Meningococcal typing serves a variety of purposes (16
). Whereas the so-called fine type, which includes serogroup, PorA type, and FetA type (11
), and the sequence type provide a framework for strain discrimination and phylogenetic assignment (19
), prediction of antimicrobial resistance (37
) and vaccine strain coverage (18
) assists with clinical management and preventive measures. Vaccine antigen typing especially needs to be flexible due to various approaches to meningococcus serogroup B subcapsular antigen vaccine development (42
In Europe, for logistic and financial reasons, many national reference laboratories seem to be overburdened by the effort to fulfill the typing requirements of the European Centre for Disease Control and Prevention (ECDC), which include serogroup, PorA and FetA types, and the sequence type (16
). Running of seven PCRs and 14 sequencing reactions for a complete MLST scheme, despite all possibilities of automation, is an obvious challenge if done on hundreds of isolates. Not surprisingly, with the advent of deep sequencing technologies such as the Illumina technology (5
) and 454 technology (29
), interest in replacement of time-consuming PCR combined with Sanger sequencing by genome data acquisition has increased considerably but until now was hampered by cost and demands for rapid data processing. A further major advantage of whole-genome sequencing would be to archive abundant strain information for rapid retrospective reanalysis, if necessary.
This report describes the first application of 200 base reads for the Ion PGM platform. Increased reading length improves substantially the results of the de novo assembly. Longer reading length and scalability are the discriminating features in comparison to the just-released Illumina MiSeq platform. Because of PGM's pyrosequencing procedure, quite a high number of indel errors due to homopolymers were observed. However, indels did not affect the results of this study, and further editing was not necessary. Furthermore, this systematic error can, in principle, be well compensated when employing a genome-wide gene-by-gene analysis coupled with allele reference databases like BIGSdb. It is important to note that substitution errors, which are not compensable by comparison to allele reference databases, appeared at a very low rate.
The semiconductor technology and the related 454 technology are sensitive to homopolymeric tracts. We therefore wondered to what extent homopolymeric tracts in the meningococcal genome will cause difficulties for the typing approach. In fact, the meningococcal genome contains a variety of long intra- and intergenic homopolymeric tracts whose erroneous replication causes phase variation (31
). Fortunately, none of the typing loci addressed in this study belonged to the category of contingency loci. Most surprisingly, the de novo
sequence was highly robust and no further editing of sequences was required. Furthermore, the detection of numerous indels, which were incorrectly identified by the novel sequencing technology, was without consequence for the typing of three strains, as no indels were identified in the numerous loci addressed for typing. This is an important finding, as manual editing consecutive to genome sequence assembly would otherwise be detrimental to broad application of genomic typing.
The availability of the Neisseria
sequence typing home page powered by BIGSdb greatly facilitated the approach (23
). Typing data for thousands of strains have been compiled herein, and the query platform allows the interrogation of multiple loci within a negligible amount of time. The concept behind BIGSdb was a prerequisite for this study. It should be highlighted that comparable database structures are also needed for the application of genomic typing to other organisms of public health importance.
Genomic typing of microorganisms such as Neisseria meningitidis
does not alter the typing philosophies per se
; it simply facilitates data acquisition. Typing of meningococci by MLST with seven loci is sufficient to define the clonal framework of a strain. eMLST of 20 loci mostly serves refined phylogenetic analyses (9
). The combination of serogroup, PorA type, and FetA type has been validated for its discriminatory power to identify possible epidemiological links between cases (10
). For 1,616 strains isolated over a period of 42 months, Simpson's index was high, at 0.963. Discriminatory power does not need to be extended by inclusion of other targets for most purposes. However, genomic typing greatly facilitates the portfolio for on-the-fly analysis of vaccine antigens and antimicrobial resistance genes. It provides the unique possibility of data storage for retrospective analysis of strains with regard to antigen-encoding genes included in future generations of vaccines. Currently, retrospective analyses of this kind are regularly initiated for investigational vaccines and require novel repetitive sequencing of hundreds of strains (4
). It will greatly facilitate the search for specific markers in the event of emergence of a new, highly virulent clone, such as the so-called electrophoretic type 15 (ET-15) clone (1
), which is typed by a single nucleotide polymorphism and an insertion element (13
). Maintaining physical strain collections will continue to be an indispensable requirement in the future, because besides the antigenic variant, protein expression is another predictor of strain coverage by bactericidal antibodies elicited by vaccines. Nevertheless, constant readdressing of stored genome data will greatly speed up analyses and facilitate vaccine implementation.
Taken together, our first experience with the use of the Ion Torrent PGM for genomic typing of meningococci was very positive with respect to speed, accuracy, and the lack of necessity of further data editing. For broad use in many reference laboratories, cost for both hardware and consumables must be within the range of average budgets for laboratories. Laboratory partnering is a possible model to offer service to small countries. Alternatively, efficient networking, as exemplified by PulseNet (14
), might help to distribute the technology at a large scale in the future. Furthermore, bioinformatics tools that enable nonspecialists to perform data processing at all steps of the procedure need to be developed.