Question: STAR --quantMode GeneCounts always gives 0 counts
0
gravatar for mgmohsen
13 months ago by
mgmohsen0
Stanford
mgmohsen0 wrote:

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?

alignment • 456 views
ADD COMMENTlink modified 13 months ago by swbarnes28.1k • written 13 months ago by mgmohsen0
1
gravatar for swbarnes2
13 months ago by
swbarnes28.1k
United States
swbarnes28.1k wrote:

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

ADD COMMENTlink written 13 months ago by swbarnes28.1k

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

ADD REPLYlink written 13 months ago by mgmohsen0
1

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

ADD REPLYlink written 13 months ago by genomax87k

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.

ADD REPLYlink written 13 months ago by mgmohsen0
1

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.

ADD REPLYlink modified 13 months ago • written 13 months ago by genomax87k
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