I am doing some RNA analysis and am having issues trying to generate count data. I mapped my reads to a reference genome fasta file (genbank fasta file from ncbi) using bbmap and .sam files as the output.
I am now trying to use featurecounts to generate count data but am running into issues with my gtf file. The gtf file is also from ncbi and corresponds to the alignment fasta file used for mapping. This is it's structure:
#gtf-version 2.2
#!genome-build ASM14559v1
#!genome-build-accession NCBI_Assembly:GCA_000145595.1
CP002120.1 Genbank gene 1 1362 . + . gene_id "SAA6008_00001"; transcript_id ""; gbkey "Gene"; gene "dnaA"; gene_biotype "protein_coding"; locus_tag "SAA6008_00001";
CP002120.1 Genbank CDS 1 1359 . + 0 gene_id "SAA6008_00001"; transcript_id "unassigned_transcript_1"; gbkey "CDS"; gene "dnaA"; locus_tag "SAA6008_00001"; product "chromosomal replication initiator protein DnaA"; protein_id "ADL64089.1"; transl_table "11"; exon_number "1";
CP002120.1 Genbank start_codon 1 3 . + 0 gene_id "SAA6008_00001"; transcript_id "unassigned_transcript_1"; gbkey "CDS"; gene "dnaA"; locus_tag "SAA6008_00001"; product "chromosomal replication initiator protein DnaA"; protein_id "ADL64089.1"; transl_table "11"; exon_number "1";
CP002120.1 Genbank stop_codon 1360 1362 . + 0 gene_id "SAA6008_00001"; transcript_id "unassigned_transcript_1"; gbkey "CDS"; gene "dnaA"; locus_tag "SAA6008_00001"; product "chromosomal replication initiator protein DnaA"; protein_id "ADL64089.1"; transl_table "11"; exon_number "1";
CP002120.1 Genbank gene 1640 2773 . + . gene_id "SAA6008_00002"; transcript_id ""; gbkey "Gene"; gene "dnaN"; gene_biotype "protein_coding"; locus_tag "SAA6008_00002";
CP002120.1 Genbank CDS 1640 2770 . + 0 gene_id "SAA6008_00002"; transcript_id "unassigned_transcript_2"; gbkey "CDS"; gene "dnaN"; locus_tag "SAA6008_00002"; product "DNA-directed DNA polymerase beta subunit"; protein_id "ADL64090.1"; transl_table "11"; exon_number "1";
CP002120.1 Genbank start_codon 1640 1642 . + 0 gene_id "SAA6008_00002"; transcript_id "unassigned_transcript_2"; gbkey "CDS"; gene "dnaN"; locus_tag "SAA6008_00002"; product "DNA-directed DNA polymerase beta subunit"; protein_id "ADL64090.1"; transl_table "11"; exon_number "1";
CP002120.1 Genbank stop_codon 2771 2773 . + 0 gene_id "SAA6008_00002"; transcript_id "unassigned_transcript_2"; gbkey "CDS"; gene "dnaN"; locus_tag "SAA6008_00002"; product "DNA-directed DNA polymerase beta subunit"; protein_id "ADL64090.1"; transl_table "11"; exon_number "1";
CP002120.1 Genbank gene 3163 3399 . + . gene_id "SAA6008_00003"; transcript_id ""; gbkey "Gene"; gene_biotype "protein_coding"; locus_tag "SAA6008_00003";
CP002120.1 Genbank CDS 3163 3396 . + 0 gene_id "SAA6008_00003"; transcript_id "unassigned_transcript_3"; gbkey "CDS"; locus_tag "SAA6008_00003"; product "conserved hypothetical protein"; protein_id "ADL64091.1"; transl_table "11"; exon_number "1";
CP002120.1 Genbank start_codon 3163 3165 . + 0 gene_id "SAA6008_00003"; transcript_id "unassigned_transcript_3"; gbkey "CDS"; locus_tag "SAA6008_00003"; product "conserved hypothetical protein"; protein_id "ADL64091.1"; transl_table "11"; exon_number "1";
CP002120.1 Genbank stop_codon 3397 3399 . + 0 gene_id "SAA6008_00003"; transcript_id "unassigned_transcript_3"; gbkey "CDS"; locus_tag "SAA6008_00003"; product "conserved hypothetical protein"; protein_id "ADL64091.1"; transl_table "11"; exon_number "1";
CP002120.1 Genbank gene 3396 4508 . + . gene_id "SAA6008_00004"; transcript_id ""; gbkey "Gene"; gene "recF"; gene_biotype "protein_coding"; locus_tag "SAA6008_00004";
CP002120.1 Genbank CDS 3396 4505 . + 0 gene_id "SAA6008_00004"; transcript_id "unassigned_transcript_4"; gbkey "CDS"; gene "recF"; locus_tag "SAA6008_00004"; note "Required for DNA replication; binds preferentially to single-stranded, linear DNA"; product "DNA repair and genetic recombination protein"; protein_id "ADL64092.1"; t$
CP002120.1 Genbank start_codon 3396 3398 . + 0 gene_id "SAA6008_00004"; transcript_id "unassigned_transcript_4"; gbkey "CDS"; gene "recF"; locus_tag "SAA6008_00004"; note "Required for DNA replication; binds preferentially to single-stranded, linear DNA"; product "DNA repair and genetic recombination protein"; protein_id "ADL640$
CP002120.1 Genbank stop_codon 4506 4508 . + 0 gene_id "SAA6008_00004"; transcript_id "unassigned_transcript_4"; gbkey "CDS"; gene "recF"; locus_tag "SAA6008_00004"; note "Required for DNA replication; binds preferentially to single-stranded, linear DNA"; product "DNA repair and genetic recombination protein"; protein_id "ADL640$
CP002120.1 Genbank gene 4518 6452 . + . gene_id "SAA6008_00005"; transcript_id ""; gbkey "Gene"; gene "gyrB"; gene_biotype "protein_coding"; locus_tag "SAA6008_00005";
CP002120.1 Genbank CDS 4518 6449 . + 0 gene_id "SAA6008_00005"; transcript_id "unassigned_transcript_5"; gbkey "CDS"; gene "gyrB"; locus_tag "SAA6008_00005"; note "TIGRFAM: DNA gyrase, B subunit PFAM: DNA gyrase, subunit B domain protein; ATP-binding region, ATPase domain protein domain protein; TOPRIM
When I try to run featurecounts in R using this code:
featureCounts("s1_mapped.sam", annot.ext = "GCA_000145595.1_ASM14559v1_genomic.gtf", isGTFAnnotationFile = T)
I get the following error:
The chromosome name contains unexpected characters: "CP002120.1 Staphylococcus aureus subsp. aureus str. JKD6008, complete genome" (77 chars)
featureCounts has to stop running
FATAL Error: The program has to terminate and no counting file is generated.
Error in featureCounts("s1_mapped.sam", annot.ext = "GCA_000145595.1_ASM14559v1_genomic.gtf", :
No counts were generated.
Do I need to format my gtf file in some way? I have also tried the code with GTF.featureType = "gene" but I get the same error.
samtools -H your_file.samshould do this or look at the first lines of the sam file in a text editor. The chromosome names start with @SQ $\endgroup$