Question: Strange MA plot from DESeq2
0
gravatar for Yuyayuya
11 months ago by
Yuyayuya40
USA
Yuyayuya40 wrote:

Hello guys,

I got a strange MA plot like this.

The code I used was simply like the manual:

plotMA(res)

Does anyone know the reason and how to fix it?

Thank you in advance!

rna-seq • 860 views
ADD COMMENTlink modified 11 months ago • written 11 months ago by Yuyayuya40
2

It looks like it's plotting virtually the same thing against itself. The log ratios should neither be that high, and one would expect both positive and negative log ratios.

How did you produce res (go through all of the DESeq2 commands)?

ADD REPLYlink written 11 months ago by Kevin Blighe28k

Here's the code I used:

lncap_de <- DESeq(data_deseq_1)
lncap_de_out <- results(lncap_de)
res <- results(lncap_de, alpha = 0.05)

And, I checked the result by:

head(res[ order(resSig$log2FoldChange), ])

It looks like (log2FoldChange seems normal):

I tried demo data from DESeq and got the same strange MA plot as well.

dds <- makeExampleDESeqDataSet()
dds <- DESeq(dds)
res <- results(dds)
plotMA(res)
ADD REPLYlink modified 11 months ago • written 11 months ago by Yuyayuya40
2

I repeated your commands for the demo data and my MA plot looked normal.

You may want to respond to Devon's comment, and provide your session's info via sessionInfo().

I run R version 3.2.5 (2016-04-14) on Platform: x86_64-pc-linux-gnu (64-bit), under Ubuntu 14.04.5 LTS, with DESeq2_1.10.1

ADD REPLYlink written 11 months ago by Kevin Blighe28k

Thanks for replying!

Here's the session's info: R version 3.4.0 (2017-04-21) Platform: x86_64-apple-darwin15.6.0 (64-bit) Running under: macOS Sierra 10.12.6, with DESeq2_1.16.1

ADD REPLYlink modified 11 months ago • written 11 months ago by Yuyayuya40
3
gravatar for Devon Ryan
11 months ago by
Devon Ryan84k
Freiburg, Germany
Devon Ryan84k wrote:

It looks like your model lacked an intercept and you didn't specify a contrast. You'll need to show the full set of commands (specifically, how you made your original DESeqDataset and the contents of any sample sheet involved). Also, you'll need to post the output of sessionInfo().

ADD COMMENTlink written 11 months ago by Devon Ryan84k

Hey Devon, you may want to take a look above!

ADD REPLYlink written 11 months ago by Kevin Blighe28k

Thanks for replying!

I generated the DESeqDataset by combining countData and colData by DESeqDataSetFromMatrix( )

Here's the entire code I used:

 > dat = read.csv(file = "/User/Desktop/gene_count_matrix.csv", header = T, row.names=1)
 > colData = read.table(file = "/User/Desktop/colData.txt", header = T, row.names=NULL)
 > n_count <- rowSums(dat) 
 > data <- dat[n_count > 0, ]
 > rownames(colData) <- colnames(data)
 > data_deseq_1<- DESeqDataSetFromMatrix(countData =
            data, colData = colData,
            design = ~ Genotype + Trt + Time)
 > lncap_de <- DESeq(data_deseq_1)
 > lncap_de_out <- results(lncap_de)
 > res <- results(lncap_de, alpha = 0.05)

Here's how input .csv and .txt data looks like:

I don't understand why even the MA plot of demo data also looks strange.

ADD REPLYlink modified 11 months ago • written 11 months ago by Yuyayuya40
1

At this point I haven't a clue what's going on. I suspect there's simply something installed incorrectly on your system. I suggest you post this on the bioconductor site and wait for Mike Love (the DESeq2 author) to reply. Perhaps he has more insight as to where the problem might be.

ADD REPLYlink written 11 months ago by Devon Ryan84k
1

Thank you, Sir! I'll do that. It may be true since I did have some error when I installed DESeq2.

ADD REPLYlink written 11 months ago by Yuyayuya40
2
gravatar for Yuyayuya
11 months ago by
Yuyayuya40
USA
Yuyayuya40 wrote:

For those people encounter the same problem like me.

Type plotMA in R and check the working environment. For example, I got

> plotMA
function (object, ...) 
UseMethod("plotMA")
<environment: namespace:limma>

It means the plotMA() I used isn’t DESeq2’s plotMA function. In this situation, use DESeq2::plotMA() instead of plotMA() to make MA plot.

ADD COMMENTlink modified 11 months ago • written 11 months ago by Yuyayuya40

Thanks for sharing the update! That's certainly something about which to be careful in the future.

ADD REPLYlink written 11 months ago by Kevin Blighe28k
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