Bioconductor Forum

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

microRNAincats

updated 3.2 years ago • sofia.hanas

Dear all,  <span style="line-height:1.6">I am analyzing my RNA-seq results with DESeq2 and I have a question about model matrix design and interpretation of the results. I have two cell types in two conditions...that is 4 groups in total. I would like to know the difference if I use factor design or pair-wise group comparison, and the interpretation of it. </span> …

deseq2 rnaseq

updated 10.1 years ago • shin.jx

have time to help! </span> <span style="background-color:white">Is the estimation of size factors included in VST normalization? I assumed according DESeq2 manual that VST normalization includes the estimation...of size factors but I noticed that the size factor after VST normalization was same for all libraries, though the library size varied...I then tried the VST normaliz…

deseq2

updated 9.5 years ago • hanna.sinkko

and would like to look at different comparisons for differential expressed genes. I combined the factors for easier interpretation. However when I look at the PCA plot (see attached), I do see a lot of variation within D6. https...interaction terms), and main effects as well (for D2 and D6). So my question is in this multi factor comparison, should I Run DEseq2 with all samples and remove…

deseq2

updated 5.8 years ago • new_user6

BiocManager", quietly = TRUE)) install.packages("BiocManager") BiocManager::install("DESeq2") library('DESeq2') library('RColorBrewer') countFilePath = './count-1.txt' countData = read.table(file = countFilePath, header...FALSE, sep = '\t', row.names = 1,stringsAsFactors=FALSE) ##make first row the colname colnames(countData) <- countData[1,] countData <- countData[-1, ] ##if n…

DESeq2

updated 3.6 years ago • salehe.ghasempur

I'm running DESeq2 for my expression analysis and have a question on the design formula. I have 2 cell lines, 2 time points, and 4 treatments...significant genes and their ranks. Why is that? Are these two models not the same? 1. dds$group <- factor(paste0(dds$cell, ".", dds$time, ".", dds$treat)) design(dds)<- ~ group + batch dds<-DESeq(dds) 2. design(dds)<- ~…

deseq2

updated 9.3 years ago • Dr Wei Chen

Hi, I am experimenting with the use of size factors in DESeq2 (i.e. the s\_ij factors). Essentially, I would like to show that the use of gene-specific s\_ij factors, derived...Hi, I am experimenting with the use of size factors in DESeq2 (i.e. the s\_ij factors). Essentially, I would like to show that the use of gene-specific s\_ij factors, derived from external data, improves the model's fit.…

deseq2

updated 7.8 years ago • Jonathan Cairns

Hello guys. I am using the rlog transformation from DEseq2 (see first figure) and it produces a strange graph. 1th graph are random numbers. 2nd are the raw counts. 3rd are normalized...In the 5th graph- Why are the red dots so close to the x axis? It should more or less look like the first graph. Any ideas on whats causing this?   <img alt="" src="http://abload.de/img/screenshotfr…

deseq2 normalization variancestabilizingtransformation

updated 8.5 years ago • john

Hello,   I have a question about the best DESeq2 experimental design matrix for my dataset.   I am working with 75 RNAseq samples from the same tissue, but classified...the groups of) late-stage disease - not the genes specific to C/D/E/F, which is what I'm after. My first question is, is there a better design matrix that I could use to account for this comparison? For exam…

rnaseq deseq2 design and contrast matrix

updated 10.1 years ago • m.fletcher

to compare the translation efficiency with and without treatment. We thought to have a model with 2 factors and the interaction between the 2 factors. The 2 factors: 1. treatment: with/without 2. library prep: total/ribosome footprint...in the cell, and also the ribosome bound RNA. We thought that in this situation the assumptions of DESeq2 normalization are violated. Therefore we thought …

deseq2 translation efficiency spikes ribosome profiling

updated 8.3 years ago • GFM

I have grown diseased cells (with 3 different genotypes, type A, B, and C) and healthy cells (WT) and put them in two different culture dimensionalities, 2D and 3D culture. I then have 8 groups: 2D healthy, 3D healthy, 2D diseased (of genotype A, B, C) and 3D diseased (of genotype A, B, C). Additionally, the samples are derived from two experimental iterations (E1 and E2), performed at different …

deseq2

updated 4.4 years ago • Nick F

as the primer that I use for first strand cDNA synthesis is specific to the target set of genes. I have three replicates for each condition. Based on the...sure that it is not biological. I think there are several possible causes, one of which might be the DESeq2 analysis, and so I would like to rule that out if I can. A colleague recently informed me that DESeq2 is not well suited...but my l…

DifferentialExpression DESeq2

updated 3.1 years ago • dadca596

span style="line-height:1.6">RUVg uses the first call of edgeR to find the factors of unwanted variance. The design matrix is constructed with the possible batch effect...y <- DGEList(counts=counts(set), group=x)</pre> If I know that I have a batch effect between first replicates (Ctl1, Trt1) and second replicates (Clt2, Trt2). Would it make sense to introduce it already in t…

ruvseq

updated 9.2 years ago • tonja.r

Hi Michael,     I'd like to ask a question related to a previous post: "Question: DESeq2 - factor base level changes the DE genes."     I ran the following code on DESeq2 (version 1.4.5): \#\# START CODE sampleTable...lt;- DataFrame(sample=sampleTable$SampleName,             group=factor(sampleTable$g…

deseq2

updated 9.4 years ago • Victor Missirian

package. I have ran the analysis as explained in the vignette and calculated the estimated factors of unwanted variation. I have got the following factors <pre> > pData(set1)[,2] [1] -0.4209557 -0.3984871 -0.4039097 0.4393171...0.4079229 0.3761126</pre> I wanted to use these factors to analyse the dataset using DESeq2, but `` DESeq `` accepts only positive values as `` s…

deseq2 ruvseq sizefacotrs

updated 9.8 years ago • Assa Yeroslaviz

of "Interaction" and presence of albumin. For 1., I concatenate Albumin and Time columns, run DESeq2 controlling for Interaction, and get the comparisons using contrasts: pre_dds <- DESeqDataSetFromMatrix(yeast...For 3., I concatenate Albumin and Interaction columns, and use this new column as a blocking factor in formula design i.e. ~Albumin_Interaction+Time. Then I retrieve time p…

DESeq2

updated 2.5 years ago • gtechbio

myself.   tldr: it seems that after \`collapseReplicates\` the levels of newly created factors are shifted affecting the results. \*\*If\*\* this observation is correct and not a due to a mistake i my design, I suggest adding...for having to source the sample data but it was the minimal example I could use) <pre> library("DESeq2") source("https://gist.githubusercontent.com…

deseq2 bug

updated 6.2 years ago • António Miguel de Jesus Domingues

I performed a Nascent RNAseq experiment, which yielded TotalCounts and NascentCounts (Tc) for each gene. I have two conditions (Control vs Tx), each with 3 biological replicates. I analyzed the data with DESeq2 using posts about [ChIP-seq][1] and [Ribo-seq][2] as a guide. My results surprised me: the top genes with significant LFC are the...I have two conditions (Control vs Tx), each with 3 …

DESeq2

updated 3.0 years ago • vanbelj

I am trying to find differentially expressed genes using DESeq2 on some RNA-seq data. In the pheno data, there is a column named 'condition' with factored values of 'Treated' and 'Untreated...I want to use just this column for my experiment. However, when I use the results() function in DESeq2 with my data, I get the first line as: 'log2 fold change (MLE): condition Untreated vs Treated' …

RNASeqData DESeq2

updated 2.9 years ago • treebig44

Hi Guys, My first question here. I'll make it as short and concise as possible. Please correct me if I am wrong (which I very well might be). I...Hi Guys, My first question here. I'll make it as short and concise as possible. Please correct me if I am wrong (which I very well might be). I would like to obtain between sample normalized, within sample normalized gene expression values, e.g. siz…

rnaseq deseq2 tximport salmon normalization

updated 7.7 years ago • kofoed

problem reading in a CSV file (hope this is an appropriate forum as it is for use directly with deseq2!) I am reading in a CSV count matrix, with first column as gene names. I am trying to make the first column a rownames columns...so that that deseqfrommatrix function will work straight from the first sample! The code I am executing is as follows: countdata<-read.csv(file = "non-norm\_c…

deseq2 countdata

updated 7.0 years ago • A

I am trying to use the DESeq2 package on my count data from a targeted RNA sequencing project. I have a two factor design and was wondering about...I am trying to use the DESeq2 package on my count data from a targeted RNA sequencing project. I have a two factor design and was wondering about the correct way how to formulate the design formula. My `` colData(dds) `` looks somet…

deseq2 R rnaseq

updated 9.6 years ago • Christoph

control (DMSO) and I have 3 times. I have 3 replicates for each combination. I am trying to use DESeq2 to find the differentially expressed genes. First, may I double check with you that the formula looks OK: ~treatment\*time...I am wondering why you would use this formula. Is there something special I should now about DESeq2 formulas?) So, using a pooled analysis with all the samples at once a…

deseq2

updated 9.8 years ago • AurelieMLB

Dear list, I am not sure the correct way to interpret the nbinomLRT results in multiple level factors condition. Here is a toy example. I would to find DE genes after controlling batch effect in the experiments, in which...there are multiple levels. ## make data library(DESeq2)dds <- makeExampleDESeqDataSet(m = 18)colData <- data.frame(row.names = rownames(colData(dds)), sample = co…

updated 10.5 years ago • shao

Hi there, My question is rather theoretical and regards the wording used to describe a multi-factor design in the vignette of Deseq2 package. For example in the following design: ```r ~genotype + condition ``` The vignette mentions...controlling for differences due to `genotype`. However when I start reading some books about multi-factor design and interaction, including here one book written …

DESeq2

updated 3.6 years ago • Alexandre

Dear Prof. Love: There have been numerous questions about “not full rank error” in DESeq2. The vignette addresses the issue and I understand when the error is generated. What I would like to find out is why you...decided to include that feature in the first place. The point is that both SAS (PROC GLM, MIXED, GENMOD with Negative Binomial response) and R (lm) are quite comfortable...with incom…

deseq2

updated 5.0 years ago • Nik Tuzov

Hey Mike, a couple of questions on DESeq2, but first of all, some code to make my questions reproducible: <pre> library(airway) library(DESeq2) library(magrittr) dds_airway...lt;- DESeq2::DESeqDataSetFromMatrix(assay(airway),                &nbsp.…

deseq2

updated 8.3 years ago • Federico Marini

Hi, I'm quite new to R and DESeq2 and need some help troubleshooting! I'm working on analyzing a dataset comparing 3 groups: patients before and after...a treatment against a healthy control (not given treatment). For the first analysis I wanted compare MDD patients before and after, so I originally subset the data-frame to include just the MDD...patients, and then created a factor to descr…

DESeq2

updated 3.2 years ago • Sophie Nicole

Hello! I am using DESeq2 to analyse allele specifc expression in two different assays. I one of the assays I have six samples with no particular...treatment, in the other assay I have 'colony' and 'body part' as blocking factors. Both assays have been analysed separately. My approach up until now has been to perform the analysis normalising...only by either sample, or by the blocking f…

deseq2 deseq normalization allele specific expression rnaseq

updated 6.7 years ago • c.martinezruiz

Hello,  I am new to DESeq2 analysis and wondered if I could get a direction about the correct design for my experiment I have counts tables (generated...I split it into separate designs? I am also concerned about if have enough power for multiple factor design (3 biological replicates)?   Thanks

deseq2 differential gene expression multiple factor design

updated 6.3 years ago • galozs

div class="preformatted">Hello, I've a rather general question related to factors that I'd like to use in linear models (for siRNA design): I have two nucleotid positions for a gene (say NC1 and NC2), and 'A...position (NC1 and NC2 and possibly with interactions). I see two possibilities for coding these factors: Two factors NC1 and NC2 each with levels A T C and G or 8 factors with level…

updated 19.9 years ago • Arne.Muller@sanofi-aventis.com

former participant of the CSAMA event in 2015. After some month of waiting, I finally received my first RNA-Seq data, so I wanted to test DESeq2 to perform my DGE analysis. What I'm trying to do is to find differentially expressed...where found using DeSeq2 with default parameters. Moreover, when looking at the adjusted po-valuue, ALL values are set to 1 Just to output some...of the DeSeq2 resul…

deseq2 multiple factor design

updated 9.0 years ago • wariobrega

Hello, I have a very large dataset which I downloaded from TCGA for pancreatic cancer. It has one normal sample but 184 patient samples. So, It has 186 columns (no technical replicates) and 20500 rows for all the genes. I am trying to get log2fold change data from this using deseq2. To begin my analysis I have extracted four patient samples(raw reads) and the normal control and now have 5 separa…

deseq2 cancer R

updated 7.5 years ago • dorothy.jrobbert

Dear Michael, i have a question regarding the usage of covariates in DESeq2 modelling. I am thinking about including a covariate, e.g. gender. 1. The model with covariate gender: <pre> condition &lt...factor(c("CTRL","CTRL","CTRL","CTRL","Disease","Disease","Disease","Disease")) sex <-factor(c("Male","Male","Female","Female","Male","Male","Female","Female...pre> 2. The…

deseq2 covariates

updated 7.6 years ago • markus.schulze

1] in the section 3.3.1 pages 35-36. I would like to use all the experimental factors combined into one combined factor (group column) then I would like to create a contrast like in the example: DrugvsPlacebo.1h...Drug.1h-Drug.0h)-(Placebo.1h-Placebo.0h). Reading the DESeq2 manual I can use contrast using a list of two character vectors or a numeric contrast vector. Both requires to use…

deseq2

updated 4.8 years ago • ribioinfo

Hi, I would like to ask for help in the design component of my Deseq2 RNA-seq analysis. My data set consists of 6 samples: 3 treated samples (gene overexpression) and three untreated samples...are the same plant leaf cut in half and exposed to the two different treatments. Another confounding factor in this sequencing experiment seems to be a batch effect. My bio rep #1s comes from a first rou…

deseq2 design multifactor

updated 5.1 years ago • sxp585

Hi, I am analysing the dataset with two independent factors - genotype (WT, MUT) and age (pup, adult). I have 4 samples for each condition. - mouse1 MUT pup - mouse2 MUT adult - mouse3 WT pup - mouse4 WT adult etc. What I want to check is how being the MUTANT affects gene expression across the age and if the impact of mutation is age dependent. What I need is to buil…

LRT DESeq2 interaction

updated 3.9 years ago • annamariabugaj

Hi, I'm trying to analysis a time-series RNA-seq data using DESeq2, I have 15 time points, 2 conditions, and 2 biological repeat. I perform LRT test first and the full model is ~batch + condition...condition:time, while the reduce model is ~batch + condition + time. All variances are recognized as factor. I choose the threshold fdr < 0.01 and get ~5000 different expressed genes.&nbsp…

deseq2 timecourse multiple time points time-series

updated 6.6 years ago • zhangfei-123

Hi, I am brand new to bioinformatics and differential expression analysis, trying to assimilate a lot of new technical and conceptual information. I am working with a dataset that used BioSpyder’s TempO-seq technology to analyze a cancer cell line using a targeted gene set of 500, where 10 chemicals were tested under two conditions. Biospyder has provided the raw read counts for each of the 500 …

deseq2 ruvseq

updated 7.8 years ago • grashow

1379 1591 3056 2799 2268 I'm doing the calculations BY HAND of the 3 steps of the DESeq2: 1. Estimate Size Factors 2. Estimate Dispersions 3. Negative Binomial GLM fitting and Wald statistics For first step...lessons/02_DGE_count_normalization.md Using that link, i was able to make the stimation of the size factors in only 4 steps. That's what I need right now. An eas…

DGE DESeq2 Differential

updated 3.5 years ago • Zeke Smith

Hi, I am trying to understand and compare the DESeq2 model and the BNB-R (https://github.com/siamakz/BNBR) model. The corresponding references are: - DESeq2: Love, M. I., Huber, W., &amp...binomial regression for differential expression with confounding factors. Bioinformatics, 34(19), 3349–3356. https://doi.org/10.1093/bioinformatics/bty330 My understanding of the BNB-R model...is…

DESeq2 BNB-R

updated 6.0 years ago • Homer

Dear community, I am using an "external" normalization method (not available in DESeq2) to obtain size factors for each sample in my dataset. Obtained size factors : sfGMPR >sfGMPR >V10T0 : 0.8564351 >V10T14...gt;V11T0 : 13.7131949 >V11T14 : 47.9961821 > ... 2) Using a DESeq2 dds object (setting my own si…

DESeq2 normalization size factors

updated 5.9 years ago • erwan.scaon

Hello, First off, I appreciate everyone reading this and providing insights. I'm aware what I'm asking is not statistically sound...Hello, First off, I appreciate everyone reading this and providing insights. I'm aware what I'm asking is not statistically sound by any stretch of the imagination, however the current data I have is three samples (one replicate each). The conditions are such t…

deseq2 replicates error DESeqDataSetFromMatrix

updated 5.9 years ago • Yonatan Amzaleg

manual and bioconductor support, there doesn't seem to be a way to specify a custom normalization factor in DiffBind when analyzing using DESeq2. I'm wondering if there is a relatively easy "hacky" way to do this? Thanks in advance

diffbind

updated 4.3 years ago • C T

Hello, i am using DESeq2 to analyse an experiment with two conditions and three groups, as described in "Example 3" in the ?results help page. I...Hello, i am using DESeq2 to analyse an experiment with two conditions and three groups, as described in "Example 3" in the ?results help page. I started the analysis by testing for an interaction effect with the following code: <pre> ## Exa…

deseq2

updated 9.9 years ago • hwildha

Before performing any differential testing, I wanted to check if the normalization with the size factors from DESeq2 will improve my data or show some possible experiment errors etc. Also, I am intended to use normalized...counts (by size factors) outside the DESeq to check for differential analysis and also VST and rlog transformed data as input for PEER.&nbsp...normalization with the sizeFa…

deseq2

updated 9.3 years ago • tonja.r

nbsp; Hi, Being new to DESeq2 and differential expression analysis, I am a bit confused by the setting up of the design matrix of my experiment, as...see, I don't have the R genotype at treated timepoint 10 (i.e. R\_10\_R1, R2 & R3).) __My first question is:__ do I need to include replicates as a factor? These are just biological replicates, not independent experiments...to investi…

deseq2 experimental design timecourse rnaseq model

updated 8.3 years ago • athief

I have a dataset with 3-factor levels (A, B, C). If I use the data of A, B, C to perform a pairwise comparison between A and B, I got a result very different from...that when I just use the data of A and B. Is there a bug in `DESeq()` of DESeq2? In the example, `deg_output.tsv` has 1857 non-NA padj `deg_output2.tsv` has 16209 non-NA padj this is a quite big difference...sprintf('~%s'…

deseq2

updated 4.9 years ago • megapode32559

I'm working with 6 samples from two different patients, so 12 samples in total.  There are two factors, Cell line (HF2303 or HF 2927) and Treatment (untrt or trt).  I'm trying to include a continuous covariate into the design...MGMT) to control for the effect of the factors based off of MGMT presence in the cell.   <pre> > colData(se) DataFrame with 12 …

covariate deseqdataset Deseq2 multiple factor design

updated 9.6 years ago • Lillyhchen

Firstly, this is my first time posting to this website, so I hope that I don't miss anything important. This is more of a question for advice of what...multiple batches and some have strong GC-bias, I chose to use salmon and tximport for processing in DESeq2. I have a number of covariates which I am interested in, as this is a control population with no sign of disease. However...from what I can …

salmon rna seq tximport ruv-seq deseq2

updated 6.8 years ago • mad6kj

Hello, I'm using DESeq2 on a non-standard dataset where I wish to supply my own normalisation factors. I'm getting an error message from&nbsp...lt;- normfacs dds <- DESeq(dds, test="Wald", parallel=TRUE) using pre-existing normalization factors estimating dispersions gene-wise dispersion estimates: 16 workers mean-dispersion relationship final dispersion...called estimateSizeFac…

deseq2

updated 8.8 years ago • enricoferrero

pre> vector <- factor(colData$vector) cell <- factor(colData$cell) design_Subtypes <- model.matrix(~0+ vector + cell )</pre>   ___DESeq2___...__PCA plot__ I also generated a PCA/MDS plots (here is the PCA plot using the rld counts from DESeq2 and an intgroup="vector"). The empty vector is represented by the first color in the legend. PCA2=1% and PCA1=9…

normalization limma deseq2 counts

updated 9.1 years ago • Radek

Hi, I need a little guidance on the interpretation of multi-factor analyses. I have the factors Stage (Q & W) + Treatment (treated & control) I created the dds object with: >ddsT <- DESeqDataSetFromMatrix

deseq2 multi-factor

updated 5.8 years ago • m.harrison

differentially present amongst different treatments. When I compare using a condition with 2 factors (Treatment: A, B), I use the following command: *library(DESeq2) dia=phyloseq_to_deseq2(physeq_object, ~Treatment) dia=DESeq...is more suitable for my type of analysis? Also, when I have a condition with more than 2 factors (Regie: treatment1, treatment2, treatment3, treatment4), I use …

deseq2

updated 4.9 years ago • pyveronneau71

Hi, I've recently started using DESeq2 with complex experimental designs and I've stumbled across some facts which I cannot wrap my head around. One of the...for the sh and some more for the wt-controls. I've decided to use a simple design with a factor representing the treatment and sh condition together as suggested in the documentation and another one to control...out these genes if I wo…

deseq2 biostatistics

updated 7.5 years ago • Elena Grassi

I am currently using DESeq2 in conjunction with the SVA package in an effort to remove latent factors from RNA-Seq data.  I have questions about...in two separate growing seasons and, as such, we expect to see signals from many environmental factors that are unaccounted for.  There are 4 genotypes sampled at 5 timepoints in each of 2 years and 2 biological replicates...directory…

deseq2 sva

updated 8.2 years ago • brookb

slices from control group the slices from treatment group may come from the same fish. I'm using DESeq2 package in R, so my colData looks like: samples fishGroup expGroup 1 Sample12 3 control 2 Sample18 4 control 3 Sample25...5 treat 11 Sample37 6 treat 12 Sample50 8 treat If I perform DESeq2 based on independent …

rnaseq deseq2

updated 7.7 years ago • zhxiaokang

Hi Everyone I have a DESeq2 design problem, where I have the counts for reads mapping to maternal and paternal allele, for the wildtype and knockdown...Hi Everyone I have a DESeq2 design problem, where I have the counts for reads mapping to maternal and paternal allele, for the wildtype and knockdown samples. So the design is like this, where TF = transcription factor, rep=replicate and KD=knoc…

deseq2 rnaseq

updated 9.8 years ago • Vivek.b

inspired by this post \#http://seqanswers.com/forums/showthread.php?t=31036 \#install.packages("DESeq2") \#\# try http:// if https:// URLs are not supported \#source("https://bioconductor.org/biocLite.R") \#biocLite("DESeq2") \# Set working directory...lt;- read.csv("rpkm.csv\_0\_filter\_atleat\_200\_count.csv", header=TRUE, row.names=1) library(DESeq2) \# Convert to matrix cou…

rnaseq deseq2

updated 9.1 years ago • prp291

Hi, I am trying to run paired and multi factor comparison by DESeq2 and keep getting errors. I have read other posts such as [DESeq2 paired multifactor test](https...support.bioconductor.org/p/62357/) and [Yet another DESeq2 nested design contrast matrix question](https://support.bioconductor.org/p/62428/) but still cannot figure out...stats4 stats graphics gr…

deseq2

updated 10.3 years ago • shoko1018