Characterization of HSPs using HANDS. (A) Illustration of Homeolog-Specific Polymorphisms (HSPs) in the T. aestivum genome. Bases that match the reference sequence are shown in grey and base substitutions (versus the reference sequence) are shown in other colors. Positions containing a HSP are highlighted in red boxes. (B) An example of using HANDS to assign subgenome-specific base-identity at HSP positions. HANDS uses the read alignments from the sequencing alignment/mapping (SAM) file of the polyploid in conjunction with the bases in the diploid progenitor-relatives at HSP positions to assign variant bases to the polyploid subgenomes. In the example shown, the polyploid genome contains HSPs at positions 5 (A/C), 12 (C/T), 40 (C/G), 55 (A/C/T) and 92 (C/G). The reference genome is depicted in black and the RNA-seq reads aligned against the reference genome are shown as arrows below the reference genome. Bases in the reads matching the reference are shown in grey, while nucleotides at polymorphic sites are indicated by a capital letter. Read alignments for each gene are considered in turn from the SAM file and are processed in five steps: (1) creation of base patterns from aligned reads, (2) removal of potential sequencing errors and embedded base patterns, (3) assignment of base patterns to subgenomes, (4) assignment of bases to subgenomes using the previously assigned base patterns, and (5) assignment of bases to subgenomes using base patterns discarded in step 4. These steps are explained in greater depth in the main text and illustrated in the Note S1 in Additional file 1.