Hi, If you don't have a home made design you can find it like in the Thermo Fisher web site like : https://www.ampliseq.com/tmpl/view.action?tmplDesignId=48744395#/?listAction=tmplCoverageSummaryList&tmplDesignId=48744395&wrapperId=ajaxTableWrapper In this case you have to sign in to get it.

Dear Titus, the panel is already designed.

I want to do known annotation file, with mutations known in the amplicons, not the panel amplicons design: Partial example of a HotSpots BED file below:

track name="HSMv12.1" description="AmpliSeq Pool HSMv12.1" type=bedDetail

chr1 43815007 43815009 COSM19193 REF=TG;OBS=AA AMPL495041 
chr1 43815008 43815009 COSM18918 REF=G;OBS=T AMPL495041 
chr1 115256527 115256528 COSM585 REF=T;OBS=A AMPL30014 
chr1 115256527 115256528 COSM586 REF=T;OBS=G AMPL30014 
chr1 115256527 115256529 COSM33693 REF=TT;OBS=CC AMPL30014 
chr1 115256527 115256529 COSM30646 REF=TT;OBS=CA AMPL30014 
chr1 115256527 115256530 COSM53223 REF=TTG;OBS=CTT AMPL30014 
chr1 115256528 115256529 COSM583 REF=T;OBS=A AMPL30014 
chr1 115256528 115256529 COSM584 REF=T;OBS=C AMPL30014 
chr1 115256528 115256529 COSM582 REF=T;OBS=G AMPL30014 
chr1 115256528 115256530 COSM12725 REF=TG;OBS=AA AMPL30014 
chr1 115256528 115256530 COSM579 REF=TG;OBS=CT AMPL30014

The REF=;OBS= field is required, as is the track line.