNfm8GR_lkJOhWFr&export=download", sep='\t', row.names=1, header=1) corData1 <- read.table("https://drive.google.com/uc?authuser=0&id=1MDWVP2jBsAAcMNcNFKE74vYl-orpo7WH&export=download", sep='\t', row.names=1, header=1)
Next, create a list of data used for correlation. The list should contain a vector of column names from the correlation data for each of the pathways that are being optimized. The gene list length that has the largest average correlation for the columns in the correlation list will be the optimized gene list.
#this is a list of pathways and columns in the correlation data that will #be used for correlation corList <- list(akt=c("Akt","PDK1","PDK1p241"))
Finally, run the ComBat batch correction procedure and run the
#run the batch correction procedure between the test and training data combat.data <- ComBat.step2(testData, pcaPlots = TRUE) #run the default optimization procedure optimization_results <- optimizeGFRN(combat.data, corData, corList, run="akt")
ASSIGN will output the results for each gene list length in the
current working directory. The
optimizeGFRN function returns a list of
optimized gene lists which can be used on other datasets and correlation
results. Additional options and documentation is available in the
optimizeGFRN function documentation.
If you use ASSIGN in your publication, please cite:
Shen, Y. et al. ASSIGN: context-specific genomic profiling of multiple heterogeneous biological pathways. Bioinformatics 31 (11), 1745-1753 (2015). doi:10.1093/bioinformatics/btv031
Please see the ASSIGN reference manual for full descriptions of functions and the various options they support.
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