Bioconductor Forum

nbsp; Hello, I'm very sorry for posting again a question regarding RNA-seq with no replicates. I've read most of the posts and still don't know what's the best way to proceed. My experimental design was: two industrial...and we took samples every 24 hours for a total of 4 samples. Due to a fail on the fermenter I have replicates only for the first time points. So My first approach was to use the…

rnaseq edger DGE no replicates

updated 8.3 years ago • Stefany Solano

experiments performed in different batches several months apart. Both experiments include technical replicates of the same 10 samples from condition A (with identical sample names: A1, A2, ..., A10). Exactly same control samples were...2: 10 samples from condition A and 10 samples from condition C. How can I use the technical replicates (condition A) to normalize for batch effects and perform …

limma DESeq2 edgeR

updated 11 months ago • anna.cot.anna.cot

I am new to RNA-Seq, Linux and R. I have raw counts from single-end RNA-Seq data for 8 different treatment conditions, with 3 biological replicates for each. I used the featureCounts command on Rsubread and have the raw counts matrix for all 24 samples at the meta...have raw counts from single-end RNA-Seq data for 8 different treatment conditions, with 3 biological replicates for each. I used the…

differential gene expression biologicalreplicates raw_counts

updated 7.2 years ago • tasnif.rahman

design is. I suspect this is why you haven't got replies yet. I can see that you have technical replicates, but I'm not quite clear that there is any biological replication. It's also unclear to me why you need a single channel...czyk <mjonczyk at="" biol.uw.edu.pl=""> > Subject: Re: [BioC] Loop design - biological, technical replication > and contrasts > &…

limma limma

updated 15.8 years ago • Gordon Smyth

but not with in a plate. Unfortunately my plate design is different. I have 384 well plates with 3 replicates for 32 genes in first 4 rows of the plate. Likewise other 3 samples in the next 3 sections of 4 rows. So in total I have

updated 15.8 years ago • jeremy wilson

Hi, I'm working with time series RNA-seq data, containig 6 time points. There are no replicates and there is no control groupe. I'm interested to find differential expressed genes between the first timepoint...3 groups like this: condition A: 1,2  condition B: 3,4,5  condition C: 6 (but they are not replicates). I first tried to do a multifactor design with the design (in orde…

deseq2 time-series without replicate

updated 7.4 years ago • bahlv

Hi I have a question regarding the Limma code. I have control samples (6 biological replicates, and each of the inturn have a technical replicate), I need to include all of them (12 samples) in contrast analysis...color:rgb(247, 247, 244)">In  the targets file, Control11 and control21 and technical replicates and so on.</span> So, I need  to compare 12  contr…

limma microarray block paired linear model

updated 11.0 years ago • vas

analysis with limma. My dataset includes related sample pre and post drug treatment. I have three replicates for each sample (technical replicates). I am using the treatment (yes or no) as a blocking variable. This is the code

limma technical replicates

updated 10.0 years ago • Eleni Christodoulou

div class="preformatted">Hi We have a collaborator with RNA-Seq data with no replicates - and yes, I understand the limitations of this. In the egdeR manual, one solution suggested to estimating common

updated 13.1 years ago • WATSON Mick

of a transcription factor. I have corresponding ChIP-seq data (one ChIP and one Input sample, no replicates unfortunately) and microarray gene expression data before/after forced expression of the TF. I'm considering...Am I doing something wrong / misunderstanding the output or is this simply related to the absence of replicates for the ChIP-seq data? Any help or ideas would be very much apprec…

Transcription Rcade Transcription Rcade

updated 11.8 years ago • André Faure

Date + mutations + treatment, data=pheno) block<-pheno$block \# this refers to the technical replicates dupcor <- duplicateCorrelation(rmat,design,block=block) dupcor$consensus \# gives 0.193 fit1 <- lmFit(rmat,design...lt;-model.matrix(~treatment, data=pheno) block<-pheno$block \# this refers to the technical replicates dupcor <- duplicateCorrelation(y,…

Limma removebatcheffect() duplicatecorrelation

updated 9.1 years ago • John

the number of samples and the number of model coefficients are equal, i.e., there are no replicates to estimate the dispersion. use an alternate design formula Calls: DESeq -> refitWithoutOutliers -> estimateDispersionsGeneEst...same number of samples and coefficients to fit, estimating dispersion by treating samples as replicates. read the ?DESeq se…

software error deseq2 metagenomics

updated 9.0 years ago • pro_zac

30 May 2003 17:28:45 +0100 >From: "Crispin Miller" <cmiller@picr.man.ac.uk> >Subject: [BioC] replicates and low expression levels >To: <bioconductor@stat.math.ethz.ch> >Message-ID: > <baa35444b19ad940997ed02a6996aae00b1448...gt; >I wanted to canvas opinion as to whether people feel we need to do this if >we have replicates and are us…

probe affy probe affy

updated 22.5 years ago • Eric Blalock

I have a dataset like this: <code>rep1_group1 rep1_group2 rep1_group3 rep2_group1 rep2_group2 rep2_group3 rep3_group1 rep3_group2 rep3_group3<br/>     18.26426    18.50355    17.87981    18.14181    18.12318    18.37539&…

pcamethods r

updated 9.6 years ago • stolarek.ir

am using edgeR for Gene expression analysis, my data is time-course transcriptome but no biological replication. I also know that there is meaningless without biological replication, but I have no choice. Now I want to analyze...keep.lib.sizes=FALSE] y <- calcNormFactors(y,method = 'TMM') I have no biological replication, here set dispersion = 0.01 fit <- glmFit(…

edgeR no replication glmLRT(fit coef=2)

updated 6.0 years ago • hong1ang

them uniform. In the tutorial HTSFilter is used , but it can not be performed on data-sets with no replicates. My problem is that i really want to use all the data-sets. What is the ( most likely ) best way to get to do this ? I am very

meta-analysis rnaseq metarnaseq

updated 9.1 years ago • bioinformatics

treatment and time x treatment effects ... under consideration of 1) sample pairing and 2) technical replication. My first (simple) approach neglecting treatment is as follows: ----------------------------- Line 1 > biolrep <- pData$Replicate Line 2 > timep...model where I can incorporate time AND treatment under consideration of sample pairing and technical replication at t…

updated 11.6 years ago • Otto, Benjamin

fold-changes in gene expression between treated and untreated cells, each group consisting of three replicates. The boss would very much like me to display some estimate of error in fold-change for genes of our immediate interest

edger rnaseq

updated 10.5 years ago • tstueve

<div class="preformatted">Dear list, I have a smallRNA sequencing data from two samples, without replicates. I tried using DESeq to identify differentially expressed gene following the manual, and the code is as below: &gt...div class="preformatted">Dear list, I have a smallRNA sequencing data from two samples, without replicates. I tried using DESeq to identify differentially exp…

Sequencing miRNA SmallRNA DESeq Sequencing miRNA SmallRNA DESeq

updated 15.0 years ago • Wu, Xiwei

was added to the cells and we want to know how expression changed along time). There are no replicates. Not having replicates seems to cause quite a lot of problems. I assume I'm just not using the right packages/calls

limma limma

updated 13.4 years ago • Guest User

div class="preformatted">If you really have good replicability, you should be able to use a gene-by-gene 2-sample t-test. --Naomi At 03:26 AM 2/19/2004, Matthew Hannah wrote: >Thanks...gt;to see what people think. > >Basically the data is highly reproducable between biological replicates but >there is a big 'treatment' effect (this is what we want!?). For example…

limma gcrma limma gcrma

updated 21.8 years ago • Naomi Altman

div class="preformatted">Hello! I am digging through the control intensities for technical replicates obtained with an Affymetrix GeneChip Rat Gene 2.0 ST array. I have 3 technical replicates of the same sample. The...across each row in my data set, which corresponds to each X,Y intensity measurement across my replicates for each probe. I see an average coefficient of variation at 22% just …

probe probe

updated 11.8 years ago • Matthew Thornton

suggested in edgeR manual. However, I was wondering is there anything specific I need to do for no-replicate dataset when applying  estimateGLMTrendedDisp and estimateGLMTagwiseDisp functions? Should I leave...it in ? I was also wondering if this is the best approach for time series analysis with no replicate ? In EdgeR manual also mentioned that a reduced model matri…

edger

updated 6.1 years ago • sirintra

Dear all, I'd like your opinion on the best way to compare two technical replicates. First, I give you some preliminary information on the experiment. I isolated total RNA from the same sample (mammalian

RNASeq

updated 3.6 years ago • Marianna

is not the same number of copies for each probe in this block. Then we have not regularly spaced replicate spots on the same array. > Please, check the gal file by human Agilent microarrays sent as Email attachment, in which

probe limma probe limma

updated 17.5 years ago • Gordon Smyth

div class="preformatted">Dear list, I am trying analysis without replicates for a large data set of small transcripts and I am getting the following error Error: condB %in% levels(conditions

updated 13.3 years ago • Andreia Fonseca

data-sets. In many forums and their manual also, they declared that if you do not have "any replicates", you should use __Fisher's exact test__ instead of __t-test__. I changed properly all the functions and parameters

bsmooth without replicate dimension

updated 7.4 years ago • nilaycan

my example, I compared 2 different peak callers in 3 cell lines: SampleID,Tissue,Factor,Condition,Replicate,bamReads,bamControl,Peaks mES_H3K27me3_m,ES,H3K27,mES_H3K27me3,1,reads/H3K27me3/ES_H3K27me3_m.be d,reads

updated 13.2 years ago • Paolo Kunderfranco

I have a set of 10X scRNA-seq data from 8 samples which include 4 biological replicates for each of the 2 conditions. The samples were processed in 3 days - 1 control sample on the first day, 1 control and

scRNA-seq Seurat DE

updated 6.2 years ago • ilee8

stat.math.ethz.ch=""> > Subject: [BioC] Using Limma for peptide array analysis - technical > replicate issues and false positives > > Hi everyone, > > I am using Limma for analysis of peptide microarrays but have...fdr", n = 50) > > > The questions I have are: > > 1. I incuabted a technical replicate for e…

limma limma

updated 15.5 years ago • Gordon Smyth

Hi all , We want only the Gene Expression for the experiment without replicate, So i just want to confirm that is this code is wright or not . I am providing code below. And what should be the dispersion

edger

updated 9.9 years ago • Sushant Pawar

Hi all, I have a few questions regarding Limma. 1) I am asking a question with reference to https://support.bioconductor.org/p/48601/\#102609. This is a case where there are no replicates for samples, and we cannot run eBayes or toptable. I understand that running fit2$coefficients will bring out logFC...to https://support.bioconductor.org/p/48601/\#102609. This is a case wher…

Limma

updated 8.1 years ago • Nithisha

Dear Bioconductor list members, I am analyzing a dataset with both biological and technical replicates. There is 1 factor with 8 different levels, denoted A-H Biological replicates of a given level are denoted by 1,2, ... Technical...replicates corresponding to a given biological replcate are denoted 2a,2b.. I wish to compute the statistics of each of levels

Survival cdf annotate affy limma gcrma matchprobes affyio Survival cdf annotate affy

updated 18.2 years ago • Richard Friedman

other is Treatment. which package can be used for differential analysis between only two samples? No replicates. How can I do that? Thank you

r bioconductor limma edger deseq2

updated 7.9 years ago • Biologist

p-adj values are 1 when I call results and contrast "C" and "U." I am guessing that I have too few replicates (3 technical replicates) to observe statistically significant differences. In my PCAplot the samples were clustered

deseq2

updated 6.4 years ago • dennism9251

td> </tr>  </tbody> </table> </td> </tr> </tbody> </table>   We do not have replicates for any model (I hope we can still get some data from this model) we would like to compare changes in gene expression...dexseq and what are the steps that we need to consider before we do this analysis, would not having replicates …

deseq2 timecourse rnaseq

updated 8.4 years ago • novicebioinforesearcher

not the difference between the patients. The problem is >>how to deal with different levels of replicates and how to create a >>correct target file since I have no common reference? >>This is how the experimental...as the original MA object. For the patients with two biopsies I >>averaged over the technical replicates (dye-swaps) and put the…

limma limma

updated 21.6 years ago • Johan Lindberg

2 conditions, at 8 unevenly spaced timepoints with some missing data (NA) and variable number of replicates (2-3): nReplicates <- c(3,3,3,3,3,3,2,3, 2,3,2,3,2,2,3,3) replicates <- rep(c(1:16), nReplicates) timePoints <- rep(rep(c(2,4,8,16,30,45,60...expressed between the two conditions, dupfit <- duplicateCorrelation(data,design,block=replicates) fit <- lmFit(…

updated 11.3 years ago • Linus Schumacher

Hey! We analyzed some data in primary human macrophages however our upstream response to different cytokines has huge variation in the absolute level of expression of the genes.  We used DeSeq2 package to calculate the differential gene expression. However we are wondering if we lose on certain genes because of different absolute levels of expression even though the fold changes&nb…

deseq2

updated 7.8 years ago • gupta

ebayesG, coef = 2, adjust = "fdr", n = 50) The questions I have are: 1. I incuabted a technical replicate for each of the arrays in this series. They're not included in this analysis - all the above targets are biological...replicates. However there are are already intra-array replicates accounted for. I seem to remember reading somewhere that...limma can't handle both types of replicates at th…

limma limma

updated 15.5 years ago • K.Z.Nambiar@bsms.ac.uk

Hi I was analysing RNA Seq datasets of an experiment selected from GEO datasets. Alignment to reference genome was done using STAR algorithm and quantification of transcripts was done using Subread package. The output 'counts.txt' was fed into edgeR for performing differential expression. The data exploration step (MDS plot) revealed a considerable amount of divergence among the replicates of sa…

edger plotmds

updated 8.2 years ago • fawazfebin

Hi all,  I am using Limma for the first time to analyze my two-color microarray data. So, I would like to know the best way to proceed to create my Targets file. My experiment has the following technical replicates:         CY3                     &…

microarray limma TARGET FILE

updated 8.3 years ago • Sanches

I use deseq2 for rna-seq data in treated and untreated group. I have no replicate in each group. The result I get ordered by pvalue: log2 fold change (MAP): condition treated vs untreated  Wald...I use deseq2 for rna-seq data in treated and untreated group. I have no replicate in each group. The result I get ordered by pvalue: log2 fold change (MAP): condition treated vs untreated&…

deseq2

updated 10.4 years ago • Rui Guo

GSVA can be applied in my experiment. My experiment have 8 time point, and each time point have 2 replicates. The total n is 16 here, but I am wondering whether I can use GSVA in this 16 samples. Best wishes Guandong Shang

GSVA

updated 4.3 years ago • Guandong Shang

div class="preformatted">Hi BioC, I have 3 groups but I have only 2 replicates for last group. so, group 1 and 2 has only one Affy CEL file. I Did..LIMMA as below and I got some Exciting results: #---------------------------------- design...I got EXCITING results, what could be the logic,since i have 2 replicates for 3rd group only ? Could anyone point me out ? I highly appreciate your hel…

affy affy

updated 20.6 years ago • SAURIN

duplicates were from different sites within the same mouse. I had previously averaged technical replicates (same site, sequenced 2+ times). If I do: design = ~ MouseID + Sex + Condition I get an error. My understanding is that this doesn

deseq2

updated 5.9 years ago • mdsutcliffe5

considering differentiation in a cell line. About 7 time points for both control and treatment, no replicates I would like to use limma to find the differentially expressed genes between time points for control and treatment

Microarray limma Microarray limma

updated 13.7 years ago • Chunxuan Shao

most likely to have significant results from looking at the MDS plot. Can anyone suggest a single replicate analysis pipeline that would allow me to do this? I cannot do it with limma as it cannot do the statistical analysis

minfi methylationArrayAnalysis

updated 4.7 years ago • chay

Hello, I am working with Bsseq to find DMRs in single replicate samples (Control and Expt) from WGBS. The bsseq object creation and smoothing worked well. Although, I encounter...I using the command right? I am having hard time understanding if bsseq has options to handle single replicate data. If it can, which steps/options require changes? The documentation does not mention any special…

bsseq Bsmooth

updated 10.8 years ago • unmeshj

I read in the achieves of this list that median polish was not a good idea when there are no replicates, and that rlm would be better. In order to use rlm, one needs an expression set, which means I have to specify some sort

Microarray Microarray

updated 20.8 years ago • McGee, Monnie

Hello, today I had some problem in DE analysis results replication. I analyzed the same dataset, with exactly the same code, same DESeq2 version but i got different results in a comparison

RNASeqData DESeq2

updated 2.4 years ago • Matteo

Hi all, I'm sure you have already talked about how to treat biological replicates in RNA-Seq differential expression but I can't find information about how  to incorporate RIN values into...288pt"> <tbody> <tr> <td>Sample</td> <td>RIN</td> <td>Group</td> <td>Patient</td> <td>Clone</td> <td>Replicate</td&…

deseq2 differential gene expression covariates rnaseq

updated 8.2 years ago • pmoranlosada

containing all conditions of 1 experiment (eg containing both patients and healthy people) or within replicates of each condition (eg on data from patients and on data from healthy people separately)? Thanks indeed. </div

Normalization Normalization

updated 13.5 years ago • Ali Mohammadian

Hello, I have downloaded some data from TCGA. Some of the participants in the project TCGA-LUAD have multiple BAM files for RNA-Seq. Are these technical replicates? How can I figure out what these files are or can I used them all in a differential expression analysis? The left column...the participants in the project TCGA-LUAD have multiple BAM files for RNA-Seq. Are these technical replicates? …

gdc data TCGA gdcdownload

updated 6.9 years ago • coyoung

<div class="preformatted">I am trying to use the DEseq package without replicates. When I look for significant hits all of them are False. Could this be a real result? Below are the commands that I enter...div class="preformatted">I am trying to use the DEseq package without replicates. When I look for significant hits all of them are False. Could this be a real result? Below are the com…

DESeq DESeq

updated 13.7 years ago • Melissa.Martin@lshtm.ac.uk

each stimulus was conducted at the same time for all the donors so I assume there are no technical replicates, but I need to take in account the "donor effect" (or biological replicates). In the "Limma" documentation there are 2...lt;-topTable(fit2, number=1000, adjust="BH") *2/The second method I used is by treating donors as replicates separately:* I used the exact same "targets" data.frame,…

Microarray limma PROcess Microarray limma PROcess

updated 11.8 years ago • Santy Marques-Ladeira

For most of the patients i have a sample for the end of treatment time point. All samples have no replicates. After reading a lot about different approaches to do an analysis without replicates, i ended up with the manual

edgeR RNASeq multi

updated 4.1 years ago • kevin

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 • rgentleman

Hi, I would like to know what are the limits of analysis for the following dataset I have been given: __1 cell population__ subjected to the following conditions: * Untreated * Drug vehicle * Drug A 1uM * Drug A 5uM * Drug A 10uM * Drug B 1uM * Drug B 5uM * Drug B 10uM (There is an additional control cell population sample, which was not subjected to any of the above condition…

deseq2

updated 7.3 years ago • rowcyclecamp

2005-04-26 at 22:55 -0400, Naomi Altman wrote: >> Significance should be based on biological replication. If the 2 chips for >> group 3 are technical replicates, then the variance estimate for the test >> is probably...too small. > >> In theory, statistical tests need only 2 replicate in a single condition, >> as the nu…

Microarray GO Regression affy limma Microarray GO Regression affy limma

updated 20.6 years ago • Gordon Smyth