Bioconductor Forum

I have 2 groups, Group A (3 biological replicates) and Group B (4 biological replicates). Will the uneven of sample amount between two groups lead to unreliable results

DESeq2

updated 3.4 years ago • Yu

and technical variation, if it does, how do I tell BitSeq which files are belong to which biological replicate I am looking to use BitSeq to analyze my single end illumina data.  I have a combinatorial design (3x2x2) where...each condition has 3 biological replicates, and each biological replicate was split between 3 lanes to have 3 technical replicates.  So I can use BitSeq...…

BitSeq bitseq rnaseq

updated 10.2 years ago • Sam McInturf

Hi, I have a question about biological replicates and technical replicates: In my project, there are four animals for each genotype (hom, het, wt). For each animal, 3 to...this case, I do not know for the samples from the same animal, should I consider them as technical replicates and use the DESeq2 `collapseReplicates` function to collapse data? e.g. For the Hom group, there are No.2/7/8/…

DESeq2 Transcriptomics

updated 3.1 years ago • Yijing

this information are useful when we want to perform some downstream t-tests. For example, three replicates (after drug) comparing with other three replicates (before drug), if given one gene, most detect values are "A" in all...these 6 replicates, we will think the t-test doesnot make sense. So how to deal with this situation? Can we incorporate with detect call

gcrma gcrma

updated 19.0 years ago • Wang mingyi

the same treatment at different doses : - exp 1: control vs exposed dose 1 and dose 2 with 5 replicates each (2 replicates control and exposed from lab 1 , 3 from lab 2 - sequenced together) - exp 2: control vs exposed dose 3...with 5 replicates each from lab 2 - done week 1 - exp 3: control vs exposed dose 3 with 5 replicates each from lab 2 - done week 2 - exp 4: control...vs exposed do…

DGEs edgeR RNASeqData

updated 13 months ago • Philippine

I am working on time course data with replicates. My dataset looks like the following: WT: 6 timepoints with 3 replicates each KO: 6 timepoints with 3 replicates each...RE: : 6 timepoints with 3 replicates each Total: 54 samples. I have the following questions: 1. While performing WGCNA, should I do it for separate genotypes

WGCNA Normalization RNASeq

updated 9 months ago • Vishnu

basic structure of my experiment is as follows: I have 8 samples: Samples 1 and 2 (biological replicates) : Cell line A; treated with vehicle Samples 3 and 4 (biological replicates): Cell line A; treated with drug Samples 5...and 6 (biological replicates): Cell line B; treated with vehicle Samples 7 and 8 (biological replicates): Cell line B; treated with drug The primary

DESeq2

updated 6.4 years ago • msubramanian1

After I ran cuffdiff and got the DE results, I was trying to visualize the results with cummeRbund package. When I ran the following command, an error occurred. Typically it was normal and worked well without the replicates=T parameter, but when added some errors just came out. How can I fix this? Thank you. > genes.MDS <- MDSplot...I ran the following c…

rnaseq cummerbund R software error

updated 8.8 years ago • wastdc

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. ``` I'm new in learning DESeq2. Can anyone help

deseq2

updated 5.2 years ago • takisliako

0" cellspacing="0" style="width:195pt"> <tbody> <tr> <td>Condition</td> <td>Replicate</td> <td>SampleName</td> </tr> <tr> <td>Control</td> <td>R1</td> <td>AB3131</td> </tr> <tr> <td>0.03ppm</td> <td>R1</td> <td>AB3132</td> <…

deseq2 extracting contrasts

updated 8.8 years ago • laianavarromartin

raw count data set of a viral infection consisting of 3 time points and a virus free control with 3 replicates each produced with featureCounts. Normalization after running template_script_DESeq2.r: ![enter image description...across samples are stabilized. Apparently there were some problems with controls, especially replicate A. Since I am working only on the viral side of the replication cyc…

DESeq2 viral controls

updated 3.1 years ago • f99942

of outliers and others. But after i spent more time on data i found something because i have three replicates for each experiment. i have significant amount of values in iBAQ (for my protein of interest) but LFQ has nothing...two replicates were zero one replicate has some values). Can you please suggest me which one to consider and what is the real difference

Proteomics DEP limma

updated 4.4 years ago • barrypraveen

use deseq2 question3:  Regarding the no replicated situations(both treated and controled no replications;    controled no replications;    treated no replications), I should choose deseq2 or deseq or edger

deseq2

updated 8.6 years ago • 247896572

2005 16:48 > > To: bioconductor > > Subject: [BioC] Using limma with contrast matrix ,replicate > > spots, donor > > effects > > > > > > This question is because I am misunderstanding how certain things

HIV limma HIV limma

updated 20.9 years ago • Pita

wrote: > Hi everyone, > > Comments from Naomi and Gordon (below) about the technical replication in > the 2x2 factorial loop experiment are very close to an issue I have been > struggling with for several...When (if ever) is it OK to treat > technical replicates as biological replicates? Often this is done when > there is more than one random effec…

Microarray limma Microarray limma

updated 19.6 years ago • Gordon Smyth

Hello, I have the RNA and Ribo-seq dataset for a biological process for 5 time points in 2 replicates. I am performing 3 LRT tests, for RNA, Ribo-seq and translation efficiency (TE = Ribo / RNA + pseudo-count) respectively...colData = metadata_rna, design = ~replicate + stage) dds_rna <- DESeq(dds_rna, test="LRT", reduced = ~replicate) dds_rpf &…

DESeq2 timecourse time

updated 22 months ago • Anastasiia

all (and hopefully Rory would see this. Thanks for the great program) I have ChIP-seq data of 3 replicates of wild type and 3 replicates of my mutant. I'm looking for loci where my protein binds in WT samples (so WT has peaks...38 (but with minOverlap as 3), and I found out that this gives the list of loci, where all 3 WT replicates are present but it didn't consider mutant peaks. This on…

DiffBind

updated 3.1 years ago • Junsik

Dear Bioconductor community, I am confronted to a puzzling question. I can't find specific answers on biostars or other websites, and can't figure it out. I have been working on RNA from olfactory epithelium. I had to extract the full sample provided as olfactory receptors can be locally distributed (I had to be sure I have everyone). Some tissues were too "big" so I extracted them in two batch…

limma

updated 6.6 years ago • mouton.alice

div class="preformatted">Hi Jo?o Here's how I did it on a set of arrays with 3 replicates. ID is a column that has identical values for replicate spots. spotaverage<-function(x) { # at max 1 NA, and have low variability...have a MAList and I want to exclude probes that show a > standard deviation above 0.1 between the replicate values. > The number of within-array repl…

probe probe

updated 18.8 years ago • Oosting, J. PATH

div class="preformatted">Hello, For a factorial design with some replicates at each level one could assume that the replicates within each level are more similar to each other than across...especially if the factor is something like "patient" or "animal"). One could normalize within the replicates 1st and then a 2nd round of normalizaton across the factor levels. Would this make sense (at al…

updated 21.7 years ago • Arne.Muller@aventis.com

I'm trying to overlay alignment tracks in the Gvis package. I have data for 3 technical replicates from 2 different cell types, so 6 alignment datasets in total.  I have managed to overlay the alignment tracks...for the technical replicates in each cell type, so I have 2 composite alignment tracks in total, each with 3 overlaid alignment tracks; one from...each technical replicate. Howe…

gviz alignment tracks

updated 7.7 years ago • camerond

What is the proper way to replicate the quick select lists seen on the Web UI when using the R cBioPortalData package? I can replicate the TCGA PanCancer

cBioPortalData

updated 3.3 years ago • ryan.hagenson

would like to know if my point "it is better > to have more time points per experiment than more replicates per time > point and less time points per experiments" is a good one. > >Because I am "replicating" my data, but collecting...another kind of info >(temporal info) also. Well, collecting more time points isn't replication. You're asking whether it is bett…

updated 22.3 years ago • Gordon Smyth

Setup details: Let's assume we have a time course dataset with 1000 genes, 8 time points and 3 replicates per time point. Here each time point is a 'condition' amounting to a total of 8 conditions. The total number of samples...estimate per gene (hence 1000 dispersion values). * Take a gene (let's say BRCA1) and a given replicate (let's say replicate 1). * Take the 8 Negative Binomial distri…

deseq2 R makeexampledeseqdataset deseq

updated 7.6 years ago • kjkjindal

peak.caller = "narrow", sampID="WT1_75", tissue = "K562", factor = "H3K9Me3", condition = "75", replicate = 1) wt <- dba.peakset(wt, peaks = system.file("/home/labs/shlush/avivdm/chipseq_313_75/9_macs_broad/wt2_ip_chip_7...peak.caller = "narrow", sampID="WT2_75", tissue = "K562", factor = "H3K9Me3", condition = "75", replicate = 2) wt <- dba.peakset(wt, peaks = system.file("/h…

DiffBind consensus

updated 6.5 years ago • aviv.de-morgan

normal sample, and 3 tumours. These relate to the samples taken at 'surgery', so not just technical replicates of same library sequenced multiple times (hence I believe I should not use `` collapseReplicates ``). As you can also

DESeq2 design and contrast matrix

updated 7.0 years ago • bruce.moran

you are comparing a cell line in stimulated and unstimulated conditions, and you have two biological replicates. Suppose the first replicate gives you 10-fold up regulation in the simulated condition, and the second replicate...the stimulated and unstimulated conditions, but that there is a lot of variability between the replicates. This is exactly what the log-ratio analysis would tell you. On …

limma limma

updated 20.6 years ago • Gordon Smyth

examples in the section "Time course RNA-seq experiments of Drosophila melanogaster" are technical replicates. So, the reads can be summed by sumTechReps(). If I have biological replicates, how to sum the reads for the time course

edgeR

updated 2.1 years ago • SuperDad

effects of different viral strains in a complex experiment. Factors: 1) Patient (p3, p4, p5) 2) "Replicate" (a, b, c) 3) Viral Titer (continuous integer variable) 4) Viral Strain (O, F, S) Although all 3 of the "replicates" per patient were...treated the same way, there are significant differences in the amount of virus recovered from each "replicate", and that appears to have a significant eff…

updated 14.8 years ago • Anand Patel

takes up in the plasmid library (and thus was differentially expressed). I performed 6 biological replicates of RNA-seq (independent transfections), and 6 replicates of sequencing library preparation from the plasmid library...that was used for transfection. The issue I'm running into now is that the dispersion in the RNA-seq replicates will be much higher than the dispersion in the plasmid libr…

deseq2

updated 9.6 years ago • dsg16

with different tissue samples (with a different purified cell type in each case), and one technical replicate per tissue. For each tissue, a pool of 12 mice was used. As you can see no biological replication was performed. My question...vs technical variability ratio, but in the case they were of the same order, could the technical replicates be considered in order to construct typical statistic…

limma LPE limma LPE

updated 20.5 years ago • Ariel Chernomoretz

preformatted"> Hi, I'm analyzing a dataset of 50 samples. Each of these samples has a dye-swapped replicate. All the replicates are side by side in the dataset (1, -1, 2, -2, 3, -3, etc...) I'm interested in some contrasts between predefined...1 0 0 -1 ... .. But this is not enough to proceed the analysis, because the 1s and -1s are replicates. So I tried to estimate the correlation …

updated 16.3 years ago • Giulio Di Giovanni

has some time to look at them and can offer some suggestion as to what might be happening. I have 3 replicates experiments for two different Transcription Factors. In both cases, two of the three replicates look real screwy...cbio.mskcc.org/~lianos/files/bioconductor/bad1.Log2Ratio.png Here's the funky MA plot for the same replicate expt: http://cbio.mskcc.org/~lianos/files/bioconductor/bad1.pn…

Microarray Biophysics Microarray Biophysics

updated 17.2 years ago • Steve Lianoglou

questions here but still I'm not sure if my matrix is correct. ```r SampleID Factor Condition Replicate sample1 eprint siNeg 1 sample2 eprint siNeg 2 sample3 eprint siFUS 1 sample4 eprint siFUS 2 sample5 input siNeg...sample2` and `sample6` `sample3` and `sample7` `sample4` and `sample8` I'm not sure if my `Replicate` column is correctly accounting for the paired samples if code…

DESeq2

updated 4.4 years ago • Alexandre

and I have som basic question on my setup. I have normalized data in a time series with two replicates. \*my samples: \* <pre> colnames(hela.bc) [1] "I.0h" "I.1h" "I.2h" "I.3h" "I.6h" "I.9h" "I.12h" "I.15h" "I.18h" "I.21h" "I.24h" "II.0h" [13] "II.1h" "II.2h...II.24h"</pre> I would like to compare "I.0h" and "II.0h" against all the other time points. (…

limma

updated 11.1 years ago • R

used to analyze continuous data sets. My experimental design does not include any "treatments" nor "replicates". Its simply a gene expression counts along an environmental gradient (with more than 200 samples, spanning along...including some zero salinity measurements). Should I bin the data into discrete values with "replicates" of similar doses, or edgeR can analyze this data set without any …

edger

updated 6.3 years ago • avihai.zolti

will be the expression values for the genes and the columns will be the case which represent the replicates. I am trying to find the differentially expression value for each gene between the two files. first file contains...300 replicates and the second file contains more than 400 replicates. I read the user guide SAGE profiles of normal and tumor tissue

differential gene expression differential expression edgeR

updated 10.6 years ago • Safana

My experiment is an easy one. - I have 3 replicates as control. No treatment done. Then, I did a treatment and collected three replicates at different days time: - 3 replicates...after 4 days - another 3 replicates after 7 days - and another 3 replicates after 14 days These are my samples > samples Experiment Run 1 Control_1

DESeq2 design coldata

updated 19 months ago • arfranco

<div class="preformatted">Hello edgeR users, I have been checking around to find out how to best analyze our data as well as remedy our experiment design. It's hard to decide so I will try my best to explain what we have, what we want in a rather long message. Please bear with me. FIRSTLY SOME WORDS ABOUT OUR SAMPLES: We did experiments with two groups of test animals namely treated "T" a…

Sequencing edgeR MEDIPS Sequencing edgeR MEDIPS

updated 11.3 years ago • Vang Le

<div class="preformatted">Dear BioC List Members: I have a data set that I would like to analyze with the limma package. I am having trouble figuring out how to make the design and contrasts matrices, and I was hoping that someone would advise me. The data set contains the results from 18 Affymetrix hybridizations. The table below explains the experimental design: Sample …

Normalization limma Normalization limma

updated 21.1 years ago • Jim Breaux

I have two conditions: negative control and treatment. Each one has 6 replicates. Then, from PCA analysis and heatmap, I found out that one negative control replicate is very different from the...in the other. Is that right? What should I do here? EDIT: Fishpond version is 2.6.0 EDIT2: These replicates are biological, rather than technical. EDIT3: I will include the PCA plot below ![PCAplot]…

rna-seq salmon fishpond swish

updated 2.5 years ago • samuelcjx

nmfDecomposition, ..., nReplicates = 1) According to manual nRplicates is: nReplicates: How many replicates should be used for assing a single value of 'nSigs'?  For decomposition methods with random seeding...values greater than 1 are reasonable but i would like to know if <span style="line-height:1.6">replicates means biological replicates. What if i …

somaticsignatures

updated 10.3 years ago • Asma rabe

div class="preformatted">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 analysis though seem to se…

updated 21.3 years ago • H. Han

find Significantly differential Expression gene across Time series 24, 48 and 72 hours. I have two replicates for each Time steps. I have used below design for analysis. dds <- DESeqDataSetFromMatrix(countData = cts, colData...I am using correct design Matrix for such time series analysis? 2. Since I am having two replicates as time Period so if…

deseq2

updated 6.7 years ago • syednajeebashraf

Hi!! I'm having trouble to do a contrast using DESeq2. I have 12 samples, AA(2 replicates), AB(4 replicates), BA(4 replicates) and BB(2 replicates). I wanted to compare AB (or BA) with the mean of AA and BB, so I found

deseq2

updated 8.2 years ago • zhuozhu132

lines (clA, clB) (but a similar if not same behavior is expected and therefore they might be used as replicates). Each experiment consists of four measurements at different points in time (6h, 24h, 72h and 144h) and for each of...I'm not sure if this is a realistic amount or if it is because of the design or the lack of further replicates (array quality checks have already been performed on the d…

GLAD maSigPro GLAD maSigPro

updated 15.5 years ago • Matthias Boeck

with the following experimental design: - 3 donors, each treated with: - placebo - 3 technical replicates - drug - 3 technical replicates ``` sample_id patient_id treatment 1 A placebo 2 A placebo 3 A placebo 4 B placebo 5 …

limma

updated 20 months ago • andrea.rodriguez-martinez13

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updated 19.7 years ago • Pedro López Romero

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updated 18.1 years ago • Ingrid H. G. Østensen

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updated 19.7 years ago • Heather Good

for that peak. The second "count" heatmap gives a much less biased view of how samples, including replicates, are correlated, as it takes into account the read density at every site in every sample. If your replicates cluster...We have been using DiffBind on our Chip-seq data. I would like to know, to see the correlation replications.... There are two ways to generate heatmaps between replicate…

DiffBind DiffBind

updated 11.9 years ago • Rory Stark

Dear, I am new to differential ChIPseq analysis. One condition has 6 biological replicates and the other 3. I did not perform the experiment, I just perform the analysis. I started with loading the data: <pre...Dear, I am new to differential ChIPseq analysis. One condition has 6 biological replicates and the other 3. I did not perform the experiment, I just perform the analysis. I started w…

diffbind merging_peaks

updated 8.8 years ago • veronique.storme

ve an experimental design with 2 factors, batch (3 levels) and dose (5 levels) with 0 to 4 technical replicates (a dose is missing for a certain batch). I've seen that one batch has consistently lower variance within doses for...I think!). Does it make sense to "fake" a random block design by taking the mean of the technical replicates and then run a friedman test? I've never used this test befo…

DOSE DOSE

updated 21.3 years ago • Arne.Muller@aventis.com

<div class="preformatted">Respected Mam/Sir, Myself Varpal singh from india, am doing a project on microarray data analysis by bioconductor package but am not able to interpretate results. I want to analysis Affymetrix data by bioconductor. I got result by running the following commands: I have a wild and a mutant types each have two replicate i.e Two for wild and two for mutant. I got o…

Microarray Microarray

updated 15.7 years ago • varpal singh

Hi, I have the following set up: 3 replicates of control and treatment in cell type A and 3 replicates of control and treatment in cell type B. 2 of the A samples...unsupervised clustering with normalized counts in DESeq2 with design ~ 1, and all of the appropriate replicates cluster appropriately. However, I want to make sure that I control for any batch effect. If I make my model desi…

deseq2 sva

updated 10.6 years ago • Jake

TRUE, bRemoveRandom = FALSE, bCorPlot = TRUE, peakFormat = "narrow") G12_1 Dermis Central Replicate WIHN 1 narrow G12_2 Dermis Central Replicate WIHN 2 narrow G12_Combine Dermis Central Combine WIHN 3 narrow &gt...3 Samples, 999 sites in matrix (2690 total): ID Tissue Factor Condition Treatment Replicate Caller Intervals 1 G12_1 Dermis Central R…

DiffBind FRiP ATAC-Seq dba.show dba.count

updated 6.7 years ago • Gary

are merged (shared) between all the samples. So if there were two expeimental conditions and two replicates per condition and a peak was consistently found in the two condition1 replicates but not in the replicates for...t count: some peaksets are not associated with a .bam file." I have my consensus peak lines (replicates) in the dba object but there are no BAM files associated in the orig…

diffbind

updated 9.0 years ago • omiguele

Subject: RE: design matrix edge R pairwise comparison at different time points after infection with replicates > > Dear Kaat, > > 1) It is not generally true that you will find more DE genes analysing > just one time separately...design matrix edge R pairwise comparison at different time >> points after infection with replicates >> &…

RNASeq Normalization edgeR RNASeq Normalization edgeR

updated 13.4 years ago • Gordon Smyth

<div class="preformatted">Dear Heidi (and all), Thank you very much for your work on this package. I found the vignettes and the info supplied excellent! My question is about the featureData layout. My experiment is as follows. I have 96-well qPCR plates to read on a LightCycler, and for my samples I think the best arrangement is: - one gene (primer pair) per plate (with one or two plat…

qPCR qPCR

updated 11.5 years ago • Guest User

bacterial (metatranscriptome) response between 2 conditions. Each condition includes 5 biological replicates. After finishing the DESeq2 analysis I generated a volcano plot and saw that almost all the genes with the lowest...padj actually have very low log2foldChange values. All replicates in the DPS condition have zero counts for those genes while DVM replicates mostly show quite high values. …

DESeq2

updated 4.1 years ago • RitaB