Bioconductor Forum

Hiya, I am finding that if I am inputting my data in one count matrix and calling to contrast two different treatments from the four (each with 5 sample replicates) using the contrast function in DESeq2 that I get different differential expression results to that if I...one count matrix and calling to contrast two different treatments from the four (each with 5 sample replicates) …

deseq2

updated 8.9 years ago • bekah

<div class="preformatted">Dear Dr. Smith and group, apologies for simple question. I have data with simple plan. There are 8 samples (4 normal and 4 tumor). These tissue samples are from different patients. No replicate experiments were done. I wish to see genes differentially expressed between Tumors and normals. In such cases how...There are 8 samples (4 normal and 4 tumor). These tis…

limma limma

updated 19.6 years ago • Srinivas Iyyer

have the following scenario: I have a bulk RNA-Seq matrix with three conditions and three biological replicates per condition. I have used CIBERSORT to estimate the cellular composition of my sample, so now I have this value

deseq2 rnaseq

updated 7.7 years ago • cartalop

<div class="preformatted">hi, has anyone experience how good rma performs on affymetrix experiments where the data from different cell lines are compared against each other? i have 4 chips, one with cell line 1 with treatment, one with cell line 1 without treatment, one with cell line 2 with treatment and one with cell line 2 without treatment. the question is, should i normalize all them …

updated 21.5 years ago • Dipl.-Ing. Johannes Rainer

to it yet. I am analyzing a metatranscriptomics experiment with treatment and control (three replicates each) and I calculated fold changes. - What does it mean when a gene is upregulated in the treatment? Does this mean

deseq2

updated 6.1 years ago • raven

quantile normalization assumes common distribution for data sets to be normalized. I am fine with replicate normalization using this. However, for different experiments, such as data from different tissues, is the assumption

Normalization Normalization

updated 22.9 years ago • Wang, Hui

have a couple of questions on how to handle the following situations: 1. There seems to be a lot of replicate probes. Is it best to take the average or use the maximum value? 2. The gProcessedSignal contains may negative values

Microarray Cancer Microarray Cancer

updated 16.2 years ago • uthra suresh

<div class="preformatted">Dear Belisa, Your experiment has 17 different conditions, so you obviously cannot analyse it as a 2x2 experiment. (A 2x2 experiment has only 4 conditions in total.) The simplest way to analyse your experiment is to create a single factor with 25 levels, and to analyse your data as in Section 8.3 in the limma User's Guide. This allows you to test any hypothesis …

hgu133plus2 hgu133plus2

updated 13.2 years ago • Gordon Smyth

consisting in 30 tags. I have 9 column in total, consisting of 3 different samples(C-L-T) made by 3 replicates each(1-3). So, I used the filter command : > keep <- rowSums (cpm(d)>5) >=3 > d <- d[keep,] Then I looked at the output of `cpm...If I am selecting the counts that in the single row (gene) are at least grater then 5cpm in the 3 replicates of 1…

Cancer Cancer

updated 13.2 years ago • Vittoria Roncalli

groups, which represent the time since the treatment was applied. Each treatment has 3 biological replicates and this experiment setup was repeated 3 times. So there are 3 biological replicates from each experiment (E1,E2...E3), giving a total of 9 biological replicates per treatment. So for the genotype A, the breakdown is: Treatment | Biological Replicates ------------ | ------------- 0h…

deseq2 timecourse michael love multiple time points

updated 6.3 years ago • cp1015

to analyze my RIP-seq data, which includes both INPUT and IgG control samples for each of the three replicates across two conditions: treated and untreated? Is it correct to put all the samples together, considering IP, INPUT

DESeq2 RIP-seq

updated 21 months ago • Adelaide

getting these error messages when I try to do IPA analysis and try to do the differential test with replicates][3] [1]: /media/images/9b45f322-325e-44d4-88e5-9e43abc9 [2]: /media/images/6ca13fa1-a855-4637-9e27-ceac8870 [3]: /media/images

APAlyzer

updated 3.5 years ago • Sasha

I am trying to analyse some drug HTS data I didn't generate. The format of the experiment is: 485 drugs split over 2x 384-well plates For each plate pair (P1 and P2), the drugs have been tested at 4 concentrations (25nm, 100nm, 250nm, 1um) such that for each replicate I have: P1- 25nm P2 - 25nm P1- 100nm P2 - 100nm P1- 250nm P2 - 250nm P1- 1um P2 - 1um The layout of the plat…

cellHTS2

updated 4.3 years ago • yura.grabovska

list and I probably have a 'stupid' question. I'am doing SAM analysis for the one-class case on a 6 replicates per 8448 gene matrix. I get each time this message SAM Analysis for the one-class case. Warning: There are 353 genes

updated 21.8 years ago • perin

have two types of controls: input DNA and unmodified histone. I have two conditions and 6 biological replicates of each condition I wanted some advice on how to perform basic quality control on Chip- seq data using Bioconductor

Sequencing Sequencing

updated 14.3 years ago • Lucia Peixoto

<div class="preformatted">Dear All , I am analyzing the Rna-seq data from one sample. I don't have replicates. I have different cell lines (6) from one sample and I am interested in varying genes among different cell lines within...div class="preformatted">Dear All , I am analyzing the Rna-seq data from one sample. I don't have replicates. I have different cell lines (6) from one sampl…

edgeR edgeR

updated 14.4 years ago • rakhs shit

based normalization with edgeR and do you think a reliable basic normalization is possible without replicates? Thank you for your comments. Best regards Jens </div

Sequencing Normalization edgeR Sequencing Normalization edgeR

updated 15.0 years ago • Jens Georg

dataset of about 200 samples, divided into 16 treatments. For each treatment, I have 3 biological replicates and 4 time points. Because of space issues, we decided to split our samples over 3 different blocks. In each block

DESeq2

updated 4.9 years ago • Alessia

separate the 4 different conditions (time course) that are on each array? I have three biological replicates split on 3 arrays and each array contains 4 time points. Maybe I should just pipe it through VSN, but with Affymetrix

Normalization probe vsn limma gcrma Normalization probe vsn limma gcrma

updated 18.3 years ago • Ido M. Tamir

div class="preformatted">Hello, I have 37 RNA-seq datasets for 18 human tissues - some have 3 replicates, some 2 and some do not have any (sorry to say that). I'm thinking of a way to do differential exon usage analysis - using

edgeR DEXSeq edgeR DEXSeq

updated 12.6 years ago • Manoj Hariharan

I have read the edgeRNA manual and I assume this is due to fitting a lot of models (or lack of replicates) ? If so, however, I dont know which of suggested choices (2-4 ) is the best alternative? and how to do this (for exmple option

edger R limma

updated 5.9 years ago • eb.mahmoudi

DESeq is used for our RNA-Seq data analysis of two conditions, each of them has three biological replicates, we got a result better than Cuffdiff2 and CLCBio Genomics Workbench, verified by RT-qPCR. Based on the normalized

Normalization DESeq Normalization DESeq

updated 12.0 years ago • Liang, Chun

I have three groups (with multiple replicates each) from 3 different locations. How can I obtain the DEG between the three groups since I have no control to compare

anova RNASeqData tukeyTest DESeq2

updated 5.2 years ago • ecg1g15

Fixed factor) 2 Populations nested in each Invasive Status (Random factor) 2, 3 or 4 biological replicates, nested in each Population (Random factor) The number of replicates is not the same in each Population and Treatment

Microarray Microarray

updated 16.7 years ago • Aurélie Bonin

genes" "source" "M" "A" >data.abiotic<- MAW$M > mat FileName Time Replicate HF HT NLT 1 1hr_HF_1_7411_1_1 1 1 1 0 0 2 1hr_HF_2_7411_1_3 1 1 1 0 0 3 4hr_HF1_7411_1_2 4 2 1 0 0 4 4hr_HF_2_7411_1_4...TimexNLT" "Time2" "Time2xHT" "Time2xNLT" > names(design$edesign) [1] "FileName" "Time" "Rep…

limma maSigPro limma maSigPro

updated 7.4 years ago • neeraj rana

Gtype1.Time1), Levels=design.1)</pre> However, in this particular experiment there is a strong replicate effect. Biological replicates where carried out over time in four independent experiments. I would like to correct

rnaseq edger statistics model matrix

updated 8.3 years ago • David Rengel

using edgeR for analyzing RNA-seq data containing Control (CR) and two Treatments (HR and SR) with 2 replicates for each. Based on the common dispersion, BCV and the MDS plot, it looks like biological replicates of HR samples

GO edgeR GO edgeR

updated 12.0 years ago • Avinash S

preformatted">Dear Bioconductor, I am working with a loop design with both biological and technical replication, and am having difficulty incorporating both types of replication appropriately. I am looking at the both strain

updated 18.8 years ago • Heather de Glanville

<div class="preformatted">Dear Bioconductor Users, I have an experimental design where I have several samples which I wish to compare in several ways (necessitating multiple comparisons) and of course several thousand genes (necessitating multiple tests). My general strategy in for analyzing these experiments is to 1. Obtain p-values for the different comparisons for each gene co…

Normalization Cancer Normalization Cancer

updated 21.5 years ago • Richard Friedman

Hello, I have some RNA-Seq samples. These samples have no replicates, but I have lots of samples. What I am trying to do is find a trend based on a continuous variable (let's say age). I am basically...Hello, I have some RNA-Seq samples. These samples have no replicates, but I have lots of samples. What I am trying to do is find a trend based on a continuous variable (let's say age). I am…

limma edgeR rna

updated 4.8 years ago • Nirad

Hi Simon, I am working with a multi-factor experiment that, in a sense, doesn't have biological replicates (replicates were done at different times, and the differences in the chemistry/instrumentation is enough to be

updated 13.8 years ago • Ingrid Lindquist

<div class="preformatted">Hi Mick, After quantification of each library using qPCR, we prepared a 20 pM dilution in hyb-buffer. This was followed by pooling of the 12 plex sets producing a 20 pM solution of 12-plex libraries, which according to Illumina should be stable for 2-3 weeks. From this 20 pM stock solution we then prepared a fresh 7 pM solution for each sequence run (7 pM results …

Sequencing qPCR Cancer Sequencing qPCR Cancer

updated 15.7 years ago • Jakob Hedegaard

below) > Dear Heidi, > > I have TLDA data in the following format which has 3 biological replicates > per file (here is the header) > > SDS 2.4 RQ Results 1.2 > Filename 7.10.10 8A.2.sdm > Assay Type RQ Study &gt...Max. "40.00" "40.00" "40.00" "40.00" > > > But if I try and assess …

qPCR GO HTqPCR qPCR GO HTqPCR

updated 15.3 years ago • Heidi Dvinge

together and doing a wilcoxon test. that's all fine, but my boss was surprised to see that our replicates (=quality controls) looked better in the "single patient normalization way" than after normalizing all patients...thanks, jo Quoting Naomi Altman <naomi@stat.psu.edu>: > Dear Johannes, > Actually, technical replication is of little interest when you have > biologi…

Normalization probe affy limma gcrma DOSE Normalization probe affy limma gcrma DOSE

updated 21.0 years ago • Dipl.-Ing. Johannes Rainer

the world over. Solid, employable kind of work. > Incidentally, distinguishing between technical replicates and > biological replicates can make a huge different to ANOVA test scores, > so I think we should insist that...I agree with this observation. Certainly there's a difference between biological and technical replication, and I've seen instances where the v(B)>V(…

GO Clustering GO Clustering

updated 22.1 years ago • Chad Shaw

Hi all, I have a fairly complex ChIP-seq experiment with lots of treated/untreated groups, 2 replicates each. All the 'replicate 1s' were generated together, separately to the 'replicate 2s' and I can see that that effect

diffbind batch effect

updated 9.5 years ago • Jon Manning

Hi, I am running an RNA Seq analysis for differential gene expressions for the following Biological Replicates: 3 Control, 3 Treatment A, 3 Treatment B, and 3 Treatment C. I need to make the following comparisons: Treatment A vs Treatment...I am running an RNA Seq analysis for differential gene expressions for the following Biological Replicates: 3 Control, 3 Treatment A, 3 Treatment B, a…

RNASeqdataanalysis DESeq2

updated 2.4 years ago • Sreeja

all, i was wondering if anyone could help mw. I am trying to calculate the correlation between two replicates spots. I triied following the manual and the bioC website but have not managed to understand why it is not working

updated 18.6 years ago • Houeix, Benoit

div class="preformatted">HuEx 1.0 st v2 Two replicates each at 0h 3h 6h 12h 24h 48h I've extracted my summarized data from aroma.affymetrix as a data frame, I now need to

Normalization limma Normalization limma

updated 17.6 years ago • Roberts, Raymond

in the manual to test for the significance of gene retention between groups of samples (multiple replicates). my design has both two categorical and 5 scaled continuous variables After running DESeq, I found a message saying

deseq2

updated 5.9 years ago • Do it!

div class="preformatted">Dear All, I've got two conditions and three replicates per condition: A1 A2 A3 B1 B2 B3 To test the INTRA VS INTER group variance, I compared the fold changes within group

updated 19.0 years ago • Emmanuel Levy

<div class="preformatted">When trying to replicate the function of plotPCA using prcomp as I use normally I noticed that I could only get it to match if I set scale=F in...div class="preformatted">When trying to replicate the function of plotPCA using prcomp as I use normally I noticed that I could only get it to match if I set scale=F

Cancer Cancer

updated 11.5 years ago • Daniel Brewer

Hi, I am trying to compare between 10 groups each with two replicates (RNA-seq data). I wish to determine the genes which distinguish each individual apart from one another. So far looking

edger multiple comparisons anova glm

updated 10.6 years ago • chickenchamp4

of features measured in small sample sets. For instance, 2  or more groups; 4 biological replicates each, 150 genes/metabolites. Can we use empirical Bayes moderation in this situation? If yes that would be great

limma metabolite data moderated t-test

updated 8.5 years ago • iglezer

Computer Science and Dep. of Biotechnology and Bioengineering). Obviously it would not make sense to replicate Bioconductor and R efforts (that we think are excellent), our intention is to develop mainly interfaces more intended

updated 22.4 years ago • Andrea Splendiani

I have PRO-seq data with the following conditions: untreated (time 0), vehicle, and treatment (3 replicates each, 4 time points for each vehicle/treatment combination). I would like to evaluate treatment effects as a function

deseq2 differential gene expression

updated 7.6 years ago • warrena

22:24, ) : undefined columns selected" The error prevents halts the analysis, and I can't replicate what is shown in the above-mentioned tutorial. I have tried uninstalling and re-installing ChAMP, to no avail... Any

ChAMP

updated 10.5 years ago • junyupub

RPKM. What is the easiest way to do this? For example, GSE165500 has RNAseq data for 3 UNX_BMDM replicates (both raw counts and RPKM). How do I take these files and generate a simple expression graph of 1 gene in 1 condition

RNASeqData RNASeq GEO RNASeqR HELP

updated 4.8 years ago • hgalmoore

the count matrix, just leaving the first column key as a reference followed by the counts in all the replicates in the matrix: <pre> dds<-DESeqDataSetFromMatrix(countData = countData, colData = colData, design = ~ sex + treatment

deseq2 deseq diffbind differential binding analysis

updated 8.1 years ago • rbronste

size factors? 3)How reliable is DESeq on ~10 samples per condition with no biological nor technical replicates. I've read in a previous post there is no support for this scenario. Sequencing is expensive, does it means that it

deseq2 rnaseq normalization

updated 7.9 years ago • xavi

Dear Bioconductor community, I am currently struggling with fitting a linear model to Illumina BeadChp HT12.v4 data. I have different cell generations (G1, G2, G3 etc) taken form the same clone. I assume these as biological replicates. For each generation I have different measurements. I assume these as technical replicates. Microarray data were measured in two different batches. Finally, I have…

limma microarray duplicatecorrelation technical replicates linear model

updated 10.0 years ago • Eleni Christodoulou

for this dual-sequence setup. **Experimental Design:** - Organism: Plant. - Samples: 3 replicates of Condition A and 3 replicates of Condition B. - Data: Paired-end RNA-seq reads (150 bp, 30 millions reads for sample

edgeR DESeq2 haplotype limma

updated 8 months ago • Paulito

be the more appropriate method? The *tximport* docs say that it can and will import inferential replicates - does DESeq2 at all utilise these inferential replicates from *tximport*? I have used R version 4.20, Bioconductor

tximport DESeq2 edgeR swish miRNA

updated 3.6 years ago • BG

the contrast tests, so this also does not recover the ANOVA. I have not tried this computation with replicate spots (only replicate arrays) but either: 1) the usual ANOVA is right and duplicateCorrelation is doing something

limma limma

updated 20.5 years ago • Naomi Altman

<div class="preformatted">Hi List, I am analyzing my RNA-Seq data with edgeR. The next is my experimental design: d.GLM An object of class "DGEList" $samples group lib.size norm.factors R4.Hot HotAdaptedHot 17409289 0.9881635 R5.Hot HotAdaptedHot 17642552 1.0818144 R9.Hot ColdAdaptedHot 20010974 0.8621807 R10.Hot ColdAdaptedHot 14064143 0.893279…

edgeR edgeR

updated 14.1 years ago • Miguel Gallach

a samplesheet.csv describing my samples and it looks like this: SampleID,Tissue,Factor,Condition,Replicate,bamReads,bamControl,Peaks,P eakCaller meio.1,meiocytes,H3K4me3,N,1,M_meiocytes_H3K4me3.bam,InM_input_meiocyt...gt;H3K4.B73 2 Samples, 38870 sites in matrix (45304 total): ID Tissue Factor Condition Replicate Peak.caller Intervals 1 meio.1 meiocytes H3K4me3 N 1 …

DiffBind DiffBind

updated 12.4 years ago • Guest User

to find differential expression using RNA-Seq > data (this is the first time I have biological replicates). > I apologize in advance for my lack of knowledge. > > I have 10 samples, 5 biological replicates of each condition

edgeR edgeR

updated 13.5 years ago • Gordon Smyth

Hi, I want to correlate two matrices of data. First matrix is metabolomic data with 8 biological replicates and 51 metabolites. Second matrix is the transcriptomic data with 8 replicates and 64 transcripts. Metabolomic

correlation

updated 7.6 years ago • adriana.gallego.02

div class="preformatted">Dear all, Thank you for the very interesting discussion on the topic of "replicates and low expression levels" in the last few day. I am facing a related problem regarding normalization and would...experiment was done on blood macrophages and hybridized to affymetrix chip HGU-133A. There were 3 replicates and 3 time points (0, 2, 48 hour). The main problem is that at…

Normalization Normalization

updated 22.7 years ago • Adaikalavan Ramasamy

Hello to everyone, I am trying to find gene fold change for transcription data in R via limma package.As I am bit new to this, just want to make sure that what I have been doing is correct or no. I am bit more concern on design matrix that i have created correct or not and also with the contrast matrix.  My target is to get relative gene fold change from control vs treatment at 3 time p…

bioconductor affymetrix microarrays differential gene expression design and contrast matrix

updated 8.6 years ago • raju.prasad.phe12