Bioconductor Forum

Hello! I'm using edgeR on DMR mode. My data are: five different conditions (without a clear reference) in two different replicates (condA_1, condA_2, cond_B1, cond_B2...). there is a clear batch effect based on the replicate so i want to include a replicate column in the design matrix. I build the matrix like this: ```r model.matrix(~ 0 + groups + rep) groupsGII groupsGV groupsR groupsS g…

edgeR

updated 6 months ago • Francesco

here. Basically, I have always known that when using tsv files, there should not be any inferential replicates considered. But now, in the latest version of tximport, I guess something has changed? > txi.kallisto.tsv <- tximport...abundance summarizing counts summarizing length summarizing inferential replicates I would like to ask if, in this way, infer…

tximport

updated 6.5 years ago • Mozart

DESeq2 used the median of ratios for normalization count, instead of CPM and TPM I have three replication for EMT and EWT. Is it possible to average EMT1,2,3 to represent one normalization count number? Thanks in advance

DESeq2

updated 4.6 years ago • Yosapol

Hello -  <span style="line-height:1.6">I have a series of 12 seq datasets with two replicates and would like to perform clustering analysis of the rlog-normalized data. However, after rlog transformation...Hello -  <span style="line-height:1.6">I have a series of 12 seq datasets with two replicates and would like to perform clustering analysis of the rlog-normalize…

deseq2 rnaseq

updated 11.1 years ago • snf

noel0925 at="" sbcglobal.net=""> > Subject: [BioC] Question regarding handling technical replicates for > Affy arrays > To: bioconductor at stat.math.ethz.ch > > Hi All, > > There seems to have been much discussion...regarding > the statistical issues surrounding handling of > technical replicates in gene expression data u…

GO Clustering affy limma GO Clustering affy limma

updated 19.5 years ago • Gordon Smyth

I would like to normalize some RNA-Seq samples without replicates using different techniques than RPKM. Thanks for any suggestions.  Humberto

normalization

updated 9.5 years ago • humberto_munoz

differential expression analysis, specifically focusing on a scenario where one group has replicates while the other does not. I would like to generate a comparison between these two groups and understand whether...package. I'm wondering if the NOIseq package supports this kind of analysis where one group has replicates and the other does not. Can I perform a comparative analysis between t…

NOISeq withoutreplicate Noiseq

updated 2.3 years ago • Rutuja

<div class="preformatted">I have tried to search the BioC archives but have not found posts dealing with both paired samples and technical replicates in a setup like mine. I have 3 patients A, B, and C from which I have 4 samples: M.early, nM.early, M.late, and nM.late. Each sample has a technical replicate resulting in 24 arrays (Affymetrix). biolrep Patient M.early nM.early M.lat…

updated 18.2 years ago • Charlotte Schjerling

Each biological sample has multiple sequencing runs, one sample has two runs, resulting in technical replicates. I'm seeking guidance on the optimal strategy to incorporate these replicates into my differential expression...analysis. Specific Questions: Merging Technical Replicates:Should technical replicates (multiple sequencing runs from the same biological sample) be merged: before al…

DESeq2 TechnicalReplicates RNASeq

updated 8 months ago • questionanswer

I have basically to compare WT vs KO data. The microarray was done first with 3 true biological replicates and later with 4 technical replicates with a pool of RNAs. > My design is the following: >> targets > FileName...using ebayes with limma I realized I could not treat biological (WT1,2,3-KO1,2,3) and technical replicates (WT4-KO4) the same way. > I tried to …

Microarray Normalization Microarray Normalization

updated 13.6 years ago • James W. MacDonald

div class="preformatted">Dear All, In our experiments we performed 3 technical and 8 biological replications. But how can I handle the bad spots by using the relications? Please give me some details. Zhao</div

updated 21.8 years ago • zhao luo

Naomi Altman; bioconductor at stat.math.ethz.ch >> Subject: Re: [BioC] Limma -time course - no replicates >> >> You can use LIMMA. The key is to have time be a continuous variable, >> not a factor. So time (or a function...Schaffer; bioconductor at stat.math.ethz.ch >>> Subject: Re: [BioC] Limma -time course - no repli…

Regression limma Regression limma

updated 15.3 years ago • Yair Benita

RMA, not multi- array, not robust and MM as PM) [See> The plots and statistics -- Variance across replicates plot, page 4] But I do not know, is to use which instructions. Can someone help me? Thanks, Mohammad </div

updated 20.3 years ago • Mohammad Esad-Djou

I have an RNA-seq data set (8 treatments, 2 replicates) that I would like to process by `` voom `` transformation for analysis in `` limma ``. I also would like to include sample...I have an RNA-seq data set (8 treatments, 2 replicates) that I would like to process by `` voom `` transformation for analysis in `` limma ``. I also would like to include sample-specific quality weights, so I know usi…

limma edger voom voomWithQualityWeights limma-voom

updated 8.0 years ago • Guido Hooiveld

Dear all I am having Affymetrix Dataset(.cel) with replicates for different time points.(1hr- 4cntrl,4expt, 4hrs-4cntrl,4expts, and 1day-4cntrl and 4 expt). Do i want to run limma...Dear all I am having Affymetrix Dataset(.cel) with replicates for different time points.(1hr- 4cntrl,4expt, 4hrs-4cntrl,4expts, and 1day-4cntrl and 4 expt). Do i want to run limma separately

limma

updated 7.3 years ago • devarajsaranya

<div class="preformatted">Hello, I need to analyze an experiment with the following design: - Mutant vs WT - WT-treatment vs WT - Mutant-treatment vs Mutant We have 2 replicates of each experiment or chip. What method is the appropriate one to carry out this analysis type?. We have carried out...the following design: - Mutant vs WT - WT-treatment vs WT - Mutant-treatment vs Mutant We hav…

updated 20.5 years ago • Pedro Botías

into account the paired samples: "Pairs" column - The last thing is that some samples are technical replicates (Sample1 with Sample2, Sample3 with Sample4, Sample5 with Sample6) and I would also like to take this into account...design,block=targets$Pairs,correlation=corfit$consensus) In theory to take into account technical replicates I would use: > biolrep <- c(1,1,2,2,3,3,4,5,6,…

limma limma

updated 12.2 years ago • sbcn

cell lines (each derived from 1 individual) and I ran the following code: ``` exprdata<-read.csv("Replicates Merged.csv", row.names=1) metadata<-read.csv("Metadata.csv",row.names=1) metadata$ID<-as.factor(metadata$ID...1] The most variation is attributed to the different cell lines. However, some of the technical replicates do not cluster well. I thought it was best to me…

limma DifferentialExpression variancePartition dream

updated 12 months ago • abadgerw

that someone mentioned this before, but wouldn't find it in the archive. I normalized 9 arrays ( 3 replicates for each of 3 conditions) used GCRMA. Surprisingly, I found that for one gene (or more) that the normalized expression...GCRMA do something like "flooring". I checked RMA normalized data, it has identical values for two replicates for only one condition. If I use gcrma with fast=FALSE, …

affy gcrma affy gcrma

updated 20.3 years ago • Fangxin Hong

<div class="preformatted">Dear Sarah, Just put Pairs in the design matrix, for example model.matrix(~Groups+Pairs) and use duplicateCorrelation() for the biolreps. Best wishes Gordon > Date: Tue, 1 Oct 2013 17:55:34 +0200 > From: Sarah Bonnin <sarah.bonnin at="" crg.eu=""> > To: "bioconductor at r-project.org" <bioconductor at="" r-project.org=""> &a…

limma limma

updated 12.2 years ago • Gordon Smyth

<div class="preformatted">Dear Charlotte, Paired samples are normally handled as described in Section 8.3 "Paired Samples" of the limma User's Guide. There shouldn't be any conflict between this and your technical replicates. Best wishes Gordon >Date: Wed, 05 Sep 2007 17:54:42 +0200 >From: Charlotte Schjerling <araneus at="" mrna.dk=""> >Subject: [BioC] LIMMA: …

limma limma

updated 18.2 years ago • Gordon Smyth

I am doing a DE analysis comparing two strains, one wild type and one in which a repressor is overexpressed with 3. We expect that its downstream effectors will have very little expression and should come up as top hits in the DE analysis. We did the experiment in the presence and absence of drug and with 3 replicates per strain per condition. Without the drug, we see that all the counts for one …

DESeq2

updated 13 months ago • nicolettacommins

I have a read count for 34 samples including biological replicate. I need to make dendogram from read count to show correlation between biological replicates. Please share biocundutor

bioconductor

updated 8.5 years ago • nabiyogesh

<div class="preformatted">Hi All, I've been asked for analysis of qRT-PCR data. I don't have raw data (from qPCR machine) but excel file (which I planned to transform to tab-delimited text file). I've read manuals for r-packages available, ReadqPCR package seems most promising (taking into consideration data import). There are instructions about columns needed (sample, detector, Cq). I do…

qPCR ReadqPCR qPCR ReadqPCR

updated 12.8 years ago • Maciej Jończyk

of the system. I wanted to canvas opinion as to whether people feel we need to do this if we have replicates and are using statistical tests - rather than just fold-changes - to identify 'interesting' genes. Does the statistical

affy affy

updated 22.5 years ago • Crispin Miller

of interest). There are also many lines marked with "EMPTY" . There is only one GPR file, and no replicates at the moment. (I'm trying to help a friend deal with an existing situation, which is less than ideal - but that's what...eBAyes" and "topTable" do not work (as explained in many other threads, they will not work without replicates). Despite the fact that it's inadvisable to continue wit…

updated 12.0 years ago • Guest User

genetically identical mice for which we can keep the biological coefficient of variation between replicates down to about 10%. For these experiments, three or four biological replicates per group works well. If you can keep...bioconductor at="" r-project.org=""> > Subject: [BioC] Expt. design question on optimal number of replicates > (in edgeR or else where) > &am…

RNASeq Organism edgeR RNASeq Organism edgeR

updated 13.5 years ago • Gordon Smyth

Greetings I am working on analyzing the data from an experiment with multiple technical replicates but no biological replicates. FWIW, it's the same data set I have posted about in recent weeks. I think the experiment...would benefit greatly from some biological replicates. The question I have is this: given that the dispersion estimator algorithm is able to 'share' data among samples...experime…

updated 12.5 years ago • Michael Muratet

div class="preformatted">Dear Venu, limma requires replicates. Pretty much any statistical testing method requires replicates. You can run lmFit() without replicates, which...question over the years. I am a bit puzzled what you expect limma to do for you in the absence of replication. Best wishes Gordon > Date: Fri, 12 Oct 2012 10:06:40 +0100 > From: Venu Pullabhatla <ve…

GO limma GO limma

updated 13.1 years ago • Gordon Smyth

working on a ChIP-Seq data set. I want to compare two groups having only one sample each group. (no replicates in both group) I generated count matrix which element is the number of reads within gene region for each data set...not helpful because it is just comparing two numbers. Which package is better for the case without replicate based on your experiences? Thanks for your help in advance. S…

edgeR DESeq edgeR DESeq

updated 14.4 years ago • Woo, Sangsoon

<div class="preformatted">I have microarray data, which is 2 colour agilent human of 3 technical replicates. Green dye case and Red dye control. I have analysed in Limma, normalising within arrays and between arrays using aQuantile normalisation. I also have some Next gen RNAseq data that has been mapped to the Refseq transcriptome and I have these raw counts. However there are no replicate…

RNASeq Microarray limma RNASeq Microarray limma

updated 14.4 years ago • john herbert

since they are all so different. Due to the world being an imperfect place, I only have technical replicates, so all of my statistics are based on these technical replicates. (In the past, from similar experiments, I've been

affy affy

updated 20.6 years ago • Ken Termiso

animals have been replicated three times resulting in a total of 28 slides. How should this replication be handled in the analysis using LIMMA? Looking forward hearing from you! Jakob ------------------------------------------------------------------- Jakob Hedegaard Danish Institute

Microarray Genetics Microarray Genetics

updated 20.8 years ago • Jakob Hedegaard

style="line-height:1.6">) explains very nicely, with nice examples, what to do when there are no replicates.</span> For example, for at section 2.10, sub-section 2 (that is picking a dispersion value, based on your experience

edger roast

updated 11.0 years ago • Daniel

Hi, Is there a way to compare significant changes between sample without replicate ? Regards, Attis

fourcseq

updated 10.4 years ago • julienpontis

michael.watson at="" bbsrc.ac.uk=""> > Subject: [BioC] Limma, dye-swaps and uneven numbers of replicate > arrays > To: <bioconductor at="" stat.math.ethz.ch=""> > > Hi > > A simple question really - I have five biological...replicate RNA samples. > Each will have cells untreated vs treated, so that's five arrays. I > w…

limma limma

updated 19.5 years ago • Gordon Smyth

an adj_p_value < 0.0001). I am also having difficulty understanding how I should consider the replicates, whether as technical or biological replicates. Am I doing something wrong in setting up my model? This is an example

RNAs limma Differen

updated 21 months ago • alessandro

div class="preformatted">Dear All: I am trying to evaluate the correlation between biological replicates. Can I use cor() function in R to calculate correlation of M value after normalization? Is this method appropriate

updated 21.8 years ago • Hua Weng

conds <- c( "Sample1", "Sample1","Sample2","Sample2") since they are > only >> technical replicates, not biological. But I'm not sure what to do. > > If you set up your test this way, DESeq will assume that the variance...gt; between the replicates is all there is. Hence, roughly speaking, it > will > call a difference significant if it is l…

RNASeq DESeq RNASeq DESeq

updated 14.1 years ago • Michael Muratet

challenges that this design would pose for statistical analysis and therefore provided 3 biological replicates for sequencing for each tissue and growth condition (at lower depths than for the pools). We would then use the replicate...manual from edgeR (Chapter 2.12) and thought we could use the common dispersion estimated from the replicates for the pools. This would mean that the same dispersio…

edgeR RNAseq

updated 2.8 years ago • k.roelfs

the reference and the 8 individuals from each of the 3 treatments used in only one hyb (no technical replicate). For each treatment, 4 biological replicates would be labelled in cye 3 and 4 biological replicates would be labelled...design in terms of dye labelling for the reference and the treatments, but no dye swap/ technical replicate for a specific fish. The goal is to capture as much biologi…

Microarray limma Microarray limma

updated 14.5 years ago • Aubin-Horth Nadia

for Cut&Run data. It is from rare types of tumors that unfortunately was not possible to create replicates. However I have data from different tumors subgroup (1, 3 and 4) as showed in the sample sheet below. > diff SampleID

DiffBind

updated 4.4 years ago • Danielle

please correct me when I’m wrong during the conversation.I have 5 libraries (unfortunately without replicate) as below,  Control-Sensitive cell line __(CS__) Treatment-Sensitive cell line after 12 hours __(TS12__...hours __(TT12__) Treatment-Tolerant cell line after 24 hours __(TT24)__ I’m aware of the replication importance, however, at this momen…

edgeR dispersion value replication

updated 9.3 years ago • Sara

div class="preformatted">Hi The title says it all really - how do I average over replicate spots from an MA object in limma? Thanks Mick</div

updated 21.5 years ago • michael watson IAH-C

functions in R to use bioconductor for more powerful analysis. We have many RNAseq libries with out replicates. And I read edgeR document and understand, not much use of doing any significant analysis. But, now, we are in a position...to have biological replicates. But, we are trying to decide the number. I understand more is merrier. But, what is a good number? If that is too vaguge...to sugges…

RNASeq Organism edgeR RNASeq Organism edgeR

updated 13.5 years ago • gowtham

gene set analysis using global test or another method (suggestions?) but I only have 3 biological replicates for each condition and I am not sure which method is the most powerful to use in this case. Also, has anyone compiled

Pathways Pathways

updated 19.4 years ago • Anthony Bosco

gt;Cufflinks>CummeRbund etc. I'm having real trouble working out how to handle my biological replicates, as there doesn't seem to be much documentation or discussion on these newer tools. It seems like most people would

ballgown stringtie

updated 8.7 years ago • bhawley1991

I am using DESeq2 to estimate fold changes between two conditions neither of which have any replicates. Will it be of any utility to use log2fold shrunken changes over the non-shrunken ones in this case

deseq2 rnaseq

updated 7.8 years ago • bn3301

and use Interactions to direct desired comparisons, if the datasets contain a differing number of replicates and different treatments?** These datasets contain a differing number of biological replicates (2 to 4) and different...Is this possible when the datasets have varying treatment conditions and a varying number of replicates? Or will I have to create independent sample tables for each…

Interactions DESeq2 Design

updated 4.9 years ago • vanbelj

ChIP-enriched regions (chers) that I hope someone can clarify. Specifically, I have four biological replicates and I find that, while cher finding on the smoothed reporter signals forms nice "peaks" in the found chers, biological...replicate correlation of probe intensities within the cher is not considered for ordering, and therefore many show bad reproducibility...of parameters miss positive co…

Ringo Ringo

updated 16.2 years ago • Diego Villar

<div class="preformatted">Dear all, I have normalized two technical replicates (same RNA) with marray. After normalization, should a scatter plot of one array against another yield a slope of 1...div class="preformatted">Dear all, I have normalized two technical replicates (same RNA) with marray. After normalization, should a scatter plot of one array against another yi…

Normalization Cancer marray Normalization Cancer marray

updated 21.3 years ago • Richard Friedman

Seq dataset produced on a Illumina GA and wonder how to account for both technical and biological replication using edgeR. The dataset consist of 48 individual samples (time series with 4-6 biological replica per time point

edgeR edgeR

updated 15.6 years ago • Jakob Hedegaard

replicates. > I assumed I can get the toptable output, but without the statistics due to > lack of replicates. Can you please...gt; On 14/10/12 23:47, Gordon K Smyth wrote: >> Dear Venu, >> >> limma requires replicates. Pretty much any statistical testing method >> requires replicates. >> >> You can…

GO limma GO limma

updated 13.1 years ago • Gordon Smyth

Hi, I am new to RNA-Seq analysis so apologies in advance if anything is not clear. I want to compare the gene expression of a mutant (knockout) strain with a wild-type strain. I have 3 replicates for my mutant (Da) and 2 for my wild-type strain (WT). As you can see in the image below, after running DESeq2 on galaxy I...want to compare the gene expression of a mutant (knockout) strain with…

DESeq2 Galaxy pca

updated 2.8 years ago • mbxdn1

it appears to be the only published and packaged method for correcting batch effects using technical replicates profiled across batches. However, I must be doing something wrong because I can't get it to work. I have a non-ideal...C + the same 12 samples with condition B So, the 12 samples with condition B are technical replicates. I'm interested in comparing the 40 samples with condi…

RUVSeq ruvseq RUVs DESEq2 deseq2

updated 6.8 years ago • enricoferrero

I haven't been able to find information about how to deal with the following issues: 1) technical replicates We have two biological samples, two libraries (of different insert size) were prepared for each of them. so I have...vector as conds <- c( "Sample1", "Sample1","Sample2","Sample2") since they are only technical replicates, not biological. But I'm not sure what to do. 2) Paired…

RNASeq DESeq RNASeq DESeq

updated 15.6 years ago • Jinfeng Liu

Dear Dr. Patro, developers of Salmon and BioC community, I have put great amount of effort to find out answer to this question on internet. Couldn't find it anywhere (bioconductor, biostars, seqanswers, github etc.). I have 3 replicates for each sample and one of the replicates from each sample is single-end and other two are paired-end as SE and PE were processed at different facilities (I kno…

salmon tximport limma-voom

updated 8.2 years ago • Sandip Darji

<div class="preformatted">Dear Neeraj Rana, You targets file is ok, although your samples are really normal and tumour rather than wt and mutant. You can use design <- model.matrix(~factor(targets$Cy5)) colnames(design) <- c("NodNeg","PosvsNeg") ... topTable(fit, coef="PosvsNeg") Best wishes Gordon > Date: Fri, 25 Jun 2010 13:55:46 +0530 > From: …

Cancer Breast limma Cancer Breast limma

updated 15.4 years ago • Gordon Smyth

I am using **vst()** from *DESeq2* and I need to estimate shrunken log2 fold changes between condition A and B(neither of which have any replicates). ```vst_data <- varianceStabilizingTransformation(dds)``` I am using **vst()** since low counts have a higher relative variance...I need to estimate shrunken log2 fold changes between condition A and B(neither of which have any replicat…

DESeq2 log2FC vst()

updated 17 months ago • lifeonmars

<div class="preformatted">Dear list, could anybody with more experiencing in the analysis of Affymetrix exon arrays comment on the need of technical replicates. We would be analyzing around 100 samples from breast cancer patients in search for putative biomarkers and would...with more experiencing in the analysis of Affymetrix exon arrays comment on the need of technical replicates. We wou…

Cancer Breast Cancer Breast

updated 16.2 years ago • Marcos Pinho