Bioconductor Forum

Hi, This is related to the DESEQ2 Normalization. First I would describe my experiment design, I have two groups, GroupA and GroupB. Group A has 39 replicates...sure that the samples from both groups should be included in each batch, therefore, I can use the deseq2 design during analysis. I have run DESEQ2 and run differential expression analysis. I have found around 6700 genes...That was surp…

DESeq2

updated 2.2 years ago • MAHESH

Micheal, I am trying to understand the directionality of the log2 fold change that is reported by DESeq2.   I see in the manual that it says that the first level of the factor will be taken as the dominator for calculations...by DESeq2.  I also see that the order of the factor levels matters when results() is called.  Can you elaborate on how those...testing between l…

deseq2

updated 8.3 years ago • adam

Dear bioconductor community, First of all, Deseq2 and tximport are great tools and offer a lot of to the research community. After reading through the user...guides and various online posts, one point is still unclear (for me). Normalization factors, size factors and how they are calculated/accessed/implemented in tximport and Deseq2. So usually I import my samples...txi,colData = samp…

tximport DESeq2 normalizationfactors sizefactors

updated 3.7 years ago • svenbioinf

Hi, I'm trying to do two-factor DEG analysis using DESeq2. ```r colData(dds) condition batch infection metabolite sizeFactor <factor> <factor> <factor> <factor...numeric> Mock1 Mock first Not-Infected Non 0.904435 Mock2 Mock first Not-Infected Non 1.060912 Mock3 Mock second Not-Infected Non 0.8645…

DESeq2

updated 5 weeks ago • JY

Hello everyone, Following reading the DESeq2 vignette and multiple posts in this forum, I still need to post my question regarding analysis design. In the experiment...run by my colleagues, there are three factors: **Species** (A, B) **Treat** (Mock (M), Inoculation (I)) **Time** (T1,T2,T3,T4,T5,T6,T7,T8) Each of the two Species has both Treats at each Time...B_M_T5) In doing so,…

deseq2 Time-course

updated 5.6 years ago • h.valipour.k

I'm analyzing an RNA-Seq dataset with three different factors: genotype, treatment, and time. I need to do a pairwise comparison of each time to time zero for every genotype and treatment...for sub-groups is to create a new column in ```coldata``` pasting together two or more of the factors together, so each combination of factors is treated as one separate group (here I have named this new colum…

deseq2

updated 4.7 years ago • aharkey

Hello,   I am fairly new to RNAseq analysis and am wondering how to do analysis with a 3 factor treatment in DESeq2. I have plants infested with insect over days. My design is as follows: 2 genotype plants x 2 treatments...infested and control) x 3 days (plants sampled over 3 days). I ran DESeq2 with the following design: design = ~ genotype + treatment + day I now want to look at wi…

deseq2

updated 7.6 years ago • ANNA

Dear all, First of all, I would like to inform you that I'm new in RNA-seq analysis and the DEseq2 package. Also, I have (very) basic knowledge...gMDSC_Ascites With sample names put as rownames. 1, 2, 3 and 4 are the 4 levels of my "origin" factor, and correspond to the different research group that isolated the cells The way I understood the Deseq2 design formula...is "you choose the fa…

deseq2 annotation normalization

updated 4.7 years ago • christophe.vanhaver

Many people were confused to handle three-factors with DESeq2 though many posts on this website. Three factors are temperature(A, B); water content (C, D); co2 concentration...warm:drought) --------------Error in checkFullRank(modelMatrix) My R version is 3.4.2 (2017-09-28), Deseq2 version is 1.18.1

deseq2 multiple factor design

updated 6.9 years ago • marburg2107

the results from RUVSeq(specifically RUVg) into the time series analysis of DESeq2. My RNASeq experiment is a time course (4 points) of two different strains, similar to the fission yeast dataset used...the DESeq2 example. We used the Thermofisher ERCC RNA Spike In mix for use as a negative control to normalize our samples.&nbsp...two sample comparison in DESeq2, the documentation includes t…

deseq2 ruvseq spike-in time course time series

updated 7.0 years ago • staceyborrego

I appreciate in advance any and all advice and guidance. I am attempting to conduct a paired multi-factor analysis in DESeq2 but I seem to be detecting a lot of differentially expressed genes that are pushed into siginificance...count outlier that was responsible for shoving those genes into significance) and re-ran the DESeq2 analysis. I then got no significant genes. I know that DESeq2 has an …

deseq2 differential gene expression multiple factor design paired design

updated 7.8 years ago • gregory.l.stone

Please ignore - I found the problem. Thanks     Hello, I have a question on 2 factor design. I have a design with 2 factors, with duplicates. Here is the design: <pre> strain time strain time sample1 mut a sample2...b sample5 mut a sample6 mut b sample7 wt a sample8 wt b Can this be run using DESeq2 in order …

deseq2

updated 9.4 years ago • GFM

factors.  First I used raw counts as input and called DESeq function directly. DESeq2 estimated size factor and dispersion...file/d/0B0LC8cC9lS-ycVU3cTdGcy1RY0k/view?usp=sharing> Then I used a set of gene-wised normalization factors. In order to keep the raw counts at a similar level, those normalization factors have been divided by their geometric...d) <code>gc=d[,1:(dim…

deseq2

updated 7.4 years ago • dustar1986

Hi everyone, I am trying to help a colleague who uses DESeq2. He has two factors for explaining the variability of genes and he wants to identify if there is an effect of each factor...to performing generalized linear models with different R packages where we can easily specify two factors with their interaction. Then there are statistical tests for determining if each factor has a global effec…

DESeq2

updated 15 months ago • mjd275

Hi, I am using DeSeq2 for a set of metatranscriptome data for which I also do have estimations for the number of total transcripts per L...Hi, I am using DeSeq2 for a set of metatranscriptome data for which I also do have estimations for the number of total transcripts per L within...on absolute instead of relative expression data. My idea was to multiply the automatically generated DeSeq2 size…

deseq2

updated 8.0 years ago • sara.beier

I realized that I get different results for one factor dependent on the order of levels within another factor. In principle, I have 2 factors with 2 levels: Age (young, old), and...Type (Treated, Control) I have performed a DESeq2 model with ~ Age + Type + Age*Type When I use 'relevel' to change the order of the levels of the Age factor in the design matrix...I get different results for the …

deseq2

updated 4.8 years ago • dfab

Hi there, Can I please ask a question I get very different results when I perform Deseq2 by choosing 3 factor levels under 1 FACTOR vs performing Deseq2 individually 3 times to compare the three factor levels...to each other So, I am unable to say which is the correct result. DeSeq2 Run1 FACTOR 1: TREATMENT Factorlevel1: conditionA Factorlevel 2:conditionB Factorlevel 3: conditionC …

DeSeq2_result DeSeq2_dispcripancy_3factorlevl_vs_individualcomparison

updated 3.3 years ago • gayathrig

Hello, I'm using DESeq2 to analyze RNA-seq data. The experiment consists of 2 factors - "concentration" with 3 levels and "strain" with 5 levels. I...want to test the effect of just one factor (say, "strain") by using the likelihood ratio test.  I defined a full model and a reduced...model. I am now lo…

deseq2

updated 9.0 years ago • nofar.chen

Screenshot%202018-11-05%2016.21.34.png?dl=0  I split my data to 4 groups. 1- Disease group: First and last TP, 2- Control groups: First and last TP, 3- First TP: Disease and control, and 4- Last TP: Disease and control groups. I...have 2 columns in my metadata/factor table. First coldata Group::First and Last. Second column Condition and each 2 samples from one paitient paired in one..…

DESeq deseq2 microbiome gut microbiome

updated 6.1 years ago • FaisalH

Hi, I have a couple of questions about DESeq2: 1) in the DESeq2 workflow, to test the dex treatment effect, the following command was used: dds <- DESeqDataSet(se, design...cell + dex) And I guess the only factor here is "dex" and it might be more straightforward to use: dds <- DESeqDataSet(se, design = ~ dex) 2)If I am performing an ANOVA

deseq2

updated 8.8 years ago • tony.fox2016

Hi Everyone, I am trying to run DESeq2 package in R. After running the package I have few questions. I will start with this. `` baseMean : It is defines as ``__average...of the normalized count values, dividing by size factors, taken over all samples in the _DESeqDataSet___ After this step of the analysis, <pre> ddsMat <- DESeqDataSetFromMatrix...above calculation I see __estim…

r deseq2

updated 8.6 years ago • gv

had a secondary component (sex of the individuals) after I had already processed it without that factor. I went back and reran the analysis including the second factor (see META file below); however, the results are a bit perplexing...CTRL2 M SC\_rep2 CTRL2 M SC\_rep3 CTRL2 M SC\_rep4 CTRL2 F SC\_rep5 CTRL2 F SC\_rep6 CTRL2 F \#First time run without 2nd factor dds <- DESeqDataSetF…

deseq2

updated 6.7 years ago • wlorenz

span style="line-height:1.6">Dear DESeq2 community, I am trying to use DESeq2 to detect DEG for my RNAseq data because it can deal with multi-factor designs...although there is not only multi-factor analysis in my experiment data. Let me clarify my datasets firstly. For my first and second dataset, t…

deseq2

updated 9.9 years ago • xfwang

timecourse experiment, particularly regarding model selection and comparisons (either in edgeR or in DESeq2). My dataset consists of three closely related species sampled at three timepoints (3 reps for each timepoint:species...with the factors as main effects (~stage + species), with interactions (~species + species:stage, ~species\*stage), and also nested (~species + species...often the terms …

deseq2 edger timecourse

updated 7.7 years ago • anna_stavrinides

lfcShrink`. Which suggests that it is sufficient to replace the design or overwrite the condition factors. However, it also states: > You should only change the factor levels of variables in the design > before running the...DESeq2 analysis, not during or afterward. Which seems contradictory to the above: cnts <- matrix(rnbinom(n=1000, mu=100, size=1...0.5),…

deseq2 DESeq2

updated 5.3 years ago • krassowski.michal

and C the control samples. Here are examples of the content of each sample (I am showing the first lines of T1 and C1 only, but the other datasets are all similar): T1 gene1 331 gene2 74 gene3 50 gene4 1676.27 gene5 496.99...59 gene3 30 gene4 1906 gene5 639 gene6 12 ... In the package DESeq2 1.8.2 on Galaxy, I am us…

rnaseq galaxy deseq2

updated 8.7 years ago • Marcelo Pereira

of my metadata file for reference: ![image contains headers (Tissue, Genotype, Treatment, Sex) and first 6 lines of sample information from allMetadata][1] For now, I am mainly concerned with genotype, tissue, and treatment...worked through the Harvard Chan Bioinformatics Core github course, and referenced the Bioconductor DESeq2 Analysis guide, however my dataset includes more factors than…

DESeq2

updated 10 months ago • KW

variance (ANOVA) to investigate which OTU's significantly different in abundance among experimental factors after Bonferroni correction. We received the comments from the reviewers: R1. ANOVA is not the most appropriate...test for identifying OTUS that differ significantly between experimental factors, as there are issues with the effects of uneven sequencing depth and subsequent raref…

deseq2 microbiome fungi fungal microbiome

updated 6.5 years ago • david.gramaje

Hi, I am quite new to RNAseq and this is my first analysis and I am not sure whether I did my analysis correctly in my pipeline. I have been searching online to see whether...Hi, I am quite new to RNAseq and this is my first analysis and I am not sure whether I did my analysis correctly in my pipeline. I have been searching online to see whether people have similar experiment layout lik…

DESeq2

updated 2.1 years ago • Grace

I am trying to do differential abundance in DESeq2 on my 16s rRNA amplicons. The design of my experiment is Y ~ treatment (as fixed effect factor) + body_site (as fixed effect...factor) + treatment*body_site + block (random effect factor). I can't see any argument on the vintage for assigning a random effect...factor in my model. Is it even possible to do linear mixed effect model in DESeq2…

StatisticalMethod DESeq2 phyloseq

updated 3.8 years ago • zardbid

kchait@tx.technion.ac.il] Sent: Monday, March 17, 2014 10:57 AM To: Olga Karinsky Subject: RE: using DESeq2 with multi factor data Hello all, I am trying to use the DESeq2 package to perform RNA-Seq analysis on a data containing...several factors. I have been closely following the emails between Ming Yi and Michael Love, because I think that my problem is very...my specific data. Just as an over…

DESeq2 DESeq2

updated 10.7 years ago • solgakar@bi.technion.ac.il

which is quantitative (say, the weight of the individuals). Is it possible/a good practice to use DESeq2 for this? One one hand the vignette clearly describes factors (qualitative) variables, but, on the other hand there is...this sentence, p8: "If the variable of interest is not a factor, the log2 fold change can be interpreted as the amount of doubling observed on average for every unit of cha…

DESeq2 DESeq2

updated 11.2 years ago • Marie Sémon

regrowth comparison for each line. So I should investigate the interaction, it is not hard for two-factor analysis but for three-way it is above my skills... So I wanted to use method proposed in [DESeq2 section on contrasts][2] Where...DESeq2 developers combine factors into one factor 'group'. Indeed it makes pairwise contrasts very convenient. So here is an...pool.plant letter tissue …

DESeq2

updated 5 weeks ago • boczniak767

Hello,  In a multi-factor DESeq2 design, it accounts for the changes in one group while testing for changes in others. In section 1.5 of the vignette...controls for type while testing for differences in condition. I assume it calculates a normalization factor + size factor and then adjusts the counts accordingly? In that example in the vignette is it possible to access the counts...whi…

deseq2

updated 9.5 years ago • feargalr

three diagnosis groups (call them A, B, and C) and age (a continuous variable, not grouped into factors). The code works fine with DESeq2 1.6.3, but not with 1.10.1. I checked and diagnosis is definitely a factor and age is...definitely an integer. <pre> design <- data.frame(Sample=paste(1:24,"D",sep=""),Diagnosis=factor(rep(c("A","B","C")),times=c(11,7,6)),Age=c(78,75,54,58,54,75…

deseq2

updated 5.9 years ago • hmgeiger

grt and cells dgrt. Now the idea is to find the differences in this 2 cell populations inducing the factors of number of patients , batches, so the structure matrix of a dataset which resembles as below. I want to use the multifactorial...2 batches are u and i for the facilities. Now Since samples belong to same patient I also wanted to factor in the patients as well to also capture if patient s…

deseq2 deseq

updated 8.2 years ago • vd4mmind

Hi, I am trying to use DESeq2 to perform differential analysis on a large 16s microbiome dataset of around 600 samples. It is clear from our current...interest are associated with one or more unwanted covariates. We want to use a parametric model like DESeq2 to account for these. As with many OTU tables, it's extremely sparse. I find that there is a lot of variation in the size...factors as well…

deseq2 metagenomeseq rnaseq 16s microbiome

updated 8.2 years ago • jbatsx

to the introduction of coldata information in the matrix before running DESeq2 when using featureCounts data. 1) using Galaxy with 2 factors (2 batches/ 2 discinct studies from the litterature), 3 levels...in each factor that are not the same. so 2 batches and in first batch I have non-treated, treated 1h and selected population treated 1h...second batch I have 3 populations selected that I th…

DESeq2

updated 4.0 years ago • NGS_enthusiast

nbsp; I understand from the manual section 3.3 that for a standard model matrix (and since my factors only have two levels, I believe this is correct), if I had two factors factorA and factorB, I would use the following code...to extract results: results(dds, contrast=c("factorA","level1","level2")) to get the effect of factor A for the reference level of factorB.    My fir…

deseq2

updated 9.2 years ago • gw.sarah

Hi there, When I using DESeq2, can I drop the bottom 10% and top 10% of read counts to calculate the size factor by using median-of-ratio (which means exclude...the high and low expressed genes from calculating the size factor)? Best, Keren

median DESeq2

updated 2.5 years ago • KEREN

Hi, I am confused about the calculation of the size factors when the gene has at least one zero count in the samples when using the default params. Though I have checked the source...code of deseq2, I did not get the point. Because I found that if there is at least one gene without zero among all of the samples, the deseq2...the sfType = "poscounts", which I think is proper for the data that have…

DESeq2

updated 11 months ago • jiakang

I recently updated to R version 3.4.2 and noticed that DESeq2 results now returns an error if I relevel the factors.   <pre> library(DESeq2) cnts <- matrix(rnbinom(n=1000, mu=100...I recently updated to R version 3.4.2 and noticed that DESeq2 results now returns an error if I relevel the factors.   <pre> library(DESeq2) cnts <- m…

deseq2

updated 7.1 years ago • Chris Stubben

But not all 8 of them have both tissue samples. 5 have both tissue samples, 2 have only the first kind of sample and 1 has the second kind but not first. Additionally, one of the samples also has two technical replicates...How do I factor all this into DESEQ2 design? 1. Can I do design = ~patient + sample, despite not all of my patients having both kinds of samples

deseq2 RNASeq

updated 4.7 years ago • divyak

Dear all, I am trying to use DESeq2 to analyze my data. I have 2 conditions (wild type and mutant), and 3 samples for each condition. My samples are paired: wt1...with mutant1, wt2 with mutant2, wt3 with mutant3. I am using R version 3.2.0 (2015-04-16) and DESeq2\_1.8.1.The first script I wrote starts as followed: <pre> d <- read.delim(infiles) m <- c(rep("MU",3),rep("WT"…

deseq2

updated 9.4 years ago • Estel Kitsune

Hi,  I was trying to visualize the normalized count of my data, however, I was running into errors: normalized.count <- as.data.frame(counts(DESeq2, normalized=TRUE)) Error in as.data.frame(counts(DESeq2, normalized = TRUE)) :    error in evaluating the argument 'x' in selecting a method for function 'as.data.frame': Error in .local(object, ...) : …

deseq2

updated 9.5 years ago • yuan.qing

I have RNA-seq dataset with 3 factors: * Treatment: control , treatment (2 levels) * Cell lines: ABC.1, Colo320DM, HCT.8, OU31, OVCAR4, RKO (6 levels) * Groups: I, II (2 levels...A, but not to the bulk of cell lines belonging to one group.  Is there a way of doing it in DESEq2? &nbsp

deseq2 multifactorial design

updated 8.6 years ago • ofonov

TN.DBP+FITC 1890-17 CD11b+.UT.r.2 2 UT CD11b+ CD11b+.UT</pre> The DESeq2 manual suggests that combining the two factors is an appropriate way to do the analysis: _Many users begin to add interaction...terms explicitly to the design formula is to perform the following steps:_ _1. combine the factors of interest into a single factor with all combinations of…

deseq2 differential gene expression multiple factor design

updated 9.0 years ago • David Eccles (gringer)

This is the first time I've tried to import Kallisto output using tximport for use in DESeq2. I want to get the sizeFactors calculated by...DEseq2 and use them for normalizating counts from another experiment. But I am only able to get size factors for each gene individually

deseq2

updated 6.7 years ago • Jon Bråte

B = 0) and an *interaction* effect if δ ≠ 0. I used the table of counts for all genes and I used DESeq2 to normalize. Then I used contrast to determine δ This is my code: ```r data <- read.table("total_counts.txt",header=T,row.names...1) groups <- factor(rep(c("CTL", "mutantA", "mutantB", "mutantC"), each=3)) sampleInfo <- data.frame(groups,row.names=colnames(data)) dds &a…

synergy DESeq2 RNA-seq contrast

updated 3.3 years ago • slcno

Hello to all, I am starting to do DGE analysis with DESeq2 with a small example, as a first experiment I am going to compare two groups as example with DESeq2. I have two groups (ND...count. I am using this command in R for making groups, I am not sure they be correct; <pre> gr <- factor(c(rep("ND", 2), rep("NF", 2))) colData <- data.frame(group=gr, type="paired-end") cds…

deseq2

updated 6.3 years ago • lkianmehr

Hello, I have a bulkRNAseq dataset which looks like this. The variable `Litter` (factor) is nested in the variable `Treatment`. What I would like to know is how to formulate a design for this experiment - gene...I read [Group-specific condition effects, individuals nested within groups ][1],[?results from DESeq2][2], [tutorial_deseq2_contrast][3], [RNA-Seq workflow: gene-level exploratory analys…

nested_design RNASeqData DESeq2

updated 2.5 years ago • JK Kim

Hi I am kind of new at DeSeq2. And I want to understand how I can play with size factors a bit.. I have a data set dataset composed of multiple conditions...Data is raw RNA counts. I have counts for around 5000 different RNA's. Say I want to calculate size factors with a different method than estimateSizeFactor function provides. Now I have a vector of 52\*3 numbers, corresponding...to my size…

deseq2 estimatesizefactors

updated 9.7 years ago • umut.caglar

not extract size factors from the DESeq2 object I imported the estimated counts from kallisto tsv files as such: kallisto_Counts <- tximport...kallisto_Counts, colData = sampleTable, design = ~ condition) And computed the sizes factors using the DESeq function dds <- DESeq(ddskallisto) estimating size factors using 'avgTxLength' from assays(d…

deseq2 tximport

updated 4.6 years ago • zroger499

Hello! I'm running DEseq2 on an experimental design of two conditions. From reading the docs, what I understand is that if I do not run "factor", than...TRUE, sep = ",") metaData <- read.csv("metadata.csv", header = TRUE, sep = ",") # create DEseq2 object dds <- DESeqDataSetFromMatrix(countData=countData, colData=metaData, …

DESeq2

updated 16 months ago • adi.rotem

Dear the bioinformaticians, I am using DESeq2 to normalize data from 100 samples from 100 patients. The 100 samples are classified into 3 groups (group A; 30 samples...to compare significance of gene expression among each group. In statistics, we use ANOVA, but in DESeq2 there is no option to compare gene expression among more than three groups. 1. So, my first question is "Is it ok to 1) obt…

DEseq2

updated 19 months ago • Changsuk

I'm running DESeq2 on RNAseq data and my experimental design has three factors: Carbon_Source, Time_Point, and Amoxicillin. I'm having...I'm running DESeq2 on RNAseq data and my experimental design has three factors: Carbon_Source, Time_Point, and Amoxicillin. I'm having trouble extracting the comparisons that I need. Here is a portion of my sample metadata: ![][1] ``` dds = DESeqDataSe…

DESeq2 RNASeq

updated 4.0 years ago • microPhD

some bits of important data from my analyses. I have RNASeq count data and a design with three factors: age at 5 levels, sex at 2 levels, and condition at 2 levels. My design is set as `~0+age*sex*condition`. code sample: `age <- (rep...at the intersection of age and condition, both with and without sex included. That is, for example: first, what is the difference between conditionExp a…

deseq2 contrasts reference level

updated 5.0 years ago • ananda2

RNA-Seq count size factors are defined in [formula 5 of Anders & Huber (2010)](https://genomebiology.biomedcentral.com/articles/10.1186/gb...11-10-r106#Sec2) ***With pre-specified geometric means, are size factors supposed to be the same for identical samples regardless of total count matrix context?*** That is, if I calculate the...size factor for a single sample or if I extract th…

size factors deseq2

updated 4.3 years ago • Megatron

stabilizing transformation, etc) should not be used. I in this situation, I wonder if using DESeq2 is correct and possible (using default configuration) or if it's better to adjust it somehow. According to DESeq2 and...DESeq papers, the size factors calculation with the median of ratios solves the problem of having "a few highly and differentially expressed genes...when the overall distribu…

deseq2 quantro normalization

updated 6.2 years ago • Victor Barrera

Hi, I am trying to do some differential expression analysis with deseq2 at the moment and I have samples with four different conditions, two different batches and five different mouse. Here...Hi, I am trying to do some differential expression analysis with deseq2 at the moment and I have samples with four different conditions, two different batches and five different mouse. Here is the colD…

DESeq2 DEG multiplefactors deseq2

updated 5.1 years ago • yura.song