Bioconductor Forum

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 8.6 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 14 months 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 14 months 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 6.9 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 10.8 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 7 months 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 6.9 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 6.5 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 6.9 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 6.6 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 5.9 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 17 months 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 4.1 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 3.5 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 4.8 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 10.8 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 17 months ago • Soniya.sherpa.lama

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

featurecounts

updated 22 months 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 5.8 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 10.0 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 16 months 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 how is genecounts defined? Can I use the transcript-count…

deseq2

updated 5.5 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 6.5 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 9.0 years ago • Ryan C. Thompson

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 7.8 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 7.2 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 5.1 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 21 months 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 8 weeks 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 4.0 years ago • sekhwal

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 10.8 years ago • Wei Shi

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 7.9 years ago • myprogramming2016

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 6.3 years ago • martin.weihrauch

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 8 months 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 4.8 years ago • algenubi81

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

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 5.3 years ago • memoly101

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 4.6 years ago • ajajoo

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 6.8 years ago • Morris

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 9.5 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 6.0 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 2.0 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 7 months 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 2.4 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 8.3 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 5.0 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 3.3 years ago • Anubhav

vs control). Briefly, I mapped the RNA-seq reads to the reference genome using STAR and I used featureCounts to quantify the reads mapped to the reference genes and found the gene counts. Before I start the deferentially

deseq2 rnaseq differential gene expression featurecounts rnaseqGene

updated 5.3 years ago • melkile26

to import each single cells. It would be nice if also something like this would be possible with featurecounts() . Thanks!   <pre> Filter <- list(1_cell.barcode) names(Filter) <- "CB" BamParam <- ScanBamParam(what=scanBamWhat

Rsubread featurecounts featurerequest

updated 6.3 years ago • alessandro.pastore

5, minOverlap=1) # get counts (mapQCth=0 was kept to match with results from DESeq2/featurecounts, summits=FALSE for counting over full peak ) obj <- dba.count(obj, minOverlap=1, score=DBA_SCORE_READS, summits...rather it is using raw counts to plot PCA. I rectified it separate analysis with quantification with featurecounts/DESeq2 and plotting PCA on both vst transformed coun…

DiffBind PCAplot

updated 5 weeks ago • Ankit

I try to analyse RRHP data. I think I have to count my reads for each of my samples. So I used featurecount on my bam files, but I had low coverage... Is anybody have already count reads from DNA seq? Thanks a lot!!! Christelle

RRHP

updated 5.6 years ago • christelle.ledantec

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

Dear All, I am using GTF file to use as input of FeatureCount along with .bam file, but I am getting error. I read many post on forum related to this query but could not get proper

FeatureCounts

updated 20 months ago • KMS

Based on the manual, featureCounts function in Rsubread set countPrimaryAlignmentsOnly=FALSE by default. By setting countPrimaryAlignmentsOnly...Based on the manual, featureCounts function in Rsubread set countPrimaryAlignmentsOnly=FALSE by default. By setting countPrimaryAlignmentsOnly=TRUE, as alignments used are less, the counts seem to be less too. But I go…

rsubread

updated 8.3 years ago • Likai Mao

e -B -G ${GTF} -o transcripts.gtf -A gene_abundances.tsv input.rmdup.bam **Deseq2 (using featureCounts counts)** featureCounts -T $threads -p -F GTF -t exon -g gene_id -s 2 -a ${GTF} -o out.featurecount input.rmdup.bam **FPKM values

deseq2 StringTie RNA-seq normalisation

updated 4.4 years ago • JindrichK

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

normal）, 2 experimental groups（Esophageal cancer），The result is Figure 1.![Figure 1][1] I use featurecounts to count genes, this is my [counts array][2] [1]: http://chuantu.xyz/t6/702/1558315882x2890202416.png [2]: http://223.94.4.98

deseq2 cancer

updated 5.0 years ago • xuyao15927402897

Hi Everybody, I 44 samples and >76000 transcripts in my RNA Seq data which was counted using FeatureCounts. I'd like to reduce the number of transcripts by filtering out the transcripts in which 80% of the samples (35...Hi Everybody, I 44 samples and >76000 transcripts in my RNA Seq data which was counted using FeatureCounts. I'd like to reduce the number of transcripts by filte…

deseq filter reads

updated 9.3 years ago • ccheung

with not problems. Thanks in advance for any help. Best regards, Christian Mertes ``` > featureCounts(file="HG00356.2.M_111215_6.bam", annot.ext=ranges, isLongRead=FALSE) ========== _____ _ _ ____ _____ ______ _____ ===== / ____..._____/ \____/|____/|_| \_\______/_/ \_\_____/ Rsubread 1.34.7 //=======================…

Rsubread counting RNA-seq

updated 4.6 years ago • mertes

Hi, The options `` read2pos `` and `` readExtension3 `` are usefull for counting fixed fragments (). Recently I considered one condition of counting reads based on their 5' most base, whose position was shifted. For example, the 5' most base is first shifted by a fixed number based on POS and CIGAR fields, then reads are summarized. It is useful if the middle point of fragments are considered in…

featurecounts subread

updated 5.7 years ago • Leon