Bioconductor Forum

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 6.9 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 7.6 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 9.8 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 2.3 years 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 11.0 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 8.6 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 7.2 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 3.1 years 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 12.4 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 12.1 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 9.0 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 11.5 years ago • Oscar Rueda

pre> > dxd = estimateSizeFactors(dxd) Error in estimateSizeFactorsForMatrix(featureCounts(object), locfunc, : every gene contains at least one zero, cannot compute log geometric means > head(dxd) class

DEXSeq DESeq2 dexseq deseq2 alternative splicing

updated 10.2 years ago • Fahmi Nazarie

generating a count matrix from this input using Rsubread package? The following code using `featureCounts` works, but I believe it is improper to use on single cell data? ```r countsmat<-featureCounts( files = c("LN.bam","blood.bam

Rsamtools Rsubread

updated 2.9 years ago • Christopher

Hello everyone, I'm having this problem with subread 2.0.3 featureCounts in Linux OS. This gtf file is converted with gff3 file using gffread. This is my using code. ``` featureCounts -T 10...__| | ========== |_____/ \____/|____/|_| \_\______/_/ \_\_____/ v2.0.1 //========================== featureCounts setting ===========================\\ Input files : 1 …

Subread GTF featureCounts segmentationfault

updated 2.9 years ago • yao hua

feature counts as implemented in Rsubread on my mac My script is: library(Rsubread) b1.res<-featureCounts(files="b1.res.bam",annot.inbuilt="hg38",isPairedEnd=TRUE,requireBothEndsMapped=FALSE) I get an output file

featurecounts hg38

updated 8.6 years ago • raf4

Thanks for sending through the data which helped us to identify the problem. The problem was that featureCounts did not allow the chromosome names in the provided annotation file to be longer than 48 characters. We have...increased this limit to 100 characters. This solved the problem and featureCounts worked fine for your data now from our testing. We have committed the changes to bioc devel re…

Annotation GO BSgenome cdf BSgenome Rsubread Annotation GO BSgenome cdf BSgenome

updated 12.4 years ago • Wei Shi

I got an error using subread v 2.0.6 Assertion failed: (NULL == pairer -> bam_margin_table -> appendix2), function SAM_pairer_finish_margin_table, file input-files.c, line 4672. zsh: abort featureCounts -O -T 8 -a stringtie_output/concat_output/concat_output.gtf -o I checked the integrity of my bam file and it looks...gt; appendix2), function SAM_pairer_finish_margin_tab…

NewWave

updated 2.3 years ago • Kilian

Dear Bioconductor list: I have aligned some paired end reads to the human genome assembly CRCh38.p7 with R subread. I am now trying to count the reads with feature counts. It is taking over an hour on my mac, and as I recall it should run faster. This is teh first time that I have used featurecounts with a GFF file as distinct from the built in annotation, and I am wondering if th…

rsubread featurecounts gff Grch38.p7 vs hg38

updated 8.6 years ago • raf4

Hi, I'm about to analyze ATAC-seq data for the first time and my approach atm is: 1. Calling peaks 2. Merge the narrow peaks files into one 3. Convert the merge bed to saf format for input in featureCounts 4. Using featureCounts (Rsubread package) to get counts 5. Use the counts as input to DESeq2 The thing is that I'm stuck at step 4. Since I cant make sense of the output I get. If I try …

Rsubread atac-seq narrowpeaks featurecounts

updated 8.1 years ago • j.bergenstrahle

There is some problem with the format of the GTF files? I used the following commands: <pre> x<-featureCounts("accepted_hits.bam", annot.ext="transcripts_exon.gff", isGTFAnnotationFile + =TRUE, GTF.featureType + ="exon

rnaseq

updated 8.6 years ago • sancharisircar24

Hi everyone, I have exonic counts for 344 samples for 15 genes that include some house-keeping genes like GAPDH and Actin genes using dexseq\_count. I am trying to normalize them like this: <pre> dxd = DEXSeqDataSetFromHTSeq( countFiles, sampleData = sampleTable, design= ~ sample + exon + disease_type:exon, flattenedfile = flattenedFile) head(sampleTable)   &…

dexseqdatasetfromhtseq dexseq_count dexseq

updated 8.3 years ago • komal.rathi

and one of my main goals is to map lncRNAs (long noncoding RNAs). I am using STAR for alignment and featureCounts for mapping (counting). For the lncRNAs, I think I need to use the annotation file gencode.v28.long\_noncoding..._RNAs.gtf.gz.  I will pass this annotation file on to the counter featureCounts.   I have already created a Genome Index for STAR but using the differen…

lncRNA featureCounts STAR annotation

updated 7.6 years ago • inah

to summarize counts to the gene level." These are the parameters that I am using: ``` mock1 <- featureCounts(files="mock1_Aligned.sortedByCoord.out.bam",isPairedEnd=TRUE, GTF.featureType="exon", GTF.attrType="gene_id

Subread RNASeq featureCounts quantification

updated 3.1 years ago • Sara

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 5.8 years ago • Konstantinos Yeles

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 5.1 years ago • juls

I am using featureCounts for my RNA-seq data to get count matrix. but m facing some problem with my output file. Most of the file is fine...I am using featureCounts for my RNA-seq data to get count matrix. but m facing some problem with my output file. Most of the file is fine but

rna-seq featureCounts SubRead

updated 6.4 years ago • hafiz.talhamalik

for sending through the data which helped us to identify the > problem. The problem was that featureCounts did not allow the chromosome > names in the provided annotation file to be longer than 48 characters. We &gt...have increased this limit to 100 characters. This solved the problem and > featureCounts worked fine for your data now from our testing. > > W…

Annotation GO BSgenome cdf BSgenome Rsubread Annotation GO BSgenome cdf BSgenome

updated 12.4 years ago • Alan Smith

So I'm basically getting these 'successfully assigned reads" of around 30 - 45%. When I did STAR I got alignment reads of like 70-80% which is really good. I know that FeatureCounts counts the amount of reads (achieved in STAR) appeared to overlap with known genes. And so it's normal that it's...30 - 45%. When I did STAR I got alignment reads of like 70-80% which is really good. I know that Featu…

LowAssignedReads STAR featureCounts

updated 3.1 years ago • Soniya.sherpa.lama

featureCounts setting ===========================\\ || || || Input files : 1 BAM file || || || || DBV07.sort.bam || || …

featurecounts

updated 3.5 years ago • 哲

Hello,  I am working on quantifying RNASeq data at the exon level, which is intended for use in the DEXSeq package. I followed the documentation provided in the DEXSeq tutorial (http://bioconductor.org/packages/release/bioc/vignettes/DEXSeq/inst/doc/DEXSeq.pdf, page 23), as well as the commands on Subread to DEXSeq helper code (https://github.com/vivekbhr/Subread\_to\_DEXSeq). …

subread featurecounts dexseq rnaseq

updated 7.5 years ago • jennyl.smith12

I have used Cufflinks RPKM values, but if I want to use edgeR, is this a valid way of doing it using featureCounts: fc <- featureCounts(files=targets$Targets,nthreads=8, isGTFAnnotationFile=TRUE, GTF.attrType="gene_id", GTF.featureType...x) expr_norm <- rpkm(expr, log=FALSE,gene.length=x$genes$Length) # Getting gene length from FeatureCounts, using rkpm() in the edgeR package, not…

RNASeq edgeR RNASeq edgeR

updated 11.7 years ago • Sindre

RNA star (to obtain Star counts similiar to the new TCGA pipeline) and afterwards I used the featurecounts function. The data from the featurecounts function I combined into one excel file (countsm) and I also created...Subsequently I performed the DESEq2 analysis as described in the pipeline. Since I used the featurecounts function, I followed the "countsmatrix" vignette steps. After I obtai…

DESeq2 star differentiallyexpressed

updated 3.0 years ago • nico

Hi, When one have generated counts using FeatureCounts it generates a list of transcripts and note genes. I understand the procotol as "gene"-counts should be used...Hi, When one have generated counts using FeatureCounts it generates a list of transcripts and note genes. I understand the procotol as "gene"-counts should be used - however...Hi, When one have generated counts using FeatureCounts…

deseq2

updated 7.2 years ago • SannaG

in both tools are the same.  While reading a bit more I came across the paper introducing "[featureCounts](https://academic.oup.com/bioinformatics/article/30/7/923/232889/featureCounts-an-efficient-general-purpose...program)". When they compared featureCounts with summarizeOverlaps and HTSeq in section 5.2, the results of summarizeOverlaps and HTSeq also slightly

R summarizeoverlaps htseqcounts

updated 8.2 years ago • Walter F. Baumann

In the documentation for featureCounts, it doesn't say whether endpoints are included or excluded. So which of Start and End, if any, are included in the

rsubread featurecounts saf

updated 10.6 years ago • Ryan C. Thompson

Hi, I have a question regarding the quantification summary file that featureCounts produces. My paired-end RNAseq data includes some read pairs of which only one mate is mapped, while the other...Hi, I have a question regarding the quantification summary file that featureCounts produces. My paired-end RNAseq data includes some read pairs of which only one mate is mapped, while the other o…

featureCounts Rsubread

updated 17 months ago • Anke

machine. So they have to divide into 2 batches for sequencing. Following sequencing, I used Tophat2-featureCounts-DEseq2 pipeline to analyze them.  My question is: should I merge the FeatureCounts result into one file...tr> <tr> <td>F.3</td> <td>ConditionF</td> </tr> </tbody> </table>   Can I run Tophat2-featureCounts…

deseq2 featurecounts rnaseq

updated 9.4 years ago • EJ

Hi, is there a way to automatically convert the featureCounts result matrix into a GRanges (IRanges) object? by feeding it into GRanges? I ran featureCounts for the data set...is smaller than the end position of the gene.  <pre> countsTable <- read.delim2(file = "../featureCounts/featureCounts.geneLevel.txt", header = TRUE, sep = "\t", quote …

genomicranges iranges featurecounts counts rnaseq

updated 8.9 years ago • Assa Yeroslaviz

Hello, I'm processing RNA-seq data (sampleA, sampleB) with featureCounts, and DE analysis with DESeq2. In the DESeq2 output, there are only baseMean, log2FoldChange... I would like to extract...the expected counts calculated by DESeq2 (already normalized, not the raw read counts from featureCounts). Is there anyone knowing how to extract the expected counts value for each sample? For…

deseq2

updated 6.8 years ago • cwliu2014

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 dataset

RNASeqData DESeq2

updated 3.4 years ago • btadfaramin

When running FeatureCounts with -R (report reads) the output bam file reads are tagged with XS, XN and XT. I have bam files aligned with HISAT2...as SAMTOOLS view -d tag filtering fails to correctly filter by XS tag. Is it possible to modify the FeatureCounts XS tag to another tag ID? Thanks in advance, Chris

FeatureCounts Rsubread

updated 22 months ago • chris2.a.white

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 5.6 years ago • sekhwal

Hi, I work on plant species. I have used STAR to map RNAseq reads and featureCounts to get expression values. I would like to counts the number of reads map to the genes and outside of genes. Is...Hi, I work on plant species. I have used STAR to map RNAseq reads and featureCounts to get expression values. I would like to counts the number of reads map to the genes and outside of genes. Is there

star featurecounts edgeR RNAseq

updated 9.6 years ago • myprogramming2016

using Rsubread. The number of transcripts in the annotation > file would not be an issue -- our featureCounts function supports > unlimited number of annotations and chromosomes/transcripts (actually, at > most...We >>> have increased this limit to 100 characters. This solved the problem and >>> featureCounts worked fine for your data…

Annotation GO BSgenome cdf BSgenome Rsubread Annotation GO BSgenome cdf BSgenome

updated 12.4 years ago • Wei Shi

the log-RPKM values ``` plotMDS(logRPKM, gene.selection="pairwise") ``` #Creating a DGEList from featureCounts If the count matrix is created using `Rsubread::featureCounts` then the output can be transformed to a DGEList...directly, without any need for intermediate data files: ``` fc <- featureCounts( ... ) y <- featureCounts2DGEList(fc) ``` The resulting DGEList object will aut…

rpkm edgeR featureCounts

updated 2.3 years ago • Gordon Smyth

Hello! I can't run featureCounts with gff3 file. Please, help! My **gff3 file** structure is: ``` chr1 . miRNA_primary_transcript 17369 17436 . - . ID=MI0022705...ID=MIMAT0027619;Alias=MIMAT0027619;Name=hsa-miR-6859-3p;Derives_from=MI0022705 ``` **Command line:** ``` ./featureCounts -F -a ./../annotation/hsa_miR.gff3 -o ./../../../projects/vesicles/1_repeat_vesicles/counts_K.txt ./../..…

subread featureCounts gtf/gff/gff3

updated 6.5 years ago • algenubi81

Hi, I'm using featureCounts() to generate the count matrix of my RNAseq experiment and edgeR to do the DGE analysis. Now, during the filtering...step I am removing low-count genes like this: fc <- featureCounts(..) counttable <- fc$counts y <- DGEList(counts=counttable) __keep <- rowSums(cpm(y) > 1) >= 4__ \# Keep genes only if...but those lincRNAs …

bioconductor edger rna-seq

updated 8.0 years ago • martin.weihrauch

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 4.3 years ago • jbnrodriguez

so I'm using feature counts from Rsubread and I have the following ERRORs after running: > featureCounts(BAMfiles, annot.ext="//path/to/gff/", isGTFAnnotationFile=TRUE, nthreads=16, isPairedend=TRUE, allowMultiOverlap...multi-overlapping features. Error in file(file, "rt") : cannot open the connection Calls: featureCounts -> read.delim -> read.table …

annotation Rsubread featureCounts

updated 7.0 years ago • memoly101

Hi, I installed Rsubread and now I want to extract raw counts from RNAseq bam files. Could you please give me the command line to use in order to analyse one or more bam files? thank you

featurecounts

updated 8.5 years ago • Morris

I can filter bam file from above command to pass it to feature count. This is what I use right now. featureCounts -p -T 5 -a hg38GENCODE.gtf -o $filename.txt $filename.bam & I am running data in bulk, that requires bwa mem with...above parameters so do not want to use resources for e.g. STAR alignment etc for passing it to featureCount. I am very new to this field, so please d…

bwa mem featureCounts

updated 6.3 years ago • ajajoo

Hi everyone, I'm trying to export the featureCounts output results of Rsubread to a .xls format (excel), but I'm not having success. I have got the counts of the reads...Hi everyone, I'm trying to export the featureCounts output results of Rsubread to a .xls format (excel), but I'm not having success. I have got the counts of the reads from my RNASeq experiment, but I can't export the result. I…

rnaseq rsubread featurecounts r

updated 11.2 years ago • gustavoborin01

but I decided to use DESeq2 for that. I used MACS for peak calling and I had BAM files. Then used featureCounts to generate counts matrix. To this count matrix, I used DESeq2.   Everything looks fine but I am not sure is

chipseq deseq2 featurecounts differential binding analysis

updated 7.6 years ago • hkarakurt

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 3.7 years ago • Chise

Hi I am running featureCounts() function from Rsubread package with my aligned dataset. I am using HPC clusters to perform the task. my Rscript...looks something like this. ``` fc <- featureCounts(files=fileNames, annot.ext=file.path("/lustre/project/jfang5", "Homo_sapiens_GRCh38", …

featureCount Rsubread HPC

updated 2.3 years ago • kthapa

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 4.0 years ago • Chise

to continue with): __dxd__ = DEXSeqDataSetFromHTSeq(...) __dxdn1__ <- dxd\[rowSums(cpm(featureCounts(dxd))>1) >= 4\] __dxdn2__ <- dxd\[rowSums(cpm(featureCounts(dxd))>2) >= 4\] __dxdn5__ <- dxd\[rowSums(cpm(featureCounts

DEXSeq rownames pre-filtering

updated 9.9 years ago • arom2

packages/Rsubread/versions/1.22.2/topics/align I wonder whether the options of the **featureCounts** function could matter too... https://www.rdocumentation.org/packages/Rsubread/versions/1.22.2/topics/featureCounts

align rsubread roche454 rnaseq

updated 6.7 years ago • Raito92

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 5.0 years ago • Anubhav