Hi,

I am doing ribo_seq analysis and I aligned my files (R1+R2) to the rRNA, then I took the resulting reads that were not aligned to the rRNA (turned the sam to one fastq file) and aligned them with tophat2 to the genome. This is my cmd:

tophat2 -G BioMart.GRCh37.75.gtf -o ./output 3  -p 3 --no-novel-juncs -g 5 --prefilter-multihits hg19  sample.fq

The thing is - I used the " --prefilter-multihits" but my summary is this:

Reads:
          Input     :  10803641
           Mapped   :   5993137 (55.5% of input)
            of these:   1915741 (32.0%) have multiple alignments (3861832 have >5)
55.5% overall read mapping rate.

This seems to mean I have not been successful in removing multihits...

Any idea where I might have gone wrong? Did I miss something with the cmd - or earlier?

Thanks,