featureCounts low rate and strange output

Question

[Yi]

Hi, I'm new to RNAseq data analysis. I used HISAT2 for alignment and the rate is high (~90%). Then I used samtools to convert sam to bam, and then sorted. Then I put the sorted bam to featureCounts, but the alignment rate is very low, like 20%. Also, the output is a bit strange. Why is there repeated locations for the same gene?

ls *sorted.bam | while read i; do (featureCounts -T 24 -p -t exon -g gene_id -a /hpctmp/renyi04/reference/Homo_sapiens.GRCh38.105.gtf -o ../counts/${i:0:11}_counts.txt $i); done

Any idea how I can modify the script and improve the alignment rate? Appreciate your help!!

[iraun]

What do you mean by "the output is a bit strange"? Have you had a look to the other posts already about this topic in biostars? Such as:

• Feature Counts output interpretation

• How to generate a count matrix with featurecounts

[Yi]

Thank you iraun for the useful information