I have RNA-sequence data where the RNA was isolated using the ribo-zero protocol. I (presumably) have the reverse complement of the transcript. I aligned them using Hisat2 with
hisat2 -x index -U input.fastq -S output.sam --rna-strandness R
However, when viewing it in IGV the reads are always aligned to the wrong strand of the annotated genes. For example in the figure IL18RAP is on the + strand, but the reads get aligned to the - strand (top sample, blue reads are - strand with <- direction, red reads are + strand with -> direction. IL18RAP has -> direction).
I tried doing the same but aligning to forward strand as I thought maybe I don't have the reverse complement of the transcript
hisat2 -x index -U input.fastq -S output.sam --rna-strandness F
but this also aligns to the wrong strand (middle sample).
Finally, I took the reverse complement of the reads and did the alignment with
--rna-strandness F, and now they did align to the correct strand. However, I thought that this should be the same with
--rna-strandness R, as the documentation says
'R' means a read corresponds to the reverse complemented counterpart of a transcript. How can I correctly allign to the reverse complement strand with Hisat?