Bioconductor Forum

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 9.7 years ago • elpsakkas

I am trying to perform differential RNA-Seq analysis from plasmodium species. I am using R command lines and I was trying to include the estimates of cell stages obtained from CIBERSORT deconvolution alongside batch effect estimate using RUVr. I managed to do the differential analysis with the code below just by including the batch effect estimate but I found a difficulty including the proportio…

DeconRNASeq limma

updated 16 months ago • ij283

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 8.3 years ago • tcalvo

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 6.6 years ago • gtechbio

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 19.0 years ago • Jianping Jin

in this image: ![enter image description here][1] I was wondering using the formula ```design = ~ batch + condition``` would be sufficient enough to control for batch effect considering the fact that the control group is not...represented in the other batch. How should I proceed with further analysis? Surprisingly, Limma was able to somewhat correct the data to cluster broadly

DESeq2 BatchEffect RNASeq

updated 19 months ago • sap275

Due to experimental limitations, the samples have been treated in the same way but in 15 different batches. Each batch includes 8-40 different donors each, and 2 donors are present in every batch (donor A and B), while others are...present in a variety of batches (some in 2, some in 3 or 4). So the majority of donors are present only in one batch, and there is not a clear baseline. Each donor...h…

limma RNASeq DifferentialExpression

updated 20 months ago • mperalc

the removeBatchEffect() function so that it will now accept continuous covariates as well as batch factors. Just enter BSCE as the 'covariates' argument. The updated function will be on the devel version of limma, and I have...gt; > function (x, batch, batch2 = NULL, design = matrix(1, ncol(x), 1)) { > x <- as.matrix(x) > batch <- as.factor(batch) &g…

limma

updated 5.0 years ago • Gordon Smyth

A.22 - A.0) ? This design is completely described in the paragraph “9.7 multi-level experiment” of limma user’s guide. My problem is that the samples were processed in batches before I was in charge of this study, and I could just...t2 p11 B6 23 A t1 p12 B6 24 A t2 p12 B6</pre>   The last batches B5 and B6 have the same proportion B/A, here : …

limma batch effect

updated 9.9 years ago • eleonoregravier

Dear Bioconductor community, I encountered a difficult problem in correcting for batch effect in repeated measurement data, which has troubled me for a long time. This is not an experimental design usually...by years elapsed between 2 visits) to assess this 'variability'. However, there seems to be some batch effect as shown in the PCA plot below: ![PCA plot for vst normalized counts][1] …

miRNA BatchEffect RNASeq limma

updated 24 months ago • Wending

class="preformatted">Dear list, I got 12 Affymetrix arrays for 4 RNA samples, 3 replicates(from 3 batches individually) for each sample. I want to look at the differential expression between these samples. While after clustering...we found obvious batch effect within the data, so we decided to add the batch effect into the linear model. I set up the design matrix as followings...is any proble…

Clustering Clustering

updated 16.1 years ago • Yuan Hao

Hi all, I've used RemoveBatchEffect function from limma package for my previous datasets but it's limited to two batches only. I'm working with a data-sets that contains more than...two batches. How can I do? Many thanks, Yi

limma RNAseq removebatcheffect()

updated 9.3 years ago • yi.huang

surfaces: water, glass, PET and PE I have 5 locations: A, B, C, D and E I also seem to have a batch effect as locations B&D was ran on the sequencer a different day than A,C,E. I was trying to correct for this by designing...that incorporated the location as an effect and then duplicate correlation to compensate for the batch effect. See code below: ```group<-factor…

duplicatecorrelation BatchEffect limma corfit

updated 4.9 years ago • Katherine

DESeq2 manual: ```r mat <- assay(vsd) mm <- model.matrix(~condition, colData(vsd)) mat <- limma::removeBatchEffect(mat, batch=vsd$batch, design=mm) assay(vsd) <- mat plotPCA(vsd) ``` - Did I define the mm right? (The condition...associated with it (my condition control vs patients)? - Also, here in mat how can I define the batch=vsd$ ```r mat <- limma::re…

DESeq2 BatchEffect limma

updated 2.2 years ago • Sara

Hi, I have read blogs on **how to remove batch effects in deseq2** and how to **visualize** it using **limma voom remove batch effect PCA**. I'm not sure if the batch effect was...attached output, I'm skeptical because in heat map they seem to cluster separately (with respect to batches). Analysis: 51 samples with two batches (Batch1 includes NYGC in the label and Batch2 doesn't include NYGC in…

BatchEffect DESeq2

updated 4.5 years ago • vinisha

We have applied batch corrected on mouse data using MNNCorrect and scMerge. The corrected matrix included non-integer and negative values...Assuming that the batch-corrected data are nonintegers and include negative values, how to apply limma-trend or voom to batch corrected single

MNNCorrect scMerge limma-trend

updated 4.5 years ago • hainct

be statistically legitimate in order to account/correct for the batch effect : **1 -- use CCA (in seurat) or MNNcorrect (in scran) to account for the batch effects** **2 -- followed by TSNE and network_based_clustering...the cells into CLUSTERS** **3 -- and perform differential expression (with wilcoxon test, limma, edgeR, etc) between the CLUSTERS** We know that CCA or MNNcorrect onl…

limma deseq2 edger scRNAseq combat

updated 6.2 years ago • Bogdan

Hello everyone, I was wondering if there is a way to remove batch effect from an expression matrix (raw counts) for downstream analysis (like pca, clustering, machine learning) using...Hello everyone, I was wondering if there is a way to remove batch effect from an expression matrix (raw counts) for downstream analysis (like pca, clustering, machine learning) using the...Deseq2 package.&nbs…

deseq2 batch effect removebatcheffect() limma

updated 7.9 years ago • lirongrossmann

I have RNAseq from three treatments (A, B, C), each with biological replicates ran in two batches (batch1 with A & B; batch2 with C). Unfortunately, the batch effects are fully confounded with the condition (e.g., to compare...A vs. C). Here, I understand it is impossible to separate the batch effects from the treatment, regardless of what statistical method I use. I thought of methods su…

batch effects limma batch-correction RNAseq RUVseq

updated 5.5 years ago • bhakti.dwivedi

http://www.biostat.jhsph.edu/~jleek/papers/framework.pdf and for Combat, an approach for removing batch effects when the source of batch is known as described in the paper: http://biostatistics.oxfordjournals.org/content...A full description of how to use the methods, including how to use the sva package with limma, removing batch effects with linear models, biological versus technical batch ef…

Regression limma sva Regression limma sva

updated 13.9 years ago • Jeff Leek

I realize that the topic of batch effect removal in RNA-seq has been addressed many times. I am not sure if this particular aspect of it was, but I may have...Specifically, there is a note: > If there is unwanted variation present in the data (e.g. batch > effects) it is always recommend to correct for this, which can be > accommodated in DESeq2 by including in the desig…

BatchEffect DESeq2 RNASeq

updated 4.9 years ago • igor

condition and 3 time points for each condition as follow: FileName Targets Time Treatment Batch F21.CEL I18 18 I 3 F22.CEL I18 18 I 3 F25.CEL N18 18 N 3 F26.CEL N18 18 N 3 F23.CEL R18 18 R 3 F24.CEL R18 18 R 3 F19.CEL S18 18...biological replicates not technical. Note that some of the rep…

updated 19.7 years ago • Ron Ophir

Hi all : - When I use limma to identify differentrial expression genes , I want to correct batch effect also. - My command and data information is...Hi all : - When I use limma to identify differentrial expression genes , I want to correct batch effect also. - My command and data information is : ``` > table(cluster) cluster control treat 368 957 > ta…

limma batchcorrect warnings

updated 6.6 years ago • xingxd16

4 time points (1, 2, 3, and 4). For unavoidable reasons, samples were prepped in several different batches; however, all samples from each patient (all time points) are in the same batch (i.e. no patient has samples in more than...one batch). The following is the format of the sample metadata: <pre> PatientID SampleID Time Disease Batch p1 p1-1 1 A …

limma rnaseq batcheffect

updated 10.4 years ago • cfreije

Lesional and Non-Lesional are taken from the same patients. Most of the Controls were processed in batch#2. To batch-correct, two technical replicates of lesional samples and two technical replicates of non-lesional samples...were also included in batch #2. All samples have **Patient ID** and **Sample ID**, like so: | Sample ID | Sample Type | Patient ID | Batch | |----------------|---------…

RUVSeq sva limma

updated 2.1 years ago • Jonathan

that in a linear model, gene expression is given by the sum of modelled variables (i.e. biological/batch factors) you have to take into account. So, in the simplest scenario: gene expression ~ factor_1 + batch Now, let's say I have...the following condition (where batches **I, II and III** are different days of the year): factor_1 batch …

deseq2 batch effect batch

updated 6.4 years ago • Mozart

Hello,   I have a question about a differential expression analysis that I am doing using Limma on RNA-seq data. Basically, I want find differentially expressed genes between tumour and normal samples (in men and...Hello,   I have a question about a differential expression analysis that I am doing using Limma on RNA-seq data. Basically, I want find differentially e…

Limma differential gene expression batch effect correction covariates

updated 7.4 years ago • abelsousa

Hi, I wonder if I can regress out batch effects from my single-cell data using following strategy. Though the tutorial suggested using limma to remove batch...countData = sub_count_matrix, colData = cell_metadata, design = ~ Batch + Condition ) # size factor was pre-estimated using scran sizeFactors(dds) <- size_factors dds <- DESeq( …

DESeq2

updated 18 months ago • kys91240

donor: design <- model.matrix(~Donor+Dose*Time) Usually I would prefer to correct for the batch effect in the design matrix, rather than pre-correcting with ComBat, because it ensures that the degrees of freedom...that fact that batch correction the has been done. Best wishes Gordon > Date: Fri, 22 Apr 2011 13:49:59 -0400 > From: somnath bandyopadhyay <genome1976...…

Microarray limma DOSE Microarray limma DOSE

updated 14.5 years ago • Gordon Smyth

this:  Older data - 4 separate conditions (control, negative, intermediate, positive), 5 batches Newer data - 1 completely different condition (diffuse), 1 batch We know there are batch effects in the older data and...batch of the newer data. The approach I've tried takes the old data, utilizes removeBatchEffects from the limma package, forces...to the controls within the older data d…

rnaseq limma sva batch effect voom

updated 9.0 years ago • schrist1

<div class="preformatted">Hi All, I am new to limma and R programming. I have the CEL.files uploaded, normalised as an expression set, and have put together the target.txt file containing the following layout: Name FileName Target Date 5 0207_DaB7_H_ea_Blast1.CEL blastocyst 13/02/2007 6 0207_DaB8_H_ea_Blast2.CEL blastocyst 13/02/2007 4 …

limma limma

updated 12.1 years ago • Helen Smith

I had to perform WGCNA. Initial PCA and hierarchical clustering of samples showed the existence of batch (expected) and was removed using `limma::removeBatchEffect()` for DESeq2 normalization. On performing WGCNA, I obtained...that this might be due to presence of strong driver of variation ([ref1][1]). As the known source of batch was correctly removed, I wanted to try on RUVseq or SVA to remove…

limma deseq2 wgcna RUVseq

updated 5.9 years ago • ag1805x

Hi, I am trying to determine whether batch correction is necessary before performing GSVA. I have a bulk RNA-seq dataset that includes two batches due to library...preparation performed on separate occasions. My conditions of interest are present in both batches. For PCA, I applied batch correction using the `removeBatchEffect` function from limma. Should I use this log-transformed...normal…

GSVA GSVAdata RNAseq

updated 15 months ago • Lucy

nbsp;h.dor.acychow.pac    2 There was ome difference in the genes expressed between batch 1 and 2, so I merged the raw count matrices in R and changed an NAs to zeros. When I ran Limma-Voom with weights and without...that the glitch in the first graph is due to batch effects. I then tried batch adjustment in Limma, using batch as a covariate. MDSplot\_by…

limma voom batcheffect covariate

updated 8.5 years ago • raf4

div class="preformatted">Hello, I've a question regarding the removeBatchEffect function in limma. I basically have a model expression_signal ~ organ + species + organ:species in which species can be considered a batch...does it make sense at all to consider an interaction as an experimental factor for which one part is "batch" and the other isn't? thanks for your thoughts, Arne …

limma limma

updated 12.2 years ago • arne.mueller@novartis.com

sva run on it and have two surrogate variables (which are continuous variables ofcourse) for hidden batch effects in my RNAseq data. Now I am aware of combatseq package and limmas removebatcheffect() function but they don't take...continuous variables. What are my options or probably I am missing something regarding continuous batch variables(in this case surrogate variables from sva). Please he…

combatseq RNASeqData sva limma

updated 2.3 years ago • amnahsiddiqa

Hi there: I have hundreds of samples with reads counted on genebody or 2kb sliding windows. I'd like to use these matrix to build binary classification and prediction models using elasti net. I plan to do the following things: First, do normalization (logCPM) in total samples to check wether batch exists then split the matrix into training set and test set. Second, use limma to find…

limma

updated 4.2 years ago • hxlei613

<div class="preformatted">Dear List, I do know that this question has been approached before, but am not sure about the reliability of the list of genes. I have data from an experiment that was run in two batches (3 groups per batch, 4 samples per group, 2 array-chips per bacth), these need to be combined and analysed. Thus there are 6 groups, and I know from the QC that there is a stron…

limma limma

updated 14.5 years ago • Natasha

vsd <- varianceStabilizingTransformation(dds, blind = FALSE) Although I tried to include the batch (Institution) in the design argument, I still see batches in my PCA plot. [This section][1] of the vignette described a solution...for this that I have pasted below: mat <- assay(vsd) mat <- limma::removeBatchEffect(mat, vsd$batch) assay(vsd) <- mat …

deseq2 vst

updated 5.2 years ago • le2336

Hello, I am trying to remove a 3 known batch effects from my data, so I can use it to perform some clustering. In total I have around 150 patients. 2 of the batch effects...male & female) and Technician(A,B &C)) and one is continuous (Age). Usually, when using limma::removeBatchEffect, I create a model matrix that includes all my batch effects like this and then pass this t…

limma BatchEffect

updated 3.4 years ago • nhaus

browser showed no sign of excessive noise (see picture for example). However, samples from second batch were sequenced much deeper (bottom, ~25 million reads) than ones from first batch (top, ~7 million reads). <img alt="IGB view" src...IGB.png" style="height:375px; width:700px"/>   In order to check for consistency between batches, I generated an MDS plot from normalized cpm co…

edger differential gene expression

updated 7.1 years ago • rfenouil

Hi! I'm new to RNAseq analysis and had a few questions in regards to batch effects. 1) I have RNAseq data for two conditions obtained from two different batches that I am trying to analyse. I have...included batch as a covariate while creating the DESeq object, but I am not sure if I am supposed to remove batch effects using limma, etc...for it in the DEseq object is enough. 2) When doi…

DESeq2

updated 8 months ago • A

experimental samples are much more heterogenous, as seen from the PCA plot (created after removing batch effect with ComBat). This is expected since the experimental samples are a result of a more stochastic process and therefore...bAAewAu.jpg" style="height:201px; width:200px"/> For differential expression analysis in limma between the groups, I account for the batch in the design matrix, ho…

limma removebatcheffect() ComBat microarray

updated 7.7 years ago • jaro.slamecka

I also have a patient variables matrix with the columns including sample name, disease subtype, batch, age & sex. I wish to adjust for batch (A-E), as well as sex and age. I read the Limma user guide and searched the Bioconductor...Is it possible to extract a fitted expression matrix from the lmfit function in limma (post batch adjustment) which can be used for PCA? Or is the only poss…

limma

updated 2.4 years ago • sarah.kelliher

need to be put together. I have learned that the best way to accomodate for this is including it as batch effect in the design formula of the DE packages (e.g. DESeq, edgeR). However, we would still need to batch correct the counts...for additional analyses (e.g. PCA, clustering), and we were thinking on using limma::removeBatchEffect. Here comes the question: on which data can we use this fun…

batch limma

updated 2.7 years ago • luca.s

hybridised about half of the samples when he realised he needed more Affymetrix chips. The second batch of chips arrived with the instruction to add DMSO in the hybridisation cocktail, which he followed. The first batch did...not have such instruction. Therefore we believe that the two batches are not directly comparable. A posting to GeneArray mailing list had a reply (http://bfx.kribb.re.kr/ge…

updated 20.9 years ago • Adaikalavan Ramasamy

Identify housekeeping genes. 3. Pre-normalization visualization via RLE plot and PCA plot. I found batch effects in the expression set. 4. I used `RUVSeq`, `DESeq2` and `limma` packages to remove batch effect and normalize the data...5. I used dataset in `vsd.after` to draw RLE and PCA plot again to check if there were still batch effects. It showed the batch effects were removed. According …

GSVA Normalization BatchEffect GSVAdata NanoString

updated 2.8 years ago • Zhijie

I have a performed a typical differential expression analysis using limma voom and I want to extract the log cpm values to draw ROC curves. But I want to extract the log cpm values produced specifically...by limma because they are normalised and free of batch effect that was added to the design matrix. Is there a function to do that

limma edgeR RNASeq

updated 18 months ago • Shaimaa Gamal

can be done by including the donor ID in the model matrix. The samples had to be processed in 3 batches (no known variables are confounded with batch).  My question: What is the best way to incorporate the pairwise analysis...and account for batch effects? Include both in the model matrix? Does the order matter? I don't think I can remove the batch effects with Limma

deseq2

updated 8.6 years ago • jogiles

Dear Community, I would like to ask for a rather challenging scenario of putative batch effect correction for differential expression analysis. Very briefly, I have pre-processed (TMM normalization, log2CPM...pre-process similarly. Thus my crucial questions are the following: 1) Is possible to perform any batch effect correction approach ? like removeBatchEffect from limma or even ComBat ? …

batch effect limma rna seq DE

updated 5.5 years ago • svlachavas

div class="preformatted">Hi, I was exploring batch effects in my data. At this stage, I don't want to introduce the 'variable of interest' (i.e. 'cancer' in the vignette/example...function (and get combat_edata2)? Will this correctly reflect the expression data corrected for batch effects? ###### Code ##### library(sva) library(bladderbatch) data(bladderdata) library(pamr) library(limma) p…

sva sva

updated 13.5 years ago • Tim Smith

Microarray Database (SMD) data. Problem : A single study in SMD often utilises different print batches - sometimes with twice as many unique features in one batch than other batches. This is compounded by the fact there...are multiple copies for some genes. Current solution : I am aware that marray has read.SMD(), limma has read.maimagenes( ..., source="smd"), merge.MAList() and someone else pr…

Microarray limma marray Microarray limma marray

updated 19.6 years ago • Adaikalavan Ramasamy

<div class="preformatted">Hello everyone I was wondering if anyone has used a tool to adjust for batch effects. I am comparing two experiments that were done the same way over two years. eg. Population 1 Treatment Control Year 1 Population 1 Treatment Control Year 2. The same experiment, just repeated in different years. I normalise all the cel files together (from both years) usin…

limma limma

updated 16.8 years ago • Sherosha Raj

div class="preformatted"> I have carefully read the Limma users guide but still have not sorted out how I design the contrasts for the following Project. In want to compare the...for subject. However, in addition, I would like to block for center. I.e. the center is like a batch effect. Is it possible to block for two factors, suject and center, in the same test in Limma? /Ingrid -- output…

limma limma

updated 12.4 years ago • Guest User

HI everyone, I'm running DESeq2 to identify genes that are differentially expressed between two general phenotypes: fruits and vegetables. Here's my design: ```r general specific batch =============================================== 1 fruit apple apple 2 fruit apple apple 3 fruit pear pear…

DESeq2 deseq2

updated 4.3 years ago • Ania

Good morning, I am currently trying to analyse a small-RNA sequencing dataset using limma package and the voomLmFit function. However, I am experiencing some issues with the p-value distribution, with some...Good morning, I am currently trying to analyse a small-RNA sequencing dataset using limma package and the voomLmFit function. However, I am experiencing some issues with the p-value distrib…

BatchEffect limma

updated 2.3 years ago • Eleonora

potentially in a different manner (for example single end vs paired end). I am trying to model the batch effect in DESeq2 in order to account for effects by batch but im running in to trouble and am unsure why: I have two datasets...specified. As far as i am aware these are not the same as batch is simply batch 1 and batch 2, while the groups has multipls groups, control, trt 1, trt2, trt3…

deseq2 batchcorrection

updated 6.1 years ago • A

For a dataset I'm working on, we would like to use the limma package for differential expression analysis. We are using `limma::removeBatchEffect` (doing it for downstream analysis...before using limma for differential expression analysis. Would it be okay to still include the batch covariate in the model to account

limma batch effect differential expression

updated 6.6 years ago • jmoon1194

I don't know if I'm doing it right. I've two factors 'batch' and 'time' listed below: > batch [1] NEW NEW NEW OLD OLD OLD OLD PRG PRG PRG NEW NEW NEW OLD OLD OLD OLD PRG PRG [20] PRG Levels: NEW...over time but within each batch. Therefore I'm constructing the following model matrix (time nested within batch, i.e. 04h and 24h at each level of batch...design.time <- model.matrix( …

probe affy limma probe affy limma

updated 21.6 years ago • Arne.Muller@aventis.com

Hi I have a quick question about a paired samples and batch effect correction. Especially I am seeking a solution for edgeR or limma/voom. A few days ago, I fount the following post...https://support.bioconductor.org/p/92753/ In his case, subject and batch are identical, so "There is no need to do any batch correction. The baseline differences between subjects, including any...batch effect, is …

edgeR paired samples batch effect

updated 7.0 years ago • NGS-seeker