Bioconductor Forum

Hi, I have a quick question on DEseq2 replicates. A good number of genes were marked as outliers (912, 5.7%) in my DEseq2 summary(res) output. I am wondering what that...flags genes which contain a Cook's distance above a cutoff for samples which have 3 or more replicates....__When there are 7 or more replicates for a given sample, the DESeq function will automatically replace counts...by the s…

deseq2

updated 7.6 years ago • Xianjun Dong

div class="preformatted">Dear friends, In my experiments there are 3 technical replications and 8 biology replications without dye swap. I have processed location and scale normalization for each print...tip-groups on 3 technical replications respectively. But next step what can I deal with them? Would I process scale normalization for 3 replications...and then take average or median of the …

Normalization Normalization

updated 21.8 years ago • zhao luo

treated conditions obtained from 4 different days. This SRA dataset obtained from NCBI doesn't have replicates but have mentioned in their article that they have merged the biological replicates drawn during sequencing...sample preparation section. When I try to run DESeq, I am getting error because of the absence of replicate sample read counts. How can obtain the differential expressed genes b…

deseq2 R bioconductor RNA_Seq replicates

updated 6.0 years ago • aishu.jp

Hello everyone, I would like to know if you could help me about someting in DESeq2. In the documentation, they talk about technical replicates ad biological replicates. From what I understand: - biological replicates are for example samples that do not come from the same people/animal/cellular culture. - technical replicates are for example samples that were sequenced twice on the same o…

DESeq2

updated 4.3 years ago • leo.dagata

if anyone knew of a function within bioconductor or method to provide some sort of metric for replicate quality analysis. I have typically been using multipanel graphs (having each replicate plotted vs each other) to...get a sense of the replicate qualities. However, I have not been able to find metric that would be good at determining how good a replicate is? I...using various outlier eliminati…

updated 22.5 years ago • Park, Richard

class="preformatted">Dear Users, I have Affymetrix Data. There are 5 animals and I have 2 technical replicates. I am trying to compare these technical replicates to ensure they are clean. I was wondering if anyone could please...or other factors caused a change in the values (readings) obtained between the technical replicates. So I would like to do some data analysis to compare these and ma…

updated 20.8 years ago • Shruti Subramaniam

Hi, I have just been passed a set of affy data that consists of 3 states, two technical replicates of each state (6 chips overall) 1. whats the best way (normalisations, algorithms) to leverage technical replicates...2. how do you tell algorithms such as rma which are the replicates? (I presume the phenoData in the AffyBatch specifies this, but the examples are in a binary format so you cant open

affy affy

updated 21.9 years ago • William Kenworthy

I did DE analysis on 80 libraries with DESeq2, the number of biological replicates is 2 (40 samples). For some genes, there is a big difference between read counts for 2 replicates, for example r1:2 and...r2:2436. If the number of replicates was more than 2, these cases were removed as an outlier. How to solve it with 2 biological replicates? Thanks

deseq2

updated 5.3 years ago • f_rahmdani

Hi, I have a set of data (cut and run) with 2 samples, three replicate of each. One set of replicate are separated from the other two replicates in sample heat map while the other two set...of replicates are segregated by sample (treatment replicate 1 next treatment replicate 2 and control 1 next to control 2). My question...is how do I go about to use mask to create a dba object just containi…

DiffBind

updated 13 months ago • Haiping

I have a question about how to analyze a mix of biological and semi-technical replicates. The experiment I am analyzing consists of 3 cell lines X 3 replicates of each cell line X 2 conditions. The 3 replicates...same cell line, but independently treated, processed and sequenced, so they aren't "hard" technical replicates, but they are not biological replicates as the 3 cell lines. They show …

limma technical replicates biological replicates paired analysis

updated 6.6 years ago • paul.alto

with 6 conditions (5 serial dilutions of a single drug and 1 untreated control) with 5 biological replicates for each condition. Our study uses low drug concentrations and so we expect to see only a moderate number of DEGs...due to condition. In fact most of the variation comes from replicate (generally global gene expression increases or decreases). Our coldata has Gene ID, Count, Condition and …

DEseq2 replicate DESeq2 normalization

updated 4.0 years ago • michael.morash

been using affy, affylmGUI, and limma for the analysis. Some of the samples in the latter group have replicates and I'd like to know how to handle these. My questions are: 1) In one case, the replicate is drawn from the same sample...but at a later time point. Is this a technical replicate (because it is the same sample but different chip), biological replicate (i.e., the different time point ma…

GO affy limma affylmGUI GO affy limma affylmGUI

updated 20.5 years ago • Jonathan Arthur

using the default 1-on-1 (pairwise) comparison for unpaired samples. My samples have both biological replicates and technical replicates. Shall I treat them the same way? Woud that be a little improper? Heyi </div

gage gage

updated 15.0 years ago • heyi xiao

have three samples with a simple design formula ~condition: sampleA - control sampleB - treatment (replicate 1) sampleC - treatment (replicate 2) DESeq2 returns one fold-change for control vs. treatment, but it is possible to consider...are three samples (A,B,C) for read counts Kij. Of course if geneX had extremely low counts in replicate 1 but not in replicate 2, we would expect strong var…

DESeq2

updated 4.4 years ago • P1000

class="preformatted">Dear List, I have a question regarding the use of biological versus technical replicates in a microarray experiment: I have two groups of samples, before treatment and after treatment. For some samples...I have biological replicates for before and after treatment (bucal swabs collected with a small brush) For some patients one of my biological...replicates usually afte…

Microarray Microarray

updated 15.0 years ago • Marcos Pinho

Looking at the oncogene2013 data set, they say they used 2 cell lines, three treatments, and 4 replicates. That's 24 samples, which is the number of columns in exprs(). What's not discussed is the number of technical replicates...which I assume they must have done. Can nondetects impute a dataset that includes technical replicates? Or do you just toss the replicates that don't work an…

qPCR nondetects

updated 2.1 years ago • Ed Siefker

I have an RNA-seq experimental design which includes replicates that are not exactly technical replicates and not exactly biological replicates. I compare tumor samples, but...I have an RNA-seq experimental design which includes replicates that are not exactly technical replicates and not exactly biological replicates. I compare tumor samples, but the same tumor is implanted sometimes in two or…

DESeq2 RNASeq limma

updated 3.8 years ago • Eyal

Hello, Here:[ https://imgur.com/z7Fkyjv](https://imgur.com/z7Fkyjv) you can find the PCA of the experiment that I am analyzing. Cond1 e cond2 were designed to have an over expression of two different genes. The sample cond1 near to controls has an expression of that gene much less than the others biological replicates. In this case I know that in that sample something …

deseq2

updated 7.5 years ago • ribioinfo

hi it is possible to use all dexseq script without replicates? ty efrat

replicates

updated 10.3 years ago • efratdahan21

analyzing some cDNA data; in the simplest case there are a total of 6 arrays, with three biological replicates; for each biological replicate, the arrays are duplicated and arrayed using dye-swap. Of course, for some genes there...understand that my two options are: a) take the easy way out, and compute a mean or a median of the replicates; b) "adapt" dupcor.series to my situation to get an esti…

Cancer limma Cancer limma

updated 22.3 years ago • Ramon Diaz

<div class="preformatted">Dear Dr. Smyth, I have "two groups Affymetrix" experiment with 5 biological replicates in one group and 2 technical replicates in other. In other words one group has all biological replicates and the other has all technical replicates. I would really be thankful to know how to use LIMMA (to identify differentially expressed genes) for such kind of design. After r…

limma limma

updated 17.0 years ago • Garge, Nikhil

How does HTqPCR handle technical replicates? Suppose I have 4 samples, 8 primer sets, and 3 replicates on a 96 well plate. "n.features indicates the number of features...read from each file. " 8*4=32 But I have 96 lines of data? Do I have to make each technical replicate it's own feature? How then would I indicate that they are replicates if they each have their own feature nam…

HTqPCR

updated 2.2 years ago • Ed Siefker

Hi I want to use the DESEQ package between a control (3 biological replicates) and treatment (1 biological replicate). IN DESeq I herefore used the following code, and got 266 genes with padj &lt

DESEQ deseq2 deseq replicates biologicalreplicates

updated 10.0 years ago • wd

Hi Michael, I am having hard time with one of the replicates in a three replicate experiment. This is a control sample. But the other replicates look good on PCA plot and also...Hi Michael, I am having hard time with one of the replicates in a three replicate experiment. This is a control sample. But the other replicates look good on PCA plot and also on pairwise scatter plot comparison. Can I …

deseq2

updated 11.0 years ago • Prasad Siddavatam

with the following issue that I have been struggling with. My experiment consists of 3 biological replicates per genotype (total of 3 genotypes; thus, 9 biological replicates) per time point. I have two technical replicates...per biological replicate. In my case, technical replicates were **not** made by running the library multiple times and/or on different lines; instead...replicates are mer…

edgeR

updated 4.7 years ago • mo17

I have 4 biological replicates of mRNA samples. For my dds, I am comparing type but have also added in the replicate factor to account for the biological...replicates. Is this correct? I have a column specifying the replicate number (1, 2, 3, or 4) per sample as they do not have the same names...dds <- DESeqDataSetFromMatrix( countData = counts_data, colData = metadata, design …

DESeq2

updated 12 months ago • shreya.kanade

Hi there, For some reason, one of the replicates of my htseq-count files is not normalizing correctly when run through DEseq2. I have attempted to change settings...re-map the original .fasta file, and all of my efforts still lead to the same replicate not normalizing. I believe that it has something to do with the htseq-count values of that replicate, but I haven't

deseq2 normalization

updated 7.5 years ago • mpw183

div class="preformatted">Dear limma experts, I have direct experiments with two biological replicates and two technical replicates. In each array sots are printted in 4 replicates. In duplicateCorrelation help it...this time it is not possible to estimate correlations between duplicate spots and between technical replicates simultaneously." The question is it possible to average on both techni…

limma limma

updated 20.6 years ago • Ron Ophir

ovarian primary tumor. After reading information about the package, I am still confused by the word , "replicates". In the context of my data, each RNA sequencing sample represents a unique patient's primary tumor, and DESeq2 would...treat these as individual replicates of the primary tumor condition, correct? Therefore, I can conduct analysis on this data using this tool? I want to

DESeq2 RNASeq

updated 21 months ago • kcarey

The thing is that i need to compare 5 conditions (stages) versus "control", each condition with two replicates. My question is... can i compare all in one run or do i need to compare by pares? And... how can i manage the replicates? I started...a count matrix (with data from the htseq count) comparing one condition versus control (with the 2 replicates of each one), and this command line:&nb…

deseq2 bioconductor R

updated 7.2 years ago • anaQ

upgraded to R version 2.9.1 and CellHTS2 version 2.8.1. Using my previous version I could miss replicates from the plate list file without generating an error - for example : Filename Plate Replicate plate_1_1.txt 1 1...3 plate_3_2.txt 3 2 plate_3_3.txt 3 3 Where plate 3, replicate 1 is missing. However, with my current ver…

Cancer cellHTS2 Cancer cellHTS2

updated 16.4 years ago • Becky Saunders

Dear Marcelo, Only one of your biological replicates has technical replicates, which isn't enough for `duplicateCorrelation()` to give a reliable estimate of the technical...0300 > From: Marcelo Laia <marcelolaia at="" gmail.com=""> > Subject: [BioC] limma technical replicates and biological replicates > in the same design > To: Bioconductor <…

limma SANTA

updated 6.5 years ago • Gordon Smyth

<div class="preformatted">Hello all, I am looking for a quantitative measure of replicate quality to know if one should be tossed or not. Right now, its a gestalt from pairs and mva plots after running rma on...div class="preformatted">Hello all, I am looking for a quantitative measure of replicate quality to know if one should be tossed or not. Right now, its a gestalt from pairs and …

GO GO

updated 22.7 years ago • Luckey, John

f1_A2 f1_B1 f1_B2 f2_C1 f2_C2 f2_D1 f2_D2 Here f1 and f2 are two families A1 and A2 are technical replicate. same for B1 and B2,C1 and C2, D1 and D2. A,B,C,D are biological replicate, say A,C are affected while B,D are unaffected. Noted...have more than 1 affected or unaffected samples. How does LIMMA take the two-level (technical replicate and familial relationship) dependence into account? C…

limma limma

updated 15.6 years ago • kuangqin

Hi, I am finding the differential peaks using DiffBind, but my sample has no replicates. And my `SampleSheet` is ``` > dbObj <- dba(sampleSheet="SampleSheet.csv") trisomy_21 fibroblasts trisomy_21 trisomy_21...33153 sites in matrix (47495 total): ID Tissue Factor Condition Treatment Replicate Caller Intervals 1 trisomy_21 fibroblasts trisomy_21 trisomy_2…

DiffBind

updated 6.1 years ago • zhangdengwei

<div class="preformatted">Hi, Does anybody know how to merge replicate spots in R Bioconductor? I use home-made spotted arrays and every probe is printed in triplicate. I normalize my data...div class="preformatted">Hi, Does anybody know how to merge replicate spots in R Bioconductor? I use home-made spotted arrays and every probe is printed in triplicate. I normalize my...using limma pa…

probe limma probe limma

updated 16.5 years ago • Barbara Cegielska

by family. I also tried method like GEE to > take familial relationship by averaging technical replicates. At this point, > looks the methods like LIMMA/GEE can not? two-level/type of dependence (like > technical replicate...deleted' vs 'undeleted'. > > In your point 2, what do you mean "combine families as biological replicates > and do cases vs. control…

Cancer limma Cancer limma

updated 15.6 years ago • Steve Lianoglou

a lot about testing on datasets with batch effects, but in all cases the effect is on biological replicates and not technical replicates. Just some quick terms for a better understanding: technical replicates: same sample...and loading onto the machine I noticed two different types of variation between technical replicates: A) systematic/plane shift in PCA --> batch effect due to…

rnaseq deseq2 differential expression batch effect replicates

updated 6.2 years ago • Nik dAK

wrote: > From: Leutz Buon <lb089 at="" research.dfci.harvard.edu=""> > Subject: Biological replicates with Gage > To: "Weijun Luo" <luo_weijun at="" yahoo.com=""> > Date: Sunday, July 24, 2011, 8:01 PM > > Hi, > I'm doing a gene set...samples at different time points : > 1hr,8hr,16hr,24hr. > For example: > c1r1 (co…

gage gage

updated 14.3 years ago • Luo Weijun

Hi, For input data for WGCNA, can I input all my data including the replications for each samples, or use average of the replications for each sample? Also to have a better chance to find modules

WGCNA

updated 5.6 years ago • bahmanik@msu.edu

<div class="preformatted">Hello Bioconductorers, I have a question regarding biological v technical replication in a continuous culture model. These are very expensive experiments to carry out so are not normally repeated to get the usual biological replication required. So the following experimental design has been suggested. Cells are grown in a continuous culture fermenter...div cla…

updated 18.5 years ago • PeadarÓGaora

Hi Rory, I very well realize the importance of replicates as well as your advice in this forum multiple times to other questions earlier that no meaningful statistics...can be done without replicates. However, the data I have received to analyze has bad replicates. There are three matched pairs for two conditions...and the biologist says that they can be loosely considered as replicates, b…

DiffBind ATACseq replicate

updated 5.0 years ago • CodeAway

vs. mut, with one gene knocked out in mutant. For one of the transcription factors, I only have one replicate as the second replicate has failed. As a result, I am getting errors from diffBind. Any advice on how to proceed? Thank

diffbind chipseq

updated 6.3 years ago • danielaperry2015

Hi, I have 6 samples each with 6 replications. One replication is missing from one sample.  T1\_LD\_FW1-6 T1\_LD\_SW1-6 T2\_LD\_FW1-6 T2\_LD\_SW1-6 __T3\_LD\_FW1

deseq2

updated 9.7 years ago • John

div class="preformatted">I'm a very new user of bioconductor. How shell I evaluate replicates quality? Which is the library to look for it ? Thanks , best regards Marco Gentilini </div

updated 20.2 years ago • Marco Gentilini

I have 10 "summit" bed files from macs2 peak-calling, with 5 factors with 2 replicates each. I added them all one-by-one to a dba object "dbObj" that looks like this: 10 Samples, 3202 sites in matrix (234027...I have 10 "summit" bed files from macs2 peak-calling, with 5 factors with 2 replicates each. I added them all one-by-one to a dba object "dbObj" that looks like this: 10…

diffbind chipseq

updated 5.7 years ago • hkitano

div class="preformatted">Hi, Is there a way to use DEXSeq with no biological replicate? I used DESeq as explained in the manual to use data with no biological replicate. In the manual of DEXSeq, I could not...find a way of using data with no biological replicate. Thank you, SangChul</div

DEXSeq DEXSeq

updated 13.7 years ago • Sang Chul Choi

nbsp;   I am using edgeR for Gene expression analysis. My question is about unequal no.of replicate. If we have 4 replicate of Control and 1 Case Sample then is it fair to do analysis or we should go with edgeR without...replicate like 1 case and 1 Control

edgeR replicate

updated 9.1 years ago • Sushant Pawar

everyone, I want to do differential exon usage analysis with DEXseq. I have 3 affected (2 technical replicates for each individual, so 6 samples total) and 3 unaffected individuals  (also 2 technical reps for each sample...6 samples overall). My question is how should I take into account technical replicates in this experiment with DEXseq? Should I add it in the formula somehow? And how…

dexseq

updated 7.4 years ago • chipolino

and the blocking argument to handle the technical replicates. If there is a marked batch effect between the replicates (because the replicates were conducted at different...duplicateCorrelation() for the technical replicates. If you want to do this, post again and I will advise you how to contruct the design matrix. BTW, I recommend including...at r-project.org, jana.asselman at ugent.be &…

Microarray limma Microarray limma

updated 12.3 years ago • Gordon Smyth

I am a frequent user of DESeq2 (when I don't have technical replicates of my libraries). When I have technical replicates I usually go for Sleuth. I have seen people that conduct analysis...I am a frequent user of DESeq2 (when I don't have technical replicates of my libraries). When I have technical replicates I usually go for Sleuth. I have seen people that conduct analysis in DESeq2 with ONLY…

deseq2

updated 7.8 years ago • jovel_juan

We are using gscreend for analysis of pooled CRISPR screen data. We have multiple T0 replicates, however gscreend only accepts 1 T0 sample. Is there a recommended way for us to consider all of our T0 replicates...So far, we have run the analysis with each T0 replicate and all T1 samples, and simply noted how many times each hit occurs across all comparisons. Thank you

gscreend

updated 3.4 years ago • pk_labrador

Hi all, If I define technical replication using the 'replicate.id.column' option in RnBeads, I get some helpful plots in the exploratory analysis. But...Hi all, If I define technical replication using the 'replicate.id.column' option in RnBeads, I get some helpful plots in the exploratory analysis. But I...in Limma), or should I deal with them myself? Only a subset of my samples have these repl…

rnbeads replicates

updated 10.0 years ago • Jon Manning

div class="preformatted">Dear Members, Do we need replicates to carry out analysis with AgiMicroRna package in bioconductor? I have one control and one treated samples. Can

AgiMicroRna AgiMicroRna

updated 13.5 years ago • Karthik K N

Hello all,   I am analyzing an RNAseq with 6 conditions using deseq2. 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? &nb…

deseq2 differential gene expression

updated 10.1 years ago • ea1402

<div class="preformatted">Hi As I have varying numbers of replicates, and they are not regularly spaced on the array, and given that I would like a list of differentially expressed genes...div class="preformatted">Hi As I have varying numbers of replicates, and they are not regularly spaced on the array, and given that I would like a list of differentially expressed...which is averaged…

updated 21.3 years ago • michael watson IAH-C

Dear all, I am working with 19 RNAseq sample, 10 of which have 2 technical replicates (same individuals in a population). I am trying to correct for a clear batch effect using the RUVs approach as described...blob/master/Peixoto_Additional_File_1.Rmd) . How should I combine these technical replicates after normalisation to avoid pseudo-replication when I do the differential expression part? D…

rnaseq ruvnormalize normalization technical replicates

updated 10.0 years ago • thomas.wolfe

<div class="preformatted">Hi all, 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…

updated 14.7 years ago • Wendy Qiao

Hi, I have a dataset where each experimental condition has 3 biological replicates and each biological replicate has 2-3 technical replicates. When I use `` lmFit() ``, I can account for correlations between...technical replicates using the `` correlation ``parameter. However, `` camera() `` does not seem to have such a parameter. Do I need to somehow aggregate...technical replicates prior to r…

limma camera

updated 9.0 years ago • Yury Bukhman

<div class="preformatted">Hello, I am a graduate student and fairly new to RNA-Seq. In my study I have a 2x2 design with each group containing 4 bio reps and each bio rep has 3 technical replicates (same library prep, but each sample is processed in 3 different lanes). After studying my frequency count data I have...my study I have a 2x2 design with each group containing 4 bio reps and eac…

edgeR edgeR

updated 11.7 years ago • Neha Mehta