Bioconductor Forum

<div class="preformatted">Hi, I am trying to estimate the variability technical replicates from different microarray platforms. The few papers I found on this topic and current opinion seems to suggest that technical replicates are not required for microarrays. However, I like to estimate the distribution of variablity of the genes and confirm...div class="preformatted">Hi, I am tryin…

Microarray Microarray

updated 17.6 years ago • Adaikalavan Ramasamy

div class="preformatted">Hi, I have an affymetrix experiment with the following target file: Replicates FileName Target 8 SA100Hu133plus.CEL LR 4 SA101Hu133plus.CEL LR 4 SA102Hu133plus.CEL LR 4 SA103Hu133plus.CEL...SA98Hu133plus.CEL LR 28 SA99Hu133plus.CEL LR where files with the same number under replicates column are blocks …

updated 20.6 years ago • Ron Ophir

and is it statistically sound, to use diffHiC for differential HiC analysis when no biological replicates are available? I know edgeR lists a few options for calculating dispersion with biological replicates, but none...certain about whether it is possible (or statistically sound) to create the "fit" object from non-replicate data which diffHiC relies upon for the rest of the analysis (Ch. 6 onwa…

diffhic estimate dispersion value glm no replicates deseq

updated 8.7 years ago • hwick1

div class="preformatted">Hi all, I have an experiment with several control and several experimental replicate chips, and the chips are not actual biological replicates, but rather represent replicate hybridizations or amplifications...value for the control side and one for the experimental side than it would be to input all of the replicate chip data? It seems that using all of the replicate …

Clustering Clustering

updated 20.6 years ago • Ken Termiso

Differentially Expressed Genes (DEGs). Basically I want to do statistical analysis from one replicate. I have the following design: 1. Group A (time point 1)      2 replicates 2. Group B (time point 2)&nbsp...nbsp;    2 replicates 3. Group C (time point 3)      2 rep…

deseq2 edger limma differential gene expression

updated 11.0 years ago • r.aprianto

elementMetadata = NULL, metadata = list())), colData = new("DFrame", rownames = c("Control Replicate 1", "Control Replicate 2", "NotchRNAi Replicate 1", "CphUp Replicate 1", "CphUp Replicate 2", "CphRNAi Replicate 1", "CphRNAi Replicate...2", "NotchCphRNAi Replicate 1", "Control Replicate 1 (3')", "Control Replicate 2 (3')", "Notch Replicate 1 (3')", "Notch Replicate 2 (3')"), n…

DESeq2 DifferentialExpression

updated 17 months ago • nhaus

generated using RMA analysis. Previously, we used to perform a comparison expression analysis of the replicates first (3 replicates for each condition) in order to determine the false positive rate. Is there a way to do something

updated 16.1 years ago • K Tre

Dear All, I have a question about taking into account technical replication in my model. I will greatly appreciate any help from your side! About my data set: * These are proteomics data (originating...corresponds to a particular protein and columns contain log-transformed protein intensities for each replicate. * There are in total 8 biological replicates, 4 …

limma proteomics replicates

updated 7.3 years ago • ivansilbern

a control treatment (Ctr) and 5 Cy treatments (Cy). For each comparison Cy-Ctr, I have 2 biological replicates per treatment that are dye swapped on the array. These biological reps are also in duplicate (so each sample is...was replicated, not the array, so new biological samples). So I actually have three "levels of replication": 1. technical replicates 2. biological replicates within a single …

Microarray limma Microarray limma

updated 12.3 years ago • Guest User

Hi, I am performing differential expression (DEG) analysis using DESeq2. I have no replicates, and I understand that without replicates, it is useless to conduct this analysis. However, I have no other option...I have created an R script for cases with no replication. Could you please review it and provide your suggestions? It will be great helpful for me to complete my work # Load

DESeq2

updated 13 months ago • kuttibiotech2009

Hello, Is there any guide or tutorial to process RNA-seq sample with biological replicates? I have 2 conditions, normal and tumor. The total number of sample is 20 with each normaland tumor has 5 biological...replicates. Thanks

rnaseq deseq2 differential gene expression

updated 7.1 years ago • bharata1803

tutorial http://www.bioconductor.org/help/workflows/rnaseqGene/ and the heatmap shows all replications. I would like to do heatmap on the average of replication. Could you please advice? Best, John

deseq2

updated 9.8 years ago • John

<div class="preformatted">Dear Gordon, I am analysing a simple two-colour microarray experiment camparing strain A vs. strain B with 3 biological and two technical replicates (dye swap). Hence, my experiment is similar to your example in section 11.1 of the limma user's guide. Our array has four...two-colour microarray experiment camparing strain A vs. strain B with 3 biological and two te…

Microarray limma Microarray limma

updated 20.2 years ago • Pie Muller

I´m using ProteoMM for my shotgun proteomics analysis. Unfortunatelly, I only have 2 biological replicates. I did a firts analysis with ProteoMM: 1) Data missing: Eliminated the data in which one value is 0 for one replicate...to estimate dispersion? ProteoMM has a model to calculate the LFC? and it's valid to use it with 2 replicates? can i use the statistic step? in conclusión, i corret …

shotgunproteomics Proteome proteomics Proteomics ProteoMM

updated 3.7 years ago • ary.lech

Is it statistically inappropriate to include a regression parameter representing biological replicate? Day D1:  performed experiment via protocol P1, obtained 3 technical replicates per condition. Day D1:  performed...experiment via protocol P2, obtained 3 technical replicates per condition.   Later on, i performed a biological replicate of these experiments... &am…

limma

updated 9.5 years ago • map2085

Hi, I'm really new to PCA analysis and just trying to understand how it works and the underlying stats. My problem is that when I run it for all my 3 biological replicates (3 Controls and 3 treatment) all 3 controls seem to correlate well but one of the "treatment" samples is really far off the other 2 (which correlate well with each other). So, I decided to try and re-run it after removin…

bayseq PCA

updated 6.7 years ago • bsad

were pooled and hybridized onto 3 separate slides. Of course the 3 slides are not biological replicates. They are not pure technical replicates either. How should I set up a design matrix for limma model analysis? Thanks

updated 19.3 years ago • Jianping Jin

interpretation correct? Does it make sense to use nested interaction formulas when I have only one replicate for each level of Time and Treatment. In your example placebo.0h has two samples.  My study has one sample for...interaction formulas, all genes reported FDR = 1. I wonder if this result is due to the absence of replicates.  Might you suggest, alternatively, other ways…

cancer

updated 9.2 years ago • rea

Hello, I have seen examples of design formulas that have the replicate factor and others that do not use it. **I was wondering when one should put the replicate factor in the design formula...have a dataset that has independent inoculations of whole plants in the greenhouse. We expected the replicates to be variable and the PCA showed it (of course they are less variable than the effect of the…

DESeq2

updated 4.7 years ago • DcL-A

Hello! When I do DE analysis of my RNA-seq data I often have 3-4 replicates of each condition. So, I want present heatmap contaning only condition1\_vs\_condition\_2. I realize that it will...not contain information about variations between replicates but I think it will be easy for understanding.   Thank you in advance, Alexander Gopanenko

deseq2 edgeR heatmap.2

updated 9.1 years ago • alexandr.gopanenko

that I have to analyze in order to find differentially expressed genes. I have 10 biological replicates, and each biological replicate has two technical replicates which appear as dye swapped. So in total I have 20 arrays...limma to do my analysis. I know at the moment it is not possible to treat duplicate spots, technical replicates and biological replicates, but I though if I use the duplicateC…

limma limma

updated 15.7 years ago • Ana Staninska

for the following questions that I did not understand during my data analysis. I have used 4 bio-replicates for treatment and control each. After following the pipeline to process data, I got FPKM value from cufflink. My...questions are: 1. When I looked at the raw FPKM value across the sample bio-replicates for ~1700 genes, I noticed that very few genes have FPKM value for all 4 bio-replicates …

rnaseq_analysis

updated 8.5 years ago • fakhter1

on Affymetrix chips. Could you suggest the best way to analyze this experiment? Is averaging both replicates and picking genes based on fold change good?? Thanks for any suggestions. Sohail Khan Scientific Programmer COLD

updated 19.7 years ago • Khan, Sohail

patients, where some patients have triplicate stem cell lines. Some of the triplicates are technical replicates (the same cell culture line was sequenced three times), while others are biological replicates (stem cells from...patient were independently cultured and sequenced). How should I deal with the different kinds of replicates? Should I merge all replicates or only the technical replicat…

DESeq2 RNASeqData

updated 12 months ago • Nikita

File4 pra3 banha3 banha is my reference and pra is my tester. Only the two first is technical replicates. The others two are biological replicates. How I could dealing a design for my situation? Thank you very much. -- Marcelo

limma SANTA limma SANTA

updated 6.5 years ago • Marcelo Laia

Hi! I´m 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...Hi! I´m 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.…

technicalReplicates BatchEffect limma RNASeq

updated 19 months ago • catv

hi, I have a question regarding technical replicates of the same biological sample. I have for a sample the following: sample1-rep1 sample1-rep2 Sample2-rep1 Sample2...rep2 etc up to 12 The individual samples are biological replicates while the the sample (rep1 and 2) are technical replicates from the same tissue. My question is, for DESeq2, should.…

deseq2 normalization

updated 5.2 years ago • A

Hi all, Can someone enlighten me as to the justification for summing counts across technical replicates in DESeq2, especially with respect to the collapseReplicates() function? I would have thought that statistically...the correct thing to do would be to build a column into the design matrix to account for technical replicates and include all samples. This effectively doubles (or x N for N tech…

deseq2 rnaseq replicate differential expression

updated 7.7 years ago • kieranrcampbell

Hello, First off, I appreciate everyone reading this and providing insights. I'm aware what I'm asking is not statistically sound by any stretch of the imagination, however the current data I have is three samples (one replicate each). The conditions are such that two of the samples share a condition (8 weeks vs 3 days) and two of them share another condition (hypoxia vs normoxia). I was a…

deseq2 replicates error DESeqDataSetFromMatrix

updated 6.7 years ago • Yonatan Amzaleg

div class="preformatted">Hi, We are trying to use Limma code for a Time Course Experiment without Replicates for 4 times. From looking at the manual, looks like don't Need replicates. But the ebayes routine complains about...no residual Degrees of freedom. Can we use Limma for Time Course without replicates? Lana </div

limma limma

updated 15.5 years ago • Lana Schaffer

experiment with 2 treatments (S and F) in a loop design. For each treatment I have 3 biological replicates (S1 to S3 and F1 to F3) and for each biological replicate there are 2 technical replicates (a and b). Biological reps

updated 20.1 years ago • Cecilia McGregor

DESeq. Each column represents a treatment, or condition, that has the mean counts of two technical replicates; each row represents the FPKMs (count reads) obtained from CuffCompare after our RNA-seq data was processed through...Bowtie and Cufflinks. In our experiment we used a technical replicate for each condition and, according to the user guide provided by Simon Anders, we must sum up their c…

GLAD DESeq GLAD DESeq

updated 13.2 years ago • Andres Eduardo Rodriguez Cubillos

Hi all, Please provide me the commands of ___"edgeR" ___for differential express analysis without Replicate

edger without replicate

updated 10.0 years ago • Sushant Pawar

I have managed to sequence the transcriptome of single replicates of very rare samples with low yield so technical or biological replicates are almost impossible to obtain. I have...used salmon to quantify the expression and inferred 10 bootstrap replicates of each Which approach is the best one to use for downstream analysis? I know that significance without variance

DESeq2 NOISeq edgeR

updated 4.9 years ago • Themis

itself. Below are a number of issues I can see: 1) First and most importantly, you have only 2 replicates per condition. Although PLGEM is capable of dealing with such a dataset, it is far from being an optimal case. You...should try to have a least 3 or 4 replicates for at least one of your experimental condition (e.g. the baseline condition). 2) Secondly there are only 802 proteins...in your …

plgem cycle plgem cycle

updated 15.2 years ago • Pavelka, Norman

Hi, Does anyone know if I can use FourCSeq on my 4Cseq data in case that my data doesn't contain replicate samples? Thanks, Tal

fourcseq

updated 9.5 years ago • tlgolan

You still need to indicate the biorep and use it as a block, even if only one biorep has a replicate. --Naomi At 09:55 PM 9/21/2009, Marcelo Laia wrote: >Hi, > >On the limma usersguide I found the example: > >FileName...banha3 > >banha is my reference and pra is my tester. > >Only the two first is technical replicates. The others two…

limma SANTA limma SANTA

updated 16.2 years ago • Naomi Altman

the counts belonging to one biosample. I understand the meaning of this if the number of technical replicates per biosample is the same for all samples. Please tell me what should I do if the number of technical replicates...the arithmetic mean is not very reasonable. For example, I have counts of 181 and 2 for different replicates of the same biosample. Note: this situation is not observed fo…

DifferentialExpression RNASeq DESeq2 replicates StatisticalMethod

updated 4.1 years ago • poecile.pal

<div class="preformatted">Dear all, We have a mix of cell-lines run by 12 different labs (more than 150 samples in total) and we have found a strong batch effect by laboratory that we would like to correct. From those 12, there are 3 labs that are bringing just one cell-line with no replicates at all (1 sample). If we remove the samples from those 3 labs, we are able to run ComBat, but we…

updated 12.1 years ago • Pedro Furió Tarí

program. Currently I have six Chip-Seq samples, all of different condition. There are no biological replicate and no control sample. So when I try to run edgeR, it does not run for there is no biological replicate used for statistical...I know the program should run up till calcNormFactor and give normfactor but without biological replicate, it cannot calculate dispersion and the whole program fa…

edgeR edgeR

updated 12.8 years ago • Guest User

<div class="preformatted">Dear Alex, Do you have the same number of replicate spots for every gene of interest and are the replicate probes identical? If so, see the case study in limma User's Guide on "Within array replicate spots". If only some of your genes are replicated, or if the probes are not identical, I would strongly advice you not to attempt to pre-emptively average the spots.…

Normalization limma PROcess Normalization limma PROcess

updated 20.1 years ago • Gordon Smyth

I would like to analyze RNAseq with 6 conditions using limma/voom. I have 6 biological replicates for control, however for the other 5 conditions (say C1,C2,C3,C4,C5) I have no replicates. Ideally, I would like to do a...variance estimation using the 6 replicates for the control, and using it compare any two condition C1 vs C2. Does the standard workflow takes care of this? &nbsp

limma voom

updated 10.1 years ago • ea1402

div class="preformatted">Hi, I have a problem with an exprSet, that consists of 16 samples with 2 replicates each, i.e. 32 arrays. Using > exprs(eset) I get the expression values for each gene in each array, with the two replicates...columns, like this: A1 A2 B1 B2 C1 C2 D1 D2 ... I would like to calculate the mean of the two replicates for each gene and generare a matrix of the me…

updated 20.0 years ago • Georg Otto

Hi, I have a question about dealing with a design that is a mixture of technical and biological replicates. It's 4 two color-arrays with the dye-swap structure: 1,-1,1,-1 array 3 and 4 are technical replicates of the same biological...source, so the biological replicate structure is: 1,2,3,3 This doesn't exactly fit the examples given in chapter 8.2 of the user's guide, maybe someone could

updated 16.4 years ago • Georg Otto

versus undifferentiated cells) were compared directly on two-color oligo array, with 3 biological replicates (different cell sources) and 4 technical replicates (arrays) per biological replicate (12 arrays altogether). In...every set of technical replicates two arrays are dye-swap. I am not sure how to handle the technical and biological replication when trying to fit...12.gpr D3 N3 Where …

Genetics oligo Genetics oligo

updated 17.3 years ago • Kateřina Kepková

contains two histone modifications (me1 and me2), both mapped in WT and mutant cells. I don't have replicates for any of those. Before I do more replicates, I want to test if there are any significant differential binding events...for both histone modifications. Can I analyze this data using DiffBind even though I don't have replicates?    Thank you!&nbsp

diffbind bioconductor differential binding analysis without replicate chipseq

updated 8.0 years ago • Hesh

div class="preformatted">Hi, How do I write the design matrix to specify technical replicates in multi-group experiment on a single-channel array (data coming from lumi)? I have 3 groups of samples (A, B, C), each group...has 2 technical replicates (so I have 6 expression vectors: A1, A2, B1, B2, B3). How do I write the design matrix to tell limma that A1 and A2 etc are technical...replicates…

limma limma

updated 15.7 years ago • Adam Kiezun

am testing the DEXSeq package with a public RNA-Seq data. Unfortunately this data set does not have replicates, only two set of data with two conditions. I tried DEXSeq but got error. I checked the estimateDispersion function...but there is no similar option like in DESeq where we can use for non replicate data. Is there any way to overcome this disadvantage of the data and finish the DEXseq anal…

DEXSeq DEXSeq

updated 13.9 years ago • Duke

I got an RNA-Seq project where the data looks like as in the PCA plot below - 4 biological replicates of 4 conditions, and there are 4 batches with each batch consisting of 1 sample from each condition. In essence...I got an RNA-Seq project where the data looks like as in the PCA plot below - 4 biological replicates of 4 conditions, and there are 4 batches with each batch consisting of 1 sample f…

BatchEffect DESeq2

updated 2.5 years ago • gtechbio

like to know if it is possible to use deseq for data with multiple groups (more than two) with no replicates. I went through the vignette but it only illustrates the case for two groups with no replicates. If it is not possible...method to study similarity/dissimilarity of gene/protein count data based on multiple groups with no replicates. I appreciate your feedback. Yolande [[alterna…

DESeq DESeq

updated 14.7 years ago • Yolande Tra

turned molecular geneticist?) I have a question that concerns analyzing arrays that contain replicate spots using limma. I am having trouble with the function dupcor that calculates the correlations between the...replicate spots. My replicate spots are not near one another on the physical array ? when I printed the array I printed my 25 print...plates containing my probes twice, I wanted to tw…

probe limma probe limma

updated 16.8 years ago • Kevin J Emerson

information... The guide rightly points out that we must not treat arrays that are technical replicates as true replicate arrays as they are not independent. Sections 10.1 and 10.2 then go on to explain how technical...replicates can be handled using either duplicateCorrelation or by manipulating the contrasts matrix. However, both these...data from a common reference design, comparing two gro…

Microarray GO limma Microarray GO limma

updated 20.2 years ago • michael watson IAH-C

aspect for differential expression analysis. For some practical reason, we could not have biological replicates. However, in order to perform mRNA-seq, we used technical replicates. Technical replicates here mean that: From 20...and powder was made. From this pooled sample, three samples were derived, which we called Technical Replicates and mRNA-seq was performed. Overall our experimental desig…

DESeq2

updated 2.8 years ago • Muahammad

I performed variance stabilized transformation on raw gene counts. Now for the future purpose, I want to combine replicates and calculate the log fold change values between control and experimental condition (I don't want to perform differential...variance stabilized transformation on raw gene counts. Now for the future purpose, I want to combine replicates and calculate the log fold change value…

DESeq2

updated 2.9 years ago • Jayesh Kumar

<div class="preformatted">I have a matrix like following > test=matrix(c(1:15),nrow=3,ncol=5) > colnames(test)=c("A","A","B","B","B") > test A A B B B [1,] 1 4 7 10 13 [2,] 2 5 8 11 14 [3,] 3 6 9 12 15 I want to calculate the average of each replicates, ie. I want the output to be A B [1,] 2.5 10 [2,] 3.5 11 [3,] 4.5 12 I can do this by looping through ea…

updated 14.7 years ago • Wendy Qiao

watson \(IAH-C\)" <michael.watson at="" bbsrc.ac.uk=""> >Subject: [BioC] Dealing with technical replication with a common > reference design >To: <bioconductor at="" stat.math.ethz.ch=""> > >Hi > >I have to say the limma...gt; >The guide rightly points out that we must not treat arrays that are >technical rep…

Microarray GO limma Microarray GO limma

updated 20.2 years ago • Gordon Smyth

is printed in triplicate. I normalized my data using limma package and I got mean from these three replicates by using duplicateCorrelation function: design<-modelMatrix(targets, ref="HL60") duplicateCorrelation(MAl.pAq...this function does not change object dimmensions (plot shows all spots, not only mean from all replicates), so I try to use avedups function (a<-avedups(MA.normAq…

probe limma probe limma

updated 16.4 years ago • Barbara Cegielska

I was told the following problem can be modeled in R/ S_PLUS. I have a data set with 3 replicates for the control and 3 replicates for experimental samples for a particular experiment. I performed a t.test and

Microarray Microarray

updated 19.6 years ago • Khan, Sohail

of RNA- seq data containing Control (CR) and two Treatments (HR and SR). One of the biological replicate for HR treatment failed leaving me with 2 replicates for CR and SR and 1 for HR. DESeq document described "Working...partially without replicates" however, that is not in the DESeq2 documentation. I want to check if I could use the code below to analyze data with...no biological replicate fo…

GO edgeR DESeq2 GO edgeR DESeq2

updated 11.8 years ago • Avinash S

I don't have replicates for two of the species I'm working on, I can't use Voom as it requires biological replicates. What is the alternative

limma edgeR Voom

updated 2.9 years ago • Mohammad