Tag: gffread

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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Converting GFF to GTF

Converting GFF to GTF 0 Hello, I am having trouble transferring my gff file to a gtf. I have tried using gffread, gffcompare, and rtracklayer, which all have left me with the same or no output. Here are my files. Please help! gff gtf • 38 views • link updated…

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TPM from STAR output without re-allign the file using RSEM or Salmon

Hi, I want to get the TPM files from aligned files generate with STAR and reading I found out that the easiest way is using RSEM or Salmon. My code for the alignment is /Users/c/STAR/bin/MacOSX_x86_64/STAR runThreadN 4 –genomeDir /Users/c/Desktop/Human_genome_index –readFilesIn /Users/c/Desktop/test/C1D20_R1_001_paired.fastq /Users/c/Desktop/test/C1D20_R2_001_paired.fastq –quantMode TranscriptomeSAM GeneCounts –outFileNamePrefix C1D20 –outSAMtype BAM SortedByCoordinate…

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gffread outputs empty gtf file

gffread outputs empty gtf file 1 Hi, I’ve been trying to convert my prokka output in gff format to gtf format to be able to use for my hisat-stringtie analysis. However, using gffreads to convert yields an empty gtf file. Im not sure if im going wrong somewhere. Any help…

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Salmon error (ex. transcript id appears in the BAM head, but was not in the provided FASTA file)

Salmon error (ex. transcript id appears in the BAM head, but was not in the provided FASTA file) 1 Hello. everyone I am trying to count aligned reads using Salmon in alignment-based mode. My command is: salmon quant -t transcript.fa -l A -a aligned_result.bam -o salmon_quant I am encountering the…

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Investigating open reading frames in known and novel transcripts using ORFanage

O’Leary, N. A. et al. Reference sequence (RefSeq) database at NCBI: current status, taxonomic expansion and functional annotation. Nucleic Acids Res. 44, D733–D745 (2016). Google Scholar  Frankish, A. et al. GENCODE: reference annotation for the human and mouse genomes in 2023. Nucleic Acids Res. 51, D942–D949 (2023). Google Scholar  Pertea,…

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Stringtie problem

Stringtie problem 0 I have found a problem while doing the part of estimation using stringtie technique, I work on TB bacterium genome and its Gff file ( I only found annotation file in gff on ensemble) , it gives me an erro that tells” can not find gene ID…

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gff to gtf missing gene id

gff to gtf missing gene id 1 Hi, I was trying my hand at annotating a genome using prokka, and I’ve converted the output gff file to gtf (gffread file.gff -T -o file.gtf) and this is what my gtf file looks like: CP001095.1 prokka transcript 210 1712 . + ….

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Live-attenuated vaccine sCPD9 elicits superior mucosal and systemic immunity to SARS-CoV-2 variants in hamsters

Ethics statement In vitro and animal work were conducted under appropriate biosafety conditions in a BSL-3 facility at the Institut für Virologie, Freie Universität Berlin, Germany. All animal experiments were performed in compliance with relevant institutional, national and international guidelines for the care and humane use of animals and approved…

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GFF/GTF file error / featureCounts

Hi all, I am trying to generate a count.matrix for sorted bam files, using featureCounts on linux. I have a non-modal organism (bacteria), so I generated the annotation.file using both PROKKA and RAST. I used all the following files in featurecounts; PROKKA.gff, RAST.gff RAST.gtf gffread converted-PROKKA.gtf file But still facing…

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Converting an output de-novo transcriptome assembled with Trinity to a .gff3 file

Converting an output de-novo transcriptome assembled with Trinity to a .gff3 file 2 Hello! I’ve de-novo assembled a transcriptome from Trinity, resulting into Trinity.fasta, whose headers look like this: >TRINITY_DN29256_c0_g1_i1 len=323 path=[0:0-322] Followed, in the next line, by the sequence. To run an external downstream analysis with a R script,…

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How to get human cDNA sequences together with UTR regions?

How to get human cDNA sequences together with UTR regions? 2 Dear all, I have downloaded the human genome and gtf files from Gencode. Based on these two files I want to generate a fasta file that has cDNA sequences including 5′ and 3′ UTRs for protein-coding genes only. What…

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PROKKA.gff file is not compatible with featureCounts

Hi all, I am trying to count the number of reads that map to each gene using FeatureCounts. (RNA-Seq PE, linux) my input; GFF. file generated using Prokka GTF.file generated by NCBI annotation Sorted.bam files generated by bowtie2 and samtools. When I used gtf.file generated by NCBI, featurecounts run without…

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Sort gff3 on chromosome, position and then featuretype (gene, mRNA, exon, CDS)

Sort gff3 on chromosome, position and then featuretype (gene, mRNA, exon, CDS) 1 Is it possible to sort a gff3 on chromosome, position and then featuretype (gene, mRNA, exon, CDS). The order of the featuretypes is important when converting a gff file to a gtf file with gffread. If the…

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“Error parsing strand (?) from GFF line” happenning in gffread, stringtie and cufflinks

“Error parsing strand (?) from GFF line” happenning in gffread, stringtie and cufflinks 0 Hi! I’m working with various genomic data and while trying to use gffread, stringtie and cufflinks I went through the same error: Error parsing strand (?) from GFF line: NC_037304.1 RefSeq gene 58315 59481 . ?…

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error making Txdb from GTF and fasta files

Hello, I would like to use ORFik to map Ribo-reads to different ORFs in the maize genome. The latest version of the genome is Zm-B73-REFERENCE-NAM-5.0.fa. The annotation file is a GFF3. I have the genome fasta file, the fasta fai file, and the GFF3 file. The ORFik package uses GTF…

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Exon number between GTF file and ENSEMBL site does not match

Based on the ENSEMBL GTF file, I counted the number of exons per gene as follows: awk ‘$3 == “exon” {print}’ Homo_sapiens.GRCh38.108.gtf | gffread -F –keep-exon-attrs –table “gene_id”,”transcript_id”,@numexons | sort -k1,1 | datamash -sg1 max 3 > gene_lists/exons_per_gene.txt An extract of the output is: ENSG00000034677 11 ENSG00000034693 12 ENSG00000034713 4…

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Accurate assembly of multi-end RNA-seq data with Scallop2

Trapnell, C. et al. Transcript assembly and quantification by RNA-seq reveals unannotated transcripts and isoform switching during cell differentiation. Nat. Biotechnol. 28, 511–515 (2010). Article  Google Scholar  Guttman, M. et al. Ab initio reconstruction of cell type–specific transcriptomes in mouse reveals the conserved multi-exonic structure of lincRNAs. Nat. Biotechnol. 28,…

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Low transcript quantification with Salmon using GRCm39 annotations

Hi everyone, first time working with mouse samples and unfortunately, there are fewer resources available for the latest mouse Ensembl genome than I was expecting. What I’ve done: I performed rRNA depletion on total RNA extracted from mouse tissue and created Illumina libraries using a cDNA synthesis kit with random…

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How to extract fasta sequences from assembled transcripts generated by Stringtie

How to extract fasta sequences from assembled transcripts generated by Stringtie 4 Hi all, I used STAR and stringtie for mapping reads to reference genome and assembly. As you know, the generated assembled transcripts by stringtie are in gtf format. Now, I want to have fasta sequence of assembled transcript….

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genbank to GTF in galaxy

genbank to GTF in galaxy 0 Hi all, I am working on galaxy and have a genome file in genbank format. To use featurecounts for my RNAseq, I need to convert the genbank format to a GTF format because that’s the format the featurecounts tool in galaxy expects. Now, I…

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Convertion Of Gff3 To Gtf

Convertion Of Gff3 To Gtf 3 How do I convert GFF file to a GTF file? Is there any tool available? gtf gff • 79k views The easiest way is to use the gffread program that comes with the Cufflinks software suite (Tuxedo) gffread my.gff3 -T -o my.gtf See gffread…

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Multi-fasta file for gffread

Multi-fasta file for gffread 0 Hey Guys, I’m having a problem trying to extract the transcripts from a merged StringTie .gtf file with gffread. I have downloaded the cDNA fastq file from ENSEMBL and tried to run the following command: gffread -w transcripts.fa -g /path/to/genome.fa transcripts.gtf However I’m getting the…

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Splice sequence indexing failed with err =127

Tophat2 Error: Splice sequence indexing failed with err =127 0 I’ve been trying to map my RNA-seq results onto an entire genome, and I’ve encountered a problem with splices. The script.pbs I submitted to cluster servers is: #PBS -N tophat_cufflinks_1 #PBS -o tophat_cufflinks_1_out.txt #PBS -e tophat_cufflinks_1_error_out.txt #PBS -l nodes=cu01:ppn=24 export…

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GTF upload error UCSC related to stradedness

GTF upload error UCSC related to stradedness 0 Hello, I’m having issues with uploading a .gtf file to the UCSC browser. I am getting the following error: “Error GFF/GTF group STRG.155047.1 on chr12+, this line is on chr12-, all group members must be on same seq and strand” I have…

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Haplotype divergence supports long-term asexuality in the oribatid mite Oppiella nova

Significance Putatively ancient asexual species pose a challenge to theory because they appear to escape the predicted negative long-term consequences of asexuality. Although long-term asexuality is difficult to demonstrate, specific signatures of haplotype divergence, called the “Meselson effect,” are regarded as strong support for long-term asexuality. Here, we provide evidence…

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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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Blank output When converting GFF3 file to GTF using either gffread or AGAT

Blank output When converting GFF3 file to GTF using either gffread or AGAT 1 Hi, I am trying to convert gff3 file (please see below) to GTF. I used two tools suggested here gffread and agat here. #gff-version 3 Bg_94-1_CX35|chr01_10700000_16500000 Liftoff gene 1 1345 . + . ID=gene_1;Name=Os01g0293800 gene;coverage=0.997;sequence_ID=0.982;extra_copy_number=0;copy_num_ID=gene_1_0 Bg_94-1_CX35|chr01_10700000_16500000…

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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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