The procedure for construction, expression and screening of random viral peptide libraries using yeast surface display is shown in Fig. S1 (see also Materials and Methods for details). In step 1, the full length HA gene of the pandemic 2009 A(H1N1) influenza virus (including the core HA protein of 519 aa, the signal peptide of 16 aa, and the inter membrane domain of 36 aa) was digested and re-assembled by PCR to form a pool of fragments enriched in 100-500 bp [24] . In step 2, the fragment library was ligated into the yeast display vector pCTCON-T (derived from the yeast surface display vector pCTCON-2 [23] shown in Fig. 1 ), transformed into yeast EBY100 cells and induced for expression, generating a typical library of 1610 5 -10 6 clones to cover most of the possible fragments [25] . Because there are 3 stop codons on pCTCON-T downstream of the peptide inserts to terminate every ORF, the theoretical possibility for in-frame fusions is 1/3 for the N-terminal end of the peptide inserts. Thus, the theoretical yield for cells expressing in-frame peptides is 1/6 (the fragments can be inserted in forward and reverse directions). Subsequently in step 3, the cell library was incubated with antisera, labeled with secondary fluorescent antibody and subjected to FACS in step 4, after which the cells harboring antibody-binding peptides were distinguished from those with non-binding peptides. In step 5, we isolated plasmids directly from the pool of yeast cells containing positive peptide sequences, which were retransformed into E. coli for sequencing. This reduced laborious plasmid extraction from individual yeast clones. Plasmids containing in-frame sequences were transformed back into yeast for individual flow cytometry (FCM) verification. Retransformation and FCM were also performed for plasmids containing out of frame sequences (false positive). Antigenic peptide sequences were aligned with the HA sequence in step 6 (see also Figs. 2 and 3). In step 7, statistical analyses both on the sequence and structural levels were then performed to map out the antigenically important regions of the HA protein, by summarizing the frequency of each amino acid residue appearing in the antigenic peptides (see also Fig. 4) .