This field specifies the alleles involved in variant calls, using this format:

REF= reference_allele; OBS= observed_allele

Examples:

• A TT insertion with 1-base prior at reference C: REF=;OBS=TT

• A TT deletion with 1-base prior at reference G: REF=TT;OBS=

Notes:

• 6-column format

  • The elements can be empty: "REF=;" or "OBS=;". Empty means deletion.

  • An additional element ANCHOR=base_before_allele can be provided for backward compatibility, but is completely optional. In fact, it is recommended that the ANCHOR key it is NOT provided for TS >= 4.2.

  • Insertion alleles should have the same start and end position, and that position corresponds to a region between two bases. SNV, MNV, deletion, and complex variants should correspond to the reference bases that are spanned by the event.

  • The REF and OBS should be on the forward genomic strand. There should be one alternative allele per line.

  8-column format

  • The +/- strand notation in the hotspot file refers to the orientation of the Ion AmpliSeq™ design input sequence, not to the reference sequence. REF and OBS alleles must always be reported on the forward strand of the reference sequence.

  • HotSpotAlleles are always reported based on the allele information from the positive strand of the reference sequence. Even if the allele strand is negative, the REF and OBS bases still report the alleles on the positive strand.

  For example, if there is a hotspot either on the positive strand or on the negative strand on a genomic coordinate, the strand information makes no difference to what is reported on the HotSpotAlleles column. HotSpotAlleles column always reports the alleles on the positive strand. In the following example, the strands are different, but the reported alleles are always from the positive strand:

  chr 143815007 43815009 ID1 0 - REF=TG;OBS=AA AMPL1

  chr 143815007 43815009 ID2 0 + REF=TG;OBS=AA AMPL2