Bioconductor Forum

one below, where I am having counts per exon? I guess it would be possible to get sth like this with featureCounts. However, when I tried, I do not get the numbering for each exon (I get sth like this: ENSMUSG00000025902.7, without

splicing Exon featureCounts subread DEXSeq

updated 23 months ago • NIK

time points (T1 and T2). The mapping was done with STAR aligner and the quantification was done with FeatureCounts. When I perform the paired analysis in F: T2 vs T1 and NF: T2 vs T1. I create a design matrix including the variables

limma lmfit R DifferentialExpression

updated 23 months ago • Dimitris

I am trying to perform a count per gene analysis using featureCounts in R. I have downloaded the gtf file and edited it within R to only contain the gene ID, chr, start, end, and strand...within a data frame (df2). I am trying to run featureCounts on df2 with my 6 bam files, however, I am unable to download the package Rsubread. ``` library("Rsubread") Error in library

featureCounts RNASeq Rsubread R

updated 23 months ago • margo

Right now, the result objects from `Rsubread::featureCounts` can easily read into most DE Analysis packages (at least for `DESeq2` and `edgeR`, which should cover most use cases...Right now, the result objects from `Rsubread::featureCounts` can easily read into most DE Analysis packages (at least for `DESeq2` and `edgeR`, which should cover most use cases). However, at this time, it appears to me…

tximport Rsubread

updated 23 months ago • johnmcma

As I know, to use DESeq2 you need raw counts. but in my case I have featureCounts. is the raw counts like feature ones ? if not is there a way to convert featureCounts to raw counts in R

deseq featurecounts counts DESeq2

updated 23 months ago • Hicham

featureCounts: 0% successfully assigned fragments on PE .BAM files I am facing same problem, performed every suggestion but

featureCounts

updated 23 months ago • KMS

Hello, I am using "featureCounts" in Rsubread package for analyzing bulk RNA-seq of drosophila. Since there is no inbuilt annotations of drosophila...Rsubread) library(limma) library(edgeR) targets <- readTargets("wholeflyseq.txt") fc <- featureCounts(files=targets$OutputFile,annot.ext="genes.gtf",isGTFAnnotationFile=TRUE, isPairedEnd=TRUE) ``` I was able to

Rsubread featureCounts

updated 2.0 years ago • Chise

Hello, I would like to confirm if the low assignment ratio (54%) is normal, and please check the possible reason I found. I used Hisat2 to assign paired-end strand-specific transcriptomic sequences (rRNA removed) to a reference genome. Because I filtered out the unmapped sequences in advance, the overall assignment ratio displayed by Hisat2 was 100%, and the multi-mapping ratio was only 0.3%. T…

FeatureCounts Rsubread Subread

updated 2.0 years ago • Sarah_piggy

I am using Rsubread to analyze human FASTQ files from the ENA. I downloaded a GRCh38 FASTA from ENCODE to create and index. I used the featureCounts(bam.files, annot.inbuilt = "hg38") function to count mapped reads for genomic features. Everything seems to work...FASTQ files from the ENA. I downloaded a GRCh38 FASTA from ENCODE to create and index. I used the featureCounts(bam.files, annot.i…

Ensembl Rsubread

updated 2.1 years ago • agustin.gonvi

Enter the body of text here I did RNAseq of a Drosophila cell line. By using RNASTAR in Galaxy I am getting high number of reads mapped to too many loci. (uniquely mapped 60 %, mapped to multiple loci 10 %, mapped to too many loci 24 %). Later when I am doing FeatureCounts there is only 40 % assigned, unasigned : unmapped 20 %, unassigned : mapping quality 35 %. Should I consider this as …

RNASeqData

updated 2.1 years ago • vasa.broz

the alternative splicing events by R. Here is the Syntax I used. ``` library(edgeR) fc <- featureCounts(files=c("file control1.bam", "file control2.bam", "file control3.bam", "file treated1.bam", "file treated2.bam", "file

diffSpliceDGE RNASeq DifferentialSplicingWorkflow AlternativeSplicing edgeR

updated 2.1 years ago • zhaoqiao1027

and have been following the Galaxy pipleine for my RNA seq data. At this stage, I have featureCounts count tables. In one one of the DEG experiments, I have to compare WT baseline data (2 males + 2 females) vs WT at 1h

DESeq2 RNASeq Galaxy

updated 2.2 years ago • István

Hi, I have a couple of questions regarding parameter choices for featureCounts. I have stranded 150 bp paired-end reads. I have currently chosen the following settings. As far as I understand...have set my STAR mapping to output all alignments with the best score as primary alignments). ```r featureCounts -p -B −−countReadPairs -C -F GTF --verbose -M --fraction --primary -A chromosome_name_ali…

subread Rsubread featureCounts RNA-seq

updated 2.2 years ago • Lucy

there, Excellent package! I am using it to do RNA-seq. But I encountered a small problem when using featureCounts(). The code is as follows: ```r featureCounts( "A1.raw_1.fastq.gz.subjunc.BAM", annot.inbuilt = NULL, annot.ext = "GCF_015227675.2_mRatBN7.2_genomic.gtf...__| | ========== |_____/ \____/|____/|_| \_\______/_/ \_\_____/ Rsubread 2.8.0 //========================…

Rsubread

updated 2.2 years ago • Zhaoxu

gtfFeature 'exon' --gtfAttr 'gene_id' ``` 2. quantify the reads based on the exons ``` featureCounts -a $gtf -t exon -g exon_id -p -T 12 -o featureCounts.exonLevel.txt subjunc/*.bam ``` 3. upload the data into R, create a `DGEList

diffSpliceDGE edgeR DifferentialSplicing

updated 2.3 years ago • Assa Yeroslaviz

analysis. What files should I use as the input file? is it just gene count files in tabular form by featurecount? Thank you

RNASeq

updated 2.3 years ago • cam

Hello, I am using "featureCounts" in Rsubread package for analyzing bulk RNA-seq of drosophila. Since there is no inbuilt annotations of drosophila...library(limma) library(edgeR) targets <- readTargets("wholeflyseq.txt") fc <- featureCounts(files=targets$OutputFile,annot.ext="genes.gtf",isGTFAnnotationFile=TRUE) (genes.gtf is in Drosophila_melanogaster

featureCounts Rsubread

updated 2.4 years ago • Chise

for analyzing Illumina `RNASeq` datasets. I follow the below steps; 1. Derived raw counts (from featureCounts) > Imported counts to DESeq2 2. Normalised the counts via an estimation of size factors `(counts(dds, normalized

RNASeq DESeq2 vst rlog Normalization

updated 2.4 years ago • mohammedtoufiq91

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...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

ExomeSeq DESeq2

updated 2.4 years ago • adR

anyone know where to download the human Annotating Genomes with GFF3 or GTF files or the function featureCount download it automatically in the command line. I did the unpaired end alignment using hg38 reference genome...My objective is to quantify read counts in the bam file using featureCounts. featureCounts -t exon -g gene_id -a annotation.gtf -o counts.txt mapping_results_SE.bam …

ExomeSeq featureCount

updated 2.5 years ago • Do it!

Hello, Does anyone know if theres a way for featureCounts to analyse bam files in parallel? I have been running featureCounts for my bam files and its taking so long ( currently

counts subread RNASeq featurecounts

updated 2.5 years ago • Beth_b

Hello- I'm trying to use `Rsubread`'s `featureCounts` function in a single-cell ATAC-seq data set, to count the number of reads in each cell that map to each peak. My...RG` tag and added `@RG` header lines for each. `featureCounts` is segfaulting when I try to read this file with `byReadGroup = TRUE`: ```plain ========== _____ _ _ ____ _____ ______ _____ …

Rsubread

updated 2.5 years ago • mruffalo

View(bam.files) # Ver lista de archivos del directorio (.BAM) bam.files[12] head(bam.files, n = 12L) #### FeatureCount: Programa que cuenta las lecturas de los bam mycount = list() for (i in 1:12) { mycount[[i]] <- featureCounts(files = bam.files...splitOnly = FALSE) } > for (i in 1:12) { + + + mycount[[i]] <- featureCounts(files = bam.files[i], + …

Rsubread

updated 2.5 years ago • v.merino.nicolas

a few samples with high counts of rRNA genes in the third replicate. My pipeline was HISAT2 --> featureCounts --> DESeq2. When clustering replicates 1 and 2, the replicates of each condition clustered closely together

DESeq2

updated 2.5 years ago • jac

Hi, I did my RNA-Seq analysis using the Galaxy platform with the following pipeline: HISAT2 --> featureCounts --> DESeq2. Now I want to recreate the PCA plot in RStudio. In the DESeq2 manual, the command line for this is: plotPCA

DESeq2

updated 2.5 years ago • jac

I did below is correct. library(DESeq2) count_file_names <- grep("counts",list.files("featureCounts"),value=T) specimen_type <- c("Age0","Age2","Age4","Age6","Age8","Age10","Age12") sample_information <- data.frame(sampleName...count_file_names, fileName = count_file_names, condition = specimen_type) Note- I switched to featureCounts instead of HTSeq …

DESeq DESeq2 R

updated 2.7 years ago • jbnrodriguez

I am using featureCounts to try and create a count table for some RNA-Seq data I collected using an Oxford Nanopore platform. I have .sam...files aligned with minimap2, and am running the following command to try to get a count table: ``` featureCounts -a knownGene_v36.gtf -o countFile *.sam -L ``` Both the GTF file I am using, and the reference genome that the alignments...problem with t…

RNASeq featureCounts

updated 2.7 years ago • nklier38

Here is my process: 1) Feed raw count data of all the predicted genes within the sample (use **featurecounts** table). 2) Tranform the data to deseq2 compatible format and provide metadata 3) i have two samples (control vs

DESeq2

updated 2.7 years ago • Shail

in a feature even if they have soft-clipped and inserted bases. Is there a simple solution to make featureCounts include these reads? I'm thinking to hack the bam file to replace cigar strings containing S or I (but not containing...amp; $1 !~ "^@" && ($6 ~ "I" || $6 ~ "S")) {$6 = length($10)"M"} print $0}' > out.sam featureCounts --fracOverlap 1 -O -f -a genes.gtf -o co…

subread featureCounts

updated 2.7 years ago • dario.beraldi

Hi, I am new to Deseq analysis. Recently i conducted metatranscriptomics analysis of bacterial community in two different conditions. My aim to explore the overall significent transcript expression and also some selective transcripts (related to specific function). I used raw featurecounts output to run Deseq and created heatmap on VSD transformed data on to 20 features. But, due to vast …

DESeq2

updated 2.8 years ago • Shail

Hi, I'm using featureCounts (subread v2.0.2) to generate a count matrix for an RNAseq experiment. I have aligned paired-end .sam files sorted...Hi, I'm using featureCounts (subread v2.0.2) to generate a count matrix for an RNAseq experiment. I have aligned paired-end .sam files sorted by read name and have specified the -p flag and the −−countReadPairs parameter (please see code below). Whe…

subread featureCounts

updated 2.8 years ago • s.muroy

rv))) { stop(paste0("Error: the file path to '", opt, "' contains the internal splitor of featureCounts (\\027). The \\027 character is unallowd in the file names or in the paths.")) } if (any(grepl("\t", rv))) { stop(paste0("Error: the file...rv))) { stop(paste0("Error: the file path to '", opt, "' contains the internal splitor of featureCounts (\\026). The \\026 …

Rsubread featurecounts coercion

updated 2.9 years ago • Konstantinos Yeles

pre-processing tools for alignment, counting, or sequence analysis (e.g., Cell Ranger, STAR, HTSeq, featureCounts, salmon, GATK, etc.) and R packages designed for the above-mentioned methods (e.g., DESeq2, limma, Seurat, etc.). A solid

JobPosting

updated 2.9 years ago • hannah.baskir

AZM", and "TOB". I have provided the output of the first 10 rows of the raw counts obtained using featurecounts below. I have also provided the metadata table I used for the analyses below: ``` head(DESeq2_data, 10) Geneid BHI_1

DESeq2

updated 2.9 years ago • Naphtap92

nthreads=8) bamlist_splicing=list.files(pattern=".subjunc.sorted.BAM$") counts_splicing <- featureCounts(bamlist_splicing, annot.ext="dmel-all-r6.38.gtf", isGTFAnnotationFile=TRUE, GTF.featureType="exon", useMetaFeatures

edgeR Rsubread

updated 3.1 years ago • jbono

If not, what approach could I follow? Please note that so far, I have fragment counts from featureCounts for all my libraries; nevertheless, there are big differences between technical replicates sometimes (e.g

edgeR

updated 3.1 years ago • mo17

The size of the bam files, generated by STAR , ranges from 1G to 27GB. Now I am using featureCounts to count reads to genes. I run featurecounts in shell command line with 24 thread machine. This is code that I...used sinteractive --cpus-per-task=24 --mem=120g module load subread featureCounts -p -T 24 -t exon -g gene_id -a genes.gtf -o P11_AM_Left_BAL_6h.txt P11_AM_Left_BAL_6h.Aligned…

RNAseq123

updated 3.1 years ago • demirkalecy

forward-stranded or rev-stranded and PE or SE)? Like it possible to do, for instance, using featureCounts from Rsubread... Unfortunately, I didn't find that information in the reference manual. Thanks in advance, Best

ribosomeProfilingQC countReads

updated 3.2 years ago • alexandr.gopanenko

____ \| |__| | ========== |_____/ \____/|____/|_| \_\______/_/ \_\_____/ v2.0.1 //========================== featureCounts setting ===========================\\ || || || Input files : 18 BAM files || || o bulk_trimmedAligned.…

featureCounts Rsubread subread CellBiology

updated 3.2 years ago • Jason

file which i converted to BAM file format. Now i want to count the reads by using Rsubread function featurecounts, This requires gtf annotation file. how can i generate the gtf file for this. Thank you

SeqGate TarSeqQC ExperimentHubSoftware projectR

updated 3.2 years ago • Ashish

Hi, I want to remove X and Y chromosome genes from my bulk seq data. I don't know how to proceed with that. Do I need to remove the genes from the counts after featurecounts or using the Deseq2? I also tried using the code from the previous post (https://support.bioconductor.org/p/67237...seq data. I don't know how to proceed with that. Do I need to remove the genes from the counts after featurec…

DESeq2 RNASeq

updated 3.3 years ago • Archit

9th column and "exon" from 3rd column of the GTF file, followed by counting of each GTF file using featureCounts (or HTSeq) and subsequent input of the count matrices into R for DEXSeq analysis. 2. Run featureCounts (or HTSeq

DEXSeq

updated 3.3 years ago • Anubhav

we have any contamination by ribosomal or other repetitive sequences in our RNA-seq data. However, featureCounts is running extremely slowly on this dataset. Specifically, with only two normal-sized RNA-seq samples, featureCounts...was still running after 16 hours, at which point I aborted it. In contrast, when I run featureCounts on the set of all human exons grouped by gene (a similar-sized ann…

rsubread featurecounts

updated 3.4 years ago • Ryan C. Thompson

is related to the introduction of coldata information in the matrix before running DESeq2 when using featureCounts data. 1) using Galaxy with 2 factors (2 batches/ 2 discinct studies from the litterature), 3 levels in each factor...error lie? Or at least what this row.names length error refers to? 2) I then tried to retrieve my featureCounts datasets from galaxy so that I can do deseq2 myse…

DESeq2

updated 3.4 years ago • NGS_enthusiast

HISAT2 using the correct --rna-strandness parameter (RF, R, F, FR). Now I would like to use featureCount to create a count matrix.  This leads to the question - How should I set these two arguments? __isPairedEnd

rsubread

updated 3.4 years ago • juls

I have RNA-Seq data with raw counts extracted from bam files using `featureCounts`. I wanted to convert counts to tpm, because I need TPM for some analysis. Initially, I got the coding length for

rnaseq counts tpm GenomicFeatures kallisto

updated 3.5 years ago • m93

HI, I'm using featureCounts (a sw provided by the Subread package) to count reads taken from a BAM file without the SEQ - QVAL fields. The read...summation routine gives the expected results with the following parameters: featureCounts -M -a data/basicAnnotation.gtf -o result/basic_M.FC data/basic.bam. I’m wondering if the absence of the SEQ/QVAL...be an issue when using any of the other …

Rsubread featureCounts

updated 3.5 years ago • martinag.work

Starting from featureCounts generated raw counts file, I used edgeR to estimate the DE analysis and it went well. Now I use CPM normalized

Normalization edgeR RPKM

updated 3.5 years ago • anikng

Hello, I have counts from Salmon that I have imported using Tximport for WT and KO samples. I want to use ERCC spike-in data for normalizing these counts. To do this, my strategy involves estimating size factors using ***only*** the **spike-in data** (function: Deseq2::estimateSizefactors) and using those normalize my counts (*Mus musculus* transcripts). What I have done so far: step1: I …

deseq2 Tximport Sizefactors normalizationfactors spike-in

updated 3.8 years ago • hina.abbas.bandukwala

package. I routinely deal with mixed paired end and single end data. The Rsubread function featureCounts had a isPairedEnd argument that used to accept a logical vector containing information of which files are

software error Rsubread

updated 3.9 years ago • barrel0luck

data. I am not sure, what type of file is required for DESeq2. I have output file (count.txt) from FeatureCounts. Here is some lines of the counts.txt file (output of FeatureCounts). Is that seem correct as input of DESeq? Geneid

edger deseq2

updated 4.0 years ago • sekhwal

I'm using the featureCounts function of Rsubread to assign aligned reads to features. Something like ~20% of my reads are unassigned (in...I'm using the featureCounts function of Rsubread to assign aligned reads to features. Something like ~20% of my reads are unassigned (in the

Rsubread

updated 4.0 years ago • am39

Using the `chrAliases` flag in Rsubread featureCounts is not working as expected. Single-end reads mapped to genome file ftp://ftp.ncbi.nlm.nih.gov/genomes/all...Using the `chrAliases` flag in Rsubread featureCounts is not working as expected. Single-end reads mapped to genome file ftp://ftp.ncbi.nlm.nih.gov/genomes/all/GCA...GCA_000001405.15_GRCh38_full_analysis_set.refseq_annotation.gtf.g…

Rsubread

updated 4.0 years ago • kathleen.e.grogan

AT5G27710.2, AT5G45240.2 ``` And it can make the Txdb lose 6 genes compared with my featureCounts result ``` > genes(Tx_At) GRanges object with 37330 ranges and 1 metadata column: seqnames ranges strand | gene_id...ATMG09980 ------- seqinfo: 7 sequences from an unspecified genome ``` ``` # My featureCounts result > head(data) …

genomicranges genomicfeatures

updated 4.0 years ago • shangguandong1996

Hello, I am analyzing RNA-seq data of arabidopsis samples in response to three treatments (let's call them WT, RT and DT). These treatments have some components in common, so I want to find genes that respond specifically to the unique components in each treatment. In summary, I have performed the Differential Expression analysis of the three treatments versus the non-treated sample (NT), a…

edger complexheatmap R clustering

updated 4.0 years ago • eggrandio

obtained my list of DE transcripts. The functions I used to align .BAM files to features are featureCounts and summarizeOverlaps respectively, the latter resulting in higher expression levels for different transcripts...and with the following parameters: For edgeR fc <- featureCounts(all.bam, annot.ext="pathofmygff3", isGTFAnnotationFile=TRUE, nthreads=16, GTF.featureType="tra…

edger deseq2 rnaseq de

updated 4.0 years ago • Raito92

Hi, I am trying to use the Bioconductor package in R 3.6.3 on Windows 64-bit platform to analyse my RNA-seq data with Apis mellifera. I am facing difficulty from an error described below. I have checked the available GTF file, using readGFF, the gene_id comes in the 10th column. But that is how I have downloaded the file from: https://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/003/254/395/GCF…

featureCounts

updated 4.1 years ago • chatterjee.arumoy

BAM" ` to get the reads. > EDIT: feature counts code nc_RNA_list[1] %>% map(~ featureCounts(files = bam_file, annot.ext = gtf_files[str_detect(gtf_files,str_glue("_{.x}_"))], isGTFAnnotationFile =TRUE, GTF.featureType

featurecount rsubread GenomicAlignments

updated 4.1 years ago • Konstantinos Yeles

I have a featureCounts results file that looks like the snippet at bottom. I have another file for the parent that looks similar. How...link looks like: ``` > library(DESeq2) > setwd("~/example_data/practice_rnaseq_data/featurecounts/") > counts=read.csv("counts2.txt", sep="", head=T, skip=1, row.names = "Geneid") > colnames(counts)[6:11] > colnames(co…

DESEq2

updated 4.1 years ago • chunter

samples into a single BAM, called peaks (Genrich), and counted reads per peak region for each sample (featureCounts). I then ran limma following common guidance (example code below), and the resulting MA plot shows many more significantly

limma edger atac atac-seq

updated 4.2 years ago • pgugger