Bioconductor Forum

this. In brief, I have a time serise with 12 Times(unevenly spaced), and for my Treatment I have 6 replicate Control individuals plus 8 replicate Affected individuals (14 people total, with repeated measurements over...the df? The limma example suggests 3-5, but it has 16 Control + 16 Treatment individuals with no replication.  I could select 12 knots for my 12 timepoints (df=14, or …

limma spline design and contrast matrix covariate

updated 10.5 years ago • Hilary

gt; Furthermore, the spots themselves have been duplicated side-by-side. > Thus, each slide has 4 replications of the same EST, occuring in 2 > adjacent pairs that are equally separated. My reading of the limma > vignette...with accounting for > duplicate spots, I haven't been able to find a solution for the > replication of the arrays on the same slide, where …

Microarray limma Microarray limma

updated 19.3 years ago • Gordon Smyth

Heidi, > > I have received a data set from a qPCR miRNA set where I have > > sample1 - 2 replicates from the same RT reaction > 1 replicate from an independen RT reaction cell_type:A > sample 2 - cell type : A > sample2...B > sample 4 cell_type:B > > I saw that I should prepare one file per sample, but the replic…

miRNA qPCR limma miRNA qPCR limma

updated 15.8 years ago • Heidi Dvinge

<div class="preformatted">Dear Roman, I know that your question was specifically about the DESeq package, but you have previously asked about the edgeR, bayseq and DESeq packages on SEQanswers: http://seqanswers.com/forums/showthread.php?t=4349 so I thought you might appreciate a more complete answer. I think that edgeR is the only package on Bioconductor that analyses RNA-Seq experim…

RNASeq edgeR baySeq DESeq RNASeq edgeR baySeq DESeq

updated 14.8 years ago • Gordon Smyth

of samples and coefficients to fit, so estimation of dispersion is not possible. Treating samples as replicates was deprecated in v1.20 and no longer supported since v1.22

DESeq2

updated 2.9 years ago • nishant raj

Has anybody already done this? I have a start on the task using the XML package, but don't wish to replicate work already done. Thanks! - Paul</div

Pathways Pathways

updated 15.7 years ago • Paul Shannon

Deseq2 for Differential gene expression analysis. I have two controls ( but they are not technical replicates) and one patient. I designed the experiment with two controls together. I was wondering what Deseq2 does when two

Deseq2 RNASeq

updated 7.0 years ago • tanyabioinfo

questions regarding the plotTranscript function. 1) what is it actually plotted? If I have several replicates, is this a plot of the total number of reads aligned or the average? 2) I assume that the two y axes are one for the RFs

riboseqr

updated 10.9 years ago • antonvila.s

with DESeq2, for dataset that has 4 Genotypes (G) in control and treated conditions (C) each in 3 replicates. I would need all levels plus G*C interactions. If anyone has similar code please share it. Many thanks, DR

DESeq2 anova condition genotypeeval

updated 5.1 years ago • Dan

within each group. My current design formula is: ~ material + allele_combination + material:replicate + material:allele_combination I included replicate as interaction term nested within material, but there has

Bioconductor

updated 3 months ago • Rita

would be greatly appreciated. I have 2 cell lines (sensitive and resistant), with 16 samples (2 replicates each= so 32 samples) at 4 times points (S1 (1hr), 12hr and 24hr) and 4 treatments (X, Y, Z). Each cell line has a baseline and each...each treatment to baseline at every time point for both cell lines. The model I tried to build was ~Replicate+ Condition+ Condition:Factor+Treatment (for e…

timecourse DiffBind DESeq2

updated 4.3 years ago • Kavi

<div class="preformatted">hii... this is Deepak.... i am working on analysis of microarray data. We tried using this code snippet phenoData(spikein95) <- new("phenoData", pData = pd, varLabels = vl) but it said that phenoData is now defunct and we need to use AnnotatedDataFrame. And i am not able to proceed because of the following error AnnotatedDataFrame(x1)<-new("Annota…

Microarray Microarray

updated 13.9 years ago • Deepak Datta

most of the genes not being differentially expressed. 2. 5 known conditions. 3. 4 biological replicates per condition. 4. To be run in a single batch. Questions: 1. Are there possible advantages to normalizing RUV as distinct...paper, I am not clear on this point (my fault). 3. If RUV-III should be used do I need technical replicates for each condition, or for only 1 condition?…

RUV Nanostring RUV-III

updated 5.7 years ago • raf4

Enter the body of text here 1: How to extract the counts of each replicate after dba.count while doing the occupancy analysis? I would like develop a genomic ranges object that consist...peaks (chr, start, end,) and in addition to score two column showing the normalized counts of each replicate in only these consensus peak set. 2: Also, why counts(48923) are different than peak set (52772…

DiffBind

updated 4.4 years ago • faiza

Hi all, This is more a general (philosophical?) question: 1. Say I have a dataset analyzed with DeSeq default, and a vst normalized dataset is obtained. 2. The VST is then used to generate a final predictive ML model (binary) with only few genes from the VST dataset. 3. Now I have a few samples that I re-rerun end-to-end with the exact same pipeline as above. 4. After importi…

DESeq2 Normalization Clustering replicate

updated 3.4 years ago • Giorgio

Hi everybody, We have scRNA seq data from 1200 cells from SMART-seq2 obtained from 3 runs: - 1st run - 400 cells - 1st condition - 2nd run - 400 cells - 2nd condition - 3rd run - 200 cells 1st condition + 200 cells 2nd condition. The cells from the 3rd run where already in the 1st or 2nd run. Which controls do you think we have to check? Is it ok to start the analysis with all the data without…

SingleCellExperiment scRNAseq

updated 4.1 years ago • Núria

Hello everyone, I was comparing the graph generated by the function plotMA() from DESeq2, with the graph that I generated supposing I extracted the data with this function:

ds2data <- as.data.frame(results(dds,cooksCutoff = FALSE))

and plotted the result with ggplot2 using similar margins and color code. The points seem to be at the same positions, however, the colors are sometimes not m…

deseq2 plotma

updated 8.4 years ago • Yvan Wenger

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updated 20.2 years ago • Carolyn Fitzsimmons

What is the significance of this error? What does this tell me about these two samples?. I added the coeficients for the donor then re-calculated the fit. > donor.fit = lmFit(MA, design.III) Coefficients not estimable: HISS0039 HISS0050 The design matrix I used was as follows, you can see that there is a column of 1's that represent that the first 16 samples are for donor HISS0033, the res…

updated 21.0 years ago • Pita

Hi, I'm dealing with quite an unusual study design. Originally (due to unfortunate and inevitable circumstances) we had all "high_risk" and "medium_risk" samples on batch 1 and "low_risk" samples on batch 2. Then we discussed the batch effect and decided to re-hybridize some randomly selected samples from each risk group on batch 3. The resulting study design looks a bit like like this (in reali…

updated 12.5 years ago • Essi Laajala

Hello, I have cDNA, I tried limma to do normalizeWithinArrays, but the results looks like they did not aveage spots. Each spot was printed twice in my cDNA array, how limma average them? I have all the log ratio from every printed spot(twice printed). When I did correlation between replicated, their coef is very low, which compared to arrayTool analysis, is significant different. When I looked at…

limma limma

updated 21.9 years ago • Joyce Gu

Dear list: I am working on a small data set that came from cytokine membranes. Each membrane contains 23 specific cDNA fragments spotted in duplicates. The experiment design includes two treatment of interest, stimulant (pre-activated or non-activated cells) and inhibitor (w/ or w/o inhibitor). After manual normalization based on control spots on each membrane, I like to run limma as factorial…

Normalization limma Normalization limma

updated 20.6 years ago • Jianping Jin

Dear Bioconductor users, I want to analyze a set of spotted arrays with an irregular spotting design, so avereps seems to be appropriate to get averaged expression values for each transcript. However, applying this to my data > dim(my.arrays) [1] 11520 18 > is(my.arrays) [1] "MAList" "list" "LargeDataObject" "vector" produces the following error: > average…

updated 17.2 years ago • Claus-Jürgen Scholz

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updated 19.1 years ago • João Fadista

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updated 19.0 years ago • João Fadista

some customized single channel arrays having only 40 genes including control probes. I have enough replicates to work with. Can anybody suggest a method for background correction and normalization for these kind of arrays

Normalization background correction

updated 10.8 years ago • Bandyopadhyay, Somnath Som

from Agilent CpG Islanfds, but i dont know if this is relevant because i have 4 groups ( one replicate for each group) ?? Can you please suggest me a method to analyze my data ? Any help will be appreciated Regards [[alternative

limma limma

updated 15.6 years ago • Mohamed Lajnef

A, B, C, D) and two conditions (time1 and time2). Each genotype in each condition has three replicates. I  would like to compare whether log2(A/B) equals log2(C/D) or not. Does someone have suggestions on how to implement

deseq2 compare two log2-transformated fold changes

updated 9.9 years ago • an.anand233

bowtie2: `-X 2000 --end-to-end --no-mixed --no-discordant --score-min L,-0.4,-0.4` How can I replicate them with Rsubread? `-X` --> `maxFragLength` `--end-to-end` --> ? `--no-mixed` --> ? `--no-discordant` --> ? `--score-min L,-0.4,-0.4` --&gt

Rsubread

updated 6.3 years ago • dktanwar1991

levels? This would be helpful when trying to obtain significant fold change from experiments without replicates. Thanks for your ideas. Lana [[alternative HTML version deleted]]</div

updated 20.8 years ago • Lana Schaffer

nbsp;0.9521476    0.9521476 One quick note:   Unfortunately I do not have replicates for everything and that may be  the problem:   For each depth, at Time 00d I just have 1 replicate fo the control...nbsp; (C\_00d). At time 2 days I have 3 replicates for the control (C\_02d), 2  replicates for the DOM treatment (DOM\_02d) a…

deseq2

updated 11.3 years ago • sandramg82

generated two sets (two tissues) of histone modification CUT&RUN data, each consists of three replicates of WT and three replicates of a mutant. I successfully made a heatmap using dba.plotProfile and now understand

DiffBind

updated 3.1 years ago • Junsik

is there a way to apply soft or general filter instead of using specific no. of minimum array replicates. Furthermore, I did-not find any specific filter for Affymetrix arrays in the manual. Please assist. ## Illumina Arrays...expressed. We keep probes that are above background on at least four arrays (because there are four replicates of each treatment): IsExpr <- rowSums(y$…

R filter limma Normalization Microarray

updated 2.8 years ago • Abdul

Hello, I have a RNAseq experiment where I have 2 conditions and 3 replicates each and replicates are taken from different individuals. So my design is as follows: Sample Condition Individual

deseq2

updated 10.3 years ago • genomica

Hi, I am relatively new to both Limma and R so excuse me if this is going to sound trivial to some. I am trying to do a meta-analysis on miRNA data from 4 different studies, each with its own caveats, to name a few: - Only 2/4 studies use the same Microarray technology (and chip design) - All studies have additional clinical labels (e.g. a measured tumor grade). Some label are available f…

limma

updated 6.8 years ago • shaybenelazar

expected with the typical academic microarray study, there are too many time points and not enough replicates. Within the problem study I have 19 arrays, containing 38 samples. The data looks pretty good throughout but a single...don't wish to remove the whole array since the paired data is of considerable value (through lack of replicates!). Would it make more sense to remove this data channel …

Microarray Clustering Microarray Clustering

updated 18.9 years ago • Stephen Rudd

<div class="preformatted">hii... this is Deepak.... i am working on analysis of microarray data using bioconductor and i am rank amateur in R I tried using this code snippet phenoData(spikein95) <- new("phenoData", pData = pd, varLabels = vl) but it said that phenoData is now defunct and we need to use AnnotatedDataFrame. And i am not able to proceed because of the following error…

Microarray Microarray

updated 13.9 years ago • Deepak Datta

1. estimateCommonDisp() and estimateGLMCommonDisp() now return dispersion=NA when there is no replication. This is to force users to make a decision for themselves about what action to take in this case. This means that...any subsequent function that uses the common dispersion will fail when there is no replication. If you want to reproduce earlier behaviour, you need to explicitly enter disper…

edgeR edgeR

updated 13.9 years ago • Gordon Smyth

Hello, I'm trying to do some gene set enrichment analysis with the `` gage `` package. I am using the output of `` DESeq2 ``'s variance stabilizing transformation as the expression matrix for `` gage ``. Unlike the example(s) in the `` gage `` vignette, I don't have a 'control' and 'target' sample but rather three biological replicates of four different cell types. Therefore, I would like to fi…

gage gsea rnaseq

updated 10.8 years ago • Tom

on at least n arrays, where n is the minimum group size. For example, if we compare wt (with 2 replicate arrays) to a mutant (with 3 replicate arrays), we filter probes that are Present on fewer than 2 arrays. This is because...If a probe is expressed in one of the conditions, then it should appear consistently across the replicates for that condition. Best wishes Gordon > Date: Mon, 28…

probe probe

updated 14.9 years ago • Gordon Smyth

Hello, Would someone please help me verify if i'm on the right track in correcting for technical replicates using limma and also if there is a better approach to use (duplicateCorrelation() vs lmer()) for my protein expression...patients per group (DiseaseA, DiseaseB). There are 2 biological samples(_1,_2) with 4 technical replicates each (rep1-4). Quite confused on what to include in the *blo…

limma lmer duplicateCorrelation technicalrepeats

updated 14 months ago • john

I repeated this scheme four times, with dye swap (two labelings in both directions). Technical replication is within each replicated scheme. So within scheme all instances of each sample (samples: dlc, dlk, dhc, dhk) is from...three times in different hybridizations). Here is my design file (powt_b is a number of biological replicate): Array Dye Sample linia temp powt_b dhk…

maanova oligo maanova oligo

updated 15.9 years ago • Maciej Jończyk

to mouse and drosophila genome separately. And now I am trying to get normalization factors for each replicate using DiffBind (v. 3.8.3), which I would like to use for differential peak analysis as well as scaling bigwig files...it. To my naive thinking, this option better controls for IP efficiency (~signal/noise ratio) per replicate than library size or background bins (assuming the antibody a…

DiffBind SpikeIn Normalization ChIP-seq csaw

updated 3.1 years ago • spg

<div class="preformatted">Hi all, I have done an differential expression analysis using DESeq on a set of data and got a lot of differential expressed genes at the end. I would like people to check the code and the assumptions I made. A-Samples I am analysing 6 samples in total from 3 different cell types (E, S and H), S1and S2 ( biological replicates) H1, H2 and H3 ( biological replicat…

Sequencing PROcess DESeq Sequencing PROcess DESeq

updated 13.9 years ago • nac

which mentions that "More complex schemes, such as forming a consensus peak set separately from the replicates for each condition and then taking the union of these, are also possible." Is there guidance somewhere to do this

DiffBind

updated 20 months ago • Ian D.

on the two different platforms is the same (it is clearly not). The samples are all biological replicates. Any suggestions? Thanks, Sean </div

updated 19.9 years ago • Sean Davis

about the multiple RNAseq libraries comparison (i.e more than 2 conditions with or without replicates) and if this feature is supported by DESeq. Best regards Roman </div

RNASeq DESeq RNASeq DESeq

updated 14.8 years ago • Mr.B

are the steps to get significantly up regulated and down regulated genes from samples which are not replicates? By doing this step "dds <- DESeq(dds)" I am getting a warning message "same number of samples and coefficients to fit

deseq2 without replicate no replicates

updated 7.7 years ago • Tony

<div class="preformatted">Hi all, I performed normalization on my array data (I use qPCR array of 384 wells), and if I normalize within replicates of all conditions(each condition normalized separately) , I get more differentially expressed genes that if I...performed normalization on my array data (I use qPCR array of 384 wells), and if I normalize within replicates of all conditions(each…

qPCR Normalization qPCR Normalization

updated 13.5 years ago • Ali Mohammadian

about the multiple RNAseq libraries comparison (i.e more than 2 conditions with or without replicates) and if this feature is supported by DESeq. Best regards Roman </div

RNASeq DESeq RNASeq DESeq

updated 14.8 years ago • Bruno Roman

Hi, i'm trying to get the normalized counts only, for all the replicates and genes of my data (named "14vs48)".  I'm trying this command: table\_counts <- counts(results14vs48, normalized

deseq2 normalized counts

updated 7.1 years ago • anaQ

determines the prior degree of freedom of residual variance?`` ``I am guessing it is the number of replicates per experimental conditions. Thank you

limma mroast fry

updated 7.5 years ago • siajunren

could go is in the platelist file. In the plateconf file, we don't have the notion of plate replicates or samples any more. Attached you find a slightly modified readPlateList function which evaluates an (optional...file), sep="\t", header=TRUE, as.is=TRUE) checkColumns(pd, file, mandatory=c("Filename", "Plate", "Replicate"), numeric=c("Plate", "Replicate", "Channel", "Batc…

GO Cancer cellHTS cellHTS2 GO Cancer cellHTS cellHTS2

updated 17.3 years ago • Florian Hahne

preformatted">Hello all, I have a special Agilent 4x44 K microarray analysis problem. There are 4 replicates of 2-colour arrays comparing two cell types. I want to add to this 4 replicates each of 3 other cell types, but they

Microarray limma Microarray limma

updated 15.2 years ago • Edwin Groot

hierarchical clustering and make heat maps for all samples. I have 2 groups (M and WB), 3 biological replicates in each group, and 2 technical replicates for each sample, totaling 6 hybes. I used a reference design and lmFit: design

Microarray Clustering Microarray Clustering

updated 17.5 years ago • Lin Huffman

ONE, Jefferey and al. BMC bioinformatic 2006,7/359 ) that it work better in experiment with few replicate per conditions. I perfom the statistical analysis on the whole data set ( more than 37 000 genes ), but I have high corrected...s paper that we can filter on the overall variance or on the overall mean, but in my case, with few replicates, how can I do ? In more, in this paper, it is not rec…

Microarray limma oligo Microarray limma oligo

updated 14.6 years ago • Stephanie PIERSON

is the best way to allocate the pooled samples to each chip. For example if I want to do 3 array replicates each for the mutant and control. Is it better to pool enough samples for 3 arrays and then separate the pooled sample...in 3 portions for hybridization or just pool different individual samples for different replicates? It seems to me that the first way is like getting a group expression a…

Microarray affy Microarray affy

updated 22.0 years ago • YUK FAI LEUNG

to amend it? Furthermore, I have a column of 474 gene names. They are non-unique since I have six replicates per gene. Thus I have used used spotIdentifier = "Name" which is one of my column names. I get the line non-unique identifiers...names have been added to the exprSetRG in normalise however when I call qPL anything to do with replicate spot returns a value of NA? Hope you can help. …

vsn vsn

updated 20.8 years ago • pmt1rew@leeds.ac.uk

NA values. > 2, I use loess algorithm to normalize my data, and did correlation between > replicates, they all have pretty good correlation, but I found that among > different samples(not replicates), their correlation

updated 21.9 years ago • Jean Yee Hwa Yang

0.000 1265.967 For the rest of the genes the values are pretty consistent in each pair of replicates. I thought that this gene won't be considered significantly changed, but in fact it shows up in the result as significant...DESeq that will help to increase the stringency of the analysis and exclude these genes, or with two replicates there is nothing that can be done? Thank you in advance…

deseq2

updated 5.8 years ago • ninova