Bioconductor Forum

it very confusing. The problem I'm gonna solve using Polyester is that I have a count matrix of 4 replicates in 4 time points from a real experiment. This is a time course study. I have downloaded the FASTA and GTF files from...Now I want to simulate a count matrix for DE analysis. I want this simulated count matrix to have 4 replicates in 4 time points as the real count matrix does. But the prob…

rna-seq polyester simulation time course

updated 8.0 years ago • fa.gholizadeh89

I am having issues with the design fomula for DESeq2 analysis of time-course data. I have a pilot experiment with 6 samples from blood, 3 of each condition, no replicates, and at the time-points of 0, 12 and 24 hrs. I've gathered from the DESeq2 vignette and reading forums that I should be...of time-course data. I have a pilot experiment with 6 samples from blood, 3 of each condition, no replicat…

deseq2 design design formula time course lrt

updated 6.0 years ago • ac_bioinformatics

<div class="preformatted">Dear Endre, I suspect the reason you didn't get a rsponse to your original posting is that your questions are not very specific. Assuming that you have three replicate arrays comparing a control to a treatment, your analysis looks fine. I can't give you any response to the problem reading...to your original posting is that your questions are not very specific. …

limma limma

updated 17.8 years ago • Gordon Smyth

I have two replicates for each time points(0,2h,4h). How to design an experiment with deseq2. &nbsp

deseq2

updated 7.4 years ago • edu.hsv.crs

Hi folks, I know that to "run edgeR without replication" has been a popular question, but I'd like to specify the situation I'm in. I'd like to compare DGE from pair tumor/normal...is to validate the MSMS data obtained from the same tissues. Therefore I don't have a typical replicates. However, I have data from multiple patients. I wonder if I can take full advantage of the sample size, …

edgeR StatisticalMethod

updated 4.3 years ago • Field -Ye

per each condition (P4_i, P4_n, P5_i, P5_n) whereas I'd need the log2 centered intensities per replicate/sample. On the actual plot `plot_single(dep, proteins = "Saa4", type="centered", plot=TRUE)` I can see the different replicates

DEP

updated 4.6 years ago • hele7

suggested to calculate ratios for each experiment (within each experiment), using the treated replicates (3 reps) per experiment versus the control per experiment (the average of the 3 control reps). This results in 3 ratio...replicates per experiment and dose. This way one would get rid of the grand differences between the experiments. Then run an

affy DOSE affy DOSE

updated 21.6 years ago • Arne.Muller@aventis.com

Dear all, I am using DESeq2 to estimate fold changes of treatment versus control in a time series experiment (with biological replicates). I am a little puzzled about the standard deviations of the fold changes provided by DESeq2. These are almost constant over all timepoints, although it doesn't look like this when I judge the biological replicates in the output of plotCounts. Can …

deseq2 log2fc standard error timecourse

updated 7.9 years ago • Verena

regarding the MDS plot. For example, for my data sets: F0a, F0b P3a, P3b P6a, P6b where a and b are replicates. when I plot them on MDS, what does the dimension1 and dimension2 mean in the MDS plot (and what is the units on x and...y-axis). If I understand correctly, Dimension 1 is the variation (tell how different are the replicates -- eg., F0a and F0b) and Dimension 2 tells how different are t…

edgeR edgeR

updated 14.8 years ago • Sridhara Gupta Kunjeti

<div class="preformatted">I have microarray data, which is 2 colour agilent human of 3 technical replicates. Green dye case and Red dye control. I have analysed in Limma, normalising within arrays and between arrays using aQuantile normalisation. I also have some Next gen RNAseq data that has been mapped to the Refseq transcriptome and I have these raw counts. However there are no replicat…

RNASeq Microarray limma RNASeq Microarray limma

updated 14.5 years ago • john herbert

Dear all, I'm working through the edgeR user guide and would like to understand the reasoning behind using `` glmLRT() `` over `` glmQFTest() `` to detect DE genes, specifically within the oral carcinoma Case Study (p40 onward). I see from the user guide and also [here](https://support.bioconductor.org/p/68629/) and other sources that for cases where the number of replicates is…

edger design matrix glmlrt() glmQFTest() paired design

updated 7.2 years ago • oakhamwolf

boxplot,the blue, red, and green ones stand for three different hybridization groups. There are 6 replications in each group. I want to carry out between-array normalization in the three groups respectively and then combine...Is this reasonable? In my opinion, we can always carry out between-array normalization between the replications of the same hybridization group (unless there is dye swap). …

Microarray Normalization limma Microarray Normalization limma

updated 18.2 years ago • De-Jian ZHAO

So my coldata looks like this: sample condition 1 RDw15_9SIV1 A 2 RDw15_9SIV2 A 3 RDw15_9SIV3 A 4 RDw15_9c1 B 5 RDw15_9c2 B 6 RDw15_9c3 B 7 RDw15_24SIV1 C 8 RDw15_24SIV2 C 9 RDw15_24SIV3 C 10 RDw15_24c1 D 11 RDw15_24c2 D 12 RDw15_24c3 D 13 RFc16_9SIV1 …

deseq2

updated 5.7 years ago • joag0001

Hello, I am new to this forum so please forgive me if my question is a bit naive :) - I have several tissues: A, B and C (and 2 replicates per tissue) - I have many genes: gene1a, gene1b, gene2, gene3... - genes 1a and 1b are paralogs In edegR I can do multiple comparisons of gene expression for each of those 4 genes between the different tissues and the different replicates, which is great!…

count rnaseq rpkm fpkm edgeR

updated 10.7 years ago • thibault.lorin

compare to each other. # Description of my dataset: Control: No treatment and time zero (total 6 replicates) Treatment A: time1, time2, time3 and time4 (3 replicates each, total 12) Treatment AB: time1, time2, time3 and time4 (3 replicates...each, total 12) Treatment AC: time1, time2, time3 and time4 (3 replicates each, total 12) Treatment ABC: time1, time2, time3 and time4 (3 replicates each,…

updated 13.1 years ago • Guest User

Hi, Is it a way to identify significant interaction without replicate and inter-sample comparison ? Thank you for your help. Attis &nbsp

fourcseq

updated 10.4 years ago • julienpontis

I have some ATAC-seq data from a published paper. They come in the form of BED files, a result of MACS peak detection. I only have region coordinates, no p-values, no enrichment scores. There are two biological conditions and 8 replicates each. I've been tasked with finding transcription binding sites for a small set of 5 genes and comparing these sites...region coordinates, no p-values, no enric…

motifmatchr

updated 3 months ago • Marek Gierlinski

be different now in the new version as the design setting has changed. I would like to block for replicate (or individual patient in my case) whilst also using a design that includes Tissue as confounder: ~Tissue + Condition...However, when I do that it says that block is ignored when design is used. Should I just include replicate in my design instead of as block or is there another way to anal…

DiffBind R ATACSeq

updated 4.4 years ago • fleur_p90

a particular vector is present, and treated means treated with an antibody] I do not have any replicates for any of the conditions. **I am looking to perform an Anova test using edgeR**, to see the gene expression at different...and performing the GLMQL tests. I am confused as to how to proceed in this case,as I have no replicates. I don't know how to define the groups or estimate dispersion …

edger anova gene expression Dispersion

updated 6.5 years ago • ilovesuperheroes1993

other tumors (1509 and 1701) for the analysis. From the cel files, it doesnt appear that we did replicates for the tumors, just one each, the rationale at the time being that we had wanted to first quickly scan the tumors...to look for the low hanging fruits.??I am not sure how do I go about doing this since I have 3 replicates for the control but 1 each for different tumors. What should be the…

Pathways GO affy Pathways GO affy

updated 13.7 years ago • hsharm03@students.poly.edu

Hi, I am considering to remove the batch effect in my samples to decrease the variation among the replicates. I find RUVseq is great and easy to use. It has very specified details about how to use RUVseq in the DE genes analysis...and use RUVs function to remove the unwanted variations (because for each conditions, I have three replicates.). 3. use normCounts function to get the corrected gene …

rnaseq ruvseq

updated 9.3 years ago • sooby

I am trying to analyze the differential peaks in my ATAC-seq data. I have ATAC-seq analysis on 2 biological conditions with 3 replicates in each condition. Below is my sample info: ``` ID Factor Condition Replicate Intervals 1 ATACctrl1 ctrl1 ctrl 1 8654 2 ATACctrl2 ctrl2 ctrl 2 6750 3 ATACctrl3 ctrl3 ctrl 3 5962 4 ATACe…

DiffBind DESeq2 ATACSeq

updated 20 months ago • lonn

Hello, I am analyzing RNA-seq data using DESeq2 I have 5 groups of samples each with 4 replicates. Below is a PCA plot of the VST transformed value: ![enter image description here][1] as shown in the PCA plot, replicate...R1) are separate from the others on the upper half of the PCA plot. There are some clustering of the replicate 2 samples as well (triangles) at the bottom. I ran des…

deseq2

updated 6.6 years ago • C T

My experimental design is the following: <table border="1" cellpadding="1" cellspacing="1" style="width:500px"> <tbody> <tr> <td>Replicate</td> <td>Tissue_type</td> <td>Genotype</td> </tr> <tr> <td>1</td> <td>A</td> <td>HE</td> </tr> <tr> <td>2</td> <td>A</td>…

deseq2 bioconductor bioinformatics statistics

updated 7.0 years ago • casikecola

Hello, Reading more on how dispersion shrinkage is working, I wanted to clarify what my understanding is as well as address a couple of questions. I have read both the DESeq2 and DDS papers that talk about dispersion shrinkage.    My understanding: The point of dispersion shrinkage is to share information (dispersion estimates) across genes with similar average expression le…

deseq2 DDS

updated 8.3 years ago • rrcutler

amount of material, it is better to increase the number of time points > instead of the number of replicates by time points. And the temporal > information can be used as "replication" (the 4 time points for each > sample are...create a design and contrast matrix that use the time points as > replicates, but in a more flexible way. For instance, taking the first &g…

Microarray Normalization Classification limma PROcess Microarray Normalization limma

updated 22.4 years ago • Gordon Smyth

comparison. Is this right? However, I have a further question. In fact, each chip has 3 technical replicates. In order to simply the anlaysis, I just average the replicates and then use limma to do the job. How could I include...such technical replicate information and repeat measurement information together with using Limma. Could Gordon or someone give me some...hints or example? Thank you ve…

limma limma

updated 15.6 years ago • Xiaokuan Wei

and G1E treated with 24 hours of estradiol.  I had following data: dataset A G1E, 2 replicates, single end reads G1E-treated, 2 replicates, single end reads dataset B G1E, 2 replicates,paired end reads G1E-treated...2 replicates, paired end reads I did differential analysis within each dataset and for following MA plots. Number of DE genes

deseq2 normalization edger

updated 9.9 years ago • tonja.r

<div class="preformatted">> Date: Thu, 30 Jun 2005 11:44:02 +0000 > From: Carolyn Fitzsimmons <carolyn.fitzsimmons at="" imbim.uu.se=""> > Subject: [BioC] duplicateCorrelation and design matrix > To: Bioconductor list <bioconductor at="" stat.math.ethz.ch=""> > > Hello, > > I need an explanation of how the design matrix inf…

limma limma

updated 20.5 years ago • Gordon Smyth

are from 3 patients and 3 gender and age matched controls. For each individual there are 3 technical replicates, except for one patient with 6 technical replicates. So there are 21 samples in total. I have processed the data following...is paired with a control. The phenotype data is as below: <pre> sampleID case_control pair replicate S10 patient pair2 p2 S11 patie…

ballgown linear models

updated 7.7 years ago • Yong Li

set where the cells are either unstimulated or stimulated with one single treatment and I have three replicates for each class. When I use the GLM framework with tagwise dispersion to fit a model for the effects of the single...treatment and plot the GOF, it is distinctly non-linear. When I remove one replicate from each class by looking at the replicate most distant from its counterparts in the …

Sequencing graph edgeR Sequencing graph edgeR

updated 13.2 years ago • Thomas Frederick Willems

transcriptomic. I prefiltered my dataset to contain at least 4 raw count and at least 3 biological replicates to be included in the analysis, leaving 14,332 genes in the dataset and I would like to analyze for DEG to compare...the two group with 8 biological replicates. I run a standard DESeq2 analysis in R and found >6000 "NA" values in the Padj. When I checked the genes with "NA" padj

DESeq2

updated 20 months ago • Agung

and in the same run with similar mapping rates, I thought they can be treated as pseudo technical replicates, hence used the collapseReplicates function in DESEq2 after checking through PCA plot to see that variation...DEGs analysis as a result of such collapsing of counts/workflow or treating R1 & R2 as technical replicates? Look forward to hearing from you soon, thanks heaps in adva…

RNASeq DESeq2

updated 4.6 years ago • Amali Thrimawithana

all, I have a large RNA-seq data set with 63 samples in 12 condition with different number of replicates. I want to run R on the server to make it faster, but I have this error. Would you please help me. error: "Error in lf\[\[1\]\] : subscript...factor(c("CD\_SIG\_b", "CD\_SIG\_nb", "CD\_TILE\_b", "CD\_TILE\_b", "CD\_TILE\_nb", "CD\_TILE\_nb")),replicate=factor(c("1","2","1","2","1","2"…

DEXSeq

updated 10.4 years ago • rahel14350

I want to remove are most often a total lack of signal as the peak is missing. I do have five replicates of each treatment I am looking for something that could remove only the extreme outliers (sample nr nine in the...0.00120032 4.786810001 The data is structured as a matrix with one line per peak and the replicates as individual columns (much like micro array data). Thanks for any suggestions…

Alignment Alignment

updated 18.1 years ago • Johannes Hanson

couple of questions about plier a) With justPlier it is possible to specify as an input vector the replicate structure of the batch we want to analyze ('replicate' input option). Can anybody tell me how this information is used

plier plier

updated 20.5 years ago • Ariel Chernomoretz

was a special way to handle duplicate spots. However the layout of the array had to be such that one replicate was printed first for all spots and then the other replicate, because somewhere you had to say something like there

limma limma

updated 17.4 years ago • Cecilia McGregor

calculate all genes with 2x change or more. This sections uses rowMeans to calculate the average of replicates-"rowMeans(e[, index])". However, since the expression values in eset are in log2, is rowMeans the correct way to calculate...of normal numbers, not log2 numbers. If this method is not correct, how should the averages of the replicates be calculated? Thanks, Mary A. Allen from the manua…

BRAIN BRAIN

updated 13.1 years ago • Mary Ann Allen

FileName Cy3 Cy5 File1 wt mu File2 mu wt File3 wt mu File4 mu wt *(four replicates of two groups (wt , mu) of which two replicates in each group is labeled by one color(red) the other two is labeled by another

GO limma GO limma

updated 19.8 years ago • Ron Ophir

8 testing gene over expression and 8 testing a gene knock out. Each group of 8 has 2 biological replicates each of which has 2 technical replicates each of which is dye-swapped. That is to say, I have plates: 1 OE_ctrl OE1 2...KD2 15 KD2 KD_ctrl 16 KD2 KD_ctrl Note that the controls from each set of 8 are technical replicates. Plates 4 and 9 have 0-weights due to low quality in 60% a…

limma limma

updated 18.9 years ago • mik mik

I want to determine DGE analysis with my data that don't have any replicate. I just want to know that can I use glmQLFit and then glmLRT as I am not getting any DE genes by glmFit?&nbsp

edger differential gene expression glmlrt()

updated 9.0 years ago • simarsidhu25

Naomi Altman p.s. I also strongly suspect that if you do dye-swaps, you should do it on biological replicates. (Is there any evidence for biological rep x dye interaction?) If the limiting factor is arrays (not RNA samples) then...biological replicates are more effective than technical replicates. Introducing technical replication requires a random effect...by most of the software for microarr…

Microarray Normalization Microarray Normalization

updated 22.1 years ago • Naomi Altman

conditions. We pseudobulked the data, since each condition has 70 samples that we treated as replicates, because they're the exact same cell type. We are now trying to run DESeq2 using this pipeline: https://hbctraining.github.io...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 scRNAseq

updated 4 months ago • rj1435

Hi I have RNA-seq time course data consisting of 11 individual time points. I however do not have replicates for each time point. I am trying to fit a simple linear model of the form to detect oscillations: time <- seq(2,22,by...My question is can my large residual degrees of freedom compensate for my lack of biological replicates at each time point. In other words, can I use the standar…

edger time course

updated 8.9 years ago • BharathAnanth

assessment criteria? If so, should all spots associated with this probe be removed across all replicates or should it simply be the spot in question specific to that array? The former approach would effectively remove...probes that are expressed above background on at least n arrays, where n is the smallest number of replicates assigned to any of the treatment combinations." What does this mean? …

Normalization probe limma Normalization probe limma

updated 11.9 years ago • Guest User

data. It includes  before & after infection data of the same individuals and also replicates of non-infected other individuals as a kind of  control to these infected ones . There are 15 individuals and...the codes below to identifiy genes differently expressed, because when i cluster the datasets, the replicates do not group together:   RG\_2= backgroundCorr…

microarray agilent limma

updated 10.4 years ago • evoke

pathway-analysis-with-sailfish-deseq2-gage-and-pathview/> but I have three samples with replicates (and more in future). I am able to get the pathways but not clear to which sample they belong to and also unable to get...down-regulated genes in the pathway. I think I need to troubleshoot the input for many samples with replicates. I have posted details of my code and output at:&n…

pathview gage rna-seq R sleuth

updated 9.0 years ago • bioinf

span style="line-height:1.6">I have quite a strong batch effect in my data (paired design,i.e. first replicates of treated and control were done together and second replicates of treated and control also together). Additionally

ruvseq

updated 9.9 years ago • tonja.r

I try 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, and

edgeR edgeR

updated 13.4 years ago • Lucia Peixoto

We are using DESeq2 to analysis some Ribo-seq data pointwise and try to replicate the dispersion estimation procedure. That is, we focus on the reads on each codon other than total count in gene...We are using DESeq2 to analysis some Ribo-seq data pointwise and try to replicate the dispersion estimation procedure. That is, we focus on the reads on each codon other than total count in gene. Thus..…

DESeq2 Ribo-Seq

updated 6.2 years ago • li.keren.cn

<div class="preformatted">Dear Memebers, I want to be able to reference individual arrays in an abatch like so abatch[,1], abatch[,2] etc. This seems to work fine using the original pData for the abatch after using ReadAffy(): sample N10S.CEL 1 N11S.CEL 2 N12S.CEL 3 N7S.CEL 4 N8S.CEL 5 N9S.CEL 6 P1S.CEL 7 P2S.CEL 8 P3S.CEL …

updated 17.7 years ago • Paul Geeleher

vector2 = intensities for negative controls > wilcox.test(vector1,vector2) > > If I have 5 replicate arrays, I can perform the test as above just by combining intensities across experiments and comparing experimental...to controls as two vectors. > > I'm not sure this is really an appropriate way to handle replicates. Is there another function like wilcox.test, …

Organism vsn limma Organism vsn limma

updated 19.1 years ago • Wolfgang Huber

<div class="preformatted">Dear Sridhara, Not exactly. Dimension 1 is the direction that best separates the samples, without regard to whether they are treatments or replicates. Dimension 2 is the next best direction, uncorrelated with the first, that separates the samples. It may turn out that one dimension separates treatments and one separates batches, but this depends entirely on your…

edgeR edgeR

updated 14.8 years ago • Gordon Smyth

48c49480@micron10> Content-Type: text/plain Hi: Does anyone has input on compatibility of "replicate-only" vs. "all- sample" quantile normalizations? I'd assume that "true significant" genes would be picked up by either...replicate-only" or "all-sample" method, though the latter is surely more conservative (by forcing the same distribution across...all samples, replicates or not). My an…

Normalization Cancer PROcess Normalization Cancer PROcess

updated 21.3 years ago • Mark Reimers

I am new to RNA-seq. I have output from `tximport` and I want to use `DESeq2`. I have used five replicates for each of two treatments and I want to check the difference. I am not sure which design to choose for `DSeq2`. Thank

deseq2

updated 5.8 years ago • zen

number of samples to compare? Can I just compare 1 treatment against 1 control, each with multiple replicates? Regards

minimum samples edgeR

updated 6.9 years ago • jzhan067

there a package capable of visualizing time series data a la Gene Spring? How about for normalizing replicate time series data points? -- Mike</div

updated 22.3 years ago • Mike Schaffer

of human lung adenocarcinoma cell lines][1] It seems it uses the inferential (Bootstrap/Gibbs) replicates to measure the assignment uncertainty of each transcript count. My question is does edgeR also considers transcript...while computing. I see `Swish` another differential transcript expression tool adjusts transcript replicate counts for sample-specific transcript length and sequencing dept…

edgeR Transcript RNASeq

updated 5 months ago • mohammedtoufiq91

for `reconstructed` value after fastMNN is not readable. For example, here is my interesting gene in replicate 1 ![enter image description here][1] and here is in replicate 2 ![enter image description here][2] As you can see, I can see

scater batchelor scRNAseq fastMNN

updated 3.4 years ago • Guandong Shang

Dear list, I have used BaySeq to my RNA-Seq data to extract DE genes and I have several biological replicates for each condition(20 replicates for each condition). The point is that if I rerun the BaySeq over my dataset then

baySeq baySeq

updated 13.1 years ago • Fatemehsadat Seyednasrollah