Hi all. First things first, sorry for posting one more question about experimental design and time series in DESeq2.
We have performed...whether there would be any way of getting this comparison directly with the design formula of DESeq2.
However, I have doubts regarding two different questions.
First, I don't know whether this is a real time-series experiment
Order by: Relevance
particularly the section on interactions (https://bioconductor.org/packages/devel/bioc/vignettes/DESeq2/inst/doc/DESeq2.html#interactions), I decided to add a third factor which is a combination of treatment and timepoint...i.e.:
```r
sample_metadata$group <- factor(paste0(sample_metadata$timepoint, "_", sample_metadata$dose))
```
Then I can extract the results for each comparison by..…
updated 13 months ago • drew
Hi Bioconductor community!
I'm using DESeq2 to visualize which taxa are significantly enriched in one treatment over a control and realized that [the vignette...bars. Is this because what the error bars represent (standard deviations) get wrapped up in DESeq2's p-values or another statistic?
First I get the results from a `` DESeqDataSet `` object "`` DDSfbps1spooled ``" g…
updated 7.3 years ago • lauren.czaplicki
Hello everyone!
My question is related to RNA-seq analysis utilizing DESeq2.
Input:
I try to analyze DEGs between normal HEK293 cells and HEK293 depleted by one protein. So, I have 3 biological replicates...obtained in one day, and the third one in a week - condition was the same). I analyzed my data using DESeq2.
The problem:
When I produce the PCA plot (top 500 genes) I have the s…
updated 4.8 years ago • alexandr.gopanenko
Hi
I want to use the DESEQ package between a control (3 biological replicates) and treatment (1 biological replicate).
IN DESeq I herefore used the following code, and got 266 genes with padj < 0.05:
table <- read.delim("test.txt")
row.names(table) <- table$Feature\_ID
count\_table <- table\[, -1\]
conds <- c("ctrl", "ctrl", "ctrl", "treatment")
c…
updated 9.0 years ago • wd
Just followed the vignette to run Swish for the first time using inferential replicates computed by salmon, on data from an F1 cross of two non-B6 mouse strains. It seemingly...separately like so:
```
y <- se[,se$condition %in% c("control1","treated1")]
y$condition <- factor(y$condition, levels=c("control1","treated1"))
y <- scaleInfReps(y)
y <- labelKeep(y)
y <…
Hello,
I finished running DESeq2 on my dataset that includes 6 timepoints and 3 biological replicates per timepoint. I would like to run a PCA analysis...unsure which count transformation method I should use, vst or rlog.
The calculated the size factors for my dataset (below). Based on this, there does not appear to be a large variation in sequencing depth (dynamic range...of size fac…
updated 6.5 years ago • agdif
of rhdf5 directly but
got no response from him. My qualm lies with the writing and re-
reading
of factor vectors using rhdf5. In the current release they are simply
written as integers and upon reading the HDF5 files the...levels are
obviously forgotten.
Of course, I could convert the factors to character vectors before
writing but I wanted to ask whether there is a plan to implement
better
fact…
I am new to DESeq2 and I'm trying to analyze some RIPSeq data.
I have 2 conditions: tagged and untagged
I have 2 assays: input and IP
I want...I am new to DESeq2 and I'm trying to analyze some RIPSeq data.
I have 2 conditions: tagged and untagged
I have 2 assays: input and IP
I want to...untagged IP
untagged_IP_rep2 untagged IP
>coldata$condition <…
DESeq2 assumes negative binomial distribution for counts distribution. That refers to the distribution of counts for a...across one sample? Each sample is normalized based on the geometric mean of all counts (or size factor). That sounds like it does not take into account the distribution. For example, two samples have the same number of reads
voom` function, once before and once after the execution of `duplicateCorrelation`. The blocking factor is not provided within the design formula in both cases.
This is different to the later described `dream` workflow. Here...executed, the blocking variable is part of the design formula. Can someone briefly explain why?
A factor defining individual patient/subject effects, which are "block…
updated 5.2 years ago • Ben
the edgeR package,
which
will still be relatively slow but faster than the glm routines in
edgeR
(or DESeq2).
Best wishes
Gordon
> From: Iddo Ben-dov <iddobe at="" ekmd.huji.ac.il="">
> Subject: edgeR and DESeq2: model design and estimation...at 4:51:51 PM GMT+3
> To: bioconductor at r-project.org
>
> hi,
>
> in both edgeR and DESeq2, estim…
two pairwise comparisons, A vs B and B vs C. I have 1000 genes.
I can provide contrasts to edgeR/DESeq2 for each of the pairwise comparison, thus I will have two contrasts.
The first time I test A vs B, I will have a list of q-values
Hello all,
I am writing to learn how to set up DESeq2 when my samples have large variation in gene counts. For example below is one row from my gene count table.
<table border...of reads at gene240880, but some have zero. When I feed the whole table (with ~25k genes) to Deseq2, using pretty much default settings recommended in the DEseq2 tutorial, and looking at comparisons between condition…
updated 6.0 years ago • Helene
Hi Michael or DESEQ2 community,
First, sorry, I'm not sure why the images don't load, but if you right click on them they will.
When I use the DESEQ2...Hi Michael or DESEQ2 community,
First, sorry, I'm not sure why the images don't load, but if you right click on them they will.
When I use the DESEQ2 results() function I get a variable called *stat*, which is useful for GSEA.
), the mean of
the
> counts divided by the size factors.
>
> The transformations involve much …
updated 10.8 years ago • Michael Love
Hi all,
I have been trying DESeq2 to analyse my RNAseq data. Data is from sequencing a cell line treated with a drug at different time point intervals...like this:
<pre>
DataFrame with 5 rows and 2 columns
treatment time
<factor> <factor>
cl_0h untreated 0
cl_2h treated 2
cl_4h treated 4
cl_6h treated …
updated 9.8 years ago • anand m t
Hey all,
I'm analysing a 16S microbial community dataset, and am using DESeq2 to test for differential abundances. When I do this, I supply raw count information to DESeq() as per the vignette rationale...ordination of my samples, I might consider using rLog transformation or VST to standardise my data first.
The [PICRUSt](http://picrust.github.io/picru…
Hello everyone,
I'm currently trying to perform DESEQ2 with my own RNA sequencing results. I performed whole transcriptome RNA sequencing and the subsequent analysis...Hello everyone,
I'm currently trying to perform DESEQ2 with my own RNA sequencing results. I performed whole transcriptome RNA sequencing and the subsequent analysis I...performed via galaxy.
I performed first fastp on th…
updated 23 months ago • nico
Hello there, I am trying to analyse a dataset using deseq2 with kallisto via tximport. I am using the following code:
tximport(files, type = "kallisto", tx2gene = tx2gene, ignoreTxVersion...suggested ways of importing estimates for use with differential gene expression (DGE) methods. The first method, which we show below for edgeR and for DESeq2, is to use the gene-level estimated counts from …
community over there. However, I still have some remaining doubts, and maybe I should have come here first since it seems it is indeed an issue of how I'm using DESeq2 rather than previous points in the RNAseq analysis.
Quick summary...each one in triplicate. The pipeline goes as such: trinity -> salmon -> tximport -> deseq2. We want to use a common pipeline for both transcr…
updated 6.1 years ago • Emiliano Canton
patients with 2 conditions and 2 time-points from each patient. The patients associated to the first condition are different respect to the second one. The experiment consist of 24 patients, 12 of them who respond to one...342.62 314.02 295.18 228.01 244.49 86.41
And so on....
And my DESeq2 code:
ListRNAseqTimeSeries<-"raw_data_matrix_allSamples_RvsNR_ne…
updated 24 months ago • perceval.vellosillo
For each cell type I have 2 biological replicates - each represent a different pool of mice.
I first ran DESeq2 without setting the 'Mice' factor, meaning without pairing the 3 cell types of the same mice pool.
The colData...c("CellType", "type1", "type2"), alpha = 0.05)</pre>
Then, I have created a data set with the 'Mice' factor, creating the following colData:
<table border="0" ce…
updated 7.7 years ago • solgakar@bi.technion.ac.il
This is a follow-up to my post about DESeq2 with 2-factor design [here][1].
Nascent RNA-seq experiments analyzed by slamdunk report a Conversion Rate for each gene...This is a follow-up to my post about DESeq2 with 2-factor design [here][1].
Nascent RNA-seq experiments analyzed by slamdunk report a Conversion Rate for each gene, which is the ratio of Nascent Reads to Total Reads for a given…
updated 2.7 years ago • vanbelj
I got a list of paired end bam files, and want to do DESeq2 analysis. The first thing is to count the reads at gene level. I tried feature counts. However, it requires reorder of the...also takes very long time.
Is there any tools could quickly count the paired end reads for DESeq2 at gene level? Thanks
updated 8.6 years ago • tszn1984
a little bit confused about the count data transformations and
the Principal Component Analysis in DESeq2.
In the last vignette, the example on pages 18-19 shows a PCA plot of
the samples, obtained with regularized log transformed...no transformation is applied, axis 1 = pathology (patients
vs control cases) and axis 2 = unknown factor
- when transformed with r-log (rld), axis 1 = unknown f…
This is my code :-
library(DESeq2)
library(ggplot2)
countData <- read.csv('/home/keshav/Downloads/gene_count_matrix.csv', header = TRUE,sep = ",")
countData...lt;- countData[ , 1]
countData = as.matrix(countData[ , -1])
head(countData)
(condition <- factor(c("Normal","Tumor","Normal","Tumor")))
(coldata <- data.frame(row.names=colnames(countData), condition))
dds <- D…
updated 2.4 years ago • srikar
expression changes between two conditions over the course of three timepoints. I attempted to use DESeq2's LRT option in order to identify genes with condition-specific temporal expression changes, but only had one significant...lt;- read.csv(file.choose(), sep = "\t", row.names = "Samples") # select coldata.tsv
Time <- factor(coldata$Time, levels = c("3","7","14"))
Condition &am…
div class="preformatted">Dear all,
I will open the first line without opening the entire file because my
file is large?
thanks
ML
--
Mohamed Lajnef
INSERM Unit? 955.
40 rue de Mesly
updated 15.5 years ago • Mohamed Lajnef
Hi!!
I'm having trouble to do a contrast using DESeq2. I have 12 samples, AA(2 replicates), AB(4 replicates), BA(4 replicates) and BB(2 replicates). I wanted to compare AB (or BA) with...header = TRUE, row.names=1)
colData <- DataFrame(row.names=colnames(countData), condition=factor(c(rep("AA",2), rep("AB",4), rep("BA",4), rep("AA",2))))
dds <- DESeqDataSetFromMatrix(countData…
updated 7.2 years ago • zhuozhu132
Hi,
I am using DESeq2 to find Differentially expressed genes. I am using the following code:
<pre>
conds<-c("kd1","kd1","kd2","kd2","GFP","GFP")
Design<...the last line, I get the following:
<span style="background-color:Yellow">using pre-existing size factors
estimating dispersions
found already estimated dispersions, replacing these
gene-wise dispersion…
updated 8.2 years ago • ta_awwad
2]. All preprocessing steps are copied from the official guide.
Result: log2Foldchange from DESeq2 without fold change shrinkage is not identical to what is calculated from normalized count matrix. And sign inversion...read.csv(pasAnno, row.names=1)
coldata <- coldata[,c("condition","type")]
coldata$condition <- factor(coldata$condition)
coldata$type <- factor(coldata$type)
…
updated 3.9 years ago • Yanling
TIN) has been developed in part to address this problem and one of the ways they did this was to first calculate a median TIN value for each sample and then correct the gene read counts based on this. In the paper they regressed...way/if it is even possible to implement this sort of normalization in a Salmon -> tximport > DESeq2 pipeline.
I guess my main question would be, on wha…
Hi,
I'm using DESeq2 to analyse some RNA-seq data. I have different treatments (XXX, YYY and ZZZ) at different time points (2h, 4h, 8h), with DMSO...Hi,
I'm using DESeq2 to analyse some RNA-seq data. I have different treatments (XXX, YYY and ZZZ) at different time points (2h, 4h, 8h), with DMSO used as a control for the treatment. 'se' is my SummarizedExperiment object containing the raw counts…
updated 10.1 years ago • enricoferrero
in gene expression. Can someone offer some suggestions on how to approach this analysis using DESEQ2? For the drug vs placebo, would a design that includes the factors treatment, week, and patient be applicable and allow
updated 9.5 years ago • rvinisko
Hi everyone,
I have a question regarding how to correctly set up a design matrix for DESeq2 with high heterogeneity between study subjects inside a group.
So we are looking at data from two different groups...Hi everyone,
I have a question regarding how to correctly set up a design matrix for DESeq2 with high heterogeneity between study subjects inside a group.
So we are looking a…
updated 20 months ago • a.wag
show(countdata)
countdata <- as.matrix(countdata)
head(countdata)
condition <- factor(c(rep("x", 2), rep("y", 2),rep("z", 2),rep("t", 2)))
show(condition)
library(DESeq2)
coldata <- data.frame(skip=colnames(countdata), condition
updated 5.4 years ago • trumbia
My dataset from a microbiome study consists of two factors, one with two levels and the other with three. I am interested in both the fixed effects and the interaction. The first...factor is fire with levels burnt and non-burnt. The second factor is depth with levels 0-5, 35-40 and 95-100.
I used the phyloseq wrapper...phyloseq_to_deseq2` to convert my phyloseq object to one compatible with D…
updated 4.3 years ago • Aditya Bandla
Hello,
I have rnaseq data generated from brain tissues of 100 individuals, half of them were affected by a disease. All the samples were mix of two different brain regions, obtained from 4 different providers. I am trying to run DESeq2 to identify
differentially expressed genes using the model like: ~age + sex + tissue + PMI + condition, where PMI is post-mortem interval. My colData…
updated 7.9 years ago • fizer
Hello I'm having issues generating a Manhattan plot in Deseq2 with the following script . Every time I run the script no Manhattan plot is generated despite using the following...head(countdata)
# Assign condition (affected versus unaffected)
condition <- factor(c("affected","affected","affected","unaffected","unaffected","unaffected"),levels=c("affected","unaffected…
Dear Bioconductor/DESeq2 community,
I am trying to analyze RNAseq data with DESseq2 and have a question about contrasts and interactions. I...packages/devel/bioc/vignettes/tximport/inst/doc/tximport.html). Initially, I was performing DESeq2 analysis at each time point separately. For example, here is the sample file for testing for differential expression...5,infected.2hr,2<br/>
6,infecte…
updated 6.4 years ago • Amit Indap
Greetings!
I have a dataset comparing the effects of two different treatments on cell line line, including both control and combination treatment samples:
- Vehicle
- Treatment A
- Treatment B
- Treatment A + B
One of my goals is to determine which genes _uniquely_ respond to each of the treatments, e.g. `A & !B & !AB`.
A simple approach to this is to perform the in…
updated 5.0 years ago • Keith Hughitt
Suppose I have run deseq like this:
```
my_coldata$varA = factor(mycoldata$varA, levels=c('control', 'case')
my_coldata$varB = factor(mycoldata$varB, levels=c(0,1))
dds = DESeqDataSetFromMatrix...Suppose I have run deseq like this:
```
my_coldata$varA = factor(mycoldata$varA, levels=c('control', 'case')
my_coldata$varB = factor(mycoldata$varB, levels=c(0,1))
dds = DESeqDataSetFr…
div class="preformatted">Hi ,
I am using Deseq2 for differential gene expression calculations. I
have two conditions Condition a (T) and Condition b (P),
when I run the...lt;- read.delim ("48_96_filtered.txt", header=TRUE,
row.names=1)
> pdata = data.frame(condition = factor(c( "T", "T", "T", "P", "P",
"P", "P")))
> library (DESeq2)
> dds <- DESeqDataSetFromMatrix(c…
We used DESeq2 a lot at our research projects. Recently I was asked about specific experimental design for DESeq2.
We have 10 subjects...surgery, and 3 follow up time points.
All 10 subjects were collected at the pre, during, and the first follow up.
Only 7 were collect at the 2nd follow up and 6 were collected at the 3rd follow up.
This is a question of paired
updated 6.2 years ago • shengqh
Hi,
I am new to using DESeq2 and would appreciate some advice for a somewhat complex experimental design (for a non-statistician anyway). I have...a dataset with 3 factor variables, each with 2 levels, as follows:
trt: control, treatment
geno: A, B
time: 6h, 12h (no time 0h)
Each combination of factor
updated 7.1 years ago • red_bricks
div class="preformatted">hi Marine,
Note that you are using an older version of DESeq2 and Bioconductor
(DESeq2 v1.4 was released in April 2014). So if you are searching by
Google for documentation, it might...not apply to your software (we
print the version number on the first page of the vignettes). You can
always get the correct documentation by using:
browseVignettes("DESeq2") and ?result…
which comes to me when I compare the output of these 2 approaches to (in appearance) do the same in DESeq2:
* First way:
AvsB <- results(dge, __alpha = 0.05, lfcThreshold = 1__, contrast = c("cond","A","B"))
AvsB\_tb <- AvsB %>%
data.frame() %>
updated 6.5 years ago • pablo_garcia05
Hi,
I'm trying to run Deseq2 analysis (R-4.1.1, Deseq2 1.31.16) on RNA Seq data. We did targeted RNA Seq on 10 genes for expression data (only 10 amplicons...cases and finally the 2^-(ΔΔCt).
I would like to get the equivalent for my RNAseq data.
I tryied Deseq2 with tutorials but I only obtained log2fold change by genes... I don't know how to obtain fold changes by cases.
Important...informati…
manged to successfully complete some basic analysis of my RNAseq data. I now wanted to do some multi factor analysis but wasn't sure where to stat. Here is the layout of my samples:
RA - resistant, 0 dpi
RB - resistant, 1dpi
RC - resistant
updated 3.5 years ago • pthom010
Hello,
I find myself with RNA-seq data where we want to look at differential gene over time. I've read DESeq2 vignette and performed LRT test just as propose.
My question is about my data set.
I have 6 patients from which I have 3...I find myself with RNA-seq data where we want to look at differential gene over time. I've read DESeq2 vignette and performed LRT test just as propose.
My que…
updated 4.2 years ago • andree-anne
as compared to "t0", most genes will be under-expressed in "t4". Yet, as far as I read, EdgeR and Deseq2 default normalization of read-counts expect symmetrical under/over expression.
attached a diagnostic histogram...mean of all samples. I am suspecting, though cannot know for sure, that the true scaling factor is different (e.g., possibly red line should be shifted even more to the r…
contaminating all of our other cell types, which is why we did this decontamination step in the first place. However, I am now trying to run deseq2 to find DEGs for each cell type. When I use the regular count matrices, I get TONS...se, design = design, ignoreRank) :
some values in assay are not integers"
I know that Deseq2 is supposed to use raw counts, but what if raw counts are biologicall…
Dear ALL.
i would like to perform some __transcription factor binding site enrichment analysis(/promoter analysis)__ but without sequences but rather geneIDs from 2 expression...Dear ALL.
i would like to perform some __transcription factor binding site enrichment analysis(/promoter analysis)__ but without sequences but rather geneIDs from 2 expression microarray datasets. Is t…
updated 8.0 years ago • svlachavas
Hello,
I am trying to import transcript abundances (from kallisto .tsv files) to analyze with DESeq2.
My goal is to do a gene-level and a transcript-level analysis of differential expression. In my kallisto files however...does not allow me to directly compare the gene-level with the transcript-level analysis performed in DESeq2 (since the gene-level approach will only use the transcripts with …
Hi good day,
This is my first time working on differential gene expression analysis using DeSeq2. I am having a problem to construct my model matrix...for DeSeq2 since I have a very complex experimental design (3 factors design) with the following information;
ID Ewed Lambd Sex...Maternal diet
Lambd: offspring diet
Could anyone suggest me what can or should I do? I read the DeSeq2 vigen…
updated 5.8 years ago • sharmila.ahmad
First, my apologies if this has been covered already. I thought for sure it would have been, but I can't find the relevant info with...my searches.
It's pretty obvious from the DESeq2 vignette how to test whether a gene is differentially expressed (DE) and how to do so at various LFC thresholds, but what...power to make a determination, how do I do that? Is there something more sophisticated …
Recent ...
Replies
Comment: lfcShrink and alpha value
by
cropero
•
0
Thank you for your fast answer, I have everything clear now
Answer: DESeq2 - Comparing Comparisons
by
James W. MacDonald
67k
Assuming that WC in your equation #2 is different in KD1 subunit1_vs_WC and SCR subunit1_vs_WC, this is just an interaction, and the easies…
Comment: Differential Expression Genes as Independent Variables
by
James W. MacDonald
67k
An alternative to that is to use the `glmnet` package to fit a regularized regression using all proteins at once.
Comment: Differential Expression Genes as Independent Variables
by
abadgerw
•
0
Thanks. The proposal was not to include all proteins as covariates in one model but to iterate with one protein serving as the independent …
Answer: How to annotate a limma Elist raw class object with an annotation file
by
James W. MacDonald
67k
Your first step when trying to use a function is to read the help page (`?mapIds`), which will bring this up:
```
Usage:
colu…
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