Bioconductor Forum

out how to perform Spearman rank test between expression value of a gene and a continuous outcome in DESeq2. One way I did was to extract the normalized count from counts(dds, normalized=TRUE), and then run cor.test in R. Alternatively...I put the continuous variable in the DESeq2 design formula and ran DESeq(dds). I could get the p and adjust p from the results. It seems the latter approach…

deseq2

updated 5.5 years ago • zhaoy

Hi, I'm still new to DESEQ2 and I wonder if I used everything correctly. I have several RNA-seq (stranded, PE, 30 million reads, 27 million aligned...Hi, I'm still new to DESEQ2 and I wonder if I used everything correctly. I have several RNA-seq (stranded, PE, 30 million reads, 27 million aligned, mouse...is not good enough? why and can I improve it? thanks for your help, I used the code …

normalisation DESEQ2

updated 4.3 years ago • xavier

Hi all, I know this was discussed already, but still after reading a lot here and in the vignette, I'm still not sure, I am doing it correctly. I have multiple time points (colData below) with two or three replica. I am interessted in finding genes which are differentially regulated between two specific time points (TP) and also such, which are DE over all TP. This is what I have done so far: …

deseq2 timecourse design and contrast matrix

updated 10.2 years ago • Assa Yeroslaviz

Hi,   I would like to install the package DESeq2, however it doesnt look to work with the last version of R... > install.packages("DESeq2") Warning in install.packages...nbsp; package ‘DESeq2’ is not available (for R version 3.4.3) What should I do ? Many thanks,   Leila   &nbsp

deseq2 R package installation

updated 7.8 years ago • layeb

those genes that are differentially expressed between the cell type tacking in account also the time factor. This because we are interested in those genes that are differentially expressed in at least one of the two time point...dds, contrast=list(c("Cell1","Time240.Cell1"), c("Cell2","Time240.Cell2"))) We understand the first 4 results table but we obtained exactly the same results fro…

deseq2

updated 10.6 years ago • barbara.mariotti

have an RNA-seq data set of samples that I wish to use for analysis of differential expression with DESeq2. The data has three time points; 1 day, 5 day and 10 days with two different tissue samples taken from each time point (Tissue...replicates of each. Initially to test differences between two timepoints or tissues I created a new factor (group) that was a combination of tissue\_days and car…

deseq2 timecourse multiple time points rnaseq

updated 8.5 years ago • Nicholas Owen

Hi,  I have used Salmon to quantify some 126 samples using the mouse ensembl reference cDNA transcriptome. Then I tried to obtained  abundance values using tximport as follows: <pre> txi.salmon <- tximport(files, type = "salmon", tx2gene = tx2gene, countsFromAbundance = "lengthScaledTPM", ignoreTxVersion = F) names(txi.salmon) head(txi.salmon$counts) ###C…

tximport deseq2

updated 7.4 years ago • ctl

longer than it should and then exits with this. <pre> > dds < DESeq(dds) estimating size factors estimating dispersions gene-wise dispersion estimates mean-dispersion relationship final dispersion estimates...not. Changing the design formula doesn't help either. Any help would be greatly appreciated.  ---------- DESeq2 version 1.14.1 R version 3.3.…

deseq2 R

updated 8.7 years ago • Jacob Stauber

same cell but different inhibitors. the sampleTable looks like ```r sampleTable$group <- factor(sampleTable$group) sampleTable$batch <- factor(sampleTable$batch) > sampleTable group batch DR1_01_S1 DMSOFHIN1...must be removed. Please read the vignette section > 'Model matrix not full rank': vignette('DESeq2') What am I missing? tha…

DESeq2

updated 2.3 years ago • Theo

i.e. Genotype `AB` and `BA`, respectively). We have data for both sexes in each group and I am using DESeq2 to analyse the data using the following design `design = ~Sex+Genotype`. For now I will ignore the `Sex` component as it is...relevant for my question. In a first step I am identifying genes that show different expression in `AA` versus `BB` and I can easily do so using the suggested...D…

RNASeq DESeq2

updated 4.1 years ago • Philipp.kaufmann

Hi everyone, I've been trying to use DESeq2 to look at expression differences across 4 SRR: 2967085, 2967086, 2973633 and 23351735. I've used HTseq and got 4 files...Hi everyone, I've been trying to use DESeq2 to look at expression differences across 4 SRR: 2967085, 2967086, 2973633 and 23351735. I've used HTseq and got 4 files. An example of the files: ``` uc001aaa.3 0 uc001aac.…

deseq2

updated 5.2 years ago • takisliako

I am curious about why the calculated log2 fold change value differs from the log2FoldChange of DESeq2 and want to know the cause. Result (three condition/ Total 16 samples): Condition 1 normalized counts: 0.000000 4.496866...log2 fold change? If it is difficult to tell me about the detailed method, I would like to know what factors(ex. baseMean lfcSE...) affect calculations and the ca…

deseq2

updated 6.5 years ago • cycy

I am trying to make a Deseq2 object for analysis but cannot seem to get my metadata df to properly add to the object. I've made sure that the order of...I am trying to make a Deseq2 object for analysis but cannot seem to get my metadata df to properly add to the object. I've made sure that the order...lt;- subset(spiders_metadata, select = -sample_name) spiders_metadata$collection_times &…

DESeq2

updated 15 months ago • aclehman31

Hello!   I have 2 questions. The first one is regarding the sizeFactors. I have the following sample groups: 1. control = no treatment: 5 biol. replicates 2. after...biol. replicates 3. after 2 h of the same treatment:  5 biol. replicates. (summa summarum: 1 factor, 3 levels, 15 animals)   My task was to find DE genes between the groups. So I did pairwise compa…

deseq2 betaPrior=FALSE sizeFactors

updated 10.5 years ago • elenigeorgopoulou86

my last question about PCA and transformed data. I have two other questions for you today ;-) The first question is about your new version of DESeq2 : - I found about 140 DEG when I used a previous version (v1.0.9) - Now I am using...adjustement. I would like to know why it is better to keep the previous estimates ? Naively I would first have filtered genes and then have estimated d…

DESeq2 DESeq2

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

Here my script (sorry I cannot post the data because it does not belong to me) ```r library(DESeq2) library(RColorBrewer) library(ggplot2) library(gplots) library(pheatmap) library(biomaRt) library(EnhancedVolcano...oviduct_data_matrix <- as.matrix(round(oviduct_df, digits=0)) #Import data matrics into a DESeq2 object dds_kidney<-DESeqDataSetFromMatrix(countData = ki…

DESeq2

updated 2.3 years ago • jovel_juan

MAS 5.0 suite and most of the questions here pertain to single arrays (aka Absolute Analysis?). The first 3 question is on Scaling Factors (SF) which is defined as SF = TGT / x_96 ; where x_96 is the 2% trimmed mean 1. Why does MAS 5.0 apply

affy affy

updated 22.7 years ago • Adaikalavan Ramasamy

I want to make sure I am doing things correctly on DESeq2.  Below is my design, it comprises 33 samples with 9 initial conditions. Associated with these conditions are some...I want to make sure I am doing things correctly on DESeq2.  Below is my design, it comprises 33 samples with 9 initial conditions. Associated with these conditions are some other factors such as age (old …

deseq2 multiple factor design nested design

updated 7.9 years ago • michael.steffen

div class="preformatted">Dear Tina, limma handles random factors using the duplicateCorrelation() function -- see the User's Guide Section 8.2 for an example. It is admitedly an unusual...Andreassen <b.k.andreassen at="" medisin.uio.no=""> > Subject: [BioC] nested design with random factor and limma??? > To: bioconductor at stat.math.ethz.ch > Message-ID: <4…

ExpressionData limma ExpressionData limma

updated 17.5 years ago • Gordon Smyth

Hello Experts! I encountered somewhat difficult experimental design. Briefly, blood cells from 2 individuals where treated with 2 different compounds (this compounds supposedly have to force cells switch their type from Phen1 to Phen2). Afterwards cells of different phenotypes were physically separated and sequenced. So this groups are paired, as well as each Treatment paired as applied to the …

deseq2 multiple factor design

updated 8.6 years ago • Vladimir Krasikov

Hi, This week was the week to upgrade to Bioconductor version '3.8' (from 3.7) so DESeq2 also got a facelift. So running SAME exact code with new upgraded DESeq2 now getting warnings when running DESeq() <pre...gt; dds<-DESeq(ddsHTSeq,parallel = TRUE) estimating size factors estimating dispersions gene-wise dispersion estimates: 6 workers mean-dispersion relationship fi…

deseq2

updated 7.1 years ago • Benoit.Fiset

Hello, I am following the DeSeq2 tutorial in Phyloseq and I'm using the following code to plots OTUs by Genus, coloured by Order. I am doing multiple plots...x = tapply(sigtab$log2FoldChange, sigtab$Order, function(x) max(x)) x = sort(x, TRUE) sigtab$Order = factor(as.character(sigtab$Order), levels=names(x)) \# Genus order x = tapply(sigtab$log2FoldChange, sigtab$Genus, function(x) max...x)…

phyloseq color ggplot2

updated 8.8 years ago • sarahmacdonald86

This is regarding the single factor design For example if I have Age or other continuous numerical variable how to provide that into the design formula...groups' or it can go without it? grouping numerical variable is needed prior to running it in deseq2 because here each age becomes a factor if I get it Here in case of metadata/coldata I m giving a single numerical value

DESeq2

updated 3.5 years ago • krushnach80

is compared to DIFFERENT reference levels as follows: * I have overall of 24 samples. * 6 first samples are of 6 different patients (group A) before a treatment ”A” was given. I consider the 6 patients as biological replicants...fold change of ”after treatment B” vs ” before treatment B”\].   I managed easily to apply DESeq2 for a differential expression of  ”before treat…

Deseq deseq2 differential gene expression rnaseq statistical test

updated 7.9 years ago • Marina

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..._PID, sex = samples$Sex, condition = samples$Condition, nested = samples$Nested) \#nested is a factor sampleTable$sex <- relevel(sampleTable$sex, ref = "Male") sampleTable$condition <- relevel(sampleTable$condition, ref

deseq2 differential gene expression model matrix

updated 8.9 years ago • gregory.l.stone

182). However, when I am trying to do DE, I cannot prepare the data for dds command to kick off the DESeq2 commands. I don't know what the problem is, but I get some errors while I looked for them on the internet and even went over...the DESeq2 tutorial and other examples. It might be noteworthy to declare that I do not have the raw htseq count data and I tried...I also attached my colData to …

DESeq2

updated 4.9 years ago • Pedram

Hi, I am doing a DESeq2 analysis using a data set of nine different conditions/time-points. I first ran the analysis with the complete data...Hi, I am doing a DESeq2 analysis using a data set of nine different conditions/time-points. I first ran the analysis with the complete data set in the count table and than have used the results function to extract the pair-wise results of the DE ana…

deseq2 differential expression independent filtering pre-filtering design matrix

updated 10.7 years ago • Assa Yeroslaviz

Dear limma users,   I would like to know if it possible to include two random interacting factors in the design matrix(like  block:tissue+tissue:genotype) and use in the lmFit() function.   thanks for the help

limma

updated 11.1 years ago • bobyboby

I want to install DESeq2 version 1.16.1. My Bioconductor version is 3.10. I tried to install it by downloading this version from: https://bioconductor.statistik.tu...Library/Frameworks/R.framework/Versions/3.6/Resources/library’ * installing *binary* package ‘DESeq2’ ... * DONE (DESeq2) but getting this error: library(DESeq2) Error: package or namespace load failed…

deseq2 software error

updated 6.0 years ago • joshianu2018

the phenotypic data for these plants are either susceptible or resistant according to what age. the first plant has two susceptible ages and two resistant ages while the other plant has three susceptible ages and one resistant...age. I am using the htseq-DESeq2 pipeline to do the differential expression. my goals are: first, compare the different ages S with S and R with R for plant...each pla…

deseq2

updated 8.3 years ago • Safa.A

and I used the GeneCounts option to get a count matrix. I would like to use this count matrix with DESeq2, however, I'm having trouble formatting STAR's gene count into a count matrix that is recognized and works with DESeq2...Does anyone have any tips/ideas how I should be importing/formating this matrix into DESeq2? Thanks,  Nikelle&nbsp

deseq2 count matrix star

updated 8.4 years ago • nikelle.petrillo

are in C:\\Users\\Administrateur\\AppData\\Local\\Temp\\Rtmps76hIZ\\downloaded\_packages library(DESeq2)\# again I had an error Error: le package ‘S4Vectors’ nécessaire pour ‘DESeq2’, mais est introuvable \#In addition: Warning message...R-3.5.1/library/S4Vectors/DESCRIPTION', probable reason 'No such file or directory' remove.packages('DESeq2') \# i tried to remove deseq2 and install it…

deseq2 S4Vectors

updated 7.1 years ago • roghaiyeh.safari

div class="preformatted">Dear Mike (and other DESeq2 developers/users), I came across this thread regarding the use of the GLM functions in DESeq2 with respect to time series...gt;Greetings, >>I have used the DESeq package previously and have been recently using >>DESeq2. I am particularly interested in repeated measures designs and was >>wondering a…

edgeR DESeq DESeq2 edgeR DESeq DESeq2

updated 11.9 years ago • Hickman, R.J. Richard

ERCC spike-in data for normalizing these counts. To do this, my strategy involves estimating size factors using ***only*** the **spike-in data** (function: Deseq2::estimateSizefactors) and using those normalize my counts (*Mus musculus...spike-in tx2gene file and the other with mouse Ensembl tx2gene file. step3: I then used the Deseq2::estimateSizeFactors with the **Spike-in_ddsTxi** object …

deseq2 Tximport Sizefactors normalizationfactors spike-in

updated 5.5 years ago • hina.abbas.bandukwala

wish to receive our emails, reply to this email with "REMOVE" as subject line. BioSlave Newsletter First Artificial Intelligence Trading Robot Released If you aren't aware of the Forex market or Forex trading robots then...profit. The only limitation on how much money you earn is how intelligent the robot is. This is the FIRST artificial intelligence trading robot ever made. More info Limited t…

updated 17.1 years ago • Michael Hanson

I'm analysing 46 samples hybridized to custom designed one-colour Nimblegen arrays. I have two factors: "temperature" (2 levels; 20 or 25) and "age" (3 levels; 20, 50 or 90), in a full factorial design, with 7 to 8 biological replicates...Or should I make a new variable 'treatment group' that with all 6 combinations of the 2 biological factors temperature and age? (as is done in the R / maanova t…

maanova maanova

updated 13.7 years ago • V. Oostra

Hi everyone! I have a question about the DEseq2 analysis. Can it be used to analyze a 3-way interaction? In my experiment, we have 3 variables: Diet + Date + Time, that need...Hi everyone! I have a question about the DEseq2 analysis. Can it be used to analyze a 3-way interaction? In my experiment, we have 3 variables: Diet + Date + Time, that need to...Diet + Date (0 day, 21 days) and Time (t…

deseq2

updated 7.2 years ago • idapantoja

Hello seasoned bioinformaticians and biologists: I am really new to bioinformatics and I am trying to use DESeq2 to perform differential expression analysis for my data. I have muscle RNA sequences from 60 individuals at 7 different time points (420 samples total). Running through DESeq2 with the following workflow: ```dds <- DESeqDataSetFromMatrix(countData = countData, colData = co…

DESeq2

updated 2.7 years ago • Hongru

I need to calculate log2 fold change values for lot of different experimental conditions when compared to their corresponding controls. Just to mention, I am not going to use these for differential expression analysis but for some other downstream analysis like clustering and stuff. Traditionally in my field, counts are normalized by TPM method and then fold change values are calculated by log2(T…

DESeq2 DifferentialExpression

updated 3.0 years ago • Jayesh Kumar

div class="preformatted"> Hi, I've been using the DESeq as well as DESeq2 packages for my RNAseq analysis. For one particular study alone, I find some discrepancies between DESeq and DESeq2...results. I've read in forums that the gene list is longer when we use DESeq2, which is true in my case. But what I also notice for this study is that there is no overlap between the gene list obtained...…

DESeq DESeq2 DESeq DESeq2

updated 11.9 years ago • Guest User

expression comparison between two groups introduced bias. So I would like to use normalization factors instead of size factors in DESeq2(as described in differential analysis of count data - the DESeq2 package). >normFactors...dds)<-normFactors Is it a correct manner to normalization and introduce the normalization factors in DESeq2?  Thank you for cooperation i…

normalization

updated 7.9 years ago • parvin.shariaty

Hello All,   I am trying to reproduce an analysis a former college of mine has produced as we are not able to reach him. He normalized some rna seq data using the VST in DESEQ2 using default parameters. However, if i input the same count matrix to the latest deseq2 version i don't get the same normalized values. Moreover, if i use the vst transformation in deseq i get values different …

deseq2 variancestabilizingtransformation

updated 8.3 years ago • aa1201

Hi, I have a problem with installing DESeq2. The R version is 3.2.3. I got errors as below. Cannot find xml2-config ERROR: configuration failed for package ‘XML’ * removing...ERROR: dependencies ‘RcppArmadillo’, ‘genefilter’, ‘geneplotter’ are not available for package ‘DESeq2’ * removing ‘/home/mhz/R/x86_64-pc-linux-gnu-library/3.2/DESeq2’ The downloaded source packages are in …

deseq2

updated 6.9 years ago • mhz

tried to duplicate the DESeq2 result by explicit calculation and found the result to be drasically different from the one performed with DESeq2...could please tell me if I am using the wrong formula for the explcit calculation or if I am using DESeq2 incorrectly. ```r > library("DESeq2") > coldata=read.table("data/targets.3.txt",sep="\t",header=T) > coldata$Targ…

DESeq2

updated 4 months ago • richardallenfriedmanbrooklyn

for the bug report. This is fixed in IRanges 1.17.11. Note that the data inside the rle must be a factor. Adding levels to an Rle after the fact doesn't create a factor-Rle. x = Rle(c(2,3,1,2,3,2),rep(2,6)) levels(x) = as.character(0:5) This is...2 3 1 2 3 2 > table(x) 1 2 3 2 6 4 The same is true for a numeric vector. table() on a non-factor argument will drop unused levels. v &…

IRanges IRanges

updated 13.2 years ago • Valerie Obenchain

  I have raw counts data from featureCounts. I actually wanted to do survival analysis. For a specific gene I want to classify the samples into Low and High based on expression cutoff. For that I'm using maxstat package. First I would like to convert raw counts to FPKM. So, I did like following.  <pre> sample1 sample2 sample3 sample4 sample5 A1BG…

deseq2 r fpkm() geneexpression

updated 7.2 years ago • Beginner

What was the very first paper to describe "limma-trend"? By "limma-trend" I mean the one described in Law (2014), but I am not sure that was the first time

limma

updated 10.3 years ago • Nik Tuzov

time point and 2 expression levels with three biological replicates (48 samples). When I treat each factor independently, I could find gene affacted by each factor separately. Since my result indicated one factor is strongly...afffected by the other, now I would like to also check the interaction between each factor pair on gene expression. But I don't know how to build model in Limma to solve my…

limma microarray

updated 10.2 years ago • baibing7713661

As I understand that Z' factor is used to measure the separation between the positive and negative controls and reflects the overall quality of...an assay, does having an Z' factor approaching 1 help to prevent false positives

HTS QC

updated 5.9 years ago • Happyflower

Hi, I have a dataset that uses single cell RNA seq on the same tissue (C. elegans male tails) across different conditions. Most tutorials deal with different cell types, however we have the same cell type across 6 different conditions. We pseudobulked the data, since each condition has 70 samples that we treated as replicates, because they're the exact same cell type. We are now trying to run…

DESeq2 scRNAseq

updated 4 months ago • rj1435

Hi, I decided to split [my question from here](https://support.bioconductor.org/p/74100/) into several parts, as I figured out that it is too long. I hope it was a good idea. I'm doing a time course analysis with deseq2, I have 8 different points and multiple replica. when I am looking at `` resultsNames(dds) `` in my analysis I see <pre> >resultsNames(dds) # [1] "Intercept" …

deseq2 timecourse time course design and contrast matrix coefficient of variation

updated 10.2 years ago • Assa Yeroslaviz

nbsp; Hello all, I am trying to use DESeq2 to determine the number of main effect DEGs and interaction DEGs in a two-factor experiment, where each factor has 2...I also need to obtain the other main effect an interactive term, which I can only do with the first method.  My questions are: 1) Is the inconsistency across the methods surprising? For example: disease\_V\_vs\_N comparison.…

DESeq2

updated 7.7 years ago • LR0306

Hi Michael, I've read you paper on DESeq2 (Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2) and have a question about using the...thing is we have a similar problem as you have with low read counts. So the question is can I use DESeq2 algorithm for multiplex qPCR when the Ct values are converted to read count like values? If not, can I use the same type

deseq2

updated 9.7 years ago • ilyaal

In the [Question: diffBind: differentially bound sites are highly different between using EDGER and DESEQ2](https://support.bioconductor.org/p/123248/#130498). You mentioned > Correct, the factors are only estimated by DESeq2...they are based directly on the libSizes. And I am wondering whether I can get the libSizes factor **(full library size, total number of reads in BAM/SAM/BED f…

diffbind

updated 5.2 years ago • shangguandong1996

Hello,  I would like to use only the lib size as normalizing factor in edgeR, so I am doing this: <pre> cds <- DGEList(counts = counts, group = group, norm.factors = MyNormFactors)</pre> I __skip__...nbsp; <pre> cds <- calcNormFactors(cds, method = "none")</pre> Because It will bring the norm factor back to 1.. and I continue with calculating…

edger

updated 7.3 years ago • Udi Landau

are using DESeq2 to capture differential chromatin accessibility due to age differences (adult vs juvenile) using CUT&RUN data...differential accessibility of peaks which were called using MACS2. We initially tested how the DESeq2 design model behaved both with and without sex as a confounding parameter for age (see below), and found that including...requested justification for includ…

DESeq2

updated 2.2 years ago • mphogan

I would like to investigate **DGE** and **DTE** in my samples using the R packages **DESeq2** and **Fishpond/Swish**, respectively. In my subject species, there are three phenotypes and for each of them, I have samples...from 13 different tissues for a total of 267 samples. First, I would like to compare the three phenotypes considering all the samples from all the tissues. Then, I would also lik…

fishpond DESeq2

updated 3.4 years ago • alexyz

Hello, There are two different transcription factors that it is relevant to compare their binding locations. The two factors have a Myc tag, and a ChIP seq will be performed...for each of the factors using an antibody against Myc. I was thinking that maybe each factor might have different frip, and it can cause biases

DiffBind csaw

updated 5.5 years ago • GFM

Hi,   I have RNAseq from two cancer+normal cases. I used salmon and proceeded with DESeq2. I do get the ./results/ directory out but the PDF figures are broken. The error message for example in boxplot.ENSG00000077454.15.pdf...states: "Error using packet 1 arguments must have same length". The X-axis is labeled "factor", Y-axis is labeled "ENSG.... normalized counts". $ cat sa…

deseq2 reporting tools

updated 7.1 years ago • mmokrejs

I am using limma-voom for an RNA-Seq dataset with global down-regulation of gene-expression (experimentally confirmed). On top of that there is also a small set of genes in this dataset that is massively(!) up-regulated. I can't use RLE or TMM normalization because they normalize out the down-regulation. I created a DGEList object and then set normalization factors to 1 (using edgeR's calcNormFa…

limma limma voom normalization edger

updated 8.1 years ago • biominer