Tag: HTSeq-count

HTseq reports missing attribute name

HTseq reports missing attribute name 1 Hello, I am running this htseq command htseq-count -r pos -t gene -i gene -s yes -f bam \ /Volumes/cachannel/ZebraFinchBrain/CB-4a_genomemapping/sorted_alignmentcb4a.bam \ /Volumes/cachannel/ZebraFinchBrain/GCF_003957565.2/Taeniopygia_guttata.bTaeGut1_v1.p.110.chr.gff3 > \ /Volumes/cachannel/ZebraFinchBrain/HTSEQ_withautomate/output_counts.txt However I get this error: Error processing GFF file (line 75 of file /Volumes/cachannel/ZebraFinchBrain/GCF_003957565.2/Taeniopygia_guttata.bTaeGut1_v1.p.110.chr.gff3): Feature gene:ENSTGUG00000013637 does not contain…

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Issues while running htseq-count

Issues while running htseq-count 0 My data is Candida glabrata and when i use htseq-count, no read is mapped to the gene_id. Thank you for your time and help. Foad htseq-count GSNO_SRR1582646.sam Candida_glabrata_genome.gtf > GSNO_SRR1582646.count 10975 GFF lines processed. 8843 alignment record pairs processed. head GSNO_SRR1582646.count gene-CAGL0A00165g 0 gene-CAGL0A00187g 0…

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Error with HTseq RNAseq read count – rna-seq

Hi, I am getting error while running HTseq. This is the command and the error: htseq-count -q -f bam -s yes Ac1_mapped/ac1_mappedAligned.bam /global/home/users/catalinacastro/star/genome/genomic_v2.gtf count.txt Error occurred when processing GFF file (line 637338 of file /global/home/users/catalinacastro/star/genome/genomic_v2.gtf): not enough values to unpack (expected 9, got 1) [Exception type: ValueError, raised in init.py:221]…

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Error with HTseq RNAseq read count

Error with HTseq RNAseq read count 0 Hi, I am getting error while running HTseq. This is the command and the error: htseq-count -q -f bam -s yes Ac1_mapped/ac1_mappedAligned.bam /global/home/users/catalinacastro/star/genome/genomic_v2.gtf count.txt Error occurred when processing GFF file (line 637338 of file /global/home/users/catalinacastro/star/genome/genomic_v2.gtf): not enough values to unpack (expected 9, got…

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Htseq-count reads with missing mate encountered

Htseq-count reads with missing mate encountered 0 Hello. I ran this HTseq command htseq-count -r name -t gene -i gene -s yes -f bam /Volumes/cachannel/ZebraFinchBrain/CB-4a_genomemapping/sorted_alignmentcb4a.bam /Volumes/cachannel/ZebraFinchBrain/GCF_003957565.2/ncbi_dataset/data/GCF_003957565.2/genomic.gff > /Volumes/cachannel/ZebraFinchBrain/HTSEQ_withautomate/output_counts.txt and got the error Warning: 72583723 reads with missing mate encountered. 80015507 alignment record pairs processed. Is there a setting I am…

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Htseq Count

Hello, I have ran htseq-count numerous times and continue to get the same error. That NONE of my genes are counted as seen here. ZXDC 0 ZYG11B 0 ZYX 0 ZZEF1 0 ZZZ3 0 __no_feature 70257177 __ambiguous 0 __too_low_aQual 1509790 __not_aligned 3970775 __alignment_not_unique 4277765 However, I have a very high…

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zero counts for all genes in RNAseq data of Ferret

zero counts for all genes in RNAseq data of Ferret 0 I have bulk RNAseq data from Ferret and trying to get counts per gene. to do so I used hisat2 and got the genome from here: hgdownload.soe.ucsc.edu/goldenPath/musFur1/bigZips/musFur1.2bit after aligning the fastq files I used htseq and the following command:…

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How do I write a correctly formatted gff3 file in R?

Dear all, I am trying to annotate non-coding RNA in a small RNA-seq dataset. The RNACentral gff3 file that I am using has different chromosome identifiers than the genome assembly. I have loaded the gff3 file in R where I changed the chromosome identifiers using the the assembly report and…

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Proper HTSeq usage on bacterial genome. Don’t quite understand –t

Proper HTSeq usage on bacterial genome. Don’t quite understand –t 1 Hi everyone, I’m trying to run HTSeq on a group of BAM files generated from the alignment of an RNAseq illumina reads mapped to a reference genome. The reference genome is the sequence with highest quality available and was…

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Chromatin compartmentalization regulates the response to DNA damage

Cell culture and treatments DIvA (AsiSI-ER-U2OS)19, AID-DIvA (AID-AsiSI-ER-U2OS)23 and 53BP1-GFP DIvA20 cells were developed in U2OS (ATCC HTB-96) cells and were previously described. Authentication of the U2OS cell line was performed by the provider ATCC, which uses morphology and short tandem repeat profiling to confirm the identity of human cell…

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Apoptotic stress causes mtDNA release during senescence and drives the SASP

Cell culture and treatments Human embryonic lung MRC5 fibroblasts (ATCC) and IMR90 fibroblasts (ATCC) were grown in Dulbecco’s modified Eagle’s medium (Sigma-Aldrich, D5796) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 U ml−1 penicillin, 100 μg ml−1 streptomycin and 2 mM l-glutamine and maintained at 37 °C under 5% CO2. MRC5 fibroblasts were cultured in…

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htseq-count reports count values for deleted genes

htseq-count reports count values for deleted genes 0 I am using htseq-count on BAM files from a bacterial species. We are comparing WT strains as well as two strains with genes knocked out. The knockouts have been verified with whole genome sequencing, but do retain the first 15 and last…

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Selective enrichment of plasma cell-free messenger RNA in cancer-associated extracellular vesicles

Clinical samples and plasma preparation Blood samples from control individuals and patients with multiple myeloma, liver cancer, and lung cancer were obtained from Oregon Health and Science University (OHSU) by Knight Cancer Institute Biolibrary and Oregon Clinical and Translational Research Institute (OCTRI). All samples were collected under OHSU institutional review…

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Bulk RNASeq: from counts to differential expression

This course consists of a live session on counting and differential expression analysis in R and a Q&A session to answer all the questions that arise when trying the analysis on your own data. The live session will demonstrate R tools to generate count files like featureCounts, and summarizeOverlaps are…

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Calculating FPKM and TPM by hand from htseq-count output?

Calculating FPKM and TPM by hand from htseq-count output? 0 Hello! I am counting reads with htseq-count, and wasted some hours trying to find an extant software that would calculate FPKM and/or TPM from that output, so I wrote a script myself. There is just one question mark – should…

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Help with htseq -count read counts

Hello I am doing a transcriptome analysis on Pseudomonas putida and I have been trying to do a read count using Htseq -count. The program always give an error. I have tried different genome references (fna) and annotation files (gtf ang gff) but it does not work. The mapping works…

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Dryad | Data — RNAseq data from: Medial prefrontal cortex samples of glutamate dehydrogenase-deficient mice, stress-exposed or -naive, and their Nestin-Cre+ controls

Glutamate abnormalities in the medial prefrontal cortex (mPFC) are associated with cognitive deficits. We previously showed that homozygous deletion of CNS glutamate dehydrogenase 1 (Glud1), a metabolic enzyme critical for glutamate metabolism, leads to schizophrenia-like behavioral abnormalities and increased mPFC glutamate; mice heterozygous for CNS Glud1 deletion (C-Glud1+/- mice) showed…

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Counting intronic reads in bulk RNA-seq

Counting intronic reads in bulk RNA-seq 0 My experience with single-cell RNA-seq shows that the inclusion of intronic reads improves the sensitivity for several genes of interest, which otherwise have zero expression when only exonic reads are considered. While single-cell sequencing quantifiers now often have options to count intronic reads,…

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Deferentially expressed gene with high log2foldchange by DESeq2; but not meaningful at the individual level

Hi all, I am working with the RNA-Seq data on human (24Cases-20 controls) to find differentially expressed genes. my RNA-Seq data is unstranded. Here is the comments that I used to align the fastq files: ls *_1P.fastq.gz | parallel –bar -j8 ‘R2=$(echo {} | sed s/_1/_2/) && out=$(echo {} |…

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Toxic mechanisms of MWCNTs for ocular cells.

Introduction Carbon nanotubes (CNTs) are nano-sized one-dimensional tubular structures rolled by single (single-walled CNTs, SWCNTs) or multiple layers (multi-walled CNTs, MWCNTs) of graphene with unique electrochemical, mechanical and thermal properties, which render them as attracted engineered nanomaterials for various commercial applications.1–4 An increase in the production and utilization of CNTs…

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No differentially expressed genes after multiple testing correction in mice

No differentially expressed genes after multiple testing correction in mice 0 Hi all, I am working with the RNA-seq data on mice (group A N=3 vs group B N=3). Mice are littermates, of which group A overexpresses a human transgene which I verified. I have had .cram files from mouse…

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dosen’t show options in bash ln ubuntu

htseq-count : dosen’t show options in bash ln ubuntu 1 foad@Linux:~/Example/Sam$ htseq-count -h usage: htseq-count [options] alignment_file gff_file This script takes one or more alignment files in SAM/BAM format and a feature file in GFF format and calculates for each feature the number of reads mapping to it. See htseq.readthedocs.io/en/master/count.html

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Error using BWA to map environmental transcriptome against a genomic reference

I have quality controlled paired end environmental transcriptomic data that I want to map against a reference database of 8 cyanobacterial genomes. I made this reference by joining together the fasta files of each genome. I performed this mapping with BWA v0.7.3a but noticed that when I tried to count…

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CRISPR-clear imaging of melanin-rich B16-derived solid tumors

B16 melanin(+) tdTomato cell line generation The generation and characterization of a lentivirus encoding tdTomato has been described previously22. The B16-D5-HER2 stable cell line was a generous gift from Louis Weiner (Georgetown University)13,23. Cell lines were cultured in DMEM Dulbecco’s modified Eagle’s medium (DMEM; Sigma) supplemented with 10% (v/v) fetal…

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BULK mRNA-seq with UMIs. Does it make sense to run bedtools genomecov?

BULK mRNA-seq with UMIs. Does it make sense to run bedtools genomecov? 0 Hi, I am using BULK rna-seq which includes UMI added after cDNA fragmentation, before PCR amplification (see my previous post BULK mRNA-seq with UMIs. Do I need to normalize by gene length? ). I am trying to…

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Stringtie does not work with NCBI GTF file?

Stringtie does not work with NCBI GTF file? 1 Hi all, I wanted to rerun my DGE analysis to see if there were any differences between HTseq-count -> edgeR and StringTie-Ballgown. However, when I tried to run my stringtie command using the same BAM file, I got an error: “Error:…

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HTseq no features

HTseq no features 0 I have got some problem when analyzing my RNAseq data and I would like to seek for a help. Here is the brief description of my pipeline: Obtained fasta file of 150 PE reads from Novaseq platform followed by non-stranded library prep I conducted fastQC and…

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[E::idx_find_and_load] warning in htseq-count

[E::idx_find_and_load] warning in htseq-count 0 I am running the htseq-count but get the [E::idx_find_and_load] warning. The bam files were sorted with name but without index. It is not required for index when running htseq-count, correct? *CODE: htseq-count -f bam \ -s no \ -t exon \ -m union \ -i…

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Warning: Mate records missing HTSEQ

Warning: Mate records missing HTSEQ 0 Hello everyone, At the end of the quantification with HTSEQ I get this warning, why is it? is it a serious problem? enrique@DE:~/analisis_maiz/quantificacion$ htseq-count -f bam ../alignements/SRR214880_sorted.bam -s no -i gene_id -r pos -t exon ../genome_anotacion/Zea_mays.Zm-B73-REFERENCE-NAM -5.0.55\ \(1\).gtf > SRR214880.txt Final: Warning: Mate records…

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Mapped reads not mapping to a real sequence?

Hi, I’ve been passed down some bam files from RNA-seq that I need to analyze. They were generated with pseudoalignments using Kallisto ( pachterlab.github.io/kallisto/about ) When I run htseq-count one specific bamfile crashes on a specific read: Error occured when processing input (record #149548485 in file 103805-016-011.kallisto.pseudoalignments.bam): Expected str, got…

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HTSeq error processing GFF file

HTSeq error processing GFF file 0 Hello, I am trying to run HTSeq but it tells me that I have a problem in my GFF and GTF file, how can I fix this? enrique@L:~prueba_guess$ htseq-count -f bam SRR214880.bam -s no -i ID -r pos -t exon GCF_902167145.1_Zm-B73-REFERENCE-NAM-5.0_genomic.gff > SRR214880.txt 100000…

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BULK mRNA-seq with UMIs. Do I need to normalize by gene length?

Hi, I am analyzed some BULK mRNA-seq data that included UMIs during the sequencing. I don’t have much experience analyzing bulk RNA-seq, and it is my first time dealing with UMIs there. I have more experience in single-cell RNA-seq, and I thought that the concept of UMIs will translate directly…

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Error using HTSEq_count

Error using HTSEq_count 0 Hello, I am trying to use HTSeq_count but when I run the command I get the following error: > Traceback (most recent call last): File “/usr/local/bin/htseq-count”, line 5, in <module> HTSeq.scripts.count.main() File “/usr/local/lib/python3.7/site-packages/HTSeq/scripts/count.py”, line 473, in main args = _parse_sanitize_cmdline_arguments() File “/usr/local/lib/python3.7/site-packages/HTSeq/scripts/count.py”, line 465, in _parse_sanitize_cmdline_arguments…

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smallRNA profiling using HTSeq error

smallRNA profiling using HTSeq error 1 Hello, I want to create a “count” file using HTseq. I have both BAM file and gtf file: htseq-count -f bam -s no -i AK1a_clean_Aligned.sortedByCoord.out.bam gencode.v42.chr_patch_hapl_scaff.annotation.gtf >> AK1a_counts.txt It still gives an error: htseq-count: error: the following arguments are required: featuresfilename Can someone please…

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HISAT2 and HTSEQ command

HISAT2 and HTSEQ command 0 @4fedfa78 Last seen 10 hours ago Japan Hello, For analysis of the data by rna-sequencing I selected HISAT2 and HTSeq-count for mapping and counting the genes levels, the libraryLayout is paired, I am using the below command for both but the results are not exact…

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Bash script to automate htseq-count

Hi everyone- I am trying to write a script to automate htseq-count on a large number of samples. The script runs but then throws the following error: “Please provide 2 arguments”. Does anyone see something obvious I am missing: #!/bin/bash for samples in *.sam do gtf = “Galaxy135-\[Escherichia_coli_str_k_12_substr_mg1655.GCA_000005845.2.29.gtf\].gtf” echo $sample …

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can gff2 reference used in htseq-count?

Dear all We are recently working with E.coli plasmid and tried to summarize the gene counts from our RNA-Seq samples. The short reads were mapped to E.coli plasmid using tophat which generated bam files accordingly. However, we were unable to obtain a gff3 version of our target plasmid genome, the…

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HTseq-Count: Long processing time

HTseq-Count: Long processing time 1 Hi everyone, I’m processing BAM files using htseq-count and it takes very long time to produce the counts for each file. It is about pair-end reads (around 50 million sequence each). It takes 75 minutes to count this pair; is that normal? Thanks. htseq-count –max-reads-in-buffer=24000000000…

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htseq-count error

htseq-count error 1 Hi, htseq-count -f bam -s yes ~/htseq-trial/SRR13826419_Aligned.sortedByName.out.bam ~refgen/gencode.v39.primary_assembly.annotation.gtf > counts.txt I am trying to run htseq-count with command above but in the err file [E::idx_find_and_load] Could not retrieve index file for ‘~/htseq-trial/SRR13826419_Aligned.sortedByName.out.bam’ 100000 GFF lines processed. 200000 GFF lines processed. 300000 GFF lines processed. 400000 GFF lines…

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Feature count is very low using htseq-count

Feature count is very low using htseq-count 0 Hello all, I performed bbmap on my RNA-seq paired sequence data using following cmd bbmap.sh in1=J2_R1.fastq in2=J2_R2.fastq out=output_J2.sam ref=im4.fasta nodisk The header of generated sam file is @HD VN:1.4 SO:unsorted @SQ SN:k141_1006 LN:2503 @SQ SN:k141_5512 LN:5393 @SQ SN:k141_4772 LN:4387 @SQ SN:k141_3267 LN:4531…

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Htseq is giving me 0 counts using the GFF3 of miRBase

Hello! I am trying to annotate a miRNA-seq so that it gives me mature miRNAs where I already have 5p and 3p. For this, I have used the index mm10.fa and the miRBase mmu.gff3. I have aligned with HISAT2 and am trying to count with HTSeq, however I get 0…

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The role of ATXR6 expression in modulating genome stability and transposable element repression in Arabidopsis

Significance The plant-specific H3K27me1 methyltransferases ATXR5 and ATXR6 play integral roles connecting epigenetic silencing with genomic stability. However, how H3K27me1 relates to these processes is poorly understood. In this study, we performed a comprehensive transcriptome analysis of tissue- and ploidy-specific expression in a hypomorphic atxr5/6 mutant and revealed that the…

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How to label columns in HTSeq output

How to label columns in HTSeq output 0 I’ve been working to process RNAseq data and I’ve used hisat2 to align my reads to the reference genome. When I take those output files and put them into HTSeq-count using the below code, I get a count matrix but the columns…

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htseq-count -t gene not working

I found a little problem. When I set the “-t gene”, the reads is mark “__no_feature”. But when I set the “-t exon”, the reads is mark “ENSG00000276104”. The gene “ENSG00000276104” is a single exon gene. I don’t know why this happens. reads: “TGTCTGTGGCGGTGGGATCCCGCGGCCGTGTTTTCCTGGTGGCCCGGCCGTGCCTGAGGTTTCTCCCCGAGCCGCCGCCTCTGCGGGCTCCCGGGTGCCCTTGCCCTCGCGGTCCCCGGCCCTCGCCCGTCTGTGCCCTCTTCCCCGCCCGCCGATCCTCTTCTTCCCCCCGAGCGGCTCACCGGCTTCACGTCCGTTGGTGGCCCCGCCTGGGAC”. I had aligned to hg38 by…

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htseq-count python tutorial attribute counts error

Hello, I’m following the htseq-count tutorial for RNA-seq (counting the overlapping genes and exons) here htseq.readthedocs.io/en/master/tour.html. However, when I get to the point where I need to find the overlaps in the .sam file and .gtf file, I get an error. This is the code I ran originally that gave…

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htseq-count Error ‘_StepVector_Iterator_obj’ object has no attribute ‘next’

htseq-count Error ‘_StepVector_Iterator_obj’ object has no attribute ‘next’ 0 I am trying to run htseq-count (v. 0.13.5) on a sorted and indexed bam file. The command I entered looks like this: htseq-count -f bam -r pos -s yes -t CDS -i gene_id -m union filename_sorted.bam filename.gtf I get the following…

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HTSeq is a Python library to facilitate processing and analysis of data from high-throughput sequencing (HTS) experiments.

Hello, I’m following the htseq-count tutorial for RNA-seq (counting the overlapping genes and exons) here htseq.readthedocs.io/en/master/tour.html. However, when I get to the point where I need to find the overlaps in the .sam file and .gtf file, I get an error. This is the code I ran originally that gave…

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Running htseq-count to “grab” long non coding gene_id names

Running htseq-count to “grab” long non coding gene_id names 0 hi all, new to bioinformatics. so bare with me.. I am trying find long non coding RNA from RNA-seq data. As i checked the human gtf file there are 2 different types of long non coding RNA, “lnc_RNA” and “lncRNA”,…

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gffread error

hello I am currently trying to do RNA-seq using public data in brassica juncea. To use htseq-count for making count table, I have to convert gff file which downloaded in brassica database to gtf file. So I used gffread for converting gff file with below command gffread Bju.genome.gff -T -o…

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HTseq doesn’t support Multi-Threading ?

HTseq doesn’t support Multi-Threading ? 1 Hello, everyone ! I’m looking for a way to use HTseq with multi-thread. I couldn’t find any options about multi-thread. Anybody knows how to, please ? (I know there are tools support multi-thread like STAR, HISAT2. but just wonder whether HTseq doesn’t support it.)…

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Fastqc user manual – vodosp.ru

FASTQ format – Wikipedia 06 September 2021 – by TC Collin · 2020 · Cited by 3 — Be accompanied by a step-by-step user-friendly manual, If the user performs FastQC prior to the removal of adapters (step 3), the length Both programs can be used on Linux/MacOS X machines and quite…

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does not contain a ‘gene’ attribute

htseq-count returns : does not contain a ‘gene’ attribute 1 Dear BIOSTAR community, I’m trying to make count matrix with htseq-count, htseq-count -s yes -t gene -i gene 01.sorted.sam annotation_cattle.gff > 01.txt even with –idattr=gene , it returns error: Error processing GFF file (line 1864255 of file annotation_cattle.gff): Feature gene-D1Y31_gp1…

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Mapping reads and quantifying genes

Mapping reads and quantifying genes – Metagenomic workshop 0 Hello, I am using the following metagenomic workshop tutorial to analyse my own metagenomic data. metagenomics-workshop.readthedocs.io/en/latest/annotation/quantification.html I performed the following steps: mapped reads with bowtie2 and generated .bam file with samtools sort. Removed duplicates with picard Extracted gene information from prokka…

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Error creating DESeq2 Data Set from HTSeq-Count

I am trying to run DESeq2 using gene counts generated by HTSeq-Count. I combine files for different conditions: directory <- “~/GeneCountFiles/” WT_Files <- c( “P0CTRS3.aligned.sam.genecount”, “P0CTRS4.aligned.sam.genecount”, “P0CTRS5.aligned.sam.genecount” ) KO_Files <- c( “P0CTRS1.aligned.sam.genecount”, “P0CTRS2.aligned.sam.genecount”, “P0CTRS6.aligned.sam.genecount” ) I then create the sample table: sampleTable <- data.frame( sampleName=c(WT_Files, KO_Files), fileName=c(WT_Files, KO_Files), genotype=c(rep(“WT”, length(WT_Files)),…

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how htseq-count counts unstranded RNA-seq data

how htseq-count counts unstranded RNA-seq data 1 preliminary Say I have some unstranded RNA-seq data and im mapping to the reference human genome using htseq-count (–stranded=no) My understanding (biologically) was that for a given protein_coding gene, reading DNA in the sense strand gives the protein_coding transcript, reading the gene in…

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HTSeq-count TruSeq RNA Exome Lib Prep

HTSeq-count TruSeq RNA Exome Lib Prep 0 Hello, I observed a high percentage of “no features” while running HTseq w/ the –stranded yes option enabled (>80%). The library prep kit I am using is Illumina TruSeq RNA Exome which generates stranded data. If I run HTseq-count w/ strand == “no”…

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hisat2 compatibility for long read

hisat2 compatibility for long read 0 Hi, I am trying to align PacBio transcriptome reads against the genome to count the gene number. For pair end read i used the following workflow: # convert gff to gtf /home/software/cufflinks-2.2.1/gffread xxx.gff -T -o xxx.gtf # build index /home/software/hisat2-2.2.1/hisat2_extract_exons.py xxx.gtf > xxx.exon /home/software/hisat2-2.2.1/hisat2_extract_splice_sites.py…

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