Bioconductor Forum

Hello, I am running into some perplexing behavior when using featureCounts. I would like to assign reads only to features with which they completely overlap (similar to HTSeq's intersection...requested in a [previous post](https://support.bioconductor.org/p/83968/)). From reading the featureCounts documentation, I suspected that this would be accomplished by setting the `nonOverlap` parameter, d…

featureCounts Rsubread

updated 24 days ago • isaac.vock

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

featureCounts

updated 23 months ago • KMS

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'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

div class="preformatted">Hi Ryan, Sorry for my late reply. We have added the following options to featureCounts to let users be able to extend reads and also control the overlap length between reads and featureCounts: readExtension5...readExtension3 minReadOverlap The featureCounts help page describes the meaning of these parameters and how to use them. Changes have been committed to bioc.…

updated 10.0 years ago • Wei Shi

Summary** It seems featureCounts under certain circumstances ignores the ```isPairedEnd = TRUE``` argument. **Background** I have been using featureCounts...sam2bed | cut -f 6 | sort | uniq -c 11 - 1272 + ``` So far so good. Now lets try with featureCount from the R package Rsubread **Rsubread::featureCount approach**: ``` library(Rsubread) ### Create regions to…

rsubread featureCounts

updated 5.0 years ago • k.vitting.seerup

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

Hello, I'm looking at the featureCount flag -t and the gtf file for my organism of study, and the gtf file has the following feature options: gene, exon...Hello, I'm looking at the featureCount flag -t and the gtf file for my organism of study, and the gtf file has the following feature options: gene, exon, transcript

Rsubread featureCounts RNASeq

updated 6 months ago • HS

3 of the SAM files were created from PE data, and 1 SAM file was created from SE data. I want to use featurecounts to create a count matrix, however, should I be using paired end mode or single end mode?  Thanks for the help

featurecounts rsubread

updated 6.7 years ago • nikelle.petrillo

I extracted exon information from mm9 and then used it for featureCount. Next I used an in-built mm9 annotation of featureCount and I got less exons annotated somehow. __Extraction...uc007aeu.1   Xkr4</pre>   __FeatureCount with the manual exon annotation:__ manual=featureCounts(c(bam.CTCF,bam.H3K27ac),annot.ext=exon\_info\_red,isGTFAnnotationFile=FALSE, <p…

annotation featurecounts rsubread

updated 8.6 years ago • tonja.r

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

because my bam is quite big, and already sorted by coordinates, and I takes much more time to ask featureCounts to re-order the reads to use paire end mode (and I have a lot of bam to count, onto various features, so it is taking

featurecounts rsubread

updated 9.0 years ago • samuel collombet

Hi,   I want to obtain read counts at the exon level using featureCounts. I run Rsubread and use these options: annot.ext="/home/inah/RefGTF/GRCh38/annotation/Homo\_sapiens.GRCh38.85.gtf

RNAseq

updated 6.9 years ago • inah

RNASeq analysis so I'm having a lot of difficulties analyzing the data. First of all, I got some raw FeatureCount data, and there are multiple entries of the same meta-feature (gene) all with the same or very similar counts. What

rna-seq featurecounts

updated 9.3 years ago • ccheung

I am testing the various featurecounts options from the very useful Rsubread package. I have noticed that in all tested libraries (>100) using exon

featurecounts rsubread

updated 7.1 years ago • IV

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

Dear, Currently, I would like to calculate exon counts using package Rsubread 1.32.0, function featurecounts. Inputs: 1. Bam file based on paired end RNA sequencing. 2. Annotation file in GTF format, downloaded from GenCode...Gatk.ApplyBQSR From my point of view, I should be able to run following command: featureCounts( files=inputfile, annot.ext=annotation…

R error software error Rsubread featurecounts

updated 5.1 years ago • edejong

Hi all I first use the featureCounts in-build annotation ([another post](https://support.bioconductor.org/p/102280/)) and get the result. There are...Hi all I first use the featureCounts in-build annotation ([another post](https://support.bioconductor.org/p/102280/)) and get the result. There are 20454...img alt="" src="https://i.imgur.com/4wcOQT8.png" style="height:147px; width:730px"/> …

rnaseq annotation featurecounts biomart

updated 6.5 years ago • Jack

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

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...a BAM file that initially contained cell barcodes in the `CB` tag of each read -- then I found that `featureCounts` has no mechanism to use anything but the read group to separate reads, so I copied the barcodes to each read...…

Rsubread

updated 2.5 years ago • mruffalo

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

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

Hi- I've noticed what i think is a bug in featureCounts with paired-end reads and `--countReadPairs`. When a bam is used that has been filtered post alignment so that...Hi- I've noticed what i think is a bug in featureCounts with paired-end reads and `--countReadPairs`. When a bam is used that has been filtered post alignment so that some...reads that were originally paired are no longe…

Rsubread featureCounts

updated 9 months ago • StevenE.Strong

rv))) { stop(paste0("Error: the file path to '", opt, "' contains the internal splitor of featureCounts (\\026). The \\026 character is unallowd in the file names or in the paths.")) } if (any(nchar(rv) >= .filepath_maximum_len...1 utf8_1.2.1 stringi_1.6.2 munsell_0.5.0 broom_0.7.6 crayon_1.4.1 ``` # run featureCounts ```r library(tidyverse) library(Rsu…

Rsubread featurecounts coercion

updated 2.9 years ago • Konstantinos Yeles

Hi, running Rsubread 2.8.2/2.12.0 or featureCounts 2.0.3/2.0.1, I stumbled over two issues when allowing ambiguous read assignment (-O/allowMultiOverlap) 1) regarding...assignment via minimum fractional overlap (--fracOverlap) using featureCounts stand-alone binary. 2) when combined with --/largestOverlap and --/fraction using Rsubread featureCounts function...is enabled (--/fraction), both re…

Rsubread featureCounts

updated 17 months ago • Konstantin

div class="preformatted">Hi, I am trying to use featureCounts to perform strand-specifc RNA-Seq read counting with an Ensembl GTF file. However, it seems to count reads from...the problem. First, if I specify strandSpefic = 0 I get 97.7% of the reads aligned to two genes: a = featureCounts("merged.bam", annot.ext = "Homo_sapiens.GRCh38.76.gtf", strandSpecific = 0, isGTFAnnotationFile = TRUE,…

updated 9.7 years ago • Guest User

p/249955/#250010)[,](https://www.biostars.org/p/249955/#250010) but according to the featureCounts website, I should post on this forum for help ::__ I am using featureCounts (version 1.5.2) to count the number of...reads within bins along the genome: <pre> featureCounts -R -F SAF --fracOverlap 1 -Q 1 --primary -T 20 -a Bins.saf -o readCounts.txt Input.bam</pre> A small percen…

subread featurecounts

updated 7.0 years ago • jma1991

Dear All, I am interested in counting the number of mapped reads in equal size intervals on genome (say 200 bp), my question is whether featurecounts any option for doing this. According to what I have found so far it sounds like it is more appropriate for counting reads mapping to exons, genes, and promoters. In order to use it for this purpose I define a set of features like following: --- l…

featurecounts rsubread

updated 7.8 years ago • Hadi Gharibi

i just created 16 BAM file by using Rsubread packege. When i run the featurecounts then I only get the total counts for each of the BAM file but not getting the count matrix.  when i use fc$counts

rnaseq rsubread featurecounts

updated 6.2 years ago • mmurshed

Hey, nice package. I have an error using featureCounts with paired end reads: I mapped paired end reads with STAR (trimmed reads with fastp), and wanted to try out Rsubread...counting in R. library(Rsubread) # Define paths to RNA-seq bam file and gtf a <- featureCounts(files = df$RNA[1], annot.ext = gtfAnno, isGTFAnnotationFile = T, isPairedEnd = T, requireBothEndsMap…

Rsubread paired end featureCounts

updated 4.8 years ago • hauken_heyken

I am new to R and RStudio but have been trying to work through different examples using Rsubread for my data. I have tried reading vignettes and manuals prior to posting here but I am stuck and could really use some advice. I have 7 paired-end, fastq files from Illumina sequencing of human samples. I have downloaded RStudio version 2022.12.0+353. I have installed the packages BiocManager and R…

Rsubread Rsu

updated 13 months ago • rmoorehe

with changes in exon usage that I should be able to recapitulate. My strategy was to use featureCounts (Rsubread) in order to get count data for each exon. I generated the flattened gtf using the python script provided...transcripts "ENST00000456328"; exonic_part_number "009" The issue arises when I try to use the featureCount function with these gtfs on their respective datasets. Both data…

dexseq rsubread featureCount software error

updated 4.4 years ago • seung.ho.steven.choi

I am processing paired-end RNA-Seq data. I map them using Hisat2.0.3.Next I convert SAM files to BAM, sort and index the BAM file using samtools 1.3. I subsequently use the sorted BAM files to aggregate mapped reads based on annotation using featureCounts (Rsubread package v1.20.6). After mapping the reads using Hisat2, we get some summary statistics like this: 20358841...1.3. I subsequently us…

featurecounts hisat2 rna-seq mapping

updated 7.9 years ago • Koen Van den Berge

Hi, I have RNAseq data with strand information and I'm using `` Rsubread ``'s `` featureCounts function to sum reads onto the GTF annotation. ``   `` This is the command I'm using: `` `` featureCounts(files=my.bam

Rsubread bam rnaseq

updated 7.3 years ago • rubi

Setup --- files: /path/to/file/here/file1.bam; /path/to/file/here/file2.bam countData=featureCounts(.....) ------- 1.28.1 --- colnames(countData$counts) [1] file1.bam [2] file2.bam ---- 1.34.0 ---- colnames(countData$counts) [1] X.path.to.file.here.file1.bam

Rsubread featureCounts

updated 4.9 years ago • wunderl

Hi all I am trying to learn how to use Rsubread. I have a bam file from a bacterial sequencing project, and a related gtf file.  I want to count hits to each gene and have been using the following command: _fc\_External<-featureCounts("bamFIle.bam", annot.ext= "gtfFile.gtf", isGTFAnnotationFile=TRUE, GTF.featureType="CDS",useMetaFeatures=TRUE...nbsp; I want to count hits to eac…

rsubread

updated 8.7 years ago • ross.chapman

a read is mapped to is reported in the FLAG field in a SAM/BAM file and that is the information featureCounts uses to perform strand specific read summarization. I would suggest you to have a close look at your read mapping...is set to 2, featureCounts will flip the strand of each read and then try to match it to the strands of genes. Since your two genes are on...or small counts) for the two gen…

Sequencing Annotation

updated 9.6 years ago • Wei Shi

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

I increased  nthethreads value in featureCounts but the running time didnt decrease. <pre> I used the following command: subread_res <- featureCounts(files...I increased  nthethreads value in featureCounts but the running time didnt decrease. <pre> I used the following command: subread_res <- featureCounts(files, annot.ext = GTF_file, …

rsubread

updated 6.7 years ago • shao

Hi all,  I am having a weird issue (weird to me being a new R user) using featurecounts to generate counts for genes but also for individual exons. I have successfully mapped gene counts to their...with total read mapped to genes. The code I am using is as follows: For annotating genes: fc <- featureCounts(bamDir, isGTFAnnotationFile = TRUE, annot.ext = "Mus\_musculus.GRCm38.94…

featurecounts Rsubread ggbio

updated 5.4 years ago • A

detailing the assignment of each individual sequencing read), I found a weird bug. Occasionally, featureCounts would assign reads to the identical set of isoforms, but would output different comma-separated target list...file with one of these two target lists and 100 with the other target list. Notably, when running featureCounts as detailed below on this downsampled bam file, the reads get assi…

featurecounts Rsubread

updated 3 months ago • isaac.vock

Hi, I think I might have found a bug in featureCounts from Rsubread (v2.12.3). I am trying to find reads overlapping exon junctions from a personalised reference...Hi, I think I might have found a bug in featureCounts from Rsubread (v2.12.3). I am trying to find reads overlapping exon junctions from a personalised reference, using Nanopore long read BAMs. I am afraid I cannot share fully …

Bioconductor Rsubread featureCounts

updated 12 months ago • Carlos

I'm having a very strange error when running featureCounts on a tiny test file: it says it's generating counts in the summary.txt file, but the 7th column of the counttable...0</pre>   Where did the 18 reads go?   <pre> head testYA.multicounts.txt # Program:featureCounts v1.5.0; Command:"/home/me/bin/subread-1.5.0-Linux-x86_64/bin/featureCounts" "-p" "-M" "--fr…

featurecounts software error

updated 8.2 years ago • Darya Vanichkina

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

featureCounts Rsubread subread CellBiology

updated 3.1 years ago • Jason

Hello, I did a bulkRNA-seq and now have an output gene count file from: `featureCounts -s 0 -p -P -d 0 -D 1000 -B --primary -t exon -g gene_name -a gtf -T 6 -o output bam1 bam2 bam3` (I did it via hisat2 then samtools sort...then featurecounts using linux command line) The three bam files belong to 3 cell lines and I want to do a differential analysis

DESeq2 FeatureCount

updated 20 months ago • Jiayi

Hello, I am using Rsubread's featureCounts() to quantify the genes in my RNA-Seq data. There are two options to count "genes" or "exons" that are then aggregated

RNASeq Rsubread featureCounts Quantification

updated 15 months ago • Sara

Hi, I am using featureCounts to count reads for every gene from refSeq annotation file. But it went to error: featureCounts -a ../refGeneReviewed.gtf...Hi, I am using featureCounts to count reads for every gene from refSeq annotation file. But it went to error: featureCounts -a ../refGeneReviewed.gtf -t exon -g gene\_id -p -Q 10 -o ../../result/fc\_accepted\_hits.txt accepted\_hits.bam &…

featurecounts rnaseq rsubread

updated 9.2 years ago • syrttgump

Hi, I am currently running featureCounts to count the number of reads that have been mapped to each gene. However, it is taking forever to run in R studio...Hi, I am currently running featureCounts to count the number of reads that have been mapped to each gene. However, it is taking forever to run in R studio and I am wondering if it is an issue with my code.  Here is the code I used…

featurecounts r rnaseq

updated 6.0 years ago • octopuslegs11

wild type and three knock-out mice. I use STAR 2.5.3a (built-in Partek Flow) for the alignment and featureCounts (Rsubread_1.30.9) for the quantification. The command I run featureCoutns is below. files <- c("Chuong753.bam...Chuong755.bam", "Chuong756.bam", "Chuong757.bam", "Chuong758.bam") rc <- featureCounts(files, annot.ext = "mm10ncbiRefSeqCurated.gtf", …

RNA-Seq featureCounts STAR minMQS mapping quality score

updated 5.2 years ago • Gary

Hi, I analyze 20 zebra finch RNA-Seq data, using STAR for alignment, Rsubread-featureCounts for quantification, and edgeR for differentially expressed analysis. However, I don’t know how to run edgeR...to read an R List object produced by featureCounts (the detail below). Our study is a three-factor factorial experimental design. I also show the group information

edger featurecounts

updated 5.9 years ago • Gary

2.24.0. They were then overlapped with all exons annotated in the GTF file used to run featureCounts using "bedtools intersect" [2.25.0](https://github.com/arq5x/bedtools2/blob/master/docs/content/history.rst...3, all strands are forward strands, which doesn't meet with the setting (reverse strand) used to run featureCounts. So these reads should be unsigned. However, there were some ove…

rsubread

updated 8.1 years ago • Likai Mao

This technically pertains to the command line featureCounts program in subread, but the subread webpage suggests posting here, and I'm assuming the bioconductor version...I'm trying to count only fragments that span splice junctions (per gene as the meta feature) in featureCounts using the --splitOnly option. The documentation suggests that this will capture reads with an 'N' in the cigar

Rsubread

updated 6 months ago • Gregory

hello all,  I am using featurecount for differential expression analysis. After running feature count I found out there are very less number...hello all,  I am using featurecount for differential expression analysis. After running feature count I found out there are very less number of...SAM file (aligned by STAR) showing 82% mapped reads. I tried both counting by exon and gen…

featurecounts rsubread

updated 9.3 years ago • amoltej

Hi,   I have been using featureCounts to obtain both exon- and gene-level read counts (reads were aligned with STAR). For one particular gene (ARID5B...count summed over the 12 exons is greater than the gene-based read count. This is not posssible as featureCounts uses the exon-union method for gene-level counting. Below are the relevant parameter settings for featureCounts

rsubread featurecounts exon mRNAseq

updated 6.9 years ago • inah

I'm using featureCounts (from Rsubread R/Bioconductor package) and gencode annotation file to do features sumarization, but the gene

biomart rsubread gencode

updated 5.5 years ago • YinCY

support.bioconductor.org/p/59273/): ``` I think what we can do is to add another parameter to featureCounts to let the function reduce the read to its 5' most base or its 3' most base, before carrying out read counting. With...readExtensionFrom5`) that reads can be extended from their 5' end. The requested option can make featureCounts manipulate reads more flexible, especially in the case …

SubRead featureCounts

updated 17 months ago • Leon

div class="preformatted">Hello, I'm trying to use featureCounts to extract read counts per transcript from the SAM file (generated using Bowtie2) mapped to de novo assembled...the following code, it does nothing for a while and then R crashes. library(Rsubread) counts <- featureCounts(files="AssembledTranscriptome-LMcontrol1.sam",annot="Ann otRsubreadSAF.txt",isGTFAnnotationFile=FALSE

Annotation GO BSgenome BSgenome Annotation GO BSgenome BSgenome

updated 10.7 years ago • Alan Smith

<div class="preformatted">Hello, I am using the featureCounts function in Rsubread to count RNA-seq reads. Since my BAM files are coordinate-sorted paired-end, I am using "isPairedEnd...div class="preformatted">Hello, I am using the featureCounts function in Rsubread to count RNA-seq reads. Since my BAM files are coordinate-sorted paired-end, I am using

Rsubread Rsubread

updated 10.5 years ago • Ryan C. Thompson

to paired-end data)  __    and __2.) What are the appropriate options to feed featureCounts with respect to strandedness and paired-endedness__?   So far I've tried strandSpecific=0,strandSpecific

rnaseq

updated 7.3 years ago • RRnaSeq

Start=202000000, End=202002100, Strand=1) Then I want to count the reads in this region: > featureCounts("MyReadsfile.bam", annot.ext=regions, annot.inbuilt="hg19", ignoreDup=T) I get a huge number of reads, but if I look

Annotation Cancer Breast Rsubread Annotation Cancer Breast Rsubread

updated 9.9 years ago • Oscar Rueda