Bioconductor Forum

dear all, I have more than 500 RNA-seq samples and have to compare cases vs controls. I first run SVA to remove unknown variation and found >500 surrogate variables. Is a good practice to perform a LRT test with...deseq2 where full model =~case+SV1+SV2+SVn and reduced model=~case to know how many surrogate variables should I add in order to...avoid overfitting? The idea would be to first&…

SVA deseq2 differential expression LRT

updated 7.6 years ago • aec

on RNAseq data, I made my count table using `` kallisto `` and then `` tximport `` to work with `` DESeq2 ``. My genes are a set of cDNAs, (supposed to be corresponding to all the genes of my species), but the annotation is quite bad...B, and not in my cDNA list, I expect to have less reads in A than is B and when the normalization by `` DESeq2 `` occurs, it could create a bias ? Example: A: …

rnaseq deseq2

updated 8.1 years ago • corend

Hi   I'd like to use EDAseq normalized data as input of DESeq2. I found the vignette for DESeq but not for DESeq2. Is there a way to do DESeq2 DEA starting from  EDAseq normalized

deseq2 edaseq

updated 7.7 years ago • zoppoli pietro

Hello. I am running DEseq2 analysis on published RNAseq data consisting of 4 control samples and 4 cKO samples. However my log2FC values are...compared to the original publication. I think the issue is with how I define the control group (#set factor level) as the plotted data is always appears the same irrespective of what group I select (Control or cKO). I am struggling...to find a solution her…

DESeq2 Log2FC RNASeqData

updated 3.0 years ago • Iwan

Hi Mike, According to the DESeq2 `lfcShrink()` documentation, it appears that the function should work when you provide a `DESeqDataSet` (`dds`) and corresponding...object 'coefAlpha' not found Calls: lfcShrink -> results Backtrace: █ 1. └─DESeq2::lfcShrink(dds = dds, res = res) 2. └─DESeq2::results(dds.shr, name = coefAlpha, lfcThreshold = lfcThreshold) ``` It seems…

deseq2

updated 6.9 years ago • Michael Steinbaugh

Hello, I would like to inquire about using DESeq2 package in order to compare different types of read data: 1. Would it be OK to use DESeq2 to compare read data between homologous...Hello, I would like to inquire about using DESeq2 package in order to compare different types of read data: 1. Would it be OK to use DESeq2 to compare read data between homologous genes __in different…

deseq2

updated 7.9 years ago • yair.gatt

Hello, At first, I must mention that I am a beginner in bioinformatics. I am really sorry since my questions may seem really basic. I am...Hello, At first, I must mention that I am a beginner in bioinformatics. I am really sorry since my questions may seem really basic. I am trying...with other transcriptome analyses in virus-infected plants. In this case, I am planning to use DESeq2. DESeq2…

deseq2 differential gene expression bioconductor

updated 8.0 years ago • islamshaikhul2014

Hi All, I am attempting to create a MF design that will allow me to analyze paired samples (mouse) when testing different conditions, i.e. contrast(c("condition", "E", "D")) . When I attempt to create the dds file I receive this error whether or not I include or exclude the first two samples (B10 and B6): --- > dds.Spe = phyloseq\_to\_deseq2(physeq, ~condition + mouse) converting cou…

deseq2 multiple factor design

updated 10.4 years ago • treleo

The thing is, I want to do differential expressed gene(DEG) analysis in the TCGA RNA-seq data. DESeq2 and edgeR manuals said they require __raw count data__ as input, so I downloaded _STAD.rnaseqv2\_\_illuminahiseq...frame with DEGs sorted by logFC. Here comes my question:  1. Am I using the right data for DESeq2/edgeR and is my code ok?  2. Suppose I am right on 1, now I wan…

edgeR TCGA

updated 8.3 years ago • JackieMe

Hello, I am new to RNA-seq. I have output from `tximport` and I want to use `DESeq2`. I have used five replicates for each of two treatments and I want to check the difference. I am not sure which design...Hello, I am new to RNA-seq. I have output from `tximport` and I want to use `DESeq2`. I have used five replicates for each of two treatments and I want to check the difference. I am not s…

deseq2

updated 5.8 years ago • zen

all treated vs all controls samples (I mean I do not want DE for each experiment separately).   DESeq2 is a right choice for my goal? actually I saw its tutorial but the explanation is for one experiment how can I use

deseq2

updated 9.0 years ago • elhamdallalbashi

Hello - I have an issue with genes (or OTUs in my case) which have zero expression across a condition. Seems to be the same issue as https://support.bioconductor.org/p/56190/\#56191 Works as expected (well as I'd expect) in DESeq2 1.10.1 Below are a few of the details __model = ~ Block + Treatment__ __contrast = c("Treatment","D","Control")__ Treatment is a factor with 5 levels (B…

deseq2

updated 8.0 years ago • greg.deakin

div class="preformatted">Dear All, Is the Scaling Factor (from GCOS) still neccessary for the quality assessment of Affy, eventhough we are applying RMA or GCRMA for the probe

affy gcrma affy gcrma

updated 19.8 years ago • Khan, Sohail

Hello everyone, I would like to know if you could help me about knowing if I can use DESeq2 for the following design: I have only 1 animal that has 12 timepoints. The 6 first timepoints are "before infection". The...6 last timepoints are "after infection". Is it possible and relevant for you if I use DESeq2 to compare the 6 samples before infection (group A) versus the 6 samples after …

DESeq2

updated 4.4 years ago • leo.dagata

Hi there, I am writing because I am lost in the last step after use limma::removeBatchEffect and introduce the new matrix to DESeq2. The reason I used limma::removeBatchEffect is because the design is not full rank and I can't fix my batch in the design. The PCAs from before and after batch effect look correct. Please, see my code below: > library(DESeq2) > librar…

deseq2 limma batch effect

updated 5.3 years ago • Patricia

Hello, I’m working with Deseq2 in order to analyse some RNA-seq generated with a plant species. I have a time series experiment with 8 time point and...Hello, I’m working with Deseq2 in order to analyse some RNA-seq generated with a plant species. I have a time series experiment with 8 time point and 4 replicates for each time point.   I just have a question concerning the normalisat…

deseq2

updated 7.3 years ago • Alex So

Dear Micheal, We are want to use DESeq2 using gene specific covariates (i.e. a separate set of covariates for each gene; these are also count data with the identical...this. We thought that you might have an idea? Kind regards, Christian edit: to DESeq2

deseq2

updated 9.8 years ago • c.oertlin

script. However, these counts are not integers, is there a way I could use this matrix in Deseq2 or I will have to generate it again using a method from the Deseq2 manual?</span> Thanks, Catalina &nbsp

deseq2

updated 7.9 years ago • Catalina Aguilar Hurtado

As the title states, I've got some RRBS data and am looking to evaluate a correlation between gene expression and CpG island methylation. In prepping the expression data (from a completed DESeq2 run), I'm planning to normalize the counts with the variance stabilized transformation (VST) prior to exporting and moving forward. However as I understand, the VST accounts for the library's size fac…

DESeq2

updated 3.4 years ago • Brendon Herring

Hello All, for estimating the differential expression values,what statistical test does DEseq2 use for estimating the differential expression values,is it t-test, Fisher exact test or some other

deseq2 statistics

updated 8.9 years ago • elhamdallalbashi

Hello, I have previously been using DESeq2 as part of HOMER, but I think I need more control over the analysis of this experiment, and would love some input on how...into consideration the different sampling batches. After reading around at forums and the manual for DESeq2 I am sure I am _not_ understanding how to properly decide on a design... FYI, looking at PCA plot and heatmap there is no..…

deseq2 mouse rnaseq

updated 8.1 years ago • Emilie

div class="preformatted">Hi, We recently upgraded to DESeq2, and we are trying to figure out the differences. We couldn't find an explanation in the documentation regarding the...with count 0 in a specific gene, we get a basemean of 0 in DESeq, and a basemean of 0.68586719 in DESeq2. I understand how the calculation is done in DESeq, but not in DESeq2. Can you explain please? Thanks -- outpu…

DESeq DESeq2 DESeq DESeq2

updated 11.5 years ago • Guest User

Hello, Total newbie here. Trying to analyze RNA-seq dataset with RSEM counts from TCGA. First I need to QC and normalize the dataset. For some reason, rows are patients and columns are genes...confusing. Do I need to...Hello, Total newbie here. Trying to analyze RNA-seq dataset with RSEM counts from TCGA. First I need to QC and normalize the dataset. For some reason, rows are pati…

RSEM DESeq2

updated 13 months ago • NB

A couple of weeks or months ago, a question was posted regarding DESeq2's behavior when dealing with samples with one extreme outlier. In that case, DESeq2 identified that gene as DE, although...there was only 1 out of N samples with extreme counts. Michael Love suggested a way to force DESeq2 to look at all replicates. I've been searching for the post for a while using both Google and the foru…

deseq2 outliers

updated 8.0 years ago • paul.alto

on how to use arrayQualityMetrics to identify sample outliers with DESeq data sets produced using DESeq2?  I'm having trouble generating a file that the "arrayQualityMetrics" function will accept using DESeq2 functions...for function ‘platformspecific’ for signature ‘"SummarizedExperiment""... the standard output of the DESeq2 functions I'm calling. My current work-around is to use DESe…

arrayqualitymetrics deseq2 sample outliers

updated 11.0 years ago • rachelwright8

Since looking at the row variance and DESeq2 both act as ranking mechanisms for genes, is there any sense to taking the top 1000 or 5000 genes with the highest variance...across samples from an RNA sequenced set and running the DESeq2 pipeline on that subset to look for differential genes between groups (so simple design ~condition)? Thanks!&nbsp

deseq2 variance genes

updated 8.1 years ago • hs.lansdell

Hello, I want to generate a dummy sample data but I want to do it based on DESeq2 result. Suppose, I have a gene, gene A and I have do DESeq2 analysis between cancer and normal. The result is, gene A logFold...fold change with 0.01 p-value. Is it possible to do that? I imagine something like this. Because DESeq2 use negative binomial as distribution, I just need to make  random sampl…

deseq2

updated 9.8 years ago • bharata1803

data with multiple clinical conditions from multiple patients/samples. As far as I understand, the first level that is used is the reference level that other conditions will be compared to. For example if I have the following...conditions: 1. Control 2. Treatment 1 3. Treatment 2 One option is to put the "control" as the first level that the other conditions will be compared to separately…

deseq2

updated 6.1 years ago • Matan G.

it to have a very large size.factor() when compared to libraries almost 10fold larger.But the size factor I get here is very small.  <pre> > sizeFactors(dds) A 1.0167371 B 0.9574096 C 1.0823689 D 0.9329557 E 0.9519349 F 1.0187297...library size is equal to the calculated " reference genome", but if a library is smaller,…

deseq2 estimatesizefactors

updated 9.1 years ago • Assa Yeroslaviz

txi.rsem, sampleTable, ~batch + condition) I then explicitly set the levels of factor condition to make sure every stage is compared to my control.  dds$condition <- factor(dds$condition, levels=c("Normal

deseq2

updated 7.5 years ago • spr

Hi! I have problems understanding the `contrasts` argument of the `results` function in `DESeq2`. This is my dataset: ``` dds <- makeExampleDESeqDataSet(m = 18) dds$condition = factor(c("exp", "exp", "exp", "stat", "stat", "stat", "pla", "pla", "pla

DESeq2 RNASeq

updated 4.4 years ago • sarah-spie

sample (and replicate), do I have to convert zero read counts to ones (pseudo counts) while running deseq2? <span style="line-height:1.6">I assume I don’t have to, but a quick clarification.</span

deseq2

updated 11.1 years ago • Prasad Siddavatam

 Hi all, I am using variance stabilizing transformation on my rna seq count data with Deseq2. Do I need to use quantile normalize the samples or does variance stabilizing transformation function take care of...nbsp;Hi all, I am using variance stabilizing transformation on my rna seq count data with Deseq2. Do I need to use quantile normalize the samples or does variance stabilizing t…

quantile normalization deseq2

updated 8.2 years ago • lirongrossmann

Hi all, Regarding the package DESeq2, -What are the input files? BAM/SAM files? -Does it makes the counting and the differential expression analysis, or just

deseq2 differential gene expression rna-seq

updated 9.7 years ago • cirisarri95

1) loading data from `package(airway)` ``` > # Loading data > library("airway") > library("DESeq2") > data(gse) > gse class: RangedSummarizedExperiment dim: 58294 8 metadata(6): tximetaInfo quantInfo ... txomeInfo txdbInfo...8 > # Defferential analysis > design(dds) ~cell + dex > dds = DESeq(dds) estimating size factors using 'avgTxLeng…

DifferentialExpression GeneExpression DESeq2 Design()

updated 4.2 years ago • p.l.chen

Hi everyone, A coworker used DESeq2 for a project we worked on together, but based on the results I'm not sure the contrasts performed were doing what we...tissue1:species, meta) ``` We also modified the ensuing model matrix to remove combinations of factors that had no observations: ```r all.zero <- apply(model_matrix, 2, function(x) all(x==0)) idx <- which(all.zero) mod…

DESeq2 R

updated 17 months ago • tbiewerh

Calculating dropout rate To check for zero-inflation, I did the following: ```r # dds is a DESeq2 object created from a salmon > tximeta > DESeq2 workflow # filter genes with less than 5 counts in at least 10 samples...lt;- sort(vars, decreasing = TRUE) zidds <- dds[names(vars)[1:1000],] # recalculate size factors and dispersion - is this necessary? zidds &…

DESeq2 ZINB zinbwave deseq

updated 4.9 years ago • rowcyclecamp

hoping some of you could help me with the following question about the design formula as input for DEseq2. I am analysing a knock-down experiment with of 3 biological replicates. For each experiment, samples have been taken...with either control shRNA, or shRNA 1147 or shRNA 1150. The important point here is that the first sample is taken after about 5 days after the cells first started expressin…

deseq2 rnaseq repeated measures time series design formula

updated 6.0 years ago • Dorien

I'm trying to reinstall DESeq2 with R 4.2.2. I used ```r if (!require("BiocManager", quietly = TRUE)) install.packages("BiocManager") BiocManager::install("DESeq2

DESeq2 SummarizedExperiment DelayedArray

updated 2.9 years ago • Danielle

At some point DESeq2 was updated so that LFC shrinkage was no longer performed automatically, and that functionality was moved into a...At some point DESeq2 was updated so that LFC shrinkage was no longer performed automatically, and that functionality was moved into a separate...in some samples and they have very high variance, and I decide to remove them prior to running DESeq2. I think the es…

deseq2

updated 5.2 years ago • www1124

Hello DESeq2 community, Could you please tell me whether I am interpreting the LFC calculation right.  Raw counts for geneX...69    36    41    134    114    42    19 DESeq2 Normalized counts for geneX (stored in normaizedcounts):        …

deseq2

updated 8.2 years ago • amalthomas111

Im getting the error when trying to install DESeq. I'm installing this on a university cluster interactive node.   <pre> > biocLite("DESeq2") BioC_mirror: https://bioconductor.org Using Bioconductor 3.4 (BiocInstaller 1.24.0), R 3.3.2 (2016-10-31). Installing package(s) ‘DESeq2’ Warni…

deseq2 installation RccpArmadillo

updated 8.8 years ago • chitsazanalex

div class="preformatted">Hello Simon, I am working with a multi-factor experimental design within DESeq and am confused as to how to (when to) export my results. I am wondering at what point

DESeq DESeq

updated 13.8 years ago • Ingrid Lindquist

preformatted">Hello, People! I need some help. How can I get the information about transcription factor binding sites into regulation region of certain gene? I'd like to get information only about proved sites, but not about

Transcription Transcription

updated 18.7 years ago • Ольга Камнева

Hi  I am currently working on some metagenome data and I'd like to use DESeq2 tool to find the genes that are differentially abundant in different treatment over time. Due to the type of the study...number which changes the distribution and nature of data from count to decimal. I know that DESeq2 works on the count data but I was just wondering if there is any possible…

deseq2 metagenomics

updated 8.3 years ago • ghanbari.msc

If I want to identify gene which express in one condition. Is it possible in deseq2. Because I think Deseq2 calculate logfoldchange for the gene that express in two condition  and test statistic

deseq2

updated 7.9 years ago • naktang1

Hi All, I would like to do differential abundance analysis using DESeq2 for shotgun metagenome data. I have used the MOCAT2 for generating functional annotations. Eg: For ARDB database, Do...which didn't get assigned to any of the ARDB categories - which is around 99% of the reads) for DESeq2 analysis? Will "unassigned reads" impact the sizefactorestimation calculation

deseq2 metagenome mocat

updated 6.3 years ago • anjali.kumari

Hello everyone, I am currently working on GLM for RNA-seq data in DESeq for a design with two factors and three factor levels, such as: type experiments A_1 A exper1 A_2 A exper2 A_3 A exper3 B_1 B exper2 B_2 B exper3 B_3...Can we add a paragraph on what to do if we only want to make specific comparisons, e.g. let fac be a factor with three levels A, B and C, and we want to test pairwise for di…

DESeq DESeq

updated 13.1 years ago • Dorota Herman

paired rnaseq data from multiple samples, counted with `` featureCounts ``, now planning to use DESeq2 and trying to design it. I have gone through https://support.bioconductor.org/p/67600/.  However, I would like...cts, colData = coldata, design = ~ tissue) </pre> <pre> dds <- DESeq(dds) estimating size factors estimating dispersions gene-wise d…

deseq2 rnaseq featurecounts DESeqDataSetFromMatrix

updated 8.1 years ago • bioinf

I would like to add a pseudocount of 0.0001 to genes with 0 counts. I can't add 0.0001 to the count table, because DESeq2 requires integers as input. Is it possible to replace the zeros with pseudocounts in the DeseqDataSet and then do the...add a pseudocount of 0.0001 to genes with 0 counts. I can't add 0.0001 to the count table, because DESeq2 requires integers as input. Is it possible to repla…

deseq2 rna-seq

updated 8.7 years ago • manuelw

in other lists is not statistically sound as it erases the false discovery rate control performed by DESeq2, and false positives/negatives will have an effect on what genes remain or are filtered out in the final gene list. The...correct way of doing this analysis given the research aim, through the inclusion of multiple factors or interaction terms in the DESeq2 design formula. Right now the …

deseq2

updated 5.3 years ago • d.huntley

imgur.com/a/R2ZSCt5)So, I am trying to find DE genes in a cancer-normal dataset. I am using both DESeq2 and limma/voom method. Interestingly, I found many genes that are significance (adj p value <0.05) but the log2FoldChange...sign is on the opposite. So, for example, in DESeq2 it is reported as down regulated but in Limma/voom result it is reported as up regulated. I check this because I…

deseq2 limma voom limma-voom rnaseq

updated 7.1 years ago • bharata1803

longer works and does not give me the correct contrast options. This was working before I updated to DESeq2 1.22.2 Here is my code: ``` > line.ds <- phyloseq_to_deseq2(ps.phyla, ~ Line) > diagdds <- DESeq(line.ds, test="Wald", fitType...parametric") using pre-existing size factors estimating dispersions found already estimated dispersions, replacing these …

deseq2

updated 6.8 years ago • muc345

Hi I am using Partek to analyze RNA-seq data, Partek has a median normalization step before Deseq2, which works fine. I understand Deseq2 takes raw data, but Partek has separated the normalization step recently (previously...Hi I am using Partek to analyze RNA-seq data, Partek has a median normalization step before Deseq2, which works fine. I understand Deseq2 takes raw data, but Partek has separ…

RNAseq123

updated 5.0 years ago • mmicky5050

Hi all, I have generated factors of unwanted variation (W_1) via RUVseq/RUVg (with spike-ins), but am having trouble incorporating them into ImpulseDE2...Hi all, I have generated factors of unwanted variation (W_1) via RUVseq/RUVg (with spike-ins), but am having trouble incorporating them into ImpulseDE2. The reason I would like to use ImpulseDE2 because the literature suggests it is a good mod…

ImpulseDE2 ImpulseDE ERCC RUVSeq

updated 4.2 years ago • Mallory

Hello, despite reading numerous tutorials and workshops on DESeq2 package (which are of a tremendous help!), I cannot seem to be able to have the appropriate design and be able to get out...Time: T0/T2/T4) As suggested by the tutorial, model matrix was filtered from 0 rows and provided to DESeq2 function. [https://bioconductor.org/packages/release/bioc/vignettes/DESeq2/inst/doc/DESeq2.html#inter…

DESeq2

updated 4.5 years ago • Mirin1357

function seems to turn every field into a factor in the resulting DataFrame object. Obviously, this is trivial to fix by hand on a small scale (e.g. when plotting a couple...1. Whether there are any foreseeable plan to address this point (i.e. automatically detect factor/numeric fields) 2. Independently of 1., which would be the recommended way to deal with the problem. I present my current.…

ensemblVEP

updated 9.6 years ago • kevin.rue

poor results, for example, AIC = 9547 for Akkermansia with a degree of freedom = 2. I have used DESeq2 many times before, I think it is an awesome method, but I can not figure out these results. I think I am familiar with DESeq2

deseq2

updated 5.3 years ago • wisam.tariqsaleem

Hello, Can you please direct me towards the resources needed to install deseq2 in Jupyter Notebook with a R kernel on a Mac? I have tried to install Anaconda, an R kernel, then followed the code described...anaconda to start fresh. Can you please show/direct me towards the resources I need to install deseq2 in Jupyter Notebook with a R kernal for a Mac? Thank you in advance for your tim…

deseq2

updated 6.9 years ago • david.wheeler7

Hello, Its my first time to deal with Longitudinal analysis in Limma and I would like to make sure that my code reflects my research question...gene expression, FEV1, and other covariants readings for 2 timepoints. Phenotype data ```r RID<-factor(rownames(combined_pheno)) #reading ID is different for each reading ID<-factor(combined_pheno$sid) # each subject has...4-6 depends on…

limma

updated 23 months ago • COPD_Project