number FQ312006) using SMALT version 0.6.3 software, SNPs were called and a tree generated from the SNP alignment using FastTree. Serotyping The serotype of predicted type b strains was determined
by the slide agglutination test using serotype-specific serum as described elsewhere . The results from these tests were supported by BLAST analysis of the respective Selleck MM-102 genome sequence derived in this study using published type b capsule gene sequence as a probe. Transformation of H. influenzae Genomic DNAs from strains Eagan and a spontaneous high level streptomycin resistant derivative, EaganstrR, were prepared and then used to transform strain Rd using the standard MIV protocol . Transformants were selected following growth overnight on BHI check details plates with or without added streptomycin (500 μg/ml). 200 independent colonies were selected, pooled, and genomic DNA was isolated from the respective Rd+EaganstrR and Rd+Eagan transformants. The pooled genomic DNA from each transformation was sequenced on an individual Illumina GAII flow cell at the Wellcome Trust Sanger see more Institute. The frequency of spontaneous strR mutation was calculated by plating on BHI/streptomycin plates competent Rd cells taken through the transformation procedure but without added donor DNA. Acknowledgements ERM and DWH were supported by grants from the Medical Research Council, UK and PP, SB and
JP were supported by the Wellcome Trust. The authors are grateful for
Thomas Connor at the Sanger Institute for help in producing the SNP-based tree. Electronic supplementary material Additional file 1: Figure S1. Tree indicating the relatedness of Haemophilus genome sequences based on similarities in their patterns of SNPs. Illumina fastq sequences were mapped against the reference sequence of Hib strain 10810 and the tree was generated using FastTree from the SNP alignments. Some minor differences in strain placement when compared to Mauve analysis reflects those strains with the lowest quantity (and quality) of genome sequence information. (PDF 8 KB) References 1. Boissy MRIP R, Ahmed A, Janto B, Earl J, Hall BG, Hogg JS, Pusch GD, Hiller LN, Powell E, Hayes J, et al.: Comparative supragenomic analyses among the pathogens Staphylococcus aureus, Streptococcus pneumoniae, and Haemophilus influenzae using a modification of the finite supragenome model. BMC Genomics 12:187. 2. Medini D, Donati C, Tettelin H, Masignani V, Rappuoli R: The microbial pan-genome. Curr Opin Genet Dev 2005,15(6):589–594.PubMedCrossRef 3. Hogg J, Hu F, Janto B, Boissy R, Hayes J, Keefe R, Post J, Ehrlich G: Characterization and modeling of the Haemophilus influenzae core and supragenomes based on the complete genomic sequences of Rd and 12 clinical nontypeable strains. Genome Biol 2007,8(6):R103.PubMedCrossRef 4. Fleischmann RD, Adams MD, White O, Clayton RA, Kirkness EF, Kerlavage AR, Bult CJ, Tomb JF, Dougherty BA, Merrick JM, et al.