Contrast for Limma - Voom - r

I'm doing a differential expression analysis for RNA-seq data with limma - voom. My data is about a cancer drug, 49 samples in total, some of them are responders some of them are not. I need some help building the contrast. I'm dealing with only one factor here, so two groups only.
I know it's the simplest type of data, but I'm getting most of the data as differntialy expressed (which should not be the case), only 13% is not differntialy expressed, and I think the problem has to do with the contrast. This is the design I made, with 1 or 0.
1 for NoResponse means there was no response, and 1 for Response means there was a response.
using dput:
structure(c(1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1,
1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1,
1, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0,
0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0,
0, 0, 0, 0, 0, 0, 0, 0, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1), .Dim = c(49L,
2L), .Dimnames = list(c("Pt1", "Pt10", "Pt103", "Pt106", "Pt11",
"Pt17", "Pt2", "Pt24", "Pt26", "Pt27", "Pt28", "Pt29", "Pt31",
"Pt36", "Pt37", "Pt38", "Pt39", "Pt4", "Pt46", "Pt47", "Pt5",
"Pt52", "Pt59", "Pt62", "Pt65", "Pt66", "Pt67", "Pt77", "Pt78",
"Pt79", "Pt8", "Pt82", "Pt84", "Pt85", "Pt89", "Pt9", "Pt90",
"Pt92", "Pt98", "Pt101", "Pt18", "Pt3", "Pt30", "Pt34", "Pt44",
"Pt48", "Pt49", "Pt72", "Pt94"), c("NoResponse", "Response")), assign = c(1L,
1L), contrasts = list(Response = "contr.treatment"))
And here is my code for the analysis it self:
d0 <- DGEList(rawdata)
d0 <- calcNormFactors(d0)
Voom <- voom(d0, design, plot = TRUE)
vfit <- lmFit(Voom, design)
contrast <- makeContrasts(Response - NoResponse,
levels = colnames(coef(vfit)))
vfit <- contrasts.fit(vfit, contrasts = contrast)
efit <- eBayes(vfit)
plotSA(efit, main = 'final model: Mean-Variance trend')
The bioconductor guide didn't help.
Note: The problem is not with the data. The voom plot is very good, I'm just stuck with the contrast which is (I think) making all the mess.

Related

R function to change value after a condition has been fulfilled

Participants in an experiment took a test that has a rule that says "once a participant has gotten 6 items wrong in a window of 8 items, you stop running the test". However, some experimenters kept testing past this point. I now need to find a way in which I can automatically see where the test should have been stopped, and change all values following the end to 0 (= item wrong). I am not even sure if this is something that can be done in R.
To be clear, I would like to go row by row (which are the participants) and once there are six 0s in a given window of 8 columns (items), I would need all values after the sixth 0 to be 0 too.
While the reproducible data is below, here is a visualization of what I would need, where the blue cells are the ones that should change to 0:
Pre-changes
Post-changes
Reproducible data:
structure(list(Participant_ID = c("E01P01", "E01P02", "E01P03",
"E01P04", "E01P05", "E01P06", "E01P07", "E01P08", "E02P01", "E02P02"
), A2 = c(1, 1, 1, 0, 0, 1, 1, 1, 1, 1), A3 = c(1, 1, 0, 0, 0,
1, 0, 0, 0, 0), B1 = c(1, 1, 1, 0, 0, 1, 0, 0, 1, 1), B2 = c(1,
1, 1, 1, 1, 1, 0, 0, 0, 1), C3 = c(1, 0, 0, 1, 0, 1, 0, 0, 0,
1), C4 = c(1, 0, 0, 0, 0, 1, 0, 0, 1, 1), D1 = c(1, 0, 0, 0,
0, 1, 0, 0, 0, 0), D3 = c(1, 1, 1, 1, 0, 0, 1, 0, 0, 1), E1 = c(1,
0, 0, 0, 0, 1, 0, 0, 0, 1), E3 = c(1, 1, 0, 1, 0, 1, 0, 0, 0,
0), F1 = c(1, 0, 0, 0, 1, 0, 0, 1, 0, 0), F4 = c(1, 1, 1, 1,
0, 1, 0, 1, 1, 0), G1 = c(1, 0, 0, 0, 0, 1, 0, 0, 0, 1), G2 = c(0,
0, 0, 0, 1, 1, 1, 0, 1, 1)), row.names = c(NA, -10L), class = c("tbl_df",
"tbl", "data.frame"))
Any help is highly appreciated!

Here is a solution that involves some pivoting, rollsum, cumsum, if_else logic, then pivoting back. Let me know if it works.
library(tidyverse)
library(zoo)
structure(list(Participant_ID = c("E01P01", "E01P02", "E01P03",
"E01P04", "E01P05", "E01P06", "E01P07", "E01P08", "E02P01", "E02P02"
), A2 = c(1, 1, 1, 0, 0, 1, 1, 1, 1, 1), A3 = c(1, 1, 0, 0, 0,
1, 0, 0, 0, 0), B1 = c(1, 1, 1, 0, 0, 1, 0, 0, 1, 1), B2 = c(1,
1, 1, 1, 1, 1, 0, 0, 0, 1), C3 = c(1, 0, 0, 1, 0, 1, 0, 0, 0,
1), C4 = c(1, 0, 0, 0, 0, 1, 0, 0, 1, 1), D1 = c(1, 0, 0, 0,
0, 1, 0, 0, 0, 0), D3 = c(1, 1, 1, 1, 0, 0, 1, 0, 0, 1), E1 = c(1,
0, 0, 0, 0, 1, 0, 0, 0, 1), E3 = c(1, 1, 0, 1, 0, 1, 0, 0, 0,
0), F1 = c(1, 0, 0, 0, 1, 0, 0, 1, 0, 0), F4 = c(1, 1, 1, 1,
0, 1, 0, 1, 1, 0), G1 = c(1, 0, 0, 0, 0, 1, 0, 0, 0, 1), G2 = c(0,
0, 0, 0, 1, 1, 1, 0, 1, 1)), row.names = c(NA, -10L), class = c("tbl_df",
"tbl", "data.frame")) %>%
as_tibble() %>%
pivot_longer(-1) %>%
group_by(Participant_ID) %>%
mutate(running_total = zoo::rollsumr(value==0, k = 8, fill = 0),
should_terminate = cumsum(running_total >= 6),
value = if_else(should_terminate > 0, 0, value)) %>%
ungroup() %>%
select(Participant_ID, name, value) %>%
pivot_wider(names_from = name, values_from = value)

How to plot a binary matrix without using additional packages?

I created a binary matrix and I wanna plot 1's as black square.
How can I write it without using any package?
For example, my matrix is:
m <- matrix(c(0,1,1,0,0,1,0,1,1),nrow=3, ncol=3)

Do you want this?
m <- matrix(c(0,1,1,0,0,1,0,1,1), nrow=3, ncol=3)
image(m, main = "My binary matrix plot", col = c("white", "black"))

If image doesn't suffice, we could write a generalized function using mapply like this one.
chessplot <- function(m, col=1, border=NA) {
stopifnot(dim(m)[1] == dim(m)[2]) ## allows only square matrices
n <- nrow(m)
plot(n, n, type='n', xlim=c(0, n), ylim=c(0, n))
mapply(\(i, j, m) {
rect(-1 + i, n - j, 0 + i, n - j + 1, col=m, border=border)
}, seq(n), rep(seq(n), each=n), t(m)) |> invisible()
}
Gives:
chessplot(m3)
chessplot(m4)
chessplot(m8)
Data:
m3 <- structure(c(0, 1, 1, 0, 0, 1, 0, 1, 1), .Dim = c(3L, 3L))
m4 <- structure(c(0, 1, 1, 0, 0, 1, 0, 1, 1, 0, 1, 1, 0, 0, 1, 0), .Dim = c(4L,
4L))
m8 <- structure(c(0, 1, 0, 1, 0, 1, 0, 1, 1, 0, 1, 0, 1, 0, 1, 0, 0,
1, 0, 1, 0, 1, 0, 1, 1, 0, 1, 0, 1, 0, 1, 0, 0, 1, 0, 1, 0, 1,
0, 1, 1, 0, 1, 0, 1, 0, 1, 0, 0, 1, 0, 1, 0, 1, 0, 1, 1, 0, 1,
0, 1, 0, 1, 0), .Dim = c(8L, 8L))

Chi Square Test of Independence of Whole Dataset

I have a 3185x90 dataset of binary values and want to do a chi-squared test of independence, comparing all column variables against each other.
I've been tried using different variations of code from google searches with chisq.test() and some for loops, but none of them have worked so far.
How do I do this?
This is the frame I've tinkered with. My dataset is oak.
chi_trial <- data.frame(a = c(0,1), b = c(0,1))
for(row in 1:nrow(oak)){
print(row)
print(chisq.test(c(oak[row,1],d[row,2])))
}
I also tried this:
apply(d, 1, chisq.test)
which gives me the error: Error in FUN(newX[, i], ...) :
all entries of 'x' must be nonnegative and finite
dput(oak[1:2],)
structure(list(post_flu = structure(c(1, 1, 1, 1, 1, 0, 0, 0,
0, 0, 1, 1, 1, 1, 1, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0,
0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1,
1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 0,
0, 0, 0, 0, 0, 0, 0, 0, 0, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 0, 0,
0, 0, 0, 0, 0, 0, 0, 0, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 0, 0, 0,
0, 0, 1, 1, 1, 1, 1, 0, 0, 0, 0, 0, 1, 1, 1, 1, 1, 0, 0, 0, 0,
0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 1, 1, 1, 1, 1,
1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 0, 0, 0, 0, 0, 1,
1, 1, 1, 1, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0,
0, 0, 0, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 0, 0, 0, 0, 0, 0, 0, 0,
label = "Receipt of Flu Vaccine - Encounter Survey", format.stata = "%10.0g")), row.names = c(NA,
-3185L), class = c("tbl_df", "tbl", "data.frame"), label = "Main Oakland Clinic Analysis Dataset")
I added a sample of my data with the final lines of the output. The portion of the dataset is small, but it all looks like this.

You could use something like the code below, which is similar to R's cor function. I don't have your data, so I'm simulating some. Note that I get one significant p-value, using the traditional cut-off of 0.05.
set.seed(3)
nr=3185; nc=3
oak <- as.data.frame(matrix(sample(0:1, size=nr*nc, replace=TRUE), ncol=nc))
oak
mult.chi <- function(data){
nc <- ncol(data)
res <- matrix(0, nrow=nc, ncol=nc) # or NA
for(i in 1:(nc-1))
for(j in (i+1):nc)
res[i,j] <- suppressWarnings(chisq.test(oak[,i], oak[,j])$p.value)
rownames(res) <- colnames(data)
colnames(res) <- colnames(data)
res
}
mult.chi(oak)
# V1 V2 V3
# V1 0 0.7847063 0.32012466
# V2 0 0.0000000 0.01410326
# V3 0 0.0000000 0.00000000
So consider applying a multiple testing adjustment as mentioned in the comments.

Here is a solution with combn to get all combinations of column numbers 2 by 2. Tested with the data in #Edward's answer.
chisq2cols <- function(X){
y <- matrix(0, ncol(X), ncol(X))
cmb <- combn(ncol(X), 2)
y[upper.tri(y)] <- apply(cmb, 2, function(k){
tbl <- table(X[k])
chisq.test(tbl)$p.value
})
y
}
chisq2cols(oak)
# [,1] [,2] [,3]
#[1,] 0 0.7847063 0.32012466
#[2,] 0 0.0000000 0.01410326
#[3,] 0 0.0000000 0.00000000

Heatmap error with : 'x' must be a numeric matrix

I know this question might be duplicated, but I was trying some of the solutions posted in this forum with no success, and that's why I am posting it here.
Let's start with my dataset to make it reproducible.
dataset <- structure(list(Comparison = c("SH vs SAP", "SH vs NEA", "SH vs ERE",
"SH vs ERH", "SH vs NAL", "SAP vs NEA", "SAP vs ERE", "SAP vs ERH",
"SAP vs NAL", "NEA vs ERE", "NEA vs ERH", "NEA vs NAL", "ERE vs ERH",
"ERE vs NAL", "ERH vs NAL"), DC1 = c(NA, NA, NA, NA, NA, 1, 1,
1, NA, 1, 1, NA, 1, NA, NA), DC2 = c(NA, NA, NA, NA, NA, 1, 1,
1, NA, 0, 0, NA, 1, NA, NA), DC3 = c(1, 1, 1, 1, 1, 1, 1, 1,
0, 1, 0, 0, 1, 0, 1), DC4 = c(1, 1, 1, 1, 1, 1, 1, 0, 0, 0, 0,
0, 1, 1, 1), DC5 = c(0, 1, 1, 1, 1, 0, 0, 0, 0, 0, 0, 0, 1, 1,
1), DC6 = c(0, 1, 1, 1, 1, 0, 0, 0, 0, 0, 0, 0, 1, 1, 1), DC7 = c(0,
1, 0, 1, 1, 0, 0, 0, 0, 0, 1, 1, 1, 1, 1), DC8 = c(0, 1, 0, 1,
1, 0, 0, 0, 0, 0, 1, 1, 0, 0, 1), DC9 = c(0, 0, 0, 0, 1, 0, 0,
0, 0, 0, 1, 1, 0, 0, 0), DC10 = c(1, 1, 0, 1, 1, 0, 0, 0, 0,
0, 1, 0, 1, 0, 0)), .Names = c("Comparison", "DC1", "DC2", "DC3",
"DC4", "DC5", "DC6", "DC7", "DC8", "DC9", "DC10"), class = "data.frame", row.names = c(NA,
15L))
I have tried to change the dataset to a matrix, as this been suggested in other posts. However, it keeps giving the same error
heatmap(dataset)
heatmap(as.matrix(dataset))
Error in heatmap(dataset) :
'x' must be a numeric matrix
Error in heatmap(as.matrix(dataset)) :
'x' must be a numeric matrix
I tried to convert to numeric the columns, but the error keeps. And so is the case when I remove DC1 and DC2 columns which contain NA values.
Any help to spot the problem?

dataset[, 1] is character so as.matrix(dataset) is a character matrix. This explains:
'x' must be a numeric matrix
Your probably want
heatmap(as.matrix(dataset[, -1]))
And how can I include the names of the rows on the right?
Set the Comparison variable as the rownames of the matrix:
m <- as.matrix(dataset[, -1])
rownames(m) <- dataset$Comparison
heatmap(m)
So your real issue is really Convert the values in a column into row names in an existing data frame in R although the problem is presented with heatmap.

Differences between merge and match functions in R

I everybody I remove my last post to make a reproducible exmaple of my problem. I am working with the next to data frames a1 (dput structure):
structure(list(r04_numero_operacion = c("0050475725", "0050490602",
"0050491033", "0050496386", "0050518985", "0050630090", "0050631615",
"0060235906", "0060238732", "0060241333", "0060244391", "0060245813",
"0060260056", "0060266356", "0800041441", "0800054041", "0800055382",
"0800058554", "2020200062", "2020200073", "CAR1010001706000",
"CAR1010001795000", "CAR1010001803000", "CAR1010001871000", "CAR1010001962000",
"CAR1010002002000", "CAR1010002120000", "CAR1010002189000", "CAR1010002215000",
"CAR1010002250000"), perdida3 = c(523.12, 265.43, 8371.66, 5242.13,
4960.51, 8473.27, 3743.45, 1283.32, 2229.25, 8001.27, 8653.94,
3670.13, 4536.02, 8216.55, 2481.36, 288.94, 1637.28, 4566.89,
1573.63, 11217.92, 0, 0, 0, 0, 0, 0, 0, 0, 9633.9, 0), Saldo = c(1,
1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 288.94, 1637.28, 4566.89,
1, 1, 481.59, 299.52, 258.13, 603.84, 231.61, 631.68, 220.6,
210.54, 1, 1224.44), Bvencida = c(0, 0, 0, 0, 0, 0, 0, 0, 0,
0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 603.84, 0, 631.68,
0, 0, 0, 0), Cvencida = c(0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0,
0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 1224.44),
Dvencida = c(0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0,
0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0), vencida = c(1,
1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 288.94, 1637.28,
4566.89, 1, 1, 0, 0, 0, 603.84, 0, 631.68, 0, 0, 1, 1224.44
), V1 = c(1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1,
1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1)), .Names = c("r04_numero_operacion",
"perdida3", "Saldo", "Bvencida", "Cvencida", "Dvencida", "vencida",
"V1"), codepage = 1252L, row.names = c(NA, 30L), class = "data.frame")
And a2 data frame (dput structure):
structure(list(r04_numero_operacion = c("0050475725", "0050490602",
"0050491033", "0050496386", "0050518985", "0050630090", "0050631615",
"0060235906", "0060238732", "0060241333", "0060244391", "0060245813",
"0060260056", "0060266356", "0800041441", "0800054041", "0800055382",
"0800058554", "2020200073", "CAR1010002002000", "CAR1010002189000",
"CAR1010002215000", "CAR1010002250000", "CAR1010002264000", "CAR1010002297000",
"CAR1010002401000", "CAR1010002412000", "CAR1010002436000", "CAR1010002529000",
"CAR1010002709000"), perdida3 = c(523.12, 265.43, 8371.66, 5242.13,
4960.51, 8473.27, 3743.45, 1283.32, 2229.25, 8001.27, 8653.94,
3670.13, 4536.02, 8216.55, 2481.36, 288.94, 1637.28, 4566.89,
11217.92, 0, 0, 9633.9, 0, 0, 0, 0, 0, 0, 0, 0), Saldo = c(1,
1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 288.94, 1637.28, 4566.89,
1, 317.72, 210.54, 1, 868.93, 242.91, 298.78, 120.63, 255.01,
357.68, 284.08, 308.83), Bvencida = c(0, 0, 0, 0, 0, 0, 0, 0,
0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 317.72, 0, 0, 0, 0, 0, 0, 0,
0, 0, 0), Cvencida = c(0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0,
0, 0, 0, 0, 0, 0, 0, 0, 0, 868.93, 0, 0, 0, 0, 0, 0, 0), Dvencida = c(0,
0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0,
0, 0, 0, 0, 0, 0, 0, 0), vencida = c(1, 1, 1, 1, 1, 1, 1, 1,
1, 1, 1, 1, 1, 1, 1, 288.94, 1637.28, 4566.89, 1, 317.72, 0,
1, 868.93, 0, 0, 0, 0, 0, 0, 0), V2 = c(2, 2, 2, 2, 2, 2, 2,
2, 2, 2, 2, 2, 2, 2, 2, 2, 2, 2, 2, 2, 2, 2, 2, 2, 2, 2, 2, 2,
2, 2)), .Names = c("r04_numero_operacion", "perdida3", "Saldo",
"Bvencida", "Cvencida", "Dvencida", "vencida", "V2"), class = "data.frame", row.names = c(NA,
30L))
My problem is when I use merge() and match() functions. merge() is more functional than match() related to add new variables by common one but when I use merge() I don't get the same result as match(). First I used merge() with a2 and a1 to create DF with the next code:
DF=merge(a2,a1,all.x=TRUE)
It added V1 variable from a1 to DF and I got this summary for DF$V1:
Min. 1st Qu. Median Mean 3rd Qu. Max. NA's
1 1 1 1 1 1 9
After I create a copy of a2 named DF and I made a match with r04_numero_operacion using this code to add V1 variable from a1 to a2:
a2$V1<-a1[match(a2$r04_numero_operacion,a1$r04_numero_operacion),"V1"]
It added `V1 to DF but the result is different to the merge() way. I got this summary for DF$V1 in match() solution:
Min. 1st Qu. Median Mean 3rd Qu. Max. NA's
1 1 1 1 1 1 7
My problem is I want to make the same I made with match() but using merge() function due to this function is more poweful than match(). Thanks for your help.

In using match(a2$r04_numero_operacion,a1$r04_numero_operacion) the a2$r04_numero_operacion values gets matched the coresponding column in a1 while in using merge(a2,a1,all.x=TRUE) the a1 all the matching columns get matched to the matching column names in a2. If you only match on the first column, the NA counts match up:
summary( merge(a2,a1,by=1,all.x=TRUE)$V1 )
Min. 1st Qu. Median Mean 3rd Qu. Max. NA's
1 1 1 1 1 1 7

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