Bioconductor Forum

centers and I want to analyze it using ReactomeGSA-Camera but I am not sure how to adjust for batch effect and unwanted variation on the web tool. I already performed RUVg correction for that purpose and I have a matrix

ReactomeGSA BatchEffect RNASeqData

updated 1 day ago • Shaimaa Gamal

t help and also used surrogate variable analysis (SVA) and combat_seq to try to adjust for hidden batch effect, but none has improved my the PCA. The challenge is pronounced when I attempt to determine Isoform switch analysis

DESeq2 pcaExplorer

updated 4 days ago • Eric Katagirya

in a PCA plot, there was a significant difference between EPIC v.1 and v.2 datasets. So when batch effect correction(using ComBat package) was performed, some beta values became negative or greater than 1. In this case

sesame BatchEffect MethylationArray

updated 5 days ago • Seungmin

Hello, I have quite a complex experimental design from a retrospective study of human disease progression and would like some advice on making appropriate contrasts with limma-voom. We have two cohorts of patients, A and B, sampled longitudinally (4 times on average). Cohort B develop disease at a later...study of human disease progression and would like some advice on making appropriate co…

rna-seq duplicateCorrelation limma voom

updated 9 days ago • Dylan.Sheerin

has started ##------ Tue May 7 15:17:18 2024 ------## Log fold changes are estimated using limma package ... Error in runStandardLimmaDEA(voom.results, contrast, logFC.cutoff, fdr.cutoff) : is(voom.results, "EList") is

EGSEA

updated 10 days ago • Chris

hello all, i am doing a differential gene expression analysis using limma, i have 3 conditions S0,S1, and S3. I am creating the contrasts and doing the fit function but its giving the error ( Number...S0, + S1_vs_S3 = S1 - S3, + levels = design + ) > > # Fit contrasts > fit2 <- limma::contrasts.fit(fit, contrast.matrix) Error in limma::contrasts.fit(fit,…

limma

updated 10 days ago • zzrammal

Hi all, I am trying to use Limma for proteomic analysis on a sample size of n=8 controls and n=8 experimental. The code runs without errors, but the results...Hi all, I am trying to use Limma for proteomic analysis on a sample size of n=8 controls and n=8 experimental. The code runs without errors, but the results show that nearly all 700 proteins are significant and the p-values are tiny…

Proteomics limma

updated 10 days ago • Abbey

Can anyone provide a better way to remove/trim out-of-bounds genomic ranges? I have 6349 scaffolds in the reference genome. I want 500 bp of upstream sequence from all start codons; many predicted genes are on scaffolds without 500bp of upstream sequence that need removed/filtered from the GRanges object. Ideally, I want only the genes that have 500bp of upstream sequence. I am not having any …

GenomicRanges

updated 11 days ago • mat149

to compare which pathways different between them. I ran gene set variation analysis follow up with limma, fgsea, camera. enrichGO() also rank the pathway but we can't choose which gene set we want to analyze. Each of them gives different...p/9158100/#9158140, seem p valule from fgsea is not truly significant as it is. When I search camera limma on pubmed, I found 2 papers. Thank you so much

fgsea GSVA CAMERA

updated 12 days ago • Chris

Hi Everyone! Im currently working on a basic DMP/DMR analysis using EPICV2 array data, and was running into an interesting issue in regards to specifying my model design. I am getting VERY different results between models with and without an intercept. Since my variable of interest is a factor, the results should be the same, right? My variable of interest is a factor with two levels ("Al…

limma DMRcate MethylationArray EPICV2

updated 14 days ago • Kim

genes (DEGs) detected across TCGA primary tumor and GTEx normal colon tissue samples. When using `limma` for analysis, the ```treat``` function can help address this issue by computing empirical Bayes moderated-t p-values relative

RNA-seq DESeq2

updated 14 days ago • Reza

frames in trying to fullfill the full rank To valorate different coldata alternatives, I am using limma ```r design <- model.matrix(~Time + Treat + Time:Treat) is.fullrank(design) ``` obtaining in all the cases a FALSE result ¿Any hint

DESeq2 design coldata

updated 15 days ago • arfranco

I am using limma to analyze mass spectrometry-based metabolomics data. I have a matrix of peaks (rows) by samples (columns). In this data...I am using limma to analyze mass spectrometry-based metabolomics data. I have a matrix of peaks (rows) by samples (columns). In this data, each...in this matrix might represent the ratio of 13C6-glucose to 12C-glucose across samples. Then, run limma using a s…

Metabolomics limma

updated 15 days ago • c53aba27

however, deseq2 did let me know I have 30 outliers after DE. My PCA plot doesn't show any crazy batch effects. I did the VST transformation, that I planned to use for downstream analysis. PCA: ![enter image description here

DESeq2

updated 16 days ago • kcarey

and see what's happening in the samples much better. Is that possible? Would this code give that? `limma::removeBatchEffect(counts(ddsfiltered, normalized=T), batch = ddsfiltered@colData$batch, batch2=ddsfiltered@colData...metadata-test.csv",header = TRUE) colData <- as.data.frame(cbind(colnames(countData), metadata$batch, metadata$condition, metadata$techRepl, metadata$condName, metadat…

DESeq2

updated 17 days ago • HAK

Hi all, After googling and ask chatGPT, I don't know how to fix this error. Would you please have a suggestion? Thank you so much! gene_sets_dir <- "gene_sets" c2_gmt_url <- "https://data.broadinstitute.org/gsea-msigdb/msigdb/release/2023.2.Hs/c2.all.v2023.2.Hs.entrez.gmt" c2_gmt_file <- file.path( gene_sets_dir, "c2.all.v2023.2.Hs.entrez.gmt" c2_list <- …

CAMERA limma

updated 17 days ago • Chris

and analyse it so that I have control vs trmt1, control vs trmt2, etc...Do I have to refer to batch for it? Or with batch I am clustering the timepoints? About moanin, it seems easy to set the different conditions for the

DESeq2 ImpulseDE2 timecour timecoursedata moanin

updated 18 days ago • Aurora

I utilized the R package GEOquery to download data from the GEO database, followed by DFG analysis using the limma package. However, I am uncertain whether this process is accurate. The DFG output indicates that each P-value is significant, but each adjusted P-value tends towards 1. I attempted this process with several datasets using the limma R package and encountered the same outcome consist…

GEOquery ArrayExpress limma DifferentialExpression

updated 18 days ago • SSSJec

technology, and red for two types of normal cells to compare patient with. As you can see the batch and method effect is very important... When doing a pairplots, I see that this batch effect can be seen the first and second...0.4765441 cor_W2_Dim2 -0.2840367 ``` Finally, with this code here, I tried to remove this batch effect directly and see the PCA before (first picture) and after (seco…

DESeq2 RUVSeq RUVseq BatchEffect DES

updated 22 days ago • Alexandre

colData = batch_condition_numericMeta, design = ~ batch + Subtype_mRNA) #IHW LFC_DiffervsImmuno<-results(dds,name="Subtype_mRNA_Differentiated_vs_Immunoreactive", lfcThreshold

DESeq2 RNAseq

updated 23 days ago • kcarey

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 25 days ago • hortense96

library size, cell type composition). At this point, I've controlled for these covariates using limma+voom, leaving me with residuals *r1* and *r2*. Since *r2* and *r1* have already been log transformed, I believe I can take the logFC...are there any other approaches you would suggest that might let me maintain the weights from limma+voom

limma RNASeq voom

updated 25 days ago • taur.vil

Hi all, I have a question that don't know why, hope you can help. I use GSVA (Gene Set Variation Analysis) package to calculate pathway scores. Then I compare pathway scores between 3 groups using limma. When I use a few thousand pathways, raw p value of pathway A will different (bigger) with when I use only a few pathways. I think...Analysis) package to calculate pathway scores. Then I compar…

limma

updated 27 days ago • Chris

trying to figure out if there is a way to deal with technical replicates when coming from different batches of sequencing. I do not want to remove the batch effect from the data, I want to include it in the statistical model. So...post https://support.bioconductor.org/p/59700/ mentioning the function duplicateCorrelation from limma, which seems to deal with technical replicates. Would I be able…

technicalReplicates BatchEffect limma RNASeq

updated 28 days ago • catv

Hi, Time to tackle an amateur's problem! Ha ha! I have 6 samples/ groups with 3 replicates (In total 17; one sample contains 2 replicates). the sample names are Mf, Bf, Ef, Mr, Br, and Er. I want to get DEGs (differentially expressed genes) between Mr-Mf, Br-Bf, Er-Ef. For doing that, I subsetted my data keeping either Mr-Mf, Br-Bf, or Er-Ef. And then I got DEGs for three different contra…

subsettingdata edgeR designmatrix

updated 29 days ago • prity6459

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 4 weeks ago • Shaimaa Gamal

Dear creators of ComBat-seq, for experimental reasons, I was trying to run ComBat-seq on only two batches of the GFRN dataset provided (https://github.com/zhangyuqing/ComBat-seq/tree/master/real_data_application). I cropped...long as I do not use the 'group' parameter. I made sure only to use the correct labels here. See my batch and group input: batch <- factor(c(1,1,1,1,1,1,1,1,1,1…

ComBat-seq sva

updated 4 weeks ago • schlumbohm

Hi all, When I use limma to compare pathway score between 3 groups, I tried two ways: 1. Keep 3 groups in the data and use `coef` to specific which 2...Hi all, When I use limma to compare pathway score between 3 groups, I tried two ways: 1. Keep 3 groups in the data and use `coef` to specific which 2 groups

limma

updated 5 weeks ago • Chris

Hi! I conducted DGE analysis between 2 groups of cell lines MYCN amplified vs MYCN non amplified. Cell lines in the MYCN non amplified group had these FPKM values for MYCN gene: 5.182582, 3.104376, 4.962478 Cell lines in the MYCN amplified group had these FPKM values for the MYCN gene: 101.2204, 301.8182 , 280.6712 Now visually there is a marked difference between these two groups and s…

limmaGUI edgeR limma

updated 5 weeks ago • Simran

differential expression, an appropriate design would look something like this (i am excluding all batch effects for simplicity): ``` ~ 0 + genotype + condition + genotype:condition ``` However, I usually find interactions a bit more challenging...for the combined object design <- model.matrix(~ 0+type, colData(dds_combined)) contrasts <- limma::makeContrasts( interaction= (typ…

DESeq2 DifferentialExpression

updated 5 weeks ago • nhaus

difference between for example treatment5 and the control? ``` #my design (for simplicity all batch effects are ignored) design = ~ patient.id + treatment ``` A related question is the following. Because I have so many samples...I am using sva to adjust for batch effects. Is it correct, that after using this approach, I can remove the `patient.id` from my design because the identified

edgeR DifferentialExpression

updated 5 weeks ago • nhaus

Is it possible to use GSVA to address this question or are the results likely to be influenced by batch effects? Best wishes, Lucy

GSVA

updated 5 weeks ago • Lucy

Hi all, I use limma to compare pathway score between 3 groups. Then I would like to make boxplot with overall p value and pairwise p value...significance-levels-to-ggplots/ The value from t test or anova is different from the p value from limma: topTable <- topTable(fit2, number=Inf, sort.by="none") topTableLSvsNC <- topTable(fit2, coef="LSvsNC", number=Inf, sort.by...for…

t.test boxplot limma anova

updated 5 weeks ago • Chris

So I have a group project where we must take RNAseq data and analyze it ourselves. We decided to use limma in order to do so, but are having some trouble understanding how to build the design matrix. There are two factors in the

limma designmatrix

updated 6 weeks ago • avery

I am analysing some proteomics data from an experiment with the following experimental design: - 3 donors, each treated with: - placebo - 3 technical replicates - drug - 3 technical replicates ``` sample_id patient_id treatment 1 A placebo 2 A placebo 3 A placebo 4 …

limma

updated 6 weeks ago • andrea.rodriguez-martinez13

ChAMP pipeline. I understand that the first SVD is used to identify the correlations and correct batch effects. However, I don't understand how I can correct the effect of cell types as it doesn't appear in my csv file. What should

ChAMPdata ChAMP

updated 6 weeks ago • simleopold11

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 6 weeks ago • kys91240

Hi, I'm investigating changes in gene expression after a body weight loss intervention. The data includes baseline measurements and two follow-up measurements. The 40 participants have varying body weights at baseline, and they lose varying amounts of body weight during the study. My main model (see m1 below) has the standard structure used in this scenario. However, because the participants s…

limma

updated 6 weeks ago • jari.karppinen

to visualize my pca with vst command in DESeq2, so I did the following passage (I also delete the batch effect of my RIN for the visualization): ```r mm_ALpcrRD <- model.matrix(~ condition + RIN, colData(dds_ALpcrRD)) mm0_ALpcrRD...blind=FALSE) covariates_ALpcrRD <- colData(vsd_ALpcrRD)[,c("SV1","SV2")] assay(vsd_ALpcrRD) <- limma::removeBatchEffect(assay(vsd_ALpcrRD), bat…

DESeq2 sva

updated 6 weeks ago • michelafrancesconi8

Some individuals received Vaccine A and other individuals received Vaccine B. I also have a batch effect due to library preparation (Batch 1 and Batch 2). The model I have used for edgeR differential expression analysis...is: `~ 0 + donor + batch + vaccine:timepoint` As I understand, this allows me to control for differences in baseline expression levels between...donors and for the library p…

ComBat sva edgeR

updated 6 weeks ago • Lucy

Hi all, I use limma to compare pathway score (from GSVA tool) between 3 conditions (normal control, early state, late state), I got no difference...different between normal control and late state. Would you please explain why? I thought when limma compare 3 conditions, it will pairwise compare them so if 2 conditions have different, 3 conditions also should have...I think apply limma is better…

limma

updated 7 weeks ago • Chris

as (phenodata$groups). ``` #Normalization normdata <- rma(rawdata) normexprs <- exprs(normdata) #Limma design <- model.matrix(~0+phenodata$groups) colnames(design) <- c("NRM","SVR","CTL") fit <- lmFit(normexprs, design) contrasts

limma DGEanalysis

updated 7 weeks ago • Amit

sig.probe2<-probe2[which(probe2$adj.P.Val<=0.05),] write.table(sig.probe2,file="limma..model2.significant..output.txt",sep="\t",quote=TRUE) write.table(probe2,file="limma..model2..output.txt",sep="\t",quote=TRUE...sig.probe3<-probe3[which(probe3$adj.P.Val<=0.05),] write.table(sig.probe3,file="limma..model3.significant..output.txt",sep="\t",quote=T…

Regression limma STATISITICS HPC

updated 7 weeks ago • Jitendra

Hello, I have an experiment where I'm comparing a batch A versus B (in biological triplicate: A1/A2/A3/B1/B2/B3). I create the DESeqDataSet, filter by low expression and rRNA, and...Hello, I have an experiment where I'm comparing a batch A versus B (in biological triplicate: A1/A2/A3/B1/B2/B3). I create the DESeqDataSet, filter by low expression and rRNA, and use

DESeq2 Transcriptomics

updated 7 weeks ago • Anita

lt;- readData(NORM.data, factor = PHENO) BATCH.data <- ARSyNseq(BATCH.cor, factor="Group", batch = FALSE, norm = "n", logtransf = TRUE) to remove batch effect it separates the samples on PCA . But when is use varFilter(BATCH.data...i get more number of DOWN DEG than UP DEG after performing eBayes. Is there any other simple batch correction and gene filter package availabl…

DGE limma

updated 8 weeks ago • Amit

Hi, I noticed that both edgeR and limma have function to perform differential splicing analysis based on exon level counts. I am wondering why exon level...Hi, I noticed that both edgeR and limma have function to perform differential splicing analysis based on exon level counts. I am wondering why exon level counts...used instead of splice junction counts (i.e. STAR SJ output). Would it be …

splicing limma edgeR

updated 8 weeks ago • scoops_streams_06

the multifactor design is only appropriate to use for normalizing factors such as tissue type or batch? Or could this be used to identify differentially bound regions between two main factors? Thanks for your help

DiffBind multifactoranalysis

updated 8 weeks ago • erbrocato

questions were asked many times before, yet I'm asking again. My design is a 3 x 2 design with a batch effect ( samples were not processed on the same day). I came across [this post][1] and am going to follow Dr. Smyth's suggestion...0 ~ + batch + group)`. I just wanted to know if my approach of making contrasts is correct. My meta_data. I combined two factors (`genotype...and `treat`) into one …

design edgeR contrasts

updated 8 weeks ago • JKim

to the CDF, the chip is called hugene11st. I'm wishing to perform a differential expression in ```limma``` after parsing CEL files with ```oligo```. While following limma's userguide, I faced problems while trying to annotate the

oligo limma hugene11stprobeset.db AffymetrixChip

updated 8 weeks ago • aefrainmp

I have performing DGE analysis of Microarray data from GEO having 26 samples containing around 56000 genes. After applying nsFilter() the genes reduce to around 8000. So my question is " Is it necessary to perform gene filtering step?".

MicroarrayData limma Genefiltering

updated 8 weeks ago • Amit

to add the cell number to design as covariate, and the colData as follows: ```r > coldata batch time type cell_num group 0d-Mut2 2 0d mut 1289.6 mut_0d 0d-Mut3 2 0d mut 1523.8 mut_0d 10d-Mut3_2 2 10d mut 1478.4 mut_10d...design formula as follow: ```r dds <- DESeqDataSetFromMatrix(dat_filtered, coldata, design=~batch+cel…

DESeq2 DEs

updated 9 weeks ago • feather-W

Hi, I am analyzing RNA-Seq dataset using EdgeR package, I have question while extracting co-efficient for pairwise comparisons to detect genes that are differentially expressed between group after performing dispersion estimation, fitting the model (steps below). Just checking if I am I doing it rightly? Alternatively, is there another way compare the contrasts? ``` r #> Samples Patient …

limma fit design RNASeq edgeR

updated 9 weeks ago • Sabiha

my RNA sequencing raw counts data. However, I wanted to perform a gene filtering step before running limma for batch correction (16 Tissue sites were used for data collection) and DESeq2 (DE Analysis and Normalization). I started...a value in literature. I am using high grade serous ovarian cancer data. After I filter, I plan to batch correct with limma before DESeq2. Any suggestions will be …

DESeq2 limma RNAseq

updated 9 weeks ago • kcarey

of strain `BAR` to the *overall* effects of knockout `Gene1KO`, essentially treating `strain` as a batch variable. Phenotypically, we expect them to be similar overall with potentially pronounced effects in a few specific

DESeq2

updated 10 weeks ago • Viktor Thiel

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 10 weeks ago • michelafrancesconi8

Enter the body of text here hi, I am working on a GEO microarray dataset, 5 groups were defined and assigned to the Expressionset. Beform ```vooma``` I used ```exprs(gset) <- normalizeBetweenArrays(exprs(gset))``` after log2 transformation, and used ```gset <- gset[complete.Cases(exprs(gset)), ]``` to remove missing value, as suggested in the manual. so when I run ```vooma``…

MicroarrayData limma

updated 10 weeks ago • Lily Cheang

the course of 1, 2, 3, 4 and 5 hours. As I understand, the "9.6.2 Many time points" section of the Limma user guide fits my experiment design, and I am using the following formula for design: ```r design <- model.matrix(~0+groups

limma

updated 10 weeks ago • dl17032000

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 10 weeks ago • sap275

``` # DGE Analysis with Limma. # Model matrix. design <- model.matrix(~0+PHENO$Group) fit <- lmFit(VAR.exprs, design) contrasts <- makeContrasts(G3- G1, G3...DGE Analysis with Limma. # Model matrix. design <- model.matrix(~0+PHENO$Group) fit <- lmFit(VAR.exprs, design) contrasts <- makeContrasts(G3- G1

limma

updated 11 weeks ago • Amit

I have been working on a meta-analysis of methylation data and encountered a snag. Before analysis, I wanted to normalize the data using the removeBatchEffects() function for array type and origination cohort. These variables were higher collinear (canonical correlation of 0.74) and resulted in the "coefficients not estimable" output. As such, I decided to only normalize by array type and leave o…

BatchEffect limma

updated 11 weeks ago • harrshavasan.congivaram