Bioconductor Forum

Hello, I'm working on a RNAseq dataset with two passages and no replicate! the STAR-HTSeq output with any of: <pre> dds <- DESeq(ddsHTSeq) dds <- DESeq(ddsHTSeq, minReplicatesForReplace=Inf

deseq2 no replicates high p-adj values

updated 8.2 years ago • Mahtetie

Can minfi be used for identifying differentially methylated regions for two samples having only one replicate each? Please suggest

minfi methylation illimina 450k methylation

updated 9.2 years ago • arisarkar88

td> <td> </td> </tr> </tbody> </table> I have been trying to work out how best to treat the replicate arrays from each individual. The fact that the replicate arrays from each individual are not strictly technical...replicates has made me reluctant to simply average them prior to carrying out analysis of differential methylation (although...a legitimate …

limma duplicatecorrelation replicates

updated 8.6 years ago • rwalke13

Dear all, I have a question regarding the consideration of the biological replicates inside the design formula of DESeq2. We performed 2 different approaches, one being (design=~condition) and the...Dear all, I have a question regarding the consideration of the biological replicates inside the design formula of DESeq2. We performed 2 different approaches, one being (design=~condition) and t…

DESeq2 RNASeqData RNASeq

updated 5 months ago • Christian

Hello, First, I know that DESeq2 is not meant to be run without replicates and results will not be meaningful. However, I have replicates (n=>30) in one group (N), while another group (T) has only

DESeq2 rnaseqGene DifferentialExpression GeneExpression

updated 4.5 years ago • SUMIT

message was scrubbed... From: Barbara Cegielska <barceg@ibch.poznan.pl> Subject: how to merge replicate spots? Date: Fri, 03 Jul 2009 09:13:15 +0200 Size: 1495 URL: <https: 20090703="" 8af467c1="" attachment.eml="" attachments="" bioconductor

updated 16.4 years ago • Barbara Cegielska

of chips. we are looking at the response of patients to a specific treatment, so we got biological replicates and no technical ones (as we have not enough material from the patients). at the moment i got 9 chips, 3 patients, 3 time

updated 21.4 years ago • Dipl.-Ing. Johannes Rainer

Hi all, I am trying to perform hierarchical clustering on the results of DESeq2. My experimental design has 12 different conditions, each with a number of biological replicates. To do this, I have used vst() on my deseq object, followed by scale() to get Z-scores. VST <- vst(dds_deseq, blind=FALSE) Counts <- assay(VST) Scaled_counts<-t(scale(t(as.matrix(Counts))…

DESeq2

updated 22 months ago • E

are your opinions on using limma::arrayWeights() for a *LC-MS/MS proteomics* data-set on *biological replicates* ? I performed stress treatment on six independent biological replicates of Arabidopsis leaves but the treatment...not 100% homogeneous due to fluctuating in stress intensities, so i want to weight the biological replicates, but i don't want to completely exclude single replicates. Samp…

Proteomics limma StatisticalMethod

updated 4.7 years ago • Steve

Dear all, I'm using MEDIPS to process MBD-seq data, with 2 control replicates and 3 case replicates. It seems that the MEDIPS.meth function needs same length of MSet (I got error message 'MEDIPSset...length').  Any advice for this issue? I can use the MEDIPS.mergeSets, but this will make the replicates into one. Thanks in advance, Fei

MEDIPS

updated 8.9 years ago • fmhsu0114

between the regions in a tumour in a particular tumour. So in this case I do not have any replicate (I have one library for regionA and one library for regionB in a tumour) I see you have some suggestions to run EdgeR...when there is no replicate but I am wondering how it should be performed for the methylation data, not gene expression? For example what would...if there is any difference in m…

RRBSdata edgeR

updated 22 months ago • annasoudi

Salmon with tximport and create an offset matrix. I also want to implement the pooling of technical replicates with `sumTechReps()`. Technical replicates were analyzed separate with Salmon and will have different offsets...are converted to corresponding sample_ids, but column names of y$offset still refer to the original replicate IDs (although matrix is reduced to the same dimensions as the pool…

tximport edgeR

updated 2.3 years ago • Vitor

Hi everyone, I'm running into a problem where the second biological replicates have significantly less IP efficiency and reads than the first replicate, so when I tried running the DESeq2 analysis...Hi everyone, I'm running into a problem where the second biological replicates have significantly less IP efficiency and reads than the first replicate, so when I tried running the DESeq2 analys…

chipseq DifferentialMethylation DESeq2

updated 22 months ago • rebw25

something wrong, but can you confirm me this? It shouldnt be, but does EdgeR summ the counts of each replicate and uses it for the analysis?Thanks a lot, and I wait for the answer!Sandra [[alternative HTML version deleted]] </div

edgeR edgeR

updated 11.3 years ago • Sandra Fernandez Moya

Hello,  I have checked the PCA from my sRNAseq data and there is one replicate which goes over 67% away from all my samples (1/18). I know that the PCA plots the genes with the top variance, and this...Hello,  I have checked the PCA from my sRNAseq data and there is one replicate which goes over 67% away from all my samples (1/18). I know that the PCA plots the genes with th…

pca variance deseq2 gene_id

updated 9.5 years ago • NMostajo

I would like to know whether bsseq package works with technical replicates (i.e. two whole-genome bisulfite sequencing of the same DNA sample). How bsseq deal with replicate information? The

bsseq WGBS

updated 9.0 years ago • Welliton de Souza

25, makePLOT = TRUE, target.micro, verbose = TRUE) If in a dataset there are two or more sample replicates and in the step of filtering probes I want to exclude a probe only if gIsGeneDetected = 0 in all two or three sample...replicates. I don't think by 'filterMicroRna' function provides any option for this type of filtering. Are there any suggestions

probe microRNA AgiMicroRna probe microRNA AgiMicroRna

updated 14.4 years ago • Viki S

Dear all, I have a RCBD with three treatment levels and two blocks. Within each block, I have 5 replicates for each treatment (that is, my block size is 15, 5 for each trt level). I am aware that since I have replicates within

updated 13.4 years ago • Guest User

Mutant 1) vs equivalent pools of wild types The other option would be simply to have 1 technical replicate for the mutants and test each gender in a separate chip, but I don't believe technical replicates are the best option

updated 18.1 years ago • Cei Abreu-Goodger

and DESeq2. I have a dataset of RNAseq, which gets from RSEM tool, for different samples (no replicate). I want to use DESeq2 for the normalization of the gene counts. Here is the RNAseq dataset: > setwd("./rsem_export_dataset...and some genes like **ENSRNOG00000000007_Gad1** for all 53 samples. The question is I have no replicates, and I need to put **~sample_ID** as a design for…

normalization deseq2

updated 5.7 years ago • Hadi Hosseini

Hi, I have ATAC-seq data for 8 time points of 4 conditions --> 32 samples in total. The only replicates I had were technical which were pooled early in the pipeline. There are no biological replicates. I have completed...76048/) and [here](https://support.bioconductor.org/p/61601/#61613)) regarding DEseq2, ATAC-seq and replicates....but none that address what to do when there are no biolo…

ATAC differential binding analysis no replicates DEseq2 chromatin

updated 8.7 years ago • YaGalbi

<div class="preformatted">Typically, how does one handle within-chip probe replicates on the Agilent microarray's to improve the significance of statistical tests? I am currently using limma, and probes...div class="preformatted">Typically, how does one handle within-chip probe replicates on the Agilent microarray's to improve the significance of statistical tests? I am currently using l…

probe limma probe limma

updated 13.1 years ago • Reed, Tyler

I've tried to calculate the qvalue with a dataset like wt1, wt2, mt1, mt2. Each group has two replicates. And all the qvalue I've got were 1. However, I've check some information on the website, it is said that DESeq2 requires...the minimum replicates is two. So I am confusing why all the qvalue are 1.&nbsp

deseq2

updated 9.2 years ago • zhdorothy5

Hello, I have a question on using RUVSeq for removing batch effects for 8 samples of RNA-Seq data - 2 batches with 4 samples each. The example data in the tutorial shows 2 conditions with 3 replicates each: <https://www.bioconductor.org/packages/release/bioc/vignettes/RUVSeq/inst/doc/RUVSeq.pdf> Following the steps in section 3 to estimate factors of unwanted variation using replicate …

RUVSeq

updated 9.4 years ago • sharvari gujja

this. I am analyzing the two color common reference design agilent arrays using Limma. I have four replicates and two treatments(control and PCB). I am trying to extract fold change values (PCB/control) for the four replicates...all the samples and I get one fold change value. Is there anyway I can get the values for individual replicates separtely? I want to use the replicate values in Rank Prod…

limma limma

updated 16.0 years ago • Neel Aluru

I get nice graphs (histogram) where I see reads mapped to + or - strand. I have three biological replicates of 'control' and 'treated' samples and instead of plotting all of them separately, I would like to plot mean values...of three replicates . How I do that? I thought that grouping could be solution, but unfortunately data grouping is not so well explained

gviz

updated 10.4 years ago • toomas.silla

Hi all, (edgeR Page no. 21,22 User Guide) I am running program of edgeR for without replicate . I am getting one one error so please help me in that . ==================================== <pre> > bcv=0.2 > library(edgeR) > counts=matrix(rnbinom(40,size

edger without replicate

updated 9.9 years ago • Sushant Pawar

class="preformatted">Dear list, I have 24 chicken affymetrix arrays. Half of them are technical replicates (2 different conditions, 3 time points per condition (2X3=6 RNA samples) each RNA sample was analysed twice (6X2=12...microarrays). The other 12 samples come from 6 different conditions, two biological replicates (different RNA samples) per condition. Up to now I have been analyzing t…

Microarray webbioc Microarray webbioc

updated 18.5 years ago • jane janes

Hello Everyone, For exploratory reasons we have RNAseq data without replicates, I totally understand that it is not possible to estimate any sgnificance from this data as there are no replicates...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. DESeq(dds) # Error : estim…

DESeq2 DEG

updated 3.8 years ago • priya.dalvi

Hi, I am doing DEG analysis from RNA seq experiment. I have two technical replicates originating from different libraries of the same sample. How should I deal with them?Do I have to merge BAM files

technical replicates

updated 10.5 years ago • sara.pegolo

analysis of differential abundance of OTUs using DESeq2. As an example, I have 3 samples with 3 replicates each, and want to compare whether selected OTUs are significantly different across all habitats (similar to...The count table below shows the setup. With the DESeq function (design=~habitat; where sample 1 all replicates= habitat1, sample 2 all replicates=habitat2, sample3 all replicates=hab…

deseq2

updated 11.0 years ago • jport

s or Spearman's correlation coefficient to measure the reproducibility among microarray biological replicates? I would be grateful for any hint. Best, B. [[alternative HTML version deleted]] </div

Microarray Microarray

updated 13.4 years ago • Barbara Uszczynska

I am using DESeq2 to analyse RNA seq data. I have 5 conditions each with two replicates and I am comparing these against controls again with 2 replicates. However the issue is that one of control replicates...was sequenced earlier. Thus only this control replicate belongs to a different batch from all other samples. As suggested in the vignette I added batch in my design like

deseq2 rna-seq

updated 7.3 years ago • bn3301

Differentially Expressed Genes (DEG) __even for several group__, since Limma does can run? If one replicate is not enough, how many replicates are needed to get reasonal result?   Thanks, Kevin     \#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\#\# \#  one-color simulation...for one-color gr <- c("c", "t1", "t3", "t2", "c") \#\#…

limma microarray

updated 8.9 years ago • kevin.m.hao

Hello, I have an issue with my RNAseq regarding the variance between my biological replicates. If i include them as a factor of variance in my design, i can fix the issue nicely and looking at the PCA plot when...Hello, I have an issue with my RNAseq regarding the variance between my biological replicates. If i include them as a factor of variance in my design, i can fix the issue n…

DESeq2

updated 3.1 years ago • lukas.krauss

expressed genes from five different cell types with five, five, four, four, and three affymetrix replicates each. (I will be using gcrma as preprocessing step) I seem to remember a paper that showed that T-tests did not predict...known spiked-in changes better than simple fold change until one had 8 or more replicates. Is there data with known spiked in datasets (or a consensus) that limma or s…

Preprocessing gcrma Preprocessing gcrma

updated 21.5 years ago • Luckey, John

<div class="preformatted">Hi Tyler, You can use duplicateCorrelation(), as long as the replicates are regularly spaced. If you are using one of the 4X44 Agilent chips, the Agi4x44PreProcess package has some facilities for looking at the duplicate probes, but nothing to do e.g., mixed models with them. Best, Jim On 10/29/2012 7:33 PM, Reed, Tyler wrote: > Typically, how does one h…

probe limma probe limma

updated 13.1 years ago • James W. MacDonald

div class="preformatted">Hi All, I am working on sRNA-seq data and with no replicates and using edgeR to identify differentially expressed tags and using an arbitrary BCV value (0.3) based on suggestion...are 0.4 for human data, 0.1 for data on genetically identical model organisms or 0.01 for technical replicates. " But I came across this post which mentions a conservative way to calculate …

edgeR edgeR

updated 12.0 years ago • Bade

<div class="preformatted">Dear list Members. I'd like to ask for advice. I have 2x2 factorial experiment conducted in a loop-design (two-colour data). There are two maize lines (dh7, dl3) and two temperature treatments (cold=c and control=k). Following hybridizations were conducted : dh_c vs dh_k; dh_k vs dl_k; dl_k vs dl_c; dl_c vs dh_c (forming a square on a diagram) and two (diagonal o…

limma limma

updated 15.8 years ago • Maciej Jończyk

3 conditions A, B, C and Ref is supposed to be same in all experiments. Each condition should have 3 replicates, one being dyeswap but one replicate A3 failed, so we only have 2 replicates for that particular condition. Instead...limma for the differential expression analysis of A vs. B and A vs. C and B vs.C. As i have only 2 replicates in the condition A, do the comparison or the normalization …

limma design and contrast matrix

updated 10.2 years ago • bioinfo.user15

<div class="preformatted"> Hi all, I am just wondering how to process replicate genes in an array using limma package. each gene is printed twice on an array. For example, the actual number of genes is 19000 in my dataset, since there are replicate genes, I have 38400 spots (genes) in my dataset. I am just wondering how I can get 19000 genes from these 38400 genes before...div class="pr…

limma PROcess limma PROcess

updated 21.8 years ago • p hu

<div class="preformatted">Dear Eleonora, You need to explain what you want to test for before we can advise you on the correct contrast. Also, if your dye-swaps are technical replicates, it appears that your experiment doesn't have any biological replication. If you've repeated the RNA extraction, labelling etc for the dye-swaps then all is well. If not, then the experiment is difficult …

Normalization limma Normalization limma

updated 15.4 years ago • Gordon Smyth

to have been much discussion regarding the statistical issues surrounding handling of technical replicates in gene expression data using the Limma package as pertains to two-color arrays, but less so for Affy data. As such...I am still uncertain regarding how best to handle a single technical replicate in a dataset 40 Affy arrays from 3 tumor types. Clearly, I want to model the biological varia…

GO Clustering affy limma GO Clustering affy limma

updated 19.5 years ago • noel0925@sbcglobal.net

I have been analyzing an affy microarray dataset with 2 treatments and 1 control, which do not have replication (so total of 3 chips), with several methods that do not require replication, including bgx. BGX seems to give the poorest

Microarray affy bgx Microarray affy bgx

updated 15.5 years ago • Ernest Turro

I just want to verify my code without replication using edgeR. Please check it and give your suggestions. it may help me to go forward. *******************Thanks in advance*************************** library...I just want to verify my code without replication using edgeR. Please check it and give your suggestions. it may help me to go forward. *******************Thanks in advance**************…

Transcriptomics

updated 3.0 years ago • kuttibiotech2009

transcripts which are differentially expressed (if any) among the five tissues (I do not have any replicates). I have tried using edgeR and DESeq packages but not getting even a single value to be significant. Can anyone suggest

updated 13.3 years ago • Srikanth Manda Srinivas

Hello, I'm currently trying to use Deseq2 but I'm getting confused with some of the conditions. I have multiple individuals that are either Sick or Healthy (that is the main condition I want to use for comparison) However, those individuals can be also classified into families. For example, Individual 1, 2, 3 and 4 are part of family 1 while individual 5,6,7 and are part of family 2. Ad…

deseq2

updated 5.1 years ago • benoit.j.kunath

I would like some help getting DE genes from what is turning out to be a more difficult design than I thought. The data come from elsewhere, so the design isn't mine (nor can I just repeat everything). I want to get DE genes from different times in fetal mouse development (E14 and E16), so comparing 2 different gestational ages. I also want to look at fetal sex as a variable. My proposed analysis…

edgeR RNAseq voom variability estimator

updated 5.7 years ago • hcnbox

direct experiments with two biological replicates and two > >> technical replicates. In each array sots are printted in 4 >replicates. > >> In duplicateCorrelation...how? > >> If not which least-squares analysis should I drop technical sample > >> replicates or spots replicates? > > > &…

limma limma

updated 20.6 years ago • Gordon Smyth

using the same Sample ID/Bam file but different peak callers: SampleID,Tissue,Factor,Treatment,Replicate,bamReads,ControlID,bamControl,Peaks,PeakCaller WT\_HU,WT,Rif1,HU,1,../2016\_020B\_3\_WT\_HU\_sorted\_alignment.bam...pre> data 16 Samples, 1079 sites in matrix (1896 total): ID Tissue Factor Treatment Replicate Caller Intervals 1 WT_HU WT Rif1 HU 1 ma…

diffbind

updated 8.1 years ago • Sophie Shaw

for differential expression quite a bit and I had a quick question regarding analyses without replicates, as this is something we run into when analyzing public datasets from tumors. After reading, I noticed two potential...methods with DESeq to perform a comparative analysis of expression between input samples without replicates... 1) run the DESeq analysis normally with sample name …

deseq2

updated 10.4 years ago • np

I have duplicate samples for RNAseq and just wonder if there is way to test if the replicates are good or not and what criterion to use. I remember there was a discussion about this recently in this email&nbsp

RNASeq

updated 10.7 years ago • Fix Ace

Now I want to look at differentially expressed genes.  Unfortunately, there are no replicates, and nestedness within the design.  There are 6 individuals- normal male, infected male (intersex1), infected...explanatory factors, as they are all important- and even then i still probably do not have enough replicates. I am not entirely sure what a 'reasonable' dispersion value wou…

edger differential gene expression without replicate nested design help

updated 9.4 years ago • knipehf

<div class="preformatted">Hi This follows on slightly from my experimental design thread. Having worked through the vignette for DESeq, it seems to work well. However, for the TagSeqExample.tab data set, when using an FDR cut off of 0.05, what we see is that we only find differential expression for large fold changes - an average of log2 fold change of 5 for up- regulated, and log2 fold c…

GO DESeq GO DESeq

updated 15.5 years ago • michael watson IAH-C

value <= 0.05. Now most of my genes >are replicated in duplicate on the arrays (within-array replicates) and >when I averaged over those replicates, and used that data to feed into >lmFit(), eBayes() and topTable() I got ~1100...watson (IAH-C) >Cc: bioconductor@stat.math.ethz.ch >Subject: Re: [BioC] Unequally spaced replicates in limma > > …

probe probe

updated 21.2 years ago • Jakob Hedegaard

cell sample each. I am just confused to import the data into R and hoe to normalise the biological replicates to perform differential analysis of gene expression between these conditions

DESeq2 Rtudio

updated 4.3 years ago • Nilofer Ali

a fairly new DESeq2 user and was hoping to get some help with how to correctly account for technical replicates in my dataset. Here is some example data: suppressPackageStartupMessages(library("DESeq2")) coldata <- data.frame...between groups 1 and 2, and we would like to account for the fact that each sample has two technical replicates. Initially, I thought the right w…

DESeq2

updated 2.6 years ago • sara.p

time point 2. How should I do this in DMRcate? Based on the manual, the data used has biological replicates, I'm wondering whether DMRcate still works well if there is no biological replicate available. Thanks

DMRcate WGBS biological_replicate

updated 4.2 years ago • xie186

Illumina HT-12 v3 Expression BeadChips. I am slightly concerned about the average number of bead replicates present on the Illumina HT-12 chip, as opposed to the Illumina WG-6 chip. Each bead is replicated on average 15 times...chip. Would you still suggest using VST with the Illumina HT-12 chip data, given the number of bead replicates? Thanks for your help. Christina Markunas cam27@duke.edu…

lumi

updated 9.9 years ago • Christina Markunas

different pattern of methylation between the regions in a tumour. So in this case I duo nor have any replicate I have one library for regionA and one library for region B in a tumour. I see you have some suggestions to run EdgeR...when there is no replicate but I am wondering how it should be performed for the methylation data, not gene expression? For example what would...to find out if there …

RRBSdata

updated 22 months ago • annasoudi