Hello everyone, I have human Rna-Seq data reading by MiniOn.I aligned with the human reference genome (GRCh38.p13) from that website= https://www.gencodegenes.org/human/ using minimap2.

I download annotation from the same website. I used SubRead for quantification and feature count. On the SubRead results, there is a problem with one of the bam files. I aligned again that fastq files with the same reference genome of the first one and same gtf file and also I checked fastq file of second one but I didn't see any problem. Only differences are these bam file has a different barcode

Do you have any suggestions for that problem?

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

Salmon Quant Alignment Based results

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