Questions tagged [deseq2]

Deseq2 is an R package for analyzing RNA sequencing data

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1answer
204 views

DESeqDataSetFromTximport all(lengths > 0) is not TRUE

I am suddenly running into an error when running the DESeqDataSetFromTximport ...
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1answer
47 views

Why use “robust” FPKMs?

Both DESeq2 and edgeR have an FPKM/RPKM function that by default uses normalized library sizes ("robust" option in DESeq2). FPKMs have their own issues, but I thought the main benefit was to ...
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1answer
55 views

Is there an established method for comparing expression of groups of genes (gene sets)?

I have a figure in a paper where I show the log2(fold-change) (obtained with DESeq2) between two groups (based on genotype) for genes within a specific hallmark gene set, and a reviewer is asking ...
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3answers
130 views

RNAseq biological replicates not clustering in PCA plots

I have RNAseq data from 4 samples with 3 biological replicates per sample. I am currently trying to do the differential expression analysis with DESeq2 but the biological replicates will not cluster ...
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1answer
42 views

Setting Contrast for DESeq2 results

This question was also asked on BioStars I am trying to recreate this heatmap. How would I compare the four variables: Ly49+/- and MOG/MOGSP to get a single heatmap? Thank you for helping me through ...
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1answer
46 views

Details of DESeq2 modeling a batch effect

When correcting my data for a batch effect using removeBatchEffect, some of the gene expression values become negative. When searching for differentially expressed genes, I do not use the data above, ...
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1answer
38 views

the variation between treatments is less than the variation between replicates in RNA-seq data

I have a set of RNA-seq samples from targeting different proteins in a complex with siRNAs. However, the ...
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1answer
130 views

RNASeq: Normalization, stabilization, gene length and rlog

I was thinking about the best method for normalization, which takes gene length into account (in order to compare genes)... Do you think I can do that? : - taking raw counts and dividing each gene by ...
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1answer
52 views

Within and between sample count normalization

recently I came across a situation where RNASeq sample quality was expresses trough DESeq sizeFactor. So authors reported quality of their samples with respect to some publicly available datasets as ...
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52 views

Differential Gene Expression with Replicates for some of the samples

[this question has also been posted on Biostars; some additional clarification from there has been copied into this question] I've been asked to analyse a set of samples in which their control sample ...
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34 views

a proper Design Matrix for several drug treatments with both control negative and control positive

I have a dataset of RNA-seq samples for testing different drugs on the presence of another drug. One of my samples is the normal cells with no drugs (control negative) and another is the cells with ...
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1answer
70 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 ...
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1answer
31 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 "...
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79 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 ...
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1answer
86 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 ...
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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 ...
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34 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 ...
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0answers
33 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) ...
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1answer
28 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 ...
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2answers
253 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: ...
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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 ...
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2answers
34 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 ...
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1answer
111 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 ...
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1answer
47 views

How are these definitions related to differential expression?

I have two groups of patients; for each patient I have an output file (RNA-seq) contains this information ...
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2answers
938 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 ...
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1answer
169 views

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

I have two matrices like this: ...
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1answer
30 views

Comparing factor levels in deseq2

I have sample information (sampleinfo) in this format: ...
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1answer
87 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 , ...
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2answers
234 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 ...
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1answer
386 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. ...
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1answer
98 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,...
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1answer
137 views

Correlate DEGs from DESeq2, EdgeR and Limma results

I have a lists of DEGs identified by DESeq2, EdgeR and Limma. I would like to correlate the the gene rankings in the lists to decide on a package to use in downstream analysis. I am havig a few ...
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1answer
54 views

Single sample in group: normal pipeline or Kal's Z test

As stated, which one is better for differential expression analysis? When I say normal pipeline I mean limma-voom, edgeR and DESeq2 pipeline for standard analysis. Kal's z test is mentioned in this ...
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1answer
77 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. ...
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1answer
278 views

Why does DESeq2 convert numeric columns to factor during differential expression analysis?

I'm attempting to perform differential expression analysis using DESeq2 on a dataset of ~90 samples and ~20000 transcripts. I have a number of variables I'd like to test against, some of which are ...
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1answer
96 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 ...
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547 views

DESeqDataSetFromTximport invalid rownames length

I am trying to use DESeqDataSetFromTximport function from DESeq2 package to construct dds ...
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0answers
278 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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1answer
250 views

Differential expression analysis when nested effects

I have 3 tumour samples from 3 patients from an experiment, I also downloaded 10 normal samples from TCGA. My design is like this ...
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1answer
38 views

Finding genes especific to microenvironment

I have RNA-seq .bam files for 3 patients, tumour and its matched derived model, namely organoid, but I don't have any matched normal sample. Differentially expressed genes between a tumour and its ...
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2answers
1k 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. ...
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59 views

Making a model of gene contribution in cancer diagnosis

I have raw read counts (Edgeseq technology) of 56 patients who have been ranked with Mandard score as responders and non-responders; I have done differential expression by DESeq2 and I obtained a ...
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1answer
71 views

Experimental Design for Differential expreression analysis

I have a Normal esophageal Fibroblasts (NOFs) cultured in DMEM media; The same NOF also have been cultured with a tumor sample from a patient named 005 on DMEM media; I have also Cancer Associated ...
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1answer
109 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 ...
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2answers
434 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 ...
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1answer
724 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 ...
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2answers
527 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 ...
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1answer
304 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 ...
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1answer
595 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 ...
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1answer
100 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 ...