I have human RNASeq data generated using lexogen kit. I have followed their analysis protocol (to process raw data). Then used tophat2 for alignment, observed only 53%. check unalinged using blast they are going human rRNA sequences end2end. Then i used hisat2, the % increased to 75. Then tried bowtie2 end2end parameter (which does not align junction read i assume) has given 79%.
Unaligned reads from different aligners checked against NCBI NT using BLAST they are hitting to human rRNAs/ncRNAs. Questions,
How bowtie is preforming better than junction aligners?
How do i improve the overall alignment?
Reference used was GRCh38 from ensemble
I have seen the same trend for fungi sample as well.
Any in site what could be the reason?