Hi,
I'm trying to run Deseq2 analysis (R-4.1.1, Deseq2 1.31.16) on RNA Seq data. We did targeted RNA Seq on 10 genes for expression data (only 10 amplicons...cases and finally the 2^-(ΔΔCt).
I would like to get the equivalent for my RNAseq data.
I tryied Deseq2 with tutorials but I only obtained log2fold change by genes... I don't know how to obtain fold changes by cases.
Important...informati…
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Hello,
I find myself with RNA-seq data where we want to look at differential gene over time. I've read DESeq2 vignette and performed LRT test just as propose.
My question is about my data set.
I have 6 patients from which I have 3...I find myself with RNA-seq data where we want to look at differential gene over time. I've read DESeq2 vignette and performed LRT test just as propose.
My que…
updated 5.2 years ago • andree-anne
Dear ALL.
i would like to perform some __transcription factor binding site enrichment analysis(/promoter analysis)__ but without sequences but rather geneIDs from 2 expression...Dear ALL.
i would like to perform some __transcription factor binding site enrichment analysis(/promoter analysis)__ but without sequences but rather geneIDs from 2 expression microarray datasets. Is t…
updated 9.1 years ago • svlachavas
contaminating all of our other cell types, which is why we did this decontamination step in the first place. However, I am now trying to run deseq2 to find DEGs for each cell type. When I use the regular count matrices, I get TONS...se, design = design, ignoreRank) :
some values in assay are not integers"
I know that Deseq2 is supposed to use raw counts, but what if raw counts are biologicall…
as compared to "t0", most genes will be under-expressed in "t4". Yet, as far as I read, EdgeR and Deseq2 default normalization of read-counts expect symmetrical under/over expression.
attached a diagnostic histogram...mean of all samples. I am suspecting, though cannot know for sure, that the true scaling factor is different (e.g., possibly red line should be shifted even more to the r…
Hi All,
I calculated size factors for RNAseq data using the estimatesizefactor(). They were:
1a 1c 2b 2c 1b 2c 3c
1.7260367 1.8360566 0.7666819 0.7158603
updated 5.5 years ago • mankadeep2
Hello,
I am trying to import transcript abundances (from kallisto .tsv files) to analyze with DESeq2.
My goal is to do a gene-level and a transcript-level analysis of differential expression. In my kallisto files however...does not allow me to directly compare the gene-level with the transcript-level analysis performed in DESeq2 (since the gene-level approach will only use the transcripts with …
Hi good day,
This is my first time working on differential gene expression analysis using DeSeq2. I am having a problem to construct my model matrix...for DeSeq2 since I have a very complex experimental design (3 factors design) with the following information;
ID Ewed Lambd Sex...Maternal diet
Lambd: offspring diet
Could anyone suggest me what can or should I do? I read the DeSeq2 vigen…
updated 6.9 years ago • sharmila.ahmad
First, my apologies if this has been covered already. I thought for sure it would have been, but I can't find the relevant info with...my searches.
It's pretty obvious from the DESeq2 vignette how to test whether a gene is differentially expressed (DE) and how to do so at various LFC thresholds, but what...power to make a determination, how do I do that? Is there something more sophisticated …
when I analysed mRNA data. I use TMM provided in
edgeR to normalizition my data. After I got the factors, How Do I
treat my raw counts?? In my oppion,
(raw.counts)*10^7/(librarysize*factors),Is It right?
And another question is which
updated 13.7 years ago • Sooby
hello,
I have a time-series, two factor design that I would like to analyze with DESeq2.
The factor levels are:
**Genotype** (wt, mut)
**Tissue_age** (old, young)
**Time** (pretreat...and young) was collected from the same plant in each bio rep.
**Q1.**Should I include the reps as a factor since they're derived from individual experiments? from the PCA they all group very nicely together.
**Q2…
updated 3.3 years ago • Maria
I am using DESeq2 for differential expression analysis. But I got big different results between the two versions while using the same...set of data as input and the same set of r code for data processing for the two DESeq2 versions. I would like to ask what is the key point changed between the two versions to make the results so different...I used DESeq2 package version 1.14.1 and pasilla version…
levels, "early", "late", and "untreated". I would like to know how to do a nested comparison in DESeq2 such that I compare ((early Vs untreated) Vs (late Vs untreated)).
I've pasted my code below:
<pre>
samples = read.csv("samples.csv...condition))
#attach the count data to the variable countdata
countdata = counts
#start DESeq2
library("DESeq2")
#construct your DESeq2 data se…
lower in the list.
The problem: all libraries are sequenced with rather similar depth.
Running DESeq2 and extracting normalized counts, the resulting total counts per sample vastly differ.
***Code and numbers:***
colSums...9894485 11149046 10372395 7993503 8691350 6914504
First 3 samples are Cell Type 1, next 3 are Cell Type 2. Cell Type 1 seems to have more initial read co…
updated 6.8 years ago • IV
hoping someone could explain what, exactly, the Intercept part of the results shows?
I have a two factor model. Three treatment groups (treatment 1, treatment 2, and control) in two species (species 1 and species 2).
I built the
updated 6.6 years ago • mrnsmi
do if we have a variable considered as "Batch" and we want to use `lfcshrink`.
In the vignette of [DESeq2][1] appears that the shrinkage estimator `apeglm` cannot handle contrasts but the design can be rearranged and nbinomWaldTest...pasAnno, row.names=1)
coldata <- coldata[,c("condition","type")]
coldata$condition <- factor(coldata$condition)
coldata$type <- fac…
updated 2.4 years ago • Eva
Hi,
I have read blogs on **how to remove batch effects in deseq2** and how to **visualize** it using **limma voom remove batch effect PCA**. I'm not sure if the batch effect was removed in my analysis...description here][1]
**PCA after removing batch effect**
![PCAafter][2]
**Heat map with batch factor included in design formula**
```design =~ Gender+ageRange+Batch+Cond```
![withBatch][3]
H…
updated 4.7 years ago • vinisha
Hi, I am doing some experiment and at a loss modeling by DESeq2. I want to __estimate abundance__ of genes across the three conditions (namely treatment1, treatment2 and treatment3...adjusted for covariates of age. I make DESeq object, and estimate size factors, estimate dispersions, and do Wald test as follows where data is raw count matrix.
<pre>
dds = DESeqDataSetFromMatrix...process?
…
updated 7.8 years ago • migimimi0
Hi All,
I want to compare Deseq2 with sSeq and I am getting similar log2foldchange values but the direction is opposite. I am confident with the DEseq2...nbsp; colData = coldata,
design = ~ Subject + Treatment)
dds
dds$Treatment <- factor(dds$Treatment,
&n…
div class="preformatted">hi,
in both edgeR and DESeq2, estimation of dispersion precedes negative
binomial GLM fitting.
my question is, can I use a design formula when estimating...after intervention.
thus, the full design is:
~ group*intervention + individual:group (blocking factor)
as I mentioned, estimation of dispersion with the above design is not
practical, and I thus would like to …
is returned:
<pre>
results(dds)
Error in results(dds) :
couldn't find results. you should first run DESeq()</pre>
This is right after successfully (I think) running DESeq.
<pre>
data<-read.csv("TotalRNA_reads.csv...but 89 genes
DESeq(dds)</pre>
The DESeq outputs this as it is running:
<pre>
estimating size factors
estimating dispersio…
Having trouble analyzing using DESeq2 (from phyloseq object). Goal is to test association of dietary intake with microbiome (effect of diet on microbiome...Having trouble analyzing using DESeq2 (from phyloseq object). Goal is to test association of dietary intake with microbiome (effect of diet on microbiome).
Phyloseq object `ps` contains otu_table, tax_table, sam_data, and phy_tree. Variable…
updated 5.4 years ago • zrf1
4.**Number of levels of mutation_status**: 2 (wildtype and mutant)
My design is as follows:
#first with the term:
dds <- DESeqDataSetFromMatrix(countData = countfile,
colData = coldata,
design = ~geneX_mutation_status...geneX_mutationwildtype.cancer_subtype5"
[12] "geneX_mutationwildtype…
seq (or, in my case, ATAC-seq). However, I wanted to check that some of the assumptions underlying DEseq2 do in fact translate, as some of my diagnostic plots differ from what I expected from reading the (excellent) DEseq2 documentation...for each sample. These counts (50,00 regions x 6 samples) constitute the count table I load into DEseq2. I then ran DEseq2 using design = ~ cell\_type to identi…
Dear all,
I am using DESeq2 for a design ~PAIR+GROUP and I obtained for geneX this result:
<table border="0" cellpadding="0" cellspacing="0" style...tr>
<tr>
<td>padj</td>
<td>0.06846016</td>
</tr>
</tbody>
</table>
The normalized counts by DESeq2 are these:
<table border="0" cellpadding="0" …
Hello,
I am currently using Deseq2 to perform differential analysis on my data. I have feature count data for 24 samples and 4 comparisons to do ( for each...in each of these 4 groups are from different tissues and cell types.
The issue is: when I run Deseq2 for all of the samples, specifying all 4 comparisons, I get very different foldchanges and p-values then when I split...the feature co…
rows and 5 columns
sample condition patient treatment sizeFactor
<character> <factor> <factor> <factor> <numeric>
sample1 sample1 WT 1 A 1.047160
sample2 sample2 WT 1 Ct 1.017620
sample3 sample3 tumor 1 A 1.056423...gene for which there is a specific effect of tre…
I have time series data from two condition, like 0h, 2h, and 6h by treatment A and B. I want to know FDR different between A and B at 1h fold change for example compared with their individual 0h. in other word, I want to subtract 0h expression first and then compare the two 1h secondly (take 1h for example). And I want to know every time point different compared with 0h...for example compared wi…
updated 6.4 years ago • Cindy
effect is very important... When doing a pairplots, I see that this batch effect can be seen the first and second PCs.
![enter image description here][1]
At first I wanted to correct for this effect by including the different...blueprint
```
So the idea we had was to use RUVseq, more precisely RUVg, to include correcting factor in the DESeq2 model, enabling us to correct the batch effect and c…
updated 20 months ago • Alexandre
Hi all,
I am running DESeq2 (version 1.36.0) for 7 different experimental groups. 2 of the groups only have 1 replicate.
I thought that DESeq2 does...still provide some information for the dispersion estimation.
To my surprise, after running DESeq2, I got a `DESeqResults` object for the group with only one replicate which even included some significant genes. Can...list(type = c("input"…
updated 18 months ago • nhaus
Hello,
I am seeking confirmation on manually setting contrasts for a 3-factor design in DESeq2 with multiple interaction terms
I have a 3-factor experiment, with each factor containing 2 levels...My experimental design formula is as follows:
<pre>
~Type*Shore*Sector</pre>
Base levels for all factors are: Juvenile, Inshore, Central
<pre>
And my Model Matrix column names ou…
updated 9.7 years ago • piensaglobalmente
4 replicates) and I'd like to find tissue enhanced genes. So the plan is to perform DE analysis with DESeq2 with a dataset where I included all samples twice, but the design matrix would tell that the first half of the dataset
updated 5.7 years ago • varga.torda
in two different condition. Thre was quality issue with the extrected RNA samples. Later i ran deseq2 normalization (by first using estimateSizeFactors and then variance stablizing transformation (vst) transformation...After the full deseq2 analysis i can see huge difference in replicate of one sample (many of the genes are highly expressed in one replicate...if my steps are correct (though i fo…
updated 4.4 years ago • Shail
I have a data set of six samples with 3x2 conditions.
> colData(dds)
<code>DataFrame with 8 rows and 3 columns
sample.ID condition date
<factor> <factor> <factor>
S1 S1 control d1
S2 S2 treatment d1
S3 S3 control d1
S4 S4 trea…
updated 8.2 years ago • Assa Yeroslaviz
I'm trying to use DESeq2 to perform something like a one-way ANOVA on 3
groups of samples. I've seen the same question and answer here:
https://support.bioconductor.org...I'm trying to use DESeq2 to perform something like a one-way ANOVA on 3
groups of samples. I've seen the same question and answer here:
https://support.bioconductor.org/p/61563/
However, I can't seem to g…
updated 10.2 years ago • jenniewoo
my circRNA data (have tried to look at all of the related posts on various sites)
I am using DESEQ2 for differential analysis and have my gene matrix counts for the linear genes:
[1] dds <- DESeqDataSetFromTximport...the SizeFactors and apply them to the circRNA count matrix? (If so how would I extract the size factors?)
Or
Something different?
You advice would be G…
updated 6.0 years ago • zoe.ward
Hi,
I am trying to analyze RNA-Seq Data with DESeq2 and could use some help. I have 2 genotypes and 14 timepoints. I want to find differences in gene expression between...genotype in the full and ~genotype+time in the reduced formula. Here is what I did so far:
library("DESeq2")
directory<-"............."
sampleFiles <- grep("??",list.files(directory),value=TRUE)
time <…
updated 11.2 years ago • nafeesa
Hello everybody,
I have analysed an experiment of ribodepleted samples using both DESeq2 and edgeR robust. I read that one can expect a concordance of 70-80% between both tools. Here, this is not the case.
In this...mutation (6 samples) and cell lines of controls (3 samples).
I attached the comparison of edgeR and DESeq2 for:
1/ cell lines of patient (12 samples) vs cell lines of controls (…
updated 9.6 years ago • Jane Merlevede
a very simple question but I am new to bulkRNAseq and would like to analyze paired FastQ files with DESeq2, rnaseqGene, or edgeR. Could anyone provide me with some simple code to get started? I would be happy to use any of the packaes...It seems like the first step is to convert the files to .txt files, which I can't figure out how to do
updated 3.0 years ago • vpl685
lt;- data.frame(row.names=colnames(countdata),genotype,Rep,stimulation,batch)
coldata$group<- factor(paste0(coldata$stimulation,"_",coldata$genotype))
dds <- DESeqDataSetFromMatrix(countData=countdata, colData=coldata...design=~group)
I used group for the design first but as I have sample from two different batch, I also would like to include batch effect into the model…
updated 6.8 years ago • Gyan Prakash Mishra
The documentation of the DESeq sizeFactors, as described in the supplement to the DEXSeq paper says:
"we multiply them with a suitably chosen constant c ... such that \[product of final sizeFactors\] = 1. This keeps the size factors close to unity and ensures that normalized count values ... remain close to their original values, making their interpretation, e. g. in plots, easier."
But I find …
updated 8.1 years ago • katzman
the differential analysis by comparing KD1 vs SCR, KD2 vs SCR, and 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 su…
Dear Dr. Love, dear persons knowledgeable about DESeq2
Yesterday (24. Oct.2016) I repeated an analysis of differentially expressed genes I had previously analyzed. This...from previous results that were generated at the end of August (with a presumably older version of DESeq2).
I am using a multifactor comparison, there are 24 samples with two states. In one example, a gene that was barely sign…
updated 9.2 years ago • Daniel Elsner
colnames(count\_data))
> colnames(site\_data)<-'site\_type'
>
> \#Perform Deseq2 magic
> dds<-DESeqDataSetFromMatrix(countData=count\_data, colData=site\_data, design= ~site\_type)
Usage note: the...following factors have 3 or more levels:
site\_type
For DESeq2 versions < 1.3, if you plan on extracting results for
these factors,…
updated 11.2 years ago • btemperton
Hello Michael,
I am new to DESeq2.
I have successfully used the results of function results() to do the plotMA. However, when I try
<pre>
lfcShrink...found</pre>
My codes are:
<pre>
setwd("C:/cygwin64/home/Coexpression_Nov2017")
getwd()
library(DESeq2)
library(apeglm)
library(ashr)
expected_matrix <- read.table(file = "17samples_expected_co…
updated 8.1 years ago • lychen83
where the second biological replicates have significantly less IP efficiency and reads than the first replicate, so when I tried running the DESeq2 analysis to find differential enrichment analysis, there were no significant...in the literatures.
An example is as such:
where the read counts for a certain peak for the first replicate (A_1, B_1) are both higher than the read counts for the sec…
updated 23 months ago • rebw25
2239</pre>
What I want is to asses allele-specific expression between two parents using Deseq2. My overall calculations look like
<pre>
groups<-factor(x=c(rep("par1",3), rep("par2",3)), levels=c("par1","par2"))
colData <- DataFrame
updated 8.3 years ago • gtechbio
sample size.
We would like to analyze RNA-seq expression from several hundred TCGA samples using DESeq2. One of the things we would like to correct for is tumor purity. We have the tumor purity for each of the patient ID\# ranging...help and advice on how to proceed.
Where should a covariate or possible confounding factor be adjusted for in the analysis described in here: htt…
So the question is: I do understand, that differential gene expression analysis in DESeq2 operates on the unnormalized count data and is not dependent on the assumption, that data is homoscedastic, meaning...Libraries##
```
library("tidyverse")
library("readr")
library("stringr")
library("DESeq2")
library("TCGAbiolinks")
library("DEGreport")
library("RColorBrewer")
library("pheatmap")
…
updated 6.8 years ago • Marc Saric
colData = samples,
design = ~ condition)
#...and run DEseq2
dds <-DESeq(dds)</pre>
__Question: __The orthologs for which I want to perform differential expression analysis are...with respect to the different transcript lengths (I presume that yes - also given the vignette of DESeq2::plotCounts which says that "the counts should be normali…
updated 7.5 years ago • al-ash
I am having trouble getting specific coefficient names after running DESeq2. The coefficients I get when I run resultsNames(dds) are "Treat1", "Treat2", etc instead of "Treat_B_vs_A", "Treat_C_vs_A", etc...had this issue? The code that I was running and the output are below:
```r
samples$Treat <- factor(samples$Treat, levels = c("A","B","C","D","E","F"))
samples$Treat
# A A A …
updated 4.7 years ago • jkhudyakov
Hey!
Thank you for a great resource! I am learning how to use DESEQ2 on time-series and I would like to get advice if my analysis is statistically "correct".
I have a single time-series i.e...the first time point is the control (t=-1) and the treatment starts from the second timepoint onwards (t=0). I have total of 8 timepoints
updated 3.3 years ago • Jigyasa
Hello,
I'm trying to analyse an RNA-Seq dataset using DESeq2 that has four experimental factors: disease, treatment, donor and sequencing run.
This is my colData (C is control):
<table...pre>
design = ~ disease + treatment + donor + disease:treatment</pre>
Adding the sequencing run factor to the formula gives me very few DE genes. When I run DESeq2 using the above formula, I get …
Hi, everyone.
My question is about the `` coef `` argument for lfcShrink function, and the overall experimental design formula.
I'm now trying to create a list of ranked genes for GSEA, and I ran into unexpected troubles.
My experimental design is 2 cell lines (b16,tc1a9) \* 3 replicates \* 2 conditions (treated,control).
I want to estimate the DE genes for each cell line …
Question is in the title.
I know there is a __collapseReplicates __function in DESEQ2. But I didn't succeed to use it so I did the sum up manually.
In the vignette ,
<pre>
The purpose of this function is to sum...raw counts by the number of technical replicates added because it will have an impact on the size factor ?
Thanks
updated 9.2 years ago • ZheFrench
Hi,
I am relatively new to performing DESeq2 analysis and I have a dataset where I am trying to compare mice tumor tissue with urothelium tissue. Because R automatically...Hi,
I am relatively new to performing DESeq2 analysis and I have a dataset where I am trying to compare mice tumor tissue with urothelium tissue. Because R automatically chooses a reference level for factors based on alph…
nbsp;
I am completely new to using R and DeSeq2 for RNA-seq analysis. My experimental set up is such that I have 3 biological conditions and 2 replicates per condition...count transcript per genomic interval. I then plugged this into the following script:
library(DESeq2)
countdata <- read.table("featurescountmatrix.txt", header=TRUE, row.names=1)
countdata &…
updated 7.2 years ago • a.rex
Dear all,
I'm struggeling to find the correct approach to handling batch effects. I have a longitudinal RNA-Seq experiment where patient samples were measured in 3 batches: First the samples from the inital visit (visit 0, batch "baseline") were sequenced, some had to be re-sequenced due to QC issues (batch...effects. I have a longitudinal RNA-Seq experiment where patient samples were measured i…
updated 4.9 years ago • Julia
Hi everyone,
I am using ``DESeq2`` (v1.26.0) to find DEGs between ``group1`` and ``group2``, and also between`` group2`` and ``group3``, respectly. And there are ten replicate...row.names=1)
readscount <- read.table('readscount.txt', header=TRUE, row.names=1)
condition <- factor(c(rep("group1",10),rep("group2",10)))
batch <- factor(c(rep("idv1",3),"idv2",rep("idv3",3),rep…
updated 5.7 years ago • Na Chen
![enter image description here][1]
Hi everybody,
I am working on a 16S dataset with 4 different groups (biodiversity gradient) and I successfully ran a deseq2 analysis for the phyloseq object (16S data) with this code:
```
diagdds = phyloseq_to_deseq2(phyloseq, ~ group)
diagdds = DESeq(diagdds, test="Wald", fitType="parametric")
res = results(diagdds, cooksCutoff = FALSE)
alpha = 0.01…
updated 3.2 years ago • Itsme
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