Bioconductor Forum

I am trying to run the qpcrImpute function from the nondetects package on my qPCR data, and I am running into some issues. After some experimentation, I have formatted my data with one row per gene, with each technical replicate in a separate column, which seems to match the format of the data in the included oncogene2013 data set, and I have normalized my data, so now I just need to run qpcrImpu…

nondetects software error

updated 7.9 years ago • Ryan C. Thompson

I'm using tuxedo work flow to find DGE  (ballgown) of RNAseq data control vs treated each with biological replicates. which one of the below matrix and stattest should be applied and why? https://github.com/skptic/tuxedo-nf any help would be appreciated, Thanks. 1. sample condition Bioreplicate sampleA Ctr rep1 sampleA Ctr rep2 sampleA Ctr rep3 sampleA Tre rep1 sampleA …

ballgown rnaseq differential gene expression tuxedo

updated 7.5 years ago • justinjj

Hello, I need to perform complex DESeq2 comparisons for mRNA sequencing data (I have biological replicates of each condition and subunit mentioned below). I have two knock-down conditions (KD1 and KD2), and 1 scrambled RNA

DESeq2

updated 13 months ago • sk

I have an experimental design where we change the genotype and the treatments. The replicates were collected at different times, so I am assmuing there is a batch effect. I have two questions here - 1. To build...I have an experimental design where we change the genotype and the treatments. The replicates were collected at different times, so I am assmuing there is a batch effect. I have two …

BatchEffect batch RNASeqR formula DESeq2

updated 3.4 years ago • estafana.t98

I have 99 samples (cancer cells in duplicates or triplicates, and 3 replicates of normal cells) subjected to bulk RNA seq. All the Fastqc parameters look fine + the sequencing depth is pretty...I have 99 samples (cancer cells in duplicates or triplicates, and 3 replicates of normal cells) subjected to bulk RNA seq. All the Fastqc parameters look fine + the sequencing depth is pretty similar

DESeq2 edgeR DifferentialExpression RNASeqData HELP

updated 2.8 years ago • Neha

I have 6 samples, 3 sets of 2 replicates. Not sure why the error is coming up. Something that seems off is that it says 'computing metrics for 10 samples when

ChIPQC

updated 4.6 years ago • Jonah

additive. Has anybody any solution for this problem?. One solution could be replace NA with its replicate (biological/technical) in another array, at least for the fit computation process? Toni Antonio Juli? Unitat de Recerca

Normalization Normalization

updated 21.2 years ago • Antonio

but is it possible to calculate a, say, "general experimental power value" given the number of replicates per condition is already set? Hope I make myself understandable. Thanks in advance, David </div

updated 19.8 years ago • kfbargad@ehu.es

class="preformatted">Hi, I am using edgeR for a 2x2 factorial design (Strain*Treatment) without any replicates and the estimateGLMCommonDisp and glmFit functions. When I run estimateGLMCommonDisp, I get warnings 1: In optimize

edgeR edgeR

updated 14.5 years ago • Filippis, Ioannis

div class="preformatted"> Dear mailing list, I have only one replicate of an RNA-seq time course experiment. I have transcript count, RPKM nomalized gene count, gene expression values

TimeCourse timecourse TimeCourse timecourse

updated 12.7 years ago • Guest User

preformatted">Hi All, I am in need of some basic 2-color (cDNA) microarray help. With 3-biological replicates (one of them is a dye-swap), I wish to merge ONLY the raw intensities of the 3 cDNA files. This merging should take into

Microarray Annotation GO limma Microarray Annotation GO limma

updated 17.1 years ago • santana sarma

I am having a query related to VOOM function in the limma package. I am trying to analyze proteomics data using limma to identify the differential expression between two groups which has control(n=3and tumor, n=6). There is only biological replicates and not any technical ones. I have a doubt that the matrix design is correct or not . Also limma can be used for the analysis...expression between t…

limma

updated 7.2 years ago • manubhaikp

of contamination from the other type, and vice versa. The contaminating read amounts vary from one replicate to another. Western blots confirm this contamination. How do I normalize the transcript read counts for all other

Normalization RNASeq contamination edgeR DESeq2

updated 4.7 years ago • Yuqia

I have 12 RNA-seq samples: 3 replicates each of male control, male mutant, female control, and female mutant. I want a list of genes that are significantly

deseq2 multiple factor design

updated 8.3 years ago • Mike

Hello, I have several batches of scRNA-seq data, which are biological replicates, and would like to use mnnCorrect() from the scran package to correct for batch effects. Each batch should have a...Hello, I have several batches of scRNA-seq data, which are biological replicates, and would like to use mnnCorrect() from the scran package to correct for batch effects. Each batch should have a quite

scran mnncorrect

updated 7.9 years ago • Dave Tang

<div class="preformatted">Hello, I am working on a dataset of nine Affymetrix chip hybridization data, consisting in three time-points analized in triplicates. I am using SAM (multiclass) to identify differentially expressed genes and I realized that some genes have been discarded due to one "bad" value out of three, in the replicate. I woukd like to know if there is an alternative analysi…

updated 20.2 years ago • Stefania Pilati

all rows" this means. For a single transcript, my manual mean calculation of all counts across all replicates, all conditions do not equal to the DESeq reported basemean for a single transcript. 2) log2foldchange seems to

DESeq DESeq

updated 11.4 years ago • rob yang

<div class="preformatted">I'd like to access an API from within R but I'm not sure how to do so. The API uses HTTP and supports both POST and PUT methods. I'm able to access it successfully from the terminal using the following curl command: curl --data @genes.xml http://api-url Where genes.xml is a local file containing the genes I want to submit and http://api-url is the API's address. H…

updated 16.3 years ago • Aaron Wolen

Hello everyone, I am trying to replicate a single cell RNA-Seq data analysis. Actually, the main problem is version. The old results were generated via newSCESet

singlecellexperiment single-cell rnaseq seurat scater

updated 7.3 years ago • hamza_karakurt

Dexseq, as well on Expression  plot.  I've used Dexseq-count followed by Dexseq on two replicates of human tumor vs normal RNA-Seq transcriptome data (hg19). The first column shows multiple ENSG ids followed by

dexseq

updated 7.4 years ago • yelekley7

see if they are differentially expressed between two conditions. Each condition has three biological replicates. Because I cannot define the boundaries of the candidate genomic regions exactly (they are not annotated with

deseq2 rnaseq

updated 7.5 years ago • bioinfo

Hello, My data consist of RNA counts  from 4  phenotypes  with biological replicates. The phenotypes are the combination of 2  genotypes R and A.  The four groups are  wild type, R, A and RA...Hello, My data consist of RNA counts  from 4  phenotypes  with biological replicates. The phenotypes are the combination of…

edger design

updated 11.2 years ago • alakatos

a bulk RNA-seq dataset with one factor and 12 levels (control and treatments), and 4 biological replicates per level. I was wondering how using one or multiple input sample tables would influence the genes that are found

deseq2

updated 7.3 years ago • fl

multiple P values(along Y axis) for a given ratio(or log_ratio). How does this happen? Do they pool replicate data with slightly different mean and std_dev so that Z score and P-value are different for a given ratio. I've posted

updated 15.0 years ago • B Enn

preformatted">Dear all, I have an Affy time course experiment as follows: 0hr - 2hr - 4hr, I have 2 replicates for the 2hr and 4hr and only one (chip) for 0hr. Would I be able to analyze this experiment using limma? (I know its not

affy limma affy limma

updated 19.8 years ago • Khan, Sohail

me out. i'd like to be able to cluster my arrays as a quality control measure (to make sure that replicates cluster with each other, etc.) I'm relatively new to using R and the affy package, so if anyone could help me out, i'd appreciate

affy affy

updated 22.0 years ago • J. Mcelwee

Hi, Some time ago I ran DESeq (one) on a set of data comparing two conditions of growth over 4 biological replicates per each condition. A collaborator constructed a graph of fold change from that output for approximately 15 genes...time ago I ran DESeq (one) on a set of data comparing two conditions of growth over 4 biological replicates per each condition. A collaborator constructed a graph …

rnaseq deseq2 deseq standard error confidence interval

updated 9.7 years ago • lisa.crossman

currently using DESeq_1.5.23 and need to identify DEGs using RNA- Seq read count data. As I hv no replicates wid me I had to go wid estimateDispersions command wid method="blind", and got very few DEGs (~10 only) along wid d warning

GO GO

updated 14.4 years ago • pushp priya

<div class="preformatted">Dear Paul, I'm not quite sure why you're so shocked about getting significant results. Usually people complain to me when they don't get significance :). limma is able to obtain much higher significance levels than a t-test because it (i) leverages information from the within-array replicates and (ii) borrows information across probes. These two approaches in c…

miRNA Microarray Normalization GO vsn limma microRNA miRNA Microarray Normalization GO

updated 16.2 years ago • Gordon Smyth

<div class="preformatted">> Date: Sun, 03 Jul 2005 10:13:29 +0000 > From: Carolyn Fitzsimmons <carolyn.fitzsimmons at="" imbim.uu.se=""> > Subject: Re: [BioC] duplicateCorrelation and design matrix > To: bioconductor at stat.math.ethz.ch > > Hi Gordon, thanks for your reply. I have a few more questions: > > Quoting Gordon K Smyth…

limma limma

updated 20.5 years ago • Gordon Smyth

<div class="preformatted">Dear Guillaume, I am going to assume that the main purpose of your experiment is to find genes for which the d7 vs d1 response is different between the two groups of patients. > Date: Fri, 20 May 2011 15:44:45 +0200 > From: Guillaume Meurice <guillaume.meurice at="" igr.fr=""> > To: Guillaume Meurice <guillaume.meurice at="" igr.fr="…

updated 14.6 years ago • Gordon Smyth

I'm trying to use arrayWeights for the experimental design described below which features technical replicates and the duplicateCorrelation function. I would greatly appreciate it if someone could confirm that what...I'm trying to use arrayWeights for the experimental design described below which features technical replicates and the duplicateCorrelation function. I would greatl…

limma duplicatecorrelation arrayweights

updated 10.3 years ago • willj

Hello, I'm carrying on part of a ChIP-Seq differential analysis for the first time. I have 6 replicates and 3 conditions; in each condition the first 3 replicates have been run separately from the other 3. I have a couple...score for the _dba.count_ function, a mask is created to separate conditions and the first 3 replicates from the other 3. A contrast is generated and the different…

diffbind filtering

updated 8.1 years ago • gasta88

metadata %>% select(1) %>% rename(genotype = title) %>% mutate(genotype = gsub("replicate 1", "", genotype)) %>% mutate(genotype = gsub("replicate 2", "", genotype)) %>% mutate(genotype = gsub("replicate 3", "", genotype)) %>% mutate...genotype = gsub("replicate 4", "", genotype)) #check sample information all(colnames(reads) %in% ro…

DESeq2 Log2FC RNASeqData

updated 3.0 years ago • Iwan

I am trying to use _DEseq2_ to determine diff. expressed genes between various conditions, but find it very difficult how to specify what I want to compare. My experimental design has three factor columns: * two different locations * four different conditions * either two or three replicates - the replicates are matched Here is what it looks like: <table border="1" cellpadding="1" ce…

DESeq2 multiple factor design experimental design contrast

updated 10.1 years ago • anton.kratz

normalization. But I cannot use DESeq2 for getting log2 fold change values because I don't have replicates for some of the experimental conditions and DESeq2 needs replicates to estimate log2 fold change values. So, I

DESeq2 DifferentialExpression

updated 3.0 years ago • Jayesh Kumar

be problematic for comparing between the different pipelines, since the pipelines where B has more replicates will identify more DE genes than pipeline 3 where B only has 3 replicates. Does anyone have any advice? Some things

unevensamplesizes ComparingDGEresults DESeq2

updated 2.6 years ago • reba.duncan

my question seems obvious. I am trying to compare RNA-seq data of two different conditions: LG (5 replicates) vs HG (3 replicates) by using RUVSeq to remove unwanted variation between batches. The data look like this: The columns

RUVSeq software error edger

updated 6.6 years ago • ce.jim.san

on our Biomark system. Typical runs are across multiple plates, and have multiple biological replicates, and usually 2 or more technical replicates (although we are moving away from technical reps, as the CVs are so tight

HTqPCR HTqPCR

updated 13.5 years ago • Simon Melov

and more to come. This data tends to be heterogenous, so I am trying to have higher number of replicates to identify true biological signal. Now, as this is a paired design, there is no need to worry about the batch effects...of working on normalised counts, I am working on a matrix of log2 fold changes of 40 biological replicates. I do not know if its the best way to do exploratory data analys…

deseq2 eda sva rna-seq

updated 9.4 years ago • g.atla

<div class="preformatted">Dear list, Rather than reinventing the wheel, I hope someone in the community can point me in the right direction. In a pilot study, we have looked at a single xenograft over time (0,8,24,48 hours) with or without drug treatment (7 groups). Using Illumina WG6 arrays, with biological quadruplicates and controls at each time point we have a phenomenal amount of clea…

updated 16.3 years ago • Mark Cowley

vars" argument. Data comes from Affymetrix gene chip from 2 different cell lines, 4 time points, 2 replicates at each time. I normalized with RMA, and filtered out low expressed/low changing genes, getting from initial 54k...what I exactly get? I mean biologically speaking... This is my design matrix: Time Replicates Group1 Group2 wt22_g21 21 1 1 0 wt22_g7 …

maSigPro maSigPro

updated 14.3 years ago • Andrea Grilli

In the process of analyzing ChIP-Seq replicates with DiffBind (drosophila data, 3 replicates for each sample, choosing DESeq2 as the analysis method), peaks with

DESeq2 DiffBind

updated 5.0 years ago • Sam

consists of two experimental factors, four conditions (+/+, -/+, +/-, -/-) and four biological replicate per condition. The batch effect in this experiment comes from the fact that the first replicate for all conditions

edgeR limma removeBatchEffect()

updated 11.0 years ago • Ekarl2

on the question of interest and the experimental design. E.g. when you have a very large number of replicates you don't want shrinkage, while with a small number of replicates, shrinkage is often beneficial. Best wishes, Wolfgang

Normalization Preprocessing Cancer probe affy vsn gcrma Normalization Preprocessing probe

updated 21.6 years ago • Wolfgang Huber

Hi BioC community, <span style="line-height:1.6">I work on a PCR data set designed like that :</span> <span style="line-height:1.6">- 21 patients are included in the study</span> <span style="line-height:1.6">- For each patient, his gene expression profile was measured (by PCR) at the inclusion of the study on a sample of his back which was not treated. We call th…

limma covariate block paired samples technical replicates

updated 10.1 years ago • eleonoregravier

I'm working with a circadian timeseries dataset generated from repeated blood draws from a group of subjects. So for each subject, I have gene expression data from multiple timepoints throughout the day. Looking at the documentation for the RAIN package (https://www.bioconductor.org/packages/release/bioc/html/rain.html), it looks like I can perform a repeated measures analysis for the data from e…

"repeated rain circadian

updated 3.8 years ago • Nick Lahens

I'm having difficulty understanding why this would be the case, even if there was variability in my replicates/small sample size etc. Would greatly appreciate if someone could clear this up for mem and confirm whether I would

RNASeqData DESeq2

updated 2.4 years ago • Marina

downstream to Limma. 3) Also, how good is the idea of using Limma in case if I do not have any replicates? Thank you so much in advance, Karthik [[alternative HTML version deleted]] </div

limma GeneSpring limma GeneSpring

updated 13.9 years ago • Karthik K N

co-hybridized with RNAs from uninfected host tissue (no parasite RNAs). Another is a biological replicate but with dye-swap. People want to know what parasite genes are expressed in the infected tissue. Is it possible to

Microarray oligo Microarray oligo

updated 20.0 years ago • xpeng

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. Calls: DESeq ... estimateDispersions -> .local -> checkForExperimentalReplicates

DESeq2

updated 6.8 years ago • csteely4

in using the FourCSeq pipeline for the analysis of some Capture-C data I have generated in replicate.  One question I have is how to handle the situation where only one 4-bp cutter enzyme was applied (DpnII) and not

fourcseq

updated 7.5 years ago • mmackiewicz

the design model for this experiment. There are 5 different RNA pool families. Each one with three replicates (Technical?), coming from individuals that receive three different treatments, a control treatment and T1, T2. The

updated 14.8 years ago • Luis Z

before performing significance test (say now i have 3 groups based on apoptosis,cell cycle, DNA replication) and then perform significance test within each group, will the results be same as that of conventional way ?? Is

Microarray Normalization cdf cycle Microarray Normalization cdf cycle

updated 14.1 years ago • Guest User

with my DEseq2 result and PCA plot. I have two RNAseq samples(each sample has three biological replicates) that are very close to  each other on the PCA plot. However, there is still a lot of differentially expressed

deseq2 rnaseq

updated 10.4 years ago • Emma

I''m new to R and deseq2 so apologies I'm made silly mistakes. I have three conditions with three replicates each. They are control, infection and attenuated infection. I have carried out analysis using 3 factor levels

deseq2 r filtering bioconductor

updated 9.4 years ago • rattigak

vsn2. scatterplot here: http://server7.pictiger.com/img/198354/other/vsn- bioc.png my data are six replicates of dual color tiling microarray (193290 spots), i.e. i pass a 193290 x 12 matrix to vsn. i guess that's a bug, is it? Tobias

Microarray Normalization vsn Microarray Normalization vsn

updated 18.6 years ago • Tobias Straub

is that converting these data into ranks would improve the situation (it certainly does improve replicate correlations considerably). However, I am not sure what type of tool to use for the next step: what kind of EdgeR-like

EdgeR rank

updated 9.6 years ago • knaxerova

Hello!  Since I do not have biological replicates I want to call DMRs with DSS. As far as I understand, first a basic smoothing is performed before using a beta-binominal

DSS WGBS coverage

updated 8.3 years ago • floriandeckert

expression in a 2x2 design of two populations with two treatments (a total of 4 groups with 6 replicates each). I have very interesting results. However, a colleague asked me the other day, "What does edgeR do with zero values

edgeR edgeR

updated 11.9 years ago • Laura Eierman