Bioconductor Forum

I am comparing three groups A, B and C and they are in 3 batches of library preparation. In the | Sample Name | Condition | Batch | |-----------|-------|------| | 1 | A | 1 | | 2 | A | 1 | | 3 | B | 1 | | 4 | B | 1 | | 5 | A | 2 | | 6 | A | 2 | | 7 | B | 2 | | 8 | B | 2 | | 9 | C | 3 | | 10 | C | 3 | | 11 | B | 3 | | 12 | B | 3 | Is the group...C being in only batch 3 confound…

DESeq2

updated 19 months ago • prabath_meemaduma

FLOATING LINK: https://support.bioconductor.org/p/62954/>) I resolved to try to do this using  limma batchEffectCorrection() on vst(dds). Once I plot the output as a boxplot, it does not look as distribution close to a p.value...I see resembles the p.value obtained by Deseq analysis? Here it's what I have done: <pre> library(limma) library(DESeq2) library(ggplot2) genes=r…

deseq2 limma differential expression visualization

updated 7.6 years ago • Mbosio85

species and is split across multiple library preparation batches, and two sequencing batches; individual replicates are at the truly biological level and represent different runs...of the same experimental conditions. The first sequencing batch contains multiple library preps, one of which (5) is confounded with individual. The second sequencing batch contains...only one library prep batch across…

limma sva batch effect

updated 9.1 years ago • irenegallego

I have a dataset with 10 condition vs 20 control samples and am using limma to test for differential expression. Broadly, groups are age/sex matched but have added noise due to complex medical...which are matched as best as possible but is still far from perfect. Ran through a basic analysis limma pipeline, everything worked as expected. In the same batch, I processed a number of other sample…

limma multtest

updated 12 months ago • Ali Barry

I wanted to run SC3 on normalized (scran/scater) logcounts that were then sent through limma's removeBatchEffect using the follwing script: <pre> library(scater) library(scran) library(SC3) txi.bbsplit <- readRDS...sce <- sce[keep,] sce <- computeSumFactors(sce) sce <- normalize(sce) library(limma) logcounts.sce <- logcounts(sce) logcounts(…

SC3 batch effect removebatcheffect

updated 8.1 years ago • muad.abdelhay

phenotypes___) So, from an initial investigation of the above 2 plots, it does not seem any severe batch effect regarding the origin/study (Additional HCs=control samples, SLE=ILLUMINATE-1  & ILLUMINATE-2), which could...imply an severe correction. However, to be certain for any downstream statistical comparison with limma, i should just include the batch information in my linea…

hta2.0 limma batch effect affymetrix microarrays ComBat

updated 8.7 years ago • svlachavas

to assess the quality of the data and the impact of the batch effect removal using sample to sample distances, amongst other things (as adviced in DESeq2 vignette). The batch effect...them. However, after removing the batch effect, I noticed that the sample to sample distances are the same between conditions. Here's an example of distance...lt;- limma::removeBatchEffect(x = mat, batch = non_blind…

DESeq2 RNASeq BatchEffect limma

updated 4.6 years ago • athiebaut

and tumor samples but to get the two profiles comparable. I was thinking to use removeBathEffect of limma using Organoids and WT as a batch and correct for this know source of variation. The question is: is it possible to re-use

limma removebatcheffect

updated 9.1 years ago • Keifa

I want to use BMI as a continuous outcome variable. Not to divide in groups. To do this, I use limma. But I am not sure limma is suitable to directly use continuous variable as outcome based on no divide groups. If i cann...I want to use BMI as a continuous outcome variable. Not to divide in groups. To do this, I use limma. But I am not sure limma is suitable to directly use continuous varia…

limma DEGseq

updated 23 months ago • mia

the crlmmCopyNumber step in my R code. I've set up a Phenotype file where I list my files and their Batch groups. In my example case, group1 and group2 Name FileName Batch A1 array-123.CEL group1 A2 array-223.CEL group1 A3 array.214.CEL...gr2-pheno.txt" pd <- read.AnnotatedDataFrame(myPhenoFile, header=TRUE, row.names=1, as.is=TRUE) batch <- as.factor(p…

GO GO

updated 15.2 years ago • Ricardo Vidal

CD8T ,CD4T, NK, Bcell and Mono) and how I can filter significant association? > library(limma) #phenotype data > targets<-read.table("Sample.Info.regression.txt", header=TRUE, stringsAsFactors=FALSE) > targets...logeGFR=log(targets$eGFR) > head(targets) V1 Sentrix_ID Sentrix_Position Batch Category Gender 1 DC…

limma Epigenetics STATISTICS R

updated 3.6 years ago • nabiyogesh

p/68196/\#68309), i would like to ask you about the important issue of annotation of illumina in limma. As i have preprocessed and analyzed the data with limma, i searched ways to convert the probeIDs to nuIDs in order to have...with the lumi package__". As the function __addNuID2lumi() __is only working with lumi batch object, i also search the library __lumiHumanIDMap…

limma illumina human ht-12 v4 annotationdbi lumihumanAll.db lumihumanidmapping

updated 10.6 years ago • svlachavas

know the design is terrible, but this is what I have to work with ;) Question 1: can I remove the batch effect between 2 groups if their samples do not share a batch, but some of their samples share a batch with a third group...samples come from different batches (batch bX and bY), but no sample of group gA shares a batch with samples from group gC, so a batch effect removal would be...impossibl…

deseq2 batch

updated 6.3 years ago • Solarion

to find differentially methylated CpG sites. Both of the datasets are from EPIC array, but different batch(batch 0 and batch 1). I've already tried to correct the batch effect using [Harman batch correction][1] ```r shifted_betas &lt...cex.main=0.7) shifted_ms methHarman <- harman(shifted_ms, expt=targets_pr$Sample_Group, batch=targets_pr$batch, limit=…

BatchEffect limma methylationArrayAnalysis

updated 2.1 years ago • kyj2226

The first model doesn't set the intercept to anything, because it has no intercept. Yes, the batch effect will be adjusted for if you include it in the model, and this is true for the first model or the second model. Yes...RNA3 to the average of RNA1 and RNA2. As far as I can see, there is no difficulty or problem at all. limma is doing doing the right thing for you regardless of how you para…

limma limma

updated 12.9 years ago • Gordon Smyth

experiments with 2 repeats for each of them. I want to combine the results and thought to try the batch function. Currently, I'm doing per plate normalization and no variance adjustment. I would like to do batch variance adjust...a general explanation of variance adjustment, what it does and why? Additionally, when I add the batch number to the plate list and ask the program to do a variance adju…

Normalization Normalization

updated 16.2 years ago • Baranes-Bachar, Keren NIH/NCI [V]

Hi All, I did DESeq2 on my set of samples and I got only two DE genes. When I checked I found that my samples were collected at different time points and in PCA a prominent batch effect is shown. So, I included a batch in my design ddsHTSeq <- DESeqDataSetFromHTSeqCount(sampleTable = sampleTable, directory = directory, …

deseq2 svaseq

updated 7.0 years ago • bioinfo

I am working on RNA-Seq data. I'm using DESeq2 for my analysis. I have 20 samples from 3 batches. I am testing for 2 conditions, cond1 and cond2. dds<-DESeqDataSetFromMatrix(countData=countTable3,colData=coldata...design = ~cond1\*cond2) When i performed PCA, I could clearly see some batch effect. I read in the forum that adding batch to the design in DESeq removes the batch eff…

rnaseq batch effect deseq2 combat removebatcheffect()

updated 9.6 years ago • AB

Hello everyone, I'm trying to analyze DNA methylation data with the Limma package to identify potential differentially methylated CpGs between my conditions using M-values. I would like...Hello everyone, I'm trying to analyze DNA methylation data with the Limma package to identify potential differentially methylated CpGs between my conditions using M-values. I would like to...into account the p…

DNAMethylation limma methylationArrayAnalysis

updated 21 months ago • hortense96

and 4 bystander control replicates in 4 hour. Below, im posting the code of an MDS plot and a basic limma DE analysis of some contrasts: <table cellpadding="0" cellspacing="0" style="height:253px; width:666px"> <tbody> <tr> <td> <pre> > head...filtered.2$targets) Assay.Name Sample.and.Data.Relationship.Format time Batch 1 GSM795538 i…

limma illumina microarray multifactorial design batch effect

updated 9.6 years ago • Konstantinos Yeles

div class="preformatted">Hi Keren, I finally found some time to fix the batch issue in cellHTS2. Things should work fine now in both the updated release version (2.10.1) and the latest devel version...experiments with 2 repeats for each of them. I want to combine the results and thought to try the batch function. Currently, I'm doing per plate normalization and no variance adjustment. I would …

Normalization cellHTS2 Normalization cellHTS2

updated 16.2 years ago • florian.hahne@novartis.com

low responders. I had the data randomised for them based on both factors so that they can use it in batches of three as we were going to produce same data for metabolomics in three batches. I kept the biological variation same...in three batches based on both factors and also the visits for same partcipants in same batch. Now theyt are telling me this: > The "run...we targeted > …

RNASeqData BatchEffect sva

updated 2.5 years ago • amnahsiddiqa

div class="preformatted">Dear List, I'm starting to do limma analyses on a small timecourse loop design with 2-color cDNA chips as follows: 0h vs 6h 6h vs 24h 24h vs 0h Four biological...A_24h Ref US22502600_F83_S01.gpr F_24h Ref ... and six more For my limma analysis, I have three options: *a*: use only the minimal number of chips (ie eac…

TimeCourse limma timecourse TimeCourse limma timecourse

updated 18.0 years ago • Yannick Wurm

<div class="preformatted">Dear BioC I have a set of Affymetrix chips which have a clear "process day" batch effect. This effect is only partially removed by RMA, gcRMA, vsn or invariantset "expresso" normalisation. What would you...div class="preformatted">Dear BioC I have a set of Affymetrix chips which have a clear "process day" batch effect. This effect is only partially removed by…

vsn gcrma vsn gcrma

updated 20.4 years ago • Aedin Culhane

div class="preformatted">Hi folks, I am fairly new to limma. I hope that someone could help me out with a design metrix and contrast. Factors are: 1st - drug response (responder versus...0 week-sample collected before treatment versus 24 weeks- sample collected after treatment ). 3rd - batches. My questions: 1, I would like to find the drug responding genes. 2, should this be a two factor pr…

limma limma

updated 16.2 years ago • Steve Shen

I have not seen asked yet and I am hoping for some feedback. I have an RNAseq dataset in two batches with an imbalanced study design, and I had a large batch effect. I used combatseq to 'correct' for the batch effect. PCA...from the original count matrix showed the large batch effect with PC1 ~90% of the variance. After combatseq, the batch effect is reduced (pc1 ~37%), but still appare…

DESeq2 sva

updated 3.9 years ago • Clay

now running ATAC-seq differential analysis by DiffBind with peaks from MACS2. I have 10 samples from batch 3 and 20 samples from batch 5. How could I eliminate batch effect and normalize them in DiffBind? Sorry, I'm quite new in this

diffbind atac

updated 8.6 years ago • niu.shengyong

Hello, I'm trying to remove batch effect from a dataset that include three experimental conditions (C_25,C_50,C_CTRL) and three batches (1,2,4). I prepared...table with all these information (targets) and then I tried to create a design matrix using ~0+group+batch in my model formula. I would like to know if this approach is correct or it's better to put ~batch+group. I tried also this last...pa…

edgeR model.matrix BatchEffect

updated 2.9 years ago • greta

Hello Bioconductor Gurus! (I apologize if this goes through more than once) We are currently using limma (through the voom() function) to analyze RNA-seq data, represented as RSEM counts. We currently have 246 samples (including...This brings down our transcript count from 73,761 to less than 20,000. While we do see groupings and batch effects we expect to see in the MDS plots, we are afraid we…

limma limma

updated 13.3 years ago • Mark Lawson

class="preformatted">Dear Tiandao, Dealing with multiple gal files is very tricky, but possible. In limma, you need to read in the GPR files for each GAL file separately, identify control spots separately, and normalize separately...gt;From: Tiandao Li <tiandao.li at="" usm.edu=""> >Subject: [BioC] different gal files using limma >To: Bioconductor_help <bioconductor at=…

Microarray Normalization limma Microarray Normalization limma

updated 18.4 years ago • Gordon Smyth

Hi, I'm new in biological analysis. I want to use DESeq2 to do my analysis, I have batch 1 and batch 2, and the batch equals group. ![My colData][1] If I want to remove the batch effect, should I only use **design = ~ group...condition**, or I should use **design = ~ batch + group + condition** ? And this is PCA result, is there any batch effect exist? Should I remove batch effect before WG…

BatchEffect RNASeqData WGCNA DESeq2

updated 2.6 years ago • zxc741xb

Hello everyone,  Could I please get an opinion on whether a batch correction is recommended in my data?  Background: I have performed RNAseq gene expression analysis on 2 condition...function (DEseq2)  (named "Unbatched" in the PCA plot). Subsequently, the normalised data was batch corrected using the removeBatcheffect function (edgeR) (named "Batchcorrected") Proble…

batch effect removebatcheffect edgeR rld DEseq2

updated 7.5 years ago • Antonio Ahn

transparent">, i show a specific part of my code regarding a statistical downstream analysis with limma in R, for an affymetrix microarray (after normalization and filtering, without batch effect correction):</span> <code><span...nbsp;30    150</span></code> <code><span style="background-color:transparent">table(batch)</spa…

limma batch effect design matrix microarray confounding

updated 8.7 years ago • svlachavas

differential gene expression analysis with edgeR. I have four conditions T1, T2, T3 and T4 and two batches. I am running the following codes: Count1=read.table("GENESFORANALYSIS.txt", sep="\t", header=TRUE) colnames(Count1) = paste...summary(decideTests(lrt1)) My model matrix is: (1) design=model.matrix(~ batch + conditions, data=y$samples) (Int…

edger

updated 5.3 years ago • chatterjee.arumoy

I would like to remove batch effects by ComBat but when we have one peptide missing for the whole batch 1 while we have data for batch 2, 3, and 4. But we...would like to incorporate this peptide in adjusting for batch effect of rest of the batches. What's the suggestion to deal with this peptide?  Thank you very much

batch effect missing value Combat

updated 7.3 years ago • hjleeab

Hi, I'm trying to identify DEG between different conditions at two time points. Here is my design : ```R group,ind,condition 4h,4h_cond1_1,cond1_4h 4h,4h_cond1_2,cond1_4h 4h,4h_cond1_3,cond1_4h 4h,4h_cond1_4,cond1_4h 4h,4h_cond2_1,cond2_4h 4h,4h_cond2_2,cond2_4h 4h,4h_cond2_3,cond2_4h 4h,4h_cond2_4,cond2_4h 8h,8h_cond1_1,cond1_8h 8h,8h_cond1_2,cond1_8h 8h,8h_cond1_3,cond1_8h 8h,8h_cond1_4,cond1…

DESeq2

updated 23 months ago • raniaouazahrou

of control replicates was sequenced earlier. Thus only this control replicate belongs to a different batch from all other samples. As suggested in the vignette I added batch in my design like `` ~batch+condition ``. However very few...genes remain significant after doing this as compared to when do not add batch to design. Is it recommended to add batch to design in such an experiment? Is there…

deseq2 rna-seq

updated 7.4 years ago • bn3301

class="preformatted"> Dear List, I am looking at differential methylation (from Illumina 450K) with Limma and have a situation in which I have several groups and want to make comparisons between particular groups. It seems...set to (0,0) in the previous example? Or am I way off the mark on how the intercept functions in Limma altogether? My second problem is that I was wondering if my model …

limma limma

updated 12.9 years ago • Guest User

I have a dataset with 10 condition vs 20 control samples and am using limma to test for differential expression. Broadly, groups are age/sex matched but have complex medical histories which...I have a dataset with 10 condition vs 20 control samples and am using limma to test for differential expression. Broadly, groups are age/sex matched but have complex medical histories which add...by sampling…

limma

updated 12 months ago • Ali Barry

Hello, I have a question in relation to limma and how to extract the random intercepts results after running a duplicated correlation. To summarize, I have DNA methylation...intervention is applied again. My code is as follows: design=model.matrix(~intervention+Age+Batch,data=pheno) library(limma) corfit <- duplicateCorrelation(DMFS,design,block=pheno$ID) …

limma

updated 5.3 years ago • macsue.jacques

In the *Feature selection across batches* section of the `simpleSingleCell` *Correcting batch effects* [vignette][1], genes with positive average (across batches...What is the reasoning behind that? Why aren't genes with positive biological variance in any batch selected instead? [1]: https://bioconductor.org/packages/release/workflows/vignettes/simpleSingleCell/inst/doc/batch.html

simpleSingleCell

updated 6.4 years ago • Angelos Armen

<div class="preformatted">Hello, I've been trying to use limma to identify genes from the following data: http://www.ebi.ac.uk/arrayexpress/experiments/E-GEOD-21340 - It's a simple control...div class="preformatted">Hello, I've been trying to use limma to identify genes from the following data: http://www.ebi.ac.uk/arrayexpress/experiments/E-GEOD-21340 - It's a simple...SDRF$Comment..S…

limma limma

updated 13.8 years ago • David Westergaard

format (e.g. 101178090056\_A\_Grn.idat).  At first 12 samples were processed on a chip denoted batch 1. Then later,  24 samples were processed on two chips at the same time denoted batch 2 and batch 3.  I am interested...pipeline. \#\#Processing 36 arrays (idat files) all together <pre> set.seed (12345) library(limma) library(magrittr) library(illuminaio) l…

illumina human ht-12 v4 limma pipeline

updated 9.4 years ago • alakatos

in a dataset of 6 tests and 6 control samples. The cDNA library preparation has been performed in 2 batches. The second batch contains only 2 samples and both of them are in the control group. When I am trying to correct for the...batch effect (~ batch + treatment), the results are not different than when the effect of batch is not included (~ treatment). As there

deseq2

updated 6.2 years ago • Mehdi Emam

div class="preformatted">Dear list, Now I am using sva to remove batch effect in my mircoarray data. First I used combat to remove know batch effects. The combat can output a new expression...matrix without batch effect, then I used sva to remove unknow batch effect, but it seems that sva can not output a new expression matrix. sva take...the surrogate variables into the test. How can I let s…

sva sva

updated 14.1 years ago • Fabrice Tourre

metadata below). However, 1 of the cell types (HCT116) was done in a different lab and shows a stark batch effect between its two replicates, while the other 2 cell types have a more muted batch effect between their two replicates...Overall, I see the best precision with a known set of gene targets when using the native DESeq2 batch correction in HCT116, but the other two cell types show a small …

DESeq2

updated 2.4 years ago • Hope

matrix of 1208 samples (1095 tumor and 113 normal) downloaded from TCGA. I know there are 3 batch effects: type, plateId and TSS. I've tried to correct for them with Combat but I need a little help with the model.matrix...Is there anyone who can help me? I'm a student. Thanks in advance. The code is:   <pre> batch<-as.data.frame(cbind(samples,plateId,group,TSS),as.is=T)[,…

combat sva batch effect

updated 7.6 years ago • Fp

So I was doing a differential expression analysis with RNA-Seq data using limma-voom pipeline. However, the fit for mean-variance is not like a flat line at 1 but a curve (top: plot after voom; bottom: plot...So I was doing a differential expression analysis with RNA-Seq data using limma-voom pipeline. However, the fit for mean-variance is not like a flat line at 1 but a curve (top: plot after vo…

limma voom

updated 6.3 years ago • kentfung

nbsp;to address an important question regarding a GEO a time series dataset, GSE21059. I used limma package for preprocessing. In detail, I'm asking about the specific step considering contrasts I would use. Part of my...bystander.0.5h bystander.1.0h bystander.2.0h bystander.24.0h bystander.4.0h ... irradiated.6.0h batch <- factor(final$targets$Batch) # where batch the 4 diffe…

limma multiple factor design multiple time points microarray design and contrast matrix

updated 9.2 years ago • Konstantinos Yeles

div class="preformatted">Dear list, In my mrcroarray data, there is batch effect. But I am not very clear what kind of batch effect in it. I want to using PCA to remove batch effect, but I am confused...how to from the raw expression data to get a new expression data without batch effect in it. Any suggestion will be welcome. Thanks. </div

updated 14.1 years ago • Fabrice Tourre

dds <- DESeqDataSetFromMatrix(countData = countdata, colData = colData, design = ~ condition + batch, tidy = TRUE)``` differ from an object after ```dds<-deseq(dds)```? How is the count matrix being changed by the ```deseq()``` code? 2. Specifying...my batch as a covariate in ```~ condition + batch``` did not seem to correct my batch effect, so I used Limma for batch correction. I…

DESeq2 rnaseqGene RNASeqData limma RNASeq

updated 23 months ago • sap275

from two two amplification protocols in the same laboratory for some reason. The amplification batch effect is quite obvious in the RLE and NUSE plots. I divided files into two groups and re-analyzed. The RLE and NUSE plots...got much better, especially for the first batch. I will incorporate the batch effect into limma gle with the data of GCRMA normalization for all chips. I also like to try..…

limma gcrma limma gcrma

updated 20.0 years ago • Jianping Jin

Deseq2 is required a count metric. However, after I look into my count data, I would that there is a batch effect in my count data. ![As from you see in the image, the image shown the total read count of the data batch 2 (high read count...count before using in Deseq2 analysis. What is the best option in my analysis, (1) Performing batch normalization using Combatseq --> Deseq2 --&am…

DESeq2 WGCNA Normalization NormalyzerDE BatchEffect

updated 2.8 years ago • suphamon.j

Dear Dr. Smyth, We are analyzing some RNA-seq samples collected in different batches, where the batch is a known variable. To account for that we reasoned we could use a linear model to include the batch...effect and then remove it.  Since the voom+limma approach is shown to work well for differential gene expression, we thought of estimating the weights for each observation...through …

voom limma clustering removebatcheffect() batch effect

updated 10.5 years ago • ale.breschi

Hii, I have 3 batches of samples that belong to 3 conditions/treatments i.e.. batch1(control), batch2(disease stage1), batch3(disease stage2...which belong to the same microarray platform and same tissue. 1) Is there a way to find batch effect for this case ? 2) If so, how can i perform batch adjustment in this case ? 3) Which method should i use to retain biological...variation among the bat…

combat sva

updated 9.1 years ago • vinnu260

appeal to the collective wisdom in this group on how best to solve this problem of normalization and batch correction. We are a service unit for an academic institute and we run several projects simultaneously. We use Illumina...2. Remove failed samples 3. Remove samples from other projects. 4. Normalize using NEQC from limma 5. Correct for scan date using COMBAT from sva. The other op…

Normalization sva Normalization sva

updated 11.4 years ago • Adaikalavan Ramasamy

i.e. which are differentially expressed due to factor 1 alone or factor 2 alone. (2) getting batch-free TMM-normalized FPKM values (where the batch effect has been removed). This will not be used in any differential expression...matrix with raw counts, log2 transform them, use removeBatchEffect() with a design matrix that lack batch effect factor, remove log2 transformation and use a utility wra…

edgeR limma removeBatchEffect()

updated 11.1 years ago • Ekarl2

Hi bioconductorlist, I am trying to combine microarray data from different experiments(batches) where some of my batches fall completely in one condition (normal/cancer). For this I try to use ComBat in the sva package...ensuring no batch effect or condition effect. Then I will do a quick find- differentially-expressed-genes analysis with limma. Contradictory...data(bladderdata) # Obtain phenot…

Microarray Clustering Cancer limma Microarray Clustering Cancer limma

updated 10.4 years ago • Vegard Nygaard

Are there tools implemented in Bioconductor that allow one to use technical replicates to batch correct. Meaning, if one has a group of samples that are repeated across known batches, are there tools that can use these...samples for batch correction

batch effect

updated 9.5 years ago • Juliet Hannah

and as always there are many factors (disease/control, infection, age, sex, treatment, cohort, pmi, batch/scandate).  So my question is basically a generell question concerning combat. I am interested in two biological...combat sequentially? If yes, what about the order? This does influence the outcome. Or is multiple batch correction basically overfitting the data? Thank you very muc…

combat sva limma

updated 8.9 years ago • juls