The sequencing results of the antigenic peptides alone are less informative (Fig. 2) . Nevertheless, when we perform a statistical analysis by summarizing the normalized frequency of each amino acid residue in all positive antigenic peptides (defined as the residue frequency map), a major peak with the full width at half maximum (FWHM) from residues 387-480 is revealed (Fig. 4 , panel A). We also modify the residue frequency map by taking into account the respective RAYS ratios of positive peptides, which however results in an overall rather similar frequency map (Fig.  S3, panel B) . Furthermore, even if we combine all the in-frame sequences from sorted yeast cells, a similar map (Fig. S3 , panel C) is again obtained, implying that the noises from the in-frame but The residue frequency map is then overlaid onto the crystal model of the trimeric HA of the 2009 A(H1N1) influenza virus (PDB ID: 3LZG) [27] , and annotated by colors from red (high frequency) to blue (low frequency) (Fig. 4, panel B ). This structural frequency map reveals that the major peak (corresponding to residues 387-480 within the FWHM) on the residue frequency map can be divided into two dominant antigenic regions since they have independent structural features. One corresponds to the central coiled-coil helix CD in the HA2 subunit responsible for membrane insertion, designated as R1 (residues 424-480, indicated by red, see also Fig. 5 ), while the other contains helix A in the HA2 subunit that is important for conformational change upon low pH exposure, designated as R2 (residues 387-423, indicated by faint red and white, see also Fig. 5) [28] . R1 is present in a relatively recessed surface area while R2 is located near the exterior surface area of the stem region. For the HA1 subunit, no distinct antigenic region is identified, because of the limited number of peptides (7) with relatively longer lengths (all .200 aa), which are insufficient to form peak areas.