Bioconductor Forum

I'm trying to use DESeq2 to determine if an OTU is enriched by treatments from 16S rRNA sequence data.  I imported OTU counts for each library...otu), and metadata information for each library (meta).  One of my metadata columns is a factor with 5 levels (factors).  I want to do all pairwise comparisons for all 5 factor levels.   I've tried to do it this way.…

deseq2 contrast matrix

updated 7.8 years ago • natalie.hull

sampleTreatment) dds<-DESeqDataSet(se, design= ~batch + sex + treatment) dds$group <- factor(paste0(dds$sex, dds$treatment)) design(dds) <- ~0 + group dds <- dds[ rowSums(counts(dds)) > 1, ] dds <- DESeq(dds, betaPrior = FALSE...res2_sorted = res2[order(res2$padj), ]</pre> In doing it this way I get results that show the first member…

deseq2 deseq differential binding analysis

updated 6.2 years ago • rbronste

transcriptomics. I’m currently trying to characterize how the overexpression of three transcription factors (x, y, z) acts on the transcriptome of a cell line. The design involves 5 groups of samples (x, y, z, xyz, Ctrl), each group containing...or represses some genes, as highlighted by differential gene expression analysis performed using DESeq2 (using the Ctrl condition as reference). I’ve ru…

deseq2 factominer PCA

updated 8.4 years ago • sebastiano.curreli

Hello, I am posting to report what I think is a bug in an error message in DESeq2. I suspect that `DESeqDataSetFromMatrix` misdiagnoses the input type when the user passes a factor, reporting to them...which I reproduce and inspect the issue, or a Git repo [here](https://github.com/sweeney-th/possible-deseq2-bug). edit: there is a better-formatted version on the PDF report [here](https://drive…

deseq2

updated 4.6 years ago • thadryan

Hi everyone, I'm trying to use DESeq2 to analyze some mutagenesis data and identify variants that confer a selective advantage. To do so, I'm comparing the...account for the difference in sequencing coverage (instead of downsampling Region A's data) using DESeq2's gene-dependent normalization factor. One idea I had was to assign a factor of 1 to all of Region A's mutants and a factor...all of Re…

deseq2 normalization

updated 6.2 years ago • Thomas_WILLEMS

Hi folks, The DESeq2 vst rlog size factors computations account for differences in library composition amongst other things. Library

deseq2 normalization

updated 4.1 years ago • twfs

Hello, I'm a PhD student and I'm using DESeq2 to analyze the differential abundance of 16S amplicons across a number of factors. The software is fabulous, I'm just...the role of host plant species and soil treatments on the abundance of bacterial taxa, these factors are crossed so that each host plant species is replicated in each soil treatment. I think an estimate of the log-fold...nbsp;associ…

deseq2 lfce contrast

updated 7.8 years ago • crfitzpat

A and Treatment A vs Treatment B per patient and for all patients. I used the following design for DESeq2: <pre> coldata$combine <- factor(paste0(coldata$patient, "-", coldata$treatment)) dds <- DESeqDataSetFromMatrix(countData...Jcounts, colData = coldata, design = ~ combine)</pre> I like the combination of the factors because it is easier use, you get your results…

deseq2 multiple factor design

updated 9.7 years ago • mat.lesche

Are polynomial contrasts for a time factor possible in DESeq2? If we have a design with multiple time points being measured for independent samples, and we want...Cubic: −1, 3, −3, 1 We have already compared adjacent time points using the contrast command in DESeq2. But someone asked about testing the trend over these time points. Using the linear polynomial contrast above is one

DESeq2

updated 20 months ago • dairey

Hello all, We have two variables from RNA-seq data: tissue (tissue1 and tissue2) and treatment (healthy and unhealthy). There are in total 4 healthy and 4 unhealthy animals, from which samples from tissues 1 and 2 were collected from each animal. We would like to perform a differentially expression gene analysis between healthy and unhealthy animals, considering the tissue effect (both tissues) …

edger deseq2 rnaseq differential gene expression

updated 6.1 years ago • camilla.alves

Dear community, I am running an RNAseq analysis using DESeq2, with which I want to find out what is the effect of 2 continuous factors (A and B) and their interaction (A:B; is this the same...as A*B?) on gene expression (bulk-RNA). I have scaled and centered my continuous factors. Once I've performed the LRT with each of the reduced factors, I'd like to extract the coefficients obtained for…

DESeq2 LRT continuous coeficients

updated 22 months ago • Laia

Hello, I am interested in using DESeq2 to analyze a bulk RNA-seq dataset with one factor and 12 levels (control and treatments), and 4 biological replicates

deseq2

updated 6.1 years ago • fl

<div class="preformatted"> Dear all, I have data collected from an experiment looking at expression differences related to diet and species. I have samples from six species, and for each species I have five paired clones, each pair exposed to one of two diets. The clones are genetically identical but were raised independently (see below for data structure ??? first three species only). sp…

DESeq DESeq2 DESeq DESeq2

updated 7.9 years ago • Guest User

Hi, I read many time DESeq2 doc(https://bioconductor.org/packages/release/bioc/vignettes/DESeq2/inst/doc/DESeq2.html#differential-expression...2101f12WaldWithR.pdf) which is very close to what I need, but in this case it shows Wald test for 1 factors with 2 levels. And I want to know how calculation is done with 2 factors... I read DESeq2 code, in particular about 'makeWaldTest...github: https:…

WaldTest_2factors DESeq2

updated 4.1 years ago • julie

https://support.bioconductor.org/p/79209/ and https://support.bioconductor.org/p/98476/). I use DESeq2 in order to get normalized counts from raw TCGA-HTseq Counts. My method is simple, put the data through DESeq2 - using design...all of my analysis (clustering, survival, gene expression heat maps) on the normalised counts from Deseq2. However I am running into problems with huge numbe…

deseq2 limma normalization tcga rnaseq

updated 5.1 years ago • Alex Greenshields-Watson

Hello, I am using DESeq2 on a project, where I tested various extraction volumes and types and got amplicon data out. So for instance, I have two...two extraction methods PowerWater and PowerSoil. I want to compare the resulting microbiomes. So at first I setup my experiment as: equation `~ExtractionType + Volume`; then changed it to equation `~Volume + ExtractionType` (I am getting...tested, …

deseq2 Tutorial

updated 4.5 years ago • Anna.bramucci

Hi, I would like to use Salmon count data and DEseq2 to identify differentially expressed genes with pre-defined size factors for the different samples (as total transcripts...sizeFactors(dds) <- size_factors dds <- DESeq(dds) #using pre-existing size factors #estimating dispersions #gene-wise dispersion estimates #mean-dispersion relationship #final dispersion estimates...…

DESeq2 tximport

updated 3 months ago • grothmn

Dear Michael,   I have a full factorial  experimental design with 2 factors that have 2 levels each, a total of 4 combinations. I am interested in the interaction between those two factor and that...Factor2). Afterward I got a list of contigs that are significantly interacting between the two factors. My questions are: 1) is there a way to test within the same model the in…

deseq2

updated 6.5 years ago • negroni

Hi All, I am using DESeq2 and I want to use GC content, gene length and size factor offsets using CQN package, as I do various QC visualization plots...and statistics, I have been trying to export the data with varianceStabilizingTransformation. First off I want to say I have used DESeq2 with the estimateSizeFactors function, and this \*appears\* to produce artifact free...smooth results. Howe…

deseq2

updated 8.0 years ago • kodream

NOISeq to test if the biases were corrected, I noticed that when I divided out the normalization factors by the geometric mean (as suggested in DESeq2 manual), the cqn correction becomes ineffective. Instead when I used the...factors matrix to account for GC and length bias library(cqn) cqnObject=cqn(counts = countData,x=uCovar_filt$GC_content, lengths...uCovar_filt$length, sizeFactors =si…

deseq2 cqn noiseq

updated 7.6 years ago • glmazzo

Hi all,   I have a question about testing main effects in a multiple-factor design with interaction term and another one on how to use the fdrtool. I have used it in the following way and I would...amp; abs(log2FoldChange)>=1.5)</pre>   I repeated the FDR correction steps for the main factor Parasite\_origin and for the interaction term Host\_origin:Parasite\_or…

deseq2 multiple factor design FDR fdrtool

updated 7.8 years ago • marieke

First of all, sorry if this is a trivial question, as I am totally new to using DeSeq2. I am attempting to use DESeq2 to look for differential...abundance in microbiome data from soil. As such, I have several different factors describing where the samples came from. These are Compartment, with levels "Bulk Soil", "Rhizosphere", and "Endosphere...Compartments comparing the 2-yr vs 4-yr rotation at…

deseq2

updated 4.2 years ago • Conard Lee

Hi all, First off, I'm a wet lab scientist learning to analyse my own data. I've design my experiment as such > coldata sample condition...Hi all, First off, I'm a wet lab scientist learning to analyse my own data. I've design my experiment as such > coldata sample condition litter 1 KO1_sort.bam KO A 2 KO2_so…

deseq2 normalization

updated 5.9 years ago • jonathan.limwc

genes with a low read count (let's say the bottom 25%) and then ran DESeq1. From the vignette in DESeq2 I know that DESeq2 filters out lowly expressed genes. I'm just wondering what is the better way now. Giving DESeq2 the...nbsp;      D D3         D I ran DESeq2 and extracted …

deseq2 differentialexpression

updated 10.1 years ago • mat.lesche

Hi,   I am running deseq2 on a data set with multiple factors and getting this warning: <pre> the design formula contains a numeric variable with...specifying a model with increasing fold change for higher values. did you mean for this to be a factor? if so, first convert this variable to a factor using the factor() function</pre> What difference does it make for d…

deseq2 design

updated 8.8 years ago • Assa Yeroslaviz

that occur in each tumor cell line between untreated and those treated with Treatment A using DESeq2, but i'm having trouble and keep getting a warning message when running the DESEq pipeline</span> <span style="background...is the error message I get when I run the DEseq pipeline: >dds<-DESeq(dds) estimating size factors estimating dispersions gene-wise dispersi…

summarizedexperiment deseq2 deseqdataset multiple factor design differential gene expression

updated 9.3 years ago • Lillyhchen

I have different treatments (infected and control ones). when using DESeq2, I have different results in two ways of comparisons. **first way:** factor: treatment factor level1: moribund infected factor...way:** (i compared just 2 groups to each other) factor: treatment factor level1: moribund infected factor level2: 5week infected --- factor: treatment factor level1: moribund...infect…

DESeq2

updated 2.5 years ago • zohreh.fazelan

seq and ATAC-seq, which produce reads mapping to genes and open chromatin, respectively.  I use DESeq2 to estimate scaling factors for each sample, so that I can compare e.g. the number of reads for region _r _in...Now I have 2 questions: 1. Is it valid to include the failed QC samples when estimating size factors for the ATAC-seq? Reading note S1 of doi:10.11…

deseq2 estimatesizefactors

updated 6.0 years ago • owenchapman1

Hi, In limma's documentation, the interaction term is described as: "The first of these questions relates to the WT.S vs WT.U comparison and the second to Mu.S vs Mu.U . The third relates to the difference...WT.S-WT.U) , which is called the interaction term." I want to perform the same analysis using DESeq2 (on RNA-seq data). I have 2 cell types: A and B for each cell type, I have a KO…

deseq2 deseq limma interaction term

updated 7.6 years ago • sbcn

Hi all, I have three RNA-Seq datasets to perform DESeq2 differential expression analysis. My main interest is to 1. Compare the treatment to control and see if there are differences...controlling for sex and batch effect The corresponding datasets from the simplest to complex are: First, subject sex condition 1 M treatment 1 M …

deseq2 RNA-Seq

updated 4.4 years ago • Helena

Im running DESeq2 on RNAseq data and my experimental design has 2 factors, A and B. A is categorical and has 4 different levels, B is categorical

DESeq2 Interactions

updated 3.8 years ago • cossardg

low expression (949 genes with counts=0 and 3643 genes with counts<50). When I look at the size factors for this sample, it is much smaller then what would be expected simply by taking the total number of reads. Here is a...the relative expression level, gets lower values than it should. I repeated the calculation of size factors, but used only the genes that had a minimal number of count…

deseq2 rna-seq normalization

updated 8.6 years ago • gil.hornung

Hi, This is my first time using the DESeq2 package for RNA-seq analysis. I am analyzing a study of three different time points in mice (5,10 and...think makes any biological sense. ``` Option 1: # sampleTable <- data.frame(condition = relevel(factor(rep(c("P10", "P15", "P5"), each = 3)), ref='P5'), rep1= factor(rep(c("1","2","3"), times=3)) # dds <- DESeqDataSetFromMatrix(countData…

DESeq2 Bioconductor RNASeqR

updated 3.7 years ago • Blanca

to the functions/arguments that changed: (e.g. http://www.bioconductor.org/packages/2.14/bioc/news/DESeq2/NEWS) I've been working on including more documentation in the package, but haven't gotten to adding the parts about...due to "expanded model matrices", which include a column in the design matrix for each level of a factor, as opposed to "standard model matrices" produced by model.matrix, wh…

DESeq2 DESeq2

updated 10.7 years ago • Michael Love

Dear community, I am trying to perform a differential abundance analysis on 16S data using DESeq2. **The metadata is as follow** : "Prelevement" : 2 types (P1, P2) "Extraction" : 3 types (E1, E2, E3) "NS" : Corresponds to donnor ID. Each donnor...Dear community, I am trying to perform a differential abundance analysis on 16S data using DESeq2. **The metadata is as follo…

deseq2

updated 5.5 years ago • erwan.scaon

I have a 2-factor design with 2 levels each: population: fish from MDPL or MDPP condition: freshwater (FW) or transfer experimental treatments...terms. After consulting the [vignette](http://bioconductor.org/packages/release/bioc/vignettes/DESeq2/inst/doc/DESeq2.html#interactions) and the `` ?results `` section on 'Using interaction terms', I still can’t decide for our...would be most appropriat…

deseq2 experimental design interaction model

updated 7.3 years ago • ljcohen

Hi, Im new to using bioconductor packages in R, I'm importing .csv files into R for DeSeq2 analysis but I can't seem to get the first column of my colData to match the column names of my countData. The match (vector

deseq2 bioconductor

updated 5.7 years ago • ishtarredman

UDNuc3, UDcyto1, UDcyto2, UDcyto3, DFNuc1, DFNuc2, DFNuc3, DFcyto1, DFcyto2, DFcyto3. I have run DESeq2 on UD nuc vs UD cyto samples and DF nuc vs DF cyto samples separately but I am trying to compare the RATIOS of UD vs DF. For...example I want DESeq2 to tell me if there is a difference between (UDnuc/UDcyto) and (DFnuc/DFcyto). I was trying to just manually (in R) calculate...the ratio…

deseq2

updated 4.7 years ago • AmyNeely2023

Hi, I have been reading through DESeq2 related questions, trying to find out if the design I had in mind was suitable, but I could not find a close enough match...4 | B | None | 0 | 1 | | 4 | B | Drug | 1 | 1 | *Main focus of the study* At first, without taking into account the response variable, the questions of interest would be: * How does the genotype affect...va…

deseq2

updated 4.1 years ago • lu.ne

Hi Michael and community, Thanks for the work and efforts on DESeq2! I've had this question for some time, I went through quite a number of posts, the manual, and the vignette, but can't really...TRUE", and  "transform = TRUE" arguments. So is the y axis just raw read counts divided by DESeq2-estimated size factor? or has it been logged? (loge? log2? log10?) Also, why is the psudocount …

deseq2

updated 6.8 years ago • Alan

I'm missing something: My experiment is based on the model ~ pos + ring + pos:ring the factors can take the following values: pos = {Base,Junction,Tip} ring = {Down,Up,Top} I made two DESeq2 runs, where in the first run I used...the default levels of factors (as above),and in the second run I releveled the order of ring to {Up,Down,Top}. When testing the effect of `"…

deseq2

updated 5.3 years ago • assaf www

for ecotype A, etc. and I would even like to raise one level of complexity and include the three factors, for example, difference between north and south at treatment conditions for ecotype A (etc). Following the DESeq2 manual...section 3.3 (Interactions), my solution is to define a factor as a combination of the factors I want to measure for example: condition=rep(rep(c("A.ctl","A.trea…

deseq2 multifactor pairwise

updated 8.5 years ago • aziza

Enter the body of text here Hello everyone, I have some questions about DESeq2 2-factor analysis. One of our ongoing project is about RNA-seq data from patient and control (2 genotypes) with or without...very interested in interaction between genotype and condition. I tried to carry on my analysis by 2-factor mode using DESeq2 with design ~ condition + genotype + condition:genotype with collap…

DESeq2 RNASeq

updated 3.6 years ago • hequn

Hi, I noticed that the size factor estimations when using type = "poscounts" with estimateSizeFactors() and estimateSizeFactorsForMatrix() differ slightly...different for the two functions. Is there particular reason for this, or does the difference in size factors comes from something else? I'm using DESeq2 1.30.1 with R 4.0.2. Thanks, Andreas

DESeq2

updated 2.8 years ago • andreas.gschwind

Hi folks, We are using pseudobulk DESeq2 (DESeq2_1.26.0) approach to perform DGE testing on single cell datasets. We are interested in assessing condition...a model with increasing fold change for higher values. did you mean for this to be a factor? if so, first convert this variable to a factor using the factor() function the design formula contains one or mo…

deseq2

updated 4.6 years ago • charles7744

Hi Mike, I am using DESeq2 to analyze differential translation efficiency (TE) from Ribosomal profiling (Riboseq) and RNAseq datasets...Hi Mike, I am using DESeq2 to analyze differential translation efficiency (TE) from Ribosomal profiling (Riboseq) and RNAseq datasets. Translation...conditions. There are helpful posts giving me examples for just comparing TE between single fa…

deseq2 ribosome profiling

updated 6.4 years ago • xiaozhuo

Hi, I am trying to do some differential expression analysis with deseq2 at the moment and I have samples with 3 different groups; genotype (A and B), treatment (control and treated), time (1hr vs...Hi, I am trying to do some differential expression analysis with deseq2 at the moment and I have samples with 3 different groups; genotype (A and B), treatment (control and treated), time (1hr vs 30.…

deseq2 multiple factor design differential analysis differential gene expression

updated 7.0 years ago • hsbio

Hello, I'm working on GLM model in DEseq2 with an experimental design of 4 treatments , and each treatment has 5 replicates. I have two factors: temperature (with...Hello, I'm working on GLM model in DEseq2 with an experimental design of 4 treatments , and each treatment has 5 replicates. I have two factors: temperature (with control and high temperature) and shore level (high shore and low sho…

DESeq2

updated 6 weeks ago • Bingqian

*This is a copy of this post https://github.com/drisso/zinbwave/issues/58#issue-656953981 but I thought it might reach out to more people by posting it here too.* Hi everyone, I am measuring differential expression between two conditions in 10X data, using zinbwave and DESeq2. I am using K=2 in zinbwave as there are some latent factors (e.g. library size) that I would like to infer from th…

deseq2 zinbwave 10X scRNA-seq Differential expression

updated 4.4 years ago • vincent.croset

I have a question about the proper design for DEseq2, when there is an siControl as well as siRNA for a target gene, then there is a treated and a vehicle for both conditions...I setup the factors and design like so: > dds$condition <- factor(dds$condition, levels =c("siControl","siTarget")) > dds$condition <- relevel...dds$condition, ref= "siControl") …

deseq2

updated 5.2 years ago • j.siefert

Hello, I have a question about DESeq2 designs and implementation. first of all, @mikelove you did a wonderful job with the package and vignette - thank you !. My...Hello, I have a question about DESeq2 designs and implementation. first of all, @mikelove you did a wonderful job with the package and vignette - thank you !. My RNA-Seq experiment has 24 samples: 4 models, 2 treatments (dr…

paired-samples DESeq2 interactionterm nested-design

updated 2.8 years ago • Elad

Hello, I am using DESeq2 in phyloseq to analyse my data, following the phyloseq tutorial. Previously I used the following code and this seemed...are quite different. The difference is at the point when I try to convert the phyloseq format to DESeq2 I get the error message in bold below (previously I was not seeing this error message). I have also loaded the sessionInfo...specifying a model with…

deseq2 phyloseq

updated 7.7 years ago • sarahmacdonald86

of count files). I aligned my fastq files with STAR, counted genes with featureCounts, and am using DESeq2 for differential expression analysis. My design is ~sex + sex:nested + sex:condition, where nested is the pairing factor...At first pass, I get 716 significant genes (padj < 0.05), yet many genes have count plots like this: http://i.imgur.com/2Yrd410.png

deseq2 outliers differential gene expression

updated 7.8 years ago • gregory.l.stone

0000 >> CC: bioconductor at r-project.org >> Subject: Re: [BioC] Questions about multi-factor contrast setting in >> DESeq2 > >> >> Hi, Mike: >> >> Thx a lot for the input and advice, which seems a great idea...gt; > Date: Sun, 9 Mar 2014 13:35:34 -0400 >> > &gt…

GO Visualization Clustering limma edgeR DESeq2 GO Visualization Clustering limma edgeR

updated 10.7 years ago • Michael Love

My name is Carlos. I am PhD student working on proteomic analysis from CSF. I am currently using the DESeq2 package in R to analyze the data, and wanted to ask if it is ok to use the negative binomial distribution on continuos...data? Is the correction factor in the DESeq2 package based on a ratio or on a multiplicative factor? Kind regards, CA

proteomics deseq2

updated 8.8 years ago • carandres74

a better model to answer our questions, I would appreciate any suggestions.  I use a 3-level factor analysis: __Cultivar __factor with 2 levels (cult1, cult2) __Time __factor with 3 levels (T1, T2, T3) __Treatment __factor...for each time point and cultivar. Therefore, I have nested cultivar with time point for my first question, and run full model using Wald test where I have used o…

deseq2 contrast LTR

updated 5.2 years ago • Iryna

published without any problem. I can not figure why this happens. For example: <pre> library(DESeq2) library(ReportingTools) data(mockRnaSeqData) conditions <- c(rep("case",3), rep("control", 3)) mockRna.dse <- DESeqDataSetFromMatrix...colData = as.data.frame(conditions), design = ~ conditions) colData(mockRna.dse)$conditions <- factor(colData(mockR…

publish deseq2

updated 9.9 years ago • noushin.farnoud

LogFC and P-adjust. What make me confused are: 1、I want to do GC content normalization before DESeq2 or EdgeR pipeline, Here's my source using DESeq2: counts_GC <- withinLaneNormalization(raw_count_matrix, "GC", which...As you see, I extracted the normalized counts created by `withinLaneNormalization()` and put it in DESeq2 pipeline, the same I did in edgeR. While, the us…

deseq2 normalization RUVg edger

updated 5.2 years ago • Yang WY

First, I know that DESeq2 is not meant to be run without replicates and results will not be meaningful. I am an analyst who has...Condition - | - A | 1 B | 1 C | 1 A | 2 B | 2 C | 2 A | 3 B | 3 C | 3 I ran DESeq2 with LRT of ~Group + Condition vs ~Group to identify all DE features significant due to Condition. However my collaborators...I believe this approach would still t…

deseq2

updated 4.7 years ago • august

Hello, I am a researcher working with miRNAs in cats. We have used DESeq2 to analyse our data. The differential expression analysis was performed using DESeq2 v1.32.0 on raw counts with a...and within each breed group 3 were healthy and three had heart disease. As per default in DESeq2, the adjusted p-value threshold (FDR) was set at 0.1 For each contrast, normalized counts of miRNAs with …

microRNAincats

updated 3.0 years ago • sofia.hanas