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