I have two biological replicates for this sample and runned HIAST2 to align my data to references (human) and then I used HTseq to count my reads. But the counting results looked abnormal. Could someone suggest me what would probably go wrong here? I checked QC of my data and they are good, don't have ove-represented sequences. my scripts for HIAST module load hisat2/2.1.0 time hisat2 -p 8 -q --phred33 -x GRCH37 references -1 reads1.fastq -2 reads2.fastq -S .sam
The summary file from HIAST run showed the alignment rate are above 90%.
my scripts for HTseq module load python/2.7.8 module load htseq/0.6.1p1 htseq-count -q *.sam GRCH37/genes.gtf > *_counts.txt
replicate1 __no_feature 39795601 __ambiguous 73269 __too_low_aQual 2553578 __not_aligned 822830 __alignment_not_unique 6895933
replicate2 __no_feature 37465277 __ambiguous 64709 __too_low_aQual 2337214 __not_aligned 736195 __alignment_not_unique 5628768
Can anyone give me an idea about the results?