Bioconductor Forum

Hi, I am running *DESeq2* to get DEGs from experiment below. The experimental design I am working on has three factors with respective levels...Hi, I am running *DESeq2* to get DEGs from experiment below. The experimental design I am working on has three factors with respective levels: - Time (1dpi, 4dpi) - Cultivar (J, Q, R) - Treatment (infected, control) time cultivar trea…

quinoa virus mutifactor design DESeq2

updated 6.4 years ago • n85825

all, in relation to my mail from January this year, I followed Simon's advice to do my analyses in DESeq2 instead of DESeq. I am working on an RNASeq from c. elegans. I have mapped the data with the ensembl genome build WBcel215...replica. Now I am trying to find differentially regulated genes between the two conditions using DESeq2. This is the script I am using to read my raw count table int…

RNASeq DESeq DESeq2 RNASeq DESeq DESeq2

updated 11.5 years ago • Assa Yeroslaviz

I'm running DESeq2 on a small RNA seq experiment. The coldata has 87 samples total and begins like this: <table border="0" cellpadding="0" cellspacing...I'm running DESeq2 on a small RNA seq experiment. The coldata has 87 samples total and begins like this: <table border="0" cellpadding="0" cellspacing="0" style="width:192pt"> <tbody> <tr> <td>Sample</td> <td…

deseq2 multiple factor design contrast

updated 7.5 years ago • russell.stewart.j

Hi, I have been reading the previous post about nested and multi factor analysis with DESeq2 , as well as, the vignettes dealing with nested and multi factor analysis. However, I am still getting...must be removed. Please read the vignette section 'Model matrix not full rank': vignette('DESeq2') [1]: /media/images/c5f255d7-2517-4939-892b-ced191c9

DESeq2

updated 4.6 years ago • melissa.mageroy

Hi folks, Currently I am able to extract a table of counts normalized by size factors: normalized\_counts = counts(dds, normalized=TRUE) However, these counts are still before fitting in the analysis. Is...it possible to obtain the post-fitting counts from DESeq2 analysis?  Thanks     &nbsp

deseq2

updated 9.9 years ago • pchiang5

Hi everyone I'm experiencing a problem with DESEq2. I downloaded matched tumour-normal cancer data from the TCGA (111 patients, 222 samples), and am attempting to perform...cancerous tissue, for the purpose of biomarker discovery. But as a preliminary, I wanted to use DESeq2 to visualise clustering between tumour and normal samples using all coding genes (i.e. ~30,000 genes). My code: …

DESeq2

updated 3.3 years ago • Mia

Hello, I've read [pages (23-25) of the DESeq2 manual](https://www.google.ca/url?sa=t&rct=j&q=&esrc=s&source=web&cd=2&cad=rja&uact=8&ved=0CCQQFjABahUKEwiT5NO_yLPIAhVQm4gKHYmjDhE...in a day "dayTime" (day, night)__ My design formula is: ~ __dayTime+lunar+dayTime:lunar__ At first I wasn’t really sure how to do contrasts cor…

deseq2 design and contrast matrix interactions contrast

updated 10.2 years ago • moldach

Hi everyone, I would like to use DESeq2 to process RNASeq paired samples (before and on treatment). I read very carefully the different posts and tutorial...Hi everyone, I would like to use DESeq2 to process RNASeq paired samples (before and on treatment). I read very carefully the different posts and tutorial but...I am still uncertain on how to design my experiment formula. Here are my experi…

deseq2 paired samples

updated 9.5 years ago • adesrichard

div class="preformatted">Dear Mike Love and list members, I'm running DESeq2 with a design containing two covariates: the first is dichotomous while the second is continuous. Here is the data.frame...the effect of the continuous covariate on the counts (about 15,000 features). I can compute the size factors, but when estimating the dispersions, I get the following error: > dds &lt…

DESeq2 DESeq2

updated 11.8 years ago • Hugo Varet

This is my first time using EdgeR or DeSeq2. I used HTSeq-Count to get counts for reads aligning to CDS (this is for ribosome profiling) for...reads and reads that don't align to the feature (CDS). Should I remove these before running EdgeR or Deseq2?     &nbsp

edger deseq2

updated 7.0 years ago • cottrellka

were paired-end. I want to consider the batch effects in the model by blocking them as a factor. Thus, I decided to use DiffBind, edgeR and DESeq2 and their respective procedures for blocking factors. My results are...description for blocking or multiple-factors…  So I am not sure what I am missing, I was expecting to have similar results between Edger/DESeq2…

diffbind

updated 9.8 years ago • armandorp

Disease:Treatment. While this comparison is possible using EdgeR, I cannot get it to work using DESeq2.  I keep receiving the message "Error in \`contrasts<-\`(\`\*tmp\*\`, value = contr.funs\[1 + isOF\[nn\]\]) : contrasts can be applied only...to factors with 2 or more levels".  Is there any way to make this comparison using DESeq2? My script and the error I receive is…

deseq2 edger differentialexpression

updated 11.2 years ago • asaferali

Hi, I like to confirm if the statistical model of Deseq2 is appropriate for my experiment design. Here’s my code. <pre> > dds<-DESeqDataSetFromMatrix(countData=countsTable...colData,formula(~ Transfection*Enrichment )) > colData(dds)$Transfection<-factor(colData(dds)$Transfection,levels=c("gfp","kd")) > colData(dds)$Enrichment<-factor(colData(dd…

deseq2 statistical learning

updated 10.2 years ago • hdf87ery

Hey Michael DESeq2 dose not work and I receive this message when I try to run library(DESeq2) library(DESeq2) Loading required package: S4Vectors...groups,row.names=colnames(data)) library(DESeq2, quietly=T) dds <- DESeqDataSetFromMatrix(countData = data, colData = sampleInfo, design = ~ groups) dds$condition = relevel...gt; data <- read.table("Single…

deseq2

updated 6.9 years ago • 21464321

are healthy. I have been reading about performing time series differential abundance analysis using DESeq2. I would like to answer 2 main questions: 1.- Are there differentially abundant taxa (OTUs) in the different trimesters...__ 1.- For number 1 I think I should do a simple design in which I only take into account the factor time, which is the factor that I want to test for changes. —&gt…

deseq2

updated 9.2 years ago • schulze.a

Mike Love, Simon Anders and I have been updating the DESeq package. This resulted in the package DESeq2, which is available from the development branch, and scheduled for the next release: http://www.bioconductor.org/packages...in order to not disrupt the workflows of DESeq users. For new projects, we recommend using DESeq2. Major innovations are: * Base class: SummarizedExperiment is used as th…

Normalization Classification Clustering DESeq cqn DESeq2 Normalization Classification cqn

updated 12.8 years ago • Wolfgang Huber

tr> <td>Blk2</td> <td>8</td> <td>Treated</td> <td>4</td> </tr> </tbody> </table> After reading the DESeq2 manual \[here\] ([https://bioconductor.org/packages/release/bioc/vignettes/DESeq2/inst/doc/DESeq2.html\#group-specific...this experiment. I consider this experiment as individuals nested within blocks. library(D…

deseq2 nested design

updated 7.4 years ago • xie186

from three different batches (PK, GB, and EG) and I am trying to apply batch correction using DeSeq2. I am intersted in contrasting all three condtions (i.e. A vs B, B vs C and A vs C) while taking the batch effect into account...My code is as follow: ``` # Assign condition and batch (condition <- factor(c(rep("A", 3), rep("B", 3), rep("C", 3)))) (batch <- factor (c("PK","GB",…

deseq2

updated 6.8 years ago • pkhadka

scale factor at first.And then they should divided by scale factor to normalize the technical bias effect by RNA compostion. And...library size or other normalization factors"(I quote from http://bioconductor.org/packages/devel/bioc/vignettes/DESeq2/inst/doc/DESeq2.html count transformation...but it didn't divided by the effective library size. and I think it is same as the counts() of DESeq2 i…

normalization DESeq2 edgeR

updated 6.4 years ago • 15958021290

clarification from you. As far as I know, calcNormFactors() produces two columns of information. The first is lib.size and the second is norm.factors, which multiplying these two columns together gives us an effective library...size. However, I don't understand how the normalization factor was calculated, could you please explain me in a simple way as I'm basically a biologist? From what I read,…

TMM normalization edgeR

updated 9.2 years ago • Sara

Hi, I used DESeq2 about 4 months back to analyse my data with the design: <pre> design=~species+condition+species:condition</pre> with species...condition1","condition3","condition4","conditon5")),listValues=c(1,-1/4))</pre> However, when I use DESeq2 now (yesterday) with the same design & dataset, I get different resultsNames(dds): _"Intercept", "species\_B\_vs\_A…

deseq2 resultsnames multifactorial design

updated 9.6 years ago • Ira

Hello everyone, I am using DESeq2 for DEG analysis. I have a set of experiment which consists of 4 duplicates and it's not clear how to create the design...object? Here is my condition: condition<- factor(c (group1 =rep ("treatment1",time=2), group2 = rep("treatment2",time=2), group3= rep("treatment3",time=2),group4 =rep("treatment4",time

deseq2 RNA

updated 6.7 years ago • Merlin

Dear Community, My RNA-Seq experiment has three factors: A, B and C. A and B are random effects while C is fixed effect. I would like to fit a mixed model including all of...A  + B + C  + A x B   + A x C  + B x C + A x B x C  Can DESeq2 or edgeR fit the mixed model? Thanks. When I searched "DESeq/DESeq2/e…

deseq2 edger

updated 9.5 years ago • Yanzhu Lin

fitting model and testing -- standard model matrices are used for factors with two levels and an interaction,    where the main effects are for the reference level of other factors. &nbsp...nbsp; see the 'Interactions' section of the vignette for more details: vignette('DESeq2') estimating size factors estimating dispersions gene-wise dispersion estimates …

deseq2

updated 10.3 years ago • Peter Langfelder

Using DEseq2, the `lfcShrink` function with the `apeglm` method requires a single `coef` value to be specified (i.e. it does not allow a...Using DEseq2, the `lfcShrink` function with the `apeglm` method requires a single `coef` value to be specified (i.e. it does not allow a contrast...cells, "_", treat, "_", replicate), cells, treat, replicate, group) colData$cells <- factor(co…

deseq2 model.matrix

updated 6.8 years ago • toddknutson

Hi, I am working with an RNA-seq data set that contains a lot of 0s across the genes. When I do Deseq2 to look for differential gene expression, it finds several significantly dysregulated genes. However, when I look...of a stumbling block when it comes to 0s. In my understanding, the normalisation works as follows. Deseq2 normalisation divides counts by the sample-specific size factors. …

deseq2 normalization

updated 5.3 years ago • lmrogers34

<div class="preformatted">Hi, I wanted to use a normalised read count matrix from EDAseq downstream in DESeq2 analysis. I am not very clear on how to do so from the vignette. Following are the steps I followed - ## EDAseq - normalising count matrix by GC content > dataWithin <- withinLaneNormalization(data, "pct_gc", which = "full") > dataNorm <- betweenLaneN…

Normalization EDASeq DESeq2 Normalization EDASeq DESeq2

updated 11.5 years ago • Guest User

Hi there, I am new to DESeq2 and multiple factor design. I have read the DESeq2 vignette and some forum discussions that deal with design questions...Hi there, I am new to DESeq2 and multiple factor design. I have read the DESeq2 vignette and some forum discussions that deal with design questions of multiple factors. But I’m still unsure if my approach is reasonable and/or what the best approac…

deseq2 multiple factor design

updated 7.3 years ago • sandra

I'm trying to do differential expression on label imbalanced data; my case:control ratio is 2:1. I know that regressions are at the core of DESeq2 machinery and I know regressions have internal machinery for coping with such imbalances. Specifically, you can down...on label imbalanced data; my case:control ratio is 2:1. I know that regressions are at the core of DESeq2 machinery and I know regres…

deseq2 rnaseq r regression

updated 9.3 years ago • bkellman

I have chip-seq data which I am analysing with Diffbind. I understood the "blocking factor" functionality of it can be used to remove confounding variables. In the Diffbind manual, the confounding factors used...dba object with a string variable, everything runs smoothly (the name of the column, by the way, is Factor, and then I call it as DBA_FACTOR). To do the same for age I simply substitute…

diffbind chip-seq

updated 6.2 years ago • melnuesch

at another platform. And finally reached here after the suggestion. I have read [this][2] manual by Deseq2 author. This is very well written manual on how to use Deseq2 for allele-specific analysis. I have some queries on how...I can better use Deseq2 for my allele-specific analysis. What I understood that author did not use size factor for normalization rather set...the size factor to unity a…

deseq2 rnaseq allele-specific normalization

updated 6.3 years ago • Ankit

Dear all, I'm a bioinformatichian and I'm using DESeq2 package. I have 2 requests: If I have a Multi-factor design, for ex. I have samples Normal and Treated in 3 different time

deseq2

updated 5.3 years ago • giovannaventola3es9

Hello, I am working through the vignettes and examples for DESeq2 `?results`. I had a question(s) regarding lfcshrinkage. Looking at Example 2: ## Example 2: two conditions, two genotypes, with...an interaction term dds <- makeExampleDESeqDataSet(n=100,m=12) dds$genotype <- factor(rep(rep(c("I","II"),each=3),2)) design(dds) <- ~ genotype + condit…

deseq2 lfcShrink

updated 6.7 years ago • rbutler

Hello All, I would like to generate an HTML report from DESeq2 results of RNASeq analysis. I have followed the example in the ReportingTool documentation and have no problem with...Hello All, I would like to generate an HTML report from DESeq2 results of RNASeq analysis. I have followed the example in the ReportingTool documentation and have no problem with that, but unlike that example my dese…

reportingtools publish deseq2

updated 10.8 years ago • noushin.farnoud

Hello everyone, This is my first time performing DESeq2, and even PCA analysis. I have 8 samples. (4 WT and 4 mutant) I have done PCA Analysis, and this is my output...data? What does it say? The code that I executed is this: ``` # Load necessary libraries library(DESeq2) library(ggplot2) # Enhance the synthetic count data by increasing the difference set.seed(123) # Ensure reproducibility…

DESeq2 RSTUDIO PCAtools

updated 20 months ago • Aaliya

Hi All, In the Deseq2 vignette the first step is creating the object  dds <- DESeqDataSet(se, design = ~ batch + condition) based on the RangedSummarizedExperiment

rangedsummarizedexperiment deseqdatasetfrommatrix deseq2 tximport

updated 8.3 years ago • lirongrossmann

Hello, <span style="line-height:1.6">I need some help creating a design for my dataset in DESeq2.  I have RNA-Seq data from samples that have been treated with a Control, with LPS, with LPS followed by molecule A...Hello, <span style="line-height:1.6">I need some help creating a design for my dataset in DESeq2.  I have RNA-Seq data from samples that have been treat…

deseq2 design multiple factor design

updated 10.8 years ago • asaferali

but find it very difficult how to specify what I want to compare. My experimental design has three factor columns: * two different locations * four different conditions * either two or three replicates - the replicates are matched...compare all nucleus vs all cytoplasm etc). I think I understand what to do if I have only two factor-columns, but now that I have three I don't get how to sp…

DESeq2 multiple factor design experimental design contrast

updated 10.1 years ago • anton.kratz

in one big matrix, I lose the difference in intensity between these genotypes presumably because DESeq2 calculates across the entire matrix. Instead, the normalized counts for both genotypes come up close to equal. In reality...of experiments and should not have equal normalized counts. If I normalize WT and KO separately with DESeq2 (two separate DESeq2 objects for either WT or KO), the ratio is…

deseq2

updated 9.2 years ago • snamjoshi87

I'm testing it with betaPrior = False and using lfcShrink either with the coef or contrast parameter (DESeq2 version 1.18.0). I notice a couple of things which I think need clarifcation I used a fairly common experimental design...with a factor that has 3 levels and DM is initial reference level. <pre> coldata$Cond <- factor(coldata$Cond, levels = c('DM', 'DP', 'Co')) ddsdm &…

deseq2 lfcshrink relevel

updated 8.1 years ago • mat.lesche

Hi, I want to clarify what is going on under the hood when a user runs `DESeq2::estimateSizeFactors()` with the `controlGenes` argument. So, if my understanding is correct, all the steps of size-factor...genes assigned to `controlGenes` (except for the final step, which is to apply the calculated size factor to all sample-wise genes) rather than the default of all genes supplied to `DESeq2::esti…

DESeq2 SpikeIn Normalization DifferentialExpression

updated 2.9 years ago • kalavattam

In csaw, there are two suggested ways to compute normalization factors: composition bias normalization factors, computed from large bins across the genome; and efficiency bias normalization...section 4.3.2 "Checking normalization with MA plots", the composition and efficiency factors are plotted over the MA plot of all windows between two samples, with the composition factor lining up horizontall…

csaw normalization

updated 9.7 years ago • Ryan C. Thompson

Hi, I'm analysing RNA-seq data and I switched from edgeR to DESeq2 because most of my differentially expressed genes had an outlier sample, which was not an outlier in the entire dataset...judged by PCA and overall Cook's distance). My question is related to the criteria used by DESeq2 to count the number of replicates in my experiment. I have 3 experimental groups, A,B and C, each of them wit…

deseq2 ruvseq cookscutoff covariates batch

updated 9.1 years ago • j.viana

Hi, I am trying to use DESeq2 to perform normalization of my RNA-Seq data. I am wondering if, after estimating the size factors, I can directly run the...Hi, I am trying to use DESeq2 to perform normalization of my RNA-Seq data. I am wondering if, after estimating the size factors, I can directly run the rlog or vst transformation, or if I also need to use the count function in between. Basical…

deseq2 normalization rnaseq rlog transformation variancestabilizingtransformation

updated 7.1 years ago • chiara.facciotto

Hello! I am doing DESeq2 for the first time. A bit of background: I am downloading the already public data available from ENA browser. I have been...Hello! I am doing DESeq2 for the first time. A bit of background: I am downloading the already public data available from ENA browser. I have been able to successfully do Kallisto on the paired reads. The output of such files in in .tsv format. I…

DESeq2 Kallisto

updated 23 months ago • Aaliya

3 data (rsem raw counts) for samples with matched normal to analyse differential expression using DESeq2. I was wondering the design I am using is correct. coldata looks like this.   <pre> DataFrame with 118 rows and 2 columns...condition pid <factor> <factor> TCGA.BJ.A28R.11A.11R.A16R.07 Normal_Tissue…

deseq2 tcga experimental design multiple factor design

updated 10.6 years ago • anand m t

viral RNA (= 10 genes) make up ~50% of the entire library in the infected condition. When running DESeq2 on this dataset, the estimated size factors effectively double the counts for cellular RNAs because they capture...for normalization. I am therefore highly interested in your suggestions concerning appropriate size factors for the normalization, would it make sense to simply use a co…

deseq2 normalization sizefactors

updated 7.9 years ago • René

For full disclosure I posted this question on the Phyloseq Github page but perhaps this forum is more appropriate. I have a 16S dataset from gut mucosa and want to analyse differential abundance according to a factor. I have 200 samples: 10 cases and 190 controls. Q1: is it valid to use DESeq2 to compare differential abundance with DESeq2 with such an a large imbalance between the numbers o…

deseq2

updated 6.6 years ago • dr.aj.scott

So will just list current design first and then get into the question: <pre> rangedCounts <- dba.peakset(Adult_BN_count, bRetrieve=TRUE) nrows <- 1054182 ncols...se<-SummarizedExperiment(assays=list(counts=counts),rowRanges=rowRanges, colData=colData) # DESeq2 analysis table(sampleSex, sampleTreatment) dds<-DESeqDataSet(se, design= ~sex + treatm…

deseq2 differential binding analysis deseq atac-seq

updated 7.9 years ago • rbronste

Hi there, I would like to ask about organising my data for DESeq2 - to do RNAseq analysis. How do I not include my first column (gene ID) for my rawcount data as I am unable to cross check my

deseq2

updated 5.6 years ago • angkoo

Hello, I usisn DESeq2 package in R for analysing Rna seq sngle end data. After running the DESeq pipeline dds <- DESeq(dds) I am getting this...to "environment" sets attribute to NULL; result will no longer be an S4 object Because it's the first time working with these kind of data and this package, could you please help me understand what is about this warning

deseq2 Biocodunctor

updated 6.2 years ago • aapapaiwannou

the number of DEGs for each pairwise comparison is greatly influenced by whether I construct the DESeq2 model with all the data or a subset of groups that are then compared. For some comparisons, building the model with all...the data seems to improves the power, whereas other comparisons benefit from first subsetting the specific groups and then building the model. For example, if I subset the …

DifferentialExpression DESeq2

updated 2.5 years ago • sean.mccutcheon

Hello, First, I know that DESeq2 is not meant to be run without replicates and results will not be meaningful. However, I have replicates...while another group (T) has only a single sample. This is the requirement of the project. I run the DESeq2 without any error and I found a change in the expression of ~9000 genes (padj < 0.05). I am sharing the script and don't if...I am doing the corr…

DESeq2 rnaseqGene DifferentialExpression GeneExpression

updated 4.5 years ago • SUMIT

Hello community, I'm interested in conducting a DEG analysis to obtain differentially expressed genes based on treatment per cell line. In other words, determine the effect of condition per cell line and extract DEGs based on treatment. ## Overview Experimental Design: This consists of a two-factorial design with factors: cell line (A vs B) and treatment (control vs treatment). Research Ques…

DESeq2 rna-seq

updated 2.8 years ago • genome_nerd

<div class="preformatted">Hi, I have counts of DNA for 16S with different library sizes and want to use DESeq2 to normalize the counts. However, I used Picrust to correct the 16S copy number for OTUs and the number generated by this...class="preformatted">Hi, I have counts of DNA for 16S with different library sizes and want to use DESeq2 to normalize the counts. However, I used Picrus…

DESeq2 DESeq2

updated 11.7 years ago • Manoeli Lupatini

Levels without samples" I thought I could drop the corresponding column in the model matrix and run DESeq2: <pre> > w <- which(colSums(mm)==0) > mm <- mm[,-w] > dds = DESeq(deseq.matrix, full=mm, betaPrior=FALSE) estimating size factors

deseq2 multiple factor design model.matrix

updated 9.6 years ago • cdemel

defined sizeFactors? I am using a calibrated ChIP-seq approach and can calculate normalisation factors (with DESeq2, for instance) using reads mapping to my reference genome and I would like to use these normalisation factors

diffbind normalization

updated 5.1 years ago • nfursova.msu

Hi all,   We are performing a transcriptome analysis with DESeq2. We have three factors: treatment (control and drought), climate (CO2 and ambient) and zone in the leaf (zone1, zone2, zone3...Hi all,   We are performing a transcriptome analysis with DESeq2. We have three factors: treatment (control and drought), climate (CO2 and ambient) and zone in the leaf (zone1, zone2,…

deseq2

updated 7.9 years ago • Jonas B.

I am trying to re-run a couple of new comparisons using DESeq2. I have previously used this script but something must have changed or updated in the last couple months that is responsible...se, design = design, ignoreRank) : some variables in design formula are characters, converting to factors ``` I have tried what was suggested [previously,][1] but was unsuccessfully. Any help or suggestio…

DESeq2

updated 5.1 years ago • Amy

of) normalized expression/abundance values that are "corrected/adjusted" for certain design factors. For example, using the regular lm(), I can correct for spatial variation (characterized by a bunch of variables, everything...analyses or plotting on the residuals. Is there a way to do something similar directly with DESeq2? I.e., not via using normalized and log2(x+1)-transformed in a sepa…

deseq2 residuals

updated 6.4 years ago • MWSchmid