Bioconductor Forum

external controls and spike to do the normalization. I still wondering whether I could use all the replicates on one slide. 6 of them are regular spacing and spacing is 800. the result comes out pretty reasonable. The commands...eb=eb, adjust="fdr") write.table(y, file="diff_result.txt", sep="\t ") But when I try to use 12 replicates on one slide, the result doesn't look right. What I am trying …

Microarray Normalization Bayesian Microarray Normalization Bayesian

updated 21.4 years ago • Hua Weng

generated with the Illumina human ht-12 v4 platform, which contains several technical and biological replicates. I first load the raw image data to Genome Studio and calculate the respective group and sample matrices. I am interested...differential gene analysis respectively. My question is whether i) I shall average the technical replicates in Genome Studio and use Group\_Probe\_Profile as inpu…

illumina human ht-12 v4 technical replicates

updated 9.9 years ago • Eleni Christodoulou

developers, Could I use the varianceStabilizingTransformation (VST) to treat RNA-seq data with no replications? Thanks in advance

deseq2

updated 6.9 years ago • nmgduan

div class="preformatted">Dear Gordon, Naomi, and BioC list, The issue of how to deal with technical replicates (such as those obtained when we do dye-swaps of the same biological samples in cDNA arrays) has come up in the BioC...comments. There seem to be three major ways of approaching the issue: 1. Treat the technical replicates as ordinary replicates *************************************…

GO Cancer vsn GO Cancer vsn

updated 21.7 years ago • Ramon Diaz

<div class="preformatted">Hi all, i am doing a Medicago GeneChip with six treated time points without any replication i used limma to analysis that,but the statistic part of Bayesian is not avialable in my condition. and i wanna got...preformatted">Hi all, i am doing a Medicago GeneChip with six treated time points without any replication i used limma to analysis that,but the statistic…

Bayesian limma Bayesian limma

updated 18.5 years ago • lidaof

Hi, I have a question about how to deal with semi-technical replicates in bulk RNAseq. I thought someone would've asked it but I couldn't find any. Essentially, I have 10 cell lines grown...in 2 different medium conditions with 1 replicate each (20 samples separated in 3 different batches). I have another 1 control line grown in 2 different conditions...but repeated the same way in each of the…

variancePa variancePartition limma-voom dream

updated 2.8 years ago • Le

flags genes which contain a Cook’s distance above a cutoff for samples which have 3 or more replicates. The p values and adjusted p values for these genes are set to NA. At least 3 replicates are required for flagging...as it is difficult to judge which sample might be an outlier with only 2 replicates. This filtering can be turned off with results(dds,cooksCutoff=FALSE).") I dont feel confortab…

DESeq2 Nullcook'svalues 2x3 DEGs

updated 4.7 years ago • andreia

and 1 has the second kind but not first. Additionally, one of the samples also has two technical replicates. How do I factor all this into DESEQ2 design? 1. Can I do design = ~patient + sample, despite not all of my patients having...both kinds of samples? 2. Can I pick one out the two technical replicates alone for the analysis

deseq2 RNASeq

updated 5.8 years ago • divyak

bacterial samples from different clonal complexes. Each of the samples have 3 biological replicates = 27 lanes in the RNASeq library - and these have been sequenced twice, a second set on a different set of lanes giving...us 2 technical replicate of each biological replicate = 54 lanes. I am struggling to find a way to collapse the technical and biological replicates...would be able to: …

deseq2

updated 6.9 years ago • rm284

Dear Limma/EdgeR users, I have 2 treatment groups, 3x biological replicates for each. I also have 2 extra samples, a pool of each treatment group.  I am comparing a "vanilla" analysis with...biological replicates, to an analysis with the pooled samples. I.e 1 vs. 1 sample. <span style="line-height:1.6">In the EdgeR manual, there are...4 clear ways/examples of how to do an analys…

limma voom edger

updated 9.2 years ago • Johnny H

of the data. I am encountering the following situation with RNA-seq analysis : A-- i have 2 sets of replicates, these are labelled : siCTL (replicate1,replicate2) vs siPROTEIN (replicate1, replicate2).the correlation coefficient...between replicates is 0.99 . B-- shall I take any replicate individually, and just compute the fold-changes, there are approx 1000 up-reg...genes, and 1000 down-reg g…

rnaseq limma edgeR deseq2

updated 9.2 years ago • Bogdan

function for creating a data frame (colData) needs (if I am not mistaken) data in same amounts of replicates (has that "each" condition). Can I create the coldata by colData<-read.table("file.csv"), where file.csv a table with...mixed amount of replicates for a given partition, as seen in the DSeq2 example? Best Theodoros &nbsp

fourcseq

updated 9.4 years ago • theodore.georgomanolis

depths or alignment methods. I would be surprised if MiSeq and HiSeq would generate perfect Poisson replicates of one another, especially if the read lengths from the two platform are different or the alignment and counting...the underlying theory behind dispersion estimation, you can > easily test whether your "technical replicates" really do represent > technical replicates. Spec…

Sequencing Alignment GO edgeR DESeq2 Sequencing Alignment GO edgeR DESeq2

updated 11.3 years ago • Gordon Smyth

TissueEnrich requires a table with TPM or FPKM for each tissue. However what I have is 4 biological replicates for each sample, so do you know how I would indicate that all of these samples are the same tissue? The other question...I read the vignette [here][1] and the manual but didn't see any reference to dealing with replicates or differential expression. In the vignette, it does say that f…

rna-seq TissueEnrich tissue-specific expression differential expression

updated 6.9 years ago • andrew.oler

of RNA-seq results. The problem is that I can't find easy solution how to average biological replicates. I assumed there is simple function for averaging replicates by conditions using info from `phenoData` for `ExpressionSet

Mfuzz RNASeq

updated 21 months ago • boczniak767

of the samples have been replicated on 2 Illumina platforms (HiSeq and Miseq). These are technical replicates - the library preparation is the same using the same biological replicates - it's only the sequencing which is different...post: https://stat.ethz.ch/pipermail/bioconductor/2010-April/033099.html It recommends pooling the replicates. The post seems to apply to a different case ("pure" t…

Sequencing Sequencing

updated 11.3 years ago • Nick N

at stat.math.ethz.ch Sent: Monday, May 24, 2010 5:07:54 PM Subject: microarray data without replication Hi, is there an R package (or some other software) for analyzing microarray data without replication? I have written

Microarray Microarray

updated 15.5 years ago • Ina Hoeschele

which worked well for me. Now I am using the codes on my own data but because of my lack of replicates the following code from the tutorial gives an error when I use my own data: data_diff_manual <- test_diff(data_imp...No residual degrees of freedom in linear model fits I am aware of why the error occurs(no replicates), my question is just how to change the code to treat 2 of…

DEP Proteomics Statistics limma

updated 6.3 years ago • tatabox

preformatted">I am using Limma for an experimental design with three categories, two biological replicates each and two technical replicates of each biological replicate for a total of 12 arrays. Since this goes just a...The categories are: Cyt1 Cyt2 Mock (which is control for both of the other groups) The biological replicates are indicated with A and B. The technical replicates are indic…

limma limma

updated 12.4 years ago • James Platt

I'm fairly new to Bioconductor and had a basic question about *recommended* steps to analyze replicate data. I apologize if this has been covered, but I've scoured the message archives and couldn't find any examples to...guide me. I'd like to look at 3 biological replicates of treated vs. untreated cells and determine p-values for over- or under-expression of each gene. We're using in-house

multtest oligo multtest oligo

updated 22.2 years ago • Mike Schaffer

Are there tools implemented in Bioconductor that allow one to use technical replicates to batch correct. Meaning, if one has a group of samples that are repeated across known batches, are there tools...Are there tools implemented in Bioconductor that allow one to use technical replicates to batch correct. Meaning, if one has a group of samples that are repeated across known batches, are there too…

batch effect

updated 9.3 years ago • Juliet Hannah

In the vignette ,  <pre> The purpose of this function is to sum up read counts from technical replicates to create an object with a single column of read counts for each sample</pre> It doesn't say that they divide after...sum up...I was wondering if i need to divide the raw counts by the number of technical replicates added because it will have an impact on the size facto…

deseq2

updated 9.1 years ago • ZheFrench

I have a question on the best practice for testing DEG using 10X data where we also have biological replicates between our conditions. What is the recommended way to account for this replication? If this has already been addressed

singleCellExperiment

updated 5.2 years ago • emy_66

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...to include it. I have not >> found any reason why you can not just sum 2 out of 3 technical replicates >> if you k…

edgeR edgeR

updated 11.4 years ago • Neha Mehta

The lab i am working in is interested in retrieving the average expression of genes across replicates after finishing performing bulk RNA-Seq using deseq2. Apparently, they are used to this information beside the...by size or normalization factors, i imagine this is the average expression of each gene in each replicate. Your guidance is appreciated thank you

DESeq2

updated 13 months ago • James

preformatted">Hi, All, I'm doing the analysis on my cDNA array with limma. There are irregular replicates for each spot and the spacing numbers are unknown. I'm trying to correlate the replicates with duplicateCorrelation

limma limma

updated 17.8 years ago • Wei Xu

Hi, I'm analysing RNA-seq data and I switched from edgeR to DESeq2 because most of my differentially expressed genes had an outlier sample, which was not an outlier in the entire dataset (judged by PCA and overall Cook's distance). My question is related to the criteria used by DESeq2 to count the number of replicates in my experiment. I have 3 experimental groups, A,B and C, each of them with…

deseq2 ruvseq cookscutoff covariates batch

updated 9.0 years ago • j.viana

transcript usage. Could you please point me out, where and how should I collapse technical replicates in DRIMSeq? For example, for DEG search in DESeq2 I use collapseReplicates function before running DESeq. Best

DRIMSeq R DTU technical replicates deseq2

updated 5.4 years ago • poecile.pal

transcriptome data of one control and one treatment for each 2 differential conditions. They have no replicates. So FPKM was calculated for normalization. I want to do DGE between them. I know from another post Gordon suggested...isn't 100% ideal, but is probably the best analysis available. But It doesn't work for me without replicates. Could you give me an idea to do DGE for this condition? I …

dge fpkm rna-seq

updated 4.5 years ago • lkianmehr

team, I am working on a 2-colour Agilent dataset which has both biological and technical replicates. A problem which many people already encountered as I could see from the previous posts, so my apologies for bringing...I try this! My experiment contains three treatments: "AC", "low" and "high" - each has 4 biological replicates and 2 technical replicates as indicated in the attached target fil…

limma limma

updated 13.4 years ago • Ommen Kloeke, A.E.E. van

by one array. Is it appropriate to use the  duplicateCorrelation function when some biological replicates have no technical replication? I would like to keep that array in the analysis since there are so few Lean subjects

limma duplicatecorrelation

updated 9.2 years ago • dsperley

of repeated measurements (cells from same individual measured multiple times) as well as technical replicates (for most of the samples \[i.e RNA samples are labelled and hybridized 2 times\]). For the repeated measurements I know...include this in the model), but I am actually unsure how to best utilize the info from the technical replicates.... According to some posts on this site the technica…

limma duplicatecorrelation avereps

updated 9.2 years ago • Guido Hooiveld

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...offset=10) and subsequently applied the >Linear model fit, which works…

GUI limma microRNA GUI limma microRNA

updated 17.2 years ago • Naomi Altman

<div class="preformatted">Hi Kaitlin, Incorporating the eighth timepoint into your experiment will be difficult, given that the chips used are likely quite different from the version 1.0 chips that you used for the other time points. Since you only used one timepoint with these chips you have aliased any biological differences with the chip type, so it will be impossible to determine if an…

Normalization Normalization

updated 15.7 years ago • James W. MacDonald

to the underlying theory behind dispersion estimation, you can easily test whether your "technical replicates" really do represent technical replicates. Specifically, read counts in technical replicates should follow...be the technical variability of of the replicates. For true technical replicates, the dispersion should be zero for all genes. So if you estimate dispersions using...the samples ha…

Sequencing edgeR DESeq2 Sequencing edgeR DESeq2

updated 11.3 years ago • Nicolas Delhomme

the makeContrast function. The main issue is how to handle the mixture of technical and biological replicates. For example, I wish to compare samples s_1,s_3,s_5,s_6 and s_7 vs control samples and get the top 100 significant...fit3, n=100, adjust="BH", sort.by="B") does the above correctly take into account the technical replicates? If not what is the best way to handle this? Thanks for any he…

probe affy limma probe affy limma

updated 18.7 years ago • Steve Taylor

Hi all, I want to know how to get a gene expression value for a condition with different replicates. For example, I have condition M and N, each condition with two replicates M1, M2, N1, N2 I want to get one value to represent...the gene expression value (FPKM or TPM) of M, can I just use the mean of each replicate? M=(M1+M2)/2? Is there any other way to calculate the gene expression value fo…

rnaseq gene expression edger

updated 7.9 years ago • Jack

Does anyone know if there are any functions like plotCtRep that can take more than two technical replicates and look at the SD of the reps? Best, Kee Wong [[alternative HTML version deleted]] </div

updated 15.2 years ago • Kee Wong

Do I need to merge bam files or collapse replicates? I am working on differential expression analysis and have 3 technical replicates for each sample from different

DESeq2

updated 3.6 years ago • myfam128

and zone in the leaf (zone1, zone2, zone3). For each possible combination, we have 3 biological replicates (no technical replicates). The PCA showed us that we have 2 outliers. They are not in the same group, so this leaves us...with 2 biological replicates for two of our sample groups.    I've been looking on the internet for information on how many biological...replicates I…

deseq2

updated 7.8 years ago • Jonas B.

Dear Noel, I'm sorry I didn't fully appreciate from your first email that you have only one replicate, because you didn't give the whole biolrep vector. A single replicate is simply not enough to estimate the technical...and I obtain [1] NaN. This doesn't seem sensible. > > Perhaps I have specified the biological replicates > incorrectly. The desciption of dupcor states tha…

affy affy

updated 19.5 years ago • Gordon Smyth

you say there's a problem. duplicateCorrelation() has no difficulty with technical and biological replicates in the same experiment. You might analysis your experiment by: targets$Treat <- factor(targets$Treat) design &lt...gt; To: bioc-devel at r-project.org > Subject: [Bioc-devel] technical and biological replicates in the same Exprset - Agi4x44 > > HI a…

Microarray Normalization Microarray Normalization

updated 13.5 years ago • Gordon Smyth

div class="preformatted">Dear Yanyan You can use the method described in Section 8.2 (Technical Replication) of the Limma User's Guide. Best wishes Gordon >Message: 9 >Date: Fri, 01 Dec 2006 11:52:37 -0500 >From: Yanyan Li <yli...at="" mail.jci.tju.edu=""> >Subject: [BioC] Limma with block and replicate measurement >To: bioconductor at stat.math.eth…

limma limma

updated 19.0 years ago • Gordon Smyth

blocking for body site with the contrasts D1-CTRL and D2-CTRL however I'm thinking that treating replicate control samples from different body sites as independent samples is incorrect and that I am underestimating...in the control group. Could anyone suggest the best approach to deal with this? Would adding the replicates as a random effect using duplicateCorrelation() be appropriate in this ins…

limma microarray

updated 9.2 years ago • obarton

Hi all, I have a general question about the model estimation used by DESeq.  There have been many posts on whether or not DESeq works well with a small number/no replicates, but I'm wondering if it's appropriate to use DESeq for differential analysis across two conditions, where one condition...by DESeq.  There have been many posts on whether or not DESeq works well with a smal…

deseq2 rnaseq differential expression

updated 8.2 years ago • krc3004

Hello all, I am working on a RNA-Seq project that was sequenced without any biological replicates. I know that it is bad practice to use this kind of data, but the researcher didn't seem to have a choice and this is...Hello all, I am working on a RNA-Seq project that was sequenced without any biological replicates. I know that it is bad practice to use this kind of data, but the researcher di…

deseq DESeq2

updated 11.2 years ago • solgakar@bi.technion.ac.il

from same batch > 2 batches. This should be no surprise, even though we did not have enough replication to do any formal testing. I think at minimum that you want to achieve results that would be replicable within your...sample from another 50 as replicate 2. > >Do you think that biological replicates should be grown at different time? > > >Absolutely! …

Normalization probe affy affyPLM Normalization probe affy affyPLM

updated 19.9 years ago • Naomi Altman

Is there a limit for the sequence coverage difference between replicates and among samples? I am using DESeq2 and edgeR in DiffBind to compare ATAC-seq samples with different sequencing...Is there a limit for the sequence coverage difference between replicates and among samples? I am using DESeq2 and edgeR in DiffBind to compare ATAC-seq samples with different sequencing coverage among samples,…

DESeq2 edgeR DiffBind

updated 3.1 years ago • cesar.arenasmena

oligo array and non-tech replicated affy experiment. I'd like to keep the information in the tech replicates so used duplicateCorrelation. However, I'm now concerned that the tech replication may be treated in some way like...biological variation by lmFit(). Basically if you mean the tech replicates and do the fit below without the dupCor then the p.values appear to be generally higher, particul…

affy limma oligo affy limma oligo

updated 21.1 years ago • Matthew Hannah

to be three major ways of approaching the issue: > > > > > >1. Treat the technical replicates as ordinary replicates > >************************************************************* > >E.g., Gordon Smyth in sept. 2003 > >(https://www.stat.math.ethz.ch/pipermail...gt; for some cases, shows potential problems with u…

Microarray GO Preprocessing Cancer vsn Microarray GO Preprocessing Cancer vsn

updated 21.7 years ago • Ramon Diaz

to something here and now with existing software. >1. Treat the technical replicates as ordinary replicates >************************************************************* >E.g., Gordon Smyth in sept. 2003 >(https://www.stat.math.ethz.ch/pipermail/bioconductor...least for >some cases, shows potential problems with using lme when the number of >technical r…

Microarray GO Cancer vsn Microarray GO Cancer vsn

updated 21.7 years ago • Gordon Smyth

Hello, While running the eisaR program with three biological replicates each for control and treated samples instead of two replicates (as shown in the vignette), is there any change to

eisaR edgeR

updated 5.2 years ago • nishanthemje

Hi, Section 11.3 of the limma User's guide introduces the concept of correlation across technical replicates. To address replicate correlation, some extra code is needed. The sample code is given as, > biolrep <- c(1, 1, 2, 2) > corfit

limma limma

updated 11.8 years ago • Guest User

we included some new functionality in tximport (so v1.4.0) to import *alevin* scRNA-seq inferential replicates. We just noticed there was a bug in the import code such that the cells in the inferential replicate matrices (e.g...matrices were mislabeled. Likely not too many groups were computing or importing these inferential replicate matrices, but wanted to make sure it was known to update tximp…

tximport News

updated 5.7 years ago • Michael Love

underlying theory behind dispersion estimation, you can >> easily test whether your "technical replicates" really do represent >> technical replicates. Specifically, read counts in technical replicates >> should...will be the technical >> variability of of the replicates. For true technical replicates, the >> dispersion should …

Sequencing Alignment GO edgeR DESeq2

updated 11.2 years ago • Nick N

Hi, is there an R package (or some other software) for analyzing microarray data without replication? I have written my own code (essentially based on ASE and data permutation) but am still wondering whether some

updated 15.5 years ago • Ina Hoeschele

First, I know that DESeq2 is not meant to be run without replicates and results will not be meaningful. I am an analyst who has the following design matrix: Group | Condition - | - A | 1 B | 1 C...First, I know that DESeq2 is not meant to be run without replicates and results will not be meaningful. I am an analyst who has the following design matrix: Group | Condition - | -…

deseq2

updated 5.7 years ago • august

Hi, Sorry for the lengthy post and might be naive. I am pretty new to differential gene expression analysis and also to transcriptomics. We did metatranscriptomics analysis of a mixed bacterial population in two different condition. Thre was quality issue with the extrected RNA samples. Later i ran deseq2 normalization (by first using estimateSizeFactors and then variance stablizing trans…

DESeq2

updated 4.3 years ago • Shail

one group received antibiotics, and one group as a control, and each have 3 samples, then I replicate this explements). But one of the samples was not good so I end up with 6 samples for controls, 5 samples for Antibiotics...all my samples are coming from one family. So basically, all 6 samples for each tank are biological replicate except they are from different tanks (tanks 1 three samples, tan…

DESeq2 ExpressionData GeneExpression

updated 4.8 years ago • Javad_Sadeghi

distributed into 2 conditions (cond1), 6 further conditions (cond2) each, 3 time-points and each 3 replicates. Can DESeq2 handle this low sample number and replicates as well, and give me DEGs for cond1 and time, while cond2

DESeq2

updated 2.5 years ago • Rockbar