The partial sequences were a string of 3,406 bp composed of ordered concatenated sequences (multilocus sequences, or MLS) from seven housekeeping genes as follows: atpA (627 bp), efp (410 bp), mutY (420 bp), ppa (398 bp), trpC (456 bp), ureI (585 bp) and yphC (510 bp) [58–60]. The MLS were from OICR-9429 clinical trial H. pylori strains from hosts from four continents: Africa, Europe, Asia, and the Americas (from Native American and Mestizo hosts). All sequences were available at the EMBL or GenBank database (http://www.ebi.ac.uk/) and/or at the MLST website for H. pylori (http://pubmlst.org/helicobacter/)
[59]. Whole genome sequences (WGS ~ 1.5 Mb) of seven H. pylori were available in GenBank. Four strains were from European hosts: 26695, HPAG1, P12 and G27 (accession numbers NC_000915, NC_008086, NC_ 011333, CP001173, respectively; all hpEurope); one, J99 (NC_000921; hpAfrica1) was from the US, and two Shi470 and V225 (NC_010698; CP001582; hspAmerind) were from Native Americans from Peru and Venezuela, respectively. The MLS of the 7 strains with whole genome sequences were also taken into account for the analysis, and form part of the 110 MLS
analyzed. Haplotype assignment All the sequences were previously analyzed Androgen Receptor antagonist and assigned to their correspondent populations [2, 5]. Neighbor joining clustering analysis [61] of all the strains was performed in MEGA 5.0. [62]. Frequency of cognate recognition sites The observed frequency of cognate recognition sites for 32 RMS (Table 2) that have been reported in H. pylori[25, 42, 43, 63] was INCB018424 in vitro determined in the 110 MLS (3,406 bp) and 7 WGS (1.5-1.7 Mb) using the EMBOSS restriction program (http://emboss.sourceforge.net/), by counting the number of restriction “”words”", in each sequence. We determined: 1) the number of cognate recognition sites, that is the sum of all words per strain, 2) their frequency per Kb, 2) their distribution per
Kb in the seven WGS, and 4) the RMS profile of each strain, which is the combination of the values for the 32 cognate recognition sites per strain. The expected frequency of cognate recognition sites was based on the actual nucleotide proportions in each WGS or MLS sequence (Additional file 1: Table S2), and determined by 1,000 simulations. The algorithm used Methane monooxygenase for simulating the frequencies of cognate recognition sites was created as follows: (i) a pool of 1,000 nucleotides containing the exact proportion of each nucleotide in each genome or MLS sequence was created (the “”pool-simulated sequence”"); (ii) a nucleotide was randomly chosen, from the pool-simulated sequence, k times, in which k is the length of each recognition sequence; (iii) simulated words that matched the recognition sequence were counted; and steps 2, 3 were repeated l-k times, where l is the length of the whole genome or MLS sequence. For each enzyme, observed and expected numbers of cognate recognition sites were compared (O/E ratio) values per enzyme.