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Antigenic variation occurs in a broad range of species. This process resembles gene conversion in that variant DNA is unidirectionally transferred from partial gene copies (or silent loci) into an expression locus. Previous studies of antigenic variation have involved the amplification and sequencing of individual genes from hundreds of colonies. Using the  pilE  gene from  Neisseria gonorrhoeae  we have demonstrated that it is possible to use PCR amplification, followed by high-throughput DNA sequencing and a novel assembly process, to detect individual antigenic variation events. The ability to detect these events was much greater than has previously been possible. In  N. gonorrhoeae  most silent loci contain multiple partial gene copies. Here we show that there is a bias towards using the copy at the 3′ end of the silent loci (copy 1) as the donor sequence. The  pilE  gene of  N. gonorrhoeae  and some strains of  Neisseria meningitidis  encode class I pilin, but strains of  N. meningitidis  from clonal complexes 8 and 11 encode a class II pilin. We have confirmed that the class II pili of meningococcal strain FAM18 (clonal complex 11) are non-variable, and this is also true for the class II pili of strain NMB from clonal complex 8. In addition when a gene encoding class I pilin was moved into the meningococcal strain NMB background there was no evidence of antigenic variation. Finally we investigated several members of the  opa  gene family of  N. gonorrhoeae,  where it has been suggested that limited variation occurs. Variation was detected in the  opaK  gene that is located close to  pilE , but not at the  opaJ  gene located elsewhere on the genome. The approach described here promises to dramatically improve studies of the extent and nature of antigenic variation systems in a variety of species.

The Use of High-Throughput DNA Sequencing in the Investigation of Antigenic Variation: Application to Neisseria Species