I am working with Rna seq data and for that i am using TOPHAT aligner.
I came across 2 options in tophat which i cannot understand
-g/--max-multihits <int> Instructs TopHat to allow up to this many alignments to the reference for a given read, and choose the alignments based on their alignment scores if there are more than this number. The default is 20 for read mapping. Unless you use --report-secondary-alignments, TopHat will report the alignments with the best alignment score. If there are more alignments with the same score than this number, TopHat will randomly report only this many alignments. In case of using --report-secondary-alignments, TopHat will try to report alignments up to this option value, and TopHat may randomly output some of the alignments with the same score to meet this number. --report-secondary-alignments By default TopHat reports best or primary alignments based on alignment scores (AS). Use this option if you want to output additional or secondary alignments (up to 20 alignments will be reported this way, this limit can be changed by using the -g/--max-multihits option above).
Now as stated a single read can have upto 20 alignments to a reference genome. By default Tophat reports best or primary alignments. I have not used --report-secondary-alignments option when i run my tophat. Why does in the bam file do i get sam flags like these 256 , 272
Now 256 means not a primary alignment (5th column mapping quality score is although very low) && 272 means read reverse strans and not a primary alignment
Why are even secondary alignments being reported if i have not turned on the option of --report-secondary-alignments
Am i missing something. Hope to hear from you soon