I am currently working in plant pathogen interaction and is trying to make a gene co-expression network using RNA-seq data. I used HTseq-count to get the read counts.
My dataset consist of Resis_treated (1st day, 3rd day and 7th day) samples taken under 3 time point with 2 replicated for each time point. I have a factor with 3 levels
condition
Day1
Day1
Day3
Day3
Day7
Day7
I am…
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Hello,
My recent RNA-Seq experiment had a few samples with high counts of rRNA genes in the third replicate. My pipeline was HISAT2 --> featureCounts --> DESeq2.
When clustering replicates 1 and 2, the replicates of each condition clustered closely together in a PCA. With all three...high counts of rRNA genes in the third replicate. My pipeline was HISAT2 --> featureCoun…
updated 4.2 years ago • jac
args' in selecting a method for function 'do.call': BiocParallel errors
element index: 3
first error: error in evaluating the argument 'x' in selecting a method for function 't': no right-hand side in 'b'
I can reproduce...error with a modified example dataset as used in DESeq2's vignette:
```r
# 1) Read example dataset from pasilla
library("pasilla")
pasCts <- sys…
updated 4.4 years ago • Steffi Grote
In paticular I would to compare:
`Ago miR vs Ago Control` adjusting for the `IgG factor`. The comparisons should looks like as follow: `(Ago.miR – IgG.miR) vs (Ago. control– IgG. control)`.
I was wondering if the right
updated 4.9 years ago • dequattro.concetta
goettingen.de> wrote:
> Dear Michael,
>
> I have been switching from edgeR/DESeq to DESeq2 and I'm happy to
include
> more complex experimental design to the analysis. Thanks for
developing and
> providing...for the last variable in
the
design formula, and for the last level of this variable over the first
level if this variable is a factor.â But other…
Hi there, Michael,
First, thanks for the great software and the excellent documentation you all have provided. My question is more theory-based...ortholog (i.e. summing the rpk values for all CDS within an ortholog into one row), and then running deseq2 on that table. I've spent a fair amount of time trying to get more familiar with your manual and thinking about this, and
updated 8.9 years ago • AstrobioMike
Hi,
I'm trying out DESeq2 (v1.8.2, R 3.2.2) for the first time and following the std bioconductor documentation, but am finding that the results
updated 10.1 years ago • Chris
alt="" src="https://i.imgur.com/C7S4O6L.png" style="height:475px; width:792px"/>
I thought the size factor normalization should take care of this? Removing the batch effect using limma's removeBatchEffect() is also not really
updated 7.3 years ago • ipso
div class="preformatted">Dear all,
I am trying do performed some analysis with DESeq2, using a model with
interaction therms, but I have a hard time understanding what DESeq2
is
doing exactly (I am also not...described in section "3.3
Interactions" of the vignette " Differential analysis of count data -
the DESeq2 package", i.e. one model with 3groups-2treatments, and one
with 2groups-2treatm…
dds)` for the former case, I get a NULL vector. Is there some documentation on how the size factor is calculated when the gene length is specified?
Thank you very much.
[1]: https://support.bioconductor.org/p/97936
Is it possible to compare one group with the mean of two others if betaPrior=FALSE? In previous versions of DESeq2, I could group C vs A+B using a list below, but I'm not sure how to work with the new resultsNames output like <span style="white-space:pre-wrap">trt\_B\_vs\_A.</span>
<pre>
dds <- makeExampleDESeqDataSet(n=1000, m=18)
dds$trt …
updated 7.9 years ago • Chris Stubben
Hi, I performed analysis using old 1.14 version of DESeq2. I had to repeat such analysis now with version 1.24. I've received very different results. With 1.14 I've received 63...Hi, I performed analysis using old 1.14 version of DESeq2. I had to repeat such analysis now with version 1.24. I've received very different results. With 1.14 I've received 63 hits...comes from?
Maybe I did sth wrong?…
updated 6.0 years ago • michalina.marija
and got the salmon merged files. Of these we used the transcript counts file and ran in on R using DESeq2.
Now I have a list of differentially regulated files.
I wanted to know, if this is the accepted workflow and if not what...And if possible, please provide the explanation in simple terms/ non-bioinformatics terms. (Its my first time with Bioinformatics analysis and I am having a tough time).
…
updated 11 months ago • sindhuri
pre>
# Generate example Data
dds <- makeExampleDESeqDataSet(n=100,m=18)
dds$genotype <- factor(rep(rep(c("I","II", "III"),each=3),2))
# Run DESeq2
dds1 <- dds
design(dds1) <- ~ 0 + genotype + condition
dds1 <- DESeq(dds1, betaPrior=F)
resultsNames...But then when I get meaningful differences (betas) in the call to "_results", _can I then t…
updated 9.2 years ago • Nikolaus Fortelny
Hello, I have an experiment without replicates, with three treatments (white, blue, and untreated) and three genotypes (mutant, wild type, and another genotype we call "four"):
blue wt
white mt
blue four
untr wt
white four
untr &…
Hi,
The DEseq2 vignettes is very helpful, but I'd still like to confirm if my understanding to the contrast design for correlated...expressed genes between case and control; (2) cell-type specific genes.
* __My first question is regarding the best way to do DE analysis for multiple group design. __
I could simply split the input...dataset into subsets, e…
updated 7.7 years ago • Xianjun Dong
Hi, this is my first time doing differential expression analysis and I have a few questions about the data output. My data was collected...though it was only detected in one sample.
For (A & C) these were NOT identified as a DEG with DESeq2 but were identified with edgeR's glmQLfit().
For DESeq2 I ran this with the default parameters. Any suggestions on which...also provide more examp…
updated 4.8 years ago • lnblock2
normalization techniques to account for batch effects on an RNAseq dataset. I am using DESeq2 for DE analysis, and RUVseq for normalization. For plotting purposes, I want to be able to extract the post-normalization...counts(dds, normalized=TRUE) call only extracts the counts based on the internal normalization that DESeq2 uses. Is there a way to extract (or compute)…
updated 10.4 years ago • nickp60
amp;ouid=118136492005618588020&rtpof=true&sd=true][3]
The count for s-173 was not flagged by DESeq2 (with design = ~ Group) and given a replaced count. How is this possible? And why is Cook's distance for S-173 so small?
> Below...counts and control for the variables I should control for? Is there a package I can use upstream to DESeq2 that performs refinement of outlier …
<div class="preformatted">Hello,
I'm trying to use DESeq2 for differential expression analysis of RNA-
seq
data containing Control (CR) and two Treatments (HR and SR). One of
the
biological replicate for HR treatment failed leaving me with 2
replicates
for CR and SR and 1 for HR.
DESeq document described "Working partially without replicates"
however,
that is not in the DESeq2 documentati…
Enter the body of text here
I have 3 questions about Paired Test in DESeq2.
**First**, I'd like to double check the design for the paired test. Here is the link https://support.bioconductor.org/p/58893...t-test?
```
design(dds) <- ~ patient + condition
Where 'patient' and 'condition' are factors which are columns in
colData(dds), and condition has levels: normal, tumor (wher…
updated 4.9 years ago • xiaofeiwang18266
drugB+drugC, drugA+drugC and no treatment/control arms. I have 3-5 replicates per arm.
I am using DEseq2 for analysis but I am not sure what should be the design. Is a simple model with treatment as a single factor with 8 levels...above code to study the effect of adding drugA to the combinations.
Is that correct?.
I am using DESeq2 version 1.32
Just to add
if dds_LRT is the ddsobject gener…
updated 3.3 years ago • SE
for my rna-seq experiment. I have two main groups each group has its own internal control. How can I first normalize them according to their internal control and then compare the two groups with each other?
My coldata:
![enter...image description here][1]
How can I design my coldata matrix and do the comparison with deseq2?
Thank you.
[1]: /media/images/c55ca60d-d598-4b9a-a6…
updated 17 months ago • kubracelikbas4
Hello,
I am have ChIPseq data from histone marks from 2 different condition (mock and treated with 2 biological replicas each) and the aim of my analysis is to study whether a particular histone mark is enriched in one condition versus the other.
I am new to bioinformatic and I don't have any statistical training and I am trying to teach myself how to normalise the data and perform the diff…
updated 2.8 years ago • Giulia
line-height:1.6">I have a RNA-seq dataset where I am trying to make comparisons among 3 levels of a factor - two parental genotypes and an F1. In addition to asking whether there is pairwise DE between genotypes (simple using...means) to the distribution of expression values of the F1. Is there a way to implement this test in DESeq2 - perhaps by setting a contrast between a value (mid parent m…
updated 10.5 years ago • johntlovell
nbsp;I have following output of DESeq2 on the following functions. It seems that if changes the fitType for dispersion only when I compute variance stabilized...countData=signal, colData=Design, design=~condition)
> dds <- DESeq(dds.1)
estimating size factors
estimating dispersions
gene-wise dispersion estimates
mean-dispersion relationship
final dispersion estimates
updated 10.0 years ago • tonja.r
Hello,
I am using DESeq2 for analysis of RNAseq data. I would like to ask you about the design in the DESEq2 formula. I have tissue from animals...FALSE)#a table with information about the samples
head(coldata)
str(coldata)
coldata$Treatment<-factor(coldata$Treatment)
coldata$Treatment %<>% relevel("water") #we need to have as reference always the control or untreated.…
updated 4.1 years ago • m.glymenaki
Hi
I have a question regarding different results when using contrast in DESeq2 vs when I do the analysis separately.
I have 3 groups :
A : condition A; n = 25
B : condition B; n = 25
C : controls; n = 350
I first looked...vs performing an isolated analysis for A and B, the results are not the same. (no FDR genes with the first approach vs plenty with the second)
I am wondering if the number …
updated 3.1 years ago • andree-anne
seq experiment and I was looking for the expression of a particular gene but I did not find it in my DESeq2 output table. I found this strange as I expect the gene to be expressed (perhaps not DE but expressed). So then I decided...to look into the salmon counts matrix I used to run DESeq2 and I see that the gene has indeed counts. This surprises me. Can anyone explain this to me? I have the outp…
updated 3.8 years ago • tomtom
that is incompatible
> with the current subsetting operation
>
>
> I then tried use DESeq2(1.4.5) and phyloseq (1.8.2) to run the same
codes, it worked without any issue. I also tried the combination of
DESeq2 (1.4.5...part of my question properly. What I really
meant is to compare :
> >>>
> >>> "Factor A level…
updated 11.5 years ago • Shucong Li
their expression when the
environment changes, controlling for the genetic background. I am
using DESeq2 for this, with the formula ~ genetics + environment +
genetics:environment.
However, the choice of the base level for the...factors in my
experimental design changes the genes that are significant. If I leave
the default reference levels (129 for
Hi,
This is my first time posting here. I have sequenced 16s rRNA from faecal samples.
I am looking at differential abundance analysis (DAA...using DESeq2. I wanted to know if there are significant DAA between older and younger aged people. I got over 80 OTU sequences as
being
updated 2.7 years ago • ms.roshanipatel
Hello,
I'd like to get some help on my RNAseq data analysis with DESeq2. The experiment is balanced design with multiple factors with biological/technical replicates.
<pre>
> coldata
group...model matrix is not full rank" as suggested in the manual, I used:
<pre>
> coldata$ind.n <- factor(rep(rep(1:2, each=2), 2)
> model.matrix(~ group + gr…
updated 9.8 years ago • yifangt
Hi. I'm very new to coding, but am trying to run the DESeq2 package for an expression analysis and am having trouble that I think should be relatively easily dealt with.
The...Hi. I'm very new to coding, but am trying to run the DESeq2 package for an expression analysis and am having trouble that I think should be relatively easily dealt with.
The design...treatment, all treatment regardles…
updated 6.1 years ago • tinglec
anova function in R,
I have 2 questions -
1. Does Limma allow inclusion of covariates ? How do I first adjust
the expression dataset to remove differences because of the sample
being a diseased sample and then understand...interaction term) would give me this ?
2. I was trying include both gender and group information as factors -
but when Im trying to build the model matrix -
design <…
Hi everyone -
I'm running a Differential Expression analysis via DESeq2 (DESeq2\_1.16.1) .
I have three levels in the conditions (SNP\_A, SNP\_B, and control), and I set as reference the "control" level...well. However, when I try to run the lfcShrink function for the same contrast, only work for the first two (the ones comparing each SNP with the control), but not for the contrasts between SNP…
div class="preformatted">Hi,
I used DEseq2 v. 1.3 under R-devel because I have many different
values for my design (celltype) and it worked ok. I upgraded my
libraries...colData names(2): sizeFactor celltype
> genex<-DESeq(genex)
using pre-existing size factors
estimating dispersions
gene-wise dispersion estimates
Error in relevel.factor(coldata[[v]], as.character(coldata
where W\_1 are the coefficients of unwanted variance
In edgeR manual batch effect should be the first in the design matrix:
`` design <- model.matrix(~Batch+Treatment) ``
Also, when I perform DESeq2 analysis as with the
`` DESeqDataSetFromMatrix...vs WNN<br/>
Wald test p-value: condition WEN vs WNN</code>
What would be a correct way to include factors of unwanted va…
Hi,
I'm a fairly new DESeq2 user and was hoping to get some help with how to correctly account for technical replicates in my dataset. Here is some example data:
suppressPackageStartupMessages(library("DESeq2"))
coldata <- data.frame(
'group' = factor(c(rep('group1', 4), rep('group2', 4))),
'sample' = factor(c(1, 1, 2, 2, 3, 3, 4, 4)),
'tech_re…
updated 2.6 years ago • sara.p
Hello,
I'm relatively new to DESeq2 and having problem with multiple factors.
I'm analyzing 16S data from patients with 3 different treatments (FOS, PN, ABX...gt; colData(dds)
DataFrame with 39 rows and 3 columns
abxgmposneg study_day FOS_pn
<factor> <factor> <factor>
ch10163 gmneg 7 yesFOSnoPN
ch10164 gmneg …
updated 5.2 years ago • Daniel
factors: fertiliser (ft) is nested within variety (vr) which is nested in crop protection (cp).
- based on this experimental design...bl) # meta_field is the metadata file from my phyloseq object, strata adds 'block' (bl) as random factor
```
![Permanova results][1]
and found out that I have a significant different microbial composition (*p* = 0.014) with the interaction...strata = 'bl')
![…
updated 4.2 years ago • M.seeliger2
to fix the error message I'm getting (see below)
This is the code I'm using:
```
library(DESeq2)
directory <- "/path/to/RNAseq"
sampleFiles <- grep("genecounts",list.files(directory),value=TRUE)
#I made columns for each...sampleTreatment,
Origin = sampleType)
sampleTable$Microbiome <- factor(sampleTable$Microbiome)
sampleTable$Region &…
updated 5.6 years ago • tjk30
214 samples(paired). I used following code(pretty much default). By setting like this, I expect DEseq2 giving me a result using the pairing information as well as 107 patient as biological replicates, is this appropriate...I feel not confident because I use 107 different values for the factor-- patient.
library('DESeq2')
directory<-"tBRCA"
sampleFiles <-c…
updated 10.1 years ago • luren
given 3 different treatments (control, drug1, drug2).
We are testing drug 1 against control in the first instance:
We also observed that there is some variation among isolates - i.e., batch effect
We load the data into DESeq2...Code should be placed in three backticks as shown below
```r
configure = data.frame(drug=factor(c("control","control","control","drug1","drug1","drug1")), isolate=c("…
updated 4.0 years ago • Jonas
Hello, I `ve conducted DEG analysis by Deseq2 and I just want to make sure that I´m right. I have to following information for the design:
```info:```
```region: region1, region...species and region. Within each region/species I have 5 replicates.
Is it ok to use the combined factor (group) to test for my comparisons of interest:
--> differences in species: region1_species1 vs…
updated 4.8 years ago • sj
alt="PCA" border="0" src="https://i.ibb.co/nBy0jmY/PCA.png"/></a>
I have analyzed the data with DESeq2 with designs either accounting for pairing (SampleID factor) or not, with dramatically different results. Here are...the design formula:
One Factor (1F) model, no pairing: ~Fraction
Two Factor (2F) model, control for sample pairing: ~SampleID + Fraction
Looking at the histog…
updated 6.0 years ago • SeqGoblin3
Hello,
I want to use DESeq2 for DE analysis. I tried to perform a nested comparison. My data has two different factors: the mice are either 1. Uninfected
analysis by comparing KD1 vs SCR and KD2 vs SCR, but then SCR vs WT.
I have created my DESeq2 condition vector as follows, so that SCR is used as the baseline for the comparisons.
```r
metadata$Condition <- factor...Negative SCRvsWT log2fc respectively).
I saw in another discussion post that the calculation in DESeq2 is more complex than manual calculation, and am not sure if my …
updated 7 months ago • sk
manager to do it). Start R again. Try the original installation code:
<pre>
>install.packages("DESeq2")
>library(DESeq2)</pre>
Doesn't work. So I try:
<pre>
>source("https://bioconductor.org/biocLite.R") #aiming to continue with...gt;<span style="line-height:1.6">biocLite("DESeq2") and </span><span style="line-height:20.8px">>…
updated 10.1 years ago • carman1979
input raw counts file containing all the groups and replicates (e.g. A1, A2, A2, B1...) using the DESeq2 result function like this:
res<- results(dds, name = "condition_B_vs_A")?
Or, is it better to first create the file containing
updated 6.4 years ago • sstankovic
I am going through the manual of DESeq2, there are some places I still don't understand:
dds <- DESeqDataSetFromMatrix(countData =countdata, colData = coldata...in the manual it is:
dds <- makeExampleDESeqDataSet(n=100,m=12)
dds$genotype <- factor(rep(rep(c("I","II"),each=3),2))
design(dds) <- ~ genotype + condition + genotype:condition
…
updated 5.3 years ago • Kai_Qi
I am working with a complex experiment design [shown here][1]. I followed the [DESeq2 manual][2] and suggestions in [another post][3] to remove the unpaired samples from my analysis and got the DESeq2 running...I am working with a complex experiment design [shown here][1]. I followed the [DESeq2 manual][2] and suggestions in [another post][3] to remove the unpaired samples from my analysis and go…
updated 6.6 years ago • sutturka
Mut3_2 Mut3_3
# colData names(2): condition sizeFactor
resultsNames(dds) # the results I have from DESeq2 saved in the dds
# [1] "Intercept" "condition_Mut1_vs_Ctrl"
"condition_Mut2_vs_Ctrl" "condition_Mut3_vs_Ctrl"
When I...to create the html report of the DESeq2 results obtained,
I
do the following:
# Creating the html report using ReportingTools:
desReport <- HTMLReport…
updated 11.9 years ago • Dimitra Alexopoulou
I would like advise about how to make a design for my experiment using DEseq2. In principle my question is straightforward, I'd simply like to find differentially expressed genes between two...I would like advise about how to make a design for my experiment using DEseq2. In principle my question is straightforward, I'd simply like to find differentially expressed genes between two conditions. How…
updated 6.9 years ago • cintapq
I’m working on RNAseq data using the DEseq2 R package. Most of the analysis will be differential expression between 2 (or 3) groups of samples with at least 3 biological...differences in total mapped reads between samples I use the default method for normalization in the DEseq2 package (the estimateSizeFactors function with default arguments, where a size-factor per sample is calculated as...for …
for your prompt response and patient
support.
Now the samples can be be classified with few other factors
like Gender( 'G') , Hypertension ( H ), Drinking ( D ).
So if the samples are labeled using these factors as well, it looks
like following...0 0 1
...
...
If I want to include other factor (e.g. Age, smoking habit ) in the
comparison (i.e…
for the inconvenience, I was hoping that @mikelove in particular could help me.
On the same dds DESeq2 object, I performed a DGEAnalysis with DESeq2, through 2 approachs :
- One all-steps-in-one
```r
dds <- DESeq(dds)
```
- and one step...see what to modify ?
And (if you have the time) another question, what is officially the default DESeq2 normalization method **name**, Means of Ratio Me…
updated 21 months ago • RL
counts. <span style="line-height:1.6">I receive this error when I run the DESeq function in DESeq2:</span>
<pre>
estimating size factors
Error in estimateSizeFactorsForMatrix(counts(object), locfunc = locfunc, :
every gene
updated 9.5 years ago • pjgalli2
Hi all,
I had a question about DESeq2, or a little sanity check.
In my case I'm only interested in comparing how expression differs between conditions at...Hi all,
I had a question about DESeq2, or a little sanity check.
In my case I'm only interested in comparing how expression differs between conditions at specific...time-points. I believe I can do comparisons with combined factor…
updated 2.5 years ago • smac97
users and developers,
I have 2 questions regarding a gene ontology analysis of RNA-Seq data.
I first used the DESeq2 package to perform the differential analysis
of my data and it resulted in a list of about 500 DEG. Now...are defined as "all genes for
which RNA-seq data was gathered for your experiment".
As you known, DESeq2 filters out low counting genes and outliers. So I
wonder if I should…
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