Questions tagged [deseq2]

Deseq2 is an R package for analyzing RNA sequencing data

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How does DESeq2 "collapseReplicates" work on read counts data?

Comparing read counts from an RNA-seq experiment for a couple select genes before and after using DESeq2's collapseReplicates function yields interesting results: ...
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Batch Effects that are Close to Collinear with Main Effect

Two years ago, I started working on a project uses both RNAseq and ATACseq. It's supposed to be a simple Healthy Control (HC) vs disease study. The sample collection was done in 2 phases, and it was ...
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How to remedy a DESeq2 collapsing technical replicates error?

Goal: To ensure "the sum of the counts for [my samples] is the same as the counts in the [samples] columns in ddsColl" after collapsing replicates using ...
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1answer
22 views

Use of "baseMean" in DeSEQ2

The basemean is described as the "mean of normalized counts of all samples". My question is, how would we interpret differences in basemean between genes, pertaining to reliability and such? ...
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1answer
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Visualizing top expressed genes

I have a spreadsheet of CPM values I want to visualize the top 20 genes expressed across my samples This is how my data look like ...
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1answer
84 views

Best R program to use for RNA seq analysis

We have an RNA seq taken from a the same tissue in two different developmental stages. We have one gene, with the number of reads from stage A and stage B, and we want to see which genes are ...
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35 views

The correct design for time series experiment

I have a treatment and control in two time points like this ...
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18 views

How to simulate replicates for DGE analysis?

I am prototyping with data visualization of DGE results, and I would like to work on the analysis pipeline before the real data is available. Currently, I only have 3 samples for wild type and 1 ...
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81 views

Help with performing PCA analysis on data from 3 different datasets of normalized counts data for RNA-seq experiment

So, I have limited knowledge of R but I need to do a PCA analysis of 3 different datasets of gene expression as a result of combined growth or mono-culture growth. The 3 different datasets I performed ...
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1answer
31 views

Understanding the different designs in DESeq2

I am using DEseq2 and trying to understand the results obtained using different models. I have a data design with 2 genotypes and 2 time points. ...
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94 views

Different contrasts in DESeq2

I have a treatment and control in two time points like this ...
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74 views

DEseq2 contrast in two time points

I need help with some clarifications please I have control versus treatment in two time points like ...
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28 views

Differential miRNA expression using RPM

I have microRNA (miRNA) expression data in RPM. I would like to do differential gene expression between two groups. How can I do this? I have considered edgeR and DESeq2 in R, but it looks like they ...
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1answer
273 views

Adjusted P-value from DESeq2

What does the padj really mean? I believe it's a BH method, where it attempts to quantify the likelihood of a "false positive" but I am not sure. Also, what does it mean if the resulting ...
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1answer
54 views

DESEQ2 Question about results()

I am currently learning to perform Differential Analysis via DESEQ2 R Package, and I believe I've made progress, able to format the data correctly [maybe] for DDS(). When I run the results function to ...
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Beginner with DESeq2 having issue with analysis

Hi there I am brand new to using RStudio and trying to run DESeq2 analysis on my featureCounts output counts table. I ran the following code: ...
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1answer
60 views

DESeq2. Which is better: using the altHypothesis argument in the results function or manually filtering for your desired P-value and Log2FoldChange?

In DESeq2 you can identify your differentially expressed (DE) genes using the results function. I noticed people in my lab using the results() function with the minimum number of arguments supplied (...
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1answer
60 views

How does FDRtool work?

I have a question about using FDRtool. In the below code (on RNA seq data whose p values were acquired using Deseq2), the FDRtool was first used and thereafter p.adjust using the benjamini hochberg ...
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186 views

Calculating most abundant transcript from RNA-Seq data

vcf2maf uses VEP to annotate variants, and I believe selects the default Ensembl transcript to use for annotation. Sometimes the ...
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2answers
163 views

DESeq2 multiple treatments, multiple time points, multiple cell lines

Note: this question has also been asked on Bioconductor Support I know there are a lot of questions asking similar things here on this forum and I checked the vingette and did a lot of other research ...
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2answers
127 views

Un-normalize DESeq2 counts

I obtained a matrix of RNA-seq count data that has been normalized by DESeq2's median of ratio method. I know that DESeq2 wants to take in un-normalized counts, but I do not have access to those data. ...
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2answers
37 views

Bulk RNA-Seq Read Length Normalization across different samples

I have 20 samples out of which 14 are 100 bp in length and 6 are 150 bp. Is there a way to normalize the read length for cross-sample differential expression comparison? What would be the best way to ...
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1answer
28 views

Differential expression analysis for a subset of transcripts

I want to check the differential expression of a specific class of transcripts (say, long non-coding RNAs) using DESeq2. Now, I know that the normalization step takes into account the total number of ...
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Adding matrix as a confounder in EnrichmentBrowser

Brain data, particualrly human brain data have very heterogeneus cell types, so it's important to normalise by cell type/add proportoins of different cell types to the formula when performing ...
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the model matrix is not full rank : This is a classic question which a biologist face without clear understanding of the model design

Saw this answer biostar. Tried to make my metadata as such but still the "Error in checkFullRank(modelMatrix) : " This is my coldata ...
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146 views

What is a sensible number of gene/observations to explain PCA variance?

I am working with a set of RNASeq dataset. I have about 4000 observations (genes) on 20 samples and plotting a PCA I found the clustering doesn't vary much when I use different number of genes, but ...
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107 views

Question of Padj value (very high, infinite) got from dds

After I got dds from my count matrix by DESeq(), I use results() method to get Padj and log2 fold changes for volcano plotting, then I found some gene's Padj values are infinite and the P-value is ...
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73 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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74 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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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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88 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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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
75 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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131 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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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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663 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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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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741 views

DESeqDataSetFromTximport all(lengths > 0) is not TRUE

I am suddenly running into an error when running the DESeqDataSetFromTximport ...
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1answer
45 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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2answers
341 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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298 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
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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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2answers
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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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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
70 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
38 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
215 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
54 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
2k 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
37 views

Comparing factor levels in deseq2

I have sample information (sampleinfo) in this format: ...