Sentative for each graph (which represents gene families) was saved. This final step, where gene families sharing 95 homology are condensed to gene families sharing 80 homology was necessary to address the problem presented by triangle inequality. For example, if the iterative approach is used to capture gene families which share greater than 80 homology without this final step, the input order of genomes will profoundly affect the final number of genes estimated in the pan genome. Consider the following simplified three gene scenario using a similarity threshold of 80 : Gene A matches gene B and gene C at 81 identity, although genes B and C match each other at 79 identity. If gene A is encountered in the first iteration, it can be get GS-4059 compared to either genes B or C next, and finally retained as the sole representative of this gene family in the pan-genome (even though genes B and C only match each other to 79 , since in this scenario genes B and C are never directly compared). However, if gene B is encountered first, it can be compared to gene A, where gene B will then be retained in the pan-genome. Then, in the next Synergisidin web iteration where genes B and C are compared, both these genes are retained in the pan-genome since they match with an identity 1 below the required threshold. This hypothetical scenario (but drawn from problems we encountered) represents a discretisation problem which is difficult to resolve without an all-versus-all approach, which is provided for by the final step the purpose of the iterative steps is to broadly capture genes which share greater than 95 homology in order to limit the number of genes used in the final all-versus-all comparison. At each stage, the genomes in which these genes could be detected was tracked, which allowed the data to finally be transformed into a binary presence/ absence matrix for further investigation. To investigate the size of the core or pan-genomes of phylogroup A or MPEC strains, for each data point we randomly sampled (with replacement) n number of strains from our pan-genome presence absence matrix data for 10,000 replications, where n is an integer between 2 and 66. For the core genome, for each data point a gene was counted as `core’ if it was present in n-1 genomes. For the pan genome, a gene was counted if it was present in at least one genome.Estimation of the phylogroup A pan-genome.Determination of the specific MPEC core genome.To determine the genes that could be detected in all MPEC (core genes), but which were not represented in the core genome of a similarly sized sample of all phylogroup A genomes, first we modelled how the numerical abundance of a gene in the phylogroup A populationScientific RepoRts | 6:30115 | DOI: 10.1038/srepwww.nature.com/scientificreports/affected the probability that this gene would be captured in the core genome of 66 sampled strains. To do this, we simulated random distributions of increasing numbers of homologues (from 1 to 533) in 533 genomes over 100,000 replications per data point. For each replication, we sampled 66 random genomes and counted how many times a gene with that numerical abundance in 533 genomes appeared in at least 65 of the 66 sampled genomes. We then fit a curve to this data using the `lm’ function within R using the third degree polynomial. Since our data intimated that randomly sampled E. coli could be expected to be as closely related to each other as MPEC are 15 in 100,000 times, we set the lower limit of the number.Sentative for each graph (which represents gene families) was saved. This final step, where gene families sharing 95 homology are condensed to gene families sharing 80 homology was necessary to address the problem presented by triangle inequality. For example, if the iterative approach is used to capture gene families which share greater than 80 homology without this final step, the input order of genomes will profoundly affect the final number of genes estimated in the pan genome. Consider the following simplified three gene scenario using a similarity threshold of 80 : Gene A matches gene B and gene C at 81 identity, although genes B and C match each other at 79 identity. If gene A is encountered in the first iteration, it can be compared to either genes B or C next, and finally retained as the sole representative of this gene family in the pan-genome (even though genes B and C only match each other to 79 , since in this scenario genes B and C are never directly compared). However, if gene B is encountered first, it can be compared to gene A, where gene B will then be retained in the pan-genome. Then, in the next iteration where genes B and C are compared, both these genes are retained in the pan-genome since they match with an identity 1 below the required threshold. This hypothetical scenario (but drawn from problems we encountered) represents a discretisation problem which is difficult to resolve without an all-versus-all approach, which is provided for by the final step the purpose of the iterative steps is to broadly capture genes which share greater than 95 homology in order to limit the number of genes used in the final all-versus-all comparison. At each stage, the genomes in which these genes could be detected was tracked, which allowed the data to finally be transformed into a binary presence/ absence matrix for further investigation. To investigate the size of the core or pan-genomes of phylogroup A or MPEC strains, for each data point we randomly sampled (with replacement) n number of strains from our pan-genome presence absence matrix data for 10,000 replications, where n is an integer between 2 and 66. For the core genome, for each data point a gene was counted as `core’ if it was present in n-1 genomes. For the pan genome, a gene was counted if it was present in at least one genome.Estimation of the phylogroup A pan-genome.Determination of the specific MPEC core genome.To determine the genes that could be detected in all MPEC (core genes), but which were not represented in the core genome of a similarly sized sample of all phylogroup A genomes, first we modelled how the numerical abundance of a gene in the phylogroup A populationScientific RepoRts | 6:30115 | DOI: 10.1038/srepwww.nature.com/scientificreports/affected the probability that this gene would be captured in the core genome of 66 sampled strains. To do this, we simulated random distributions of increasing numbers of homologues (from 1 to 533) in 533 genomes over 100,000 replications per data point. For each replication, we sampled 66 random genomes and counted how many times a gene with that numerical abundance in 533 genomes appeared in at least 65 of the 66 sampled genomes. We then fit a curve to this data using the `lm’ function within R using the third degree polynomial. Since our data intimated that randomly sampled E. coli could be expected to be as closely related to each other as MPEC are 15 in 100,000 times, we set the lower limit of the number.