Genotyping
Genotyping determines differences in the genetic composition by comparing the DNA sequence of an organism to that of a reference organism. Genotyping identifies variations in the genetic sequences of organisms, such as single-nucleotide polymorphisms (SNPs). CCUG relies heavily on genotyping for determining the identifications of microorganisms that we receive from the hospital and from deposits to the collection. CCUG is using sequencing of the 16S rRNA gene and other housekeeping genes for most of the identifications we do.
We extract DNA and perform PCR and Sanger-based sequencing in the CCUG laboratory. For most of our gene sequencing protocols, we use a rapid boiling-prep method, which may include enzymatic pre-treatments or bead beating, depending upon the microorganism. PCR-amplification of the target genes are employed, using a library of taxon- and gene-specific amplification primers that have been accumulated over the years. The PCR-products are purified and used as templates for Taq cycle-sequencing with fluorescent dye-labelled dideoxynucleotides, using Applied Biosystems sequencing technology (Thermo Fisher Scientific).
CCUG is employing whole-genome sequencing (WGS) for more definitive identifications as well as analyses of antibiotic resistance and virulence. DNA extraction for WGS analyses is performed in the CCUG, using automated systems or a modified Marmur extraction protocol. The library preparation and sequencing, using Ion-Torrent NGS technology (Thermo Fisher Scientific), are performed at the NGS-Lab of Clinical Microbiology, Sahlgrenska University Hospital.
Today the Sanger sequencing protocol for the 16S rRNA gene for the identification of bacteria and recA for the identification of Achromobacter species are accredited (ISO 15189), and we have an extensive list of different housekeeping genes that we use for the identification of different taxa.
The identification of a species is always dependant on the similarities to the Type strains of species and significantly lower similarities to the Type strains of all other species. We use primarily GenBank, NCBI for matching and comparisons and critically searching for Type strain matches. For reliable species identifications, using the 16S rRNA gene, the similarity must be higher than 98.6% to the Type strain of a given species and, at least, 0.5% lower than the best match to all other species. If these limits are not reached, we either determine the sequence of another housekeeping gene or we report that we are unable to make a species-level identification, and an identification to a genus-level or complex /group is given.