Bioconductor Forum

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, described...to 0. For the rest of the post, I will be discussing the results when using the following call to featureCounts (where the goal was to assign reads to a gene with which it completely…

featureCounts Rsubread

updated 18 days ago • isaac.vock

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 20 days ago • Ankit

the 4 count files but omit any row that do not appear in all 4 files. Any help would be appreciated. ``` featureCounts -T 5 -p -t exon -g gene_id -a panTro6.ncbiRefSeq.gtf -o panTro6_counts.txt bam1 bam2 bam3 bam4 bam5 bam6 bam7 featureCounts...t exon -g gene_id -a panPan3.ncbiRefSeq.gtf -o panPan3_counts.txt bam1 bam2 bam3 bam4 bam5 bam6 bam7 featureCounts -T 5 -p -t exon -g gene_id -a ponAbe3.…

rnaseqGene

updated 22 days ago • Christian

genes across species. I was planning to filter the table of raw counts (previously obtained with featureCounts) to obtain the raw counts of these ~2500 orthologs across species replicates in condition B, normalize them

DESeq2

updated 23 days ago • Laura

alteration between conditions of **mice** samples. I made a workflow with Rsubread to align and featureCounts to count. For splicing I switched to the subjunc() function. It worked well. Then I discovered the "reportAllJunctions

Rsubjunc Rsubread

updated 24 days ago • thomas.heigl.ibk

I am using v2.0.6 of `featureCounts` on a Linux machine. I am running the following command: ``` $ subread-2.0.6-Linux-x86_64/bin/featureCounts -a fc_test.saf...NC_086317 ``` And run the following command: ``` $ subread-2.0.6-Linux-x86_64/bin/featureCounts -a fc_test.saf -F SAF -o fc_test.counts.out -T 2 -p -A aliases.txt fc_test.bam ``` Still, I see that none of the alignments...from th…

featureCounts subread Rsubread

updated 28 days ago • vkkodali

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 6 weeks ago • chris2.a.white

Hi! I did the code for exactTest and for glmQLFTest for my 153 samples. I used raw counts (DGE) for the DE analysis. Resuts from the exactTest: 10 genes with FDR<0.05 and 1651 genes with p-value< 0.05. With glmQLFTest results, all the p-values are >= 0.99 and all the FDR values are =1. Why are the results so different? glm approach ```r library(edgeR) library(tidyverse) l…

edgeR

updated 7 weeks ago • fr8712ca-s

analysis; I got the mouse GTF file from NCBI). I have attached screenshots of the summary of the featureCounts process as well as the .txt file I received as an output. Most of the columns of the .txt file look normal but there...txt to .xlsx in R. Am I missing something in this conversion or is there something wrong with my featureCounts output file? As you can see from the attachment, it did no…

GenomeWideAssociation countsimQC RNASeq

updated 7 weeks ago • ryann

Hi, I am a master student in biomedicine at Lund University in Sweden. I am using edgeR for differential expression of genes. I am a beginner in the use of edgeR. ``` setwd("C:/Users/Analysis") library("edgeR") library(tidyr) data <- read.csv("C:/Users/Analysis/Data/Case_Control_Phenotype_Features.csv") group <- as.factor(data$T2D) #group include individuals with T2D (YES) and n…

RBioinf

updated 8 weeks ago • fr8712ca-s

Hello, I'm trying to use featureCounts to annotate a long read dataset obtained from nanopore sequencing. I noticed from the results reads that...Hello, I'm trying to use featureCounts to annotate a long read dataset obtained from nanopore sequencing. I noticed from the results reads that are...reads were assigned to all 3 ACTG1 isoforms. ![enter image description here][1] I was runni…

featureCounts Rsubread

updated 10 weeks ago • Alex

When featureCounts is used to count miRNAs present in genome aligned bam files, it gives miRNA size outputs, as well as long fragments...ie 8kb). I think this is happening because overlapping miRNAs exist, and thus featureCounts cannot distinguish where an individual miRNAs starts and ends in this situation (when there is an overlap...some genomes there are 0 known miRNAs). Thus I have the follo…

featureCounts Rsubread

updated 11 weeks ago • tjgray4

Hello, I am trying to use featureCounts() to assign RNA-seq reads to the set of annotated transcript isoforms with which they are completely consistent...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...bam file. I am hostin…

featurecounts Rsubread

updated 12 weeks ago • isaac.vock

bam_file <- "path/aligned_reads.bam" gtf_file <- "path/to/annotation.gtf" counts <- featureCounts(files = bam_file, annot.ext = gtf_file, isPairedEnd = TRUE)" what could be wrong???? GTF file is absolutely fine i did

Rsubread BAMfiles GTFfile

updated 12 weeks ago • mirjanakessler

the reads back to genes found in the assembly and counting up the reads mapped per gene using featureCounts, I then divided the total reads by gene length, and now would like to sum up these coverages by KO to get a sense

DESeq2 medianofratiostransformation StatisticalMethod Normalization dispersion

updated 3 months ago • Linton

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 5 months ago • HS

and two time points for uninfected samples. I have a count matrix that was generated using featureCounts after alignment of reads using bowtie2, and the matrix includes reads mapping to the bacteria as well as the

RNASeq DESeq2

updated 6 months ago • Sean

Hello, I have a rather simple question: featureCounts has a long-read mode ("isLongRead" parameter). How does this parameter affect the counting behavior of featureCounts

Rsubread LongRead

updated 6 months ago • mmisak

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

pulled down the protein and sequenced both IP and input. I got the reads assigned to each gene using featurecount and use that as input to Deseq2, and I have two replicates for IP and input. After differential expression analysis...Log2FC from Deseq2, however, the log2FC is not consistent with the RPM I calculated (RPM calculation: featurecount assigned reads/total reads *1000000). Here is an e…

RNASeqData DifferentialExpression SmallRNAData DESeq2

updated 6 months ago • Shihui

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 6 months ago • Kilian

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 6 months ago • kthapa

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 7 months ago • Gordon Smyth

Since I had forgotten to output the BAM file, I mapped again using only STAR. However when I run featureCounts, for the same sample: ``` featureCounts(files = BAM1, isPairedEnd = TRUE, allowMultiOverlap = TRUE, countMultiMappingReads...and the log looks the same as mentioned above. This time I outputted the .genome.bam file, and for featureCounts: ``` feat…

RNASeqData Rsubread mapping featurecounts

updated 8 months ago • vitoriastavis

transcripts into a single feature, just like what `bedtools merge` does. 2. When I use the `featureCounts` of `Subread` package (v2.0.3, command-line version), will `-g gene_id -t transcript` and `-g gene_id -t gene` give me same

Rsubread ensembldb

updated 8 months ago • Ya

I am a new learner of DESEQ2 and have seen similar posts on this forum but could not find clear answers. What would be the best way to create heatmaps specifically with 2 log fold change(base 2) in DESEQ2 or R in general? In DESEQ2, heatmaps are created based on VST (Variance Stabilizing Transformation) values. The count data I've used is unnormalized raw counts from FeatureCounts. I would…

DESeq2

updated 9 months ago • bio2249

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

tool on a local instance of Galaxy (I find the GUI really easy to use, plus I can upload batches of featureCounts files rather than combining them manually). Today, I went to check my results by running it in R. Both were fresh

DESeq2

updated 11 months ago • murphytho1401

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 11 months ago • Carlos

Hello all, From my understanding of the featureCounts manual it should, by default, count reads that align to the features (exons) of a meta-feature (gene). However my...Hello all, From my understanding of the featureCounts manual it should, by default, count reads that align to the features (exons) of a meta-feature (gene). However my output...file is at the exon level (sorry for the…

GeneExpression

updated 12 months ago • CDSPARKS

Hi All, I want to extract the counts that are arising form pre-mRNA (i.e non-split reads). For the sigle-end library kind of easy but for the paired end the situation is bit different. Because FWD reads in the exon and reverse reads are in the introns so i don't know how to extract this information. I have searched quiet a lot but not able to find the information. My read size is 60 nt and …

rsubread featurecounts Bioconductor countsextraction

updated 12 months ago • barrypraveen

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

sample represent one cell. I have used `STAR` to map the samples against my indexed genome and `featureCounts` to quantify the reads onto the genes. If I do a pre-filtering before running the DESeq function only very little

DESeq2 SMART-seq

updated 13 months ago • Assa Yeroslaviz

Ensembl/Gencode GRCh38 for mapping total RNA-Seq projects with STAR, and then counted reads with the featureCounts command from Subread or Rsubread. We are attempting to set up new pipelines with the latest release 43 (GRCh38.p13...differ (NC_060930.1). What (if any) modifications can be made to run Subread or RSubread featureCounts on the new NCBI release and obtain counts data for all ~57 th…

CHM13v2.0 featureCounts T2T Rsubread

updated 13 months ago • Hilary

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

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

Before using this pipeline I used to get started from the raw gene counts from `featureCounts` then use in EdgeR. ```salmon.merged.gene_counts_length_scaled.rds salmon.merged.gene_counts_length_scaled.tsv

salmon edgeR tximport nf-core gene_counts

updated 14 months ago • mohammedtoufiq91

converting counts to integer mode Warning message: 0 aggregate geneIDs were found truncated in featureCounts output Error in `$<-.data.frame`(`*tmp*`, "dispersion", value = NA) : replacement has 1 row, data has 0 Calls: estimateDispersions

DEXSeq

updated 14 months ago • Ben J

Hi, I am running my bulk RNA-seq analysis, I use feature counts from subread package to generate my raw counts from bam files using paired-end option. Do I still need to give library type when I use DESeq() function to get my DEGs? I appreciate your help!

DESeq2 featurecounts

updated 14 months ago • Bo

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

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 15 months ago • nico

files with czid-dedup - Used HISAT2 for allignment with GRCm38 as the reference genome and used FeatureCounts to summarize counts for genes - Used Bayesian method based on mRNA counts provided by DESeq2 for differential

DESeq2

updated 15 months ago • Yongqing

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 16 months ago • Soniya.sherpa.lama

assignment via minimum fractional overlap (--fracOverlap) using featureCounts stand-alone binary. 2) when combined with --/largestOverlap and --/fraction using Rsubread featureCounts function...or an issue? Please find below my test Bash script which generates BAM and SAF files and runs featureCounts stand-alone or Rsubread featureCounts via Rscript. It requires SAMtools, Rscript and featureCou…

Rsubread featureCounts

updated 16 months ago • Konstantin

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

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

Hello, I am having trouble getting an output file in Rsubreads using featureCounts. I want to set up my data to run analysis of differential expresssion in EdgeR. I'm running about 40 .bam files...Hello, I am having trouble getting an output file in Rsubreads using featureCounts. I want to set up my data to run analysis of differential expresssion in EdgeR. I'm running about 40 .bam files in...…

RNAseq R Rsubread featurecounts RNASeq

updated 17 months ago • Jon

Using featureCounts v2.0.1, I don't see any effect in changing `-d` and `-D` options (minimum and maximum fragment/template length, respectively...Here's example output using silly values of `-d 10 -D 10000` and `-d 10000 -D 10`. No difference: ``` featureCounts -d 10 -D 10000 -a genrich/ATACseq/single/atac_merge.gff -o tmp.tsv -t peak -g locus_id -p bwa/Ap2oGFP1o.pb.bam cat...0 Unassigne…

subread featureCounts

updated 18 months ago • dario.beraldi

Hi, I am currently running FeatureCounts following mapping meta-transcriptomic data back to various reference genomes. I have predicted genes using...CDS 47 1186 127.9 - 0 ID=1_1;partial=00;[…] I looked to run featurecounts using parameters -t CDS, -p, and -g ID for this file, and it did not map any reads. However, when I use the NCBI downloaded...and this file maps 52% of th…

RNASeq FeatureCounts Rna

updated 18 months ago • rridley3

infection consisting of 3 time points and a virus free control with 3 replicates each produced with featureCounts. Normalization after running template_script_DESeq2.r: ![enter image description here][1] It does not look

DESeq2 viral controls

updated 18 months ago • f99942

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 19 months ago • KMS

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 19 months ago • Jiayi

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 20 months ago • btadfaramin

Essentially, due to low input RNA (issue with sequencer), there's a high number of 0s in the gene count matrix with featurecounts, and this gives a poor dispersion estimate (shown below, with ~60,000 genes) ![enter image description here][1] Then to deal with this, I filtered out the lowly expressed genes (with idx <- rowSums( counts(dds, normalized=TRUE) >= 5 ) >= 3)…

dispersionestimates dispersion DESeq2 estimates deseq2

updated 20 months ago • Julia

Hello! I am trying to run featureCounts v1.6.2 to use for DESeq2. I am running the following command: ./featureCounts -T 8 -s 2 -t gene -o /bighome/lastarr/Practice_DA_RNAseq...featureCounts/input.counts.txt -a /bighome/lastarr/c_elegans.PRJNA13758.WS285.canonical_geneset.gtf /bighome/lastarr

DESeq2

updated 21 months ago • lastarr

Hi, I read in this site that gff3 format is also acceptable for featureCounts and I don't have gtf but only gff3 file, but it doesn't work. ``` featureCounts -T 6 -t exon -g gene_id -Q 30 -F GTF -a medtr.R108_HM340.gnm1.ann1.85YW.gcv_genes.gff3

gtf/gff3 subread featureCounts

updated 21 months ago • selmanurkeskin

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

featurecounts

updated 21 months ago • 哲

very-sensitive-local` or others such as miRDeep2. When aligned to the genome, one can use e.g. `featureCounts` with mature miRNA coordinates from miRBase to assign features and when aligned to miRase sequences directly

tximport DESeq2 edgeR swish miRNA

updated 22 months ago • BG

of genes in this cell line. Fastq files for all these 8 experiments were processed using Hisat2 and featureCounts to get gene counts. Replicates from each exp vary from 1 to 3. To complicate matters, half the exp were performed

RNASeq BatchEffect DESeq2

updated 22 months ago • Alexandre

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