[Caitriona McEvoy]

Hi Michael, Thank you for your reply. I installed packages as suggested, and it's working. Thanks! Caitriona

[Michael Love]

hi Caitriona

It looks like you have a mix of out-of-date and new packages. These often generate conflicts. This can occur if you install Bioconductor packages using install.packages() rather than with biocLite().

Try this:

source("http://bioconductor.org/biocLite.R")
biocValid()

[Caitriona McEvoy]

Thanks for replying. I am using DESeq2 1.6.3.
First I made dds:
dds <- DESeqDataSetFromMatrix(countData = countData,
colData = colData,
design = ~ Organ + Fibrosis)
then:
rld = rlog (dds)
I went on to examine rld using: head(assay(rld)), and it is listed in my environment as a large SummarisedExperiment.
Caitriona

[dpryan]

How did you make "rld" and what version of DESeq2 are you using?

[Caitriona McEvoy]

Hi all,
I am very new to r & DEseq2 but have been following the excellent vignette. I have encountered a problem though. I can create the object rld as per the command in the vignette, but when I try to run PCA as suggested I repeatedly encounter the following error:

plotPCA(rld, intgroup=c("Fibrosis", "Sample.ID"))

Error in (function (classes, fdef, mtable) :
unable to find an inherited method for function ‘plotPCA’ for signature ‘"SummarizedExperiment"’
I have also tried:
plotPCA( DESeqTransform(rld ) )
and get the following:
Error in plotPCA(DESeqTransform(rld)) :
error in evaluating the argument 'x' in selecting a method for function 'plotPCA': Error: could not find function "DESeqTransform"
Can anyone advise?
Caitriona

[apredeus]

Hello

single cell data is very peculiar, and as a rule I'd say, no - you can not use regular methods for DE analysis of single-cell data. Look at SCDE by P. Kharchenko et. al:

http://pklab.med.harvard.edu/scde/index.html

Overall, people more often use PCA than DE with single-cell data. As you work with them some more, you probably will see why

[amolinaro]

Hi all,

I'm doing single cell RNAseq on a heterogeneous population of cells & I would like to do DE analysis to identify different subpopulations. I plan on using the tophat/cufflinks/monocle pipeline since it was specifically designed for single cell data, but I would also like to apply a raw count method to verify the results. Can DESeq2 be used for DE analysis of single cell data? If so, how would I go about doing that and what would the output look like? I'm very new to bioinformatics so apologies if these are silly questions...

Thanks in advance!

[Joe Dever]

The first step is the only step which matters how you generate the counts. This is the step when you build the DESeqDataSet. So you only need to follow the htseq steps up until DESeqDataSetFromHTSeqCount(). After that, you have the DESeqDataSet, which you can save as "dds", and then you can follow the rest of the steps below. You can either follow this instruction in the vignette, or you can also read the help file for this particular function by typing ? and the function name into R and press enter:

?DESeqDataSetFromHTSeqCount

Note that you can name the object however you like. To follow the rest of the workflow you can choose 'dds':

dds <- DESeqDataSetFromHTSeqCount(...)

where you will fill in ... with the appropriate code from the vignette and help.

Thanks for your reply, I'll try again.

[Michael Love]

The first step is the only step which matters how you generate the counts. This is the step when you build the DESeqDataSet. So you only need to follow the htseq steps up until DESeqDataSetFromHTSeqCount(). After that, you have the DESeqDataSet, which you can save as "dds", and then you can follow the rest of the steps below. You can either follow this instruction in the vignette, or you can also read the help file for this particular function by typing ? and the function name into R and press enter:

?DESeqDataSetFromHTSeqCount

Note that you can name the object however you like. To follow the rest of the workflow you can choose 'dds':

dds <- DESeqDataSetFromHTSeqCount(...)

where you will fill in ... with the appropriate code from the vignette and help.

[Joe Dever]

Hi,

I am not familiar with R but I used DESeq in the past following its vignette.
I can't understand how to use DESeq2. I tried to follow the section for HT-Seq input but then it switches to loading the Pasilla dataset.

Is there any tutorial anywhere showing clearly how to use DESeq2 with HTseq-count, like the DESeq vignette did? Please advise.

[enrico16]

See: https://support.bioconductor.org/p/68277/

--

Hi Michael,

Is the Cook's distance-based flagging of p-values performed when using a continuous variable?

I don't see how the condition:

"At least 3 replicates are required for flagging, as it is difficult to judge which sample
might be an outlier with only 2 replicates."

can realistically be met for continuous variables.

If that's the case, how can I remove genes that display such a behavior?
Please let me know if this needs clarification.

Thanks!

[rozitaa]

I ran a code like this (hope it is the correct one?!):

dds_LRT_liver <- nbinomLRT(dds[, dds$tissue=='liver'], full=~gut_microbiota, reduced=~1)
dds_LRT_PC <- nbinomLRT(dds[, dds$tissue=='PC'], full=~gut_microbiota, reduced=~1)
dds_LRT_Si5 <- nbinomLRT(dds[, dds$tissue=='Si5'], full=~gut_microbiota, reduced=~1)

[rozitaa]

Hi,

I have recently have changed from DESeq to DESeq2. So I need some help in designing my contrast for the binomial tests. This is my design table:

sample	tissue	gut_microbiota
1	5231	Si5	GF
2	5231	PC	GF
3	5231	liver	GF
4	5232	Si5	GF
5	5232	PC	GF
6	5232	liver	GF
7	5233	Si5	GF
8	5233	PC	GF
9	5233	liver	GF
10	5234	Si5	GF
11	5234	PC	GF
12	5234	liver	GF
13	5161	Si5	mono
14	5161	PC	mono
15	5161	liver	mono
16	5162	Si5	mono
17	5162	PC	mono
18	5162	liver	mono
19	5163	Si5	mono
20	5163	PC	mono
21	5163	liver	mono
22	5164	Si5	prevExci
23	5164	PC	prevExci
24	5164	liver	prevExci
25	5164	liver	prevExci
26	5165	Si5	prevExci
27	5165	PC	prevExci
28	5165	liver	prevExci
29	5166	Si5	prevExci
30	5166	PC	prevExci
31	5166	liver	prevExci
32	5167	Si5	prev
33	5167	PC	prev
34	5167	liver	prev
35	5168	Si5	prev
36	5168	PC	prev
37	5168	liver	prev
38	5169	Si5	prev
39	5169	PC	prev
40	5169	liver	prev
41	5170	Si5	prev
42	5170	PC	prev
43	5170	liver	prev
44	5171	Si5	mono
45	5171	PC	mono
46	5171	liver	mono
47	5172	Si5	mono
48	5172	PC	mono
49	5172	liver	mono
50	5173	Si5	mono
51	5173	PC	mono
52	5173	liver	mono
53	5174	Si5	prev
54	5174	PC	prev
55	5174	liver	prev
56	5175	Si5	prev
57	5175	PC	prev
58	5175	liver	prev
59	5176	Si5	prev
60	5176	PC	prev
61	5176	liver	prev
62	5177	Si5	prevMono
63	5177	PC	prevMono
64	5177	liver	prevMono
65	5178	Si5	prevMono
66	5178	PC	prevMono
67	5178	liver	prevMono
68	5179	Si5	prevMono
69	5179	PC	prevMono

and this is how I have designed my count data:
dds = DESeqDataSetFromHTSeqCount(sampleTable=sample_table, directory='~/htseq/', design= ~ tissue + gut_microbiota)

Now I would like to test if any gut_microbiota level has significant effects on any of my genes at each separate tissue; (i.e. running the test for each tissue separately). How should I design my contrast formula? and How I should specify specific tissue at each test?

Thanks!

[TomHarrop]

Perfect, thanks.

[Michael Love]

The fitted means, mu_ij in the paper, for the full model are in assays(dds)[["mu"]]. Not that these include the size factor scaling, so you would have to divide those out to get the q_ij in the paper. See the formula in the vignette or paper for more details.

We don't store the reduced design fitted means, but you can generate these easily by running the GLM without a log fold change prior:

dds.reduced <- dds
design(dds.reduced) <- ~ batch
dds.reduced <- nbinomWaldTest(dds.reduced, betaPrior=FALSE)
assays(dds.reduced)[["mu"]]