Computational protocol: Characterization and Distribution of the autB Gene in Neisseria meningitidis

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Protocol publication

[…] As the genes flanking the autB gene are conserved in N. meningitidis, we gained the autB gene and its flanking sequences by PCR and sequencing of the PCR products. The dominant CCs of N. meningitidis in China are different to other countries, and the primers were redesigned according to the sequences of the smpB (NMB1526) and glcD (NMB1524) from the whole genome of N. meningitidis MC58 (Accession number NC_003112) (Tettelin et al., ; Shao and Zhu, ). The primer sequences are listed as follows: autB-F: 5′-GAAGGCTGGGAAGTCAAAG-3′ and autB-R: 5′-CGAAAACGACATCAACAGCAC-3′. To amplify the autB gene, TaKaRa LA Taq (RR02MA, Takara, Dalian, China) high fidelity polymerase was used and the annealing temperature was set to 57°C. The length of the products of MC58 ought to be 3,156 base pairs (bp) but it might fluctuate in other strains according to the number of AAGC repeats. In autB-null strains, the length of the products was considered to be ~1,539 bp according to the whole genome sequence of N. meningitidis 053442 (Accession number NC_010120). Both the PCR products with the positive and negative strains were sequenced by Beijing Tianyi Huiyuan Bioscience & Technology Inc, China. The alleles were aligned with the reference autB sequence of MC58 or the sequence of the spacer region in 053442 by MEGA (version 6.0) (http://www.megasoftware.net/). We then obtained the correct sequence of autB by excising the spare sequence (Tamura et al., ). The sequences of the spacer regions flanking the autB gene were obtained using the same method. The correct reading frame of autB was predicted by altering the number of AAGC repeats and removing the premature stop codons and frameshift mutations. Using the SignalP web tools (http://www.cbs.dtu.dk/services/SignalP/) to predict the cleavage site of the N-terminal signal sequence of AutB and neighbor-joining in MEGA, the phylogeny of the N-terminal domain of the passengers was analyzed (Arenas et al., ). […]

Pipeline specifications

Software tools MEGA, SignalP
Applications Phylogenetics, WGS analysis
Organisms Neisseria meningitidis, Homo sapiens
Diseases Haemophilus Infections, Sprains and Strains