Tag: deseq2

Deseq2 Output Explained With Code Examples

Deseq2 Output Explained With Code Examples In this session, we are going to try to solve the Deseq2 Output Explained puzzle by using the computer language. The code that follows serves as an illustration of this point. Column Description 1 Gene Identifiers 2 mean normalised counts, averaged over all samples…

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DESeq2. Multi-factor Interactions designs

DESeq2. Multi-factor Interactions designs 0 Dear all, I want to get your feedback, please !! I have gene expression data from two tissue(Tissue_1 and Tissue_2) in three conditions(A, B and C). I want to get genes upregulated in Tissue_2 in condition C vs A+B. I was trying this, but not…

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Same results in Limma Toptable as in DESEQ2 results

Same results in Limma Toptable as in DESEQ2 results 1 Dear all, I am trying to generate exactly the same results in Limma for my result table as I did in DESEQ2 (Differential Expression Analysis). With the toptable function I am not getting lfcSE and basemean. Can anyone help me…

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Multiple conditions DESeq2

Multiple conditions DESeq2 1 Hi, I am working on differentially expressed genes analysis of RNA-seq data extracted from multiple but related experiments: experiment DESH21_E DESH40_S DESN21_E DESN21_S DESN40_E DESN40_S ES21_E ES21_S ES40_E ES40_S where, DES and ES are different in vitro cells; 21/40 are concentration (low/high), and H/N are additional…

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deseq2 – Creating multiple phenotype datasets using bootstrap method “Bootstrap-samples-by-column-of-a-data-frame-in-r” for DEG analysis

I am working on a datasets and after some discussion with my group, we doubt that maybe one or more of our controls are different than the other controls. The motivation is to see if one or more controls have been effected differently by the solvent they were kept in….

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Exporting DESeq2 results to CSV files

Exporting DESeq2 results to CSV files 1 Hello there. I need a help with this situation. I’m following this vignette and I’m getting an error on the item “Exporting results to CSV files”: write.csv(as.data.frame(resOrdered), file=”condition_treated_results.csv”) In my case i get this: write.csv(as.data.frame(resOrdered), file = “myres_counts.csv”) error in evaluating the argument…

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Evolution of stickleback spines through independent cis-regulatory changes at HOXDB

Darwin, C. On the Origin of Species by Means of Natural Selection (John Murray, 1859). Owen, R. On the Archetype and Homologies of the Vertebrate Skeleton (Richard and John E. Taylor, 1848). Stern, D. L. & Orgogozo, V. Is genetic evolution predictable? Science 323, 746–751 (2009). CAS  PubMed  PubMed Central …

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Log2FC values slightly higher in some genes after DESeq2 shrinkage

Hi, I have a question about DESeq2 LFCshrinkage: Is it possible that some genes have a slightly higher LFC after shrinkage? It happened during my RNAseq DE analysis, I have very deeply sequenced samples with large base means. I tried visualizing using MAplot check, and it looks fine. I’m mainly…

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NODULIN HOMEOBOX is required for heterochromatin homeostasis in Arabidopsis

NDX is a heterochromatin-associated factor To understand the genome-wide regulatory roles of NDX, we first analyzed its genomic distribution. For this, we performed chromatin immunoprecipitation sequencing (ChIP-seq) in 10-day old Arabidopsis seedlings expressing N-terminally and C-terminally tagged NDX fusion proteins (flag-NDX/ndx1-1(FRI)/flc-2 and NDX-GFP/ndx1-1(FRI)/flc-2, respectively) expressed from their endogenous promoter21,32. The…

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Long non-coding RNA SNHG6 couples cholesterol sensing with mTORC1 activation in hepatocellular carcinoma

Chu, B. B. et al. Cholesterol transport through lysosome-peroxisome membrane contacts. Cell 161, 291–306 (2015). CAS  Article  Google Scholar  Luo, J., Yang, H. & Song, B. L. Mechanisms and regulation of cholesterol homeostasis. Nat. Rev. Mol. Cell Biol. 21, 225–245 (2020). CAS  Article  Google Scholar  Luo, J., Jiang, L., Yang,…

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Time course RNA seq with DESEq2

Hi all, Hope you are well. I am running time course experiment to examine differentially expressed genes from tumour tissues between two conditions, radiotherapy and sham-radiotherpapy groups at 4 time points post radiotherapy. Each time point had size matched untreated controls. I used the following code to run model with…

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TPM normalization starting with read counts

Hello everyone I have multiple bulk RNA-seq datasets that I need to apply the same pipe line on. I want to normalize them from counts data to TPM. In all datasets, I have the genes as rows, and samples as columns. Unfortunately, I don’t have the fastq files, all I…

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Feature selection for a binary classifier using mixed cancer type samples

Feature selection for a binary classifier using mixed cancer type samples 0 Hi all, I am not an expert in machine learning (ML) and have a few specific questions regarding the design of a binary classifier. I have bulk RNA-seq data for the samples from 6 different cancer types. These…

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Targeted inhibition of ubiquitin signaling reverses metabolic reprogramming and suppresses glioblastoma growth

Cell culture Human glioblastoma cells (U87MG and U87MG-Luc) and human embryonic kidney cells (HEK293) were obtained from the American Type Culture Collection (Manassas, Va.). Cells were cultured in Dulbecco’s Modified Eagle Medium supplemented with 10% fetal bovine serum (Gibco™ Fetal Bovine Serum South America, Thermo Scientific Fisher-US), 2 mM l-glutamine, 50 U/ml…

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I have a query regarding differential gene expression using limma-voom.

I have a query regarding differential gene expression using limma-voom. 1 @28946033 Last seen 1 day ago India I used the following pipeline for RNA Seq Analysis Fastq-Trimmomatic- Hisat2(gtf file was annotated)-featurecounts After featurecounts I tried to do limmavoom, but I get error saying this An error occurred with this…

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DESeq2 aggregated single cell data

Hi, Im aiming to use aggregated single cell data to perform a pseudobulk analysis to assess differential expression between those with sarcopenia and those without, termed “status_binary” with the levels “yes” and “no”. # DESeq2 —————————————————————— dds <- DESeqDataSetFromMatrix(y$counts, colData = y$samples, design = ~Sex+age_scaled+status_binary) # Transform counts for data…

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Batch effect correction in 2 conditions + 2 genotypes + interaction term design

Dear all, I am analyzing RNAseq data with DESeq2 for a dataset that resemble the type “2 conditions, 2 genotypes and interaction term”; specifically I have healthy donors and patients for both male and female population. I am interested in obtaining: 1- genes modulated in male patients as compared to…

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DESeq2 analysis has not been run for this contrast

DESeq2 analysis has not been run for this contrast 1 @e16bdcce Last seen 10 hours ago Hong Kong error :DESeq2 analysis has not been run for this contrast DB.DESeq2 = 0 I think until this step everything is ok here is my file↓ here is my code ↓ # dbObj_contrast…

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lncRNA analysis tutorial

lncRNA analysis tutorial 2 hello, I would like to ask if there is some good tutorial concerning analysis of lncRNA along RNAseq analysis? like practical example. thank you for your help. lncRNA RNA-seq • 458 views Generally speaking, doing differential expression (DE) or transcript discovery on lncRNA is no different…

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Seeking help in multifactor differential gene expression analysis using DESeq2. Can not get significance difference while comparing 3 time factor (0,2 and 4h) with 3 group (1 control, 2 treated)

Dear Experts, I have RNAseq data from 6 different plant samples (2 control, 2 Sensitive, and 2 tolerance), and different location of one species. I am trying to see the effect of the different groups at different time points, but after going through all the posts and vignettes I am…

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Hisat2 – stringtie – deseq2 pipeline for bulk RNA seq

Software official website : Hisat2: Manual | HISAT2 StringTie:StringTie article :Transcript-level expression analysis of RNA-seq experiments with HISAT, StringTie and Ballgown | Nature Protocols It is recommended to watch the nanny level tutorial : 1. RNA-seq : Hisat2+Stringtie+DESeq2 – Hengnuo Xinzhi 2. RNA-seq use hisat2、stringtie、DESeq2 analysis – Simple books Basic usage…

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Getting the best of RNA-Seq

Forum:Getting the best of RNA-Seq 0 This is not a banal discussion. I am facing some problems with the analysis of DE genes in mouse. Most methods of analysis of DE genes must face two considerations or challenges. The first needs to take into consideration the existence and the different…

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Difference in number of DEGs from Deseq2 and limma-voom

Difference in number of DEGs from Deseq2 and limma-voom 0 Hello, I have RNA-seq data from two different treatment groups (F and NF ) at 2 different time points (T1 and T2). The mapping was done with STAR aligner and the quantification was done with FeatureCounts. I run differential expression…

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Multiple disease condition vs Normal

DESeq2: Multiple disease condition vs Normal 0 Hello! I have an mRNA dataset with one cell type and 3 different conditions (Metastatic, Primary Tumor, and Solid Tissue Normal). I would like to compare the two diseased conditions with the normal. I am using the following code but getting the understated…

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Multifactor analysis

Multifactor analysis 1 @66dd6104 Last seen 1 day ago United States HI every one I am new to RNA seq analyisis using R. I Am usig DESeq 2 for my analysis. I have four different Samples with three replicates each and I want to compare all of them with control…

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Running DESeq2 from rpy2 – Askdevz

If you open R and type: you will see that the assay function is not actually coming from DESeq2, but from its dependency which is called SummarizedExperiment: > assay standardGeneric for “assay” defined from package “SummarizedExperiment” function (x, i, withDimnames = TRUE, …) standardGeneric(“assay”) <bytecode: 0x5586a354db90> <environment: 0x5586a3535e20> Methods may…

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rna seq – R – [DESeq2] – How use TMM normalized counts (from EdgeR) in inputs for DESeq2?

I have several RNAseq samples, from different experimental conditions. After sequencing, and alignment to reference genome, I merged the raw counts to get a dataframe that looks like this: > df_merge T0 DJ21 DJ24 DJ29 DJ32 Rec2 Rec6 Rec9 G10 421 200 350 288 284 198 314 165 G1000 17208…

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DESeq2 and WGCNA

DESeq2 and WGCNA 0 I am currently performing an RNAseq analysis with a dataset from a GeneAtlas where I’ve identified DEGs from different comparisons. I want to now do a co-expression analysis with these comparisons and was wondering if anybody had suggestions of tutorials I could be directed to. I…

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Deseq2 Multifactor Design – Design Forum

Deseq2 multifactor design – In fact, deseq2 can analyze any possible experimental design that can be expressed with fixed effects terms (multiple factors, designs with interactions, designs with continuous variables, splines, and so on are all possible). We have searched different posts in different forums and i can’t figure out…

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InteractiveComplexHeatmap on DESeq2 object with more than 2 groups

InteractiveComplexHeatmap on DESeq2 object with more than 2 groups 1 Hello all, I’m writing with the hope someone can clarify a doubt I have about the differential heatmap generated by the package InteractiveComplexHeatmap via the simple command interactivate(dds). I read the package documentation at bioconductor.org/packages/release/bioc/html/InteractiveComplexHeatmap.html, but I couldn’t find the…

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tReasure: R-based GUI package analyzing tRNA expression profiles from small RNA sequencing data | BMC Bioinformatics

tReasure (tRNA Expression Analysis Software Utilizing R for Easy use) is a graphical user interface (GUI) tool for the analysis of tRNA expression profiles from deep-sequencing data of small RNAs (small RNA-seq) using R packages. The whole analysis workflow, including the uploading of FASTQ files of small RNA-seq, quantification of…

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DESeq2 how to specify contrast to test difference of differences

I am trying to take the “difference of differences” in contrasts from two factors (sex and group). We have male and female animals (sex factor) that were untrained or trained for 1, 2, 4, or 8 weeks (group factor, i.e., “control”, “1w”, “2w”, “4w”, “8w”). I want to know the…

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Error in SummarizedExperiment

I have installed DESeq2 version 1.36.0 samples <- colnames(txi$counts) group <- as.factor(c(“control”,”control”,”control”,”control”,”control”,”diet”,”diet”,”diet”,”diet”,”diet”, “control”,”control”,”control”,”control”,”control”,”diet”,”diet”,”diet”,”diet”,”diet”,”diet”)) coldata <- data.frame(samples, group, stringsAsFactors = F) coldata <- coldata[,c(“samples”,”group”)] coldata$samples <- factor(coldata$samples) coldata$group <- factor(coldata$group) rownames(coldata) <- sub(“fb”, “”, rownames(coldata)) all(rownames(coldata$samples) %in% colnames(txi)) all(rownames(coldata) == colnames(txi)) TRUE library(DESeq2) ddsTxi <- DESeqDataSetFromTximport(txi, colData = coldata, design =…

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Genes looking differential abundant are not accoring to DESeq2

Genes looking differential abundant are not accoring to DESeq2 0 I have a metagenomic dataset crossing three time points from which I have mined CAZymes and am using DESeq2 to identify differentially abundant CAZymes from using trimmed mean depth generated my CoverM (very similar to Q2Q3 contig coverage). From this…

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Extremely different results for both EdgeR and DESeq2 analysis

Extremely different results for both EdgeR and DESeq2 analysis 1 @373f98d7 Last seen 23 hours ago Singapore Dear all, Upon comparing my results for the analysis between DESeq2 and EdgeR, I have realized that the 2 results obtained after DEG analysis are extremely different from each other. The thresholds I…

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RNA interference triggers that target SARS-CoV-2 genome

Coronavirus disease 2019 (COVID-19) vaccines have played a critical role in reducing transmission of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission. However, with emerging reports of waning vaccine efficacy, there remains an urgent need to develop prophylactic measures against COVID-19. In a recent study published on the bioRxiv*…

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Can Differential Isoform expression analysis can be performed using DESeq2 package

Can Differential Isoform expression analysis can be performed using DESeq2 package 0 @03ddb485 Last seen 9 hours ago India Hello, I am want to perform differential isoform expression (DIE) analysis for RNAseq data from human. Can I use DESeq2 for this by inputting the transcript level abundance and getting differential…

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Counts from recount3 for DESeq2 analysis

Hi, As the topic title suggests, I want to use RSE objects from recount3 for differential expression analysis with DESeq2. This is an ongoing project that I took over. Looking at what has already been done, there are two points I wonder about : 1 – I found that the…

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deseq2 problem

deseq2 problem 0 Hi I am trying to draw a PCA plot with DESeq2 but somehow I cannot use DESeq2 functions. It is a really simple code i wil be pasting below. > transform <- DESeq2::rlog(eliminated_data, blind = TRUE) Error in (function (classes, fdef, mtable) : unable to find an…

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scrnaseq – Normalization methods to combine scRNA-seq experiments with different sequencing depths

I don’t think you need to complicate the idea of normalisation by introducing machine learning classifiers as a necessary component. Normalisation is common when comparing different datasets for all differential analysis. If you have single cell data, have a look at integration techniques in the Seurat workflows: satijalab.org/seurat/articles/integration_rpca.html If you’ve…

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DESEQ2 Results log2fold and pvalue changes after removing genes

DESEQ2 Results log2fold and pvalue changes after removing genes 1 @bine-23912 Last seen 9 hours ago UK Dear all, we have been discussing internally but couldnt find an answer, maybe you could help us. The following happened: I run my DESEQ2 analysis with the full dataset and got genes with…

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different results between DESeq model with multiple groups or with specific groups

different results between DESeq model with multiple groups or with specific groups 1 @b295d7f1 Last seen 3 hours ago Italy Hi! I am working with DESeq2 to perform a differential expression analysis between different treatments.I have 4 conditions and 4 four biological replicates for each conditions. I’ve performed differential expression…

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Tximport in usegalaxy

Tximport in usegalaxy 0 Devon Ryan: Please help in resolving this issue. How to use tximport in usegalaxy to convert transcript ID(DESEQ2-SALMON) to gene ID. I want to get gene ids from the results of deseq2(salmon) . Which GTF should be used for tximport. Iam getting the following error in…

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About outliers and non -separated samples in PCA

About outliers and non -separated samples in PCA 0 Hi all, I have plotted PCA for my samples(Tumor and Normal) in some cancer types. I have used the HTSeq-counts data from TCGA. Then I’ve normalized them by DESeq2 and the total normalized counts are in cnt dataframe. Head of cnt:…

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How to merge miRDeep2 counts for DeSeq2 analysis?

How to merge miRDeep2 counts for DeSeq2 analysis? 0 Hi, I am trying to identify DE miRNAs using small RNA-Seq data. I ran miRDeep2 tools mapper.pl an also miRDeep2.pl scripts and got counts for my cases. How can I merge count.cvs files of multiple cases for DE miRNA detection using…

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What is the relationship between the value of the bigwig file generated by MACS2 and the read count?

What is the relationship between the value of the bigwig file generated by MACS2 and the read count? 0 What is the relationship between the value of the bigwig file generated by MACS2 and the read count? When using Deseq2 or edgeR to identify the differential peak, because the value…

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Three Differential Expression Analysis Methods for RNA Sequencing: limma, EdgeR, DESeq2

A detailed protocol of differential expression analysis methods for RNA sequencing was provided: limma, EdgeR, DESeq2. Three differential expression analysis methods for RNA sequencing:limma, EdgeR, and DESeq2. Open the RStudio program and then load R file, DEGs. The file can be acquired from supplementary files.One. Downloading and pre-processing of data.1.1….

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Error in DESeq2

Error in DESeq2 1 @4bede5c6 Last seen 11 hours ago India Hi, All I am new to Rstudio , i Have to study expression data for the following But my DESeq2 data is having an error of Error in h(simpleError(msg, call)) : error in evaluating the argument ‘x’ in selecting…

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How to identify differential enhancers?

How to identify differential enhancers? 0 Deseq2 or edgeR only allow the input of reads count to identify differential regions, but I get less differential enhancer with reads count, can I use the signal from the bigwig file as input to deseq2 or edgeR to identify the differential enhancer? EdgeR…

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DESeq2 (or EdgeR) Exploratory Analysis with no Replicates

DESeq2 (or EdgeR) Exploratory Analysis with no Replicates 1 My pipeline so far is hisat2->featureCounts->DESeq2. I have generated heatmaps after rlog and log2 transformation of the genes with the most variance, which is somewhat meaningful. What I really want to do is compare everything to the control sample and take…

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Advice on gene expression method through heatmap

Advice on gene expression method through heatmap 0 Hello! I want to kindly get advice on a method I am using for showing gene expression on a heatmap. I have three groups: Negative Control (4 samples) Control with injury (4 samples) Treatment with injury (4 samples) I took the intersecting…

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deseq2 datasetfrommatrix

deseq2 datasetfrommatrix 1 @235c1561 Last seen 1 day ago United Kingdom countsTable <-read.csv(“counts.csv”, header = T, stringsAsFactors = FALSE) head(countsTable) sample_info <-read.table(“sample.txt”, header = T, stringsAsFactors = FALSE) #rownames(countsTable) <-countsTable[,27902] #countsTable <-countsTable[,1] class(sample_info) sample_info$Type <- as.factor(as.character(sample_info$Type)) library(DESeq2) rownames(countsTable) <-countsTable[,1] countsTable <-countsTable[,-1] (dds<-DESeqDataSetFromMatrix(countsTable[,c(1:128)],colData = sample_info,design = ~Type)) i get this error…

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DESeq2: Error in checkFullRank(modelMatrix) | Tech Programing

I want to build a DEseq2 model matrix using this coldata but it shows this error: Error in checkFullRank(modelMatrix) :the model matrix is not full rank, so the model cannot be fit as specified.Levels or combinations of levels without any samples have resulted incolumn(s) of zeros in the model matrix….

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TCGA transcriptome data to R (DESeq2)

This seems to be frequently asked question, so here is a robust method to fully recapitulate the counts given by TCGA and port it to DESeq2. Why the long way? Tanya and I noticed via TCGA-Biolinks and Firehose did not generate the full count matrix. ~5-10% of genes were missing…

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Isolate contrasts using DESeq2 when use a combination of categorical and continuous variables

Dear community members, I am currently using DESeq2 to analyze RNASeq data from plant roots that were colonized by a fungus. But I am facing some problems regarding contrast with a continuous variable. If you have any suggestions or comments it would be great if you can let me know….

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need some help on how to use DESeq2 for TCGA data

need some help on how to use DESeq2 for TCGA data 0 Hello, I am sorry for this newbie question, but I spent all morning trying to find it out but can’t find a clear answer anywhere. I want to normalise RNA seq data from TCGA using DESeq2. I use…

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Error with hicdcdiff function in HiCDCPlus package

I am trying to find differential interactions from my allvalidpairs data. I was able to create an index file with the union of significant interactions but when proceeding to the next step to perform differential analysis with hicdcdiff function I am getting the following error. Error in gi_list_read(path.expand(prefix)) : Some…

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Paired sample design in DESeq2

I am analyzing a dataset that is looking into the effects of age on cancer using patient samples within one cancer type. In short, we are interested in finding genes involved in tumorigenesis that are altered by age. Each patient is classified as young or old, and had tumor tissue…

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Map mouse RNA-seq Ensembl gene ids to “org.Mm.eg.db”, but get some “UNKNOWN” gene symbol, like “NA..25729”, is it normal?

Map mouse RNA-seq Ensembl gene ids to “org.Mm.eg.db”, but get some “UNKNOWN” gene symbol, like “NA..25729”, is it normal? 0 Hi I was trying to map mouse RNA-seq Ensembl gene ids to “org.Mm.eg.db” database, but get some “UNKNOWN” gene symbol, like “NA..25729”, is it normal? How to explain it? >…

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DEseq2 replicate normalization

There are maybe two questions going on here. Normalizing by “replicate” Unless there is something common among the different replicates in your experiment, I’m not sure that including the replicate term in your model as you are doing and trying to normalize is possible/reasonable. Allow me to outline a few…

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Deseq2 dataframematrix error

Error in DESeqDataSet(se, design = design, ignoreRank) : some values in assay are negative I have raw counts of rna seq data with approxx 68 samples, im getting error in deseq dataset can you guys help me countsTable <-read.csv(“counts.csv”, header = T, stringsAsFactors = FALSE) head(countsTable) sample_info <-read.table(“sample.txt”, header =…

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Installing packages by bioconductor version 3.14

Installing packages by bioconductor version 3.14 1 @dfefb504 Last seen 5 hours ago Vienna I installed the latest R version 4.1.2, but I got problems with installing the latest bioconductor version 3.14. I can not install any package by bioconductor. Can anyone tell me the reason? THANKS! > if (!requireNamespace(“BiocManager”,…

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values (NA) in p value Deseq2 (reopen)

Hello how are you? I reopen this question because the following has happened: I am doing a differential expression exercise using the hisat2, stringie & DESeq2 workflow. Finally I use the python prepDE.py script recommended in the StringTie manual to extract the counts. So far so good, I have rows…

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Help with replicating results of differential expression analysis in DESeq2 : bioinformatics

**Also posted this on Biostars** Hi everyone! I’m fairly new to bioinformatics (self-taught for my MSc) and I’m trying to replicate a study using publicly available transcriptomic data (GSE107934). I’m struggling to get the same results as the authors. I’m following the study’s methods and using DESeq2 to conduct the…

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Differential expression analysis – issue with replicating results

Hi Biostars! I’m fairly new to bioinformatics (self-taught for my MSc) and I’m trying to replicate a study using publicly available transcriptomic data (GSE107934). I’m struggling to get the same results as the authors. I’m following the study’s methods and using DESeq2 to conduct the differential expression analysis. Most of…

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Errors when ajusting pvalue on R

Errors when ajusting pvalue on R 2 Hello. I’m trying to ajust the pvalues from the file I’ve got from Deseq2. But I keep getting error mesages: p.adjust(dds, method = “bonferroni”) Error in as.vector(x, mode = “numeric”) : no method to coerce this S4 class to vector Then I tried…

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Alignment of de novo assembled transcripts to reference transcriptome?

Alignment of de novo assembled transcripts to reference transcriptome? 0 Hi all, I started with 40 samples of raw (but trimmed) RNASeq samples. I used these as inputs for SPAdes-rna and Trinity, to assemble them without a reference. I now have 80 assembled transcriptomes (?), and I tried to follow…

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Normalization by variance stabilizing transformation VST

Normalization by variance stabilizing transformation VST 1 @6c372dab Last seen 8 hours ago Sweden Hello! I am a bit confused about the normalization performed by the DESeq2 varianceStabilizingTransformation() and vst() functions in addition to the actual variance stabilization. My understanding is that the normalization by division by size factors (which…

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Time series RNA seq design query

Hello, I am working with Deseq2 on RNA seq time-series data. I have RNA seq data at baseline before any intervention (timepoint0), RNA seq data with two different interventions (Placebo, and Condition) at TP1 and TP2. I went through the time-series tutorial for Deseq2 and don’t think the same example…

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Error when running ‘DESeqDataSetFromMatrix’ from Deseq2

Error when running ‘DESeqDataSetFromMatrix’ from Deseq2 1 Hello. I’m having problem at the very beggining of the DESeq2 pipeline. When I try to run the command dds <- DESeqDataSetFromMatrix(countData=countData, colData=metaData, design=~dex, tidy = TRUE) I keep getting the error mesage: Error in `.rowNamesDF<-`(x, value = value) : duplicate ‘row.names’ are…

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Biostar DESEQ2

Showing : DESEQ2 • reset 2 days ago • updated 1 day ago adR &utrif; 10 10 days ago • updated 8 days ago adeler001 • 0 15 days ago • updated 9 days ago Cheryl • 0 updated 17 days ago by swbarnes2 &utrif; 920 • written 18 days…

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Treat time variable as continuous or discrete?

Hello, I have two questions regarding an RNA-seq experiment with 10 time points and 20 replicates per time point. So far I’ve run an LRT using the following models: design = ~ timepoint reduced = ~ 1 Throughout the experiment, RNA was extracted at days 3, 4, 15, 16, 44,…

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DESeq2 input file from featureCount

DESeq2 input file from featureCount 0 Hello. I am analysing RNA-Seq data for 8 samples (2 treatments, 4 repetition each). I just finished the gene count using featureCount (Subread). How do I generate a matrix to use on DeSeq2 from the files (.RDS) genereted from featureCount? Do I just create…

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Is it not possible to use LFCshrink on the “intersect” coefficient?

Is it not possible to use LFCshrink on the “intersect” coefficient? 0 I have been trying to use the LFCshrink function on the intersect coefficient. I thought this would be possible on any coefficents from resultsNames() but it gives an error: Error in apeglm::apeglm(Y = Y, x = design, log.lik…

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normalizing EXOM-seq read using DESeq2

normalizing EXOM-seq read using DESeq2 0 @do-it-23093 Last seen 9 hours ago Germany, München Hi, I have exom read aligned to hg38. I used featureCounts to quantify the reads. Therefore, I would like to normalize the reads in the different sample using DESeq2. May I know if there especial consideration…

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just trying to launching my DE-seq2 by csv but isn’t work…

hello sir, im korean and currently work in dpt, bioenginearing… first of all, my major is basically experiment and im suddenly transferred into Industry-University Cooperation Foundation. its my first time coding r script. so i have to run DE-seq2 so that make my MA-plot(and have no idea any of r…

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no DE genes found in subset

Troubleshooting DESeq2 Analysis: no DE genes found in subset 1 @8e2958c3 Last seen 5 hours ago United States Hi, I’m quite new to R and DESeq2 and need some help troubleshooting! I’m working on analyzing a dataset comparing 3 groups: patients before and after a treatment against a healthy control…

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Read Counts from miRDeep2 for posterior DE analysis

Hello everyone, I would appreciate some help with my data: I’ve been analysing some NGS miRNA data in order to do Differential Expression profiling. Using the miRDeep2 program and algorithm, I’m in trouble when dealing with results to make a proper table in order to use it as input for…

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GOStats “genes being tested do not have corresponding GO terms”

Hi all, Has anyone ever had problems with the org.Hs.eg.db database not picking up any GO classifications for a list of genes? For example, I have a list of 21 genes, and when I try referencing the org.Hs.eg.db database, I get “genes being tested do not have corresponding GO terms”…

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Normalizing exonic reads and then log transforming or variance stabilising

Normalizing exonic reads and then log transforming or variance stabilising 0 @a-14337 Last seen 38 minutes ago United Kingdom Hi, I have raw counts per exon matrix and want to normalize and do a VST or log2 transform without running any differential expression (for downstream usage separate to DE), so…

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Genome sequencing of the multicellular alga Astrephomene provides insights into convergent evolution of germ-soma differentiation

1. Smith, J. M. & Száthmary, E. The Major Transitions in Evolution (Freeman, 1995). Google Scholar  2. Queller, D. C. Relatedness and the fraternal major transitions. Philos. Trans. R. Soc. Lond. B Biol. Sci. 355, 1647–1655 (2000). CAS  PubMed  PubMed Central  Google Scholar  3. Grosberg, R. K. & Strathmann, R….

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Question: DESeq2 multiple factor design

I have an experiment where I got 2 factor, the first one is the cell line, and the second one is the treatment. In this, one cell line is derived from the other, the derived cell line received a inducible gene. I then treated both cell lines with the inducer…

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Using normalised counts for pairwise gene correlation (pearson) across samples

Using normalised counts for pairwise gene correlation (pearson) across samples 0 Hi, I have carried out gene pairwise correlations between every pair of genes in my RNAseq data. I have done this with non-log transformed normalised counts generated from estimatesizefactors in DESeq2, as well as with VST counts and log2…

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Filtering Gene by mean expression

Filtering Gene by mean expression 0 Dear All, Sorry for another post. I have RNAseq data and used DESeq2 pipeline for differntially expressed genes. Now I am doing WGCNA and the author suggest to filter genes based on mean expression rather than variance. I am wondering whether anyone has ever…

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Elastic Net for RNAseq data

Elastic Net for RNAseq data 1 @1c386ab3 Last seen 1 hour ago Canada I have RNAseq results from NovaSeq and after doing some differential gene expression with DESeq2, I will like to explore the Elastic Net model to select features that differentiate my two conditions. However, I haven’t been able…

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Deseq2 model matrix error

Deseq2 model matrix error 1 I have trying to do Deseq2 analysis for mainly mutants vs controls. I have two covariates influencing the dataset – Gender and Genotype. This is how my sample annotation file looks – sample condition genotype gender SK01 mutant hr F SK02 mutant hr F SK03…

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DESeq2 – VST data

DESeq2 – VST data 1 @3a675fdc Last seen 4 hours ago United States I have been using DESeq2 package for RNA-sq data analysis and really like the VST data in log2 units. But was unsure about usage of VST data for certain analyses. Specifically, can the VST data be used…

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How to define the gene length for RPKM calculation

How to define the gene length for RPKM calculation 4 Hi guys, I would like to calculate the RPKM of my RNA seq experiment. To do this, as from the formula, I need to know the gene length. My starting point are the row reads (single end) counts resulting from:…

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Difference between two groups of conditions

Deleted:DESeq2: Difference between two groups of conditions 0 Hello. I intend to perform three contrasts including: i) mock.24h vs mock.4h; ii) S2.24h vs S2.4h and iii) the difference between these two comparisons (S2.24h vs S2.4h) – (mock.24h vs mock.4h). The design of my colData is as follows condition time group…

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Calculating gene expression dominance coefficient via DESeq2

I am interested in calculating dominance coefficients of gene expression. For this we have RNAseq data consisting of two homozygous lines (i.e. Genotype AA and BB, respectively) and of the heterozygous crosses between them (i.e. Genotype AB and BA, respectively). We have data for both sexes in each group and…

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Retrieving and interpreting specific contrast results from an interaction experiment in DESeq2

I have an experiment with 2 different groups, and several different time points (for simplicity, let’s say it is 2 time points), and I’m trying to find the best way to model both group and time effects. Note that time is a categorical variable. # in dds <- makeExampleDESeqDataSet(n=100,m=32) dds$time…

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S4Vectors, strange metadata behavior

S4Vectors, strange metadata behavior 0 @hauken_heyken-13992 Last seen 12 hours ago Bergen Is this internal logic intentional? makes no sense to me. # Goal: insert b or bmeta in index 1 of a # Will fail if metadata is of type GRangesList and length > 1. library(GenomicRanges) b <- bmeta…

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betaPrior and lfcShrink

Hi, I am using DESe2 (v 1.33.4) to compare three shade avoidance phenotype in Arabidopsis. I observed that the betaPrior in current version of DESeq2 was set to FALSE. If this is the case, we can compare C vs A and B vs A but we can’t compare C vs…

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Annotating DESeq2 Differential Expression Results

Annotating DESeq2 Differential Expression Results 1 @adeler001-21743 Last seen 11 minutes ago Canada Hello I am trying to annotate a DESeq2 significantly differentially expressed genes results table with the gene start (bp) , gene end (bp) and SNP ID info using the biomaRt package on RStudio, when I look at…

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filtering most variable genes

filtering most variable genes 1 Dear All, I am trying to filter most variable genes for my specific analysis. I have normalized count from DEseq2 attached here with row for genes and column for sample ID. I have found code chunk in Biostar with the following **data$variance= apply(data, 1, var)…

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How to get DEGs compare one sample to another?

How to get DEGs compare one sample to another? 0 I have a count matrix data (23 samples with different condition), and coldata is shown above. I want to get DEGS like this: I tried this code: # Read data data_matrix <- read.csv(“matrix_data”, row.names=1) coldata <- read.csv(“data/coldata.csv”, row.names=1) # Creating…

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When I change samples grouping for normalization by DESeq2 I get different results, Why?

When I change samples grouping for normalization by DESeq2 I get different results, Why? 0 Hi all I have downloaded the raw counts of RNA-Seq from TCGA for DE analysis. the number of primary tumor samples was 223 and the normal adjacent tissue was 41. I have performed the normalization…

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Which interactions terms to choose?

Which interactions terms to choose? 0 I would appreciate your help with choosing the right structure of result names to answer my research question. I defined 2 conditions: Strain (WT is the reference level) cell-Fraction (FractionA is the reference level) I want to calculate the difference between FractionA and FractionB…

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Which rlog should I use for DESeq2 analysis

Which rlog should I use for DESeq2 analysis 0 @rafaelsolersanblas-22935 Last seen 1 minute ago Alicante I have a question regarding the rlog normalization. I have many samples to compare, with only one factor. A vs Treat, B vs Treat, C vs Treat … So, should I put everything in…

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DEseq2 contrast in two time points

I need help with some clarifications please I have control versus treatment in two time points like > y X condition time 1 CTRL_24_hrs_replicate1 control 24 2 CTRL_24_hrs_replicate2 control 24 3 CTRL_24_hrs_replicate3 control 24 4 treatment_24_hrs_replicate1 t 24 5 treatment_24_hrs_replicate2 t 24 6 treatment_24_hrs_replicate3 t 24 7 CTRL_48_hrs_replicate1 control 48…

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