Bioconductor Forum

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 7.0 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 7.9 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 21 months 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 7.3 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 3.6 years 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 3.3 years 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 9.9 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 6.0 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 4.1 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 6.6 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 11.0 years ago • ccheung

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 7.3 years ago • Leon

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 6.3 years ago • mertes

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 3.8 years ago • agustin.gonvi

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 3.4 years ago • Julia

was done by sequencing facility using STAR) and **1) Generated read counts with RSubread featureCounts** ``` counts<-featureCounts(files=c("WGTR_0001_Le_n_WT_OT1DTAM1.merged.sorted.bam", "WGTR_0001_Le_n_WT_OT1DTAM2.merged.sorted.bam

edger

updated 6.5 years ago • melanie.girard

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 4.0 years ago • cam

Hello, I would like some assistance in figuring out why Rsubread and the bash subread are giving different feature count outputs. I came across this issue because I wrote a wrapper script for a pipeline used by a colleague, and during the validation process we have been trying to resolve why the wrapper feature count table differs from hers. The differences drastically affect the statistical anal…

Subread Rsubread

updated 13 months ago • Aimee

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 4.2 years ago • jac

after aligning using the subjunc function. Is it acceptable (to save processing time) to apply featureCounts() to the subjunc BAM files? Will this produce different feature counts than I would get if I used the align function

rsubread subjunc

updated 10.4 years ago • Stephen Piccolo

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 4.2 years ago • v.merino.nicolas

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 4.8 years ago • alexandr.gopanenko

Hi, I was doing repeat analysis from RNA seq data and learn that featureCounts() function ("Rsubread" package) can provide fraction counts for multi-mapping reads. However, DEseq2 doesn't take

deseq2

updated 10.1 years ago • tg369

down). Below are the codes of how I obtained length info and plots, as well as the plots 1) Using featureCounts results: featCountsData <- read.table("charolais\_145A\_TGACCA.featCounts.txt", header=TRUE, comment.char...nullp(DEgenes = genes, bias.data = testLenData, plot.fit = TRUE) PLOT 1: Length info obtained from featureCounts: https://drive.google.com/file/d/0B6Ho35U9KAepTGxuR0Fx…

goseq

updated 9.7 years ago • mrodrigues.fernanda

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 5.8 years ago • chatterjee.arumoy

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 3.8 years ago • vasa.broz

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 5.5 years ago • barrel0luck

Hi , For the following : > fCountsList = featureCounts(props, annot.ext="/patho/to/gtf/GRCh38.gtf", isGTFA nnotationFile=TRUE, nthreads=16, isPairedEnd=TRUE, allowMultiOverlap

normalization

updated 6.9 years ago • memoly101

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 5.0 years ago • Archit

nbsp;and in Genome Browser that some exons do not belong (or are not included in the analysis of featureCounts from rsubread package) to the gene that is specified by those packages.</span>   <pre> mm9 = TxDb.Mmusculus.UCSC.mm9.knownGene...3 are Xkr4.  I do not know why but only one transcript is included in the in-built version of FeatureCounts of Rsubread p…

rsubread featurecounts

updated 10.3 years ago • tonja.r

Hello, I am trying to combine multiple bulk RNA Seq datasets which contain multiple conditions in all of them. I obtained the counts matrix using featurecounts and I am not sure if I should normalize each dataset and then combine them using ComBat or Limma or combine them...Seq datasets which contain multiple conditions in all of them. I obtained the counts matrix using featurecounts and I am not…

RNASeqData DESeq2 Normalization

updated 18 months ago • Tanvi

Hello, I have a question relative to the featureCounts function, from the Rsubread package: from the documentation: "minOverlap: integer giving the minimum

Rsubread minOverlap featureCounts

updated 9.7 years ago • c.merce

rejecting overlapping reads if I assign them to all isoforms in which they are falling i.e. by using featurecounts and then find differential expression using DESeq. Do you think the results which I got after it will be significant

DESeq ASSIGN DESeq ASSIGN

updated 11.4 years ago • Gourja Bansal

case. Another approach I have used to get the normalized TMM value for upstream regions is to use featureCounts program and then normalize the reads using DESeq TMM normalization. But in that case the value of total counts...here or any parameter values I need to change because for the same dataset with default parameter featureCounts gives me counts that I can further normalize using DESeq (Kind…

chip-seq TSS TMM csaw

updated 9.6 years ago • saadmurtazakhan

Hi Everyone,   I'm trying to analyze some RNAseq results, but one of my samples is a pretty bad outlier by PCA and by clustering over the entire transcriptome. I have 4 groups with 3 biological replicates.  These samples were run in 2 batches. When I try to summarize my reads using RSubread's featureCounts, the outlier has a very low assignment %, with a high % of multiple a…

rnaseq outlier statistics batch effect

updated 9.6 years ago • grastalt27

RNA-Seq data for 8 different treatment conditions, with 3 biological replicates for each. I used the featureCounts command on Rsubread and have the raw counts matrix for all 24 samples at the meta-feature(gene) level. I was wondering

differential gene expression biologicalreplicates raw_counts

updated 7.3 years ago • tasnif.rahman

Hi I essentially have a counts data frame from featurecounts (Rsubread) and its giving me the error seen above..... Does anyone know how I can check input counts file "test4", find

deseq rna-seq

updated 10.8 years ago • rodriguez.varenka

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 3.2 years ago • f99942

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 2.4 years ago • Ya

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 4.4 years ago • Shail

nbsp;org.Mm.eg.db (org.Mm.egENSEMBL) to annotate the differential expressed genes (tophat(mm9)-featureCounts-RUVseq-edgeR). Although the results are nice some genes could not well being annotated and I guess it is is because

edgeR ruvseq rnaseq isoforms

updated 10.6 years ago • sergio.espeso-gil

to a genome using the Gencode M17 comprehensive annotation .gtf file. Then I did read-counting using featureCounts, but I supplied a subset of the annotation, containing only lncRNA annotations. This leads to  only 1-2% of

edger rnaseq

updated 7.7 years ago • martin.weihrauch

res$ensembl, mart = ensembl ) fc$counts are gene IDs and counts from featureCounts function

biomaRt getBM

updated 6.7 years ago • angelica.lindlof

35096 34391 24142 38428 30607 28948 45369 35688 36033 25738 20554 44144 `` The parameters used in featureCounts function are: <pre> countPrimaryAlignmentsOnly=TRUE, isPairedEnd=TRUE, annot.ext='gencode.v24.chr_patch_hapl_scaff.annotation.gtf

rsubread

updated 9.8 years ago • Likai Mao

I am using DESeq2 to run a multifactor paired analysis to determine the change in gene expression between men and women due to a treatment on 70 males and 45 females. Because I have a differing number of samples for each condition, I'm using the following steps to create a custom design matrix: https://support.bioconductor.org/p/63134/\#63146 Everything is fine until estimateDispersionsGeneEst(d…

deseq2 differential gene expression model matrix

updated 8.9 years ago • gregory.l.stone

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 4.0 years ago • mohammedtoufiq91

site led me to this [discussion](https://support.bioconductor.org/p/65604) on the performance of featureCounts for that purpose but it has no details about how the annotations are input to the software. i'm familiar with

GenomicFeatures repeatmasker GenomicAlignments

updated 8.2 years ago • Robert Castelo

I want to plot trends between genes across different conditions from my normalized count matrix (featurecount-EdgeR). I was using "csCluster" in cummerbund. Now I switched to this pipeline and I am wondering, if similar pacakage

edger clustering visualization

updated 7.3 years ago • thindmarsmission

nbsp; I'm wondering whether I should be using the raw counts output from read summarisation (eg featureCounts output) or say the "E" matrix of normalised log2 counts from the EList object after voom transformation, or if

limma rnaseq geneexpression voom counts

updated 11.2 years ago • Peter Crisp

all reads are reversed with respect to the genomic sequence of the transcript and annotation. With featureCounts I would have to use -s 2 option, for example, to get a 70+% rate of assignments to transcripts. But now I am very concerned

easyrnaseq rnaseq strand

updated 8.6 years ago • Michael Dondrup

Hi all - Hoping I can get some guidance on an issue I'm having with the _featureCounts_ function in the _RSubread_ package. I've successfully aligned 150bp paired-end reads to the mouse genome and am trying to summarize the reads to features. My problem is that if I summarize with strandSpecific=1, I get the following output showing less than 3% of fragments assigned to features: …

rsubread featurecounts rnaseq

updated 10.7 years ago • beiting

More perplexingly, they have 100% sequence identity with a single gene. My question, then, is why featureCounts is failing to map these sequences? I've thought to change the stringency settings, but I doubt that will make

featurecounts rnaseq subread

updated 7.4 years ago • rogangrant

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 2.8 years ago • Assa Yeroslaviz

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 2.3 years ago • Shihui

packages/release/bioc/html/Rsubread.html) aligner and were then summarized to genes using the [featureCounts](http://bioconductor.org/packages/release/bioc/html/Rsubread.html) program. This package includes the gene

seqc rsubread featurecounts subjunc ercc News

updated 11.1 years ago • Wei Shi

with DESeq2 using the least variable Drosophila genes (dm6) and applied it on my human counts (from featureCounts -t exons). ``` dds_dm6 <- DESeqDataSetFromMatrix(countData = counts_dm6, colData = sample_info, design = ~ treatment

SpikeIn DESeq2 Normalization

updated 16 months ago • ADopico

RNA Seq data from human biopsies (2 groups, 50 samples each). My pipeline is trimmomatic-star-featureCounts-edgeR-voom/limma (big fan of limma, being a veteran from array days..). <span style="line-height:1.6">I am looking into

limma edger sequencing

updated 9.4 years ago • blofeld

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 21 months ago • Laura

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 2.9 years ago • mohammedtoufiq91

The featureCounts manuscript mentions that for RNA-Seq multi-overlapping reads must not be counted and the reasoning seems

RNA-Seq featureCounts Rsubread

updated 10 months ago • Arindam