Questions tagged [deseq2]

Deseq2 is an R package for analyzing RNA sequencing data

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16
votes
2answers
5k views

Understanding DESeq2 design, contrast and results

I have a set of high-troughput experiments with 2 genotypes ("WT" and "prg1") and 3 treatments ("RT", "HS30" and "HS30RT120"), and there are 2 ...
13
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2answers
3k views

How can I extract normalized read count values from DESeq2 results?

The results obtained by running the results command from DESeq2 contain a "baseMean" column, which I assume is the mean across samples of the normalized counts for ...
10
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2answers
542 views

Can I model technical replicates in DESeq2?

I’d normally use collapseReplicates (or do the collapsing upstream) to handle technical replicates. However, in my current RNA-seq experimental design, samples ...
10
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2answers
613 views

*very* unbalanced group sizes for DE

I downloaded some publicly available RNA-seq data and want to compare those samples carrying a mutation (~4) against the rest (~800!). I ran both EdgeR and DESeq2, and the first results in an ...
7
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2answers
416 views

differential gene expression complex design no replicates

We have an experimental design as seen below Where we administered drug at 0 min for each mouse genotype and took them down at given below intervals. wt and ko mouse models were administered only ...
7
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1answer
745 views

Trouble using biomaRt to retrieve hgnc symbols from Ensembl transcript ids

I have a matrix of gene counts which I'm going to use as input for DESeq. Right now, each gene is labeled by its Ensemble transcript ID, but I'd like to convert these to their HGNC symbols before I ...
6
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2answers
689 views

Running differential expression analyses on count matrices with many zeroes

Note: I also posted this issue (with less context) in the bioconductor support site: https://support.bioconductor.org/p/97424/ I'm working on a snakemake workflow that identifies various small RNA ...
5
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1answer
318 views

What are the count units in DEseq2?

This is a pretty silly/simple question, I suppose. What units are DESeq2 counts? Or are the units arbitrary but internally consistent estimate of actual reads?
5
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1answer
583 views

What are the ways to process a list of differentially expressed genes?

We are studying six different human macrophage/dendritic cell types isolated from healthy skin. They all differ from each other in a few cell surface markers. We are interested in the characteristics ...
5
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1answer
69 views

design formula question

I am not sure I am building the proper design formula for the question I want to answer I have the following samples with three factors; clone, the structure and the condition. ...
5
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2answers
477 views

Is it possible to create a DESeqDataSet with a user-provided design matrix?

I'm trying to run a differential gene expression analysis using DESeq2, with counts coming from kallisto. I have imported them using tximport and I'm creating the ...
5
votes
1answer
153 views

How can I specify to DEseq2 to only perform comparisons on which I am interested with?

I am currently performing a large RNA-seq analysis from mice PBMCs. The dataset contains around 6,000 transcriptomic profiles and I would like to use DESeq2 to identify the sets of differentially ...
4
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1answer
100 views

LRT or LRT-like test on cyclical (Sleep) data

I have RNA-Seq data from 4 time points (3 hours awake, 9 hours awake, 3 hours asleep, 9 hours asleep). I'm interested in doing something similar to a LRT where genes are found to be significant if ...
4
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1answer
57 views

How to design DESeq2 LRT model with individuals nested in 2 levels?

We have a complicated experimental design that we would like to perform LRT analysis for. Our main goal is to discover significant genes for the "Injection:Social" interaction term across ...
4
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1answer
95 views

plotMA function issue: can not circle genes

I am trying to make MA-plot for my bulk-RNA-seq dataset and experiencing issues with it. I just copy-pasted the code that can be ...
3
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2answers
1k views

gene-level versus transcript-level analysis

Traditionally, RNA-seq data was quantified on gene level. Newer methods quantify on transcript/isoform level. For example, Kallisto only outputs transcript-level abundances. From the DESeq2 vignette: ...
3
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2answers
209 views

PCA plot shows big difference but not many differentially expressed genes are found

I got a PCA plot of bulk RNA-seq experiment that looks the following way: It was generated by the following code: ...
3
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2answers
888 views

Installing DESeq2 in Ubuntu

I am trying to install DESeq2 in my Ubuntu with R version 3.5.1. According to the package repository in Bioconductor the version should be 3.5. ...
3
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2answers
509 views

DESeqDataSetFromTximport invalid rownames length

I am trying to use DESeqDataSetFromTximport function from DESeq2 package to construct dds ...
3
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1answer
167 views

Removing genes with less than a correlation cut-off between two matrices

I have two matrices like this: ...
2
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3answers
1k views

Install DESeq2 through anaconda

I tried running conda install -c bioconda bioconductor-deseq2 in a conda environment, but when I run ...
2
votes
1answer
353 views

Salmon tximport

I ran bulk RNA-seq experiment and got quant.sf file. Now, I am struggling with understanding what ...
2
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3answers
186 views

Selection of differential expressed genes

I'm working with RNA-seq data. I have 40 tumor samples and 5 Normal samples. Differential analysis with Deseq2 based on Fold change > 1.2 and alpha < 0.05 gave ...
2
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1answer
1k views

Error in checkFullRank(modelMatrix) deseq2

Trying to use deseq2 for differential expression analysis (rna-seq) between three groups and also account for batch effect as the control were sequenced at a different time point. control: con ...
2
votes
2answers
32 views

Is there a computational tool or possibility to identify mRNA isoforms from the count matrix of a bulk RNA sequencing dataset?

I have the counts matrix of an RNA sequencing dataset of fibroblasts and I wish to identify isoforms of a particular gene of interest in it. Can anyone please hint me on a bioinformatics method to ...
2
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2answers
39 views

Can I ask DESeq2 which variable alone explains which gene's behavior the best?

Imagine I have a dataset of highly periodic gene expression, taken at densely spaced timepoints. I know that the expression is periodic, but different genes have different offsets. It is trivial to ...
2
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2answers
559 views

STAR quantMode vs RSEM vs Kallisto

I recently discovered this Snakemake pipeline for RNASeq that uses STAR's quantMode to quantify gene expression for DESeq2 differential expression analysis. In the past I've always seen the workflow ...
2
votes
1answer
90 views

How to get log2 fold change of RNA-Seq data for time series experiment?

I know if there is one control and one treatment group it is pretty straight forward to interpret log 2 fold change. But, I have time course experiment. I have infected cells with viruses and I ...
2
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1answer
648 views

How to get the corrected matrix after SVA batch effect correction

I ran SVA to remove batch effects for my bulk RNAseq experiments, but now I need to somehow correct my data matrix in order to ...
2
votes
1answer
172 views

Why do I obtain very strange results with DESeq2?

I am using DESeq2 to perform a differential expression analysis, but I obtained very strange results for some genes. For some genes, I have very high log2FoldChange with very low p-value as displayed ...
2
votes
1answer
52 views

Should biological replicates be the most similar pairs in an RNAseq experiment?

The attached figure (from deseq2) shows the sample to sample distances of a RNAseq experiment with 4 conditions (A,B,C,D) at 2 time points (0h,4h) with 2 biological replicates. I am a little bit ...
2
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1answer
276 views

How to input data for DESeq2 from individual HTSeq count?

I am comparing the gene expression of 2 bacteria under 1 condition. I have now the count tables for 3 tech. replicates for each bacteria. ...
2
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0answers
30 views

vst() from DESeq2 vs voom() from limma

I have used both to transform my leukaemia RNA-Seq data for subsequent hierarchical clustering. The result is quite different. Some subtypes of leukaemia only form a cluster (or at least sit closer) ...
1
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2answers
32 views

Identify differentially covered genes only between two samples

I have a question about finding differentially covered regions (coverage represents methylation level which goes from 0 to several thousands). I'm using enrichment based method which can be summarized ...
1
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2answers
360 views

levels of factors in the design have non-unique level names after make.names() is applied

When I try to import the data using DESeq2 package: dds <- DESeqDataSetFromTximport(txi, sampleTable, ~Group + Cre) I am ...
1
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1answer
91 views

Comparing RNA-Seq and Ribo-Seq

I'm attempting to compare total RNA-seq with Ribo-Seq, to determine if changes in Ribo-Seq are due to changes in transcriptional expression (akin to the analysis performed by Anota2Seq). I am, however,...
1
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1answer
508 views

DESeq2 complicated design - effect of replicated samples

I have RNAseq data from a relatively complicated experimental design with variables = genotype, treatment, time, and batch. I have 2 biological replicates for each genotype/condition, however ...
1
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1answer
25 views

DESeq2 compare within a condition

This might be a really stupid question, but I can't figure out how to do this. I've read through the DESeq2 vignette and manual pages but couldn't find an answer. I have a bunch of samples split up ...
1
vote
1answer
64 views

How do I differentiate outliers from in-group variations in a DESeq2 PCA plot of 9 samples distributed into 3 conditions?

I have a PCA plot from DESeq2's plotPCA(vsd, intgroup=c("conditions")) function. I have 9 samples distributed in to 3 groups of 3 biological replicates each. My ...
1
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1answer
285 views

Compare clusters from different datasets

I have two scRNAseq datasets, and I select a cluster from one of them, and another cluster from the other. I want to find differentially expressed genes between the ...
1
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1answer
84 views

Negative binomial modeling of RNA-Seq data

A common way to model RNA-seq data is using a negative binomial distribution, where each sample-gene pair is modeled by a different negative binomial distribution with mean $\mu_{ij}$ where $i$ and $j$...
1
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1answer
28 views

Is it conflicting: significant difference according to Wald test but not in PERMANOVA and ANOSIM to compare species abundances of metagenomic samples?

If there is no significant difference between two groups of metagenomic samples according to "Multivariate differential abundance tests" for example PERMANOVA and ANOSIM, does it makes sense to use "...
1
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0answers
234 views

Is it advisable to remove X and Y chromosome genes in a bulk RNA-seq dataset at the level of the count matrix?

From this link remove X and Y chromosome genes in RNA-seq data using DESeq2 pipeline, I have learned that depending on context, it is perfectly valid to remove X and Y chromosomal genes in an RNA-seq ...
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0answers
454 views

Likelihood Ratio Test in DEseq2

I have a RNA seq data which I am trying to identify DEGs. Dataset contains: Untreated - 2 replicates (time point 1st day) Treated - 4 replicates (2 replicates at 3rd day & 2 replicates at 7th ...
0
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2answers
48 views

Ranking metric in GSEA

I am using fgsea in r to calculate and plot a bunch of GSEA graphs. My question is whether it is acceptable to use the Wald statistic from DeSeq2 to rank the gene list? I have seen in the GSEA ...
0
votes
1answer
42 views

How to incorportate RIN values as covariate in the design matrix?

I have been following the last DESeq2 pipeline to perform an RNAseq analysis with a dataset with low rin samples in the experimental (or treated) and high rin on the control ones. I read a paper in ...
0
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2answers
182 views

Interpreting this PCA plot for RNA-seq

I have RNA-seq from two sequencing batches; Lab technician says that he has run the RNA expression quantification two times in bathes 1 and 2 for example ...
0
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1answer
975 views

Highly variable genes analysis by DESeq2

In the following article. There are two ways of getting highly variable genes: ...
0
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1answer
46 views

DeSeqDataSet experimental design: column with integer values

I am creating DeSeqDataSet and I am unsure whether I need to include the number of mice into the design formula. Basically, the samples look like that: I have 4 ...
0
votes
1answer
85 views

How I deal with this kind of gene expression comparision

I have RNA-seq from two sequencing batches; Lab technician says that he has run the RNA expression quantification two times in bathes 1 and 2 for example tumor 1 in batch 1 and tumor 1 in batch 2 , ...