Computational protocol: Nontypeable Pneumococci Can Be Divided into Multiple cps Types, Including One Type Expressing the Novel Gene pspK

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Protocol publication

[…] PCR for MLST was performed as described elsewhere (), and the DNA sequences of the PCR products were determined by the Genomics core facility at University of Alabama at Birmingham (UAB). All the DNA sequences were submitted to the pneumococcal MLST website (http://spneumoniae.mlst.net), which identified the sequences either as existing alleles or as new alleles with new allele numbers.A neighbor-joining tree () was created with concatenated DNA sequences (2,751 bp) of six of the seven genetic loci used by MLST (ddl was removed) for the 17 NT pneumococcal isolates using MEGA version 4 (). The sequence types of these 17 isolates are in bold in . To include reference pneumococcal and nonpneumococcal species in the tree, the corresponding DNA sequences of 39 S. pneumoniae strains, 42 S. pseudopneumoniae strains, 25 S. mitis strains, and 8 S. oralis strains were retrieved from the GenBank or the MLST database. The sequences in these two databases were deposited by others (, , ). The statistical significance of a branching was determined by performing 1,000 bootstrap replications with the minimum evolution algorithm. The tree was drawn to scale, with branch lengths measured in the same units used for the evolutionary distances on the phylogenic tree. [...] To detect genes encoding pneumococcal autolysin (lytA), pneumolysin (ply), cpsA (wzg), aliC, aliD, pspK, and the intergenic region, PCR was performed as described elsewhere () with the primer sets listed in . The PCR products were visualized by agarose gel electrophoresis (). To amplify the entire cps locus, a long-range PCR was carried out using the primers located at dexB and aliA () with TaKaRa LA Taq (TaKaRa Bio United States, Madison, WI).The DNA sequences of the PCR products were determined by the Genomics Core Facility at UAB. The DNA sequences were analyzed and aligned using DNASTAR Lasergene version 6.0. Insertional sequences and the open reading frames (ORFs) in the DNA sequences were identified using a BLAST search at the National Center for Biotechnology Information website (http://www.ncbi.nlm.nih.gov/BLAST), Softberry (http://www.softberry.com) free web-based software, and IS Finder (http://www-is.biotoul.fr). […]

Pipeline specifications

Software tools MEGA, DNASTAR Lasergene
Databases MLST.net
Applications Phylogenetics, Sanger sequencing
Organisms Mus musculus, Streptococcus pneumoniae, Mycoplasma pneumoniae, Homo sapiens
Diseases Pierre Robin Syndrome