Performance comparison of different approaches in genotyping MHC-DRB: the contrast between single-locus and multi-locus species

Abstract

Major histocompatibility complex (MHC) genes are widely recognised as valuable markers for wildlife genetic studies given their extreme polymorphism and functional importance in fitness-related traits. Newly developed genotyping methods, which rely on the use of next-generation sequencing (NGS), are gradually replacing traditional cloning and Sanger sequencing methods in MHC genotyping studies. Allele calling in NGS methods remains challenging due to extreme polymorphism and locus multiplication in the MHC coupled with allele amplification bias and generation of artificial sequences. In this study, we compared the performance of molecular cloning with Illumina and Ion Torrent NGS sequencing in MHC-DRB genotyping of single-locus species (roe deer) and species with multiple DRB loci (red deer) in an attempt to adopt a reliable and straightforward method for future deer genotyping projects. For high-throughput data processing, we applied the Amplicon Sequencing Analysis Tools aiming to avoid complex bioinformatic analyses. Our results show that all methods work similarly well in roe deer, but we demonstrate non-consistency in results across methods in red deer. With Illumina sequencing, we detected a maximum number of alleles in 10 red deer individuals (42), regardless of the number of reads used for data analysis. Other methods were somewhat less accurate as they scored 69–81% of alleles detected with Illumina sequencing.