Evaluation of two approaches to genotyping major histocompatibility complex class I in a passerine-CE-SSCP and 454 pyrosequencing
Abstract
MHC is linked to individual fitness and is important in evolutionary ecology and conservation genetics. Gene duplication and positive selection usually lead to high polymorphism in the MHC region, making genotyping of MHC a challenging task.
We compare the performance of two methods for MHC class I genotyping in a passerine with highly duplicated MHC class I genes: CE-SSCP analysis and 454 GS FLX Titanium pyrosequencing. According to our findings, the number of MHC alleles detected by CE-SSCP is significantly lower than detected by 454.
To resolve discrepancies between the two methods, we cloned and Sanger sequenced a MHC class I amplicon for an individual with high number of alleles. We found perfect congruence between cloning/Sanger sequencing and 454.
Thus, in case of multi-locus amplification, CE-SSCP considerably underestimates individual MHC diversity. Numbers of alleles detected by both methods are significantly correlated, although the correlation is weak (r=0.32).