70 ± 0.04 0.18 ± 0.01 3.53 ± 0.01 0.11 0.05 HpyCH4V TGCA 3.85 ± 0.75 3.70 ± 0.03 3.45 ± 0.03 Selonsertib datasheet 3.53 ± 0.03 1.04 0.98 HpyCI GATATC 0.00 ± 0.03 0.31 ± 0.01 0.02 ± 0.00 0.33 ± 0.00 0.01 0.07 HpyF10VI GCNNNNNNNGC 2.70 ± 0.35 1.96 ± 0.04 2.97 ± 0.09 1.43 ± 0.02 1.38 2.07 HpyF14I CGCG 2.26 ± 0.46 1.96 ± 0.05 1.55 ± 0.05 1.43 ± 0.02 1.15 1.08 HpyF2I CTRYG 1.16 ± 0.17 0.92 ± 0.01 0.37 ± 0.01 0.88 ± 0.00 1.26 0.42 HpyF36IV GDGCHC 0.20 ± 0.21 1.22 ± 0.03 0.31 ± 0.01 0.93 ± 0.01 0.16 0.33 Hpy44II GGNNCC 1.21 ± 0.38 1.96 ± 0.05 0.44 ± 0.00

1.43 ± 0.02 0.62 0.31 HpyII GAAGA 2.29 ± 0.23 2.14 ± 0.03 2.87 ± 0.02 2.16 ± 0.00 1.07 1.33 HpyIP CATG 4.63 ± 0.25 3.70 ± 0.03 4.43 ± 0.04 3.53 ± 0.01 1.25 1.25 HpyIV GANTC 1.70 ± 0.25 3.70 ± 0.04 1.66 ± 0.02 3.53 ± 0.01 0.46 0.47 HpyNI CCNGG 2.04 ± 0.30 1.96 ± 0.05 0.87 ± 0.02 1.43 ± 0.02 1.04 0.61 HpyPORF1389P GAATTC 0.01 ± 0.05 0.31 ± 0.01 0.11 ± 0.00 0.33 ± 0.00 0.03 0.32 HpyV TCGA 0.95 ± 0.25 3.70 ± 0.03 0.18 ± 0.00 3.53 ± 0.01

0.26 0.05 HpyVIII CCGG 1.92 ± 0.30 1.96 ± 0.04 1.06 ± 0.02 1.43 ± 0.02 0.98 0.74 aRestriction endonucleases with palindromic recognition sites are indicated in bold. bO/E ratio indicates the LCZ696 observed/expected (O/E) ratio values. cExclusively underrepresented

in hpEurope GDC-941 MLS. The observed/expected (O/E) ratio indicates deviation from the expectation based on G + C ratio. O/E ratios were highly similar for the WGS and MLS (R2 = 0.87, p < 0.001), without any differences by haplotype. Analysis of the hpEurope and hspAmerind sequences showed that 10 of the 32 cognate restriction sites were underrepresented in MLS and 6 of those sites were also underrepresented in WGS (defined as O/E ≤ 0.5 and Chi Square p-value ≤ 0.005; Table 2). One exception, Hpy166III (cognate site: CCTC) was exclusively underrepresented in hpEurope MLS, but not in the hspAmerind nor in WGS. The underrepresented sites Branched chain aminotransferase varied in their C + G content from 33.3 to 75%. Most (9) of those 10 underrepresented sites were palindromic [28–30] (Table 2). Conversely, only one cognate recognition site: Hpy99III (cognate site: GCGC), was strongly overrepresented (O/E ≥ 2 and Chi Square p-value ≤ 0.005) in both hpEurope/hspAmerind MLS and WGS (Table 2). Overall, similar results were found when analyzing hspEAsia and hspWAfrica strains (data not shown). In summary, the H. pylori genome has mostly evolved to avoid RMS cognate recognition sites.