The motivation of the development of DESeq and DEXSeq is being able to estimate biological variability between replicates, and take this into account to call differentially expressed genes or exons. If you don´t have replicates, you do not know if the changes that you are observing are due to biological variation or due to the differences in your genotypes. In any experiment is crucial to do replicates, this is the only way to guarantee reproducibility on your differential expressed calls. For more details, you could check:
the discussion of our DEXSeq paper:
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Originally posted by areyes View PostHi gokhulkrishnakilaru,
The error talks by its own: "Underdetermined model; cannot estimate dispersions. Maybe replicates have not been properly specified.", you do not have replicates. Sorry that I can not help.
Alejandro
So no dexseq could work without replicates?
Is that the conclusion?
Is there a possibility to change the declaration while specifying the replicates in this section
Code:samples = data.frame(condition = c("WT", "KO"),replicate=c(1,1),row.names=c("WildType", "KnockOut"),stringsAsFactors=TRUE,check.names = FALSE)
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Hi gokhulkrishnakilaru,
The error talks by its own: "Underdetermined model; cannot estimate dispersions. Maybe replicates have not been properly specified.", you do not have replicates. Sorry that I can not help.
Alejandro
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Any thoughts anybody?
Sorry mods, for bumping up posts.
Urgent task. So, had to.
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Originally posted by areyes View PostYou could, but they are also deleted automatically in the function "read.HTSeqCounts"!
I was successful in making the counts file as you suggested. I ran the script. The following are my errors. Any pointers that could be of help?
Code:ecs<- estimateSizeFactors(ecs) > ecs<- estimateDispersions(ecs) Dispersion estimation. (Progress report: one dot per 100 genes) Error in FUN(c("ENSMUSG00000000078", "ENSMUSG00000000134", "ENSMUSG00000000182", : Underdetermined model; cannot estimate dispersions. Maybe replicates have not been properly specified. In addition: Warning messages: 1: In .local(object, ...) : Exons with less than 11 counts will be discarded. For more details read the documentation, parameter minCount 2: In .local(object, ...) : Genes with more than 70 testable exons will be kicked out of the analysis. For more details read the documentation, parameter maxExon
Last edited by gokhulkrishnakilaru; 10-10-2012, 06:35 AM.
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You could, but they are also deleted automatically in the function "read.HTSeqCounts"!
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Originally posted by areyes View PostBy the way, where can I download the annotation files you used?
That is where I got the one that worked for me.
You can use genome.ucsc.edu and go to tables section. Choose mouse and refseq genes and then refFlat or refGene. Select format to be GTF and if you are successful in preparing the annotations file. Please upload it somewhere or I can invite you to my dropbox. So, that way I have a refseq annotation file.
Thanks for the support, my friend.
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Originally posted by areyes View PostI see, I think the files you are using as input are causing some problems with the output of our htseq python scripts. I will check what is going on. In the meantime you can reformat your files to look more like this:
Code:FBgn0000003:001 0 FBgn0000008:001 0 FBgn0000008:002 0 FBgn0000008:003 0 FBgn0000008:004 1 FBgn0000008:005 4 FBgn0000008:006 1 FBgn0000008:007 18 FBgn0000008:008 4 FBgn0000008:009 16
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I see, I think the files you are using as input are causing some problems with the output of our htseq python scripts. I will check what is going on. In the meantime you can reformat your files to look more like this:
Code:FBgn0000003:001 0 FBgn0000008:001 0 FBgn0000008:002 0 FBgn0000008:003 0 FBgn0000008:004 1 FBgn0000008:005 4 FBgn0000008:006 1 FBgn0000008:007 18 FBgn0000008:008 4 FBgn0000008:009 16
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Originally posted by areyes View Postups, my bad in the gtf extensions thing...
does your files contain NAs also?error in scan(file what nmax sep dec quote skip nlines na.strings line 1 did not have 3 elements
I deleted those lines and it gives me another error sayingError in round(countData) : Non-numeric argument to mathematical function
Any pointers to these issues. This is my counts file's head
Code:"ENSMUSG00000000001" :001 1 "ENSMUSG00000000001" :002 0 "ENSMUSG00000000001" :003 0 "ENSMUSG00000000001" :004 1 "ENSMUSG00000000001" :005 0 "ENSMUSG00000000001" :006 0 "ENSMUSG00000000001" :007 0 "ENSMUSG00000000001" :008 0
Last edited by gokhulkrishnakilaru; 10-10-2012, 05:54 AM.
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Originally posted by areyes View PostHave you checked the size of your first file? Looks like you are replacing your input file with the output file.
Could you please include a reproducible code for your R code? with the output of the sessionInput()? Also, I would not have many hopes in the results without replicates
Alejandro Reyes
Yes, I did check the size of the input file. I am changing the extension. The input file has GTF and the output has GFF as its extension.
My R code is as follows
Code:library(DEXSeq) options(digits=3) setwd("/test/dexseq/") library(DEXSeq) rm(list=ls()) annotationfile = file.path("/test/dexseq/Mus_musculus.GRCm38.68.gff") annotationfile samples = data.frame(condition = c("WT", "KO"),replicate=c(1,1),row.names=c("WildType", "KnockOut"),stringsAsFactors=TRUE,check.names = FALSE) samples fullFilenames<- list.files("/test/dexseq/",full.names=TRUE,pattern="DEXSEQ.txt") fullFilenames ecs<- read.HTSeqCounts(countfiles = fullFilenames,design = samples,flattenedfile = annotationfile) head(counts(ecs)) head(fData(ecs))
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Have you checked the size of your first file? Looks like you are replacing your input file with the output file.
Could you please include a reproducible code for your R code? with the output of the sessionInput()? Also, I would not have many hopes in the results without replicates
Alejandro Reyes
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DEXSEQ Prepare Annotation File and R output
Hi Folks,
I have downloaded the DEXSEQ package from Bioconductor.
When I try to make the annotation file using the dexseq_prepare_annotations.py script, a gff file is generated but is of zero KB in size.
I tried with the following files
Mus_musculus.NCBIM37.64.gtf
Mouse_UCSC_Refgene.gtf (Refgene from UCSC)
Mouse_UCSC_RefFlat.gtf (Refflat from UCSC)
The command I tried was
Code:python dexseq_prepare_annotation.py Mus_musculus.NCBIM37.64.gtf Mus_musculus.NCBIM37.64.gff
Also, the R output from running DEXSEQ using Mus_musculus.GRCm38.68.gf and one wild type and one knock out sample (no replicates) didn't give me anything. All I see is NA in all the columns. I am using the bam files from Tophat aligned to Refseq annotation.
What am I doing wrong? Any pointers are highly appreciated.Tags: None
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