Bioconductor Forum

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 6.9 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 7.3 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 9.6 years ago • sebastiano.curreli

Hi Michael, I am having some trouble fitting my model in DEseq2. In my design, I am missing one condition (A-5); see below: stiffness shear A 1 A 1 A 1 A 2 A 2 A 2 A 2 A 3 A 3 A 4 A 4 B 1 B 1 B 1 B 1 B 2 B 2 B 2 B 3...Hi Michael, I am having some trouble fitting my model in DEseq2. In my de…

DESeq2 design 0substract nofullrank

updated 3.1 years ago • Laia

First mid 1.1230212 N1R3 Rachis Meristem Second low 0.7712608 N1R4 Rachis Meristem Second low 0.7368674 N2R1 Primary Branch...Meristem First high 0.8212009 N2R3 Primary Branch Meristem Second low 0.6173428 N2R4 Primary Branch Meristem Second low 0.7801434...N3R1 Secondary Branch Meristem First high 1.4397311 N3R2 Secondary …

deseq2

updated 10.8 years ago • Tom

Hi all, I am a microbiology grad student new to bioinformatics, conducting some RNA-Seq experiments to determine differentially expressed genes after antimicrobial treatment. I have an untreated control and 4 different antimicrobial treatments all conducted on the same cell type/microorganism with 3 biological replicates for each. The only factor changing between these groups is the treatment a…

deseq2 design and contrast matrix rnaseq

updated 8.0 years ago • dbouzo

Hello, I am working with RNA-seq data and using DESeq2 to perform differential expression analysis. My design has a single factor, diagnosis, which is a multi-level factor...in comparing each diagnosis against 'Craniopharyngioma'. To do so, I tried to relevel the 'diagnosis' factor: ``` dds$diagnosis <- relevel(dds$diagnosis, ref = "Craniopharyngioma") ``` However, when I run the …

DESeq2

updated 2.4 years ago • Amdom

Hi everyone, I have a bit of a problem with DESeq2 which I have already seen described and answered before, but I think I’m missing some key information to correctly...Hi everyone, I have a bit of a problem with DESeq2 which I have already seen described and answered before, but I think I’m missing some key information to correctly tackle it. To summarise the experiment, I have a set of sa…

RNASeq DESeq2

updated 4.5 years ago • jehimi

nbsp; Dear Mike: I am using DESeq2 to analyse RNA-seq data and very appreciated its comprehensive functionality. In my data, the counts are affected...doing this. One is adding the interaction term explicitly in the formula, the other is combing two factors into one factor named group. I run the examples provided in the help page and found that two options output different

deseq2

updated 9.0 years ago • zkwu2011

Hello, As far as I understand the DESeq2 package is automatically loaded when R is started up. I am not interested in using DESeq2 anymore, so how can I stop it...as sometimes I need to restart my session so that the package I am attempting to install would first update an already existing package. Because DESeq2 masks so many packages and loads first, I get basically stuck in a...Loading requir…

r deseq2 error

updated 7.3 years ago • mdi3

Hello, I have a few questions regarding the normalization performed by the DESeq2 package: * I understand that the normalization is working under the assumption that most of the genes are not DE and...Hello, I have a few questions regarding the normalization performed by the DESeq2 package: * I understand that the normalization is working under the assumption that most of the genes are no…

deseq2 deseq

updated 10.9 years ago • solgakar@bi.technion.ac.il

two separate runs. I wonder if I can merged the two raw counts tables and normalize them using the DESeq2 size factors method, or do I need to normalized each table separately with DESeq2 package and then use a quantile normalization

RNAseq normalization deseq2

updated 7.6 years ago • uguy

makeContrasts(a-(b+c)/2, a-b, levels=design)</pre> I'm trying to replicate my analysis with DESeq2. Here is some test data, and code to show I can extract DEG results between group a and b, b. However, How do I get DEG between

deseq2

updated 8.0 years ago • chang02_23

cultures, co-cultures and triple co-cultures. . My question is to do with the design formula for DeSeq2. I have a number of comparisons to make. These comparisons can be single factor and or additive between various combinations...of the factors. My question is would I need to make a new design formula and re-run the Deseq2 process each time for each unique comparison...or can I code up the disti…

StatisticalMethod DESeq2

updated 4.5 years ago • Saif

then be converted to BAM format. I ran ```featureCounts``` on the BAM files and now I'd like to run DESeq2 on this count-matrix of edited reads. There are a few potential problems with this. The treatments had different overall...few questions about how best to proceed. - For the edited-read count matrix, should I input size factors based on the original library sizes, or based on the number o…

deseq2 circRNA rna-editing

updated 6.0 years ago • maxhenryhills

I am attempting to use DESeq2 to model differential abundance in microbiome count data. I have been performing my other analyses in phyloseq, and...I am attempting to use DESeq2 to model differential abundance in microbiome count data. I have been performing my other analyses in phyloseq, and I used the phyloseq\_to\_deseq2() conversion prior to attempting differential abundance analysis: …

deseq2 microbiome phyloseq

updated 8.2 years ago • couchc

Hello,   I have a question about the DESeq2 package. My experiment is comprised of eight different treatments, which I am analyzing in pairs. I did the DESeq2 analysis...twice for each pairwise comparison: 1) The first time only the counts files for the two compared treatments were taken in consideration for both the normalization...double the amount of statistically significant dif…

deseq2

updated 7.7 years ago • meshi.barsheshet

to my earlier question https://support.bioconductor.org/p/85766/ - thank you Aaron) <pre> pop <- factor(rep(1:6, each=4)) lat <- factor(rep(c("N", "S"), each=12)) treatment <- factor(rep(rep(1:2, each=2), 6)) design <- model.matrix(~0 + pop + lat:treatment...latN_treatment2" "latS_treatment2"</pre> Yet, I actually have the same question regarding DESe…

multifactor nested design deseq2

updated 9.1 years ago • swaegers.janne

in the vignette on interactions (http://master.bioconductor.org/packages/devel/bioc/vignettes/DESeq2/inst/doc/DESeq2.html#nested-indiv), I combine the gender and condition to a single column and use it in the following...model dds$group <- factor(paste0(dds$convexity, dds$gender_cond)) dds$group= leftM_dis1, leftF_dis1, rightM_dis1 and so on. Am I correct in the design

deseq2 multifactor interaction term gender effect 3-factors

updated 6.5 years ago • kmu004

Hi Michael, I have a couple of questions about DESeq2. 1) Could we use it for repeated measured outcome? Do we only need to add a time variable in the design as below? dds <- DESeqDataSetFromMatrix...2) For the condition variable, could we use continuous outcome here? Or we can only use the factor variable for condition due to NB distribution behind the DESeq2? 3) I saw you men…

DESeq2

updated 4.4 years ago • lovelymaoqin

unable to find an inherited method for function 'exprs' for signature '"DESeqTransform"' > DESeq2::plotPCA(rld,intgroup=c("name", "condition")) Error in .local(object, ...) : the argument 'intgroup' should specify columns of colData...DataFrame with 40 rows and 2 columns condition sizeFactor <f…

software error deseq2

updated 5.4 years ago • yueli7

was more or less ignored in limma, edgeR, and DESeq between 2004 and 2013 until it was added to DESeq2 in 2014. DESeq2 paper (Love et al, 2014) mentions three other Bayesian papers that moderate LFC, but they are all dated 2012...was applied only to the 2nd order (variance) parameters, and my question is why.   The first version of limma (Smyth, 2004) provides an answer but it's very p…

limma edger deseq deseq2

updated 8.1 years ago • Nik Tuzov

Hi. I have two RNa seq datasets generated from same cell line at different time for same treatment of drugs. First dataset is vehicle only vs Treated with drug for 2 days , and another dataset in same cell line where vehicle only and 6...two RNa seq datasets generated from same cell line at different time for same treatment of drugs. First dataset is vehicle only vs Treated with drug for 2 day…

org.Mmu.eg.db DESeq2 TimeCourse DMSOvsdrug RNASeqData

updated 7 months ago • Cosmic

recommend tools preferably in Bioconductor for the following problem: I have a list of transcription factors. At first, I must find all the possible downstream gene targets, then retrieve all the possible interactions (edges...to build a regulatory network. All the tools I found do the opposite: finding transcription factors for a list of genes. Your help and advice are highly appreciate…

transcription factors regulatory gene network gene targets

updated 6.1 years ago • alakatos

span style="line-height:1.6">While running DESeq2 on a Human dataset (commands given below), I have noticed the following error. How I can handle my data with this case...p/63845/>? Could you tell me how to solve the problem, please? > library("DESeq2") > setwd("/folder/to/path/") > sampleFiles =grep("count",list.files("/folder/to/path/"),value=TRUE) …

deseq2

updated 10.7 years ago • ygchai2015

data (2 males + 2 females) vs WT at 1h (2 males + 2 females). I am using "Timepoint" as my primary Factor, and the 2 groups mentioned before as my Factor Level 1 and 2 respectively. Thus, my primary comparison will be between...tutorials and info pages, I was drawn to the conclusion that I can introduce "Sex" as a secondary factor, and contrast 'Males' - Factor Level 1 with 'Females' - Factor Lev…

DESeq2 RNASeq Galaxy

updated 3.7 years ago • István

Hello, I apologize if this question was answered many times before. I'm a little bit confused by DEseq2 coefficient. I have two factors, genotype (WT, KO) and batch (B1, B2, B3) variables and would like to see gene expression between...or overall difference between genotype. i.e. sample 1~6 (WT) vs sample 7 ~ 12 (KO). ```r library(DESeq2) library(tidyverse) dds <- makeExampleDESeqDataS…

DESeq2 coefficient

updated 20 months ago • J

Hello, I'm developping package with DESeq2 and agricolae as dependencies. But DESeq() function seems to not work anymore when package agricolae is called. ``` library...agricolae) library(DESeq2) ## DESeq() Example commands cnts <- matrix(rnbinom(n=1000, mu=100, size=1/0.5), ncol=10) cond <- factor(rep(1:2, each=5)) # object construction

deseq2 dependendies conflict agricolae

updated 5.6 years ago • etienne.rifa

Hi, I want to combine DESeq2 and CQN to normalize the count matrix of ATAC\_seq data. I follow this step: cqnOffset <- cqn.subset$glm.offset &nbsp...object\_cqn) <- cqnNormFactors object\_cqn <- DESeq(object\_cqn,parallel=T)   But DESeq2 mannul describes that normalization factors, if present, will always be used in the place of size factors. <span…

deseq2 cqn

updated 8.4 years ago • 984366176

in information. My question is, "What is the appropriate way to use this spike-in information with DESeq2?" Is it appropriate to use the spike-in information with the `DESeq2::estimateSizeFactors()` `controlGenes` argument (e.g...somehow exclude these genes from the counts matrix in the *DESeqDataSet* object prior to running `DESeq2::DESeq()`—is that correct? Another thing we've considered is c…

DESeq2 SpikeIn Normalization

updated 2.8 years ago • kalavattam

about RIP-Seq by email, and wanted to post a reminder about how to test for ratio of ratios using DESeq2. Suppose we have two assays: Input and IP, and we have two conditions: A and B. Then using a design: '~ assay + condition + assay...replicates), then there are not enough residual degrees of freedom to estimate the dispersion, and DESeq2 will automatically use a design of ~ 1 for es…

deseq2

updated 9.6 years ago • Michael Love

div class="preformatted">Hi, Please can someone explain what is the difference between Z-factor and Z'-factor as reported in cellHTS2? Many Thanks, Steve </div

updated 17.9 years ago • Steve Taylor

mind to run "RUV_total" function. But, I have no idea which one is more reasonable, or better ideas? First, I can merge the codesets, and then run "RUV_total" function on the merged one. For example, there are 241 Endogenous + 20 (hk...ruv normalization. Second, I can do normalization on codeset1 and 2, respectively. Then, the RUV factors can be combined to be pData and passed to DESeq2. In …

RUVSeq DESeq2

updated 3.4 years ago • xiaofeiwang18266

I use DEseq2 to analyse the RNA-seq data out of an animal experiment (two conditions and two gender). The conditions include HS and...header = F, row.names = 1) countall <- as.matrix(countall) #load libraries library(DESeq2) #Assign condition condition <- factor(c(rep("Control",5),rep("HS",8),rep("Control",7),rep("HS",9))) sex <- factor(c("female","male…

deseq2 ~group

updated 5.4 years ago • weichengz

Hi, I am trying to understand the DESeq2 design formula.  I am new to RNAseq, new to DESeq2 and new to writing design formulas/matrix (not a statistician or...from each technical replicate. I used  <pre> sampletable <- data.frame (temperature= factor (rep(c("low", "high"), each = 12)</pre> But in this design it did not take into account the technical repl…

deseq2

updated 7.9 years ago • deeptiptl74

Hi, We are relatively new to RNA-seq data analysis and we have some questions regarding DESeq2 although we have extensively read the paper and the vignette. 1)we wonder how and when DESeq2 takes specified covariates...actual» fold change from this type of variable. Also we would like to know if there is a function in DESeq2 where we can get «intermediate» output file from different analysi…

deseq2

updated 6.4 years ago • andree-anne

to use other additional data to compare.   So I put the multi-factors into the design command. I thought it would affect other results if I included multi-factors, as opposed to just Group...nbsp;  77   Below is the code I have bee trying to run. > coldata.clinic$Group <- factor(coldata.clinic$Group, levels = c("Control","Case")) > coldata.cli…

DESeq2 multiple factor design design model design matrix rna-seq

updated 6.9 years ago • iammiso

__  Please read the vignette section 'Model matrix not full rank':__ __  vignette('DESeq2')__ I read in the DESeq2 manual that DESeq works on duplicates. My questions are: 1) Is there any case where I can use DESeq2...it? 2) I tried the option __ignoreRank = TRUE__, but it still didn't work. Is this necessary in the first place? Thank you

deseq2

updated 7.7 years ago • csijst

I am trying to take the "difference of differences" in contrasts from two factors (`sex` and `group`). We have male and female animals (`sex` factor) that were untrained or trained for 1, 2, 4, or 8 weeks (`group` factor...1w - male:control) - (female:1w - female:control) ``` However, I can't figure out how to do this in DESeq2. Specifying this contrast in limma would be straightforward, but I wa…

DifferentialExpression contrasts interaction DESeq2

updated 3.5 years ago • Nicole

Hi. there are two issues that I have. 1. Raw read count used as input data for deseq(). Is it really okay? I referenced that information on following website.[http://bioconductor.org/packages/devel/bioc/vignettes/DESeq2/inst/doc/DESeq2.html#why-un-normalized-counts][1] But, I want to try normalizing raw count into RLE(Relative Log Expression). 2. What does default statistical method of DEs…

normalization deseq2 StatisticalMethod

updated 2.6 years ago • SH

please help me solve this issue?</p> <p abp="10403">I am flummoxed.</p> <p abp="10405">I have ran DESeq2 and have my count matrix and now want to rlogTransform to visualise the data on a PCA.</p> <p abp="10407">However I get the following...em></p> <p abp="10417"><em abp="10418">## the formula of "design" defines the interested factor i…

deseq2

updated 8.5 years ago • helen.cwp

Hi all , I am new to this field of Transcriptome analysis , so please help me in this.          I just want to know that can we run DESeq2 for without replicate of treated and Untreated .If yes then what should the change in the syntax of DESeq2 .Below is the syntax i am using for with Replicate samples. And if no , then w…

without Replicate deseq2

updated 9.9 years ago • Sushant Pawar

2 samples (replicates) each. I have 4 sample classes since the experimental design has two different factors with two conditions for each factor. <table border="1" cellpadding="1" cellspacing="1" style="width:500px"> <tbody> <tr> <td>sample...refers to miRNA expression from exosomes. My main doubt is how to test these contrasts with DESeq2. Initially I supposed to create…

deseq2 mirna-seq experimental design differential expression

updated 8.5 years ago • francesco.gandolfi

Hi, I am new in RNA-seq analysis via Deseq2. I want to study differential gene expression pattern in two different genotypes in two different time points with...stringsAsFactors=TRUE) cond <- as.data.frame(cond) sapply (cond, class) ##all should be factors cond[,1] <- factor(cond[,1]) cond[,2] <- factor(cond[,2]) cond[,3] <- factor(cond[,3])</pre

bioconductor deseq2

updated 7.2 years ago • suren.neupane04

Hello, I have some questions about releveling to reset the factor level. I am running an experiment in DESeq2 with 4 stages that I want to compare to each other under one treatment. My goal...is to change which factor is the reference for generating result outputs. I understand that the default is the 1st factor, by alphabetical...ways: <pre> dds.t0$stage <- relevel(dds.…

deseq2 relevel resultsNames multifactorial design

updated 9.7 years ago • rclaire

was only done in duplicate, so another two replicate were done at a later date. I am using deseq2 for my analysis. I have included the batch effect as a factor in my deseq2 design. After accounting for the batch, my contrast...want a batch corrected PCA plot and I am struggling to get it.__ My intuition is to use deseq2's rlogTransformation function to generate the normalized c…

deseq2 DE rnaseq sva combat

updated 9.8 years ago • mbio.kyle

the included target file <pre> target<- readTargets("targetPT.txt") head(target) Genotype <- factor(target$Genotype) Disease<- factor(target$Disease, levels=c("stageA", "stageB", "stageC"))</pre> I have performed a paired samples...genes differentially expressed between stages A and B for example but I noticed that the first patient and the…

limma design matrix limma

updated 10.6 years ago • Riba Michela

Hello, I have some questions about running a likelihood ratio test in the DESeq2 package in R. My experiment has two factors: sex and population, with 8-12 replicates per condition. I wish to identify...countData), sex = c("M", "F", "M", cont... ) pop = c("A", "A", "B", cont... ) library("DESeq2") dds <- DESeqDataSetFromMatrix (countData=countData, colData=stickleDesign, …

deseq2 LRT differential gene expression

updated 9.8 years ago • newsomew13

Hi there, I encountered a problem using DESeq2 adding variables created with RUVSeq aiming to remove unwanted variation in my dataset. I set up my DESeq dataset...Hi there, I encountered a problem using DESeq2 adding variables created with RUVSeq aiming to remove unwanted variation in my dataset. I set up my DESeq dataset like...countData = counts, colData = sample_info, design = ~Bat…

RUVSeq DESeq2

updated 2.3 years ago • anne.hoffrichter

Hi, I have two experiments in the first I have cells of type A treated and cells of type A not treated. In the second I have cells of type B wild type and cell of type...and compare the FC between the two analyses of some genes. What is the best method to do this using DESeq2? Should I perform two separate analyses with DESeq2 and then compare the FC? Or should I load the four conditions in t…

deseq2

updated 8.8 years ago • ribioinfo

Hi , Could someone please suggest a probable reason for the following contradiction I see with DESeq2? DESeq2 reports high logFC but the same gene expression median across condition in both normalized counts (obtained...from DESeq2) and raw counts in matrix is not difference as reported. For example, DESeq2 reports a logFC as 5 and I see literally 0 difference...and groupB. Code used is…

deseq2

updated 5.2 years ago • Raj

customizable_ figures). Tximport were used to load the count data from Kallisto into the DESeq2 pipeline. The first _runs_ with DESeq2 were pretty consistent with the Sleuth results (considering Sleuth...is more conservative than DESeq2) with 19 DETs for treatment1 and 34 for treatment 2. The highest/lowest log fold change was around 6.6 and -3. __BUT then...__&nbs…

deseq2 tximport rnaseq kallisto R

updated 8.4 years ago • birgittenilsson

Hello, I have seen examples of design formulas that have the replicate factor and others that do not use it. **I was wondering when one should put the replicate factor in the design formula and when...and without the replicate, I cannot get to see any interesting DEGs but if I put the replicate factor in the design formula, I see the genes that are expect to move. Then, we have another dat…

DESeq2

updated 4.6 years ago • DcL-A

I can get some clarification on our design. I am trying to specify the appropriate model in DESeq2 for our RNA-seq data. We have 8 pairs of corals that each came from 3 reefs (1, 3, and 4 pairs per reef), and each one in the pair...time and which do not. We are not interested in pair-specific expression; in fact I would like to factor OUT pairs by comparing primarily within pairs and then among …

deseq2 rnaseq model design

updated 6.9 years ago • swillis4

I am doing a differential gene expression (DGE) analysis using DESeq2 v1.24.0 with ~200 bulk RNA-seq samples (whole-blood) with a continuous variable of interest. I am interested in applying...count-based outlier detection and replacement using Cook's distance. For factors, this functionality is applied by default when calling `DESeq()`. However, based on the [DESeq2 vignette](https://bioconducto…

deseq2

updated 6.0 years ago • bryancquach

Hello! I am working on a project where I have a fully-crossed two-factor design, with two levels per factor and four biological replicates per treatment combination (16 biological replicates...temperature, and which genes have expression patterns that depend on the interaction of those two factors (i.e., for which genes does the effect of testing temperature depend on prior rearing temperature…

DESeq2 RNASeqData deseq2 ExperimentalDesign

updated 2.0 years ago • Sam

Hi all, I'm analyzing the cancer sample using DEseq2 but I encountered some errors when using interaction terms. My Coldata is below. ```{r} > coldata sample condition patient...want to compare with 1) sample pairwise like P03_pre and P03_post for minimizing sample compounding factor and 2) use interaction terms. To 1) compare with sample pairwise and 2) correct batch eff…

DESeq2 interaction pairwise

updated 4.2 years ago • Lucas

Hi,  it seems like with the new version of DESeq2 (Version 1.10.0) is always using the standard model matrix as default rather than the expanded model matrix for design...expanded model matrix by doing the following, but got an error. Please help. my experiment has two factors (Type with 2 levels and Day with 3 levels) experimental design I use is ~Type + Day + Type:Day > dds …

deseq2

updated 9.9 years ago • KELVINLEE

Hello, My team and I have a question regarding the addition of covariables in DESeq2 analysis. We ran DESeq2 with a first model and obtained adjusted p-values for all genes. However, when we tried to add 2...Hello, My team and I have a question regarding the addition of covariables in DESeq2 analysis. We ran DESeq2 with a first model and obtained adjusted p-values for all genes. Howev…

deseq2

updated 5.3 years ago • andree-anne

I am looking for advice about how to correctly use the "coef" argument to lfcshrink when I am using DESeq2 with a grouping variable. I am analyzing an RNAseq dataset with multiple factors and variables using DESeq2: * SurgeryGroup...results and shrink the LFCs by specifying my contrasts of interest. > dds$group <- factor(paste0(dds$SurgeryGroup, dds$TimePoint)) &a…

deseq2 lfcshrink

updated 7.2 years ago • kendralec