Bioconductor Forum

div class="preformatted">Hi Simon I've been using DESeq2 to analyze RNA-seq data I was given for a multi-factor experiment, three factors with two, three and three levels each...manual or on the list, I'd appreciate your input. Should I use a weighting scheme via normalization factors? Thanks Mike Michael Muratet, Ph.D. Senior Scientist HudsonAlpha Institute for Biotechnology mmuratet at h…

Normalization DESeq2 Normalization DESeq2

updated 12.0 years ago • Michael Muratet

the effect of three treatments (8 animals/treatment) on the metagenome level and Now I want to use DESeq2 tool to find the specific genes that are significantly affected by treatment per gut section. For example Effect...of treatment, A compared to Treatment B in Ileum.  Therefore, I used DESeq2 with the following design:  <pre> <code>dds$group <- …

deseq2 metagenome

updated 7.6 years ago • ghanbari.msc

Hi all, I have extracted raw count data from raw reads file, and my data set is as follows: The first column is the gene name, and the next four rows are counts for each sample, the first two being control data, and the next...have a "summarized experiment" object: https://www.bioconductor.org/packages/devel/bioc/vignettes/DESeq2/inst/doc/DESeq2.pdf https://www.bioconductor.org/help/course-mat…

deseq2 counts

updated 8.6 years ago • uzer

DESeq2 but am considering switching to edgeR or limma. The biological questions I am hoping to address are:   1. What genes...males and females overall?   I’m looking for help with the following questions: 1. Is DESeq2, edgeR, or limma more straightforward for this type of analysis? 2. How should I design my differential expression model...interaction terms for Surger…

differential expression deseq2 edger interactions limma

updated 7.5 years ago • kendralec

I am trying to read in a Sample Table into DeSeq2, where the first column is all my gene names and the row names are my individual samples and their corresponding counts

deseq2

updated 6.8 years ago • Marc

answer my previous question. Further to my previous question, Can we do pair-wise comparison on a DEseq2 object from likelihood ratio, considering that it is similar to ANOVA?. i.e ``` dds_LRT <- DESeq(ddsobj, test="LRT", reduced...1) ``` then use this object to do pairwise comparisons for the 8 levels of the factor ``` res_LRT <- results(dds_LRT, lfcThreshold = 0, contrasts…

DESeq2

updated 2.6 years ago • SE

Hi. how can I normalize raw counts using RLE option? I've looked into deseq2 manual, but there's no information about RLE normalization. It seems it has removed or changed. Here's my code below. Code...DESeqDataSetFromMatrix(countData = Counts, colData = coldata, design = ~condition) # Estimate size factors using RLE method dds <- estimateSizeFactors(dds, type="relative") **Error in match…

RLE normalization deseq2

updated 2.1 years ago • SH

want to compare all of them against baseline so time 0 and then pull the results table after running DESeq2 with all the contrast 0vs2, 0vs6, and 0vs8. I get the error/warning when trying to do the design, listed below. ```>samples...time <- relevel(samples$time, ref='0')``` I also tried with factor to compare: ```>samples$condition <- factor(rep(c('0_days','4_d…

deseq2 contrast design

updated 5.5 years ago • luciest.rodriguez

<div class="preformatted">Hi, I am analysing a patient dataset for diff. expression with DESeq2 (v 1.2.10). We have several patients, and for each patient we have several samples. I am running this analysis on different combinations of samples. My design has two factors : patient (multilevel) and a second factor (proliferation, true/false) so : design ~ patient + proliferation I am inte…

DESeq2 DESeq2

updated 10.9 years ago • Carl Herrmann

Hello, I am performing DGE analysis using DESeq2. I have two groups to compare: CTRL and SA, and I have performed a group comparison using DESeq2 and there's no issue with...lt;- DESeq2::counts(DESeq2::estimateSizeFactors(DE_dataset_pre_sva), normalized = TRUE) write.csv(median_of_ratios_normalised_counts...SummarizedExperiment(as.matrix(filtered_rawcounts), colData = metadata) DE_dataset_wi…

sva DESeq2

updated 16 months ago • marinaw

Is it somehow possible to do a chipseq experiment with two factors (treatment, no treatment), (antibody of interest, control Ig antibody)? I believe that one could do this with edgeR utilizing...group representing each individual chipseq run, and then creating a design matrix to calculate norm factors, estimate dispersion, and perform glmQLFtests (following p.8 of the edgeR manual

diffbind edger

updated 7.2 years ago • james.dalgleish

Hi, I am performing differential expression (DEG) analysis using DESeq2. I have no replicates, and I understand that without replicates, it is useless to conduct this analysis. However, I have...suggestions? It will be great helpful for me to complete my work # Load required libraries library(DESeq2) # Read in the count data count_data <- read.csv("count.csv", header = TRUE, row.names …

DESeq2

updated 5 months ago • kuttibiotech2009

I have a question regarding contrasts in a multi-factorial design in Deseq2. I have a design that have 1 factor (Inoculation) with two levels (Inoc or No.Inoc) and another factor (Crop) with 4 levels...Co, Mix 1 - M1, and mix 5 - M5). I set No.Inoc and Fa as the reference levels for their respective factors. The design was the following: ```r design(degtreatMF) <- formula(~ Crop + I…

DESeq2

updated 2.6 years ago • Pablo

vs Untreated\_GFP(-) (4) Untreated\_GFP(-) vs Treated\_GFP(-). I have different results when I first use esimateSizeFactors then run DESeq, compared if I run first DESeq then estimateSizeFactors.  a part of the R script

estimatesizefactors deseq2

updated 9.5 years ago • Mehmet Ilyas Cosacak

Hi Michael,   I’ve tried to apply DESeq in two sample comparison, and it works well. It is really great for the NGS data analysis.   Now I tried to analysis multiple groups. Though I checked the section about glm model and refer to limma package and look for some answers in BioC posts, I was still stuck and confused. Will you help me to clarify it?   The first que…

deseq2 deseq

updated 7.8 years ago • skyoflcz

Help me In deseq2 design I have factors sex, 2species, 5stages I want to know differential expressed genes between male and female of

DESeq2 genes Differential expressed

updated 23 months ago • Syed Zaheer

and timepoint2, which is basically the difference in contrasts of contrast a and contrast b. The two factors could be coded as <pre> treat=rep(c(0,1,0,1),each=3) time=rep(0:1,each=6)</pre> In edgeR, after fitting a model with ~treat+time+treat...with the null hypothesis: Is any of the three contrasts false?  How could this be done using DESeq2

deseq2

updated 8.6 years ago • Koen Van den Berge

realised it probably belongs here. Sorry about that. I have a table of FPKM values generated by DEseq2, and I'm trying to find out what DEseq2 uses as gene lengths when these are not supplied (I'm trying to assess to what extent...a given row of object, e.g., the union of all basepairs of exons of a given gene." Does that mean DEseq2 directly uses the ranges obtained by using rowRanges(dds…

deseq2

updated 5.0 years ago • hasse.bossenbroek

I have a 3-factor experiment, each factor having two levels: * __gt__: Genotype: XX vs XY * __sex__: Sex: M vs F * __tmt__: Treatment: Letrizole (let...1. Average main effects of genotype, sex, and treatment across all other levels of other factors 2. Interaction effect of gt:tmt across all levels of sex 3. Interaction effect of sex:tmt across all levels of gt Then

deseq2 deseq interactions multiple factor design

updated 9.2 years ago • Stephen Turner

Hi I'm trying to use DESeq2 to find differentially expressed genes between sample with replicates and sample without replicates. I have 2 condition...control and tumour. I am new to Deseq2 package, do you think my command lines are OK for my experiment?   Thanks in advance   code for find DE in tumour...feature/Cont_control_34.txt", header=TRUE, row.names=1) > pda…

deseq2

updated 8.3 years ago • Bob

of running the following in an R session install.packages("BiocManager") BiocManager::install("DESeq2") library(DESeq2) countData <- as.matrix(read.csv("count.csv", row.names="id")) head(countData) condition <- factor(c(rep

DESeq2

updated 2.8 years ago • Md. Faridul

Hi Mike, I am trying to design a DESeq2 model using solely a __continuous variable__. However, it is treated as a factor inside the model, even after I specify...nbsp; colnames(metadata3) <- "cont\_var" metadata3 __\# DESeq model, treats 'cont\_var' as factor, why? dds3 <- DESeqDataSetFromMatrix(countData = cts3,           …

deseq2 mikelove

updated 7.3 years ago • NinaKons

I have RNAseq with multiple factors such as 4 genotypes, 2 treatment and 4 time points. The top few lines look like, Samples Genotype Treatment Time S1 VN1...Stress 3hr S8 VN1 Stress 3hr . . Now I am estimating DEGs using DESeq2 package. My intention is to make pairwise comparisons to see time effect and treatment effect using different combi…

DEGs DESeq2

updated 4.3 years ago • 2021yearsold

Hi everybody, Recently I have been doing differential gene expression analysis. I am new to this, and I struggle a bit with my design. I have 4 different cell lines: A, B, C and D. In previous experiments I found that cell lines A and B are resistant to radiation, while cell lines C and D are sensitive. I am interested in the differential gene expression in the resistant vs sensitive group. H…

DESeq2 DifferentialExpression

updated 3.7 years ago • d.s.h.andel-3

on some RNA-seq materials. We have both raw count data matrix and also TPM data of all samples. At first, We performed DE analysis on DEseq2 using the raw count data, and we got some results. Then, to make sure the signals we found...we found that the two analysis was not consistent. We expect that, to some extent, for signals in DEseq2 analysis, they should also have low p-values. Or maybe they …

limma deseq2 Tutorial

updated 4.5 years ago • yuemolinn

I have a data set from the following Design of Experiment(DOE) ``` sample batch condition FRT19A_se se FRT19A_Control_2 Hinfp_A_G_se se Hinfp_A_G_point_mutant Hinfp_M2_11_se se Hinfp_M2_11_Deletion_mutant W1118_se se W11…

deseq2

updated 5.8 years ago • aimin.at.work

left to ask: The way I see it, there are 4 ways described to obtain normalized data: - The first one is to extract data, normalized using the normalization factors for a gene x sample matrix, and size factors for a single...vst(dds, blind=FALSE) rlog(dds, blind=FALSE) When I just got started, I used the the first function (counts(dds, normalized=TRUE)), to obtain the normalized data, wh…

deseq2

updated 5.6 years ago • Jonas B.

So I output a matrix from DiffBind as my input into DESeq2, with the chromosome positions being removed from the count matrix, just leaving the first column key as a reference...colData, design = ~ sex + treatment)</pre> Following this I ran DESeq2 and have output the results as follows: <pre> write.csv(as.data.frame(results), file="results.csv")</p…

deseq2 deseq diffbind differential binding analysis

updated 7.3 years ago • rbronste

Hello everybody! I am trying to install DESeq2 and I keep getting errors. At first I had about 10 errors regarding packages I couldnt install. The main problem of that...From searching I managed to install RCurl but I still get the bellow error. > install('DESeq2', lib = '/home/kostas/R/x86_64-pc-linux-gnu-library/3.6') 'getOption("repos")' replaces Bioconductor standard repo…

DESeq2

updated 3.0 years ago • tsomakiank

I have missed any essential components in my script for differential gene expression analysis using DESeq2. I first filtered my data by adjusted p value (0.05) and ranked those by fold change. The following is an example of my output...I be filtering something else? Have I missed something? Here is the R script: ##Load DESeq2 R package library(DESeq2) ##Load in raw re…

deseq2

updated 5.7 years ago • umajs

Dear users, Instead of counts (non-negative integers) required by DeSeq2, I have negative numbers and fractions, and I need to use DeSeq's statistics to find out significantly up/down genes...Dear users, Instead of counts (non-negative integers) required by DeSeq2, I have negative numbers and fractions, and I need to use DeSeq's statistics to find out significantly up/down genes. Since...for…

deseq2 logfoldchange

updated 8.4 years ago • rraadd_8

I am analysing a bacterial 16S dataset for differential abundance / correlations with phyloseq and DESeq2. I have on factor with 4 levels and ~10 samples in each (for which I'd like to control for) and 1 continuous variable for which...otu_table(OTU_nifh, taxa_are_rows = F), sample_data(FuncDat)) d2 <- phyloseq_to_deseq2(ps, ~Factor + Variable)   Sigdiff <- DESeq(d2, …

deseq2 r phyloseq

updated 8.4 years ago • fabian.roger08

I am working with RSEM outputs of RNA-seq data that I downloaded from a repository as .tsv files. I would like to apply VST to this data by first using tximport to import the RSEM outputs. However, the RSEM outputs from this repository only include the lengths and...data that I downloaded from a repository as .tsv files. I would like to apply VST to this data by first using tximport to import the…

tximport deseq2 rsem

updated 4.7 years ago • le2336

Dear all, I believe I am using the DESEQ2 Formula correct but after getting some unexpected results I would like to double check with you: I want to check the...Dear all, I believe I am using the DESEQ2 Formula correct but after getting some unexpected results I would like to double check with you: I want to check the effect of Vitamin D on patients and I have a group who takes Vita…

DESeq2

updated 4.1 years ago • Bine

I'm using DESeq2 to analyze 7 patient-matched samples (*1 tumor and 1 normal from each patient*) and I want to identify DEGs with padj&lt...file to include a column for "Source" (tumor or normal) and "Patient ID" (listed as A-G). The DESeq2 vignette says to use a multi-factor design which includes the sample info in the design formula to analyze paired samples...It seems that the program is i…

DESeq2

updated 3.8 years ago • rln0005

<div class="preformatted">Hi I have a group of samples for which I'd like to ascertain if differential binding is detectable based on a "condition" binary variable (stored in DBA_CONDITION). However, these samples have been processed in 4 batches (each batch has at least 3 samples). I would like to run a multifactorial analysis to regress the batch effect first, and then possibly analyse…

updated 10.8 years ago • Giuseppe Gallone

column, `` Dose ``, that we use for this now. The thought was to change that to an ordered factor which better represents the data (or not). I created a new column as an ordered factor, `` DoseFactor ``, for this purpose, which...contains the same information, just stored as a factor instead of a numeric. <pre> Dose DoseFactor <dbl> <ord> 1 …

deseq2

updated 6.9 years ago • chris

2013 at 10:56 PM, Jon Lees <jonglees@gmail.com> wrote: > Dear Prof. Love > > Im using the deseq2 package, its giving us really nice results. > and seems to fix some issues over deseq2 with our data. > > I just had quick...question, after I scale the counts by the scale factors, > and calculate the log2 fold change of case versus control, I g…

DESeq DESeq2 DESeq DESeq2

updated 12.0 years ago • Michael Love

In my RNA-seq experiment, I have subjects at two doses (high and low) and three time-points (pre-treatment, after treatment 1, after treatment 2).  I want a subject-level baseline to account for subject-to-subject variability. Thus, my design matrix has three columns: `` dose ``, `` time ``, and `` subject ``.  The design i want is essentially `` dose*time + subject ``.&nbs…

deseq2 interaction model nested design

updated 8.1 years ago • glocke01

I have done the following: 1. Divided the reads in each condition by the corresponding DEseq size factor 2. Subtracted the vehicle mean from the treatment values at the corresponding time point (assuming additive effects...3. Subtracted the min over the whole expression matrix (new min = 0) I would like to use DESeq2 for DEG analysis, but clearly cannot enter the matrix of interest as counts. …

deseq2 differential gene expression

updated 6.8 years ago • warrena

Hello DESeq2 community, I am using DESeq2 package to find DEG. I read vignette but could not find answer to my question. My experiment...has three factors. I designed my experiment in two different ways:main effect and interaction effect. 1. design=~Time+Trt+Tissue 2. design...were significant. I think it could be because the second design use the reference level of other factors even in…

DESeq2

updated 4.0 years ago • menuka

Hello, I have a multifactor RNA-seq experiment I’m trying to analyze with DESeq2 (Vs. DESeq2\_1.8.1). I am not sure how to setup the contrasts to find the right subset of deferentially expressed genes...is the design I use: <pre> design = ~ treatment + genotype + genotype:treatment</pre> Treatment factor has 2 levels: “Tret” or “Mock” (Mock is the base level) Genotype factor has …

deseq2

updated 9.6 years ago • morgene

like above; doo I need to have a separate model matrix for this interaction? ``` TEMP<-factor(paste(design$TEMP)) GENO<-factor(paste(design$GENO)) design_model <- model.matrix(~ GENO+TEMP) ``` **2. How to add a blocking factor...still control for this effect. What would be the best way to do this in the model matrix? From the first model matrix, should I just add S…

edgeR ExpressionData RNASeqData

updated 3.0 years ago • Mafalda

Dear all I am a working with DESeq2 (version 1.22.2) on R (version 3.5.3) to analyze my data from RNA sequencing. I did some tests last week and I had no trouble...2643 ``` I have 6 replicates for OM group and 5 replicates for YM group. I am using a second factor which is numeric (taille). I checked that colData labels and colnames of countData are the same ``` > all(rown…

deseq2

updated 5.4 years ago • emilie.derisoud

Hi, I'm using DESeq2 for DE analysis. I found in the "Beginner's guide to the "DESeq2" that it's possible to perform both default independent...Hi, I'm using DESeq2 for DE analysis. I found in the "Beginner's guide to the "DESeq2" that it's possible to perform both default independent filtering (which uses mean of normalized counts as a filter statistics) and independent filtering wit…

deseq2 INDEPENDENT FILTERING

updated 10.6 years ago • Laura.Fancello

A) distributed in 2 sequencing runs. This experiment consists of 3 biological replicates, and the first replicate has 3 technical replicates, which I collapse using the collapseReplicates function. Moreover, the 2nd sequencing...samples from a different experiment (experiment B). Now I want to analyse the experiment A using DESeq2, correcting for the batch effect with design=~batch+condi…

DESeq2

updated 3.3 years ago • turtle012

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 more numeric...design = design, ignoreRank) : some variables in design formula are characters, converting to factors # perform DEA dea_1 <- DE…

DESeq2

updated 4.5 years ago • georgina.fqw

is any hidden structure in this cell population. I was thinking of performing PCA. I wanted to use DESeq2 but their workflow seems to involve computing the differential expression first: <pre> ddsHTSeq<-DESeqDataSetFromHTSeqCount...or depend on computing differential expression between 2 or more groups. How can I do this? If DESeq2 can't do this can you recommend an alternative? &…

rnaseq pca deseq2 clustering

updated 9.9 years ago • Nick N

Hi, I have a DESeq2 dataset where we want to compare patient with and without distant metastasis. As we have IHC scores for tumor infiltrating...lymphocyte scores, we will also be including this in the design formula. Each of the factors has two variables (No/Yes, and hot/cold). With TIL scores are available, we would also like to assess if there is any difference...TIL score groups separately. …

DESeq2

updated 2.7 years ago • e.v.egeland

Hi, I am doing differential expression analyses for the first time and using DESeq2. The tutorials are great, but I have a couple niche questions that I cannot find the precise answer...Hi, I am doing differential expression analyses for the first time and using DESeq2. The tutorials are great, but I have a couple niche questions that I cannot find the precise answer to. 1) If I use a truth ta…

deseq2

updated 7.2 years ago • jrlarsen

preformatted">Greetings, I have used the DESeq package previously and have been recently using DESeq2. I am particularly interested in repeated measures designs and was wondering about applications with DESeq2. I have...multiple coefficients (i.e. lrt <- glmLRT(fit, coef=5:6)). I attempted something similar with DESeq2: res <- results(dds, name=resultsNames(dds)[5:6] but I …

edgeR DESeq DESeq2 edgeR DESeq DESeq2

updated 11.8 years ago • Charles Determan Jr

Hi All, I am working on RNAseq data of parasites from three populations (p1, p2, p3) during its interaction between two different hosts (h1 and h2). so finally I have 6 conditions (3 populations X 2 hosts) and each condition have 3 replicates. It has been observed that p1h1, p2h1, p3h1 and p2h2 have same phenotype (S) while p1h2 and p2h2 have another phenotype (R).  <table border="0…

deseq2

updated 9.3 years ago • cs3jd

I frequently use Deseq2 to analyze RNAseq microbiome data. An error I commonly get is ```r dds <- DESeqDataSetFromMatrix(countData = desd, colData...design, design = ~condition) dds < DESeq(dds) estimating size factors Error in estimateSizeFactorsForMatrix(counts(object), locfunc = locfunc, : every gene contains at least one zero, cannot

Microbiome DESeq2 RNASeq

updated 6 months ago • Caroline

I am using DiffBind to create a count matrix as summarizedExperiment and then running this through DESeq2 and I was wondering about ways to filter for specific baseMean ranges within this data? My script looks like the following...pre> library("DESeq2") library("ggplot2") library("BiocParallel") library("DiffBind") parallel=TRUE BPPARAM=MulticoreParam(4) # Create a dba.count...se&am…

deseq2 diffbind basemean summarizedexperiment

updated 6.7 years ago • rbronste

I am reanalyzing some CRISPR-Cas9 screening data to look for sgRNAs effective across cell lines. countData: ![enter image description here][1] colData: ![enter image description here][2] Condition here corresponds to time (initial vs. final). I can set up the DESeqData like this, where the fold-change result is from the effect of time: `DESeq2::DESeqDataSetFromMatrix(countData = counts…

DESeq2

updated 4.1 years ago • P1000

I came across an issue when trying to import RSEM files into DESeq2 according the [online tutorial](https://bioconductor.org/packages/devel/bioc/vignettes/tximport/inst/doc/tximport.html...data: <pre> txi.rsem <- tximport(files, type = "rsem") sampleTable <- data.frame(condition = factor(rep(c('A', 'B','C','D'), each = 3))) dds <- DESeqDataSetFromTximport(txi.rsem, …

deseq2 tximport software error bug

updated 8.1 years ago • falkohofmann

tring to use the TCGA HTseq\_counts data to do some annlysis. I plan to follow the process below. First. Use the RNA\_seq dataset which containing tumor and normal tissue to find differently expressed genes by DESeq2::DESeq...Second, because i only want to normalize the tumor data, so i use DESeq2::rolg to transform the data only containing tumor data. Third, I will use the rolg transformed tu…

deseq2 rna-seq

updated 6.8 years ago • 137737756

I'm confused by your response to Ingrid last May as to how to construct a design matrix for her 2 factor experiment with repeated measures on 1 factor. I came across this post in searching the mailing list because I have...the two centers. You experiment can be analyzed as suggested in Section 8.4.2 of the User's Guide. First combine treatment and timepoint into one factor (following Section 8.…

limma limma

updated 11.7 years ago • Jenny Drnevich

Is it possible to run DESeq2 with gene-dependent binary variable such as membership in a pathway or transcription factor association? If not, in

deseq2

updated 7.4 years ago • yuri.pritykin

we've been using DESeq for some of the analysis that we usually perform and now we'd like to jump to DESeq2. We've been checking the DESeq2 manual but it is not clear to us how to switch from one version to the other. Could you please...Dox\_vs\_DMSO.csv",header=TRUE, row.names=1) condition = factor(c("DMSO","DMSO","Dox","Dox")) libType = c("oneFactor","oneFactor","oneFactor","oneFactor") …

Deseq2 Deseq

updated 9.9 years ago • Osvaldo