Bioconductor Forum

reviewers accepted the paper, and one reviewer rejected it, under the argument that having only two replicates per condition (control plants and plants infected with a fungus) does not produce reliable results. We argued...expression analysis in experiments with only 3 samples (2 degrees of freedom), and since we had two replicates per group (n=4; 3 degrees of freedom) our experimental design was…

deseq2

updated 5.1 years ago • jovel_juan

div class="preformatted">Dear all, my array contains 4 replicates of each gene. Each gene is present two times within one block and this block is present two times on the array. block...structure: gkooooo ooooooo ooooooo gkooooo ooooooo ooooooo I think that within one block (2 of 4 replicates) the "evenly spaced"-assumption holds true for all the genes. My question is, if the "evenly space…

limma limma

updated 21.6 years ago • Martin Kerick

even though it is a dye-swap experiment. Presumably this means I don't have to treat the technical replicates in any special way...? Mick -----Original Message----- From: Gordon Smyth [mailto:smyth@wehi.edu.au] Sent: 11 September 2005...gt; >The guide rightly points out that we must not treat arrays that are >technical replicates as true replicate arrays as they are not >i…

Microarray GO limma Microarray GO limma

updated 20.2 years ago • michael watson IAH-C

div class="preformatted">I have a conceptual question on normalization of replicate arrays. Let's say I have several replicate arrays. I wonder whether it is advisable or not to apply different normalization...methods to each of the replicate arrays. Let me illustrate with the following two situations. (1) By inspecting the raw data, some arrays look as if...tip lowess to some and global low…

Normalization Normalization

updated 18.0 years ago • Seungwoo Hwang

<div class="preformatted">I apologize in advance, since I only started using limma 2 days ago. I have a simple 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. Biological reps are from different plants, while technical reps are different RNA …

Genetics limma limmaGUI Genetics limma limmaGUI

updated 20.0 years ago • Cecilia McGregor

<div class="preformatted">Hi, Im new to limma, and I'm trying to determine a design matrix for the following type of experiment. I dont see an example of this sort of experiment which is becoming increasingly common. I have Diseased vs control (two color). I have 30 diseased individuals, and each individual has had 4-6 technical replicates carried out with dye swaps involved. My question is…

limma limma

updated 22.0 years ago • Simon Melov

Dear all, I am using ballgown to quantify differential gene expression in a data set after performing the hisat2+stringtie pipeline. I have read other questions about how to handle technical replicates in DE analyses but I still have some doubts on how to handle them, and the purpose of the `` adjustvars  ``option in...performing the hisat2+stringtie pipeline. I have read other question…

ballgown rnaseq differential gene expression

updated 8.1 years ago • o.diogosilva

div class="preformatted">Dear Christine, duplicate correlation in limma is only for replicate spots which are regularly spaced. The spacing value is the number of spots between replicates. Your value of 21 cannot...and 11 rows, and that is what limmaGUI is telling you. You haven't actually told us how the 4 replicates for each mir are arranged on the arrays. If your replicates really are …

GUI limma limmaGUI microRNA GUI limma limmaGUI microRNA

updated 17.1 years ago • Gordon Smyth

My question is what is the exact meaning of individuals? I have 7 bacterial colonies and three replicates for each one. Now i have 7 individuals or 21? Also i want to know is it recommended to use this package between subspecies

edger

updated 5.2 years ago • Ava_jm75

were the samples pooled? Were they pooled and then split, or are > there three distinct biologic replicates? > > The lack of dye swap IS a problem, as you will likely find dye- biased > probes (potentially MANY). > >> Of course...the 3 slides are not biological replicates. They are not pure >> technical replicates either. How should …

limma limma

updated 19.3 years ago • Jianping Jin

<div class="preformatted">Hello, We are using Affymetrix Drosophila microarrays to investigate central nervous system gene expression profiles at eight timepoints spanning metamorphosis. Each of the eight timepoints consists of three biological replicate samples. Unfortunately, our final eighth timepoint had to be hybridized to version 2.0 arrays, while all our other samples were hybridi…

Normalization Normalization

updated 15.7 years ago • Kaitlin Louise Bergfield

Dear all, I'm trying to combine the normalized counts from 3 biological replicates into single columns of the normalized count matrix. I need it in order to "absolutely" analyze the expression of...or other plots) and doing co expression analysis. I would like to emphasize that all 3 biological replicates are, in my point of view, identical, meaning, all are before any treatment, or all of them…

DESeq2 Normalization RNASeqR

updated 4.7 years ago • Marina

We have RNA-seq data for 12 samples for 12 conditions. Unfortunately, we do not have any replicates and each sample corresponds to one condition. For differential gene expression analysis, I will need at least...3 replicates (or patients) for each condition to be able to compare gene expression, which I don't have. I am interested in changes...gene expressions. Another thing I tried to solve t…

DESeq2 RESEQ RNASeqData

updated 2.7 years ago • 4214811

I am currently working on an analysis of two factors with two replicates each. I have called peaks using three different peaks callers, and would like to first derive a consensus of the...without having to create multiple DBA's? My current workflow is: - create DBA with ALL samples, replicates and peak calls (2 x 2 x 3 = 12) - generate consensus for each factor - export and save consensus…

diffbind

updated 5.8 years ago • Jim

time points in mice (5,10 and 15 days old), all mice with the same genotype, and 3 biological replicates per age. I want to evaluate differential expression as an effect of the different ages. I am confused on how I should...correct for the variability associated to the biological replicates when checking for the effect of age. I have tried both these options for the code but I always get very s…

DESeq2 Bioconductor RNASeqR

updated 4.7 years ago • Blanca

conditions). For each protein I have the protein count, and the coefficient of variance from three replicates in every condition. I want to  do pairwise comparisons between conditions to identify proteins that change...R. The problem is that I have the data (Schmidt _et al _2016) as the abundance estimate based on 3 replicates, and the coefficient of variance (standard deviation/mean) f…

deseq2 deseq differential analysis proteomics

updated 7.7 years ago • blehtine

a lot for your fast answer! I attached a schema of the slide-design, also with an example of the 4 replicates, hoping that like this I could explain it sufficiently. For my understanding the replicates are regularly arranged...As example mir 16, which we use as control mir, its 4 replicates can be found in block 5, block 13, block 21 and block 29, always with the same coordinates: column 9, row 3…

limma limma

updated 17.1 years ago • Christine Voellenkle

I have RNA-seq data sets (3 control replicates and 3 treatment replicates). I have obtained intron counts for every intron in all the 6 samples(3,3). My question is

deseq2 intron differential expression

updated 5.7 years ago • gv

affected patient against control patient. For each condition, I have three patients and two replicates from the same patient, with a total of 6 replicates for each condition (see Figure attached). ![enter image description...was wondering if for this experiment it is recommended to perform a nested analysis or a “collapsed- replicates” analysis. [1]: /media/images/2d154b51-5ffe-4381-a9dd…

RNASeq DESeq2

updated 3.5 years ago • giulia.lopatriello

limmaCtData to look for differential expression between groups, always having the same number of replicates. However, I currently have a dataset of several groups with 3 replicates and 1 group with 2 replicates. As far as I...can tell, limmaCtData expects the number of replicates to be the same between groups (i.e. there is only one 'ndups' parameter). Has anyone else encountered this issue? …

HTqPCR HTqPCR

updated 13.4 years ago • Fletez-Brant, Christopher NIH/VRC [C]

anybody know a R package or function to compare expression level (affy data) of two groups with no replicates in each group ? In fact, just compare one array to an other. The purpose is to find differentially expressed genes...We cannot used statistical test (not enougth replicates), but we can used graphical approach based on scatter plot, and outliers detection approach. Thanks for your help

updated 19.9 years ago • Nicolas Servant

<div class="preformatted">I have an experimental setup in which four strains (A, B, C and D) are given a treatment or control mock treatment, and observed (by Affy) over a post-treatment timecourse (4 timepoints); each strain/treatment/timepoint observation is performed in replicate. At the end of the day, I'd like to answer two scientific questions: 1) which probesets are consistently (a…

TimeCourse timecourse TimeCourse timecourse

updated 18.2 years ago • aaron.j.mackey@gsk.com

<div class="preformatted"> Dear Board, I know this topic has been discussed several times, yet I'm still confused on how to come up with the right design matrix when technical replicates are present, especially when dealing with affy arrays. For one thing, I know that when we have the same number of tech...<div class="preformatted"> Dear Board, I know this topic has been discussed …

affy affy

updated 19.6 years ago • Giorgi, Elena

Hi all, We have a 2-factor, 2 levels per factor experimental design with 2 replicates per covariate combination (so 8 samples total, n=2 per cell) that we are analysing using DESeq2. Normally DESeq2...Hi all, We have a 2-factor, 2 levels per factor experimental design with 2 replicates per covariate combination (so 8 samples total, n=2 per cell) that we are analysing using DESeq2. N…

deseq2

updated 6.4 years ago • stuart

relative to technical variation. This approach also allows you to compare the three biological replicates if you want. However, you might be wanting to find genes which are statistically significant relative to biological...variation, and this is harder. In principle, the three biological replicates can be treated as blocks, but limma isn't smart enough to handle the dye-swaps and the blocking…

Microarray Genetics oligo Microarray Genetics oligo

updated 17.3 years ago • Gordon Smyth

div class="preformatted"> Hi, I am working with Porcine Affymetrix in an experiment with technical replicates. I have 4 animals (2 control and 2 infected) and for each of them I have a technical replicate. Could somebody help me...on how to include the technical replicates in a correct way in the model using eBayes analysis in the Limma package, please? I am not sure if the following script

limma limma

updated 18.2 years ago • Lana Teixeira Fernandes

Hi all , I am running DESeq2 for Transcriptome analysis , Sometimes it is not possible to make replicate for each sample because it increases cost of analysis .So is it all right to simulate the replicate for case and control

deseq2

updated 10.0 years ago • Sushant Pawar

edgeR User Guide. In this experimental design I have a single factor with 3 levels. Each level has 2 replicates and each replicate had 5 samples taken. see below. ``` Treatments <- as.factor(c(rep("control", 10), rep("dose1", 10), rep("dose2...10))) Replicate <- c(rep("C1", 5), rep("C2", 5), rep("D1.1", 5), rep("D1.2", 5), rep("D2.1", 5), rep("D2.2", 5)) Sample <…

edgeR

updated 4.8 years ago • James

between these two conditions using `HiCDCplus` package. Is it possible to run `hicdiff` without replicates? if yes, how? if no, is there any other way to do so from .hic files? Thanks much Best, TA

hicdcdiff HiCDCPlus

updated 3.9 years ago • ta_awwad

Hi I'm trying to use DESeq2 to find differentially expressed genes between sample with replicates and sample without replicates. I have 2 condition, control and tumour. I am new to Deseq2 package, do you think my command

deseq2

updated 9.0 years ago • Bob

span style="line-height:1.6">1.Would it be a potential bias if I have 2 replicates for one condition and 4 replicates for the other? Or would it better to use then only 2 replicates per condition...span> Dataset 1: cond A,  2 replicates cond B, 4 replicates   2.Also, in my another dataset I have 2 replicates extracted with Long PolyA+ RNA (single...…

deseq2 edger

updated 9.9 years ago • tonja.r

However, we have encounter one bottleneck in using Ballgown, maybe because of the insufficient replicates of SRA data. The phenotype data as following, but we obtained 0 differentially expressed genes and <10 differentially...nbsp;1h control1 CdCK-2 1h control1 Cdck24 24h control24 (without any replicate) CdCd24 24h&…

ballgown differential gene expression

updated 9.1 years ago • boyashuang

Instead, I got 108 files (18 samples/per lanes with ~ 10 million reads). 12 groups with 3 biological replicates. It turned out, each sample was sequenced 3 times ( technical replicates). My understanding is that I do not need technical...replicates for RNAseq only biological replicates. When I asked why , the answer was  :"This was safer way to do the analysis...Lane 1, 2,3 and respect…

rnaseq edger technical replicates

updated 8.4 years ago • alakatos

using dual-channel microarrays. Every time-point was performed in triplicate including 3 biological replicates. Microarray analysis is performed in R with Limma using linear models (but not based on a separate channel analysis...mixed models)!!!). Comparing the boxplots for the three biological replicates on every time-point it seems as the data distribution is quite different between the rep…

Microarray Normalization Cancer limma Microarray Normalization Cancer limma

updated 18.2 years ago • Serge Eifes

treated,2)sensitive+untreated , 3)tolerant+untreated and 4) tolerant +treated each having three replicates I want to perform WGCNA to find a modules that are unique in either sensitive or resistant i.e the genes that are...differentially expressed in sensitive and not in resistant or vice versa. I have three replicates of each sample, so my question is how to handle the replicates because if I…

replicatesRNAseq WGCNA

updated 18 months ago • k200987

variation. limma has ways of making use of technical replicates, but there needs to be biological replication as well. Best wishes Gordon > Date: Fri, 26 Mar 2010 09:49:13 +0100 (CET...replicates are treated as independent samples. The technical replicates > are handled with the block argument in the duplicateCorrelation...As I understand the experimental design, however, there a…

limma limma

updated 15.7 years ago • Gordon Smyth

to cluster time-point series data from RNA-seq experiment. I have 4 time points each of 7 biological replicates in one matrix. I'm currently using `TMixClust` to perform the clustering. my questionn is: is there any option to allow...TMixClust to recognise my biological replicates? thanks much in advance best TA

TMixClust

updated 18 months ago • ta_awwad

The human Agilent chip 4x44 has most of the probes only once, but some of then appear in 10 replicates. These replicates are genes and not controls. I am using the Limma package to analyze them. I would like to define...these replicates as technical ones and leave the other probes (those that appear once) as they are. What is the best way to define only...part of the spots as replicates? Thanks …

limma limma

updated 18.4 years ago • Ester Feldmesser

that were quantified with the Alevin module from Salmon, given that we want to use the inferential replicates that it produces via bootstrapping. I know there exists the `catchSalmon` function to calculate a per-transcript...prefer to perform single-cell DE between clusters on the pseudobulk level (given one has biological replicates of course). Therefore, are there best-practices to import th…

edgeR alevin

updated 5.1 years ago • ATpoint

related to nucleotide-excision repair or junction resolution. Of course, we have few biological replicates of each condition (e. g. only two with Bloom syndrome). Despite being associated with a specific gene, nobody investigated...I want to use DESeq2 to find out differentially expressed genes. The problem is: few biological replicates in the case group and possibly cases within the same conditi…

deseq2 rnaseq

updated 11.2 years ago • daniel.silvestre

affected patient against control patient. For each condition, I have three patients and two replicates from the same patient, with a total of 6 replicates for each condition (see Figure attached). ![enter image description...was wondering if for this experiment it is recommended to perform a nested analysis or a “collapsed- replicates” analysis. Best regards, Giulia Lopatriello [1]: …

RNASeq DESeq2

updated 3.5 years ago • giulia.lopatriello

mu3 wt3 mu3 wt3 mu1, mu2, and mu3 are different mice which are biological replicates, and wt1, wt2 and wt3 are different mice which are biological replicates. > targets <- readTargets() # I try to include

updated 21.3 years ago • Ren Na

Hello, I have a small query with respect to running eisaR. I have three replicates each for control and treatment samples. I am able to generate the individual alignment files (bam files), followed...by the generation of the count table. I would like to know how to indicate the files as replicates, for the program to perform statistical analysis between the replicates of control and experiment…

eisaR

updated 5.2 years ago • nishanthemje

given for a multi-factor experiment, three factors with two, three and three levels each with three 'replicates' in each cell. I recently learned that the replicates are actually technical, not biological, and I'm looking for

Normalization DESeq2 Normalization DESeq2

updated 12.6 years ago • Michael Muratet

I have several samples , 3 biological replicates. (2015, 2016\_1,2016\_2 )  I want to compare each condition between each other. My PCA on rlog transformed values...analyse 2015 group separately. Maybe I'm wrong ! In 2015 group I have 3 conditions with 2 technical replicates each time. ([here](https://github.com/ZheFrenchKitchen/pics/blob/master/pca2015.png)) So then can I analyse the…

deseq2

updated 9.1 years ago • ZheFrench

Hello, I am using the Hisat2 - Stringtie - Ballgown pipeline that was published in nature protocols in 2016.  When I upload my ballgown data in to r, I have 4 lane technical replicates per biological sample, in addition to 3 biological samples per condition (2 conditions total). This leads to 24 "samples...in nature protocols in 2016.  When I upload my ballgown data in to r, I …

ballgown biologicalreplicates technical replicates stringtie

updated 8.0 years ago • linda.boshans

I have a 10x single-cell dataset with 6 replicates each containing cells from the same 5 donors. For the sake of simplicity, let's assume I have only two clusters, perturbed...testing, comparing the two clusters. But I want to pseudobulk **each donor** -- **not** each replicate. The complication is that each donor appears in all replicates. One way to do this is to first aggregate the replicates…

pseudobulk edgeR DifferentialExpression

updated 21 months ago • Jack S.

Hi all , I am new to this field of Transcriptome analysis , so please help me in this.          I just want to know that can we run DESeq2 for without replicate of treated and Untreated .If yes then what should the change in the syntax of DESeq2 .Below is the syntax i am using for with Replicate samples. And if no , then w…

without Replicate deseq2

updated 10.0 years ago • Sushant Pawar

a microarray data set in standard Affymetrix CEL-format consisting of only six samples without any replicates (same organism and cell type, but different individuals and different biological conditions for each individual...single-sample microarray data or from multiple samples with different biological conditions and no replicates) - or is there nothing I can do given the above limitations? Ma…

Microarray Clustering Microarray Clustering

updated 16.2 years ago • Rainer Tischler

div class="preformatted"> I want to know how to deal with data without replicates using DESeq2,can you tell me how to deal with it.You can see my codes below,but it have errors when deal with sample...without replicates. -- output of sessionInfo(): library('DESeq2') readcount<-read.delim("readcount.xls",row.names=1) readcount<-round

updated 12.0 years ago • Guest User

Hi, In lmFit(object, correlation), "correlation" is "the inter-duplicate _or_ inter-technical replicate correlation". Correlation is calculated using duplicateCorrelation(object, ndups, block), where "ndups" is the number...of spot replicates, and "block" is used to describe technical replicates. The help entry for duplicateCorrelation() says that "At this...time it is not possible to estimate…

updated 17.3 years ago • Dries Knapen

and EdgeR. In the similar package DEseq, there is the possibility of analyzing a dataset containing replicates only for one condition, but not for the other. In this DEseq case only the condition with replicates is considered

edgeR DESeq edgeR DESeq

updated 12.6 years ago • Teresa

gt;From: Jenny Drnevich <drnevich at="" illinois.edu=""> >Subject: edgeR with only 1 replicate? > >Hi Mark and others, > >Can edgeR be used if you only have 1 replicate per group? If not, do >you or anyone know

Sequencing SAGE edgeR Sequencing SAGE edgeR

updated 16.4 years ago • Jenny Drnevich

treatment. I wonder if I should treat the different samples from the same individual as technical replicates and therefore should be collapsed or consider them as biological replicates despite being from the same individual

DESeq2 timecoursedata replicates

updated 2.8 years ago • fepesanz8

differential expression analysis of digital gene expression data", 2.1.1 What to do if you have no replicates 2. Simply pick a reasonable dispersion value.   What of the bcv value is suitable for human cancer cell， 0.4，0.1

edger

updated 7.6 years ago • yueli7

is actually conservative, in that the residual variability will be larger than if you had true replicate arrays, hence you will find fewer DE genes than you might otherwise. Best wishes Gordon >Date: Fri, 31 Mar 2006 12...0200 >From: Pedro L?pez Romero <plopez at="" cnic.es=""> >Subject: [BioC] using limma with no replicates >To: <bioconductor at="" stat.mat…

genefilter limma genefilter limma

updated 19.7 years ago • Gordon Smyth

Dear all, I perform 2 color hybridization, using microRNA and Exiqon slides. slide design: 4 replicates of 1 array each array consits of 8 subarrays, each subarray of 16 columns and 11 rows each mir has 4 replicates For...offset=10) and subsequently applied the Linear model fit, which works, as long as I treat each replicate like a singl…

GUI limma microRNA GUI limma microRNA

updated 17.2 years ago • Christine Voellenkle

the limma manual and paper and several posts about using `duplicateCorrelation` with technical replicates mixed with biological replicates, I am still unsure when to use it (and why not use it). The 2015 limma paper says about...the limma R package says: Estimate the correlation between duplicate spots (regularly spaced replicate spots on the same array) or between technical replic…

limma duplicateCorrelation rna-seq technical replicates biological replicates

updated 6.1 years ago • paul.alto

am using DESEq for a DGE analysis. I have STRANDED RNA-Seq data for 4 developmental stages with no replicates but I know that to have a more reliable DGE I should have replicates. So I got (from another lab member) UNSTRANDED...RNA-Seq data with 3 replicates per stage. So my data situation at the moment is: STAGE 1 stranded STAGE 1.1 unstranded STAGE 1.2 unstranded STAGE...If so, I thoug…

Normalization DESeq Normalization DESeq

updated 11.8 years ago • Federico Gaiti

I am relatively new to GUIDEseq analysis, and was wondering how to handle multiple biological replicates. Currently, I have 3 biological replicates of a sample and they are differentiated by the P5 index. For every biological...replicate, there are 8 libraries (P7 index is different), 4 for + strand and 4 for the - strand. How should I analyze my data so that I

guideseq

updated 8.3 years ago • aamir.mir