I'm having quite abit of trouble handling my RNAseq fastq files. These were originally obtained from a single cell RNAseq experiment (probably not relevant). As usual I map back to mm10 using HISAT2 and then using a UCSC GTF I check for hits using htseq. The issue is that htseq doesn't seem to pick up any hits, it only does so in 1 or 2 genes.
I thought something might be wrong with my aligning so loaded the bam. file from hisat into IGV just to check where the reads fall and they do fall in genes, definitely more than 2 so im not sure what I'm doing wrong with my htseq settings. Any suggestions?
I've checked out this thread which was quite similar but it didn't seem to help (What to do when alignment rate is low even though the genomic data and RNA-seq data are of same stain)
This is the output I get from Hisat:
3337547 reads; of these: 3337547 (100.00%) were paired; of these: 3337528 (100.00%) aligned concordantly 0 times 12 (0.00%) aligned concordantly exactly 1 time 7 (0.00%) aligned concordantly >1 times ---- 3337528 pairs aligned conco