Genotyping strategy matters when analyzing hypervariable major histocompatibility complex-Experience from a passerine bird.

Genotyping of classical major histocompatibility complex (MHC) genes is challenging when they are hypervariable and occur in multiple copies. In this study, we used several different approaches to genotype the moderately variable MHC class I exon 3 (MHCIe3) and the highly polymorphic MHC class II exon 2 (MHCIIβe2) in the bluethroat (). Two family groups (eight individuals) were sequenced in replicates at both markers using Ion Torrent technology with both a single- and a dual-indexed primer structure. Additionally, MHCIIβe2 was sequenced on Illumina MiSeq. Allele calling was conducted by modifications of the pipeline developed by Sommer et al. (BMC Genomics, 14, 2013, 542) and the software AmpliSAS. While the different genotyping strategies gave largely consistent results for MHCIe3, with a maximum of eight alleles per individual, MHCIIβe2 was remarkably complex with a maximum of 56 MHCIIβe2 alleles called for one individual. Each genotyping strategy detected on average 50%-82% of all MHCIIβe2 alleles per individual, but dropouts were largely allele-specific and consistent within families for each strategy. The discrepancies among approaches indicate PCR biases caused by the platform-specific primer tails. Further, AmpliSAS called fewer alleles than the modified Sommer pipeline. Our results demonstrate that allelic dropout is a significant problem when genotyping the hypervariable MHCIIβe2. As these genotyping errors are largely nonrandom and method-specific, we caution against comparing genotypes across different genotyping strategies. Nevertheless, we conclude that high-throughput approaches provide a major advance in the challenging task of genotyping hypervariable MHC loci, even though they may not reveal the complete allelic repertoire.