Bioconductor Forum

to run analysis on 3 different cell types undergoing the same treatment conditions, each with 2 replicates (see 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 (PCA below). ![metadata][1] ![pca...between cell …

DESeq2

updated 2.4 years ago • Hope

affybatch and then to normalize it? I'd like to use the averaged intensities of my technical replicates to normalize against other biological replicates. Any comments and/or suggestions will be very appreciated

updated 21.9 years ago • Puhong Gao

m using DiffBind to analyze a set of ChIP sequencing data. For each sample, I have three biological replicates. I performed the ChIP calling analysis using SICER.  Now I'm interested in performing an overlapping analysis...using the three biological replicates. In the SICER output there is an enrichment value for each peak (this means an enrichment respect to the input). Is

fold enrichment values using DiffBind

updated 11.0 years ago • robertacar

a question about analysis using DESeq2. I have three samples of different time points without replicates. I want to check the genes whose expression level changes over time. I have studied time series analysis but I know...requires a comparison target such as control (untreated vs treated). I have control samples with replicates but these are not samples of different time points. Is th…

deseq2

updated 6.7 years ago • snu_jh

Hello dear community, There has been a similar post: "modeling heteroscedasticity in limma-voom for RNA-Seq data analysis" but the topic was different. I have proteomics data over time, so every row of my matrix is a protein, and the columns are timepoints and replicates. Additionally, I have two conditions (groups) which are growth phases of the cells in the bioreactor (exponential and sta…

limma splines Proteomics

updated 8 months ago • Thomas

4 substances. My code is below: <pre> pData(normalized2) condition replicate dataset_603.dat Control1 1 dataset_604.dat Control1 2 dataset_605.dat Biological1 1 dataset_606.dat Biological1...of two different batches: that is, each substance and the control is comprized of two biological replicates…

limma batcheffect batcheffectcorrection multiple factor design

updated 10.7 years ago • svlachavas

I have a time course experiment with two conditions, control and treatment, with three biological replicates for each time point except for time 0 (control conditions) that has two biological replicates. I'm using the pipeline...help me to solve this problem? I'm very grateful if you kindly tell me how to define two biological replicates for control (time 0) and three replicates for other times i…

ballgown software error

updated 8.9 years ago • Sara

using edgeR. The data consists of four different groups (A,B,C,D) that each consist of five replicates. I have a few miRNAs, that turned out to be differentially expressed, and I would like to make a plot for each individually...I am currently generating: ![enter image description here][1] Each point stands for one sample (replicate). The y axis displays the count of the miRNA in the sa…

miRNAData miRNA DifferentialExpression edgeR

updated 3.7 years ago • Sarah

Hi, I am using limma to analyse a 2-colour microarray experiment. There are 2 treatments and 4 replicates in each of these groups. Each replicate is paired to a replicate in the otehr treatment group. Each sample was hybridised

Microarray limma Microarray limma

updated 21.7 years ago • Danielle Fletcher

a 2 condition RNA seq experiment using "disease" versus "control" cells. I have 16 biological replicates in my disease group and 11 in my control. I'm using DESeq2 v 1.0.9 for the analysis. >From the heatmap and pca plots...attached) its clear that there's some variability amongst the biological replicates in my groups which id expect, but also 6 of my disease samples seem to cluster clo…

DESeq2 DESeq2

updated 12.7 years ago • Emma Quinn

Hi. I have some doubts about statistics of DGE analysis (RNAseq data). 1. Some packages use the likelihood ratio test (LRT) and other use quasi-likelihood (QL) F-test. What is better and why? Can I use qlFtest if I have replicates? Please, share with me all the bibliography references to understand it. 2. I have the following experiment: Two populations...and other use quasi-likeliho…

edger limma

updated 5.6 years ago • sandravelasco911012

after this? I do have the set parameters for the log2 count and probability. I have 6 samples: 3 replicates of treated condition and 3 replicates of the controlled condition. Can someone pls hep me out, i am really lost on

DESeq2 Kallisto

updated 23 months ago • Aaliya

the examples in the DESeq tutorial it appears that I would have to analyze these pairs as having no replicates but I'm thinking I really have four replicates. Is there a way to calculate the dispersion using all of the data and

DESeq DESeq

updated 13.9 years ago • Willie Nelson

am a bit confused whether I may be able to use the conds argument for my count dataset. When I have replicate samples I would like to get only the ones specified in the conds vector as the nodes in the dendrogram of the heatmap...possible to do using methods in the DEseq package or do I have to calculate average values for the replicates manually before I obtain the distances? -- output of sess…

Visualization Clustering DESeq Visualization Clustering DESeq

updated 14.2 years ago • Guest User

no residual degrees of freedom in linear model fits" and stopped. My guess is that my data has no replicates in the experiment. Is there any other packages I can use to find differentially expressed genes which does not...require replicates in the experiment? Thanks for your help. Chuming </div

limma limma

updated 16.0 years ago • Chuming Chen

always being compared to. How do I do this? My code is below. `# Assign conditions. I have three replicates in each group except for the fourth group I'm calling CD49a which has two replicates. (condition <- factor(c(rep("CD103

deseq2

updated 5.9 years ago • michael_sportiello

Hi, I have a count matrix with 8 samples (3 replicates each) and I performed a DESeq2 analysis. I did a PCA analysis and there was quite nice clustering of the replicates

deseq2 plotpca

updated 7.3 years ago • snp31

gene below is one of those that gives NA for both kinds of p-values. the 3 first numbers are the replicates from the first treatment and the second 3 numbers are the replicates from the second treatment: PP_2663: 4106 30886...to find differences in gene expression even between conditions that should give differences. The replicate number is low, I know, and there is variation between the repli…

DESeq2

updated 2.4 years ago • Juan Pablo

<div class="preformatted">Hello, I need an explanation of how the design matrix influences the consensus correlation of the duplicateCorrelation function when accounting for technical replicates. Here is my specific example: Design matrix: > design RJf RJm WLf WLm 1 0 0 0 1 2 0 0 0 1 3 0 0 0 1 4 0 0 0 1 5 0 0 0 1 6 0 0 0 1 7…

limma limma

updated 20.5 years ago • Carolyn Fitzsimmons

Hello, I am fairly new to DESeq2, though I have used it before it was for experiments with more replicates.  I am hoping to get advice on how to deal with DE contrasts for this experiment in which I have 15 groups, 11 groups...Hello, I am fairly new to DESeq2, though I have used it before it was for experiments with more replicates.  I am hoping to get advice on how to deal wi…

deseq2 cookscutoff

updated 8.9 years ago • Zach Roe

and samples as columns with counts. The column data for all the 8 samples look like below with replicate and cell-line information: Samples TYPE Replicate Cell-lines Cell1_HA1 Control 1 1 Cell1_HA2 Control 2 1 Cell1_foxcut11...analysis. This is the first time I'm doing differential analy…

edger r differentialanalysis rnaseq designmatrix

updated 6.7 years ago • Beginner

ratios. The design of the experiment is as follows: group1: DRG amplified RNA (4 Affymetric replicates) group2: spinal cord amplified RNA (4 Affymetrix replicates) group3: DRG unamplified RNA (3 Affymetrix replicates...group4: spinal cord unamplified RNA (3 Affymetrix replicates) I fed all data through to limma and specified the following contrasts: contrast1: group1-group2 contrast2: group3

Microarray limma Microarray limma

updated 20.7 years ago • Ilhem Diboun

the summary of my data: I have two conditions (young and old). In each condition I have 3 biological replicates (sample) and each biological replicate is replicated twice (technical replicate), in total 12 libraries. My technical...replicates are not different runs or different sequencing machines, they are two libraries generated from one single biological...replicate. All 12 samples were sequen…

edger design matrix model matrix

updated 10.7 years ago • silvia.paolucci81

I am trying to use DiffBind and having an error. The data set is just two conditions without any replicates. I set up the contrast manually as groups as suggested.I get the error in dba.analyze step. Here is the actual commands...operator is invalid for atomic vectors In addition: Warning messages: 1: Some groups have no replicates. Results may be unreliable. 2: In estimateCommonDisp(res) : The…

DiffBind DiffBind

updated 12.1 years ago • Kasthuri Kannan

Dear all, I have analyzed RNA-seq dataset, see the experimental design below. The two questions we are trying to answer are: 1. Find DE genes between different conditions. It can be seen that each condition has 4 samples (biological replicates) for which I performed pairwise differential expression using DESeq2. e.g. (A-B), (A-C), (A-D), (A-E), (B-C) .... (D-E). I can see the DE genes.…

deseq2

updated 5.9 years ago • Zohaib Anwar

Hi; Novice at creating a design for DESEq2 We have 3 conditions each with replicates: * CTL(untreated – 4 replicates) * Ce (treated w/ cerium – 4 replicates) * nCe (treated w/ modified cerium – 4 replcates) Sequences

deseq2

updated 8.6 years ago • charlesh

of contaminants and phiX, chimeras..). I have observed wrong spearman correlations between tehcnical replicates so my assumption is that the experiment did not work well, at least would not be reporducible given the weak correlation...between some technical replicates. There are more than 50 samples, some with tehcnical replicates , some others without. I´m trying to use DESeq2 to do

deseq2

updated 7.8 years ago • David

<div class="preformatted">Dear Sir / madam, I wish to have an understanding of what is One class data with cl<- c(1,1,1,1,1) in SAM and Rankproduct. I have 5 Biological replicates for a sample and I wish to apply a test to find out the significant genes. As said here no control group but only 5 biological...of what is One class data with cl<- c(1,1,1,1,1) in SAM and Rankprod…

updated 17.0 years ago • John Antonydas Gaspar

Hi! I've got two sets of replicate (x3) HiC libraries. I think I've set the min.inward,  min.outward and max.frag thresholds for them correctly...or at least for the bulk of the datasets - not sure if I should set the threshold for replicate 3 individually?). My question is: what is going on with these HiC libraries? Why do these peak heights look so different

diffhic

updated 9.1 years ago • Darya Vanichkina

design suits for analyzing the Agilent single color data using the LIMMA package without having any replicates? Instead of using the replicate of the samples we have just used the pooled samples in analyzing the data. -- [[alternative

limma limma

updated 13.7 years ago • Muralidharan V

to compare a resistant (R) and susceptible (S) plants. In this intention, we used two biological replicates for susceptible (RED – treatment, and YELLOW – control) and three replicates of resistant (GREEN – treatment and BLUE

deseq2 2x2

updated 7.6 years ago • filipematias23

for those data determined by t-test. As I currently have only those data available and not the replicates themselves, I wonder if it is sensible to use the FCs as input for the limma function `` lmFit ``? The results I get seem...uneasy about the reliability. Is this possible in some way or is my only solution to obtain the raw replicate data? Thank you for your insights

limma logfc lmfit

updated 10.6 years ago • Moritz E. Beber

div class="preformatted">Dear Limma users After finishing the analysis successfully with replicates, I am now trying to analyze the same without replicates. After following a suggestion that I can use Limma without...applying the fit2 <- eBayes(fit2) step (for without replicates), I tried to do the same for just one of the patients as a test, but it failed because of the following erro…

limma limma

updated 13.2 years ago • Venu Pullabhatla

name = expName, path = dataPath) from a platelist file that look a bit like: Filename Plate Replicate Channel Batch 1a1.before 1 1 1 1 1a2.before 1 2 1 1 1b1.before 2 1 1 1 1b2.before 2 2 1 1 2a1.before 3 1 1 1 2a2.before...represent plates processed by different experimenters. The complete set have 18 plates, with 2 replic…

cellHTS2 cellHTS2

updated 16.6 years ago • Ian Sudbery

I'm analysing low cell number ChIP-seq data (3 ChIP replicates / 3 Input replicates). The replicates are highly variable due to the low amount of starting material and the number...These generally correspond to windows which have been highly amplified randomly in one of the replicates). The windows in green are the increasing count - decreasing variance windows (These seem to be windows containing

limma voom

updated 8.1 years ago • jma1991

Hi All, I have three conditions and two replicates per condition. I'd like to make three comparisons: (1) TE vs [MP and EE] (2) MP vs [TE and EE] (3) EE vs [TE and MP] I've tried doing the...r samples <- c('TE_1', 'TE_2', 'MP_1', 'MP_2', 'EE_1','EE_2') replicates <- c('TE', 'TE', 'MP','MP', 'EE','EE') col_…

DESeq2

updated 4.2 years ago • jkanbar

is that the first two paired samples are biologically distinct from the second two paired samples. Replicate group 1 is thus 159:73, 44:24, and replicate group 2 is 0:49, 0:68 - the groups are defined on the *paired* data. This situation...data', rather than 'where are there consistent differences in the ratios of paired data between replicate groups', you can use the nullProps = 0.5 option as gi…

baySeq baySeq

updated 11.9 years ago • Thomas J Hardcastle

a simple comparison between two conditions in a multi-condition comparison. For example: if I have 3 replicates for 3 cell types (A,B,C) collected at 3 time points (0,1,3 hours) (total 27 RNA-seq datasets). How should we set the contrast...to compare the replicates of A-0 hour to A-3 hours? Thanks!!! [1]: http://bioconductor.org/packages/devel/bioc/vignettes/DESeq2/inst/doc/DESeq2.html

DESeq2 RNASeq

updated 4.4 years ago • hwu12

this type of analysis if they have any advice for experimental design, in particular, the number of replicates they have used and why (I was planning on going for all biological replicates, no technical). Thanks Mick [[alternative

edgeR DEGseq DESeq edgeR DEGseq DESeq

updated 15.6 years ago • michael watson IAH-C

and different sequencer. So I have 2 big sources of batch effect: 1. Different biological replicate (different mothers) 2. Different sequencing run (this is the biggest batch effect) I wanted to run DESeq with the following...design formula: `~Replicate+Sequencing_run+condition` but I got the error of "Not full rank model matrix". Here is the chunk of code that I run: ```r...lt;- f…

DESeq2 BatchEffect RNASeq

updated 3.2 years ago • Flavio

the sample names (BI,BM,BI2,BM2) stand for. In particular you need to tell us what are biological replicates and what are technical. If the same label appears twice in your targets frame, it is just a technical replicate of...BioC] about limma linear models > > Thank you for your reply. actually I have two biological replicates in > my experiment, and I'm confident with the…

Microarray limma DOSE Microarray limma DOSE

updated 14.7 years ago • Gordon Smyth

in toptable in LIMMA package. I know the >M in toptable is not a simple average from all the replicates, it is a >coefficient value from lmFit. I have three biological replicate array >and within each array I have...six technical replicates. My commands are the >following: > >design <- c(1,1,1) > fit <- lmFit(MA.norm, design, ndups…

limma DOSE limma DOSE

updated 20.9 years ago • Gordon Smyth

I 'm lost in the settings of the model.matrix for DESEQ2 or EDGER. I have two biological replicates with a short timeline. Treated Day1 \* 2 // Treated Day 6 \*2 // Treated Day 10 \* 2 Untreated Day1 \* 2 // Untreated Day 6 \* 2 // Untreated...model matrix in DESEQ2 or EDGER. I was wondering If I sould take account that the two biologicial replicates are linked in time. (BiolRep1 ->D0,…

DESEQ2 EDGER design

updated 9.2 years ago • ZheFrench

<div class="preformatted"> Hello, I would like to ask for your opinion on whether using replicated pools in the context of RNASeq experiments makes sense, or not. Lets say that we are interested in detecting genes that are differentially expressed in two genetic backgrounds (a certain KO mutant strain and the corresponding WT), in mouse liver. We could perform an RNASeq experiment using …

RNASeq Microarray edgeR RNASeq Microarray edgeR

updated 11.7 years ago • Guest User

<div class="preformatted">We are analysing a cDNA microarray dataset in LIMMA with the following design and we run into "Coefficients not estimable" comments. R = 2.9.0 limma=2.18 We have two groups, "A" and "D" with 6 biological replicates each (indicated in the targets file). We are interested in significant gene expression differences between A and...not estimable" comments. R = 2.9.0…

Microarray limma a4 Microarray limma a4

updated 16.1 years ago • Aubin-Horth Nadia

18282.89 16758.22 9854.22 9336.5 9091.05 for each transcript, there is three replication and two conditions. for the Mfuzzy, the input should be normalized. how can i normalize the data and how could use...the mean of three replicate as input for the Mfuzzy? Code should be placed in three backticks as shown below ```r # include your problematic code

edgeR RNASeq Mfuzz

updated 4.5 years ago • najib

outlier by PCA and by clustering over the entire transcriptome. I have 4 groups with 3 biological replicates.  These samples were run in 2 batches. When I try to summarize my reads using RSubread's featureCounts, the outlier...assignments   My question is how should I proceed with my analysis?  I don't have enough replicates to kick the outlier out.  Are th…

rnaseq outlier statistics batch effect

updated 9.6 years ago • grastalt27

or anything like it. Is that correct? If it is, would a limma analysis of one group with 1 replicate and one group with no replicates be considered sufficient (convincing, ;))? A two word response is good enough for me

limma limma

updated 20.4 years ago • davidl@unr.nevada.edu

Hi. I have ~30 biological replicate RNAseq data, and the plan is to measure the variance/dispersion of expression for each gene  and use this information...Hi. I have ~30 biological replicate RNAseq data, and the plan is to measure the variance/dispersion of expression for each gene  and use this information for the future dataset from this system for the improved inference.&a…

edger

updated 11.3 years ago • lwc628

Is there any need to set up a design or contrast matrix using limma ebayes on ratiometric replicates?  I have three replicate ratios that are log2 transformed.  I'm running ebayes:   data = read.csv(paste

ebayes limma bioconductor

updated 8.2 years ago • reventropy2003

Hi all, Please let me know how I should go about in fixing this error. <pre> > pData(bg) ids treatment genotype 1 B1C_01 control resistant 2 B1T_01 inoculated resistant > prr01_trans_result=stattest(bg_filt, feature="transcript", covariate="treatment", getFC=TRUE, meas="FPKM") Error in stattest(bg_filt, feature = "transcript", covariate = "treatment", : T…

R rnaseq Ballgown

updated 7.9 years ago • amy16

on high throughput screening (HTS) of compound libraries and have screened 125,000 compounds as 2 replicates (800 x 384-well plates as 2 sets of 400). As you know, Z score is traditionally used to normalize the data in each plate...to plate median and SD. However, since I am only 2 replicates, I am looking for a more comprehensive statistical analysis package for R, in order to identify false neg…

HTSStatisticalAnalysis highthroughputassays

updated 4.9 years ago • Hamidreza Hashemi

Error: grouping factors must have > 1 sampled level Grouping factors are: ``` group ngeneson replicate neuroblastoma.Bridge -0.791254569558456 jansky neuroblastoma.Bridge -2.1203581896722 jansky neuroblastoma.Bridge...colData(sca)$celltype <- celltype # same for donors (which we need to model random effects) replicate <- factor(colData(sca)$source) colData(sca)$r…

Mast mastR MAST

updated 22 months ago • Andrew

1 samples element names: ch1 phenoData sampleNames: 1 varLabels and varMetadata description: replicate: Replicate number assay: Biological assay additional varMetadata: channel featureData featureNames: 1, 2, ..., 288 (288...1 samples element names: ch1 phenoData sampleNames: 1 varLabels and varMetadata description: replicate: Replicate number assay: B…

cellHTS2 cellHTS2

updated 17.6 years ago • Steve Taylor

I'm having some trouble modifying my qPCR plate layout to be compatible with the R package HTqPCR. Please let me know if you have any suggestions. Unlike the example in the vignette, I have a "low throughput" plate, coming off of the ABI Viia 7 qPCR machine. I have 3 features (genes) and 5 samples in technical replicate.  I'm able to import my SDS txt file with a modified readCtData:&am…

htqpcr qPCR

updated 7.9 years ago • samantha_jeschonek

If I have two subgroups of data from two different siRNAs, lets say I have different biological replicates for two siRNAs ( 3 biological replicates each ) and compare against three control sets ( 3 biological replicates

edger differential gene expression

updated 9.9 years ago • g.atla

to use avedups() to average over the triplicate probes, and then to treat the patients as biological replicates (as blocks using duplicateCorrelation). But by doing so I do not understand how the two other replication levels...treated, that is extraction and hybridization. Is it possible to keep the information of this two replication levels in the analysis? Is it possible to set different levels…

updated 18.7 years ago • Caroline TRUNTZER

<div class="preformatted"> Hi all, i am using NOISeq with biological replicates.(i saw the Warning: NOISeq for biological replicates has not been tested yet. In case you use it, we will be very grateful to receive any feedback from you! , so, it will be a feedback in the end..) I have 2 conditions with 3 replicates for each. I normalized my data by using "tmm". i execute NOISeq as: myresul…

NOISeq NOISeq

updated 13.8 years ago • Guest User

the gene vector2 = intensities for negative controls wilcox.test(vector1,vector2) If I have 5 replicate arrays, I can perform the test as above just by combining intensities across experiments and comparing experimental...probes to controls as two vectors. I'm not sure this is really an appropriate way to handle replicates. Is there another function like wilcox.test, that can compare two matric…

Organism vsn limma Organism vsn limma

updated 19.1 years ago • Sturgill, David NIH/NIDDK [C]

dispersion? Typically using the commands (note that I compare here "B" to "C", thus samples without replicates). edgeR: countTable=read.table('mytable',header=F,row.names=1) ; dge <- DGEList(counts=countTable,group=c("A","A","A,"B","C")) ; dge...the end to estimate the dispersion) Does this correspond to the example "working partially without replicates" from the DESeq manual) ? Or should…

edgeR DESeq edgeR DESeq

updated 13.2 years ago • Yvan Wenger