Bioconductor Forum

New to DESeq2. I am using DESeq2 to normalize data for some microbiome and eDNA metabarcoding projects. My datasets are observational...ecological and there are many factors that I am interested in assessing with downstream analyses. For example, with my microbiome dataset, I am interested...species. However, I'd also like to do more analyses with machine learning on other ecological factors. …

DESeq2

updated 2.4 years ago • Devin

question if such exists. To keep this simple, I can go into more details in response, I am using DESeq2 to analyze the effect of heat stress on expression in two closely-related fish species.  I want to determine if...species vary in their response to the heat stress. So the model I used in DESeq2 was: <pre> dds=DESeqDataSetFromMatrix(countData=countmatrix, colData=coldata, desi…

deseq2 sva interaction term grouping variable deseq

updated 7.2 years ago • kevin.kingsland

Hello, I am very new to using interactions using DESeq2 and handling multi-factor design. I have two groups 'Treated' and 'Untreated' which include both males and females in both...groups. First, I am interested in looking into comparing treated vs untreated without considering sex specific effects. And in the

deseq2

updated 6.0 years ago • hrishi27n

in gene expression between the left and right hemispheres changes at each time point relative to the first time point, while controlling for the covariates (`cov1` and `cov2`). For this reason I convert `time_point` to a factor, as well...as `animal_id` and `hemisphere` (setting hemisphere `R` as baseline): ``` df$time_point <- factor(df$time_point) df$animal_id <- factor(df$an…

DESeq2 paired design

updated 3.7 years ago • dr

Hi  Im having alot of trouble applying DESeq2 to my metagenome gene abundance data , any advice regarding where I may be going wrong would be much appreciated.Early...lt;- estimateDispersionsGeneEst(dds)</pre> and get this error... <pre> Error in .local(object, ...) : first calculate size factors, add normalizationFactors, or set normalized=FALSE</pre> I tried…

deseq2

updated 7.8 years ago • brijon

I had a query regarding what DESeq2 assigns as ControlGenes (housekeeping genes). As per the manual, the Normalized Counts can be determined by estimateSizeFactorsForMatrix...controlGenes) While, geoMean is explained, controlGenes are the first 200 genes? Could anyone please explain the criteria for selecting the first 200 genes as Control? Is there a way in DESeq2

normalization deseq2 sizefactors

updated 4.9 years ago • mankadeep2

Hi all, I have RNA-seq data (5 subjects are measured on 4 time points) and would like to do a SVA first to be able to include potential confounders into the statistical model (Deseq2 pipeline). I am having troubles how to...Null model ~ SUBJECT.ID** OR **Null model ~ 1** Should the subjects ID be treated as a factor of interest or as a confounding factor? Thanks in advance! …

SVA deseq2 repeated measures

updated 5.5 years ago • sara.blocquiaux

There, I am trying to do a differential abundance analysis with microbiome sequencing data suing DESeq2 package, but I keep getting errors after trying different approaches within the package. The data is a 50 by 501 matrix...with each row being a sample and the first column being the group indicator and the other 500 columns are sequencing reads for 500 taxa. No missing data in the...Please h…

deseq2 microbiome sequencing

updated 5.7 years ago • zhigang.li

Hi all! Using DEseq2 (v 1.30.0) I try to analyze a "complex" data set with 2 factors (A and B) harboring different levels. Factor A (named hereafter...Hi all! Using DEseq2 (v 1.30.0) I try to analyze a "complex" data set with 2 factors (A and B) harboring different levels. Factor A (named hereafter "line") has two levels (infected/non-infected) and factor 2 (named hereafter "group") has 4…

DESeq2

updated 4.2 years ago • lessismore

div class="preformatted">Hi all, I have a question concerning the normalization factors in edgeR. I think that if we know which genes are none-DE genes in advance, we could calculate "true" normalization factors...based on those information. Given "true normalization factors", edgeR could find more right DE genes, or even find all the right DE genes when the simulation is simple. …

Normalization edgeR Normalization edgeR

updated 10.7 years ago • Zhan Tianyu

ways to get the condition effect for one set basing on  the examples parts of results: <pre> # first one: # the condition effect in set Z # (the interaction effect added to the main effect) dds <- makeExampleDESeqDataSet...500, m = 18, betaSD = 4) dds$set <- factor(rep(rep(c("X","Y","Z"),each=3),2)) design(dds) <- ~ set + condition + set:condition dds &…

deseq2 interactions contrast

updated 9.8 years ago • shao

Hello, I'm working on differential expression analysis with the given sample data using DESeq2. <pre> Strain=rep(rep(c("ST1","BT6","MT3","XT7"),each=3),4) Treatment=rep(c("Low","High"),each=24,1) Tissue=rep(c("Lvr","Hrt"),each=12,2) targets<-data.frame...Hello, I'm working on differential expression analysis with the given sample data using DESeq2. <pre> Strain=rep…

deseq2 edgeR multifactorial design limma voom

updated 7.9 years ago • Alan Smith

p/101210/ Below is an example of the code I am running and the errors I encounter: ``` library(DESeq2) countdata<-read.table("T1.txt", header=TRUE, row.names=1, check.names = F) class(countdata) countdata<-as.matrix(countdata...class(countdata) condition<-factor(c("Control,"Treatment")) (coldata<-data.frame(row.names=colnames(countdata), condition)) coldata dd…

DESeq2 DEG

updated 3.1 years ago • priya.dalvi

Hello, I'm running DESeq2 for my expression analysis and I'm having trouble with the design formula. I have 32 mice samples and 3 different factors...and each factor has 2 levels. So the last factor (tissue) has always 4 biological replicates. Here is an overview:   <table align="center...Another thing I could do is to run DESeq several times but exclude samples. For example for th…

deseq2 multiple factor design contrast interactions

updated 10.1 years ago • mat.lesche

Hi, it would be nice if the first factor can be names differently than 'condition'. It is really hard to figure out that this needs to be specified and even

DEXSeq

updated 8.3 years ago • bjoern.gruening

from SNP arrays. I would like to know if those clusters are enriched for any of the clinical data/factors the samples come with. One example of a result for cluster 1 could be: <table align="center" border="1" cellpadding="0" cellspacing...td> <td> </td> <td> </td> <td> </td> <td> </td> </tr> <tr&g…

R overrrepresentation factor variable factor enrichment

updated 8.5 years ago • IOM

I am planning to use DESeq2 to compare gene expression in samples from two different species of mammals with sequenced genomes. My plan is to...species after they are normalized wrt library size and average transcript length as is done in DESeq2? (2) I wanted to understand the normalization that DESeq2 does in more detail, so I dug through the DESeq2 source code and...separately implemented …

normalization deseq2 across species rnaseq

updated 4.9 years ago • rmurray2050

consider normalizing the counts for each of the samples before combining them. That is, I would use DESeq2 size factors to normalize independently the samples in m1-r1, m1-r2, m2-r1, and m2-r2. My documentation and google search...might         make  the p-values more conservative? My first questions are about the i…

deseq2

updated 7.6 years ago • bge

mouseRCB2.html and saw that the model was specified as "formula = ~ celltype + mouse", in which the factor of interest (cell type) is listed first and the blocking factor (mouse) is second. However, in every other linear model I...ve used (e.g., limma, deseq2), the blocking factor is listed first, then the factor of interest (i.e., ~ mouse + celltype). Which is the correct approach? Thank

msqrob2 ProteomicsWorkflow proteomics

updated 2.9 years ago • Jane

sampleFiles <- grep("counts", list.files(directory), value=TRUE) sampleTreatment <- factor(rep(c(rep("control",3), rep("A",3), rep("B",3), rep("A_B",3)),3), levels=c("control","A", "B", "A_B")) samplePatient <- factor(c(rep("p1",12), rep("p2",12), rep("p3",12...However, I attempted to do as the poster here did, which was to combine the patient and treatment factors and then…

deseq2

updated 5.2 years ago • jropa

genes in each treatment with respect to the different time points**. To achieve this, I compared first the treatments against the control, however, I am also interested in identifying those DEG between each time point...What I have been doing is to follow the interaction section of the DESeq2 vignette: ```dds <- DESeqDataSetFromTximport(txi, samples, ~1) colData(dds) dds$g…

deseq2

updated 6.2 years ago • Ale

and then Tximport. my question is what is the correct command line to import my txi data file as a deseq2 dataset in a  time series experiment. Is the following one correct or not??? I need to  import my txi data file for...deseq2, because I need to do quality assessment and latter deferential gene analyses. I use the following command  dds...Time + Condition:Time) b…

deseq2 tximport

updated 6.4 years ago • ahmedaalkarim

In a differential abundance modelling using DESeq2, given a single ASV (out of about 1000) with the following abundance values: | | | Seq 3 | |----|------|-------------| | 1 | A1-2 | 26250 | | 2 | A1-3 | 45729 | | 3 | A1-4 | 56033 | | 4 | A1...In a differential abundance modelling using DESeq2, given a single ASV (out of about 1000) with the following abundance values:…

DESeq2

updated 17 months ago • Roey Angel

Dear Bioconductor community, First of all, thank you for this forum that helped me many times during my PhD years. This is the first time I'm posting a question...a long time now. My problem is the following : I would like to introduce bias coefficients into DESeq2, before differential expression analysis. Basically, what I would like to do is to multiply all my raw counts in a given.…

DifferentialExpression DESeq2 BatchEffect

updated 6 months ago • CriticalPeriod

according to Deseq (not DeSeq2) within Galaxy. I compared triplicates of cells, where the protein was not induced against triplicates of cells, where...i did my experiment the very same way with the truncated protein, there was the new version of DeSeq, DeSeq2. When i run the programm, the output told me, that only 4 genes are regulated, 1 of them is the control gene, which is an enormous...ou…

deseq2 truncated protein

updated 9.8 years ago • ChIP-Tease

account differences in size factors; and in > datasets with large variation in sequencing depth (dynamic range of > size factors ≳≳4) we observed undesirable...gt; of the VST. But in the R vst function page: > The rlog is less sensitive to size factors, which can be an issue when > size factors vary widely. https://www.rdocumentation.org/packages/DESe…

rna-seq deseq2 warnings nbinomWaldTest transformation

updated 4.8 years ago • andreia

my RNAseq raw data to cDNA using Kallisto and import the result using _tximport_ as suggested by the DESeq2 tutorial (tx2gene maps ensembl\_transcript\_id to ext\_gene symbol) <pre> txi.kallisto_gene <- tximport(files, type...I looked into the <span style="white-space:pre-wrap">txi.kallisto\_gene, it shows as below, the first row does not have a name, anyone knows ho…

deseq2

updated 4.2 years ago • Raymond

Hi, I'm trying to understand the normalization for DESeq2 analysis within DiffBind.  If I run: <code>Pool1 = dba.analyze(Pool1,method=DBA_DESEQ2,<strong>bFullLibrarySize...Pool1.DB) are calculating by taking the raw counts for each peak divided by the normalization factor s\_j calculated via the median of ratios method described in (http://genomebiology.com/2010/11/10/R106)…

diffbind deseq2

updated 9.9 years ago • JasonLouisStein

Hi Michael, I got some question about the tutorial of DESeq2 and I was wondering if you could help me in: 1) normalized metric you use in Deseq is not referring to RPKM or TPKM, but: "counts...divided by sample-specific size factors determined by median ratio of gene counts relative to geometric mean per gene" or median of ratio.. so 1) creates a pseudo...geometric mean), 2) calculates ra…

deseq2 normalization

updated 5.9 years ago • santamariagianluca

Dear there, Is it expected to get negative tag count by using DESeq2? I am using HOMER (__getDifferentialPeaksReplicates.pl __http://homer.ucsd.edu/homer/ngs/peaksReplicates.html...width:876px"> <tbody> <tr> <td>Inp1_tagDir Tag Count in given bp (14503111.0 Total, normalization factor = 1, effective total = 10000000)</td> <td>Inp2_tagDir Tag Count in gi…

deseq2

updated 7.7 years ago • xiaofeiwang198266

Hello, In a multi-factor DESeq2 design, I want to extract **normalized counts adjusted by multi-factor designs**; batch, gender, etc... But **counts(dds...normalized = TRUE)** only accounts for size factor. A similar post was made 7 years ago, but at that time it stated that DESeq2 did not have such a feature. https://support.bioconductor.org...p/68123/ Is it still impossible to ac…

DESeq2

updated 2.5 years ago • s26sw

B P 11 sample_5 after B P 12 sample_6 after A P</pre> The base factor levels are "before", "A" and "N" (in `` df[2:4] ``) after re-levelling. Previously I have combined the factors into various different...and performed contrasts between the groups for all manner of combinations. <pre> df$c1 <- factor(paste(df$time, df$group, sep=".")) df$c…

deseq2

updated 8.1 years ago • vincent.knightschrijver

So, I have experiment design with 4 conditions, A, B, C, and D. I want to get the DE genes list with DESeq2 for all possible comparison (A-B,A-C,A-D,B-C,B-D,C-D). I tried voom/limma to do this and they can do it. Now, I want to try using DESeq2...I have specified conditions for design parameter in the DESeqDataSetFromMatrix. It only gave me the first and the last comparison (A-D). Anyone can help…

deseq2 rnaseq

updated 9.3 years ago • bharata1803

I(sample_name), cell_line = cell_line, stimulant = stimulant, screen = screen)) #start DESeq2 library("DESeq2") #construct your DESeq2 data set, making sure to specify the design matrix here dds <- DESeqDataSetFromMatrix...so they match the sample metadata sheet colnames(dds) <- coldata$shortname #add the combined factor group to dds, reset design to ~group dds$group &a…

deseq2 differential gene expression rna-seq

updated 9.4 years ago • erin.gill81

Hi, is it possible to compare this 7 factors (levels) at once? dds$brain_areal = factor(dds$brain_areal, levels = c(**"primary and secondary corticies","limbic and association...white matter structures", "cerebellar gray matter", "striatum"**)) Normally R uses the first and the last factor for comparison. I want to know, if I can perform everything at once? I want to create Networks …

DESeq2

updated 20 months ago • melander

I am experiencing problems with adding covariates to my paired samples design in DESeq2. We have 39 paired samples of human subjects (39 untreated samples; 39 treated samples). So in summary, it is a ‘time series...in the effects of treatment on gene expression. Based on the information available in the DESeq2 vignette, I created the following design: <pre> ~ patientID + treatment</pr…

deseq2 deseq paired samples covariates multiple time points

updated 7.5 years ago • imb

I am working on a more complex design, but I think a basic 2-factor (each with two levels) captures the spirit of my question. Namely, I am interested in creating "reasonable" mock data to...appropriately test my understanding of extracting the appropriate contrasts in DESeq2's `results` method. In the script below, I create some mock data using DESeq2's `makeExampleDESeqDataSet` method. I t…

deseq2 deseq

updated 5.0 years ago • blawney

Hi I have a question regarding RNAseq data analyses by DESeq2, I have some data with 3 conditions (A, A1, D) and 3 other factors (P, B, Sex) with 2 or 3 levels in each, my question is that if I use...ddsMF) res_A_D_July17_ncon <- results(ddsMF)</pre> 2- if not, how a two-way comparison of DESeq2 is able to tell me how different the 3 conditions are from each other, I mean if I …

deseq2

updated 6.7 years ago • soheilazareie

Hello, I run DESeq2 with multilevel. For each of the levels, I run "results" with the desired contrast between pairs of conditions. When I...Hello, I run DESeq2 with multilevel. For each of the levels, I run "results" with the desired contrast between pairs of conditions. When I run...resultsNames, I noticed that  output refers to the first level as background. Also, lfcShrink&nbsp…

deseq2 lfcshrink

updated 7.0 years ago • pierre.k

Hi, I am using DESeq2 with this model matrix (dput of the model matrix and DESeq2 command are pasted below). However, DESeq2 complains that...s1 = structure(c(2L, 1L, 1L, 1L, 1L, 1L, 2L, 1L, 1L, 1L, 1L, 1L), .Label = c("0", "1"), class = "factor"), s2 = structure(c(1L, 2L, 1L, 1L, 1L, 1L, 1L, 2L, 1L, 1L, 1L, 1L), .Label = c("0", "1"), class = "factor"), s3 = structure(c(1L, 1L, …

deseq2

updated 6.0 years ago • arielle

some advice for a complex experimental design (complex at least for me!). I have a dataset with 3 factor variables, as follows: 4 genotypes A, B, C, D  2 tissues A, H  2 infection status UI, I  Each combination of factor

deseq2

updated 7.2 years ago • julien.varaldi

Dear DESeq2 Experts, I found a miRNA that is expressed significantly different between 2 groups. Below is the output from DESeq2...at the figure generated by "plotCounts", it seems the difference is mainly driven by an outlier in first group. There is the link to the figure: https://figshare.com/articles/t1\_png/5219959   My question is, why DESeq2

deseq2

updated 7.7 years ago • nikmehr22

Hello, I'm analyzing some RNA-Seq data using DESeq2 and have a couple of questions on how to set up my experimental design. My experimental design has a control ("ctrl") and

DESeq2

updated 4.2 years ago • GlycineMax

Hi, I have a question regarding outlier replacement when including a continuous variable in DESeq2 design. I am currently analyzing Nanostring data using RUVseq normalization and DESeq2 following the method by Bhattacharya...et al. As the normalization factor from RUVseq is included in the design, outlier detection and replacement will not be performed by DESeq as described...57 out of 770 gene…

DESeq2

updated 2.6 years ago • e.v.egeland

I am using DESeq2 for a while now, but I never see people talking about the use of the function "estimateSizeFactors" with the option&nbsp...controlGenes". For what I understand, it is supposed to be used with spike-ins, in which case DESeq2 will estimate the size factors only with the spike-ins and use it to normalize the counts of the other genes.  In my

deseq2 differential gene expression controls references

updated 6.4 years ago • cris.kgs

I'm attempting to run a glm analysis with edgeR on 32 biologically independent samples, with 3 factors. I have sex, age, and genotype. I'd like to create a full design matrix, but I'm having some trouble doing this, and understanding...gt; sex = rep(c("m","f"),each=2,times=8) > time = rep(c("p0","p2","p6","p12"),each=8) > geno = factor(rep(c("WT","KO"),times=16)) > geno = …

edger glm rnaseq

updated 7.3 years ago • Denniswu

2 in age=adult. > > From what I know, there is no way to receive interaction between three factors at a time. Yes, this is true for betaPrior=TRUE, the default. I only implemented the shrinkage of effects for models with...want using only one full data set containing all the samples? > > I had an idea to combine the factor age and condition into one factor: age_condit…

updated 10.9 years ago • Michael Love

Hi all, We have a 2-factor, 2 levels per factor experimental design with 2 replicates per covariate combination (so 8 samples total, n=2 per cell...that we are analysing using DESeq2. Normally DESeq2 generates a Cook's distance for each replicate within a cell (by "cell" I mean unique combination of covariates...individual observation with the high Cook's distance, but we are not aiming fo…

deseq2

updated 5.8 years ago • stuart

Hi, I've a multifactor design (5 factors, each having two possible value (0 or 1)). One of the factor specify the type of samples (tumor, control) and the 4 others are...sample3 1 0 0 1 0</pre> I want to test the effect of these mutations (factor geneA, geneB, geneC and geneD) taking into account the sample type (factor normal indicating if it's a tumor (=0) …

deseq2

updated 8.6 years ago • Nicolas Rosewick

to ensure that I am using the correct design formula for my experimental design. I have a condition factor with a control level and four other levels in no particular order (CL, A, B, C, D), biological sampling was done in triplicate...pairs (C vs A) of conditions different from each other? **What I've tried** I've read the DESeq2 paper, the vignette, and rummaged through various post …

deseq2

updated 6.2 years ago • galen.seilis

unique gene reads) made using CLC - Read in the count table in R and got it running in the DESeq2 package - Made pair-wise comparisons using the group-functionality in DESeq2: <pre> # construction to compare specific...groups which are combinations of variables dds1$group <- factor(paste0(dds1$treatment, dds1$zone)) design(dds1) <- ~ group dds1 <- DESeq(dds1)…

DESeq2 Two-way analysis deseq2

updated 7.3 years ago • Jonas B.

I am trying to understand some results from DESeq2. The experiment is as follows: Before and after treatment for 11 individuals. The most significant gene has the following...only, except from the second individual, who has 1298 counts before treatment). The p-value from DESeq2 is: 1.49e-21 (using the default Wald test). Here I have used the DESeq vignette instructions for doing paired comp…

deseq2

updated 6.0 years ago • staaln

div id="__zsc_once"> <p>Hi,</p> <p>I am using DESeq2 for the analysis of my RNAseq data. I have the following sample set up:</p> <p>Condition      &nbsp...separation according to library prep Day1 and Day2.</p> <p>Therefore I want to include a blocking factor in the design model.</p> <p…

deseq2 model

updated 9.8 years ago • da.de

I am learning to use DESeq2 for analyzing time course experiment from [RNA-seq workflow: gene-level exploratory analysis and differential expression...about the "time" in the analysis. Should the time variable in the fission dataset be considered as factors or numeric values? I worry we would lose information if we consider time points as factors.   Many thanks for your

deseq2

updated 7.1 years ago • hwu12

Hi everyone, I’m using DESeq2 to analyze RNA-Seq data. I have basically 2 factors: Genotype and Treatment. I build the design matrix as ~ Genotype * Treatment...to capture interaction effects (Genotype:Treatment) as well. After correctly releveling the factors, I get the coefficients that I want. So far so good. The question is, is there any way to test if the interaction term itself

deseq2

updated 6.1 years ago • cihan.erkut

Hi, I'm trying to install DESeq2 on my R-server (vers. 3.6.1), and it fails with : > BiocManager::install("DESeq2") Erreur : .onLoad a échoué dans loadNamespace...version '3.8' requires R version '3.5'; see https://bioconductor.org/install I first did what explained at : https://bioconductor.org/install/ if (!requireNamespace("BiocManager", quietly = T…

deseq2

updated 5.5 years ago • eric.trezel

Dear Michael,  first of all I apologize if my questions sound silly, because I am bench researchers, but I am trying to learn R. I have help from...that are differentially expressed between the single mutants and interaction between the two genes. First I thought that with DESeq2 I can only normalize the counts and extract the normalized counts and do 2-anova afterwards...but as trying …

deseq2

updated 7.4 years ago • denius

nbsp; So here's the gist. I have replicates of three conditions (each with its own Input). What I first did, last year (when I was much more primitive in bioinformatics than I am now), I called the peaks then used a IDR to determine...a pipeline, so I followed his advice on making a Count Table of all the conditions and then run a DESeq2 a la RNA seq. Based on the discussion from his question on…

deseq2 diffbind csaw greylistchip chipseq

updated 6.3 years ago • Yonatan Amzaleg

in this question <https://support.bioconductor.org/p/87999/> "the normalized counts in DESeq2 are just provided for visualization, they aren't actually used in the DESeq2 model which uses all samples on the original...counts scale and keeps track of size factors on the right-hand side of the equations as shown in the DESeq2 paper". So, is performing one of DESeq2's transf…

deseq2 rnaseq mfuzz

updated 6.7 years ago • rbenel

Dear community, I'd need some help in the analysis of my RNAseq data using DEseq2. I'm trying to characterize the effect of the over expression of three transcription factors (x,y,z) on the transcriptome...1) Ctrl 2) x 3) y 4) z 5) x,y,z As far as I understood I can set up my analysis as a "single factor" with "multiple levels" (as in explained in the manual 3.2 paragraph). So, if I under…

deseq2 rnaseq

updated 9.0 years ago • sebastiano.curreli