Bioconductor Forum

change in the sensitive group compared to the tolerant group. However, in this approach I'd use 10 replicates only, and I've read that the analysis minimum is 15. Can anyone provide some advice/explain what type of analysis

WGCNA

updated 9.4 years ago • nicky.driedonks

needs to be at least 2 more arrays than the number of coefficients to be estimated (ie at least 2 replicates). If I were doing a 13 time point series of arrays, is it necessary to replicate all of them or could I select just a few...time points of interest? Also, better to do bio or tech replicates? B) Secondly, I used your code (written above) to average the spots, but when I tried to calcula…

updated 17.6 years ago • Sanjat Kanjilal

confused how to deal with repliactes count. Is it reasonable to merge the vst result, divided the replicates number and get the mean vst as the input of k-means cluster or some other methods about Network or cluster? Guandong

deseq2

updated 5.6 years ago • shangguandong1996

ChIPQCreport `` requires that the "Replicate" of each sample be provided during the construction of its `` experiment `` object. If it is not provided, `` ChIPQCreport...invocation of `` ChIPQCreport `` for an `` experiment `` object whose sample was assigned "Replicate" = 1.   &nbsp

ChIPQC chipqc

updated 9.2 years ago • Coby Viner

I have a question on averaging biological replicates together. the code below plots data for each sample. When and how do I combine my biological replicates for plotting

deseq2 pheatmap average

updated 7.2 years ago • chighfi

to clarify: 1.  I am doing case-control comparison (KO vs WT each has four biological replicates), do I understand correctly that  ___stattest()___ use KO/WT for log-fold change calculation based on provided...does not matter, as phenotype data basically describes the order? 2. When the number of biological replicates is not equal for example,  two KOs and…

ballgown rna-seq

updated 8.9 years ago • Ir

cannot seem to figure out why my heat map does not group my samples by condition when I use collapse replicate function. The only thing that is changed is dds --> ddsColl and vstdds --> vstddsColl. Any help would be appreciated

deseq2

updated 6.9 years ago • coyoung

The analysis involves comparing multiple samples with bead-only samples. some of the samples lack replicates while others have them, I have not encountered any errors. However, I am concerned whether this approach is appropriate

edgeR

updated 2.7 years ago • f_rahmdani

implement your DESeq2 package. However, I like to understand what is being done, so I have tried to replicate the results using DESeq2 in R in a standalone way. Unsuccessfully. The baseMean, pvalue or lfcSE values are quite...And here you have the same log2FoldChange for that genes through the DEApp software that I cannot replicate: ``` baseMean log2FoldChange lfcSE stat pvalue padj sam…

DESeq2

updated 4.5 years ago • Sara

with EdgeR for 2 RNAseq samples, with only one condition (1 treatment, 1 control), and I only have 1 replicate of each, which I know is far from ideal. Having reviewed the user manual, I've decided to use option 2 for this scenario

edgeR

updated 5.1 years ago • bertb

DeSeq R script for data analysis, and I've seen the section in the manual relative to "DESeq without replicates", so I was thinking of modifying the default TETranscript script and simply go for that.  I am not an expert in

normalization deseq2 edger

updated 7.8 years ago • giulia.pasquesi

If you are producing of analyzing ChIP-exo data (Rhee HS, Pugh BF, 2011, Cell 147:1408-19) with replicates, you might want to consider CexoR to uncover high-resolution protein-DNA interactions. It processes data data

updated 12.1 years ago • Pedro Madrigal

I am trying to write the R code for the analysis of a microarray experiment involving both technical replication and common reference design. The targets file is: --------------------------------------------------------------------- FileName Cy3 Cy5 Array1A.gpr Ref1 WT1 Array1B.gpr Ref1 WT1

limma limma

updated 18.1 years ago • Juan C Oliveros Collazos

I will be grateful for your feedback and advice. I have been tenured for some time now and appreciate that my statistics training was very different than what is available now. Can I use WGCNA to identify clusters of responsive proteins from one perturbation time series (pooled from two independent replicates)? I plan on following up with additional proteomic experiments, based on specific c…

WGCNA

updated 2.6 years ago • Adam

gt; > 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 • Gordon Smyth

<div class="preformatted"> > Hi Mark, > > I was able to run RP from the M values but have couple of doubts. Thank you for suggesting that to me. I really appreciate if you could help me out with it. First question - is it possible to use only significantly differentially expressed genes obtained from the linear model (LIMMA ) for RP analysis. I was unable to select the …

GO limma GO limma

updated 15.9 years ago • Neel Aluru

is that **the adj.P.Val are tiny** (and <0.05 in 91% of genes). Is this just due to the amount of replicates (donors) that I have, or **should I be worried**? There are ~10% with abs(logFC)>1, which is in the line of what I've seen in other...your advice. Clarification: This is pseudobulk data from scRNA-seq, so I could potentially create replicates for some donors within each batc…

limma RNASeq DifferentialExpression

updated 21 months ago • mperalc

melissa.martin ...="" at=""> writes: > > 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

DESeq DESeq

updated 13.6 years ago • Asma

Dear community, i would like to address my important questions regarding a complex experimental design in R, concerning an agilent microarray dataset with multiple time points. Briefly, after normalizing, a view of my phenotype information is the following: <table cellpadding="0" cellspacing="0" style="height:1590px; width:890px"> <tbody> <tr> <td> <pre> > …

limma design matrix multiple time points agilent microarrays

updated 9.8 years ago • Konstantinos Yeles

so for every single test I have 6 libraries (control + infected at 3 time points). Having three replicates this makes 18 libraries in total. What I did until now is look at each time point separate and calculate DEgenes

RNASeq edgeR RNASeq edgeR

updated 10.3 years ago • Kaat De Cremer

Hi all, I am new to R, I have found HTqPCR very useful, but I am having a couple of issues that maybe you will be able to clarify for me. -          When using “filterCatergory” to filter high Ct values (and change them to NA), technical replicates are all given the same value (and, specifically, the value of the f…

limma htqpcr

updated 4.9 years ago • edu.guasch

We have a time course infection experiment with 12 samples (2 technical replicates and 2 biological replicates per sample). The samples were sequenced on a HiSeq over 2 lanes, 1 x 50 bp reads. 1. 24hr\_infection1...a counts file using HTSeq for each sample. My questions now are:  1. Should the technical replicates be collapsed (add gene counts for technical replicates)?  2…

edgeR rna-seq replicates

updated 10.8 years ago • eb0906

bioconductor@r-proj ect.org="">> Subject: Re: [BioC] separate MEDIPS.createSet required for each replicate? RE: using custom BSgenome with MEDIPS Hi Kelly, No, youshould not ignore the error message. I did not fully understand...bioconductor@r-project.org="">>> Subject: [BioC] separate MEDIPS.createSet required for each replicate? RE: using custom BSgenome with MEDIPS …

Alignment BSgenome safe BSgenome MEDIPS Alignment BSgenome safe BSgenome MEDIPS

updated 12.0 years ago • Vining, Kelly

regions of the genome. I've been working through the vignette, but I find that I cannot even replicate all of the examples in the vignette. Specifically, the "rectangle" segments designed to show the positions of variants

software error ggbio circle plot

updated 9.7 years ago • davis

gmane.comp.lang.r.sequencing/1663/match=edgeR +time+serie ) I have an RNA Seq experiment with 2 replicates of 8 time points with both a control and treatment conditions. Time point 0 however represents the culture prior

updated 12.9 years ago • Marcelo

I have some RNA-seq samples from mouse, 2 conditions with 4 replicates each, read quality is good and for each sample 85-90% of reads align. The number of aligned reads in millions are: Condition...1 replicate 1: 100 Condition 1 replicate 2: 79 Condition 1 replicate 3: 52 Condition 1 replicate 4: 37 Condition 2 replicate 1: 56...Condition 2 replicate 2: 59 Condition 2 replicate 3:…

deseq2

updated 8.1 years ago • Mike

Recently I used both cuffdiff and Deseq2 to find the differential genes. The outcome is very different. Cuffdiff gives me about 1600 changed genes and Deseq2 gives me 12347 genes. The overlapping is only 25%, which is very low. I expected some number like 50%. Cuffdiff unique is about 5% and Deseq2 unique is about 70%. The huge difference actually comes out from the downregulate…

deseq2

updated 9.3 years ago • kan.jiang

my data, I have two groups: a treatment group and a control group. Both groups have two biological replicates (i.e., two replicated populations): "1" and "2". I would like to nest the replicates within the main groups OR set the replicates...a random effect when running the DESeq2. I am not particularly interested in differences between the replicates (we would like to assume that there are none)…

deseq2 Nested design random variable

updated 10.5 years ago • Silva

so for every single test I have 6 libraries (control + infected at 3 time points). Having three replicates this makes 18 libraries in total. What I did until now is look at each time point separate and calculate DEgenes

RNASeq edgeR RNASeq edgeR

updated 13.4 years ago • Kaat De Cremer

div class="preformatted">I have three biological replicates of a tissue and each biological replicates has three technical replicates. There is little difference between...biological replicates however technical replicates are almost same. I would like to use quantile normalization method between biological...replicates. matrix at present is as follow: Biological replic…

Normalization Normalization

updated 9.3 years ago • Yashwant

<div class="preformatted">Dear list, I am going to analyze data with the design shown below. I do not have replicates, but is a complete 2x3 factorial design and therefore I should be able to fit a linear model y=mean+Celltype+Treatment+error for each of gene and then do multiple testing correction, right? So, why after running, TS<-paste(raw_files$Cell_Type, raw_files$Treatment, …

Microarray qPCR Classification limma HTqPCR ASSIGN Microarray qPCR Classification limma

updated 15.9 years ago • Andreia Fonseca

guide. Please forgive my lack of statistical knowlege. 1. For the example in section 9.4 technical replication II >design<-designMatrix(targets, ref="wt1") >fit<-lmFit(MA, design) >cont.matrix<-makeContrasts(MuvsWt...gt;fit2<-contrasts.fit(fit,cont.matrix) This technique includes an effect for each biological replicate. How about technical replic…

Microarray limma Microarray limma

updated 21.1 years ago • Ren Na

I am working with an RNA-seq dataset where we're looking at the potential synergistic effects of two genes (by definition, the output in a synergistic system suggests that the two genes, when acting together as proteins on the same cell, will have a greater effect than the sum of their individual effects on a cell). All 4 mouse samples are stimulated with the proteins expressed by these genes: …

edger normalization counts RNA-seq

updated 6.5 years ago • Tima Karginov

HELLO ALL i have a question to ask, how to design a matrix i have such samples 0h, 3 replicate and 3 control 6h, 4 replicate and 1 control 12h, 4 replicate and 1 control 18h, 4 replicate and 1 control 24h, 3 replicate...and 3 control 36h, 3 replicate and 1 control 48h, 3 replicate and 1 control thank you very much -- shan gao Room 231(Dr.Fei lab) Boyce Thompson Institute

updated 13.5 years ago • wang peter

8 samples (genes as rows, samples as columns): AA - FP SC T1 (Follicular phase, solvent control, replicate 1) AB - FP T1 (Follicular phase, treated, replicate 1) AC - FP SC T2 (Follicular phase, solvent control, replicate 2) AD - FP T2...Follicular phase, treated, replicate 2) AG - LP SC T1 (Luteal phase, solvent control, replicate 1) AE - LP T1 (Luteal phase, treated, repl…

DESeq2

updated 3 months ago • snijesh

div class="preformatted">Dear Support team, I have three biological replicates of a tissue and each biological replicates has three technical replicates. There is little difference between...biological replicates however technical replicates are almost same. I would like to use quantile normalization method between biological...replicates. matrix at present is as follow: …

Normalization Normalization

updated 11.6 years ago • Yashwant Kumar

70-mer oligonucleotide microarray slides. Here is my experimental design: Control X T1 (Biological Replicate 1) T1 X Control (Biological Replicate 1) Control X T1 (Biological Replicate 2) T1 X Control (Biological Replicate 2) Control...X T2 (Biological Replicate 1) T2 X Control (Biological Replicate 1) Control X T2 (Biological Replicate 2) T2 X Control (Biological Replicate 2) My

Microarray TimeCourse timecourse Microarray TimeCourse timecourse

updated 16.8 years ago • Priscila Grynberg

I have a control set of samples, and 3 viruses, each exposed to two treatments. 5 replicates of each. In two viruses, in both treatments, the first two replicates are left shifted in the PC1 values compared...to the other replicates, example: - virus1_treatment1_Rep1 -26.21084691 - virus1_treatment1_Rep2 -23.81517717 - virus1_treatment1_Rep3...10.74887635 - virus1_treatment1_Rep5 -10.1106…

DESeq2

updated 4.6 years ago • swbarnes2

each of these (and that they are different!), but the basic outcome I am trying to achieve is to replicate the results of [ToppGene ToppFun analysis](https://toppgene.cchmc.org/) - but using R, and with species other than human

topgo biomart gene ontology goexpress pathway analysis

updated 9.5 years ago • Darya Vanichkina

<div class="preformatted">I am analyzing a set of RNA-Seq data without replicates, possibly using edgeR. (We understand limitations and are only interested in descriptive analyses.) There are three...div class="preformatted">I am analyzing a set of RNA-Seq data without replicates, possibly using edgeR. (We understand limitations and are only interested in descriptive analyses.) There are

edgeR edgeR

updated 12.0 years ago • Lisa Cohen

I now have an expression profile of an organ at various developmental time periods, such as 3 replicates in the first week, 5 replicates in the second week, 4 replicates in the third week, and 5 replicates in the fourth week...design. such ## make a single series edesign > Time <- rep(c(1,5,10,24), each = 3) > Replicates <- rep(c(1:4), each = 3) > Group &…

tissueTreg

updated 2.7 years ago • guang-hui

Hello, My recent RNA-Seq experiment had a few samples with high counts of rRNA genes in the third replicate. My pipeline was HISAT2 --> featureCounts --> DESeq2. When clustering replicates 1 and 2, the replicates of each condition...clustered closely together in a PCA. With all three replicates, samples of the third replicate clustered more closely together than to the samples o…

DESeq2

updated 4.1 years ago • jac

<div class="preformatted">Dear Naomi, Sorry I made a mistake, I really want to fit the interaction model, I was tired and I forgot to add the interaction term. If I have two factors I have to count for the effects of each and the interaction right? Why did I I runned of d.? How can I analyze this if the problem is no replicates? Can I use a random block design? thanks for your opinion Ki…

Microarray qPCR Classification limma HTqPCR ASSIGN Microarray qPCR Classification limma

updated 15.8 years ago • Andreia Fonseca

We have been trying to replicate part of the analysis of the paper titled 'An approach for normalization and quality control for NanoString RNA...We have been trying to replicate part of the analysis of the paper titled 'An approach for normalization and quality control for NanoString RNA expression data' by Bhattacharya et al. To get a feeling for the effect of RUVseq normalization compared to n…

RUVSeq

updated 2.1 years ago • myrthevanbaardwijk

div class="preformatted">Dear BioC, I have technical replicates are in dye-swap pairs with four hybs conducted in house and four hybs conducted in commercial lab. Same RNA samples...corfit$consensus is supposed to be a negative number according to limma usersguide (the technical replicates are dye-swap and should vary in opposite directions). Why am I getting a positive number in the dye-swap…

limma limma

updated 20.2 years ago • kevin Lin

main point though is that there is no need to take special action to allow for different numbers of replicates. This is accommodated automatically in the course of an ordinary analysis. Gordon Dear Gordon, Sorry for being...slow about this.... If in an extreme example you had three biological replicates A,B and C and for biological replicate A you had 12 replicates but for biological replicat…

updated 20.6 years ago • Jason Skelton

Hi I just started to work with oligonucleotide microarray and I would like to have suggestions on the methods I should use to analyse the data. I have three duplicates done in two different places, so they look very different in terms of background and signal intensities. Since I'm not going to have more duplicates I would like to get the most information possible from these, even if I do know t…

Microarray Microarray

updated 21.7 years ago • Paola Sgado'

There are times when it makes sense to have genes duplicated in both the universe and the set of interest - e.g. if the geneIds come from BLAST hits of unigenes of an unsequenced species against the genes of a sequenced species. I fiddled a bit with GOstat, but was not able to see how to change the code to allow this. (I can see where duplication was removed in the gene set but not in the unive…

updated 18.2 years ago • Naomi Altman

<div class="preformatted">Hi again, I apologise for replying to my own post, but it helps keep track if someone will be interested. I analysed my data with single channel analysis in limma, according to Chapter 9. of limma usersguide. I changed my targets file (to make it more condensed) and removed suffix which identified biological replication. So my targets looks like: >nt_trg…

limma limma

updated 15.8 years ago • Maciej Jończyk

<div class="preformatted">Thanks very much for the thorough explanation, I will add tm:trt1 to my model to see expression changes in two condition. Best wishes, On Tue, Mar 20, 2012 at 7:51 PM, James W. MacDonald <jmacdon@uw.edu> wrote: > Hi Xuan, > > > On 3/20/2012 2:13 PM, shao chunxuan wrote: > >> Hi Jim, >> >…

Microarray GO Regression limma Microarray GO Regression limma

updated 13.5 years ago • Chunxuan Shao

that have the same effect at both time points. How do we best set this up in deseq/dexseq. We have 3 replicates of each set: - 3 replicates cases/ 3 replicates controls - timepoint 1 - 3 replicates cases/ 3 replicates controls - timepoint

DEXSeq RNA-seq DESeq2

updated 2.3 years ago • Alice

<div class="preformatted">Hi, In Chapter 8.1.2 in Limma users guide there is a description how to detecting the Dye effect. The example there describes an experiment of Wt vs Mut with two dye swaps replicates as follow: FileName Cy3 Cy5 File1 wt mu File2 mu wt File3 wt mu File4 mu wt Let's say that I found a list of gene which are significant due to mutant effe…

limma limma

updated 4.9 years ago • Ron Ophir

import. My experiments has two groups (low temperature and high temperature). I have two technical replicates for each group and 6 biological replicates from each technical replicate. I used  <pre> sampletable <- data.frame...c("low", "high"), each = 12)</pre> But in this design it did not take into account the technical replicates. Could anyone help me to underst…

deseq2

updated 8.0 years ago • deeptiptl74

my scenario.  I'm using edgeR to analyze genome wide genetic screen data.  I had three replicates of my library, each infected independently, however, before the beginning of the screen one replicate became contaminated...nbsp;At the start of the screen, I took one of my remaining two replicates and split it into two, maintaining each separately for the duration of the experimen…

edger design matrix

updated 9.6 years ago • laura

there would be four copies or > > >more which inturn obviously effects spacing etc between replicates. > > >I'm not sure why they would want differing numbers of copies of genes but > > >they would like to be able

limma limma

updated 22.1 years ago • Gordon Smyth

Dear BioC people, I am analyzing a wet lab experiment on a cell line with 5 biological replicates. Each biological replicate has 3 or 4 technical replicates. Here is my targets$BiolRep entry <pre> targets$BiolRep...1] 1 2 2 1 2 1 4 4 1 4 3 3 3 5 5 5</pre> Some of the biological replicates are sensitive and some are resistant to a drug. The purpose of the experiment is to find the…

limma duplicatecorrelation

updated 9.1 years ago • Eleni Christodoulou

gene expression from an RNA-Seq experiment in which two factors were applied. I have multiple replicates among samples when just Factor 1 is considered, and there is significant clustering among the groups. When Factor...2 is considered as well, there is one group that has no replicates of Factor 2 at a certain level of Factor 1, while the other levels of Factor 1 have 2 replicates for each level…

rnaseq edger differential expression dispersion

updated 9.1 years ago • nancyjwahl7

I have a RNA seq data which I am trying to identify DEGs. Dataset contains Untreated - 2 replicates (time point 0hr) Treated - 4 replicates (2 replicates at 6hr and 2 replicates at 12 hr) I merged all the samples and tried

deseq

updated 7.7 years ago • aishu.jp

is ~200K. Similarly to a CRISPR, within each biological replicate I have internal replicates, which are amplicons with different DNA sequence that I would expect to give the same...I have looked at the internal replicates, and seen that there is a lot of variation between them as well. So I am assuming that the experimental system is...opinion about this: - would it be OK to discard the amplic…

independentfiltering DESeq2 CRISPR

updated 4.8 years ago • Rubén

relative to each other. Methods: To do this, the 3 populations were captured from four biological replicates. Thus 12 arrays. Two complete replicates were hybed on day 1- thus Hyb A, and the second two rep.s a month later- Hyb B

qPCR affy affydata vsn limma gcrma matchprobes affyPLM affyio BRAIN qPCR affy affydata

updated 19.2 years ago • k. brand