TopHat -M/--prefilter-multihits; unmapped reads

[imsharmanitin]

hi!

i am confused when to use TopHat "-M/--prefilter-multihits and when not

"When mapping reads on the transcriptome, some repetitive or low complexity reads that would be discarded in the context of the genome may appear to align to the transcript sequences and thus may end up reported as mapped to those genes only. This option directs TopHat to first align the reads to the whole genome in order to determine and exclude such multi-mapped reads (according to the value of the -g/--max-multihits option)."

the alignment of reads first to genome and then to transcriptome seems opposite to -G option

As far i know the multi-mapped reads will end up in Unmapped.bam along with the reads which would have not mapped even if have not used -M option

so do we need to discard all unmapped.bam reads?

i am struggling to understand when to use -M option and when not and how to deal with the multi-mapped reads discarded if -M option is used

[GenoMax]

Umapped reads file comes in handy if you find a large number of reads not mapping because you can then chase those down to see if you have a contamination or some other sort of a problem.

For some of tophat analyses you may want to run multiple versions with/without option combinations to see if one works better than the other (e.g. -G or -G with -T etc).