Bioconductor Forum

I'm running DESeq2 on an RNA-seq dataset with 25 samples divided into 2 clusters (9 and 16). The DESeq2 object was created without any issues...dds_select_time <- DESeq(dds_select_time ) ``` Error message ``` using pre-existing size factors estimating dispersions gene-wise dispersion estimates mean-dispersion relationship final dispersion estimates

DESeq2

updated 7 months ago • Pak

all, I do DGE analysis (sample A vs sample B) using Salmon then Tximport (without scaling) then DEseq2. Everything works fine but my problem is that sample A is contaminated with red blood cells so that half of the total...less than 10 transcripts, easy to identify) from the TXI output file before loading into DESeq2? If doing so, should I use scaledTPM values? Thank you for your help. Bruno …

deseq2 salmon tximport

updated 8.4 years ago • bruno.saubamea

Hi there, Just got a couple of questions for DESeq2: 1. To build a design including paired samples (i.e. Subject) and batch variables (as estimated by SVA, i.e. SV1 and SV2), and...the same lfcThreshold? therefore to enable a more direct comparison between RNA-seq (DGE using DESeq2) and microarray (DGE using limma) data for the same samples. Thanks Guan

deseq2

updated 6.2 years ago • guanwang179

I am trying to use deseq2 to compare differential gene expression across multiple generations of repeated treatment. I have three replicates...control vs treated - 3 time treatment: control vs treated So far, I have been running deseq2 on each generation separately since each generation has their own control. However, I want to be able to compare between...generations and was wondering if i…

rnaseq deseq2

updated 2.7 years ago • lunarskye222

Hi, I'm quite sure I have a problem with the DESeq2 independent filtering. DESeq2 doesn't flag any outliers when performing the DE analysis. We have little concern about...Hi, I'm quite sure I have a problem with the DESeq2 independent filtering. DESeq2 doesn't flag any outliers when performing the DE analysis. We have little concern about blood being in the samples as the raw counts vary from …

deseq2 filtering outliers count outliers

updated 8.4 years ago • heikki.sarin

encountered one problem when doing pair-wise transcript-level differential expression analysis using DESeq2. I used tximport to import counts from Salmon quantification results, which consists of 65 pairs of samples (130 samples...data_dir, samples$Sample_id, "quant.sf") names(files_salmon) = samples$Sample_id samples$State = factor(samples$State, levels=c("BEFORE","AFTER")) samples$State = rele…

tximport DESeq2

updated 4.8 years ago • Marcus

Hi Michael and community, As always, thank you for your devotion in DESeq2. I'd like to ask about: is it reasonable to to run DESeq2 analysis on only a "subset" of the original raw count matrix? (would...Hi Michael and community, As always, thank you for your devotion in DESeq2. I'd like to ask about: is it reasonable to to run DESeq2 analysis on only a "subset" of the origina…

deseq2

updated 7.9 years ago • Alan

will not give good results (because baselevels are different). What I think I should do, is first fit a linear regression line on expression vs time for each donor separately and then compare those slopes and regard...them as replicates? I am quite new to DESeq2 model design that involves interactions, so I was wondering if you could help me out? Here's an overview about the components

DESeq2 Timecourse

updated 5.0 years ago • K.

Hello.. I am having trouble properly installing DESeq2. Currently have R version 4.0.2, trying to install with Bioconductor version 3.11. I tried troubleshooting with some...I have tried commands for installing the package survival individually. The command library('DESeq2') results in an error message: Error in library("DESeq2"): there is no package called ‘DESeq2’. I would appreciate any help w…

deseq2 survival

updated 5.5 years ago • hall.brooke33

RNAseq data. I recently updated my R version via an uninstall/re-install and now can no longer get DESeq2 to install or run properly. When trying to install with <pre> > source("https://bioconductor.org/biocLite.R") > biocLite...DESeq2")</pre> I get the following errors:  <span style="background-color:Yellow">BioC\_mirror: https://bioconductor.org …

bioconductor r deseq2

updated 8.8 years ago • tscharschmidt

It's recommended to do RUV correction before DESeq2. However, the PCA doesn't show any clear separation between the groups. How can I know if my data needs further pre-processing...before DESeq2? Is there any metrics or plots that can guide me? ![enter image description here][1] [1]: /media/images/30ab2b39-6893-4c5e-9531

DESeq2 PrincipalComponent prin DifferentialExpression

updated 23 months ago • Shaimaa Gamal

Hello Mike, I have one question about the DESeq2 model. Given that I have 2 groups of RNA-seq, each of which has 3 biological replicates. Thus, each gene has 3 biological...replicates in each condiction. does the DESeq2 model use average gene expression or each replicate of gene to estimate paratmeters, such as beta and intercept? Thanks

DESeq2

updated 2.8 years ago • BioEpi

any plans for transcript level differential expression (I.e. Supporting fractional counts), with DESeq2?? &nbsp

deseq2

updated 10.6 years ago • andrew.j.skelton73

Hello, I am computing a differential analysis on count data per region with DESeq2. For example, comparing only the region close to the transcription initiation site. Therefore, my count table represents...interval (e.g. [TSS, TSS + 300]) for each sample. I wonder if it is correct to compute the size factors with counts in the complete interval ([TSS, pA]) and then using them for the differ…

DESeq2 median-of-ratios normalisation

updated 2.7 years ago • msrch

using diffbind to identify differential binding of CUT&tag peaks of H3K27ac. I obtained scaling factors from ChIPSeqSpikeIn package. I'm very new to bioinformatic analysis. I know the Diffbind has dba.normalize function...which you can specify a numeric normalization factor. Could anyone suggest the code to specify number for normalization method in dba.normalize using DiffBind? Thank

DiffBind CUT&Tag

updated 2.0 years ago • Hai

I am attempting to test some hypotheses about whether thermal effects on transcription (polyA RNAseq data) are tissue-specific. The experiment includes four replicate samples per tissue/temperature combination, with three tissues and four temperatures (the treatment). Ordinarily, I'd test for factor interactions in a model implemented in edgeR. But, this design involves multiple tissues with w…

DESeq2 qsmooth edgeR limma

updated 2.9 years ago • Gregory

Hi,  I'm wondering if it's possible to use the unmix() function from the latest DESeq2 release for deconvolution of global gene expression from whole blood. I know programs like CellMix can do this without...cell type heterogeneity from matched samples, but it would be great to just use this new function in DESeq2 for the task. Thanks in advance! Noah&nbsp

deseq2 bioconductor rnaseq differential gene expression

updated 8.7 years ago • nsimons

samples are nicely clustered. How do you deal this scenario? Is there any required modifications to DESEQ2?  <span style="line-height:1.6">Greatly appreciate your help.</span> Prasad

deseq2

updated 11.1 years ago • Prasad Siddavatam

The `` DESeq2 `` package manual states that the `` results() `` function "extracts a result table from a DESeq analysis", but I am wondering...The `` DESeq2 `` package manual states that the `` results() `` function "extracts a result table from a DESeq analysis", but I am wondering if

deseq2

updated 8.3 years ago • LilithElina

Hi, I am currently analyzing a rna-seq dataset for differentially expressed genes between 2 conditions (20 individuals in condition A and 6 in B, B being the reference). I ran both DESeq2 and edgeR (glmFit and glmLRT) to compare my results with both methods. I have 51 DE genes with DESeq2, 156 with edgeR, 44 are found by both. So they are globally consistent about the DE genes (good news) althou…

edger deseq2

updated 10.1 years ago • Matarit

span style="line-height:1.6">Is it possible to use DESeq2 for transposon insertion sequencing analysis?</span> The experiment is to construct a library of transposon-insertion...counts for each possible insertion sites/genes for different samples, conceptually can I use the DESeq2 package for differential analysis between different conditions/time series?    &nbsp…

deseq2

updated 10.6 years ago • yifanz119

Hello everyone, I have a doubt about the cutoffs in DESeq2 related to padj and fold change. I initially did all my analyses using the default parameters in "results" function...Hello everyone, I have a doubt about the cutoffs in DESeq2 related to padj and fold change. I initially did all my analyses using the default parameters in "results" function. Afterwards...table from those default analys…

deseq2

updated 9.3 years ago • pmpierry

<h3 id="hello-community-i-am-trying-to-load-deseq2-and-have-been-receiving-a-namespace-error-">Hello Community, I am trying to load DESeq2 and have been receiving a namespace error :</h3> library("DESeq2") Loading required package: SummarizedExperiment Loading required package: Biobase Error: package or namespace load...h3 id="hello-community-i-am…

deseq2 error namespace biobase

updated 7.0 years ago • jpcourneya

interaction as 1-way with 6 treatments and no interaction. Statistically they are equivalent, but DESeq2 will generate different fits because for 2\*3 design the main effects are not shrunk. Why was it so necessary to introduce

deseq2

updated 10.3 years ago • Nik Tuzov

nbsp; I know you can set `` parallel=TRUE `` within the `` DESeq `` and results functions of the `` DESeq2 `` package, but I'm wondering if there's any way to parallelize the `` estimateDispersions `` and/or `` nbinomWaldTest `` functions

deseq2

updated 9.2 years ago • david.watson

in the vignette: [http://bioconductor.org/packages/release/bioc/vignettes/DESeq2/inst/doc/DESeq2.html\#interactions](http://bioconductor.org/packages/release/bioc/vignettes/DESeq2/inst/doc/DESeq2.html...www.dropbox.com/s/udoafy783uz2wve/Simulated_Data.pdf?dl=0) As described in the vignette, in the first set of simulated genes (G1.1, G1.2,…, G1.10), the condition effect is consistent across …

deseq2

updated 8.6 years ago • Daniele.Muraro

lt;- rowSums(counts(dds)) >= 1 dds_F <- dds[keep,] dds2 <- DESeq(dds_F) dds2$group <- factor(paste0(dds2$genotype, dds2$treatment)) design(dds2) <- ~ group resultsNames(dds2) [1] "Intercept" "genotype_ds2.3_vs_Col.0

deseq2

updated 6.8 years ago • lihongfei93

My design consists of a blocking variable and two factors of interest. We collected samples from three plots repeatedly over time (5 time points), but we are not interested in

deseq2

updated 5.3 years ago • Aditya Bandla

I have a multifactorial design containing an integer variable (‘age’) and a boolean factor (‘qr’) which represents treatment (TRUE) vs control (FALSE). str(data.phenotypes) Classes ‘tbl_df’, ‘tbl’ and 'data.frame': 71 obs...I have a multifactorial design containing an integer variable (‘age’) and a boolean factor (‘qr’) which represents treatment (TRUE) vs control (FALSE). …

deseq2

updated 6.1 years ago • benjamin.aaron.taylor

title suggests, I want to use RSE objects from recount3 for differential expression analysis with DESeq2. This is an ongoing project that I took over. Looking at what has already been done, there are two points I wonder about...1]) was sometimes applied on the recount3 RSE objects before creating the DESeqDataSets. In the DESeq2 vignette, it is indicated : > The DESeq2 model internally …

recount3 DESeq2

updated 3.7 years ago • william.benit

Hello guys, By  calling DESeq() on a DESeqDataset it estimates the size factors (normalization) automatically. How can I access this data? I must be stored somewhere. I would like to access the normalized...counts. >dds <- DESeq() estimating size factors estimating dispersions gene-wise dispersion estimates .... I know there is the counts() function but why use t…

deseq2

updated 10.8 years ago • john

shortName ='RNAseq_analysis_DESeq2.html',title ='RNA-seq analysis of differential expression using DESeq2 ',reportDirectory = "./Reports") #publish(dds,des2Report,pvalueCutoff=0.05,annotation.db="org,Hs.eg.db") publish(dds,des2Report...pvalueCutoff=0.01,annotation.db="org.Hs.egENSEMBL2EG",factor=colData(dds)$condition,categorySize=5) finish(des2Report) and I have this error: Error in resul…

Deseq2 reportingtools

updated 11.1 years ago • jarod_v6@libero.it

I am trying to figure out why my MA plots look so different between DESeq and DESeq2.   The plot made with DESeq has the funnel-like shape that I am told to expect from MA plots (spreads out as we go...src="http://i.imgur.com/JtHIsVJ.jpg" style="height:240px; width:240px"/>   The plot made with DESeq2, however, does not seem to increase in range of fold change…

deseq deseq2

updated 11.0 years ago • najami

I have single ended RNAseq reads from an allopolyploid organism. This means that I will have groups of 2,3, or even 4 genes with high sequence homology among them. I am sure some reads will map to more than one gene. So far, my pipeline is 1. Map reads to genome using tophat2, with default options (i.e., no -M or -g) 2. count using htseq as htseq-count -m intersection-strict --stran…

deseq deseq2 edger tophat bowtie

updated 10.5 years ago • ysdel

I read the code of DESeq2 roughly and realized that for each gene, one dispersion estimate is used across all samples. My work is to compare the...to be highly expressed in one genotype but not expressed in the other. I'm curious about how DESeq2 would estimate 1 dispersion value that would fit both genotypes. I read from the [DESeq2 vignette][1] that my design formula...the way dispersion is…

deseq2

updated 5.3 years ago • changxu.fan

the software and its incredibly helpful user manual. More specifically, I have two questions. The first is whether I have chosen the correct method for analyzing my multi-factorial experiment. I have 2 factors, state (apo, sym...3629 4837 3383 4221 > #assign samples to groups, A=AC, B=AT, C=SC, D=ST > group <- factor(c("A","A","A","A","B","B","B","B","C","C","C","C","D ","D…

RNASeq edgeR RNASeq edgeR

updated 12.8 years ago • Morgan Mouchka

Hello! I'm working in an experimental design that consists of two genotypes, B6 and TCR, in four different stress conditions: saline-control, saline-maternal separation, LPS-control, and LPS-maternal separation (SAL_CON, SAL_MS, LPS_CON, LPS_MS, respectively) I'm trying to use DESeq2 to answer the following question: what are the taxa that are affected by genotype, irrespective of which ge…

DESeq2 phyloseq contrasts

updated 4.4 years ago • Mir

voom(calcNormFactors(DGEList(counts)), design) I understand that any normalization factors found in counts will still be used even if `normalize.method="none"` Alternatively, just using `voom`: voom(counts, design

edgeR limma

updated 4.5 years ago • P1000

Hi, I have a question, I am working with mRNA-Seq dataset (18 samples) corresponding to different 2 batches and 3 cell types . `Batch_1` has 2 cell types (A and B types), however, `Batch_2` has 3rd cell type (C type). I imported the dataset via `DESeq2` in R., but for some reason (as mentioned below regarding the “the model matrix is not full rank", I cannot perform differential expression and t…

DESeq2 limma Normalization R

updated 3.1 years ago • Sabiha

the code below : <pre> library(limma) conditions <- data.trusted.eset$condition condition <- factor(conditions, levels(condition)[c(2,1)]) pairs <- factor(rep(1:13, each = 2)) design <- model.matrix(~condition+pairs) fit <- lmFit...i wanted to implement a multifactorial analysis in limma to evaluate the interaction between two factors, condition(con…

limma microarray multiple factor design paired

updated 10.9 years ago • svlachavas

Hello, I have used DESeq2 for differential analysis of small RNA data. I wanted to plot jitter plot of top most differentialy expressed gene...i have my question as follows:- Will the batch effect modeled by limma will be same as that by DESeq2 and can that be used as exact proxy or is there any way to extract and plot batch effect removed modeled values

deseq2 normalization

updated 5.7 years ago • dhwani.dholakia

Hi, We use DiffBind for the differential binding analysis of our ChIP data. In this we use edgeR TMM for normalization. I would like to print out the normalization factors for this but it only ever gives me **NULL**. Any help would be really appreciated. Thank you. ``` indiff <- dba(sampleSheet="//smb.cluster/080719ATACseq/Diffbind/SampleSheets/AAG57_0v2_SampleSheet_desktop.csv")…

edgeR Normalization DiffBind

updated 4.5 years ago • Nico

Hello, I am analysing a RNA-Seq experiment using DESeq2 trying to understand the different log2Fold shrinking algorithms available in DESeq2. After reading the documentation...Hello, I am analysing a RNA-Seq experiment using DESeq2 trying to understand the different log2Fold shrinking algorithms available in DESeq2. After reading the documentation I understood that setting `betaPrior=TRUE` …

deseq2

updated 6.9 years ago • sven.schenk

Hello, I am wondering if you can advice me on my latest project. I have 3 batches of mRNA-seq with 50, 40, 100 samples (same protocol). My end goal is to use NMF to cluster the samples. --- I noticed that there are ~200 genes with very high expression values (100k+ raw counts) spread between the samples such that a gene has a very high count in only 1 sample. From the DESeq2 documentation I sa…

deseq2 combat outliers NMF

updated 8.6 years ago • noname

nbsp; Hi, I did my analysis using EdgeR and with DESeq2 with and without lfcShrink. At FDR<10% we have the following: EdgeR - 17 genes  DESeq2 without LFC shrink - 37 DESeq2...54 Only 10 genes overlap between all the 3 methods at FDR<10%. The fold change for EdgeR and DESeq2 without LFC shrink are almost the same for these 10 genes are high. But when the analysis is r…

EdgeR and DESeq2

updated 7.3 years ago • Akula, Nirmala NIH/NIMH [C]

Hi, I used DESeq2 to analysis read count data using LRT test. I have read counts at transcript level and I want to conduct LRT test using...on 1. Does it indicate a problem of model fitting? Do I need to discard the low-count transcript first? I'm now using the default independent filtering in DESeq2. Also how "good" do the MAplot and dispersion plot need to be, in

deseq2

updated 8.9 years ago • Hydrangea

Hi all, I'm using DESeq2 for the first time and ran into some problems during the LFC shrinkage step. I got this error when I tried to run the process...Hi all, I'm using DESeq2 for the first time and ran into some problems during the LFC shrinkage step. I got this error when I tried to run the process function "lfcShrink()": using 'normal' for LFC shrinkage, the Normal prior from Love et al (…

deseq2 software error

updated 5.7 years ago • junli1988

are two different species rather than control vs. treatment group). I did the following steps in DESeq2 for normalization: dds <- estimateSizeFactors(dds) sizeFactors(dds) normalized_counts <- log2(counts(dds, normalized

deseq2 rna-seq

updated 5.6 years ago • thjnant

Hello! I have a question about the results of differential expression analysis in deseq2. I did: DEgenes=results(DEcd, contrast=c("clD", "V", "VG")) and I got the table with the log2 fold change and the p values. But what I...Hello! I have a question about the results of differential expression analysis in deseq2. I did: DEgenes=results(DEcd, contrast=c("clD", "V", "VG")) and I got the ta…

deseq deseq2

updated 11.1 years ago • francesca.defilippis

I am trying to run DESeq2 with many samples (single-cell experiment). However, it seems to be choking on the amount of data. When I run `` DESeq...I am trying to run DESeq2 with many samples (single-cell experiment). However, it seems to be choking on the amount of data. When I run `` DESeq() `` with

deseq2

updated 10.2 years ago • igor

Hello, I've been running DESeq2 recently using the lfcShrink function i.e. lfcShrink(dds, coef=14, type="apeglm", lfcThreshold=0.1375). However, an error

deseq2

updated 6.2 years ago • g.wang2

Hi, I am using DESeq2 to analysis rna-seq data. When I plot PCA for all samples in two groups I can see a nice separation within two groups(500

deseq2 michael love

updated 7.1 years ago • sam.far.7

Dear Michael, I am new in the field of RNAseq analysis and I find DESeq2 a great and intuitive tool. I read your RNAseq workflow on bioconductor (https://www.bioconductor.org/packages/devel...bioc/vignettes/DESeq2/inst/doc/DESeq2.html) and I have just a few naive questions that I would like to ask you. These are focused on the startegy...gt; 1 keep <- rowSums(counts(dds) >= 10) …

deseq2

updated 5.2 years ago • luca.s

Hello, I am running DESeq2 with this metadata table - | Condition | Time | Batch | |-----------|------|-------| | Control | 0h | 1 | | Control | 12h | 1 | | Control | 24h | 1 | | Control | 0h | 2 | | Control | 12h | 2 | | Control...Wald", betaPrior = FALSE, full = m1, parallel = TRUE) using supplied model matrix estimating size factors estimating dispersio…

DESeq2

updated 13 months ago • bhandary.8590

over cluster by about 5-fold compared to the rest of the libraries. With the intention of using DESeq2 after processing the data, would you suggest subsampling to match the rest of the sample depths, or will DESeq2 handle

deseq2

updated 6.5 years ago • russell.stewart.j

my p values and you could probably help me understand. I am doing a gene expression analysis using deseq2. In total I have 5534 genes and out of them around 230 genes showed NA for both adjusted and non-adjusted. Maybe this is...For example this gene below is one of those that gives NA for both kinds of p-values. the 3 first numbers are the replicates from the first treatment and the second 3 num…

DESeq2

updated 2.4 years ago • Juan Pablo

Hiya, I find that setting mcol of a GRanges to a DataFrame one of whose columns is an ordered factor does "the wrong thing". Not sure how to approach this other than report it for now. Best, Malcolm viz: > library(GenomicRanges...5 0.45 --- seqlengths: chr2 NA > mc1<-mcols(gr1) > mc1$f<-factor('x') > mcols(gr1)<-mc1 > …

updated 12.4 years ago • Malcolm Cook

in `` results() ``? (I can only do either Drought vs Control or 4610 vs Rondo. How to consider both factor at the same time?)   Thank you, &nbsp

deseq2

updated 7.9 years ago • Yuya Liang

Hello, I happily use DESeq2 (DESeq2\_1.10.1) in R for several years now. The log2FoldChanges (lfc) returned by the _results _function are estimates...Hello, I happily use DESeq2 (DESeq2\_1.10.1) in R for several years now. The log2FoldChanges (lfc) returned by the _results _function are estimates after _estimateSizeFactors _and _estimateDispersions _(called by _DESeq _function). For cluster …

deseq2

updated 7.2 years ago • konradgru

Hi all, I'm working on RNAseq data set and use DESeq2 in order to find differentially expressed genes above a given log2FoldChange, say 2 (or -2 for downregulated genes...Hi all, I'm working on RNAseq data set and use DESeq2 in order to find differentially expressed genes above a given log2FoldChange, say 2 (or -2 for downregulated genes), by...in my two week-old results, but now its in abo…

deseq2

updated 7.4 years ago • hinkel2