Bioconductor Forum

Hello ! PhD student and working with rna-seq data, I used DESeq2 for my analysis but after finishing my work, I believe some elements were misunderstood when using the software. To...my work. So the design appropriated seem to be: design = formula(~G + B + G:B) Reference factors are : G1 and T1 intercept : base mean of G1 in T1 (reference) G2vsG1 : Genotype 2 against r…

deseq2 R logfoldchange rna-seq

updated 6.4 years ago • pierre.marin

like described in e.g. the Trinity description into one table for all samples. Striuggeling now in DeSeq2 with the function "deseqtable <- DESeqDataSetFromMatrix" we tried now the tximport package reading the manual but...54.45    0.00    17.39    0.00 without rounding it first ? Would be helpful if anyone c…

RSEM deseq2 count matrix

updated 8.9 years ago • kmeusemann

I've been using DESeq2 for differential expression analysis of microbial (meta)transcriptomic datasets and have been very happy with...I've been using DESeq2 for differential expression analysis of microbial (meta)transcriptomic datasets and have been very happy with its...in a given dataset. __My question is whether it makes sense to normalize, specifically via a DESeq2-performed size f…

deseq2 pathway analysis order normalization

updated 9.0 years ago • Matt

Hi all. First things first, sorry for posting one more question about experimental design and time series in DESeq2. We have performed...whether there would be any way of getting this comparison directly with the design formula of DESeq2. However, I have doubts regarding two different questions. First, I don't know whether this is a real time-series experiment

DESeq2 design

updated 3.8 years ago • jordi.planells

in a chromosome-specific manner, and I want to be sure I am handling them right. I am using DESeq2 to compare Hi-C contacts at loops called among these three cell types. I have an un-normalized count matrix of Hi-C contacts...1] [1]: /media/images/79219219-e1b1-4cc6-b336-2e5dabdc I also noticed that after running DESeq2, we have a higher number of differential loops on chromosomes th…

DESeq2

updated 2.5 years ago • Kathleen Reed

I would like to know if it is possible to use DESeq2 to analyze NimbleGene microarrays. I have a dataset of several conditions with enriched (IP) and control (Input) sample...cdc48 IP MJK503_FK2_2 561565A10_635.pair 561565A10_635 cdc48 Input MJK503_FK2_2 </pre> My first comparison would be between the IP and Input of the WT and of the cdc48 sets separately, But I would also like to try a…

deseq2 microarray nimblegene ratio limma

updated 8.2 years ago • Assa Yeroslaviz

Hi, I am getting error while running DESeq2, as some of the samples contain 0 so I want to add pseudo-count to dds so that I can able to run it without any error but I...Hi, I am getting error while running DESeq2, as some of the samples contain 0 so I want to add pseudo-count to dds so that I can able to run it without any error but I am...10 dds <- dds[keep,] dds …

deseq2 r

updated 5.3 years ago • nabiyogesh

The formula looks like this: ~ flow_cell + tissue*caste Where 'flow_cell' is a blocking factor accounting for batch effect. If i then run 'resultsNames' on the DESeq object I obtain: > resultsNames(dds_atlas_fc...contrasts within group? I am aware that a possibility is to group caste and tissue into a single factor, but we would rather keep the data structure as it i…

deseq2 interaction factors complex design

updated 6.6 years ago • c.martinezruiz

particularly the section on interactions (https://bioconductor.org/packages/devel/bioc/vignettes/DESeq2/inst/doc/DESeq2.html#interactions), I decided to add a third factor which is a combination of treatment and timepoint...i.e.: ```r sample_metadata$group <- factor(paste0(sample_metadata$timepoint, "_", sample_metadata$dose)) ``` Then I can extract the results for each comparison by..…

DESeq2

updated 2.1 years ago • drew

Hi Bioconductor community! I'm using DESeq2 to visualize which taxa are significantly enriched in one treatment over a control and realized that [the vignette...bars.  Is this because what the error bars represent (standard deviations) get wrapped up in DESeq2's p-values or another statistic?   First I get the results from a `` DESeqDataSet `` object "`` DDSfbps1spooled ``" g…

deseq2 metagenomics phyloseq ggplot2

updated 8.4 years ago • lauren.czaplicki

Hello everyone! My question is related to RNA-seq analysis utilizing DESeq2. Input: I try to analyze DEGs between normal HEK293 cells and HEK293 depleted by one protein. So, I have 3 biological replicates...obtained in one day, and the third one in a week - condition was the same). I analyzed my data using DESeq2. The problem: When I produce the PCA plot (top 500 genes) I have the s…

deseq2

updated 5.8 years ago • alexandr.gopanenko

of rhdf5 directly but got no response from him. My qualm lies with the writing and re- reading of factor vectors using rhdf5. In the current release they are simply written as integers and upon reading the HDF5 files the...levels are obviously forgotten. Of course, I could convert the factors to character vectors before writing but I wanted to ask whether there is a plan to implement better fact…

rhdf5 convert rhdf5 convert

updated 12.9 years ago • Moritz E. Beber

Hello, I finished running DESeq2 on my dataset that includes 6 timepoints and 3 biological replicates per timepoint. I would like to run a PCA analysis...unsure which count transformation method I should use, vst or rlog.  The calculated the size factors for my dataset (below). Based on this, there does not appear to be a large variation in sequencing depth (dynamic range...of size fac…

DESeq2 quality assesment vst rlog transformation

updated 7.6 years ago • agdif

Just followed the vignette to run Swish for the first time using inferential replicates computed by salmon, on data from an F1 cross of two non-B6 mouse strains. It seemingly...separately like so: ``` y <- se[,se$condition %in% c("control1","treated1")] y$condition <- factor(y$condition, levels=c("control1","treated1")) y <- scaleInfReps(y) y <- labelKeep(y) y &lt…

tximeta DESeq2 swish fishpond

updated 3.9 years ago • jeremymsimon

Hi I want to use the DESEQ package between a control (3 biological replicates) and treatment (1 biological replicate). IN DESeq I herefore used the following code, and got 266 genes with padj < 0.05: table <- read.delim("test.txt") row.names(table) <- table$Feature\_ID count\_table <- table\[, -1\] conds <- c("ctrl", "ctrl", "ctrl", "treatment") c…

DESEQ deseq2 deseq replicates biologicalreplicates

updated 10.1 years ago • wd

voom` function, once before and once after the execution of `duplicateCorrelation`. The blocking factor is not provided within the design formula in both cases. This is different to the later described `dream` workflow. Here...executed, the blocking variable is part of the design formula. Can someone briefly explain why? A factor defining individual patient/subject effects, which are "block…

limma voom variancePartition dream

updated 6.2 years ago • Ben

I am new to DESeq2 and I'm trying to analyze some RIPSeq data. I have 2 conditions: tagged and untagged I have 2 assays: input and IP I want...I am new to DESeq2 and I'm trying to analyze some RIPSeq data. I have 2 conditions: tagged and untagged I have 2 assays: input and IP I want to...untagged IP untagged_IP_rep2 untagged IP >coldata$condition &lt…

DESeq2 RIPSeq

updated 19 months ago • ck

DESeq2 assumes negative binomial distribution for counts distribution. That refers to the distribution of counts for a...across one sample? Each sample is normalized based on the geometric mean of all counts (or size factor). That sounds like it does not take into account the distribution. For example, two samples have the same number of reads

deseq2 rnaseq

updated 9.3 years ago • igor

two pairwise comparisons, A vs B and B vs C. I have 1000 genes. I can provide contrasts to edgeR/DESeq2 for each of the pairwise comparison, thus I will have two contrasts. The first time I test A vs B, I will have a list of q-values

edger deseq2

updated 8.8 years ago • SmallChess

the edgeR package, which will still be relatively slow but faster than the glm routines in edgeR (or DESeq2). Best wishes Gordon > From: Iddo Ben-dov <iddobe at="" ekmd.huji.ac.il=""> > Subject: edgeR and DESeq2: model design and estimation...at 4:51:51 PM GMT+3 > To: bioconductor at r-project.org > > hi, > > in both edgeR and DESeq2, estim…

limma edgeR DESeq2 limma edgeR DESeq2

updated 11.5 years ago • Gordon Smyth

nbsp; I am trying to use DESEQ2 to estimate the correlation coefficient of gene expression levels with different phenotypes. dds = DESeqDataSetFromMatrix

deseq2

updated 7.3 years ago • songeric1107

Hi Michael or DESEQ2 community, First, sorry, I'm not sure why the images don't load, but if you right click on them they will. When I use the DESEQ2...Hi Michael or DESEQ2 community, First, sorry, I'm not sure why the images don't load, but if you right click on them they will. When I use the DESEQ2 results() function I get a variable called *stat*, which is useful for GSEA. ![Resu…

deseq2 stat lfcshrink

updated 5.8 years ago • divercory

Hello all, I am writing to learn how to set up DESeq2 when my samples have large variation in gene counts. For example below is one row from my gene count table. <table border...of reads at gene240880, but some have zero. When I feed the whole table (with ~25k genes) to Deseq2, using pretty much default settings recommended in the DEseq2 tutorial, and looking at comparisons between condition…

deseq2

updated 7.1 years ago • Helene

Hello! I am a bit confused about the normalization performed by the DESeq2 varianceStabilizingTransformation() and vst() functions in addition to the actual variance stabilization. My understanding...Hello! I am a bit confused about the normalization performed by the DESeq2 varianceStabilizingTransformation() and vst() functions in addition to the actual variance stabilization. My understan…

library composition vst DESeq2 Normalization

updated 4.1 years ago • kristoffersandas

Hello everyone, I'm currently trying to perform DESEQ2 with my own RNA sequencing results. I performed whole transcriptome RNA sequencing and the subsequent analysis...Hello everyone, I'm currently trying to perform DESEQ2 with my own RNA sequencing results. I performed whole transcriptome RNA sequencing and the subsequent analysis I...performed via galaxy. I performed first fastp on th…

DESeq2 star differentiallyexpressed

updated 3.0 years ago • nico

Hey all,    I'm analysing a 16S microbial community dataset, and am using DESeq2 to test for differential abundances. When I do this, I supply raw count information to DESeq() as per the vignette rationale...ordination of my samples, I might consider using rLog transformation or VST to standardise my data first.    The [PICRUSt](http://picrust.github.io/picru…

deseq2 picrust

updated 8.7 years ago • handibles

Hi all, I have been trying DESeq2 to analyse my RNAseq data. Data is from sequencing a cell line treated with a drug at different time point intervals...like this: <pre> DataFrame with 5 rows and 2 columns treatment time <factor> <factor> cl_0h untreated 0 cl_2h treated 2 cl_4h treated 4 cl_6h treated …

deseq2 timecourse

updated 10.9 years ago • anand m t

Hello! I'm working on comparing tumor and healthy samples with DESeq2. My analysis only cares about the adjusted Fold Change values that DESeq2 computes. The input data encompasses a huge...the process up given the fact that I merely require the adjusted fold change instead of the whole DESeq2 output? The full script that I run on the data is [here at this permalink](https://github.com/TCP…

DifferentialExpression DESeq2

updated 2.2 years ago • Luca

base mean is rowMeans(counts(dds, normalized=TRUE)), the mean of the > counts divided by the size factors. > > The transformations involve much more complicated mathematics than > simply dividing the size factors and...gt; wrote: >> > Hi Michael, >> > >> > I was recently using your DESeq2 a lot, but unfort…

Visualization Clustering DESeq2 Visualization Clustering DESeq2

updated 11.9 years ago • Michael Love

Hello there, I am trying to analyse a dataset using deseq2 with kallisto via tximport. I am using the following code: tximport(files, type = "kallisto", tx2gene = tx2gene, ignoreTxVersion...suggested ways of importing estimates for use with differential gene expression (DGE) methods. The first method, which we show below for edgeR and for DESeq2, is to use the gene-level estimated counts from …

kallisto tximport deseq2 counts

updated 5.8 years ago • Mozart

For each cell type I have 2 biological replicates - each represent a different pool of mice. I first ran DESeq2 without setting the 'Mice' factor, meaning without pairing the 3 cell types of the same mice pool. The colData...c("CellType", "type1", "type2"), alpha = 0.05)</pre> Then, I have created a data set with the 'Mice' factor, creating the following colData: <table border="0" ce…

DESeq2 deseq2 rna-seq paired samples

updated 8.7 years ago • solgakar@bi.technion.ac.il

on my volcano plots. I am attaching these, here: ![enter image description here][3] I know that DESeq2 calculates the log2(fold-changes) before shrinking, which is why this may appear a little strange (referring to the...at the volcano center). However, my question lies in why these genes are not filtered out in the first place? I can do it with some pre-filtering (I have seen these genes remov…

DESeq2

updated 6 months ago • Alexander

community over there. However, I still have some remaining doubts, and maybe I should have come here first since it seems it is indeed an issue of how I'm using DESeq2 rather than previous points in the RNAseq analysis. Quick summary...each one in triplicate. The pipeline goes as such: trinity -> salmon -> tximport -> deseq2. We want to use a common pipeline for both transcr…

deseq2

updated 7.2 years ago • Emiliano Canton

patients with 2 conditions and 2 time-points from each patient. The patients associated to the first condition are different respect to the second one. The experiment consist of 24 patients, 12 of them who respond to one...342.62 314.02 295.18 228.01 244.49 86.41 And so on.... And my DESeq2 code: ListRNAseqTimeSeries<-"raw_data_matrix_allSamples_RvsNR_ne…

DESeq2 RNASeqData

updated 3.0 years ago • perceval.vellosillo

I got a  list of paired end bam files, and want to do DESeq2 analysis. The first thing is to count the reads at gene level. I tried feature counts. However, it requires reorder of the...also takes very long time.  Is there any tools could quickly count the paired end reads for DESeq2 at gene level? Thanks

deseq featurecounts paired

updated 9.7 years ago • tszn1984

This is a follow-up to my post about DESeq2 with 2-factor design [here][1]. Nascent RNA-seq experiments analyzed by slamdunk report a Conversion Rate for each gene...This is a follow-up to my post about DESeq2 with 2-factor design [here][1]. Nascent RNA-seq experiments analyzed by slamdunk report a Conversion Rate for each gene, which is the ratio of Nascent Reads to Total Reads for a given…

DESeq2

updated 3.8 years ago • vanbelj

a little bit confused about the count data transformations and the Principal Component Analysis in DESeq2. In the last vignette, the example on pages 18-19 shows a PCA plot of the samples, obtained with regularized log transformed...no transformation is applied, axis 1 = pathology (patients vs control cases) and axis 2 = unknown factor - when transformed with r-log (rld), axis 1 = unknown f…

DESeq2 DESeq2

updated 12.2 years ago • amandine.fournier@chu-lyon.fr

div class="preformatted">Dear all, I will open the first line without opening the entire file because my file is large? thanks ML -- Mohamed Lajnef INSERM Unit? 955. 40 rue de Mesly

updated 16.5 years ago • Mohamed Lajnef

This is my code :- library(DESeq2) library(ggplot2) countData <- read.csv('/home/keshav/Downloads/gene_count_matrix.csv', header = TRUE,sep = ",") countData...lt;- countData[ , 1] countData = as.matrix(countData[ , -1]) head(countData) (condition <- factor(c("Normal","Tumor","Normal","Tumor"))) (coldata <- data.frame(row.names=colnames(countData), condition)) dds <- D…

DifferentialExpression

updated 3.5 years ago • srikar

expression changes between two conditions over the course of three timepoints. I attempted to use DESeq2's LRT option in order to identify genes with condition-specific temporal expression changes, but only had one significant...lt;- read.csv(file.choose(), sep = "\t", row.names = "Samples") # select coldata.tsv Time <- factor(coldata$Time, levels = c("3","7","14")) Condition &am…

rna-seq R DESeq2 LRT

updated 5.3 years ago • luke.briody

Hi!! I'm having trouble to do a contrast using DESeq2. I have 12 samples, AA(2 replicates), AB(4 replicates), BA(4 replicates) and BB(2 replicates). I wanted to compare AB (or BA) with...header = TRUE, row.names=1) colData <- DataFrame(row.names=colnames(countData), condition=factor(c(rep("AA",2), rep("AB",4), rep("BA",4), rep("AA",2)))) dds <- DESeqDataSetFromMatrix(countData…

deseq2

updated 8.2 years ago • zhuozhu132

Hi, I am using DESeq2 to find Differentially expressed genes. I am using the following code: <pre> conds<-c("kd1","kd1","kd2","kd2","GFP","GFP") Design&lt...the last line, I get the following: <span style="background-color:Yellow">using pre-existing size factors estimating dispersions found already estimated dispersions, replacing these gene-wise dispersion…

deseq2 rnaseq differential gene expression

updated 9.3 years ago • ta_awwad

TIN) has been developed in part to address this problem and one of the ways they did this was to first calculate a median TIN value for each sample and then correct the gene read counts based on this. In the paper they regressed...way/if it is even possible to implement this sort of normalization in a Salmon -> tximport > DESeq2 pipeline. I guess my main question would be, on wha…

deseq2 tximport salmon

updated 6.3 years ago • hsbio

I was comparing various modeling methods for differential expression and found that DESeq2 mysteriously inverted the sign of a subset of fold changes. According to [this discussion][1], turning off fold change...shrinkage should make log2foldchange from DESeq2 be simply equal to (mean of normalized counts group B) / (mean of normalized counts group A). However, it seems that some degree...2]. Al…

DESeq2

updated 4.9 years ago • Yanling

Hi, I'm using DESeq2 to analyse some RNA-seq data. I have different treatments (XXX, YYY and ZZZ) at different time points (2h, 4h, 8h), with DMSO...Hi, I'm using DESeq2 to analyse some RNA-seq data. I have different treatments (XXX, YYY and ZZZ) at different time points (2h, 4h, 8h), with DMSO used as a control for the treatment. 'se' is my SummarizedExperiment object containing the raw counts…

deseq2

updated 11.2 years ago • enricoferrero

in gene expression. Can someone offer some suggestions on how to approach this analysis using DESEQ2? For the drug vs placebo, would a design that includes the factors treatment, week, and patient be applicable and allow

deseq2 annotation multiple factor design

updated 10.5 years ago • rvinisko

show(countdata) countdata <- as.matrix(countdata) head(countdata) condition <- factor(c(rep("x", 2), rep("y", 2),rep("z", 2),rep("t", 2))) show(condition) library(DESeq2) coldata <- data.frame(skip=colnames(countdata), condition

deseq2

updated 6.4 years ago • trumbia

Hi everyone, I have a question regarding how to correctly set up a design matrix for DESeq2 with high heterogeneity between study subjects inside a group. So we are looking at data from two different groups...Hi everyone, I have a question regarding how to correctly set up a design matrix for DESeq2 with high heterogeneity between study subjects inside a group. So we are looking a…

DESeq2

updated 2.7 years ago • a.wag

My dataset from a microbiome study consists of two factors, one with two levels and the other with three. I am interested in both the fixed effects and the interaction. The first...factor is fire with levels burnt and non-burnt. The second factor is depth with levels 0-5, 35-40 and 95-100. I used the phyloseq wrapper...phyloseq_to_deseq2` to convert my phyloseq object to one compatible with D…

deseq2

updated 5.3 years ago • Aditya Bandla

Suppose I have run deseq like this: ``` my_coldata$varA = factor(mycoldata$varA, levels=c('control', 'case') my_coldata$varB = factor(mycoldata$varB, levels=c(0,1)) dds = DESeqDataSetFromMatrix...Suppose I have run deseq like this: ``` my_coldata$varA = factor(mycoldata$varA, levels=c('control', 'case') my_coldata$varB = factor(mycoldata$varB, levels=c(0,1)) dds = DESeqDataSetFr…

deseq2 levels factor colData

updated 6.8 years ago • ariel

Greetings! I have a dataset comparing the effects of two different treatments on cell line line, including both control and combination treatment samples: - Vehicle - Treatment A - Treatment B - Treatment A + B One of my goals is to determine which genes _uniquely_ respond to each of the treatments, e.g. `A & !B & !AB`. A simple approach to this is to perform the in…

deseq2 deseq differential expression contrasts

updated 6.1 years ago • Keith Hughitt

Hello, I have rnaseq data generated from brain tissues of 100 individuals, half of them were affected by a disease. All the samples were mix of two different brain regions, obtained from 4 different providers. I am trying to run DESeq2 to identify  differentially expressed genes using the model like: ~age + sex + tissue + PMI + condition, where PMI is post-mortem interval. My colData…

deseq2 dexseq rnaseq differential gene expression differential exon usage

updated 9.0 years ago • fizer

Hello I'm having issues generating a Manhattan plot in Deseq2 with the following script . Every time I run the script no Manhattan plot is generated despite using the following...head(countdata) # Assign condition (affected versus unaffected) condition <- factor(c("affected","affected","affected","unaffected","unaffected","unaffected"),levels=c("affected","unaffected…

deseq2 RNA-seq

updated 5.7 years ago • adeler001

Dear Bioconductor/DESeq2 community, I am trying to analyze RNAseq data with DESseq2 and have a question about contrasts and interactions. I...packages/devel/bioc/vignettes/tximport/inst/doc/tximport.html). Initially, I was performing DESeq2 analysis at each time point separately. For example, here is the sample file for testing for differential expression...5,infected.2hr,2<br/> 6,infecte…

deseq2 extracting contrasts rnaseq interaction term

updated 7.5 years ago • Amit Indap

We used DESeq2 a lot at our research projects. Recently I was asked about specific experimental design for DESeq2.   We have 10 subjects...surgery, and 3 follow up time points. All 10 subjects were collected at the pre, during, and the first follow up. Only 7 were collect at the 2nd follow up and 6 were collected at the 3rd follow up. This is a question of paired

DESeq2

updated 7.3 years ago • shengqh

div class="preformatted">Hi , I am using Deseq2 for differential gene expression calculations. I have two conditions Condition a (T) and Condition b (P), when I run the...lt;- read.delim ("48_96_filtered.txt", header=TRUE, row.names=1) > pdata = data.frame(condition = factor(c( "T", "T", "T", "P", "P", "P", "P"))) > library (DESeq2) > dds <- DESeqDataSetFromMatrix(c…

DESeq2 DESeq2

updated 11.8 years ago • Mubarak hussain Syed

Hi, I am new to using DESeq2 and would appreciate some advice for a somewhat complex experimental design (for a non-statistician anyway). I have...a dataset with 3 factor variables, each with 2 levels, as follows: trt: control, treatment geno: A, B time: 6h, 12h (no time 0h) Each combination of factor

deseq2 rna-seq rnaseq interactions

updated 8.2 years ago • red_bricks

which comes to me when I compare the output of these 2 approaches to (in appearance) do the same in DESeq2: * First way: AvsB <- results(dge, __alpha = 0.05, lfcThreshold = 1__, contrast = c("cond","A","B")) AvsB\_tb <- AvsB %>%   data.frame() %&gt

deseq2

updated 7.6 years ago • pablo_garcia05

div class="preformatted">hi Marine, Note that you are using an older version of DESeq2 and Bioconductor (DESeq2 v1.4 was released in April 2014). So if you are searching by Google for documentation, it might...not apply to your software (we print the version number on the first page of the vignettes). You can always get the correct documentation by using: browseVignettes("DESeq2") and ?result…

edgeR DESeq2 edgeR DESeq2

updated 11.4 years ago • Michael Love

manged to successfully complete some basic analysis of my RNAseq data. I now wanted to do some multi factor analysis but wasn't sure where to stat. Here is the layout of my samples: RA - resistant, 0 dpi RB - resistant, 1dpi RC - resistant

tximport R tximportData RNASeq DESeq2

updated 4.6 years ago • pthom010