Bioconductor Forum

to analyze allele-specific count data from RNASeq experiments. In these studies, each biological replicate (n=18) has two columns: one with counts from the maternal allele and the other with counts from the paternal allele...for each gene. Thus, the data is paired since these counts are parsed from the data for each each replicate. We wish to fit a glm to the data that tests for a main effect …

RNASeq Normalization edgeR RNASeq Normalization edgeR

updated 11.5 years ago • Christopher T Gregg

regions based on smoothX using "findChersOnSmoothed". I am wondering if this function takes sample replicates into calculation. If not, how can I get the cher results based on mean values of replicates? Or the results with chers...of each sample listed. So one can count chers out of the total number of replicates for each gene that was picked up by the method. Thanks, Jianping ###############…

updated 16.8 years ago • Jianping Jin

I found a sample during QC that appears to be an outlier as it did not cluster with the other replicates in a multidimensional scaling plot. A pearson correlation also showed that it is more similar to the control...I found a sample during QC that appears to be an outlier as it did not cluster with the other replicates in a multidimensional scaling plot. A pearson correlation also showed that it …

DESeq2

updated 2.6 years ago • Sydney

hello,  I have 4 samples two control (one biological replicate) and two treated(one biological replicate). I have obtained the intensities for them called LFQ. Do you think deseq2

deseq2 proteomics label free

updated 9.3 years ago • Nemo

Hello I have a question about the consensus peakset used in DiffBind. I am using ChipSeq data to find statistically significant differential binding sites between two groups. After running Diffbind, I don't find any significant differential binding sites. However, if I take the intersection of replicates in each group, and then subtract the peaks found in the intersection of both groups. I sti…

DifferentialPeakCalling ChIPanalyser DiffBind chipseqDB

updated 2.8 years ago • narzouni

Getting gene information: FPKM Differential Expression Data Annotation Data Replicate FPKMs Counts Getting isoforms information: FPKM Differential Expression Data Annotation Data Replicate FPKMs...Counts Getting CDS information: FPKM Differential Expression Data Annotation Data Replicate FPKMs Counts Getting TSS information:…

cummerbund heatmap isoforms transcript_id

updated 8.7 years ago • domenico.somma

nbsp;the depths of DeqSeq2 manuscript) I have an experiment with 4 time points and 5 biological replicates and each biological replicate has 5 technical replicates (100 samples thus) The first two time points are really...hence we often expect less reads for some genes: After summing the reads from the technical replicates, I get this profile (OBVIOUSLY AN ARTEFACT) 0h GeneA 3 r…

deseq2

updated 7.2 years ago • xei6

I'm new at bioinformatics and use DESeq2 for the first time. I'm analyzing a RNA-seq data - 3 WT replicate and 3 mutant replicates. The MA plot show diagonal lines and I'm not sure if it's normal, or what it means. It looks too

deseq2 MAplot

updated 7.0 years ago • avitalwasser

data from Agilent using limma. The experimental design include a common reference (4 biological replicate), after 1hr treatment( 4 biological replicates) and after 3 hour treatment (4 biological replicates). The adjusted

Normalization limma Normalization limma

updated 17.4 years ago • Abhilash Venu

for my samples, but having difficulties getting started. I have 8 pairs of samples most with 3 replicates each (1 replicate each from 2 groups had to be thrown out because they didn't pass QC). So in general, each pair consists...of 3 control and 3 treated biological replicates. What I've tried to do is this: ``` group <- factor(c(1,1,1,1,1,1,2,2,2,2,2,2,3,3,3,3,3,3,4,4,4,4,4,4,5…

edger DGE

updated 6.3 years ago • ctstackh

color). My point is that I have just 1 sample TREATMENT and 1 sample REFERENCE. Neither technical replicates nor biological replicates are available. A statistical test to find differentially expressed genes between...the two conditions seems to me impossible (even the simple t-test) due to the absence of replicates. People who asked me to do the analysis were interested only in finding the genes…

Microarray Microarray

updated 13.2 years ago • Eleonora Lusito

dds, test="LRT") res <- results(dds)</pre>   Also, in another case if I have 3 biological replicates for each time point \[no treatment/condition\] and want to analyse across time points . It should be like: <pre> design...dds) <- ~replicates + time dds <- DESeq(dds, reduced=~replicates, test="LRT") res <- results(dds)</pre> Is it cor…

deseq2 rna-seq design matrix deseq

updated 7.2 years ago • thindmarsmission

errors: A. Error: could not find function "dispersionPlot" B. dend.rep<-csDendro(genes(cuff),replicates=T) Error in csDendro(genes(cuff), replicates = T) : unused argument(s) (replicates = T) C. mCount<-MAplot(genes(cuff),"hESC","Fibroblasts

cummeRbund cummeRbund

updated 13.4 years ago • Jack Luo

0.323417620577 > logFC: NaN > geneID: 38956 > > Raw data: > > Condition 1: > replicate 1: 2,372,532 good reads, female: 8.0 > replicate 2: 3,966,968 good reads, female: no signal > replicate 3: 1,389,571 good reads...male: no signal > > Condition 2: > replicate 1: 3,102,608 good reads, male: 5.0 > replica…

Normalization edgeR Normalization edgeR

updated 14.2 years ago • Gordon Smyth

GrpC2.1_healthy GrpC2.2_healthy I have different conditions within Group A biological replicates (meaning samples), only baseline condition for Group B biological replicates, and I have 2 technical replicates...that I could use the readCtData function but I am unsure of how to specify and tackle biological replicates and technical replicates or how to read all this in from one file. The ulti…

htqpcr

updated 7.8 years ago • Nithisha

trying to run DESeq on a dds (created from a gse using information from tximeta) in which technical replicates from the same library (run on two different lanes) have been collapsed, so instead of 20 samples, I now have 10. When...but the order of operations seems wrong, in that I would want to run DESeqDataSet before I collapse replicates. My design formula doesn't include 'lane' information, so…

deseq2 DESeq nrow == ncol is not TRUE

updated 5.5 years ago • maniermk

<div class="preformatted">Hi, I am trying to run DESeq2 analysis on sample design like this: sample replicate treatment A 1 no A 2 no A 1 yes A 2 yes B 1 no B 2 no B 1 yes B 2 yes (repeated for 4 different samples. eg. A-D). The interests of DE...div class="p…

DESeq2 DESeq2

updated 11.4 years ago • BJ Chen

better to complete the loop, with 4 hybridizations. I am not sure what you mean by "dyeswap and 2 replicates". Are you using 6 or 3 arrays, or is this the base design which you intend to replicate? If you plan to have 8 or 12 arrays...you have many more choices for the design. Finally, technical replication is not that informative. If at all possible, each hybridization should be done with an…

Microarray GO Microarray GO

updated 20.6 years ago • Naomi Altman

to analyse the single color data using the R+Bioconductor using the LIMMA package? We dont have the replicates instead of that we have pooled the samples before sending for the analysis. So is it possible to use the LIMMA package...without having any replicates. -- [[alternative HTML version deleted]] </div

Microarray limma Microarray limma

updated 13.6 years ago • Muralidharan V

Copying a question I received:  "_I have 10 samples each with two replicates. What is the most straightforward way to pull out a quick and dirty coefficient of variation accounting for replicates

deseq2 coefficient of variation

updated 9.1 years ago • Michael Love

a treatment.  I have 5 genotypes and would like your advice on the minimum number of biological replicates. Can I get away with 2 biological replicates per genotype per treatment? Thanks for your time, Catalina &nbsp

Deseq2 genotype experimental design

updated 8.8 years ago • Catalina Aguilar Hurtado

Hi I have the RNA-seq data and I am comparing two groups of tissue (6 replicates each) to obtain the DE genes using DESeq2. When I plot the adjusted p-values as a histogram, I get a bimodal histogram...instead of adjusted p-values. Does this indicate a problem with read counts of any of the biological replicates?? Kindly help

deseq2

updated 8.6 years ago • rdu234

Hello all, I need to ask two questions. I am new to this area. I have my epic array data from 4 different cells with 2 replicates. I have to use Minfi package. I have loaded my idat data into the environment. I checked tens of workflows and all of...to ask two questions. I am new to this area. I have my epic array data from 4 different cells with 2 replicates. I have to use Minfi package. I hav…

minfi

updated 12 months ago • Melisa Tecik

div class="preformatted">Hi, After viewing the various postings on this topic ("technical replicates (again!): a summary"), I couldnt find a solution to a pretty basic problem. I have two technical replicates per sample...something. What I have is this: 4 treatments: A,B,C,D For each treatment, I have 2 technical replicates (dye swaps), I want to average these in limma, and then look at comp…

limma limma

updated 21.6 years ago • Simon Melov

A. Taking a union of all the CCAT called peaks and calculating read count in each biological replicate OR B. Calculating the read count for each peak in each replicate whether or not it has been called in the replicate

ChIPSeq chipseq DESeq ChIPSeq chipseq DESeq

updated 11.6 years ago • Guest User

robust I means 'nearest to biology' and 'statistically acceptable'. My problem is how to summarize replicate signals for each probe before compute ratios. What do you think is the best way to have a mean signal from replicates...men? Is one of this approach wrong? Is the choose of the approach dependent from the homogeneity of replicates? Thank you Best Regards Remo Sanges BioGeM</div

Normalization probe gcrma Normalization probe gcrma

updated 21.7 years ago • sanges@biogem.it

any money; could I please ask you if the design gives what I want? You please imagine I have 1- 3 replications of fibroblast surrounding a tumor, 2- an 3 replications organoid (3D model) of this tumor and 3 - I have 3 replications

edgeR normalization cancer

updated 6.7 years ago • Fereshteh

to whatever system you are using. For example, I am looking for genes (on ATH1 array) that contain "replication" in GOterm. Good luck! Fangxin ####### #function used to search for GO terms ########### GOTerm2Tag=function(term) { GTL=eapply(GOTERM...Glen=sapply(GTL,length) names(GTL[Glen>0]) } ##Example (get GOID of the terms that contain replication) GO.rep=GOTerm2Tag("replication"…

GO GO

updated 20.1 years ago • Fangxin Hong

Now I have a bulk RNA-seq dataset, where I initially thought I could use the 3 donors as biological replicates, but it seems that the inter-patient differences are bigger than the effect that the treatment induces. Just to...illustrate: ![PCA illustration][1] So, since I don't have 3 technical replicates per donor for each timepoint (but each timepoint has 3 donors that I though I coul…

DESeq2 Timecourse

updated 4.9 years ago • K.

pre> > # the following is a partial listing! > head(metadata, 12) drug dose replicate 1 NONE NaN 1 2 A 1 1 3 A 10 1 4 A 100 1 5 B 1 1 6 B 10 1 7 B 100 1 8 C 1 1 9 C 10 1 10 C 100 …

deseq2

updated 9.2 years ago • kjo

Hello, I am hoping for some help in understanding which read normalization approach is appropriate for the biologically complex samples that I'm hoping to do DE analysis on. I'm interested in DE of a bacterial endosymbiont of an insect; specifically, I'm trying to establish which genes are differentially expressed in the bacterium when the host insect is infected with a eukaryotic parasite. So,…

deseq2 differential expression normalization

updated 8.3 years ago • RMRG

Hello all I am trying to analyze RNA-seq data at 3 different time points. I have two replicates for two time points while 1 replicate at the 0 timepoint. All the samples are with the same condition and I want to

timecourse edgeR

updated 3.5 years ago • Mug_qd

type of input data (counts vs. normalised counts) and am struggling to understand how to indicate replication. Should the replicates be averaged or separate columns with the same (?) name? Thank you very much for your help! With

TMixClust time series expression analysis

updated 5.7 years ago • kathrin.garschall

homogeneous cell culture to another that has been treated, and am using RMA. I only have 3 replicates of each. Would you recommend a 2 tailed equal variance t test? I also thought I read that with such few replicates

updated 22.0 years ago • Jason Hipp

SC1 SC2 SC3 ST1 ST2 ST3 gene1 30.2 1.9 32.2 7.4 1.1 8.4 gene2 ... SC1 = Untreated Replicate 1 SC2 = Untreated Replicate 2 SC3 = Untreated Replicate 3 ST1 = Treated Replicate 1 ST2 = Treated Replicate 2 ST3 = Treated...Replicate 3 Thnaks very much Marcelo Luiz de Laia, M.Sc. Dep. de Tecnologia, Lab. Bioqu?mica e de Biologia Molecular Universidade

updated 22.1 years ago • Marcelo Luiz de Laia

and hrp48- (red channel) against a non-specific RNAi control (green channel) in three independent replicates for each KO experiment. after reading the raw data files into an RGlist object called 'RG' i've performed background...QA/MA-plots2.png when i look to these plots i see the following two unexpected features: -in the replicates of hrp36, replicate 1 of hrp38, replicate 1 of hrp40 and rep…

Microarray Microarray

updated 16.1 years ago • Robert Castelo

Dear All, I am using package DiffBind, Due to the fact that I miss real biological or technical replicates, I am assesing the overlapping rates between different peak callers. Here is how I set up my database to read in...Cell lines, 4 Factor, 2 Replicates, for a total of 12 Conditions > rm(list=ls()) > setwd("/home/pkunderf/output/DiffBind/ES-CMN-CMA/") > test=dba(sampleShe…

CMA DiffBind CMA DiffBind

updated 13.3 years ago • Paolo Kunderfranco

Hi all! I have RNAseq data of different bacterial species in conditions A and B (three biological replicates each). I already performed differential expression analysis comparing condition B vs A for each species separately...Hi all! I have RNAseq data of different bacterial species in conditions A and B (three biological replicates each). I already performed differential expression analysis comp…

DESeq2

updated 20 months ago • Laura

Hi, I am interested in finding genes with large dispersion values in the same condition (all the samples are biological replicates of the same condition) and I do not want to make the assumption that genes with similar expression levels have similar...in finding genes with large dispersion values in the same condition (all the samples are biological replicates of the same condition) and I do n…

DESeq2

updated 4.8 years ago • raya.fai

and edgeR) for differentially expression analysis. In my current dataset I compare 3 biological replicates of control vs. 3 biol. replicates from a mutant. The resulting 4 top genes according adjusted pvalue by DESeq and...a very high variance. (The reason for this is, that this are genes located on the chrY and only one replicate of the mutant was male) My question is now, how can genes with su…

edgeR DESeq edgeR DESeq

updated 14.0 years ago • Steffen Priebe

Hi, 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. Suppose Mf, Bf, and Ef are coming

DGEList edgeR

updated 19 months ago • prity6459

div class="preformatted">hi i did two color array (agilent 44K two color) .one sample with two replicate. which test or algorithm can i use to find differentially regulated genes in R package. i have one samples with two...techincal replicate: 1.normal = cy3 treated= Cy5 = Cy5/Cy3 2.normal= Cy3 treated = Cy5 = Cy5/Cy3 thanx neeraj [[alternative HTML ver…

updated 14.1 years ago • neeraj rana

I would like to average the R / G expression values (on a Log scale) for all probes for which I have replicate spots (ideally it should work for any number of replicate spots, no replicate, or 1, 2 or more replicates). Does anybody...duplicate entries in RG$genes$GeneName? Limma can only average duplicates if you have the same nr of replicates per probe, so that doesn't work for me... For the sam…

Microarray probe limma Microarray probe limma

updated 15.7 years ago • Tom Wenseleers

with it. I am looking forward to finding the proportion of genes, whose pvalue from biological replicates are above 0.05. I have tried to use the following code: ``` BiocManager::install("genomicsclass/maPooling") ##Here, I just...the base to create pool and indiv in the following lines. Pool will contain pooled samples(technical replicates) and indiv the individual samples (biological r…

rowttests

updated 3.9 years ago • leonardorosas2001

alignment and I am starting from a gene count matrix and a colData that I made I want to collapse replicates however I am in doubt on how to deal with the mate or paired-end counts for each sample. my count matrix looks like...I have the counts for each mate, shouldn't they be just one for each sample? or should I collapse replicates of a sample but all R1(L001) replicates and R2(L002) rep…

deseq2

updated 5.7 years ago • jnaviapelaez

Dear Yong Li, This experiment has been designed in four blocks, each one representing a biological replicate of all five genotypes. The best and safest way to analyse it is to treat each of the four blocks as batches, removing...rcd11, rcd13, rcd14 and sro11) and wild- type > Col0. For the 4 mutants there are 4 biological replicates each. For Col0 > there are also 4 biological rep…

Microarray Normalization limma ArrayExpress Microarray Normalization limma ArrayExpress

updated 14.7 years ago • Gordon Smyth

to know if it is necessary or recommended to filter the data according to the Mean / SD ratio for replicates before running a comparison analysis using a test with a "FDR option" like SAM ? My understanding is that if I have...an important variability between replicates then the FDR would increase and be more than let say 5% so that these genes with such a variability would not be "selected

updated 22.4 years ago • Phguardiol@aol.com

that explains what is going on behind the scenes. How does limma calculate the logFC? I have three replicates in my data and I applied limma (contrast) to the full dataset as well as per replicate. For the most part I get the same

Limma Differential Expression Time-course Analysis

updated 6.5 years ago • mcfarn1

div class="preformatted"> Dear all, I have 4 samples of HT 430mgpm array plate. Three of them are replicates of wild types and 1 is a tumor condition with no replicates. But I do not have any other information regarding how...should I be doing to get differential expression of genes in tumor as compare to the 3 wild type replicates that I have . I am very new to this field and so I am not sur…

affy limma affy limma

updated 13.6 years ago • hsharm03@students.poly.edu

batch** to my model matrix formula, and there was **one** sample from a 2nd batch with a technical replicate from the 1st batch. The reason I have this sample to begin with is to help me do future batch correction, as [explained...here][2]. While removing the sample, combining the technical replicates via ```sumTechReps``` or fixing the formula to be **"~0 + grp + age + sex + batch"** addressed …

limma

updated 2.2 years ago • Jonathan

and 2 patients for each diet. My pData(x.norm) looks like this: sample replicates sibship A96_hA_09_113_1_base.CEL 1 Control_Diet_1 113 A96_hA_40_113_1_final.CEL 2 Treatment_Diet_1...103 My design matrix (for a paired t-test) is calculated as follows (from the Limma user guide): Replicates <- factor(pData(x.norm)$replicates) SibSh…

limma limma

updated 18.0 years ago • Groot, Philip de

has no conditions for comparison (so just the "control" condition, essentially). Three biological replicates of around 500 individuals each were set up, and about 20-30 individuals (per replicate) were taken and sacrificed...the data looks like this (with expression quantified for each `Sample` below): ``` Sample Time Replicate t0_r1 00 01 t0_r2 00 02 t0_r3 00 …

DESeq2

updated 2.9 years ago • Dunois

six different treatments (A,B,C,D,E,F) of a model organism, with four-fold biological (NOT technical) replicates. FASTQC revealed no abnormalites in the RNAseq data and after normalization (rlogtransformation) with DESeq2...articles/10.1186/s13059-016-0881-8) they state the following: "Reproducibility among technical replicates should be generally high (Spearman R2 > 0.9) \[[1](https://g…

deseq2 rnaseq PCA hierarchical clustering outlier

updated 9.0 years ago • wd

Is it possible to analyze data from barcode screens using DESeq2 (or a similar package, such as edgeR)? More details: I have a library of barcoded knockout strains (much like an shRNA or CRISPR library) so that I can look for fitness differences as measured by strain count changes after exposure to the experimental condition. After growth in liquid culture, I took an aliquot of the strain as t…

edgeR DESeq2

updated 3.4 years ago • nc2849

if this seems silly. I have recently gotten back results of two samples from a 454 and do not have replicates of either. I was reading the edgeR manual section about what to do about calculating common dispersion factors...if no replicates are available. One of the options was to use genes that are not suppose to be differentially expressed (ie housekeeping...housekeeping genes?? Or if anyone…

edgeR edgeR

updated 14.1 years ago • Tonya Mariko Brunetti

at gene expression over a time series. My design is as follows Time Replicates Group uncut_1_i 0 1 1 uncut_2_i 0 2 1 uncut_3_i 0 3 1 uncut_3_ii 0 3 1 hab00_1 0 1 1 hab00_2 0 2 1 hab00_3 0 3 1 hab01_1...over time,but the plot is difficu…

maSigPro

updated 5.8 years ago • dorothy.mitchell

<pre> What is going on here ? ChIPQCreport(dba_chip_init,facet=T,lineBy=c("Replicate"),facetBy=c("Condition","Factor"),reportName=paste0(filename,"Marks",collapse = "_"), reportFolder=dir_output,colourBy=c...pre> What is going on here ? ChIPQCreport(dba_chip_init,facet=T,lineBy=c("Replicate"),facetBy=c("Condition","Factor"),reportName=paste0(filename,"Marks",collapse = "_…

ChIPQC

updated 8.4 years ago • ZheFrench

Hi everyone, I have RNASeq Data, with 4 conditions, 2 replicates each (very far from ideal, I know). By looking at my data through heatmap and PCA, I see that there are relevant differences...within conditions, among replicates. To further analyze this dataset, I got the recommendation of using betaPrior=TRUE instead of the default FALSE

deseq2

updated 7.0 years ago • andrebolerbarros

p-values performed when using a continuous variable? I don't see how the condition: _"At least 3 replicates are required for flagging, as it is difficult to judge which sample  might be an outlier with only 2 replicates

deseq2 rnaseq

updated 10.5 years ago • enricoferrero

Hello there,  i'm doing a DEGs analysis based on RNA-seq data. I've got 2 experimental thesis and 3 biological replicates each. Here is attached the relative MDS plot for count data and i was wandering if it is a good idea to do not take into...i'm doing a DEGs analysis based on RNA-seq data. I've got 2 experimental thesis and 3 biological replicates each. Here is attached the relativ…

edger DEG mds plot

updated 8.3 years ago • baus87