featureCounts returning counts of 0 for all gene_id

Question

[krg16]

I am trying to use featureCounts to create a table of gene counts, but so far my counts are all 0. When I run featureCounts it says "Successfully assigned alignments : 0 (0.0%)". I aligned my RNA-seq files to the version 5 b73 Zea mays reference genome (GCA_902167145.1_Zm-B73-REFERENCE-NAM-5.0_genomic.fna) from NCBI and am using the annotation file for that version (GCF_902167145.1_Zm-B73-REFERENCE-NAM-5.0_genomic.gtf) of the genome also from NCBI.

I have read online that it might be that the chromosome names in the sam files I'm using as input don't match the ones in my annotation file but why would this be an issue if both of them come from NCBI?

Here is what I'm running:

featureCounts -T 5 -g gene_id -a GCF_902167145.1_Zm-B73-REFERENCE-NAM-5.0_genomic.gtf -o counts.txt SRR940299_trimmed_aligned.sam

And this is my output:

        ==========     _____ _    _ ____  _____  ______          _____  
        =====         / ____| |  | |  _ \|  __ \|  ____|   /\   |  __ \ 
          =====      | (___ | |  | | |_) | |__) | |__     /  \  | |  | |
            ====      \___ \| |  | |  _ <|  _  /|  __|   / /\ \ | |  | |
              ====    ____) | |__| | |_) | | \ \| |____ / ____ \| |__| |
        ==========   |_____/ \____/|____/|_|  \_\______/_/    \_\_____/
      v2.0.1

//========================== featureCounts setting ===========================\\
||                                                                            ||
||             Input files : 1 SAM file                                       ||
||                           o SRR940299_trimmed_aligned.sam                  ||
||                                                                            ||
||             Output file : counts.txt                                       ||
||                 Summary : counts.txt.summary                               ||
||              Annotation : GCF_902167145.1_Zm-B73-REFERENCE-NAM-5.0_gen ... ||
||      Dir for temp files : ./                                               ||
||                                                                            ||
||                 Threads : 5                                                ||
||                   Level : meta-feature level                               ||
||              Paired-end : no                                               ||
||      Multimapping reads : not counted                                      ||
|| Multi-overlapping reads : not counted                                      ||
||   Min overlapping bases : 1                                                ||
||                                                                            ||
\\============================================================================//

//================================= Running ==================================\\
||                                                                            ||
|| Load annotation file GCF_902167145.1_Zm-B73-REFERENCE-NAM-5.0_genomic. ... ||
||    Features : 496625                                                       ||
||    Meta-features : 45738                                                   ||
||    Chromosomes/contigs : 217                                               ||
||                                                                            ||
|| Process SAM file SRR940299_trimmed_aligned.sam...                          ||
||    Single-end reads are included.                                          ||
||    Total alignments : 18236415                                             ||
||    Successfully assigned alignments : 0 (0.0%)                             ||
||    Running time : 0.13 minutes                                             ||
||                                                                            ||
|| Write the final count table.                                               ||
|| Write the read assignment summary.                                         ||
||                                                                            ||
|| Summary of counting results can be found in file "counts.txt.summary"      ||
||                                                                            ||
\\============================================================================//

My corresponding count table looks like this:

Geneid  Chr Start   End Strand  Length  SRR940299_trimmed_aligned.sam
LOC103644366    NC_050096.1;NC_050096.1;NC_050096.1;NC_050096.1;NC_050096.1;NC_050096.1;NC_050096.1;NC_050096.1;NC_050096.1;NC_050096.1;NC_050096.1;NC_050096.1;NC_050096.1;NC_050096.1;NC_050096.1;NC_050096.1;NC_050096.1 34607;34607;36037;36037;36259;36259;36600;36600;36822;36822;37416;37416;38021;38021;38571;39701;39701   35318;35318;36174;36174;36504;36504;36713;36713;37004;37004;37633;37633;38482;39618;39618;40208;40208   +;+;+;+;+;+;+;+;+;+;+;+;+;+;+;+;+   3717    0
LOC100382519    NC_050096.1;NC_050096.1;NC_050096.1;NC_050096.1;NC_050096.1;NC_050096.1;NC_050096.1;NC_050096.1;NC_050096.1;NC_050096.1;NC_050096.1;NC_050096.1;NC_050096.1;NC_050096.1;NC_050096.1;NC_050096.1;NC_050096.1;NC_050096.1 41254;41297;41742;41742;42257;42257;42508;42508;42762;42762;43039;43039;43318;43318;43750;43994;45593;45593 41588;41588;41807;41807;42375;42375;42665;42665;42917;42917;43197;43197;43520;43520;44303;44303;45928;46100 -;-;-;-;-;-;-;-;-;-;-;-;-;-;-;-;-;- 2258    0
LOC103630116    NC_050096.1;NC_050096.1;NC_050096.1 82666;82984;84969   82892;84213;85376   -;-;-   1865    0
LOC103649349    NC_050096.1;NC_050096.1;NC_050096.1;NC_050096.1;NC_050096.1;NC_050096.1;NC_050096.1;NC_050096.1;NC_050096.1;NC_050096.1;NC_050096.1;NC_050096.1;NC_050096.1;NC_050096.1;NC_050096.1;NC_050096.1;NC_050096.1;NC_050096.1;NC_050096.1;NC_050096.1;NC_050096.1;NC_050096.1;NC_050096.1;NC_050096.1;NC_050096.1;NC_050096.1;NC_050096.1;NC_050096.1;NC_050096.1;NC_050096.1;NC_050096.1;NC_050096.1;NC_050096.1;NC_050096.1;NC_050096.1;NC_050096.1;NC_050096.1;NC_050096.1;NC_050096.1;NC_050096.1;NC_050096.1;NC_050096.1;NC_050096.1;NC_050096.1 92293;92293;92293;92293;94869;94869;94869;94869;95530;95530;95530;95530;96140;96140;96140;96140;96611;96611;96611;96611;98521;98521;98521;98521;98730;98730;98730;98730;98974;98974;98978;98978;99928;99928;99928;101812;101966;101966;101966;101966;107610;107610;107610;107610    94779;94779;94779;94779;95133;95133;95133;95133;95993;95993;95993;95993;96525;96525;96525;96525;97107;97107;97107;97107;98566;98566;98566;98566;98803;98803;98803;98803;99085;99085;101868;99085;101868;101868;100015;101868;107528;107528;107528;107528;107876;107876;107876;107876    -;-;-;-;-;-;-;-;-;-;-;-;-;-;-;-;-;-;-;-;-;-;-;-;-;-;-;-;-;-;-;-;-;-;-;-;-;-;-;-;-;-;-;- 12944   0
LOC109943579    NC_050096.1;NC_050096.1;NC_050096.1 109103;109340;109498    109264;109411;109616    -;-;-   353 0
LOC109942621    NC_050096.1;NC_050096.1;NC_050096.1;NC_050096.1;NC_050096.1;NC_050096.1;NC_050096.1;NC_050096.1;NC_050096.1;NC_050096.1;NC_050096.1;NC_050096.1;NC_050096.1;NC_050096.1;NC_050096.1;NC_050096.1 128767;128767;128792;129337;129818;129818;129818;144152;144152;144152;144401;144401;144401;145411;145411;145411 129054;129054;129054;129674;129948;129948;129948;144317;144317;144317;144577;145129;144577;145774;145788;145792 -;-;-;-;-;-;-;-;-;-;-;-;-;-;-;- 2034    0
LOC103630159    NC_050096.1;NC_050096.1;NC_050096.1;NC_050096.1 170798;171359;171845;173192 171274;171653;171930;173564 +;+;+;+ 1231    0
LOC115032971    NC_050096.1;NC_050096.1;NC_050096.1;NC_050096.1;NC_050096.1;NC_050096.1;NC_050096.1;NC_050096.1;NC_050096.1;NC_050096.1;NC_050096.1;NC_050096.1;NC_050096.1 187229;187240;187240;187611;187611;188917;188917;188917;189146;189146;189146;189146;189146  187640;189088;189037;187640;187640;189088;189037;188996;189581;189581;189587;189591;189591  -;-;-;-;-;-;-;-;-;-;-;-;-   2306    0

[jared.andrews07]

Please use the appropriate label for your posts. This should be a "Question" rather than a "Blog". In addition, please make use of the code formatting button (the 101010 one) in the future, as it makes posts much easier to read and follow.

I have done both of these for you this time.

[krg16]

Thank you for making me aware!

[swbarnes2]

Why don't you include the headers of your input files? That's much more useful than SUBREAD in ASCII.

Answer 1

[h.mon]

You are mixing a GenBank assembly with a RefSeq annotation. The names of the chromosomes are indeed different between them:

 zcat GCA_902167145.1_Zm-B73-REFERENCE-NAM-5.0_genomic.fna.gz | head -n1

>LR618874.1 Zea mays genome assembly, chromosome: 1, whole genome shotgun sequence
  

zcat GCF_902167145.1_Zm-B73-REFERENCE-NAM-5.0_genomic.gtf.gz | grep -v "^#" | head -n1

NC_050096.1     Gnomon  gene    34607   40208   .       +       .       gene_id "LOC103644366"; transcript_id ""; db_xref "GeneID:103644366"; gbkey "Gene"; gene "LOC103644366"; gene_biotype "protein_coding";
  

In addition to naming differently the chromosomes, there may be (usually minor) differences between the assemblies. Download and use both genome fasta and annotation gtf from the same source, RefSeq or GenBank, but don't mix them.