Computational protocol: Genome-wide dissection of globally emergent multi-drug resistant serotype 19A Streptococcus pneumoniae

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Protocol publication

[…] MLST was performed according to the standard method described by Spratt and Enright http://www.mlst.net[]. Briefly, seven housekeeping gene loci were sequenced bidirectionally, uploaded to the MLST website, and analyzed for sequence type and clonal complex associations based on the existing database. DNA sequencing of genes associated with drug resistance (pbp1a, pbp2b, pbp2x, tetR and ermB) was performed using a standard capillary gene sequencer from Applied Biosystems (Foster City, CA). The Solexa paired-end Sequencing Platform (Illumina, San Diego, CA) was used to generate reads of 50 to 75 bp (with on average greater than 100X coverage for the genome) which were assembled using NextGene (SoftGenetics, State College, PA). []. Annotation of the genome was performed using the RAST (Rapid Annotation using Subsystem Technology) server []. Additional file http://www.pillailab.com/suppdata/index.html summarizes all polymerase chain reaction (PCR) and DNA sequencing primers and associated cycling parameters used in this study to confirm WGS findings at the capsule locus, resistance alleles, and transposon insertion sites. [...] eBURST analysis of MLST sequence data was performed as described on the MLST website http://www.mlst.net[]. Genome assembly was performed using NextGene (SoftGenetics, State College, PA) and genome comparison using the Artemis Comparison Tool (Wellcome Trust Sanger Institute, Cambridge, UK). Gene ontology classification was achieved using GenoList (Institut Pasteur, Paris). Gene ontology categories were identified using AmiGO []. […]

Pipeline specifications

Software tools NextGENe, RAST, BURST, ACT, AmiGO
Databases MLST.net Genolist
Application WGS analysis
Organisms Streptococcus pneumoniae
Chemicals Amoxicillin, Ceftriaxone, Erythromycin, Penicillins