Bioconductor Forum

Hi, I like to use DESeq2 for making PCA-plots and heat maps, but on a current dataset we only have count values from cufflinks/cummeRbund (exported...using count() in cummeRbund). I know DESeq2 needs raw counts, but can I use these counts only for plotting/visualization? And can I perform the rlog-transformation

deseq2 cummerbund cufflinks

updated 10.4 years ago • Jon Bråte

Hi! I am using DESeq2 on an in-house server which is shared among a handful of users and has not a cueing system. I want to set a limit on the number...of nodes DESeq2 uses, for which I used `MulticoreParams` as follows: ```r parallelParams <- MulticoreParam(workers = 4,progressbar = TRUE...bpforceGC: TRUE bplogdir: NA bpresultdir: NA cluster type: FORK ``` However, whe…

DESeq2 BiocParallel

updated 3.2 years ago • Sebastian

by pheatmap package and I am new to this. I have a list of DEGs obtained by RNAseq analysis using DESeq2 and I only have normalized counts from DESeq2 and also FPKM values for these genes. To my knowledge, I need to plot Z-score...per sample for each gene (rows) but I dont know how to get Z-scores. Should I calculate the Z-scores first and then read it into R as a data frame or pheatmap will calc…

normalization pheatmap

updated 5.7 years ago • Hamidreza Hashemi

Hey everyone, I'm having an issue with running DESeq2 and hoping someone can help or give some advice. I'm very new to this and don't know a lot about CPU memory/parallel or series...I have a folder with 26 bam files (13 paired comparisons of 2 tissue types) and one GTF file. I first tried running DESeq2 with just one comparison, so the 2 bam files with GTF.  After the first attempt froz…

bioclite bioconductor summarizeoverlaps genomicfeatures genomicalignments

updated 9.1 years ago • BioinfStudent

I have an RNASeq experiment, and I am using DESeq2. After I get the results, I plot the MA plot. This is the output of plotMA: ![](https://s21.postimg.org/l731pcobb/deseq_maplot.png...like this? Imbalanced sampling depth at the two conditions? Why doesn't normalization (sample size factors) fix this? Also, is there a reason why DESeq2::plotMA doesn't plot the best fit line? Disclaimer: Cross p…

deseq2 deseq rnaseq

updated 8.8 years ago • ysdel

Dear community, I'm trying to use DESeq2 to test if there is a relation between the expression of any genes and X (a continuous variable). For my design I have 5...Dear community, I'm trying to use DESeq2 to test if there is a relation between the expression of any genes and X (a continuous variable). For my design I have 5 persons

deseq2

updated 9.1 years ago • sasha

of both padj and fold change ratio to define differentially expressed genes (DEGs) identified by DESeq2. Would it be valid to submit a manuscript where the DEGs are identified only with a padj threshold? Many thanks for the

deseq2

updated 5.4 years ago • marija.buljan.2

Hi,  I am trying to get direct dispersion estimates from DESeq2. As the results() does not contain the direct estimates, is there an way to get hold of the estimates ? I want plug in these

deseq2 dispersion R

updated 9.5 years ago • hiraksarkar.cs

Dear Michael, I am hoping to use DESeq2 to analyze the sequencing results of a Multiply Parallel Reporter Assay that I performed. I was wondering if you might...that DESeq originally calculated dispersion for each condition separately. My question is: __Does DESeq2 still calculate dispersion for each condition separately, such that the low dispersion of my plasmid reps will not

deseq2

updated 9.7 years ago • dsg16

I am trying to run a time course DESeq2 analysis with 16S amplicon data in a phyloseq object. All treatments in the time course have the same starting point...others and must be removed. Please read the vignette section 'Model matrix not full rank': vignette('DESeq2')</span></pre> [Michael Love has previously suggested a remedy for model matrix rank issues with time series](https://s…

deseq2 phyloseq multiple time points

updated 8.5 years ago • campbell87

Hello, I have a multi-factor design and I have successfully used DESeq2 to extract my comparisons of interest.  I wanted to use DESeq1 in order...Can we add a paragraph on what to do if we only want to make speci c comparisons, e.g. let fac be a factor with three levels A, B and C, and we want to test pairwise for di erences between A and B (without C), between A and C (without

DESeq2 deseq

updated 10.6 years ago • ramiro.barrantes

mean more appropiate than arithmetic mean for the pseudo-sample created to obtain the size factors? 3)How reliable is DESeq on ~10 samples per condition with no biological nor technical replicates. I've read in a previous...for this scenario. Sequencing is expensive, does it means that it doesn't make sense to use DESeq2 for DEG in this case, what would you recommend? Thank you so m…

deseq2 rnaseq normalization

updated 7.8 years ago • xavi

I am a bit confused on how to use normalizationFactors in DESeq2. In the manual it says that "Normalization factors should include library size normalization". Does that mean that I

deseq2

updated 9.4 years ago • vedran.franke

Hi all, a theoretical question about DESeq2. I have a dataset of sorted blood immune cell types from healthy and diseased humans. I want to calculate DE genes for...have different amounts of RNA (e.g. neutrophils vs B cells, etc.), the normalisation method that DESeq2 uses internally might give me erroneous results. Do you think I can still use DESeq2 or should I use a different package

deseq2 normalization

updated 6.1 years ago • tkapell

from this, I have 3 overlapping timepoints - baseline, d1, and d2 timepoints. My understanding of DeSeq2 is that as long as the biology is maintained (that is to say that these samples were treated the same), then the more information...I feed into it, the more accurate a model it can generate. So I am inclined to feed Deseq2 all of the timepoints, but perform differential expression analysis li…

Deseq

updated 3.8 years ago • Alex

div class="preformatted">Good morning all, I am just starting to analyse my first set of RNA-seq results and am trying to use EdgeR to do this. I have spent time reading the vignette and manual and working...gt; targets <- read.delim(file = "targets.txt", stringsAsFactors = FALSE) > Treatment <- factor(targets$Trt, levels=c("Con","Dr")) > Genotype <- factor(…

edgeR edgeR

updated 11.5 years ago • Smith H.

Hello everyone, First, I am so sorry if my post title does not correspond to my post completely. I am using scater/scran package for scRNA-Seq...and MNN for batch correction. As I know with multiple datasets; MNN integrates first and second datasets first and integrate the third one with first-and-second-integrated data set. After the analysis...space. I want to do something like this…

scater singlecell scran BatchEffect

updated 5.2 years ago • hamza_karakurt

I am trying to teach my students the using of DESeq2, and they are repeating the instructions contained into the vignette  And they have found a very small bug. This...I am trying to teach my students the using of DESeq2, and they are repeating the instructions contained into the vignette  And they have found a very small bug. This bug does not appear when the "old" DESeq is i…

software error

updated 9.2 years ago • arfranco

Hi,  I have a question about how DESeq2 estimate dispersion parameters. In the low level function of DESeq2, when estimating dispersion alpha, I think the

deseq2 dispersoin estimatedispersions

updated 7.7 years ago • xr2120

readcount matrix form TCGA. The size is 577 samples with number of genes 18.522. When I tried to run DESeq2 to calculate log foldchange, it took not that long, around 3-4 hours. After that, I want to use rlog function to get the log...Core™ i7 CPU 975 @ 3.33GHz × 8 with RAM 24 GB. I know that R can not use multiple core to calculate DESeq2. Is there any suggestion how to optimize this process

deseq2

updated 10.0 years ago • bharata1803

    Hello, I am analyzing my small RNA seq data which contains 6 samples in total and includes two controls and 3 different treatments. In addition, one treatment has two different time points, so my controls also have two different time points. My coldata looks sth like this: <table border="1" cellpadding="1" cellspacing="1" style="width:500px"> <caption>colData&…

deseq2 rna-seq mirna

updated 7.8 years ago • michael.frech

Hi, I am using DESeq2, but unsure whether or not I am using the right design. I could not find here anything similar. **subject**: A, B, C, D, E, F **cohort...Hi, I am using DESeq2, but unsure whether or not I am using the right design. I could not find here anything similar. **subject**: A, B, C, D, E, F **cohort**: ctrl...ctrl or treatment) and each subject is sampled at mult…

deseq2 nested de

updated 6.1 years ago • gaio.transposon

  <span style="line-height:1.6">Dear folks,</span>     I got stuck about how DESeq2 get inference or calculated test\_statistics for differential tests. There is a gene of my interest. I want to dot testing between 2 samples/conditions (no replicates) to see if it is significant differentially expressed. Here is the normalized count for them:   …

deseq2

updated 10.3 years ago • xfwang

class="preformatted">Dear all, I am using limma for the analysis of a factorial experiment for the first time. I would greatly appreciate your help re to few questions that I could not understand. The experiment is 2^3 factorial...all the 3 factors at 2 levels each. I have 24 samples. The 3 factors ar below, it is a balanced design genotype<-rep(c(1,2),each=12) ##here 2 represents...r…

limma limma

updated 14.9 years ago • Shaheena Bashir

May I clarify a part of the DESeq2 package? Is it true that DESeq2 counts for linear (only) transcripts and excludes any possibility in circular RNA (circRNA...decrease in circRNA has no effect on the number of counts produced in the results/output? Or does DESeq2 not able to distinguish the two, will assume circRNA as linear fragments, and include it into the counts

deseq2 circrna rsubread

updated 7.5 years ago • csijst

Hi DESeq2, I have a question with respect to DESeq2. I have one factor with 4 levels (wild-type, mutant A, mutant B, mutantA&B; the wild

deseq2 bioinformatics

updated 9.2 years ago • gmhhope

Hi, I am analyzing RNA-seq and want to put cqn offset (GC% normalization) to DESeq2 object. I followed the vignette of DESeq2 but have a question about geometric mean standardization of cqn offset. ```r

cqn DESeq2

updated 3.4 years ago • bioinfo

all together in the count matrix and later contrast to specify the sample vs  samples in DESeq2                        &nbsp...samples) in the count matrix and compare all Knock-…

deseq2

updated 8.8 years ago • deena

Hi all  for prevent p-values set to NA in DESeq2, should I offset my raw reads as same as this ? Thanks <pre> cont1 cont2 cont3 tumor1 tumor2 tumor3 geneA 64 80 36 0 0 0 geneB

deseq2

updated 8.2 years ago • Bob

Hello! I'm using DEseq2 to perform differential gene expression analysis between two conditions. When I normalized each group separately...Hello! I'm using DEseq2 to perform differential gene expression analysis between two conditions. When I normalized each group separately I find that one of the conditions has significantly larger dispersions in most genes. How can I preform a DEseq2 anal…

DESeq2

updated 2.4 years ago • adi.rotem

microarrays and RNA seq I have always formatted my matrix as you specified: groupGend <- factor(paste(group, gender, sep = "_")) design <- model.matrix(~0+groupGend) where each contrast name is easy to read as "male.diseased...gt; I have 2 questions - >> >> 1. Does Limma allow inclusion of covariates ? How do I first adjust the &…

Microarray limma Microarray limma

updated 12.3 years ago • Sam McInturf

are about the interpretation of results between LRT and Wald test. In my phenodata, there are two factors: “line” (3 levels in the following order: A, B and C) and “treatment” (2 levels: control and pollutant). The design formula is written...nbsp;      1 … * The logFC is calculated for the last level of both factors, so it corresponds to the log…

DESEq2

updated 7.0 years ago • RS1

<div class="preformatted">On 11/02/2012 11:38 AM, wang peter wrote: > thank u for your advice > i really donot know how to pick up the first element > > i only know > sapply(x,unlist) > This is the only way you know how to use sapply? So I wonder who wrote this code...11:38 AM, wang peter wrote: > thank u for your advice > i really d…

Cancer Cancer

updated 13.2 years ago • Hervé Pagès

nbsp; sce <- computeSumFactors(sce, sizes=c(20, 40, 60, 400)) \#Computing separate size factors for spike-in transcripts sce <- computeSpikeFactors(sce, type="ERCC", general.use=FALSE) \#Applying the size factors

deseq2 normalization counts

updated 8.3 years ago • herrmannmaria94

Hi, Getting an unexpected error with DESeq2.   > dds = DESeq(dds) using pre-existing normalization factors estimating dispersions gene-wise dispersion estimates

software error deseq2

updated 7.2 years ago • ashley.doane

Hello there, I guess I would like some clarifications on how DESeq2 handles multiple testing in terms of contrasts of interest along with contrasts of other variables. For example...Hello there, I guess I would like some clarifications on how DESeq2 handles multiple testing in terms of contrasts of interest along with contrasts of other variables. For example, please...It looks to me that…

deseq2

updated 5.7 years ago • g.wang2

Hi, We are currently using the DESeq2 R package for analysis of differential expression of small-RNAs over genes and we would very much appreciate if you...also affect and shift the results. This leads me to the question itself: with the intention to use DESeq2 for differential expression analysis, which counting method would you recommend to use while dealing with small

rnaseq deseq2

updated 10.6 years ago • lehouri

to-date methods and utilizing knowledge and experience gained in the 10 years since `DiffBind` was first written. The main updates are in the areas of **modelling**, **analysis**, **normalization**, and **blacklists**, as follows: **Modelling...DiffBind` now supports modelling using arbitrary design formulas, including multi-factor designs with any combination of metadata factors. Contrasts…

Normalization ATACSeq ChIPSeq DiffBind chipseq

updated 5.0 years ago • Rory Stark

Hi,   I am using version 1.16.1 of DESeq2.  According to the documentation, a Cook's distance cutoff of 0.99 is applied to the results function by default...1.16.1 in Bioconductor up to date with the source code on github? (https://github.com/mikelove/DESeq2).  I have not done much digging otherwise.    Thanks,   Will

deseq2

updated 7.5 years ago • wpoehlm

Hi everyone First of all thank you for making rna-seq data much more accessible to an average clinical doctor through the DEseq2 packages...lt;- relevel(vsd@colData@listData$acplacPOD1, ref="Placebo") vsd@colData@listData$time <- factor(vsd@colData@listData$time, levels=c(0,1)) vsd@colData@listData$chronic_pain_intensity_ACTIVITY_FU <- relevel(vsd

Dese DESeq2

updated 2.6 years ago • Asger Mølgaard

Hi there! I'm interested in DEG analysis in RNA-seq and I have a question about statistical analysis methods. I tried to analyze DEGs using three different methods as follows: 1. DEseq2 package 2. FPKM t-test in excel =T.TEST(ctr1:ctr3,trt1:trt3,2,2) 3. log2(FPKM) t-test in excel =T.TEST(ctr1:ctr3,trt1:trt3,2,2) Actually, I'm a bit confused between 2 and 3. I feel like I should do 3 to a…

DESeq2 statistics rna-seq

updated 2.7 years ago • SH

3 43 minutes ago Rafael Soler ▴ 220 I am doing a DESeq2 comparison with different levels and one factor. To do this, I have performed the analysis in two different ways. First

Factor Levels Dispersionestimates DESeq2

updated 4.2 years ago • rafaelsolersanblas

Hello all, I want to ask a question about Log2FoldChange in DESeq2. In many cases for the similar counts of Case and Control if the count shows down regulation but the DESeq2 give the output...table <span style="background-color:Yellow">__xyz1-xyz6__ </span>results are Down Regulated but the DESeq2 give the Up Regulation. where xyz7-xyz10 shows the correct result. Please help me in …

deseq2 R log2fc

updated 10.1 years ago • Sushant Pawar

For the TCGA COAD HTSeq count data matrix from UCSC Xena, what is the best way to input it into DESeq2 to do DE analysis at gene level? The data matrix is 60488x513, which seems to be not at gene level. Right now, DESeq2 vignette

deseq2 TCGA HTSeq-count

updated 5.5 years ago • zhaoy

Hi. I would like to perform differential peak analysis for my CUT&Tag data. I converted the bam files of the samples to bedgraph files. I then normalized the signal of the bedbraph files using a normalization factor calculated based on the E. coli bacterial read count. Since my Tn5 enzyme was extracted in E coli, the bacterial read count...samples to bedgraph files. I then normalized the …

DiffBind

updated 4.7 years ago • mwong043

After the DESEQ2 analysis, the baseMean values from the results range from (0.2 to 87622.00). For the selected results I have used 0.05

deseq2

updated 6.9 years ago • angajalaanusha

Hi all I'm working with DEseq2 on RNAseq data and I observe an excess of positive signals (see attached MA plot). Any idea about how to reduce the noise

deseq2

updated 6.8 years ago • mesaez

Dear all, How can I filter the  counts with low count in Deseq2?  Any suggestion on how to do? <pre> dds<-DESEq(dt) count<-counts(dds,normalize=TRUE) filter<-rowsum(count)> 10</pre

deseq2 lowcount

updated 4.9 years ago • aristotele_m

results from a DESeq analysis'   <span style="white-space:pre-wrap">dds$genotype <- factor(rep(rep(c("I","II","III"),each=3),2)). But do not understand the 'each=3),2)' in this line, as I need to modify it for my 5 genotypes. </span> Also...has in each genotype and don't have a reference genotype. Would it be better for me to use the group/factor rather than…

DESeq2

updated 7.9 years ago • Catalina Aguilar Hurtado

T3 1/3 (T1 + T2 + T3) = T4 1/4 (T1 + T2 + T3 +T4) = T5 How would I implement this contrasts in DESeq2

deseq2

updated 8.8 years ago • yohannesafew

Hi I would like to exclude lncrna and pseuodogens from DEG results (using DESeq2). At what step do I do this? Thanks

filter deseq2

updated 2.7 years ago • PD

I have a 4 level single factor dataset to analyse with edgeR. I used: tmm<-calcNormFactors(data.dge) y<-estimateDisp(tmm) et<-exactTest(y,pair...I have a 4 level single factor dataset to analyse with edgeR. I used: tmm<-calcNormFactors(data.dge) y<-estimateDisp(tmm) et<-exactTest(y,pair()) to...DE genes in the desired comparisons. However, is there…

edgeR deanalysis test fisher exact

updated 7.8 years ago • tkapell

I am unable to install DESeq2 package even after installing BiocManager. I have looked for various sources to find a tutorial video to solve this...I am unable to install DESeq2 package even after installing BiocManager. I have looked for various sources to find a tutorial video to solve this, yet I keep on receiving unable to write error. Can you give me step by step instructions what to do to g…

scRNAseq RStudio DESeq2

updated 2.4 years ago • DrFizz

Hi, I would to use DESeq2 to process three bulk RNASeq paired samples but I am trying to figure out what is the valid model to use here. I used tximport...to import Kallisto's transcript-level abundance estimates at gene level to use with deseq2. In the paired samples, the treatment is overxperssion of gene A. Sample information is as follows: ``` condition patient_…

deseq2

updated 6.5 years ago • Puks

the same problem posted a while back here: https://www.biostars.org/p/193916/ I am using DESeq2 to run a multifactor paired analysis to determine the change in gene expression between men and women due to a treatment...of samples for each condition, I'm using the following steps to create a custom design matrix: [C: DESeq2 paired and multi factor comparison](https://support.bioconductor.org/p/63…

deseq2 paired samples

updated 8.9 years ago • gregory.l.stone

Dear all, I am trying to use the `DESeq2` to conduct the differential expression analysis in time course experiments. I want to find changes of gene expression...setting in `limma-trend` is here. **input** was generated by using `DGEList()` ```r treatment = factor(coldata$Time, level=c("0h","0.5h","1h","2h","4h","8h","12h","24h")) design <- model.matrix(~ 0 + treatment) logCPM …

DESeq2 limma DifferentialExpression

updated 16 months ago • Jay

data for about 23000 genes. The values are not integers. So, when I run the dds <-  on Deseq2, I got the following error: "some values in assay are not integers" Is there any commands in Deseq2 to fix this error? I mean...how I can use normalized data directly into Deseq2? Thanks

Deseq2

updated 10.0 years ago • mheydarpour

each individual sample has an **different** age with 2 decimal digits. I defined the column as a factor and then used it in the "design" of DESeq2. I get the following error: > Error in checkFullRank(modelMatrix) : the model matrix

deseq2

updated 5.8 years ago • Matan G.

I have single cell RNA seq data from two groups. When I use DESeq2 to explore differential regulation between the two groups, I visualize them in a plotMA graph. To my surprise this...I have single cell RNA seq data from two groups. When I use DESeq2 to explore differential regulation between the two groups, I visualize them in a plotMA graph. To my surprise this shows...I have single cell RNA se…

deseq2

updated 7.0 years ago • mdroog

Hi, I'm working with whole-genome EM-seq data from an experiment with two factors: - Two genotypes (with contrasting response to a pathogen) - Infected and non-infected Only one tissue and one time analyzed...now I'm following the methylKit package tutorials. According to it, in cases like mine with two factors and two biological replicates, I can apply logistic regressions to test for…

methylKit

updated 2.4 years ago • Andrés