We have performed RNA-seq of 60 sorted T cells from human and aligned to the reference genome (GRCh38) using HISAT2. About 80% of reads were mapped to the reference genome. However, when featureCounts of Subread package was used to count mapped reads, only 20-25% of mapped reads were counted to be associated with feature provided (exon and gene_id) of the reference gtf file (Homo_sapiens.GRCh38.87.gtf). I do not know what is happening here. I have two questions:
- First what can be possible reason for this observation?
- Is it possible that we can identify to which region of the reference genome the rest of the reads mapped?
I will appreciate your help.