HTseq-count to DEseq2: Need a little help..

[sindrle]

No surprise, that worked like a charm!

Thanks again, Ill try to run DEseq2 now for diff.exp.

[dpryan]

Ah, I see what the problem is. colData is is set from SampleTable (it's all but the first 2 columns). However, there's just a single column there and you have two factors in your design. Try the following:

sampleFiles <- list.files(path="/Volumes/timemachine/HTseq_DEseq2",pattern="*.txt")
status <- factor(c(rep("Healthy",26), rep("Diabetic",22)))
timepoints = as.factor(c(1,1,1,1,1,1,1,1,1,1,1,1,1,2,2,2,2,2,2,2,2,2,2,2,2,2,1,1,1,1,1,1,1,1,1,1,1,2,2,2,2,2,2,2,2,2,2,2))
sampleTable <- data.frame(sampleName = sampleFiles, fileName = sampleFiles, status=status, timepoints=timepoints)
directory <- c("/Volumes/timemachine/HTseq_DEseq2/")
des <- formula(~timepoints+status)
ddsHTSeq <- DESeqDataSetFromHTSeqCount(sampleTable = sampleTable, directory = directory, design= des)

Something along those lines should work (I always use DESeqDataSetFromMatrix).

[sindrle]

For the first post:

sampleCondition =read.table("/Volumes/timemachine/HTseq_DEseq2/03_SampleCondition.txt", header=TRUE)
condition = sampleCondition

03_SampleCondition.txt:

Condition
Diabetic_pre-exercise
Diabetic_pre-exercise
Diabetic_pre-exercise
Diabetic_pre-exercise
Diabetic_pre-exercise
Diabetic_pre-exercise
Diabetic_pre-exercise
Diabetic_pre-exercise
Diabetic_pre-exercise
Diabetic_pre-exercise
Diabetic_pre-exercise
Diabetic_post-exercise
Diabetic_post-exercise
Diabetic_post-exercise
Diabetic_post-exercise
Diabetic_post-exercise
Diabetic_post-exercise
Diabetic_post-exercise
Diabetic_post-exercise
Diabetic_post-exercise
Diabetic_post-exercise
Diabetic_post-exercise
Healthy_pre-exercise
Healthy_pre-exercise
Healthy_pre-exercise
Healthy_pre-exercise
Healthy_pre-exercise
Healthy_pre-exercise
Healthy_pre-exercise
Healthy_pre-exercise
Healthy_pre-exercise
Healthy_pre-exercise
Healthy_pre-exercise
Healthy_pre-exercise
Healthy_pre-exercise
Healthy_post-exercise
Healthy_post-exercise
Healthy_post-exercise
Healthy_post-exercise
Healthy_post-exercise
Healthy_post-exercise
Healthy_post-exercise
Healthy_post-exercise
Healthy_post-exercise
Healthy_post-exercise
Healthy_post-exercise
Healthy_post-exercise
Healthy_post-exercise

[sindrle]

Design = des doen not work:

ddsHTSeq <- DESeqDataSetFromHTSeqCount(sampleTable = sampleTable, directory = directory, design= des)
Error in validObject(.Object) :
invalid class "DESeqDataSet" object: all variables in design formula must be columns in colData

[sindrle]

Heres my "sampleTable":
sampleName fileName Condition
1 D104.txt D104.txt Diabetic_pre-exercise
2 D121.txt D121.txt Diabetic_pre-exercise
3 D153.txt D153.txt Diabetic_pre-exercise
4 D155.txt D155.txt Diabetic_pre-exercise
5 D161.txt D161.txt Diabetic_pre-exercise
6 D162.txt D162.txt Diabetic_pre-exercise
7 D167.txt D167.txt Diabetic_pre-exercise
8 D173.txt D173.txt Diabetic_pre-exercise
9 D176.txt D176.txt Diabetic_pre-exercise
10 D177.txt D177.txt Diabetic_pre-exercise
11 D179.txt D179.txt Diabetic_pre-exercise
12 D204.txt D204.txt Diabetic_post-exercise
13 D221.txt D221.txt Diabetic_post-exercise
14 D253.txt D253.txt Diabetic_post-exercise
15 D255.txt D255.txt Diabetic_post-exercise
16 D261.txt D261.txt Diabetic_post-exercise
17 D262.txt D262.txt Diabetic_post-exercise
18 D267.txt D267.txt Diabetic_post-exercise
19 D273.txt D273.txt Diabetic_post-exercise
20 D276.txt D276.txt Diabetic_post-exercise
21 D277.txt D277.txt Diabetic_post-exercise
22 D279.txt D279.txt Diabetic_post-exercise
23 N101.txt N101.txt Healthy_pre-exercise
24 N108.txt N108.txt Healthy_pre-exercise
25 N113.txt N113.txt Healthy_pre-exercise
26 N170.txt N170.txt Healthy_pre-exercise
27 N171.txt N171.txt Healthy_pre-exercise
28 N172.txt N172.txt Healthy_pre-exercise
29 N175.txt N175.txt Healthy_pre-exercise
30 N181.txt N181.txt Healthy_pre-exercise
31 N182.txt N182.txt Healthy_pre-exercise
32 N183.txt N183.txt Healthy_pre-exercise
33 N186.txt N186.txt Healthy_pre-exercise
34 N187.txt N187.txt Healthy_pre-exercise
35 N188.txt N188.txt Healthy_pre-exercise
36 N201.txt N201.txt Healthy_post-exercise
37 N208.txt N208.txt Healthy_post-exercise
38 N213.txt N213.txt Healthy_post-exercise
39 N270.txt N270.txt Healthy_post-exercise
40 N271.txt N271.txt Healthy_post-exercise
41 N272.txt N272.txt Healthy_post-exercise
42 N275.txt N275.txt Healthy_post-exercise
43 N281.txt N281.txt Healthy_post-exercise
44 N282.txt N282.txt Healthy_post-exercise
45 N283.txt N283.txt Healthy_post-exercise
46 N286.txt N286.txt Healthy_post-exercise
47 N287.txt N287.txt Healthy_post-exercise
48 N288.txt N288.txt Healthy_post-exercise

[dpryan]

What are the dimensions of sampleTable and have you tried "design=des" rather than "design=~des"? For your first post, what were the dimensions of condition?

[sindrle]

I have also tried this:

status <- factor(c(rep("Healthy",26), rep("Diabetic",22)))
timepoints = c(1,1,1,1,1,1,1,1,1,1,1,1,1,2,2,2,2,2,2,2,2,2,2,2,2,2,1,1,1,1,1,1,1,1,1,1,1,2,2,2,2,2,2,2,2,2,2,2)
des <- formula(~timepoints+status)
ddsHTSeq <- DESeqDataSetFromHTSeqCount(sampleTable = sampleTable, directory = directory, design= ~ des)

Same error..

[sindrle]

Hi!
This is what Ive done:

sampleFiles <- list.files(path="/Volumes/timemachine/HTseq_DEseq2",pattern="*.txt")
sampleCondition <- read.table("/Volumes/timemachine/HTseq_DEseq2/03_SampleCondition.txt", header=TRUE)
sampleTable <- data.frame(sampleName = sampleFiles, fileName = sampleFiles, condition = sampleCondition)
directory <- c("/Volumes/timemachine/HTseq_DEseq2/")
ddsHTSeq <- DESeqDataSetFromHTSeqCount(sampleTable = sampleTable, directory = directory, design= ~ condition)


Im getting an error:

"Error in validObject(.Object) :
invalid class "DESeqDataSet" object: all variables in design formula must be columns in colData"