Total Alignment: 0 /results of featureCounts RNA-Seq

0

I have human RNA-Seq dataset it has two different barcodes in the different folder. I aligned with that command
minimap2 -ax splice -uf -k14 ref.fa direct-rna.fq > aln.sam
I try to quantify and counts using with Subread featureCounts function. In the subread results, there is a problem with one of the bam files. I downloaded reference and gtf files from GENCODE.
I checked the bam file with samtools view -H first.bam-second.bam I saw that I followed the same steps for each bam file.
In the IGV results, I saw matches and alignment for all bam files.

Do you have any suggestions the solve this problem? What am I doing wrong?

featureCounts -T 8 -a gencode.v38.chr_patch_hapl_scaff.annotation.gtf -g 'transcript_id' -o readcouts.txt bam/*.bam

|| Total alignments : 11214480 ||
|| Successfully assigned alignments : 4051945 (36.1%) ||
|| Running time : 2.67 minutes

|| Total alignments : 0 ||
|| Successfully assigned alignments : 0 ||
|| Running time : 2.89 minutes

I also tried with Salmon in the salmon alignment-based quantification results bam file has huge differences between each other.

`salmon quant --ont -t reference.fa -l A -a first.bam -o salmon_quant1`

Total # of mapped reads : 5465357

of uniquely mapped reads : 328808350000000

ambiguously mapped reads : 2177274
`salmon quant --ont -t reference.fa -l A -a second.bam -o salmon_quant2`
Completed first pass through the alignment file.

Total # of mapped reads : 3843632

of uniquely mapped reads : 2552463

ambiguously mapped reads : 1291169


Nanopore


featureCounts


HG38


Rna-Seq

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