Bioconductor Forum

batches, so my design is model.matrix(~batch + treatment). My understanding is that with this design, limma first compares treatment1...gives different batches equal weights when combining the results from each batch, even if batches contain different number of samples, am...I right? Additionally, most of my batches contain both treatment1 and treatment2 samples. Let's call these dual-treatment …

limma batch effect

updated 6.9 years ago • jalali4

I have 4 treatments at 4 time points, mostly in triplicate, spread approximately evenly across 2 batches. Previously I have used ComBat to reduce batch effects and then used Limma and AgiMicroRna to get DE miRNAs. However...I read on the list somewhere that it is better to add Batch as a factor to the model in Limma rather than try and remove it. ### So this time I added Batch to my targets fi…

miRNA Microarray limma PROcess AgiMicroRna miRNA Microarray limma PROcess AgiMicroRna

updated 12.4 years ago • Segal, Corrinne

div class="preformatted">Hi, Is it possible to correct for batch effects in limma when doing a paired analysis? I have pairs from two runs Batch 1 and Batch 2. There  are no pairs where...one is in Batch1 and the other in Batch 2. If I enter the Batch no. into the design matrix, no coefficients are generated as there is no difference between run no

limma limma

updated 11.6 years ago • khadeeja ismail

for. I read both on this forum and on _Biostar_ that it is generally recommended to include the batch effect in the limma model instead of using for example _Combat_seq_ to adjust for the batch effect. I wanted to make sure...that limma can appropriately control for the batch effect, so my idea was to fit a model with and without the the batch effect included...in the design and then perform a…

BatchEffect limma sva DifferentialExpression

updated 21 months ago • nhaus

Hi bioC community, Until now I used comBat to remove the batch effects present on my datasets. After reading the article "Methods that remove batch effects while retaining group...content/early/2015/08/31/biostatistics.kxv027.full.pdf>, I decided to block for batch effect in limma in the case where the groups are distributed between the batches in an unbalanced manner. But I am wondering...…

batch effect combat limma block

updated 8.2 years ago • eleonoregravier

15:08:55 +0000 >From: Adaikalavan Ramasamy <ramasamy at="" cancer.org.uk=""> >Subject: [BioC] LIMMA: testing for batch effects >To: BioConductor mailing list <bioconductor at="" stat.math.ethz.ch=""> > >Dear all, > >We...We wish to select >genes adjusting for two covariates : gender (male/female) and >experimental batch (o…

Cancer limma Cancer limma

updated 17.7 years ago • Gordon Smyth

Hi all, I wanted to know if it is possible to remove 3 batches using "limma". I have sequencing data with three possible batch effects: age, gender and working sets. As far as I understand...using limma's "removeBatchEffect" enables to remove only two batches categories by defining "batch" and "batch2" removeBatchEffect...x, batch=NULL, batch2=NULL, covariates=NULL, …

limma

updated 3.8 years ago • Matan G.

not clear on what/how to do this. Here is my experimental setup: Array Param1 Param2 Rep Batch 1 H 4 1 1 2 H 4 2 2 3 H 4 3 2 4 H 20 1 1 5 H 20 2 2 6 H 20 3 2 7 H 37 1 1 8 H 37 2 2 9 H …

limma GeneSpring limma GeneSpring

updated 16.3 years ago • Nathan Haigh

normal. We wish to select genes adjusting for two covariates : gender (male/female) and experimental batch (one/two/three). >From biological knowledge, we expect the batch effect to be significant but we wish to quantify it numerically...that we tried and the problems we faced. We searched the archives without much success. library(limma) # version 2.3.3 dd <- model.matrix( ~ …

Cancer limma Cancer limma

updated 17.7 years ago • Adaikalavan Ramasamy

Dear all, about correcting the batch effects in **LIMMA and SVA,** 'd appreciate having your comments : assuming that we have a set of RNA-seq data (no treatment, + treatment...in many distinct BATCHES, would the linear model in LIMMA : **design <- model.matrix(~0 + BATCH + Treatment, data=RNA)** suffice to correct for batch effects

limma combat sva

updated 4.4 years ago • Bogdan

Hey, Is it possible to correct for both a location and batch effect using Limma? I have 4 surfaces: PET, PE, Glass and Water, 5 locations: A, B, C, D ,E, and 2 batches: 1, 2 enter OTU<-read.csv("filterotu.csv...lt;- as.matrix(tax) group<-factor(targets$surface) location<-factor(targets$location) batch<-factor(targets$Batch) dge&…

limma BatchEffect duplicatecorrelation block batch

updated 3.0 years ago • Katherine

enter image description here][1]Hello, I am attempting to remove batch effects from my data using `limma::removeBatchEffect()`. I have two batches of samples, and there are four conditions. In the...figures below batches are color-coded. I'm wondering why the batch effect seems stronger after applying the `limma::removeBatchEffect()`. The...as follows: vst <- vst(dds) …

limma deseq2

updated 4.5 years ago • lech.kaczmarczyk

Hi everyone, I have some doubts about the removeBatchEffect function from the Limma package. I am working on association of expression data (RNAseq) with a continuous phenotype variation (instead of a...Hi everyone, I have some doubts about the removeBatchEffect function from the Limma package. I am working on association of expression data (RNAseq) with a continuous phenotype v…

limma

updated 5.1 years ago • dressa_ol

experiments (which use different platforms also). I want to compare HLT vs TUM minimizing the batch effect, thus I implemented in the design matrix both the condition and the experiment info: g = c(rep('HLT',9),rep('TUM',9)) conditions...factor(g, levels = c('HLT','TUM')) b = rep(c(rep('EXP1',3),rep('EXP2',3),rep('EXP3',3)),2) batch = factor(b, levels = c('EXP1','EXP2','EX…

batch effect limma batch effect correction

updated 4.8 years ago • ginlucks

div class="preformatted">Hi Dear BioC! I have a question concerning batch effect using Limma. I searched through the BioC archives but didn't find the answer to my question. Suppose I have the folling...the type, f2 the stage in which the sample was collected and f3 the m3 KO samples that have a clear batch effect. The idea is to try to modell this batch effect of the m3 KO mice. Hence, /de…

Proteomics limma Proteomics limma

updated 15.4 years ago • Paul ChristophSchröder

Dear List, I have microrarray data where condition is completely confounded with a time batch effect. When doing a PCA on the RMA normalized data, the first principal component separates clearly the two batches...What option do I have when I still want to compare different conditions across batches? As far as I understood I can't dissolve this batch effect neither with limma nor with e.g. ComBat…

GO limma GO limma

updated 11.7 years ago • Tefina Paloma

similar for large sample size. Now, my question is since my sample size is large and I am using limma for calculating differential methylation, should I be adjusting batch directly in limma as a covariate (or use removeBatchEffect...not sure about this, as this will again be like removing batch separately)  <pre> batch <- pheno$batch BSC <- pheno$BSC_batch group …

combat sva limma

updated 7.7 years ago • AST

Good morning I have a question regarding the function `removeBatchEffect` from `limma` package. This is my experimental design ID Patient Metastasis S1 A NO S2 A YES S3 B NO S4 B YES S5 C NO S6 C YES S7 D NO S8 D YES...gt; dds <- DESeqDataSetFromTximport(txi_rsem, colData, ~ Patient + Metastasis) Now I need the "batch effect remo…

limma deseq2 batch design batch effect

updated 3.3 years ago • Pietro

Hello! I have a question about correcting for batch effects prior to differential gene expression analysis with limma. I've read that batch effect correction functions...such as ComBat should not be used prior to differential expression analysis in limma, and that batch effects should be accounted for in linear modeling instead. However, in my case a batch effect and...disease e…

limma batch effect rna-seq

updated 6.5 years ago • adscheid3

Hi, I'm currently exploring methods for batch correction with my RNA-seq dataset from a series of clinical samples. The goal of this approach is to remove batch effects...we see patients separating predominantly by PC1 and batch separating on PC2 . The "removeBatchEffect()" command in limma appears to correct for batch effect very well, with the 5 samples...from each patient now clustering very …

limma batch effect rna-seq

updated 5.2 years ago • Mike A

I want to find the different expression genes between dissese A and B. There are two type of batch effect in my analysis. I want to use limma to remove two type of batch effect, one is gender, another is tissue type. I do as

limma limma

updated 13.0 years ago • Fabrice Tourre

We have an experiment where samples were collected and sequenced in several batch (matched ATACseq and RNAseq). We would like to include the batch in our analysis formula if possible. The problem is that...after quality control and removing substandard samples, some of the batches have only one sample in them (we have several batches with many samples in and a few with only one). We have used DE…

batch effect deseq2 limma

updated 5.6 years ago • i.sudbery

colData = colData, design = ~ Type+ batch) dds<- DESeq(dds) I understand that even though I included "batch" in the formula, but I still see a batch effect. I read this...Why after VST are there still batches in the PCA plot? The transformations implemented in DESeq2, vst and rlog , compute a variance stabilizing transformation...is possible to vi…

deseq2 batch removebatch effect

updated 3.1 years ago • Bine

div class="preformatted">Hi, I have a miRNA-Seq dataset with clear batch effect that I'm trying to correct for in order to run differential expression using voom/limma. Based on the Limma User...Guide, this can be done by adding batch as a confounding factor to the design matrix used by voom and limma fit. However, when I run an MDS plot on batch using the...voom results, it's still showing a…

Normalization limma Normalization limma

updated 9.9 years ago • Heather Estrella

is: paired samples before and after treatment, and I would also like to take into account batch effect. I am having problems with the batch effect, by reading through mails in this mailing list, I've gotten...to do: Create example date, patient (5 patients, 2 samples), treatment (A or B), and batch (three batches, 1-3) <pre> >…

limma

updated 8.5 years ago • John Burger

I have 130 individuals with their RNAseq data in 7 batches treated in a Case-Control study (Case=37 and Control=93). I would like to run differential gene expression analysis...18258 genes for this study in Limma. When I read some discussions about removing batch effect in the model through Bioconductor's forum, I found that the...best way to remove batch effect is to consider individuals as rand…

batch effect Coeficents non estimable block random effects limma design matrix

updated 6.1 years ago • mheydarpour

reps, so 54 total samples. I have a co-variate of number of mutations and a date of experiment of batch effect. The size of the gene expression matrix = 5000 genes by 54 samples. If I run Limma lmFit eBayes like so;  design...treatment",Inf)\[,5\]<=0.1) \# gives 409 genes at 10% FDR (seems good).  \# extracting the batch effect removed data like so, using my b…

Limma removebatcheffect() duplicatecorrelation

updated 7.2 years ago • John

pre> Unfortunately, as anyone can see from the pData object, my dataset consists of two different batches: that is, each substance and the control is comprized of two biological replicates-and each batch is compized of 5 CEL...also together in a different time). Thus, my main consern is if and how could i use from limma the information of the batch that each CEL belongs(normalized2$replicate)…

limma batcheffect batcheffectcorrection multiple factor design

updated 8.6 years ago • svlachavas

I want to remove a known batch effect with limma using blocking. My experiment has 36 samples from 3 donors with 12 different treatments. The batch...I want to remove a known batch effect with limma using blocking. My experiment has 36 samples from 3 donors with 12 different treatments. The batch effect is based on the different donors. I would like to remove batch effect and later compare diff…

limma batch effect

updated 8.4 years ago • Jeannette

Dear BioC community, I am analyzing microarray data from three different batches. There are many technical and biological replicates in the batches. In the past, when I had only two batches, I was using...removeBatchEffect function from limma, giving the batch of each sample as argument (with batch=...). I am not sure if this function works with more than two batches...I am alerted because the u…

microarray limma removebatcheffect()

updated 7.5 years ago • Eleni Christodoulou

from the inital visit (visit 0, batch "baseline") were sequenced, some had to be re-sequenced due to QC issues (batch "baseline_reseq") and finally the follow-up...samples from visits 1, 2, 3 came in and were sequenced in the third batch. Here is an example of the meta data matrix: | patient| visit | batch| | -------------| -----------| ----------| | s1 |0 |baseline | | s2 |0 …

duplicateCorrelation limma BatchEffect

updated 2.8 years ago • Julia

Hi All, I have a dataset which contains two batches. I wanted to remove that batch effect and further analyze the data (to perform differential analysis and clustering...Hi All, I have a dataset which contains two batches. I wanted to remove that batch effect and further analyze the data (to perform differential analysis and clustering). I used two approaches: 1. giving the batch and the biolo…

limma differential gene expression clustering removebatcheffect

updated 6.2 years ago • lirongrossmann

batch <- factor(rep(c("A","B"), each = 3)) rld <- rlog(dds) mod <- model.matrix(~ condition, colData(rld)) assay(rld) <- limma::removeBatchEffect...assay(rld), batch = rld$batch, design = mod) ``` Would it be correct to simply inverse the log2 transformation to get the desired matrix? ```r normcounts...lt;- counts(dds, normalized = TRUE) # normalized c…

DESeq2 limma

updated 2.3 years ago • jma1991

Hi my understanding is that we should not use corrected counts with something like edgeR or limma? **However, because ComBat_seq is correcting at the raw count level** and then input into edgeR for TMM follow with voom...Hi my understanding is that we should not use corrected counts with something like edgeR or limma? **However, because ComBat_seq is correcting at the raw count level** …

sva limma

updated 2.3 years ago • Ahdee

steps in the laboratory (ChIP-experiment). My first general question is how do you deal with batch effects? I found not much about it in the archive. I proceeded as follows: I used limma for computing oligos with differential...intensities between two classes. Adding a factor for batch effects is easy and reduces the R^2 of the gene-wise models in my case noticeably. I am more worried about t…

Normalization Regression vsn limma oligo Normalization Regression vsn limma oligo

updated 16.2 years ago • Hans-Ulrich Klein

I have a clear batch effect that's caused by sequencing paired-end vs. single-end on different days. I'd like to correct for this in the DESeq2...analysis as suggested in the vignette ("If there is unwanted variation present in the data (e.g. batch effects) it is always recommend to correct for this"). I added the sequencer batch effect to the design but only see a very...blind = FALSE) plotP…

deseq2 limma RUVSeq

updated 3.5 years ago • 94133

Hi, I am doing RNA-seq analysis using limma and edgeR, I have 48 samples and 2 batches. I built MDS plots (see below), and now my question is do I have a batch effect I need...to correct for or not? My understanding is if batches cluster together on MDS/PCA plot, it's an evidence that batch effects are present, am I right? <img alt="" src="https://imgur.com

limma rna-seq edger batches

updated 4.6 years ago • chipolino

are not correct, please correct it so I could understand better : Code1: library(limma) mod <- model.matrix(~sex) #model could be explained by sex svafit <- sva(geneExpression,mod) # we suspect some batch effect...variable pvals=2*(1-pt(abs(tt),lmfit$df.residual[1]) Code2 library(limma) mod <- model.…

limma BatchEffect sva

updated 10 months ago • Lenny186

Hi deseq2 is good considering batches and I can see and remove them with a command assay(vsd) <- limma::removeBatchEffect(assay(vsd), vsd$Batch) and see with plot

TargetScoreData batchelor

updated 16 months ago • Syed Zaheer

Hi I am trying to batch correct for samples from 4 different experiments that I ran in one fluidigm plate. I tried normalization with liima...using ``` norm_exprs <- limma::normalizeBetweenArrays(exprs, method="quantile") ``` followed by ``` batch <- factor(data$Experiment) # Perform ComBat batch...modcombat <- model.matrix(~ Group, data=data) combat_data <- sva::Com…

sva limma

updated 4 months ago • Khushbu

cluster into different groups under a certain gene signature, the samples are from two different batches so batch needs to be factored. Say I want to take a quarter of my samples that respond best to some treatment and the then...one for for the first cluster and one for the other cluster and run DEG analysis as outlined in the Limma Manual *https://www.bioconductor.org/packages/devel/bioc/vign…

limma voom

updated 19 months ago • jmannhei

done at different times, not just the library prep/sequencing). As a result, there is a significant batch effect, clearly visible in an MDS plot of edgeR-calculated logCPM values, and inter-batch contrasts always have many...more differentially expressed genes than intra-batch contrasts, regardless of whether I use edgeR LRT, edgeR QLFT, or limma voom. So, normally I would just say that there's …

Normalization GO limma edgeR Normalization GO limma edgeR

updated 10.4 years ago • Ryan C. Thompson

Hello all, I have a question regarding batch effect correction with LIMMA. I have some proteomics data, and when I do my PCA plot I see that samples cluster by patient...each sample with a different treatment). For this reason I would like to include patient as a batch effect in my linear model (rather than use removebatcheffects because I then want to perform differential analysis...with LIMM…

limma batch effects linear model

updated 3.8 years ago • sandra.murphy

I'm trying to adjust batch effect using `deseq2 limma::removeBatchEffect` and also `Combat-Seq`. With limma version, I can clearly see the batch effect...is removed, where I see control from Batch1 is together with the other 3 controls from Batch2. ```r ###### Batch Correction with limma removeBatchEffect ####### dds <- DESeqDataSetFromMatrix(countData = data, …

DESeq2 combatseq limma BatchEffect tpm

updated 7 months ago • vk

0300 > From: John Burger <clinicalgenomics at="" gmail.com=""> > Subject: \[BioC\] Limma design: paired samples, batch effect > To: bioconductor at stat.math.ethz.ch > Content-Type: text/plain > > Hello...paired samples before and after treatment, and I would also like to take > into account batch effect.…

limma

updated 8.5 years ago • Gordon Smyth

days 1, 4, 8, 15 and 29) in two treatments: drug and placebo. The data were collected in several batches. We know age and sex of each patient. Initially I run *limma* using a model ``` ~ treatment + day + batch + age_group + sex ``` It shows very...strong batch effects. The figure below shows the numbers of statistically significant proteins (going up - blue, and down - orange) at...mo…

limma Regression overfitting

updated 19 months ago • M.Gierlinski

Hi Michael, I'm trying to remove the batch effect from my samples doing like this: APPOGGIO: <pre> Well Barcode day bioRep RAcond TNx techRep ATTACT B4 ATTACT D5...pre> vsd <- vst(ddsIO) plotPCA(vsd, "TNx")</pre> This plot is showing me samples without the batch effetc removing... Only if I use limma::removeBatchEffect (which is actually another funt…

deseq2

updated 5.2 years ago • santamariagianluca

I'm trying to adjust batch effect using `deseq2 limma::removeBatchEffect` like below: ```r ###### Batch Correction with limma removeBatchEffect ####### dds <- DESeqDataSetFromMatrix...design = ~ Samplebatch + cond) dds <- DESeq(dds) dds dds$batch <- as.numeric(dds$Samplebatch) dds$cond dds$batch ## vst after adding batch information vsd_abc <- vst(…

DESeq2 BatchEffect tpm fpkm

updated 7 months ago • vk

Dear Bioconductor Community, based on the very interesting question on a previous post (__https://support.bioconductor.org/p/72815/__) regarding the possible batch correction methodologies, through the answers created i desided to adress a very important issue in my opinion-as im...question on a previous post (__https://support.bioconductor.org/p/72815/__) regarding the possible batch correction…

limma batch effect ComBat differential gene expression

updated 8.2 years ago • svlachavas

differentialy expressed gene using the **plotCounts** function. When i plot them i can still see the batch effect in the image generated.I then refereed to your post https://support.bioconductor.org/p/76099/. There it is mentioned...that it will model the batch effect in linear regression not remove them also in the following threads it is mentioned that we can use limma package...removebatcheffe…

deseq2 normalization

updated 3.6 years ago • dhwani.dholakia

div class="preformatted">Dear Gordon Smyth, bioconductor list, In the limma package we found a function called, removeBatchEffect, which removes the effects of batch effects or other technical...on a gene expression matrix. The code of this removeBatchEffect is as follows: function (x, batch, batch2 = NULL, design = matrix(1, ncol(x), 1)) { x <- as.matrix(x) batch <- …

limma limma

updated 7.4 years ago • Djie Tjwan Thung

generalizing. I have 36 iPSC, 9 primary CM, and 18 biopsy samples. My quality control plots showed batch effects in the data and I added 3 batches to the design matrix, using, e.g. this: https://support.bioconductor.org/p/69328...adding "batches" explicitly is introducing too many variables and there is no way to explicitly include batch effects? 2) In this case...does the regression on the sa…

limma batch effect

updated 7.7 years ago • smt8n

Dear all, I have a bulk-rna dataset which I have analyzed by DESEQ2 and I have batch corrected it using limma package and removeBatchEffect() function. I would like to calculate the FPM value for expression...deseq2. I know it will use the raw data to calculate these value. I have created a heatmap from my batch corrected data for some genew and I wanted to also plot the fpm value for these gen…

deseq2 limma

updated 4.2 years ago • Emir

Hello, I have some RNA-Seq samples. These samples have no replicates, but I have lots of samples. What I am trying to do is find a trend based on a continuous variable (let's say age). I am basically using edgeR's cpm function with log as TRUE and prior count of 3. The first question is about Normalization. Is there a better way? Should I try the following? Find genes with the lowest var…

limma edgeR rna

updated 2.7 years ago • Nirad

I have used `` limma `` trend to find DE genes between 3 groups (including batch effect in design). Now I would like to visualize the genes, and used...to get corrected logCPM values for each sample. `` logCPMc <- removeBatchEffect(logCPM, batch=targets$Batch, design=design_4_batch) `` Now I want to use the average of each group, and with `` aveLogCPM() `` from `` edgeR ``, I don't...see…

edger limma cpm

updated 5.2 years ago • b.nota

and the treatments. The replicates were collected at different times, so I am assmuing there is a batch effect. I have two questions here - 1. To build the DESeq object, I am using the ~Batch + treatment + genotype + treatment:genotype...model. DESeq2 automatically converts the treatment and genotype to factor variables, but not the batch effect. So, how would it matter if Batch was considere…

BatchEffect batch RNASeqR formula DESeq2

updated 15 months ago • estafana.t98

I have a quick question concerning the removal of the batch effect using removeBatchEffect()…   I have RNA-seq data and i have three batches. I normalize using spike in RNA and i...end i get a matrix which i log2 transform. When i do PCA using this data matrix i have a profound batch effect.   I used the removeBatchEffect function on this log2 matrix. I get a correction for …

limma rnaseq batch effect pearsoncorrelation

updated 7.8 years ago • elpsakkas

Dear all, I am having a problem with removing/accounting for the batch effect in my RNAseq experiment using DESeq2. Initially we did 1 big time-course experiment in one batch of human cells...a similar experimental design with 3 other fungal species) I realized that 3D samples have a strong batch effect (I have seen this by removing the batch effect using `limma`). For the case above, wh…

deseq2 batch_effect

updated 4.7 years ago • gtechbio

I'm currently analyzing 4 microarray experiments in an integrated manner. For this, I'm using limma accounting for the batches through the design matrix: <pre> design <- model.matrix(~0+Class+Batch, data = phenot)</pre> Since...removing the batch effects, for example, with Combat is not advisable before DEG analysis through limma. Can I correct the batch effects...combined d…

batch effect microarray plot

updated 6.4 years ago • tcalvo

div class="preformatted"> Dear list: A batch effect was observed in 16 out of 105 hgU133_plus2 arrays based on NUSE plot. I know SAM, like limma, is able to handle this...that is appropriate way for this unintentional situation. I would try to use the lmFit function of limma with batch as a factor to remove the batch effect. Then use the resulting data values for other software applications

limma limma

updated 17.1 years ago • Jianping Jin