Why Is Tophat Reporting Secondary Alignments Even When The Option To Do So Is Turned Off

Question

[Varun Gupta]

Hi Everyone

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

Regards VARUN

[cpcantalapiedra]

Are there any alignments with a different flag or better score for those reads?

[Jordan]

I think this was a bug in tophat2. Unique alignments were being reported as secondary alignments, which I think was fixed in the latest versions (from version 2.0.3). Which version of tophat2 did you use?

[Fabrice Jossinet]

I have the same problem with tophat 2.0.13 (the last version according to the website).

Answer 1

[Martombo]

if there is more than one alignment with the same best score, TopHat will randomly select one of them to be a primary alignment, while it will report all the others as secondary.