Bioconductor Forum

Suppose that for three replicates of treatment 1, the average TPM for a particular gene is 80 (and not much difference between the three replicates...And for three replicates of treatment 2, the average TPM for the same gene is 990 (again, not much difference between replicates). 80 / 990 = 0.08

DESeq2 TPM Foldchange

updated 3.7 years ago • dadca596

div class="preformatted">Hi All, In DESeq, if one data set has both technical and biological replicates, i am curious how technical and biological replicates are distinguished in that package. for instance, say, each...biological replicate has two technical replicates. T_b_1_t_1(cancer biological replicate 1, technical replicate1) T_b_1_t_2, T_b_2_t_1...of samples mentioned in DESeq manual, …

DESeq DESeq

updated 15.1 years ago • sunghee OH

I have analysed an RNA-seq dataset containing 2 conditions (control and transgenic mice) with 3 replicates for the control condition and only 2 replicates for the transgenic one (we initially sequenced 3 transgenic samples...As in the DESeq2 Genome Biology article: « experimental design with as little as two or three replicates are common and reasonable » I think this is valid t…

deseq2

updated 10.7 years ago • celine

The final format for each of these tables was as follows: Gene Treated1(Replicate1) Treated2(Replicate 2) Untreated1(Replicate1) Untreated2(Replicate2) tag_id FPKM FPKM FPKM FPKM tag_id FPKM FPKM FPKM FPKM ... I understand...or "Not Applicable"…

DESeq DESeq

updated 13.2 years ago • Andres Eduardo Rodriguez Cubillos

Hello, I'm using DiffBind to extract peaks that appear in my replicates. I have four replicates, and I wish to fetch peaks that are in at least 3 replicates. It doesn't matter which 3/4 (or 4/4...replicates they appear in. How do I achieve this? I expect it has to do with minOverlap parameter, but I can't figure out the changes

DiffBind

updated 3.8 years ago • Krista

due to NB distribution behind the DESeq2? 3) I saw you mentioned that DESeq2 only works for replicates data, here replicates data means biological replicate or technical replicate? Thanks in advance. Elena

DESeq2

updated 4.4 years ago • lovelymaoqin

Trying to generate replicate masks for the dba.count function from a .csv where I list 3 replicates under DBA\_CONDITION. Want to do dba.counts...with minMembers = 3 to include only those that are in all 3 of each replicate group for the binding matrix. Any clues on how to do this? Thanks

diffbind chipseq

updated 8.3 years ago • rbronste

data for 2013 ). - Data seasonally (4x/year for 11 years), but some years missing some seasons - Replicates vary per season/yr **Data structure for rain** = irregular time series (since I am missing data for 2012), and irregular...repeats Data matrix names are are sites x asv ID - 2012_Q1_1 (replicate 1 for year 2012) - 2012_Q2_2 (replicate 2 for year 2012) - 2014_Q1_1 (repl…

rain

updated 21 months ago • mpuli011

analyze DEGs between normal HEK293 cells and HEK293 depleted by one protein. So, I have 3 biological replicates. (2 of replicates I obtained in one day, and the third one in a week - condition was the same). I analyzed my data using DESeq2...The problem: When I produce the PCA plot (top 500 genes) I have the situation when the first two replicates have a good clasterisation and thirds ones are …

deseq2

updated 5.8 years ago • alexandr.gopanenko

combined with the first batch. Within both batches I have the same organ but different biological replicates... for example, 2 replicates of the lungs in batch 1 and 2 and the third and fourth replicate of the lungs in batch 2.&nbsp...With the metadata, replicate info and the column order of the counts table aligning with the row order of the metadata sheet, all samples model...well with DESe…

deseq2

updated 7.2 years ago • A

baySeq) if(require("parallel")) cl <- makeCluster(4) else cl <- NULL data(simData) replicates <- c("simA", "simA", "simA", "simA", "simA", "simB", "simB", "simB", "simB", "simB") groups <- list(NDE = c(1,1,1,1,1,1,1,1,1,1), DE = c(1,1,1,1,1,2,2,2,2,2)) CD <- new("countData...data = simData, replicates = replicates, groups = groups) libsizes(CD) &…

bayseq missing data

updated 10.6 years ago • k.w.lau

by SEQC package to benchmark my method, however I am a bit confused about the final counts for each replicate. In each dataset, there are libraries from different lanes and flow cells for each group's replicate. My question...I add up the counts from all lanes and flow cells to obtain the final count for that replicate? For example, is the counts for replicate …

SEQC

updated 9.8 years ago • siamak

SS <- DiffBind::dba(sampleSheet = csvFile, scoreCol = 5)``` The sample sheet has 6 bed files, 3 replicates of one sample and 3 replicates of a second sample. I made the venn diagram: ```Venn <- DiffBind::dba.plotVenn(SS, 1:3)``` everything...works great and I get the number of peaks that overlap between the first 3 replicates. However, if I go back to the original sample she…

chipseq DiffBind

updated 3.2 years ago • Nicole

1:12,c(1,6,8)]) title type source_name_ch1 GSM424759 pSUPER-scramble replicate 1 RNA HUVEC infected by retroviral vectors bearing a control scramble sequence GSM424760 pSUPER-scramble replicate...HUVEC infected by retroviral vectors bearing pre-miR-210 sequence GSM424763 pSUPER-mir-210 replicate 2 RNA HUVEC infected by retroviral vectors bearing pre-miR-2…

updated 14.3 years ago • Jing Huang

<div class="preformatted">Hello ! I am comparing the transcriptome of two yeast strains, A and B. For each strain we have 5 biological replicates. The strains are compared to a reference on two-color arrays. For each biological replicate, we used two slides, in dye-swap. So we have 20 slides : slide Cy3 Cy5 1 A1 ref 2 ref A1 3 A2 ref 4 ref …

Yeast limma a4 Yeast limma a4

updated 17.1 years ago • sylvie pinloche

I'm having difficulty conceptualizing how significance of logFC is calculated with \`exactTest()\` and \`Benjamini-Hochberg\` adjusted FDR calculation with \`topTags()\`.   Let's say you had \`100 genes\` with \`5 control replicates\` and \`5 experimental replicates\`,  how are p-values for the logFC calculated and then how are they adjusted using the \`Benjamini-Hochb…

edgeR bioconductor fdr statistics differential gene expression

updated 9.0 years ago • jol.espinoz

Hello: I have faced a problem when I´m trying to analyze my TLDA data: I have 3 or 2 technical replicates per sample, my problem is that I really don´t know how to read and asses the replicates. What I tried is to analyze my...data as individual samples and then asses the replicates as following: d.normA2= exprs(d.normA) > d.normA2.prom= (d.normA2[,1]+ d.normA[,2]+ d.normA2[,3])/3) &am…

Cancer Cancer

updated 14.9 years ago • Sandra Romero Cordoba

about the DESeq2 model. Given that I have 2 groups of RNA-seq, each of which has 3 biological replicates. Thus, each gene has 3 biological replicates in each condiction. does the DESeq2 model use average gene expression...or each replicate of gene to estimate paratmeters, such as beta and intercept? Thanks

DESeq2

updated 2.7 years ago • BioEpi

Affymetrix chips. It is basically a 2x2x4 design repeated twice using different biological replicates (the third replicate will be provided later). The plants within each biological replicate were grown at the same...time, and in that sense are related and form a block. There are no technical replicates within each block. I am using limma. In the model I calculate separate coefficients for each …

limma limma

updated 21.1 years ago • Thibaud-Nissen, Francoise

<div class="preformatted">Hi, I have the following MA experiment running on Affymetrix technology. It includes 4 condition and 3 time points for each condition as follow: FileName Targets Time Treatment Batch F21.CEL I18 18 I 3 F22.CEL I18 18 I 3 F25.CEL N18 18 N 3 F26.CEL N18 18 N 3 F23.CEL R18 18 R …

updated 19.9 years ago • Ron Ophir

Dear All, I am having trouble dealing with technical replicates in my RNAseq data analysis using Deseq2. I have generated a _SummarizedExperiment_ object for my dataset but

deseq2

updated 10.8 years ago • Adeolu Adewoye

<div class="preformatted">Dear list, I have a short question: I'm performing heatmaps on lists of genes selected as being differentially expressed on a set of 4 technical replicates (from an Agilent gene expression 2 colors experiment). As technical replicates are behaving very similarly, I am taking the median values of the replicates log2ratios, and basically trying to get a heatmaps th…

updated 15.4 years ago • Sarah Bonnin

I have a RNA seq data which I am trying to identify DEGs. Dataset contains Untreated - 2 replicates (time point 0hr) Treated - 4 replicates (2 replicates at 6hr and 2 replicates at 12 hr) I merged all the samples and tried

LRT co-expression network deseq2 time course experiment network analysis

updated 7.7 years ago • aishu.jp

Hi Michael, I am analyzing a dataset (treatment vs. control) each with three replicates. In the PCA plot one of the replicate samples (control) is positioned away from the other two replicates but the treatment

deseq2

updated 11.0 years ago • Prasad Siddavatam

I am trying to use DeqMS to do some differential enriched protein analysis. I have 4 replicates of my control condition and 4 replicates of treatment condition. The package seems require at leat 2 values in...However there are many proteins only exist in the treatment condition while missing in all 4 replicates in control condition. I assume those proteins should be "DEP " but how can I isolate t…

DEqMS

updated 4.9 years ago • Ling

I had three experimental condition each with control and experimental condition, having two replicates each T1 - control (2 replicates) - experiment (2 replicates) T2- control (2 replicates) - experiment (2 replicates) T3-control...2 replicates) - experiment (2 replicates) When analysis was perfomed for T1, T2 and T3 seperatly such as Name FileName Target T1_c1

Microarray Microarray

updated 16.8 years ago • hemant ritturaj

say I have two conditions treated and untreated (6 and 2 ), are the values of gene expression of two replicates (untreated) normalized and compared with the values of the 6 replicates (treated) ? I mean are this taken as a single...and one as reference condition? Thank you also, to make a heatmap where all the biological replicates cluster together I could take the top 1000 gene that are s…

deseq2 RNA-seq Heatmap

updated 6.4 years ago • Merlin

some queries regarding differential expression using deseq. I have time-point data with 3 biological replicates and control with biological replicates. I have generated the counts using htseq-count. I want to be able to compare...DESeq(dds) res <- results(dds) I have a few questions. - How to take care of biological replicates to identify differentially expressed genes between condi…

rnaseqGene DESeq2

updated 4.2 years ago • Shilpi

I'm comparing knockdown ChIP (3 replicates in the green) versus control ChIP (3 replicates in the orange), but the Fold (in the grey) is much lower than I expected...Could the low Fold value be caused by shrinkage adjustments due to inconsistencies between the replicates? ![enter image description here][1] [1]: /media/images/4818964b-a81c-497e-8cbb-b75be00c

DiffBind

updated 2.9 years ago • slrpatty

species E, with individual E1 and E2. So there are 2 sample in group 2, each with 2 biological replicates, while in group 1, we have 3 sample, but no replicates. We would like to test whether a gene is significantly differentially...linear models (GLMs). But a reviewer says that it may be not appropriate to manage the biological replicates in group 2 in this way. My questions are: What…

linear model

updated 11.2 years ago • wenming126

of samples, but very large number of reads in another groups? Here is an example: Samples, Sample 1-replicate 1, Sample 1-replicate 2, Sample 2-replicate 1, Sample 2- replicate 2, Sample 3-replicate 1, Sample 3- replicate 2 Gene_X

edgeR edgeR

updated 12.8 years ago • Guest User

<div class="preformatted">Dear all, I have a question on how to analyze the following Affymetrix gene expression data. - There are three groups (each representing a haplotype). - There are several persons in each group (3 persons in group 1, 5 persons in group 2, and 4 persons in group 3). - There are 4 biological replicates in each person. The experimenter took a cell from a person and cu…

updated 17.2 years ago • Seungwoo Hwang

matrix in EdgeR. How can I write the code for them to be recognized? I also need to make sure my replicates are similar to one another in each 'group' in order to effectively compare. I have been analyzing everything separately...th scope="col">Factor #2</th> </tr> </thead> <tbody> <tr> <th scope="row">Treatment #1</th> <td>4 Replicates</td>…

edger design model interaction model rnaseq

updated 7.0 years ago • cody0071

I have faced a problem when I?m trying to analyze my TLDA data: > > I have 3 or 2 technical replicates per sample, my problem is that I really > don?t know how to read and asses the replicates. > What I tried is to analyze...my data as individual samples and then asses > the replicates as following: > > d.normA2= exprs(d.normA) >> d.…

Cancer HTqPCR Cancer HTqPCR

updated 14.9 years ago • Heidi Dvinge

Hello, I have the following RNA-seq experimental design: - Control: 3 biological replicates from ther first sequencing + resequencing from the 3 same samples - Treatment1: 3 biological replicates from ther...first sequencing + resequencing from the 3 same samples - Treatment2: 3 biological replicates from ther first sequencing + resequencing from the 3 same samples * The reseque…

Sequencing sequencingDeepth BatchEffect DESeq2

updated 4.1 years ago • ary.lech

Hi all, Would you suggest how to fix this error? I have only 2 replicates for the control and 2 replicates for the diseased group. Thank you so much. result <- dba.analyze(samples, bParallel...messages: 1: No contrasts added. There must be at least two sample groups with at least three replicates. 2: No contrasts added. There must be at least two sample groups with at …

ATACSeq

updated 2.7 years ago • Chris

the Dye effect. > The example there describes an experiment of Wt vs Mut with two dye > swaps replicates as follow: > > FileName Cy3 Cy5 > File1 wt mu > File2 mu wt > File3 wt mu > File4 mu wt > > Let's say that I found...ignore them or > can I correct the dye effect? > I guess that it …

limma limma

updated 20.0 years ago • Gordon Smyth

condition) and Lactose (Induction condition) 3 epigenetic marks : H3K4me3, H3K9me3 and H3K27me3 3 replicates for each mark and each condition 2 inputs for each condition 1 mock for each condition The three replicates for...each condition are biological replicates from which I IPed my three marks : Glucose condition : Biological replicate 1 : H3K4me3 (1) H3K9me3 (1) H3K27me3 (1) Input...1)…

ChIPSeq DiffBind ExperimentalDesign

updated 4.7 years ago • SFn

BioConductor mailing list! I perform 2 color hybridization with Exiqon slides, where each mir has 4 replicates. I compare a rather small number of slides (from 2 up to 6 biologial replicates for each treatment). For analysis of...the calculation of Duplicate correlation. Seems to work well, the problem is, that not always all 4 replicates of one mir are "unflagged". It can happen that on the same…

Normalization limma limmaGUI Normalization limma limmaGUI

updated 17.0 years ago • Christine Voellenkle

<div class="preformatted">Hi Bioconductors I followed the example 10.2 in Limma's user guide which includes a coefficient for each mouse in the linear model to account for technical replication. I am somewhat puzzled because the example in the user's guide returns non-estimable coefficients and hence the...user guide which includes a coefficient for each mouse in the linear model to accoun…

updated 20.2 years ago • Pie Muller

male: no signal Condition 2: replicate 1: 3,102,608 good reads, male: 5.0 replicate 2: 3,451,983 good reads, male: no signal replicate 3: 2,892,192 good reads, male...13.6684814046 p.value: 0.0588163345523 logFC: NaN geneID: 38959 Raw data: Condition 1: replicate 1: 2,372,532 good reads, female: 5.0 replicate 2: 3,966,968 good reads, female: 5.0 replicate 3: 1,389,571 good reads, male: no..…

Normalization edgeR Normalization edgeR

updated 14.2 years ago • Nick Schurch

SAMPLE 4 MEDIA A Day 30 (triplicate) SAMPLE 5 MEDIA A DAY60 (single) SAMPLE 6 MEDIA A DAY90 (REPLICATES) SAMPLE 7 MEDIA B DAY 7 (triplicate) SAMPLE 8 MEDIA B DAY 14 (triplicate) SAMPLE 9 MEDIA B DAY 30 (triplicate) SAMPLE 10...MEDIA B DAY60 (SINGLE) SAMPLE 11 MEDIA B DAY 90 (TRIPLICATE) SAMPLE 12 MEDIA C DAY 7 (REPLICATES) SAMPLE 13 MEDIA C DAY 14 (REPLICATES) SAMPL…

limma lumi limma lumi

updated 16.1 years ago • Maria Dolores Serafica

SAMPLE 4 MEDIA A Day 30 (triplicate) SAMPLE 5 MEDIA A DAY60 (single) SAMPLE 6 MEDIA A DAY90 (REPLICATES) SAMPLE 7 MEDIA B DAY 7 (triplicate) SAMPLE 8 MEDIA B DAY 14 (triplicate) SAMPLE 9 MEDIA B DAY 30 (triplicate) SAMPLE 10...MEDIA B DAY60 (SINGLE) SAMPLE 11 MEDIA B DAY 90 (TRIPLICATE) SAMPLE 12 MEDIA C DAY 7 (REPLICATES) SAMPLE 13 MEDIA C DAY 14 (REPLICATES) SAMPL…

limma lumi limma lumi

updated 16.1 years ago • Maria Dolores Serafica

replicates, groups=groups) >> cD at libsizes = getLibsizes(cD, data=simData, replicates=replicates, subset=NULL, estimationType...gt; unused argument(s) (quantile = quantile) >> cD at libsizes = getLibsizes(cD, data=simData, replicates=replicates, subset=NULL, quantile=0.75, estimationType="edgeR") > Calculating library sizes from column totals...gt; unused ar…

Normalization Bayesian edgeR baySeq DESeq Normalization Bayesian edgeR baySeq DESeq

updated 14.0 years ago • Gordon Smyth

Dear Bioconductor Team,  We sequenced samples A with 4 replicates and B with 4 replicates last year, and C with 3 replicates this year. Since there are no overlapped samples, I can't

sva deseq2

updated 7.1 years ago • capricygcapricyg

the different genes and the columns are the different samples. There are 10 columns (5 are Control replicates and 5 RNAi replicates). I want to do a scatter plot of the log10 FPKM, with the x-axis is RNAi and y-axis is Control. Does...anyone have any recommendations on how to do this plot? Also, do I need to average all the replicates for Control together and all the replicates for RNAi together …

ggplots2 DESeq2 fpkm()

updated 4.5 years ago • Gime

Hi, For RNASeq analysis, I am generating a PCA plot for various strains with three biological replicates each. When I make the PCA plot , I get a symbol on the plot for every replicate. For a large dataset, I was wondering if...there is a way to have a single symbol (average of three biological replicates) be represented on the plot, instead of all three replicates.  In DESeq2 package …

deseq2 pca plot

updated 8.9 years ago • pkachroo

to perform a DEA between 2 groups of cell lines. In each group, I have 3 cell lines and 2 biological replicates per cell line. My colData is below: ```r sample cell.line replicate group BE2C.RNA1 BE2C 1 group1 BE2C.RNA2 BE2C 2...the groups ? I performed the analysis using only the group as factor in the model, when using all replicates or only one replicate for each cell line. ```r dds &lt…

DESeq2

updated 2.3 years ago • isabelle.dupanloup

4 MEDIA A Day 30 (triplicate) SAMPLE 5 MEDIA A DAY60 (singleton) SAMPLE 6 MEDIA A DAY90 (replicates) SAMPLE 7 MEDIA B DAY 7 (triplicate) SAMPLE 8 MEDIA B DAY 14 (triplicate) SAMPLE 9 MEDIA B DAY 30 (triplicate) SAMPLE 10...MEDIA B DAY60 (singleton SAMPLE 11 MEDIA B DAY 90 (TRIPLICATE) SAMPLE 12 MEDIA C DAY 7 (REPLICATES) SAMPLE 13 MEDIA C DAY 14 (REPLICATES) SAMPLE …

limma lumi limma lumi

updated 16.1 years ago • Maria Dolores Serafica

Can you run the DMLtest function with different replicate group sizes? For me it fails with the following output: <pre> Smoothing ... Estimating dispersion for each CpG site, this...take a while ... Error in x1 * wt1 : non-conformable arrays</pre> if called with groups of 3 and 5 replicates. It might not be the best idea to have different sample sizes in the first place but woul…

DSS

updated 9.3 years ago • aerval

Primarily, I do not want to delete this 0-byte bed file from the sample sheet, because its parallel replicates still has some peaks. But if I have to delete it (replicate no is 2), should I also change other replicate numbers, for

DiffBind

updated 4.9 years ago • ahua217

Hi there, I'm hoping to get some help with the design table for my analysis, I continue to get the model.matrix error when I put it into DESeq2 and I'm not sure how to resolve this. Below, I have included an example of my design table. Pooled and multiplexed sequencing used - there was a total of 5 Pools and within each pool 24 index's used for multiplexing. Would I be correct in saying the pool…

ExperimentalDesign RNASeq replicates DESeq2

updated 3.6 years ago • Beth_b

The proteoQC package is exactly what i was looking for to give me some QC information on proteomic samples, however i am running into two issues. One may be a bug, and the other may be me doing something weird. The first is that when i am creating the sample list to run with the mQCpipe, it will only work if i have all the headings outlined in the example (file, sample, bioRep, techRep and fra…

ProteoQC Proteomics

updated 6.1 years ago • laural710

rcd11, rcd13, rcd14 and sro11) and wild- type Col0. For the 4 mutants there are 4 biological replicates each. For Col0 there are also 4 biological replicates but for each biological replicate there are 4 technical...replicates. The targets I made is as the following: > targets FileName Cy3 Cy5 1 122.rcd1.gpr.proc rcd11.r1 Col0.r1 2 124.354.…

Microarray Normalization limma ArrayExpress Microarray Normalization limma ArrayExpress

updated 14.7 years ago • Yong Li

Mu2 Wt2 Time point 2 Apart from the time effect, I would actually like to test if the "replicates" are actual replicates, since the biological data seem to indicate they are not quite so similar. If I assume that...the replicates are actual replicates, how would the design matrix look like? and if I want to see if the replicates are behaving

updated 20.0 years ago • jg295@mole.bio.cam.ac.uk

Hello Rory, I am experiencing trouble trying to perform a dba with blocking factor (replicate in my case). My current dba object looks like this, and to my eyes, it has the correct format, indicating the Replicate...metadata in the corresponding column > Escape_all ID Condition Treatment Replicate Caller Intervals FRiP 1 Ctrl_D8_1 Ctrl 0d 1 counts…

Diffbind ATAC batch correction blocking factor

updated 6.3 years ago • rm1238

WT1945",tissue="BM B cells", factor="WT",replicate=3) dp.K4 <- dba.peakset(dp.K4, peaks=system.file("2016_K4_peaks.xls", package="DiffBind"), peak.caller="macs", sampID...Mutant2016",tissue="BM B cells", factor="Mutant",replicate=1) dp.K4 <- dba.peakset(dp.K4, peaks=system.…

diffbind macs2 chip-seq

updated 8.0 years ago • chunhong.liu07

Array A : TBVII Treat 01 Array B : TBVII Treat 02 Array c : TBVII Treat 03 (they are biological replicates) Array D : SSC15 -01 (just a sample without replicate) Array E : TBVII control 01 Array F: TBVII control 02 (They are technical...replicate) I understand that this is a Bad experimental design but a group wants to do have some preliminary information to...to decide what sort of statistical…

updated 17.0 years ago • John Antonydas Gaspar

data and i choose limma library to make normalization and statistical analysis because i only have 2 replicates per condition and i read in some paper that a moderated t test perform better when there are few replicates. The...multiple testing correction : pvalue ? log2FC ? other criteria ? I have a lot of variabily between replicates, ie, for many genes, i have a fold change <0 in one r…

Microarray Normalization Microarray Normalization

updated 14.5 years ago • Stephanie PIERSON

Hello everyone, I've been working extensively with a small RNA-seq dataset comprising 47 samples, each associated with a specific date and categorized into 'ON' or 'OFF' conditions, with three replicates per condition (except for one condition with two replicates only). This dataset encompasses the counts of 77,000 small RNA molecules detected on our reference genome using short stack. I've co…

conditions replicates SmallRNAData DESeq2

updated 24 months ago • samer