Bioconductor Forum

paper and the vignette have been very clear. However I still have a question since I am using the DESeq2 package for data other than bulk RNA-seq data. I am trying to use the DESeq2 package for analysis of bulk methylation...What I am trying to understand is if my data fits the 'assumptions' and the analysis pipeline of the DESeq2 package, since I might have more 0 counts in my -dox samples a…

DESeq2

updated 2.9 years ago • a.puchkina

I have a time series RNA-seq data for two different genotypes, and I did a Likelihood ratio test in DEseq2 to see whether there are genes showing different expression profiles between the two genotypes across time points...files.htseq,fileName = files.htseq,time = time, strain = genotype) library("DESeq2") ddsHTSeq = DESeqDataSetFromHTSeqCount(sampleTable =sampleTable, …

DESeq2

updated 6.4 years ago • yh362

Or will I have to create independent sample tables for each comparison? Thanks for any help! ``` #First I make a dds against all groups (grouped by biological replicates only) ddsTxi <- DESeqDataSetFromTximport(txi, colData...design = ~ Condition) #Then I try to define Interactions ddsTxi$group <- factor(paste0(ddsTxi$GSE, ddsTxi$Group…

Interactions DESeq2 Design

updated 5.0 years ago • vanbelj

Hi I need help the analysis of my RNA-seq samples using DESeq2. I uploaded cancer evidence for 100 samples divided into two parts, each section contains 50, the first group for a dead

DESeq2

updated 3.9 years ago • Karim

I want to run DESeq2 to identify differences in open chromatin accessibility (i.e. ATAC-seq data) between 3 different GROUPS: A, B and C. I also...was the same) for both analysis methods. Method 1: Combining all groups, using LRT while running DESeq2 to control for BATCH and SEX, and then obtaining pairwise results using contrast and the Wald test afterwards Data...Can someone please expl…

deseq2

updated 6.9 years ago • jonathan.diedrich

interested in analyzing timecourse data but would like some advice as to the experimental design in DESeq2. The experimental data include 4 ramets of a single sapling (tree) clone collected on Day 0 (before treatment) and 4 treated...but we do have controls for Day 7 and Day 14. We included spike ins in the library prep and found factors of unwanted variation (W_1) using RUVseq. Because of the…

DESeq2

updated 4.2 years ago • Mallory

Hello, I am using DESeq2 for a DGE analysis and I have been stocked on this specific error. ```r dds <- DESeqDataSetFromMatrix(countData = all.data.set_v3...counts to integer mode #Warning: some variables in design formula are characters, converting to factors dds@assays@data@listData[["counts"]] <- as.matrix(dds@assays@data@listData[["counts"]]+1) #run that in order t…

DESeq2

updated 2.9 years ago • Comp_bio97

Hi, I ran conditional quantile normalization on my RNAseq counts. I am then going to use DEseq2. However, DEseq2 only accepts non-negative integers. CQN can be coerced to be non-negative, but is still on the log scale...and cannot be coerced into integers. So because DESeq2 only accepts un normalized non-negative integers, then how can the merits of CQN be considered when calculating for...di…

deseq2 cqn

updated 6.1 years ago • nicholas.macknight

preformatted">Hello Michael. I am a graduate student neuroscience researcher attempting to use the DESeq2 package to perform differential expression analysis of my sequencing data. I am following the beginner vignette...DESeq function, but I received the following error: > dds <- DESeq(ddsFull) estimating size factors Error in estimateSizeFactorsForMatrix(counts(object), locfunc…

Sequencing Alignment DESeq DESeq2 Sequencing Alignment DESeq DESeq2

updated 10.6 years ago • Guest User

Hello, I want to do an analysis of some mice data with DESeq2. We have a total 24 samples and three factors. Factor one is the genotyp (6 wildtype, 6x knockout 1, 6x knockout2, 6x knockout3...and factor two are two different cell lines (cellA and cellB). Factor three is the sex (male and female <table border="1" cellpadding...wt-cell2, ko1-cell2 vs ko1-cell2 but also wt-cell1 vs ko1-cell1. T…

deseq2 multiple factor design group

updated 9.3 years ago • mat.lesche

I am trying to use DESeq2 to identify differentially expressed genes across different rodent species as part of a larger project on rodent...when I try to create ddsHTSeq: ``` Error in Ops.factor(a$V1, l[[1]]$V1) : level sets of factors are different In addition: Warning messages: 1: In `==.default`(a$V1, l[[1]]$V1) : longer object length is not a multiple of shorter...or whether the…

deseq2 rna-seq

updated 6.6 years ago • Emma

<div class="preformatted">Hi, My experiment consist of control and diseased samples; looking like this: colData<-DataFrame(condition=factor(c("A","A","B","B","C","C","D","D", "E","E","F","F","H","H","I","I")), type=factor(c("plasma","plasma","plasma","plasma","plasma","plasma","p lasma","plasma","tissue","tissue","tissue","tissue","tissue","tissue", "tissue","tissue")), diseased=fa…

updated 12.1 years ago • Hanneke van Deutekom

Hi, I use this code for install Biocmanager and then DESeq2 packages and I got these errors. what is the problem? > install.packages("BiocManager") Installing package into ‘C:/Users...in C:\Users\melika\AppData\Local\Temp\RtmpgVb1KM\downloaded_packages > BiocManager::install("DESeq2") Bioconductor version 3.7 (BiocManager 1.30.10), R 3.5.2 (2018-12-20) Installing packag…

deseq2

updated 5.7 years ago • melikabakharzi

strategies for scRNA-seq data. Contrary to the header, I think, I have actually two questions, the first one being: Is the computeSumFactors() philosophy really applicable to current Drop-seq data sets? The sample that initiated...for each gene across all those cells are quite low, of course, so in order to avoid getting size factors of zero, I use a very small subset of genes (around 370) where…

scran scater drop-seq scrnaseq

updated 8.7 years ago • Friederike

loci where the duplicates have significantly different expressions - I was wondering if I could use DESeq2 to do this. In particular, I was going to set up a table with samples consisting of all tissue x duplicate pairs that looks...differentially expressed loci - for example conducting a log ratio test to see if the duplicate factor is significant in the design. My question is whether this vi…

rnaseq DESeq2

updated 2.1 years ago • pl23

due to sequencing runs (see below). Is it possible even if I don't have blood samples in the first sequencing run? ![PCA plot of the 3 populations][1] ![PCA plot for each subset][2] I tried to apply the combat-seq algorithm from...the code runs but never ends, it keeps running even after several hours. ```r Population <- (factor(meta2$Population)) Tissu <- (factor(meta2$Tissu)) …

BatchEffect sva DESeq2 RNASeqData limma

updated 2.4 years ago • D-Bug

to identify all genes that are significantly differentially expressed across all the 4 groups. Does DESeq2 allow such assessment? Thank you for your time and I look forward to your response

deseq2

updated 7.9 years ago • gupta.anuj0608

When using DESeq2 in the past, count data and normalized count data, etc... retained the names of samples indicated in the first column of...When using DESeq2 in the past, count data and normalized count data, etc... retained the names of samples indicated in the first column of the colData table. I've used dds = DESeqDataSetFromMatrix(countData = countdata,     &am…

deseq2

updated 9.5 years ago • jshouse

Dear Mike Love, I just upgraded DESeq2 to get the last version (1.12.0) and I have a problem when using the estimateSizeFactors() function on a DESeqDataSet

deseq2 tximport rsem estimatesizefactors

updated 9.6 years ago • Hugo Varet

I have no code to post. This is question about the different the differing results I get when I change a comparison variable in the design formula from character, to factor. i am comparing differential expression across age groups. The data has a variable 'age' : with these values, 20,25,30,35,40...differing results I get when I change a comparison variable in the design formula from chara…

DESeq2

updated 22 months ago • James Aiden

I have rnaseq data for various combinations of three different factors (let's call them X, Y, and Z).  Some of these combinations represent controls, and the rest represent treatments. I want...is to stratify the data into subsets corresponding to combinations of the levels of two of the factors (say, X and Y), and then perform the DE analysis on each subset using `` design = ~ Z ``. I…

deseq2

updated 9.3 years ago • kjo

Hi, Until a few weeks ago, the DESeq2 function "DESeqDataSetFromMatrix" was working fine for me. However, when I use it now, I get the error, "invalid class “DESeqDataSet...can reproduced using the example dataset given on pages 5-6 of the "Analyzing RNA-Seq data with the "DESeq2" package" vignette on the DESeq2 Bioconductor page:   <pre> > library("pasilla") &…

deseq2 error

updated 10.5 years ago • alextuck

se, design = design, ignoreRank) :some variables in design formula are characters, converting to factors) which I never encountered in my DESeq2 use before. My design is already a factor(male and female). ## here is my dds dds &lt

deseq2

updated 5.6 years ago • Do it!

<div class="preformatted">Dear Michael and DESeq2 users, I started using DESeq2 three weeks ago. Even I was having some normal problems for a newby person my experience was really positive and I was and I am detecting more DEG than with DESeq(1). However suddenly I am encountering a new problem that wasn't coming up previously: > resSig <- res[ res$padj < .1, ] Error…

DESeq2 DESeq2

updated 12.2 years ago • Alicia R. Pérez-Porro

I have a RNASeq data set with ~650 samples. When I run DESeq2, I get a very poor fit of the mean-dispersion trend: <a href="https://i.imgur.com/XZeqgTN.png"><img alt="" src="https://i.imgur.com...se, design = ~condition + run) : some variables in design formula are characters, converting to factors estimating size factors estimating dispersions gene-wise dispersion estimates: 64 w…

deseq2 dispersion

updated 7.7 years ago • Frederik Ziebell

Hi,  I tried to analyze RNA-seq data using DESeq2. I have 2 sample conditions: 1 mg/mL and 2 mg/mL My control sample is 0 mg/mL.   When I tried to identify differentially...sampleTable, directory = directory, design = ~condition) > colData(ddsHTSeq)$condition<-factor(colData(ddsHTSeq)$condition, levels = c('0','1')) > dds<-DESeq(ddsHTSeq) &…

rnaseq deseq2

updated 8.2 years ago • Letícia

with my RNAseq regarding the variance between my biological replicates. If i include them as a factor of variance in my design, i can fix the issue nicely and looking at the PCA plot when removing"batcheffect" with: ``` mat &lt...is if it is acceptable to do this or if this takes away the whole point of using replicates in the first place. Thanks alot for your help and insight! Lu…

DESeq2

updated 3.1 years ago • lukas.krauss

a meta analysis and ran into this peculiarity with SVA: In at least one study ([GSE147851][1]), the first SV found by sva correlates entirely with the sample groups provided in mod1. Could one of you kind people help me explain...source final_description group_nr <character> <character> <character> <character> <factor> GSM4447088 GSE147851 GSM444708…

sva

updated 3.5 years ago • shdam

Hi, ******* Background *********** I have following design (currently test data): 4 inbred lines (A, B, C, D) 2 temperatures (x, c) "c" is a control 4 time points (possibly more in future) Each combination of the above is repeated 3 times ************************************* * possibly "c" and "x" could be joined somehow because plants grow first in "x" temperature and after say 24 h it i…

DESeq2

updated 2.8 years ago • boczniak767

list <bioconductor at="" r-project.org=""> >> Subject: [BioC] Please help! How to specify factors for a RCBD in >> edgeR >> >> Dear Tilahun, >> >> The first step is that you need to create a data frame containing...the >> experimental factors, just as you would for a SAS analysis. So yo…

edgeR edgeR

updated 13.9 years ago • Gordon Smyth

Hi, I have a quick question on DEseq2 replicates. A good number of genes were marked as outliers (912, 5.7%) in my DEseq2 summary(res) output. I am wondering what...counts with large Cook’s distance with the trimmed mean over all samples, scaled up by the size factor or normalization factor for that sample."_ Do you mean technical or biological replicates here? In my case, I only have...cell t…

deseq2

updated 7.7 years ago • Xianjun Dong

Hi there, I have been stuck with this analysis, that in first place seems a quite simple experiment but the results that gave me are weird and i dont know how to resolve this. Its not...my first time that i have problems with outliers that the DESeq2 not filter, i tried have a solid answer for my problem, but with...variable the automatic filtering is off (ref: http://bioconductor.org/packages/…

maxcooks outliers zero counts DESeq2

updated 3.8 years ago • andreia

Input Data into DESeq data <- newCountDataSet(total\_counts, conditions) \#Estimate size factors - difference in coverage between replicates data <- estimateSizeFactors(data) sizeFactors(data) \# Estimate dispersion...OQ\_YQ.Sig),quote=F) \#End DESeq (v1) example       Below is the code for Deseq2. This results in Zero DEGS: total\_count…

edger deseq deseq2

updated 8.5 years ago • michael.steffen

Hello everybody, I have one experiment with 3 conditions : cell lines of patient with one specific mutation (6 samples), cell lines of patient with another specific mutation (6 samples) and cell lines of controls (3 samples). I have 2 questions of interest: what are the deregulations between: 1/ cell lines of patient with one specific mutation (6 samples) vs cell lines of patient with another s…

deseq2 contrast

updated 9.6 years ago • Jane Merlevede

x treatment effects ... under consideration of 1) sample pairing and 2) technical replication. My first (simple) approach neglecting treatment is as follows: ----------------------------- Line 1 > biolrep <- pData$Replicate Line 2 > timep <- factor(pData...Timepoint, levels=c("Pre", "Post")) Line 3 > treat <- factor(pData$Treatment, levels=c("reagent","c…

updated 11.6 years ago • Otto, Benjamin

I am trying to perform differential analysis on gene data but I am struggling to solve the following error. After creating the `DESeqDataSet` object with `DESeq()` function I am trying to get the result tables by using the `result()` function on the `DESeqDataSet` object `ds` generated as follows: ``` results( ds, contrast = c("clinical_group", "Treatment", "Control") ) ``` but I am getti…

deseq2

updated 5.8 years ago • jrossol

levels (ctrl, A and B, where ctrl is the reference level). I have added the design below my post (first column is just the sample ID) as the dput. I am using the following formula to consider all possible variables and interaction...X time 24h phenotype A) vs (tissue X time 24h phenotype ctrl). Please note that I do know how to run DESeq2 for simple pairwise comparisons, but this is not the goal …

r deseq2

updated 6.4 years ago • arielle

since it is a rather long script. And again: splitting point of the two different outputs is the `DESeq2::results()` function, supplying different contrast codings. So based on the fact that more than 17,200 genes are completely...data = SummarizedExperiment::colData(x = dds) ) DESeq2::design(object = dds) <- model_formula dds_estimate_size_factors <- DESeq2::estimateSizeFactors( …

DESeq2 RNASeqData DifferentialExpression

updated 5.1 years ago • Ben

available I have some doubts about how to do the contrast. I run the following commands: ##Loading DESeq2 source("http://bioconductor.org/biocLite.R") biocLite("DESeq2") library("DESeq2") setwd("~/DESeq2") milk<- read.csv("global_count_genes_milk.csv...DESeqDataSetFromMatrix(countData= milk, colData= milk_design, design= ~ breed + day) dds$breed<- factor(dds$breed, levels=c(…

DESeq2 DESeq2

updated 11.6 years ago • AROA SUÁREZ VEGA

i.e. Ages 0-12. For each age, I have sequenced 3 replicate specimens. I'm completely new to DESeq2 and I was wondering if what I did below is correct. library(DESeq2) count_file_names <- grep("counts",list.files("featureCounts...formatted the featureCounts output files to reflect the HTSeq count output format. As far as I know, DESeq2 needs just counts and doesn't discriminate …

DESeq DESeq2 R

updated 4.3 years ago • jbnrodriguez

div class="preformatted">Hi! I would in a microarray experiment analyze three factors simultaneously in Limma, treatment, group (i.e. gender), and study center. Is it possible to include three factors simultaneously

Microarray limma Microarray limma

updated 12.6 years ago • Ingrid Dahlman

In other words, I would like to compare whether log2(1A/1B) equals log2(2A/2B) or not. I read [DESEQ2--compare two log2-transformed fold changes](https://support.bioconductor.org/p/79133/), and I am generating a model using...for my purpose. Does someone havs suggestions on how to design the model in this situation in DESeq2? Thanks in advance for your help. Regards, Tatsuhiko

deseq2

updated 6.4 years ago • tntntntntn

Hi, I would like to use DESeq2 for the analysis of the differential expressed gene during the tumor development. I got the expected cell number...Hi, I would like to use DESeq2 for the analysis of the differential expressed gene during the tumor development. I got the expected cell number of...Hi, I would like to use DESeq2 for the analysis of the differential expressed gene during the …

DESeq2 DEs

updated 21 months ago • feather-W

I'm doing a differential gene expression analysis and am working on getting the design model right with DESeq2. The data I'm working is split by sex and three different groups, so Males/females and groups control/E/S. I do want to analyze the interaction terms as well so from what I gather my model design should be: ```r dds1 <- DESeqDataSetFromMatrix(countData = round(countsTable), …

DESeq2

updated 8 months ago • hampsdiv

in the section "Group-specific condition effects, individuals nested within groups" of the DESeq2 RNAseq tutorial: [https://www.bioconductor.org/packages/devel/bioc/vignettes/DESeq2/inst/doc/DESeq2.html\#group...effects-individuals-nested-within-groups](https://www.bioconductor.org/packages/devel/bioc/vignettes/DESeq2/inst/doc/DESeq2.html#group-specific-condition-effects-individuals-nest…

deseq2 linear model

updated 7.2 years ago • fnigsch

I have been failing to build a DDS object using the function `DESeqDataSetFromMatrix()` Here is some randomly generated matrix I am using to test out if my DEseq2 package is working properly: x <- round(matrix(rexp(480 * 10, rate=.1), ncol=12), 0) rownames(x) <- paste("gene", 1:nrow(x)) colnames(x) &lt...DESeqDataSetFromMatrix()` Here is some randomly ge…

deseq2 null summarizedexperiment rstudio

updated 6.5 years ago • 200dumplings

Hello, I am running a glm analysis with edgeR on 64 biologically independent samples, with 3 factors. I have sex (M and F), age (P0, P7, P15, P30), and genotype (Control, KO). I have two separate questions, 1) I would like to look at the...Hello, I am running a glm analysis with edgeR on 64 biologically independent samples, with 3 factors. I have sex (M and F), age (P0, P7, P15, P30), and genotyp…

edgeR glm RNAseq

updated 5.3 years ago • jackiesalzbank

Hi! This question is regarding the creation of a contrast matrix in limma, edgeR or DESeq2, while analyzing RNA-Seq data. The question is: "How to create contrasts which represent comparisons between two factors...3,4,9,8,7,8,8,10,2,3,1,1,3,2,9,9,7,6,8,6)) # Setting levels for fixed effects. groups$treat <- factor(groups$treat) groups$time <- factor(groups$time) groups$grou…

limma deseq2 edgeR

updated 6.5 years ago • Ben

Hi Michael, I want to pass a corrected model matrix to `DEseq` following the collapse of some technical replicates and I just wanted to make sure I am doing things in the correct order and grabbing the right data. First I created an arbitrary `dds` object with a design including only 2 of the 3 elements of my design. Then I collapsed the technical replicates. Then filtered the collapsed c…

deseq2 RNAseq model.matrix collapseTechnicalReplicates

updated 6.1 years ago • rbenel

lt;- DESeq(ddsHTSeq) table_counts_normalized <- counts(dds, normalized=TRUE) samples$Group <- factor(paste0(samples$participants,samples$response, samples$intensity, samples$time, samples$gender)) dds <- DESeqDataSetFromMatrix

edger and deseq2 differential gene expression bias

updated 7.2 years ago • yusuf.zhc

in D1 and leg in D2. My current solution is a standard cell means model with site as a blocking factor: <pre> <span style="line-height:1.6">f <- factor(sampDat$joinedGroup)</span> site <- factor(sampDat$site) design <- model.matrix...0.05)</pre> Which gives me expected results. My question is in regards to the unbalanced blocking factor. I'm…

limma

updated 9.3 years ago • obarton

Hello fellow DESeq2 users,  I am having trouble figuring out how to set up the design formula for an experiment with 3 factors with up...not full rank' error. They do not appear to be a linear combination or a combination of other factor levels to me.   I am able to successfully set up the experiment without the positive controls and a design with

deseq2 multiple factor design

updated 8.4 years ago • rrcutler

Try the simpleaffy package from Crispin Miller et al. for a graphical summary of scaling factors, %present calls, background etc. for all .cel files in an experiment. Jacky From: "Khan, Sohail" <khan@cshl.edu> Subject: [BioC...Scaling Factor for Affymetrix chips. To: <bioconductor at="" stat.math.ethz.ch=""> Message-ID: <c8696843ae995f4ea4cdc3e2b83482a901878fa3...Content…

affy gcrma simpleaffy affy gcrma simpleaffy

updated 19.8 years ago • Jacky Pallas

dds)) > 1, \] \# Removes rows with zero reads in all samples dds <- DESeq(dds) \# Runs DESeq2 dds2 <- dds \# Copy dds to dds2 dds2$group <- factor(paste0(dds2$genotype, dds2$muscle, dds2$region)) design(dds2) <- ~ group dds2

deseq2 rna-seq

updated 8.4 years ago • martin.weihrauch

Hi Michael / DESeq2 developers, I’m trying to make sense of a comparison of DESeq v1.14 and DESeq v1.22 on the identical data set / condition...lfcShrink function running). This code looked like this: DESeq2_test_1.22.R: ``` packageVersion("DESeq2") # [1] ‘1.22.1’ deseqds<-DESeqDataSetFromMatrix(countData=test_counts, colData=test_conditions, design= ~ condition...and “deseq1.22_…

deseq2 shrinkage

updated 7.0 years ago • vvi

Hello,  First of, I just want to say I've never used DESeq2 before and I'm new to R. I've a counts.htseq file I've created with none of the mentioned...samples in to one file, which i've called counts.htseq. Now, i thought it would be a breeze to run deseq2, but the first thing i noticed before even running the first line of code, is that I need a sample information table or

deseq2 coldata

updated 7.1 years ago • mjrarcher

of samples in a group) across the board.</span> <span style="line-height:1.6">2.</span> I have a 2-factor RNAseq experiment (factor 1=habitat, l vs s; factor 2=watershed, a vs b) with 3 replicates for each condition, for 12 libraries...3 <span style="line-height:1.6">However, the example given in the documentation is for a single-factor experiment, so I am wondering if …

edger cpm multiple factor design independent filtering

updated 10.0 years ago • dietahanson

Hello, sir. I have a question regarding using microbiome dataset for DESeq2. I used DADA2 and got like 200 samples X 3000 RSVs table. I want to determine which RSV are differentially abundant across...Hello, sir. I have a question regarding using microbiome dataset for DESeq2. I used DADA2 and got like 200 samples X 3000 RSVs table. I want to determine which RSV are differentially abundant acro…

deseq2 dada2

updated 7.8 years ago • blastzone.heimerdinger

has three replicates except for the control group. After performing the time course experiments in DESeq2, I got over 1000 DEGs. Now, my simple question is: Are those DEGs (__1) behaving in a condition-specific way __(aka rapid is...pre> countdata=read.table("all.genes.txt", sep=" ", header=TRUE, row.names=1) condition <- factor(c(rep("control",2), rep("rapid",3), rep("slow",3), …

DESeq2 Time course RNA-seq

updated 9.5 years ago • CandiceChuDVM

the following dataset: - 26 indivuals with expressiondata (log-ratio from common ref design) (random factor: patient) - each individual has expression data from 1 to 3 different (but fixed) locations (fixed factor: location) - 14 controls...12 patients (fixed factor: group) i am mainly interested in the group effect! i tried limma, but it did not seem to work with my random factor patient

ExpressionData limma ExpressionData limma

updated 17.5 years ago • Bettina Kulle Andreassen