Creating eset object from preprocessed expression matrix? - r

I am analysing with R some gene expression data. I would like to do differential gene expression analysis with limma's eBayes (limma is part of BioConductor), but to do that I need to have my expression data as an eset object. Thing is, I have only preprocessed data and do not have the CEL files, I could convert directly to eset object. I tried searching from Internet, but couldn't find a solution. Only thing I found, was that it IS possible.
Why eBayes:
It should have robust results even with only two or three samples in some of the groups and I do indeed have 3 groups that are from 2 to 3 samples in size.
In detail what I have and want to do:
I have expression data, already as logarithmic, normalized intesity values. The data is in expression matrix. There is about 20 000 rows and each row is a gene and the rownames are the official gene names. There is 22 columns and each column corresponds to one cancer sample. I have different kinds of cancer subtypes there and would like to compare for example subtype 1 samples' gene expression to that of the group 2's. Below is a two row, 5 column example of what my matrix would look like.
Example matrix:
SAMP1 SAMP2 SAMP3 SAMP4 SAMP5
GENE1 123.764 122.476 23.4764 2.24343 123.3124
GENE2 224.233 455.111 124.122 112.155 800.4516
The problem:
To evaluate the differential gene expression with eBayes I would need the eset object out of this expression data and I have honestly no idea how to go about that step. :(
I am very grateful for every bit of info that can help me out! If someone can suggest another reliable method for small sample size comparisons, that might solve my problem as well.
Thank you!

Using an ExpressionSet seems to be quite similar to a SummarizedExperiment which is also prevalent in Bioconductor packages. From what I understand, there is nothing special about using one or the other in a package--in my experience, it's considered as a generalized container for data in order to standardize the data set format across Bioconductor packages.
From the vignette on Bioconductor:
Affymetrix data will usually be normalized using the
affy
package. We will assume here that the
data is available as an
ExpressionSet
object called
eset. Such an object will have an slot containing
the log-expression values for each gene on each array which can be extracted using
exprs(eset).
In other words, there's nothing special about the data for the ExpressionSet. An ExpressionSet is simply a bunch of related experimental data strung together into one, but it appears that I can create a new object just from the regular object:
library(limma)
# counts is the assay data I already have.
dim(counts)
# [1] 64102 8
# Creates a new ExpressionSet object (quite bare, only the assay data)
asdf <- ExpressionSet(assayData = counts)
# Returns the data you put in.
exprs(asdf)
This works on my setup.
The second part that you need to consider is the design of the differential expression analysis comparison model matrix. You will need predefined factors to go along with your samples (probably within a phenoData argument to ExpressionSet and then create a model.matrix using R's special formula syntax. They look similar to: dependent ~ factor1 + factor2 + co:related. Note that a factor1 is a factor category or dimension, not just one level.
Once you have that, you should be able to run lmFit. I've actually not used limma much before but it appears to be similar to edgeR's scheme.

Just decided to make it answer to help some other poor sod, who has the same accident. Figured the problem out myself after going through the links kindly given in comments.
ExpressionSet() does take matrices and turn them to eSet object fine. Just had to make sure the data was as matrix instead of data frame object.

Related

Output for large correlation matrices in R

From what I've seen, R cannot very easily produce usable output for large correlation matrices (50-100 variables). For instance, "corr.test" or "cor" output is horrendously wrapped (each variable should have only one row and one column, but this is certainly not the case) and does not copy well into Excel for later examination. Is there a way to produce SPSS-like correlation output in R? Namely, correlation matrices that can be copied and pasted easily into something like Excel, where each row and each column pertains to one variable (no wrapping of text), and ideally, sample-sizes and significance values are somehow available. Corr.test provides this information, albeit in an inconvenient format, and when variables exceed output viewer space in R, the output is basically unreadable. Any thoughts would be greatly, greatly appreciated, as I'm frequently working with many variables at once.
Is there anything wrong with
z <- matrix(rnorm(10000),100)
write.csv(cor(z),file="cortmp.csv")
? View(cor(z)) works for me, although I don't know if it's copy-and-pasteable.
For psych::corr.test
dimnames(z) <- list(1:100,1:100)
z[1,2] <- NA ## unbalance to induce sample size matrix
ct <- psych::corr.test(z)
write.csv(ct$n,file="ntmp.csv") ## sample sizes
write.csv(ct$t,file="ttmp.csv") ## t statistics
write.csv(ct$p,file="ptmp.csv") ## p-values
et cetera. (See str(ct).)
R's paradigm is that if you want to transfer information to another program you're going to output it to a file rather than copying and pasting it from the console ...

interpreting R code function

I would like to perform pathway enrichment analyses.
I have 21 list of significant genes, and mutiple types of pathways I would like to check (ie. check for enrichment in KEGG pathways, GOterms, complexes etc.).
I found this example of code, on an old BioC post. However, I am having trouble adapting it for myself.
Firstly,
1- what does this mean? I don't know this multiple colon syntax.
hyperg <- Category:::.doHyperGInternal
2 - I don't understand how this line works. hyperg.test is a function that needs 3 variables passed to it, correct? Is this line somehow passing "genes.by.pathways, significant.genes, and all.geneIDs to thr hyperg.test?
pVals.by.pathway<-t(sapply(genes.by.pathway, hyperg.test, significant.genes, all.geneIDs))
Code that I would like to adapt
library(KEGGREST)
library(org.Hs.eg.db)
# created named list, length 449, eg:
# path:hsa00010: "Glycolysis / Gluconeogenesis"
pathways <- keggList("pathway", "hsa")
# make them into KEGG-style human pathway identifiers
human.pathways <- sub("path:", "", names(pathways))
# for demonstration, just use the first ten pathways
demo.pathway.ids <- head(human.pathways, 10)
demo.pathways <- setNames(keggGet(demo.pathway.ids), demo.pathway.ids)
genes.by.pathway <- lapply(demo.pathways, function(demo.pathway) {
demo.pathway$GENE[c(TRUE, FALSE)]
})
all.geneIDs <- keys(org.Hs.eg.db)
# chose one of these for demonstration. the first (a whole genome random
# set of 100 genes) has very little enrichment, the second, a random set
# from the pathways themselves, has very good enrichment in some pathways
set.seed(123)
significant.genes <- sample(all.geneIDs, size=100)
#significant.genes <- sample(unique(unlist(genes.by.pathway)), size=10)
# the hypergeometric distribution is traditionally explained in terms of
# drawing a sample of balls from an urn containing black and white balls.
# to keep the arguments straight (in my mind at least), I use these terms
# here also
hyperg <- Category:::.doHyperGInternal
hyperg.test <-
function(pathway.genes, significant.genes, all.genes, over=TRUE)
{
white.balls.drawn <- length(intersect(significant.genes, pathway.genes))
white.balls.in.urn <- length(pathway.genes)
total.balls.in.urn <- length(all.genes)
black.balls.in.urn <- total.balls.in.urn - white.balls.in.urn
balls.pulled.from.urn <- length(significant.genes)
hyperg(white.balls.in.urn, black.balls.in.urn,
balls.pulled.from.urn, white.balls.drawn, over)
}
pVals.by.pathway <-
t(sapply(genes.by.pathway, hyperg.test, significant.genes, all.geneIDs))
print(pVals.by.pathway)
The reason you are getting your error is because it appears you don't have the Category package installed from bioconductor. I suspect this because of the triple colon operator :::. This operator is very similar to the double colon operator ::. Whereas with :: you can access exported objects from a package without loading it, the ::: allows access to non-exported objects (in this case the hyperg function from Category). If you install the Category package the code runs without error.
With regard to the sapply statement:
pVals.by.pathway<-t(sapply(genes.by.pathway, hyperg.test, significant.genes, all.geneIDs))
You can break this down into the separate parts to understand it. Firstly, the sapply is iterating over the elements of gene.by.pathway and passing them to the first argument of hyperg.test. The following arguments are the two addition parameters. It is a little unclear and I personally recommend that people explicitly identify the parameters to avoid unexpected surprises and avoids the need for the exact same order. This is a little repetitive in this case but a good way to avoid a silly bug (e.g. putting significant.genes after all.geneIds)
Rewritten:
pVals.by.pathway <-
t(sapply(genes.by.pathway, hyperg.test, significant.genes=significant.genes, all.genes=all.geneIDs))
Once this loop completes, the sapply function simplifies the output in to a matrix. However, the output is much more user-friendly by taking the transpose t.
Generally speaking, when trying to understand complex apply statements I find it best to break them apart in to smaller parts and see what the objects themselves look like.

CCA Analysis: Error in weighted.mean.default(newX[, i], ...) : 'x' and 'w' must have the same length

I'm very new to R and this might be a very silly question to ask but I'm quite stuck right now.
I'm currently trying to do a Canonical Correspondence Analysis on my data to see which environmental factors have more weight on community distribution. I'm using the vegan package. My data consists of a table for the environmental factors (dataset EFamoA) and another for an abundance matrix (dataset AmoA). I have 41 soils, with 39 environmental factors and 334 species.
After cleaning my data of any variables which are not numerical, I try to perform the cca analysis using the formula notation:
CCA.amoA <- cca (AmoA ~ EFamoA$PH + EFamoA$LOI, data = EFamoA,
scale = TRUE, na.action = na.omit)
But then I get this error:
Error in weighted.mean.default(newX[, i], ...) :
'x' and 'w' must have the same length
I don't really know where to go from here and haven't found much regarding this problem anywhere (which leads me to think that it must be some sort of very basic mistake I'm doing). My environmental factor data is not standardized as I red in the cca help file that the algorithm does it but maybe I should standardize it before? (I've also red that scale = TRUE is only for species). Should I convert the data into matrices?
I hope I made my point clear enough as I've been struggling with this for a while now.
Edit: My environmental data has NA values
Alright so I was able to figure it out all by myself and it was indeed a silly thing, turns out my abundance data had soils as columns and species as rows, while environmental factor (EF) data had soils as rows and EF as columns.
using t() on my data, I transposed my data.frame (and collaterally converted it into a matrix) and cca() worked (as "length" was the same, I assume). Transposing the data separately and loading it already transposed works too.
Although maybe the t() approach saves the need of creating a whole new file (in case your data was organized using different rows as in my case), it converts the data into a matrix and this might not be desired in some cases, either way, this turned out to be a very simple and obvious thing to solve (took me a while though).

Bandwidth selection using NP package

New to R and having problem with a very simple task! I have read a few columns of .csv data into R, the contents of which contains of variables that are in the natural numbers plus zero, and have missing values. After trying to use the non-parametric package, I have two problems: first, if I use the simple command bw=npregbw(ydat=y, xdat=x, na.omit), where x and y are column vectors, I get the error that "number of regression data and response data do not match". Why do I get this, as I have the same number of elements in each vector?
Second, I would like to call the data ordered and tell npregbw this, using the command bw=npregbw(ydat=y, xdat=ordered(x)). When I do that, I get the error that x must be atomic for sort.list. But how is x not atomic, it is just a vector with natural numbers and NA's?
Any clarifications would be greatly appreciated!
1) You probably have a different number of NA's in y and x.
2) Can't be sure about this, since there is no example. If it is of following type:
x <- c(3,4,NA,2)
Then ordered(x) should work fine. Please provide an example of your case.
EDIT: You of course tried bw=npregbw(ydat=y, xdat=x)? ordered() makes your vector an ordered factor (see ?ordered), which is not an atomic vector (see 2.1.1 link and ?factor)
EDIT2: So the problem was the way of subsetting data. Note the difference in various ways of subsetting. data$x and data[,i] (where i = column number of column x) give you vectors, while data[c("x")] and data[i] give a data frame. Functions expect vectors, unless they call for data = (your data). In that case they work with column names

perform function on pairs of columns

I am trying to run some Monte Carlo simulations on animal position data. So far, I have sampled 100 X and Y coordinates, 100 times. This results in a list of 200. I then convert this list into a dataframe that is more condusive to eventual functions I want to run for each sample (kernel.area).
Now I have a data frame with 200 columns, and I would like to perform the kernel.area function using each successive pair of columns.
I can't reproduce my own data here very well, so I've tried to give a basic example just to show the structure of the data frame I'm working with. I've included the for loop I've tried so far, but I am still an R novice and would appreciate any suggestions.
# generate dataframe representing X and Y positions
df <- data.frame(x=seq(1:200),y=seq(1:200))
# 100 replications of sampling 100 "positions"
resamp <- replicate(100,df[sample(nrow(df),100),])
# convert to data frame (kernel.area needs an xy dataframe)
df2 <- do.call("rbind", resamp[1:2,])
# xy positions need to be in columns for kernel.area
df3 <- t(df2)
#edit: kernel.area requires you have an id field, but I am only dealing with one individual, so I'll construct a fake one of the same length as the positions
id=replicate(100,c("id"))
id=data.frame(id)
Here is the structure of the for loop I've tried (edited since first post):
for (j in seq(1,ncol(df3)-1,2)) {
kud <- kernel.area(df3[,j:(j+1)],id=id,kern="bivnorm",unin=c("m"),unout=c("km2"))
print(kud)
}
My end goal is to calculate kernel.area for each resampling event (ie rows 1:100 for every pair of columns up to 200), and be able to combine the results in a dataframe. However, after running the loop, I get this error message:
Error in df[, 1] : incorrect number of dimensions
Edit: I realised my id format was not the same as my data frame, so I change it and now have the error:
Error in kernelUD(xy, id, h, grid, same4all, hlim, kern, extent) :
id should have the same length as xy
First, a disclaimer: I have never worked with the package adehabitat, which has a function kernel.area, which I assume you are using. Perhaps you could confirm which package contains the function in question.
I think there are a couple suggestions I can make that are independent of knowledge of the specific package, though.
The first lies in the creation of df3. This should probably be
df3 <- t(df2), but this is most likely correct in your actual code
and just a typo in your post.
The second suggestion has to do with the way you subset df3 in the
loop. j:j+1 is just a single number, since the : has a higher
precedence than + (see ?Syntax for the order in which
mathematical operations are conducted in R). To get the desired two
columns, use j:(j+1) instead.
EDIT:
When loading adehabitat, I was warned to "Be careful" and use the related new packages, among which is adehabitatHR, which also contains a function kernel.area. This function has slightly different syntax and behavior, but perhaps it would be worthwhile examining. Using adehabitatHR (I had to install from source since the package is not available for R 2.15.0), I was able to do the following.
library(adehabitatHR)
for (j in seq(1,ncol(df3)-1,2)) {
kud <-kernelUD(SpatialPoints(df3[,j:(j+1)]),kern="bivnorm")
kernAr<-kernel.area(kud,unin=c("m"),unout=c("km2"))
print(kernAr)
}
detach(package:adehabitatHR, unload=TRUE)
This prints something, and as is mentioned in a comment below, kernelUD() is called before kernel.area().

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