Qiasymphony sp automated instrument

Bacterial growth was harvested from the agar plates and pre-lysed by the recommended method for Gram-negative bacteria (Qiagen, Manchester, UK), which is effective for pneumococcus and closely related streptococci. DNA was extracted from the lysates using a QIAsymphony SP automated instrument and a QIAsymphony DSP DNA Mini Kit (Qiagen), using the tissue extraction protocol. DNA concentrations were measured using the Quant-IT Broad Range dsDNA Kit (Life Technologies, Paisley, UK) and GloMax^® 96 Microplate Luminometer (Promega, Southampton, UK). WGS was performed using Illumina methodology by the PHE Genomic Services Delivery Unit (GSDU, Colindale, UK). The resulting data were automatically analysed using an in-house bioinformatics pipeline for S. pneumoniae.

Isolates were grown on horse blood agar (PHE Media Services) and treated by the Qiagen-recommended method for lysis of Gram-negative bacteria (Qiagen, Manchester, UK), which is effective for S. pneumoniae and simpler than the Gram-positive protocol. DNA was extracted from the lysates using a QIAsymphony SP automated instrument (Qiagen) and a QIAsymphony DSP DNA Mini Kit, using a tissue extraction protocol. DNA concentrations were measured using the Quant-IT Broad Range DNA Kit (Life Technologies, Paisley, UK) and GloMax 96 Microplate Luminometer (Promega, Southampton, UK). After adjusting to a concentration of 10–30 ng/μl, DNA was sent for whole genome sequencing (WGS) by Illumina methodology. The resulting data were automatically analysed using a bespoke bioinformatic pipeline for S. pneumoniae, developed by PHE. Among other things, this (i) checks species identification by a kmer method and (ii) automatically assigns MLST sequence types (STs), identified by mapping the reads against all S. pneumoniae allele variants held in the MLST database [29 ], using a modification of the short-read sequence typing (SRST) software [30 (link)]. Resistance genes affecting susceptibility for macrolides and tetracyclines were identified, and their sequences reviewed.