STAR --quantMode GeneCounts always gives 0 counts

Question

[mgmohsen]

I'm trying to perform an alignment using STAR and extract gene count information. Here is the command I entered:

STAR --runThreadN 16 --readFilesCommand zcat --quantMode GeneCounts --genomeDir /path/to/genome/dir --readFilesIn /path/to/file_R1.fastq.gz /path/to/file_R2.fastq.gz

However, whenever I analyze ReadsPerGenes.out.tab all columns are 0 all the way down.

$ head -10 ReadsPerGenes.out.tab
N_unmapped      0       0       0
N_multimapping  0       0       0
N_noFeature     0       0       0
N_ambiguous     0       0       0
ENSG00000223972.5       0       0       0
ENSG00000227232.5       0       0       0
ENSG00000278267.1       0       0       0
ENSG00000243485.5       0       0       0
ENSG00000284332.1       0       0       0
ENSG00000237613.2       0       0       0

All of the following commands provide no output:

$ awk '$2 > 0' ReadsPerGenes.out.tab
$ awk '$3 > 0' ReadsPerGenes.out.tab
$ awk '$4 > 0' ReadsPerGenes.out.tab

In case there could be something wrong with the genome indexing, the genome was indexed using the following command:

STAR --runThreadN 16 --runMode genomeGenerate --genomeDir . --genomeFastaFiles ./GRCh38.primary_assembly.genome.fa --sjdbGTFfile ./gencode.v31.primary_assembly.annotation.gtf

Any ideas what could be going wrong here?

Answer 1

[swbarnes2]

I don't think STAR can quantify genes without you giving it a gtf to know where the genes are.

[mgmohsen]

Hm, but I've given it in a gtf in the genome indexing step. Does it also need to be specified during alignment?

[GenoMax]

Basic question - chromosome identifiers in your GTF file and your reference match, correct?

[mgmohsen]

Yes they match. And also, I just tried aligning some other fastq sets and those seem to work normally. It seems like there might be something wrong with this set of fastqs in particular.

[GenoMax]

Good to know. Grab a few reads and align them using BLAST. That would be the easiest place to start by checking if that data is from the right genome.