Hi everyone ,

I have ran this feature count command :

featureCounts  -T 4 -p -a gencode.v43.basic.annotation.gtf -o featurecount.txt *.bam

this gave me this error :

  Process BAM file UI_E2_sorted.bam.bam...                                    ||
||    Paired-end reads are included.                                          ||
||    The reads are assigned on the single-end mode.                          ||
||    Total alignments : 49308686                                             ||
||    Successfully assigned alignments : 0 (0.0%)                             ||
||    Running time : 0.26 minutes               

this error coming for all bam files.

I am using gencode.v43.basic.annotation.gtf as the annotation file but the feature count command is giving zero assigned alignments .Please help on this , I am not sure if it's the right gtf annotation file , I got it from NCBI.

I am using human ref genome hg38 for my analysis of RNA seq data .

This is how my bam file header looks by using this command :

samtools_0.1.18 view RA1_E2_sorted.bam.bam | head
NB551648:44:HK5KLBGXG:3:23507:16037:10429       419     NC_000001.11    13158   1       76M     =       183744  150203  GAAGGGGATGCACTGTTGGGGAGGCAGCTGTAACTCAAAGCCTTAGCCTCTGTTCCCACGAAGGCAGGGCCATCAG    AAAAAEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEE/EEEE  AS:i:0  ZS:i:0  XN:i:0  XM:i:0  XO:i:0  XG:i:0  NM:i:0  MD:Z:76 YS:i:0  YT:Z:CP NH:i:5
NB551648:44:HK5KLBGXG:3:23507:16037:10429       419     NC_000001.11    13158   1       76M     =       13225   143     GAAGGGGATGCACTGTTGGGGAGGCAGCTGTAACTCAAAGCCTTAGCCTCTGTTCCCACGAAGGCAGGGCCATCAG    AAAAAEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEE/EEEE  AS:i:0  ZS:i:0  XN:i:0  XM:i:0  XO:i:0  XG:i:0  NM:i:0  MD:Z:76 YS:i:0  YT:Z:CP NH:i:5
NB551648:44:HK5KLBGXG:3:23507:16037:10429       339     NC_000001.11    13225   1       76M     =       13158   -143    GGCCATCAGGCACCAAAGGGATTCTGCCAGCATAGTGCTCCTGGACCAGTGATACACCCGGCACCCTGTCCTGGAC    EEEEEEEEAEEEAEEEAEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEAAAAA  AS:i:0  ZS:i:0  XN:i:0  XM:i:0  XO:i:0  XG:i:0  NM:i:0  MD:Z:76 YS:i:0  YT:Z:CP NH:i:5
NB551648:44:HK5KLBGXG:3:12601:18920:17906       355     NC_000001.11    14360   1       2S74M   =       14414   132     GTCATCCTGCACAGCTAGAGATCCTTTATTAAAAGCACACTGTTGGTTTCTGCTCAGTTCTTTATTGATTGGTGTG    AAAAAEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEAEEEEEEEEE  AS:i:-2 ZS:i:-2 XN:i:0  XM:i:0  XO:i:0  XG:i:0  NM:i:0  MD:Z:74 YS:i:0  YT:Z:CP NH:i:2
NB551648:44:HK5KLBGXG:1:23104:13420:13760       355     NC_000001.11    14362   1       2S74M   =       14403   119     CGTCCTGCACAGCTAGAGATCCTTTATTAAAAGCACACTGTTGGTTTCTGCTCAGTTCTTTATTGATTGGTGTGCC    AAAAAEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEE  AS:i:-2 ZS:i:-2 XN:i:0  XM:i:0  XO:i:0  XG:i:0  NM:i:0  MD:Z:74 YS:i:0  YT:Z:CP NH:i:2
NB551648:44:HK5KLBGXG:3:22601:17672:18350       355     NC_000001.11    14366   1       1S75M   =       14517   228     AGCACAGCTAGAGATCCTTTATTAAAAGCACACTGTTGGTTTCTGCTCAGTTCTTTATTGATTGGTGTGCCGTTTT    AAAAAEEEEEEEEEEEEEEEEEEAEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEE  AS:i:-1 ZS:i:-1 XN:i:0  XM:i:0  XO:i:0  XG:i:0  NM:i:0  MD:Z:75 YS:i:0  YT:Z:CP NH:i:5
NB551648:44:HK5KLBGXG:3:22601:17672:18350       355     NC_000001.11    14366   1       1S75M   =       185038  150290  AGCACAGCTAGAGATCCTTTATTAAAAGCACACTGTTGGTTTCTGCTCAGTTCTTTATTGATTGGTGTGCCGTTTT    AAAAAEEEEEEEEEEEEEEEEEEAEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEE  AS:i:-1 ZS:i:-1 XN:i:0  XM:i:0  XO:i:0  XG:i:0  NM:i:0  MD:Z:75 YS:i:0  YT:Z:CP NH:i:5
NB551648:44:HK5KLBGXG:4:23409:8298:7644 355     NC_000001.11    14367   0       76M     =       184930  150180  CACAGCTAGAGATCCTTTATTAAAAGCACACTGTTGGTTTCTGCTCAGTTCTTTATTGATTGGTGTGCCATTTTCT    AAAAAEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEE  AS:i:-5 ZS:i:-5 XN:i:0  XM:i:1  XO:i:0  XG:i:0  NM:i:1  MD:Z:69G6       YS:i:-10        YT:Z:CP NH:i:5
NB551648:44:HK5KLBGXG:2:13208:13922:5858        355     NC_000001.11    14399   1       76M     =       14437   114     GTTGGTTTCTGCTCAGTTCTTTATTGATTGGTGTGCCGTTTTCTCTGGAAGCCTCTTAAGAACACAGTGGCGCAGG    A//AAEEEEEEAA/A/AEE<EEEEE6AE6//E/AA//E/EEAEEEEE/EEE/6E6E<AA<AEAE6A////EE/AAE  AS:i:0  ZS:i:0  XN:i:0  XM:i:0  XO:i:0  XG:i:0  NM:i:0  MD:Z:76 YS:i:0  YT:Z:CP NH:i:5
NB551648:44:HK5KLBGXG:3:22508:3274:10922        99      NC_000001.11    14402   1       76M     =       14492   166     GGTTTCTGCTCAGTTCTTTATTGATTGGTGTGCCGTTTTCTCTGGAAGCCTCTTAAGAACACAGTGGCGCAGGCTG    AAAAAEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEE  AS:i:0  ZS:i:0  XN:i:0  XM:i:0  XO:i:0  XG:i:0  NM:i:0  MD:Z:76 YS:i:0  YT:Z:CP NH:i:5

and this is how my gtf file looks :

##description: evidence-based annotation of the human genome (GRCh38), version 43 (Ensembl 109)
##provider: GENCODE
##contact: gencode-help@ebi.ac.uk
##format: gtf
##date: 2022-11-29
chr1    HAVANA  gene    11869   14409   .       +       .       gene_id "ENSG00000290825.1"; gene_type "lncRNA"; gene_name "DDX11L2"; level 2; tag "overlaps_pseudogene";
chr1    HAVANA  transcript      11869   14409   .       +       .       gene_id "ENSG00000290825.1"; transcript_id "ENST00000456328.2"; gene_type "lncRNA"; gene_name "DDX11L2"; transcript_type "lncRNA"; transcript_name "DDX11L2-202"; level 2; transcript_support_level "1"; tag "basic"; tag "Ensembl_canonical"; havana_transcript "OTTHUMT00000362751.1";
chr1    HAVANA  exon    11869   12227   .       +       .       gene_id "ENSG00000290825.1"; transcript_id "ENST00000456328.2"; gene_type "lncRNA"; gene_name "DDX11L2"; transcript_type "lncRNA"; transcript_name "DDX11L2-202"; exon_number 1; exon_id "ENSE00002234944.1"; level 2; transcript_support_level "1"; tag "basic"; tag "Ensembl_canonical"; havana_transcript "OTTHUMT00000362751.1";
chr1    HAVANA  exon    12613   12721   .       +       .       gene_id "ENSG00000290825.1"; transcript_id "ENST00000456328.2"; gene_type "lncRNA"; gene_name "DDX11L2"; transcript_type "lncRNA"; transcript_name "DDX11L2-202"; exon_number 2; exon_id "ENSE00003582793.1"; level 2; transcript_support_level "1"; tag "basic"; tag "Ensembl_canonical"; havana_transcript "OTTHUMT00000362751.1";
chr1    HAVANA  exon    13221   14409   .       +       .       gene_id "ENSG00000290825.1"; transcript_id "ENST00000456328.2"; gene_type "lncRNA"; gene_name "DDX11L2"; transcript_type "lncRNA"; transcript_name "DDX11L2-202"; exon_number 3; exon_id "ENSE00002312635.1"; level 2; transcript_support_level "1"; tag "basic"; tag "Ensembl_canonical"; havana_transcript "OTTHUMT00000362751.1";