Bioconductor Forum

Affymetrix data? I have a 2 x 2 design, treatment versus control at two labs. There are strong (lab) batch effect that can be seen by clustering the data. So far I have computed RMA (and GCRMA) values for each lab separately, normalized...across arrays using limma's normalizeBetweenArrays (quantile) and generated lists of potentially differentially expressed genes via lmFit...eBayes and TopTable…

Clustering Clustering

updated 20.9 years ago • Malard, Joel M

Hello all, I have performed batch correction using SVA using normalized counts generated from DESeq2. I then used a function in Limma in order to adjust...log normalized counts in so that I can output these batch corrected counts to do analysis that does not involve differential expression. Now I have batch corrected counts that...lt;- rowMeans(dat) > 1 dat <- dat[idx, ] # sin…

sva deseq2 limma

updated 7.8 years ago • rrcutler

matters. I tried removeBatchEffect() and it works well. The example project I tried had four batches, one of which consisted of only single sample. How is removeBatchEffects() able to deal with single sample batches when...ComBat does not? Here is an example: expr <- matrix( 10 + rnorm(10000*100), nc=100 ) batch <- paste0("Batch", rep( c(1, 2, 3, 4), c(33, 33, 33, 1) ) ) tissue…

Normalization limma sva

updated 11.3 years ago • Adaikalavan Ramasamy

enter image description here][1] For paired data analysis using limma with multiple factor, if I was interested in the interaction effect between Condition and Time would the structure...look like this? ```r mm <- model.matrix(~Patient + Condition*Time + Batch) ``` [1]: /media/images/b50660d0-46f4-4149-a0fb-a3a86d00

DifferentialExpression limma

updated 2.6 years ago • Michael

of samples, as follows: > a # see below for dput(a) accession cond donor batch pair A1.r1 GSM1587362 ASD AA1 1a 1 A1.r2 GSM1587363 ASD AA1 2a 1 A2.r1 GSM1587364 ASD AA2 1a 2 A2.r2 GSM1587365 ASD AA2...Label = c("AA1", "AA2", "AA3", "AN1", "AN2", "AN3", "NN1", "NN2", "NN3"), class = "factor"), …

limma paired batch

updated 6.3 years ago • english.server

div class="preformatted"> Hi, I am attempting to remove two known batch effects from my Illumina BeadChip expression data. I have the following experimental design: ID Condition Batch Parents...Within each pair of siblings I have a wt and ko. On top of this, samples were prepared in 2 separate batches (batch1 = first 4 samples). After background correction and normalisation in limma my MDS p…

limma limma

updated 11.4 years ago • Guest User

Dear R users, I have a project that needs to use linear model of limma to do the contrast analysis, but the difficult is to use numeric variable in the contrast. We would like to use Batch as...represent the post-infection) to 0 (pre-infection), I don't know if there is a way to solve this in limma or other package. Thanks a lot and appreciate it for any input. Best, Li &nbsp

limma

updated 11.2 years ago • jial2

aim of the analysis is to further subgrouping the cases by certain traits (e.g. translocation). The batch effect is very heavy and my current approach is: filtering by TPM > voom+quantile normalisation/Deseq2 vst > limma...the distribution of delta in gPCA is at 0.01. However, I am still looking for some other means to do batch removal without knowing the underlying biological facto…

batch effect unsupervised

updated 7.4 years ago • kentfung

the following code on the vsd transformed data vsd <- vst(dds) plotPCA(vsd, "batch") assay(vsd) <- limma::removeBatchEffect(assay(vsd), vsd$batch) plotPCA(vsd, "batch") and the PCA looked far better where the replicates...clustered according to their respective samples. But is it possible to remove the batch effect from raw counts or normalised c…

DEseq2 batch effect RNAseq

updated 6.4 years ago • chudar.chudar

preformatted">Hi Naomi and list, some time ago I asked a question on how to model an experiment in limma. I think I need some additional help with it as the experiment grew in complexity. I also added a factor "batch" because the...arrays were run in separate batches, and I think would be good to control for it. The dataframe with phenotypic informations ("dummy") looks like this: >&…

affy limma affy limma

updated 16.7 years ago • Paolo Innocenti

of samples in normal and disease condition. I have tested that the data also contain significant batch effects with hybridization time. However, the positive hits I obtained using the following approaches are very different...NHLBI Here eset is the expression dataset after RMA function. __Approach 1__: <pre> # Consider batch effects in the model matrix design<-model.matrix(~0+co…

Microarray

updated 8.2 years ago • Xie, Zhi NIH/NHLBI [E]

RNA-seq data and facing challenges with designing an appropriate analysis approach due to confounded batch effects and unbalanced conditions. Here is a summary of my data. ![enter image description here][1] challenges: 1. The Diagnosis...groups (e.g. healthy vs. cancer) do not overlap with the same batches, making it impossible to adjust for batch effects using the typical design matrix: `~ Dia…

edgeR BatchEffect

updated 14 months ago • Thili

I also would like to adjust my model for 4 covarites (age, sex, center, BMI) and also remove batch effect. So, I used the following design matrix: treat=factor(rep(pheno$treat, 2),levels=c("A","B")) time <- rep(c("Before", "After"), c(ncol...GE\_before), ncol(GE\_after))) design=model.matrix(~0+age+sex+center+BMI+batch+treat:time)    Could you please let me know that…

limma batch effect covariates contrast differential gene expression

updated 8.8 years ago • mheydarpour

have been using the EdgeR differential expression analysis methods with GLMs. Here, I have specified batch as a factor in the design matrix. The batch factor is uncorrelated with the other experimental factors and each batch...is evenly represented among all environments. But I have been asked to get batch-adjusted RPKM expression values for a select number of genes to make a bar chart (I of cou…

limma rna-seq removebatcheffect()

updated 8.0 years ago • Ekarl2

My recent problems with extremely high dye bias turned out to be due to a defective dye batch. While it is expensive to do, if all the arrays were done with a particular batch and an anomalous result is found, it does...Pinese <z3062573 at="" student.unsw.edu.au=""> > > Subject: [BioC] Significant dye bias using limma > > To: bioconductor at stat.math.ethz.ch &a…

limma limma

updated 20.4 years ago • Naomi Altman

same doses of a drug). I guess, if the tumours are considered as replicates one could include the batch as a factor (as you suggest below), but if they contain different tumour classes one could not separate the dmso effect...subjects and will show strong differences per se. Maybe one get some estimates for the impact of the batch by using a mixed effects model with each sample as random effect …

Cancer Cancer

updated 21.1 years ago • Arne.Muller@aventis.com

preformatted">Dear BioC List Members: I have a data set that I would like to analyze with the limma package. I am having trouble figuring out how to make the design and contrasts matrices, and I was hoping that someone...a different lot of reagents, and a different scanner. It is not surprising that we are observing a batch effect in the "C" replicates that is not removed even after normaliz…

Normalization limma Normalization limma

updated 21.2 years ago • Jim Breaux

<div class="preformatted">Hi, In Chapter 8.1.2 in Limma users guide there is a description how to detecting the Dye effect. The example there describes an experiment of Wt vs...div class="preformatted">Hi, In Chapter 8.1.2 in Limma users guide there is a description how to detecting the Dye effect. The example there describes an experiment of Wt...to correct dye effect. If dye replicates…

limma limma

updated 5.0 years ago • Ron Ophir

Hi, I'm trying to use limma to get a list of differentially expressed genes between subtypes (GCB and ABC) of B-cell lymphoma. I have a dataset from...to read my CEL files and perform normalisation on the gene expression data: ``` norm.batch = rma(batch) dat = exprs(norm.batch) ``` But I'm just not sure where to start with creating a design matrix for the limma analysis

limma

updated 5.0 years ago • d808bc07

div class="preformatted">Dear all, I am currently working with gene expression analysis in limma. I have a total of 146 samples divided into 21 groups. What I want to do is pairwise comparisons between one group (the control...code shows this for the first pairwise comparison between group B and the control group, also adding batch effects to the model. All groups are included in the "Group" …

limma limma

updated 11.5 years ago • Arvid Sondén

I am trying fit a linear model with limma, where I do adjust for a batch variable. Then I want o calculate statistics for all pair-wise comparisons. Here, I'm wondering...consider the example below and give me a feedback on that? __\# Fit many adjusted statistics with limma. Here we adjust for the day effect (a surrogate for a batch effect):__ \#\#--------------------- \#Covariate of interest:…

limma limma contrast matrix limma design matrix

updated 7.4 years ago • payam_delfani1980

Hello, I'm doing some bulk RNA-Seq analysis to identify different genes in two condition (Braccio) but before that I want to identify some not known variable and delete the batch effect (RIN) of my variable. So I'm moving in this way: ```r dds_campione <- DESeqDataSetFromMatrix(countData=raw, colData=condition_breakfast, …

DESeq2 BatchEffect sva

updated 22 months ago • michelafrancesconi8

Hi, I have a very complex paired design to test for differential expression with limma. I have a control group and intervention group (more than 100 RNA-seq samples). The intervention group received a treatment...Post1-Pre1 `` `` Post2-Pre2 `` `` Pre2-Pre1 `` `` Post2-Post1 `` But taking into account batch effects (batch1 and batch2) and sex (male/female) . But I am not sure if including bat…

limma paired samples complex-design

updated 10.0 years ago • aec

Hi, I have a question, I am working with mRNA-Seq dataset (18 samples) corresponding to different 2 batches and 3 cell types . `Batch_1` has 2 cell types (A and B types), however, `Batch_2` has 3rd cell type (C type). I imported the dataset...I cannot perform differential expression and to measure the effect of the Cell_Type, controlling for batch differences. I would like to export either norm…

DESeq2 limma Normalization R

updated 3.1 years ago • Sabiha

<div class="preformatted">Dear All, I used limma with two different parametrizations. Both approaches should be equivalent in my opinion. However, if I use weights, the...div class="preformatted">Dear All, I used limma with two different parametrizations. Both approaches should be equivalent in my opinion. However, if I use weights...below. Does someone know the reason for this? Regar…

limma limma

updated 17.2 years ago • Hans-Ulrich Klein

Hello, Its my first time to deal with Longitudinal analysis in Limma and I would like to make sure that my code reflects my research question. Research question: which gene-expressions...Hello, Its my first time to deal with Longitudinal analysis in Limma and I would like to make sure that my code reflects my research question. Research question: which gene-expressions are...Hello, Its my first t…

limma

updated 23 months ago • COPD_Project

So I have a dataset that consists of the batch correction through RUV-normalization of several microarray datasets containing tumoral and non-tumoral samples...is in Log2 RUV-normalized expression. I want to perform differential expression analysis. Is the limma package in R fit for this? From what I've read the limma package expects Log2 expression data without normalization, but

RUVnormalizeData MicroarrayData limma

updated 2.6 years ago • pg45863

well as in the heatmaps of the genes with the highest variability, I am quite sure that there is a batch effect based on the date the samples were processed. This I would like to correct by adding the date factor to the design...whether or not the date is indeed a problem I have plotted the PCA before and after removing the batch effects. The top plot is before, the bottom plot is after removing …

deseq2 batch effect removebatcheffect() design matrix

updated 8.2 years ago • Assa Yeroslaviz

As Naomi Altman as already told you, analysing an experiment such as this is straightforward with limma. I guess the problem you are having is that you are trying to use the limma User's Guide's suggestion of forming a composite...You could for example use targets <- read.delim("targets.txt") design <- model.matrix(~Batch+Sex*(Phen/Line), data=targets) This will produce a d…

affy limma siggenes factDesign maanova affy limma siggenes factDesign maanova

updated 16.7 years ago • Gordon Smyth

I have a RNA-seq dataset with two treatments A and B (+/+, +/-, -/+, -/- design) and a batch effect. Both environments are equally represented in all batches. For differential expression analysis, I have added...genes that were found to be differentially expressed. When I do this, the batch effect is visible and samples form the same batch group together (but there is a stronger grouping from tre…

limma removeBatchEffect heatmap clustering

updated 10.0 years ago • Ekarl2

controls (which are present in many but not all multiplexing runs), taking into account 1) the batch effect originating from the multiplexing runs and 2) an additional unwanted biological source of variation for which...9 and 10 nicely separating known haploid from known diploid WT-controls. B) Running RUVs() on batch-corrected vst-transformed data vsd <- vst(dds) assay(vs…

ruvseq deseq2

updated 6.4 years ago • Frederik Ziebell

div class="preformatted">Hi all, I have performed batch-normalization on my RNA-Seq data leaving the data as cpm-log2 normalized. How do I use this as input in limma? It usually

updated 11.3 years ago • Guest User

samples) are analysed in the same batch. For female, the samples have been randomized to batch, so there are some subjects which have their "before" sample in one...batch and their "after" sample in another. - We need to account for a number of covariates; age, BMI and so on. Importantly, we'd like...My current reasoning is that we cannot say anything about sex differences, since it's confound…

limma microarray design paired mixed model

updated 6.9 years ago • rasmus.agren

and tutorials I found are regarding plotting heatmaps and PCA plots, where you remove the batch effect of the paired samples from vst. Now, how do you think I should proceed regarding the normcounts? Since I am introducing...negative values, when I apply limma::removeBatchEffect it doesn't seem correct. Thank you for your time

DESeq2

updated 19 months ago • altuda

patients and healthy control In the expression dataset the biological variable of interest and batch are partially confounded. Even though batch effect is not particularly strong on Principal components 1 versus 2, I...td> <td>Cond1</td> <td>Cond2</td> </tr> <tr> <td>batch 1</td> <td>8</td> <td>0</td> <td>0</td&g…

combat differential gene expression sva

updated 11.0 years ago • AdamJ

to mention that even if you do normalize all the chips together, you are still likely to see the 'batch' (or 'block') effects. To try to assess the extent of the problem, you might cluster the samples and see if you get samples from...subjects and will show strong differences per se. Maybe one get some estimates for the impact of the batch by using a mixed effects model with each sample as rando…

Clustering Cancer Clustering Cancer

updated 21.1 years ago • Darlene Goldstein

I am trying to perform differential expression of  Agilent data `` GSE9210 `` by using `` limma. `` Following is the example code <pre> target = file.path("/path/to/file") SDRF <- read.delim(target,check.names=FALSE,stringsAsFactors...I am trying to perform differential expression of  Agilent data `` GSE9210 `` by using `` limma. `` Following is the example code …

limma agilent microarrays

updated 7.9 years ago • Agaz Hussain Wani

When trying to adjust data for batch effects using the respective limma (v3.34.9) function removeBatchEffect() I got strange values (at least to my understanding...When trying to adjust data for batch effects using the respective limma (v3.34.9) function removeBatchEffect() I got strange values (at least to my understanding). My question: In the example below wouldn't one expect row 1 to show th…

limma removebatcheffect()

updated 7.5 years ago • jf1

Hello, I have the following (certainly not ideal) RNA-seq experimental design: - Batch 1 contains 40 samples with condition A + 12 samples with condition B - Batch 2 contains 40 samples with condition C + the same...So, the 12 samples with condition B are technical replicates that have been profiled across the two batches. I'm actually not interested in condition B; I need to compare th…

deseq2 ruvseq sva edger limma

updated 6.9 years ago • enricoferrero

gt; Hi Naomi and list, > > some time ago I asked a question on how to model an experiment in limma. > I think I need some additional help with it as the experiment grew in > complexity. I also added a factor "batch" because...the arrays were run in > separate batches, and I think would be good to control for it. > The dataframe with phenotypic information…

affy limma siggenes factDesign maanova affy limma siggenes factDesign maanova

updated 16.7 years ago • Paolo Innocenti

Hi, I have a question about concerning the number of differentially expressed probes after batch combination, using ComBat from 'sva'. I have 2 data sets: one containing around 250 samples that correspond to around 50...to 2 groups (let me call them Batch2_Group1, Batch2_Group2). One of the 2 group labels in the second batch (Batch2_Group2) also exists in the first batch, so there is no confoun…

updated 12.6 years ago • Michaela Oswald

p/70664/][1]). So now I have multiple clusters in a 270 leukaemia sample cohort comprises of 3 batches. I have groups of samples according to their cytogenetics or clustering results. Some clusters are bigger (> 20) and...some are small (3), and some of these groups span across all batches and some are only found in one or two batches (some big groups can only be found in one batch). An…

limma voom group-design sva

updated 6.1 years ago • kentfung

dataset we are analyzing, and I have a general question about the quantile normalization options in Limma. Our dataset shows a relatively pronounced batch effect, so the between-array normalization is a critical step. We have...normalizeBetweenArrays(MA.Loess$M, method="quantile") Because this is not one of the built-in Limma functions, I was wondering whether there any obvious reason why this …

Normalization limma Normalization limma

updated 14.6 years ago • Wlasiuk Battagliotti, Gabriela - wlasiuk

measurements. I assume these as technical replicates. Microarray data were measured in two different batches. Finally, I have a specific mutation that I am looking into: T790M: this is present in some of the samples and not present...height:296px; width:365px"> <tbody> <tr> <td>Sample</td> <td>BiolRep</td> <td>T790M </td> <td>Batch…

limma microarray duplicatecorrelation technical replicates linear model

updated 10.0 years ago • Eleni Christodoulou

Hi, I am working with microRNA microarray data and I am facing very strong batch effetcs. In particular, I have 15 samples with a disease and 15 healthy controls. The first thing that I did was to explore...noted the presence of multiple confounding factors. Later, i looked into detail at the sources of batch effects with a PVCA, which reported that the main source of variability is due to the d…

BatchEffect sva limma

updated 3.3 years ago • Jacopo

Hi I'm using DESeq2 to analyze raw read counts. I have 5 groups and 3 batches (different projects). In order to find out which genes are expressed differentially in all groups and also adjust...batch effects I conducted the LRT test step by step from the guideline. This is my code: rna.o=DESeqDataSetFromMatrix(data...result this code gave me thousands of highly significant genes. I took no…

DESeq2 DEG ANOVA Batch effect Comparison

updated 6.4 years ago • amin.ghareyazi

Dear all, I am a bit frustrated right now. I wanted to double check my DESEQ2 Differential Expression (RNA SEQ) results in Limma and now I am getting different results. I dont know what I did differently that the results are so different. I am here interested...right now. I wanted to double check my DESEQ2 Differential Expression (RNA SEQ) results in Limma and now I am getting different resul…

DESeq2 limma

updated 3.3 years ago • Bine

I have seen quite a few different ways for constructing a design matrix with limma. My problem concerns RNAseq data, for which I want to find the top DE genes between "good" and "poor" responders. This meta...I have seen quite a few different ways for constructing a design matrix with limma. My problem concerns RNAseq data, for which I want to find the top DE genes between "good" and "poor" respo…

limma

updated 5.8 years ago • msveldhuis96

phenotypes to the same control while also blocking for `subject_ID` that are repeatedly measured in batch 1 and batch2. **Experimental design** ```r # print meta data > pb.colData condition subject_ID_unique subject_ID batch 01501_b2...with `0_0` being control and others being diseases. `subject_ID` is the identifier for every sample. `batch` is a factor I wish to regress…

covariancematrix limma

updated 18 months ago • jiachengd

Limma users guide there is a description how to > detecting the Dye effect. > The example there describes an experiment of...dye effect. If dye replicates are also biological > replicates they night also represent a batch effect. That is all first > replicates was sent in one day and the second replicates sent in another > day, which this...second option of dye …

limma limma

updated 20.1 years ago • Gordon Smyth

two plates. I have bulk RNA-seq data with conditions diseased and healthy. One plate from each batch consists of 50 per cent healthy and 50 per cent diseased. I am trying to correct for the batch effect of two plates in Deseq2...both plates. Is there a way to do this I am not aware of? Or do I just use the PCA to see if the batch correction has been done correctly? I use design = plate …

BatchEffect DESeq2

updated 3.6 years ago • lmrogers34

Hi there, I am writing because I am lost in the last step after use limma::removeBatchEffect and introduce the new matrix to DESeq2. The reason I used limma::removeBatchEffect is because the...design is not full rank and I can't fix my batch in the design. The PCAs from before and after batch effect look correct. Please, see my code below: > library(DESeq2) > libr…

deseq2 limma batch effect

updated 5.4 years ago • Patricia

11:56:17 +0100 > From: Hans-Ulrich Klein <h.klein at="" uni-muenster.de=""> > Subject: [BioC] limma - different parametrization and weights > To: Bioconductor list <bioconductor at="" stat.math.ethz.ch=""> > Message-ID...gt; Content-Type: text/plain; charset=ISO-8859-1; format=flowed > > Dear All, > > I used limma with two differ…

limma limma

updated 17.2 years ago • Gordon Smyth

class="preformatted">Dear list, Fellow the paper "Tackling the widespread and critical impact of batch effects in high-throughput data" and the code at http://rafalab.jhsph.edu/batch/, I used sva in my analysis. But I always get...more than 90% of gene with p.adjust<0.05 (compared in two cell line). When I try limma package without sva, it give me about 3000 genes with p.adjust<…

limma sva limma sva

updated 14.1 years ago • Fabrice Tourre

dataset with data from multiple sources and I have decided to use RUVSeq along with DESeq2 for the batch correction and DEG analysis. Following the information on the vignette [here](https://bioconductor.org/packages/release...dds$Type,ref = "Control") dds <- DESeq(dds,test = "LRT",reduced = ~1) res <- results(dds) #batch correction set <- newSeqExpressionSet(counts(dds)…

BatchEffect RUVSeq RNASeq DESeq2

updated 2.3 years ago • Shashank

I'm just asking here whether doing batch correction in a design with 3 batches where one of the batches only contains one sample is possible. From this post https...for clarity whether the sample is removed or kept in the analysis. This is the experiment design ``` BATCH SEX RESPONSE 1 F A 1 F A 2 F A 2 F A 2 F A 2 M A 1 F B 1 M B 3 M B 2 F B 2 M B 2 M B 2 F C 2 M C 2 M C 2 …

DESeq2

updated 2.1 years ago • Tom

Hello All, I have an RNA-seq dataset and I've performed PCA analysis to check batch effect using package edgeR. and have used raw counts from cuffnorm output. here is my code     library(edgeR...dge1)     logCPM1 <- cpm(dge1,log=TRUE,prior.count=5)     batch=c(rep("batch1",8), rep("batch2",8)) &…

rna-seq edger limma removebatcheffect()

updated 7.9 years ago • cvu

I am trying to get a better understanding of how **limma** and **dreamlet** handle random effects differently in a confounded pseudobulk scRNA-seq dataset where all of condition...1 are in one batch, and all of condition 2 in a separate batch, where batch is a random variable (rather than a fixed variable). Where do these...that if I don't set batch_id as a random variable in the formula, the…

limma dreamlet

updated 18 months ago • birb

Hello, I am using limma to determine differential gene expression between healthy and KO mice. In my design matrix, I am including several...Hello, I am using limma to determine differential gene expression between healthy and KO mice. In my design matrix, I am including several covariates that I know influence gene expression, but that I am not interested in. Specifically, it looks s…

limma DifferentialExpression

updated 3.9 years ago • nhaus

Hello everyone, I am fairly new to limma and I think I am faced with a particularly difficult design for batch effect removal. Basically, what I have done is a proteomic...are 5 male and 5 female mice. After some exploratory analysis (PCA) I noticed that there is a batch effect due to the sampling time: 4 different cohorts/batches needed for obtaining the 40 samples, 2 cohorts for model...t…

Microarray DifferentialExpression limma MicroarrayData removebatcheffect

updated 3.0 years ago • dqt