I'm having problems with a stranded, paired end RNA-seq analysis. I tried to map the reads with HISAT2 and RNA STAR but when I visualize the alignment it seems unstranded. If it helps, here the parameter I used with HISAT2:
Tool Parameters Input Parameter Value Note for rerun Source for the reference genome indexed Select a reference genome hg38 Is this a single or paired library paired FASTA/Q file #1 80: Rep1_1.fq FASTA/Q file #2 81: Rep1_2.fq Specify strand information Reverse (RF) Paired-end options defaults sum Output alignment summary in a more machine-friendly style. False Print alignment summary to a file. False adv Input options defaults Alignment options defaults Scoring options defaults Spliced alignment options defaults Reporting options defaults Output options defaults Other options defaults Job Resource Parameters no
In order to understand more what is going on I tried to run "infer experiment' on the bam files and it gives this kind of result...
This is PairEnd Data Fraction of reads failed to determine: 0.9590 Fraction of reads explained by "1++,1--,2+-,2-+": 0.0045 Fraction of reads explained by "1+-,1-+,2++,2--": 0.0365
Does anyone have an idea why this is happening?
Thank you very much